Literature context: ries Inc., Wilmington, MA, USA, RRID:IMSR_JAX:000664) and ASCâˆ’/âˆ’ (purchased from Mil
Inflammation within the CNS is a major component of many neurodegenerative diseases. A characteristic feature is the generation of microglia-derived factors that play an essential role in the immune response. IL-1β is a pro-inflammatory cytokine released by activated microglia, able to exacerbate injury at elevated levels. In the presence of caspase-1, pro-IL-1β is cleaved to the mature cytokine following NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome activation. Growing evidence suggests that ceramide plays a critical role in NLRP3 inflammasome assembly, however, the relationship between ceramide and inflammasome activation in microglia remains unknown. Here, we investigated potential mechanistic links between ceramide as a modulator of NLRP3 inflammasome assembly and the resulting secretion of IL-1β using small bioactive enzyme stimulators and inhibitors of ceramide signaling in wild-type and apoptosis-associated speck-like protein containing a CARD knockout (ASC-/- ) primary microglia. To induce the expression of inflammasome components, microglia were primed prior to experiments. Treatment with sodium palmitate (PA) induced de novo ceramide synthesis via modulation of its synthesizing protein serine palmitoyl transferase resulting in increased IL-1β secretion in microglia. Exposure of microglia to the serine palmitoyl transferase-inhibitor l-cycloserine significantly prevented PA-induced IL-1β secretion. Application of the ceramide analogue C2 and the sphingosine-1-phosphate-receptor agonist Fingolimod (FTY720) up-regulated levels of IL-1β and cleaved caspase-1 in wild-type microglia, whereas ASC-/- microglia were unaffected. HPA-12 inhibition of ceramide transport did not affect inflammasome activation. Taken together, our findings reveal a critical role for ceramide as a positive modulator of NLRP3 inflammasome assembly and the resulting release of IL-1β.
Literature context: old, mean weight 25.4 Â± 0.6 g, RRID:IMSR_JAX:000664) were obtained from Charles Riv
Cocaine is a recreational drug of abuse that binds to the dopamine transporter, preventing reuptake of dopamine into pre-synaptic terminals. The increased presence of synaptic dopamine results in stimulation of both pre- and post-synaptic dopamine receptors, considered an important mechanism by which cocaine elicits its reinforcing properties. However, the effects of acute cocaine administration on pre-synaptic dopamine function remain unclear. Non-invasive imaging techniques such as positron emission tomography have revealed impaired pre-synaptic dopamine function in chronic cocaine users. Similar impairments have been seen in animal studies, with microdialysis experiments indicating decreased basal dopamine release. Here we use micro positron emission tomography imaging techniques in mice to measure dopamine synthesis capacity and determine the effect of acute cocaine administration of pre-synaptic dopamine function. We show that a dose of 20 mg/kg cocaine is sufficient to elicit hyperlocomotor activity, peaking 15-20 min post treatment (p < 0.001). However, dopamine synthesis capacity in the striatum was not significantly altered by acute cocaine treatment (KiCer: 0.0097 per min vs. 0.0112 per min in vehicle controls, p > 0.05). Furthermore, expression levels of two key enzymes related to dopamine synthesis, tyrosine hydroxylase and aromatic l-amino acid decarboxylase, within the striatum of scanned mice were not significantly affected by acute cocaine pre-treatment (p > 0.05). Our findings suggest that while the regulation of dopamine synthesis and release in the striatum have been shown to change with chronic cocaine use, leading to a reduced basal tone, these adaptations to pre-synaptic dopaminergic neurons are not initiated following a single exposure to the drug.
Literature context: RID:IMSR_RRID:JAX:000664). In all experiments, we used m
Functional deficits in sensory systems are commonly noted in neurodevelopmental disorders, such as the Rett syndrome (RTT). Defects in methyl CpG binding protein gene (MECP2) largely accounts for RTT. Manipulations of the Mecp2 gene in mice provide useful models to probe into various aspects of brain development associated with the RTT. In this study, we focused on the somatosensory cortical phenotype in the Bird mouse model of RTT. We used voltage-sensitive dye imaging to evaluate whisker sensory evoked activity in the barrel cortex of mice. We coupled this functional assay with morphological analyses in postnatal mice and investigated the dendritic differentiation of barrel neurons and individual thalamocortical axon (TCA) arbors that synapse with them. We show that in Mecp2-deficient male mice, whisker-evoked activity is roughly topographic but weak in the barrel cortex. At the morphological level, we find that TCA arbors fail to develop into discrete, concentrated patches in barrel hollows, and the complexity of the dendritic branches in layer IV spiny stellate neurons is reduced. Collectively, our results indicate significant structural and functional impairments in the barrel cortex of the Bird mouse line, a popular animal model for the RTT. Such structural and functional anomalies in the primary somatosensory cortex may underlie orofacial tactile sensitivity issues and sensorimotor stereotypies characteristic of RTT.
Literature context: der were crossed with C57BL/6J (RRID:IMSR_JAX:000664, The Jackson Laboratory), and m
Detailed anatomical tracing and mapping of the viscerotopic organization of the vagal motor nuclei has provided insight into autonomic function in health and disease. To further define specific cellular identities, we paired information based on visceral connectivity with a cell-type specific marker of a subpopulation of neurons in the dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus (nAmb) that express the autism-associated MET receptor tyrosine kinase. As gastrointestinal disturbances are common in children with autism spectrum disorder (ASD), we sought to define the relationship between MET-expressing (MET+) neurons in the DMV and nAmb, and the gastrointestinal tract. Using wholemount tissue staining and clearing, or retrograde tracing in a METEGFP transgenic mouse, we identify three novel subpopulations of EGFP+ vagal brainstem neurons: (a) EGFP+ neurons in the nAmb projecting to the esophagus or laryngeal muscles, (b) EGFP+ neurons in the medial DMV projecting to the stomach, and (b) EGFP+ neurons in the lateral DMV projecting to the cecum and/or proximal colon. Expression of the MET ligand, hepatocyte growth factor (HGF), by tissues innervated by vagal motor neurons during fetal development reveal potential sites of HGF-MET interaction. Furthermore, similar cellular expression patterns of MET in the brainstem of both the mouse and nonhuman primate suggests that MET expression at these sites is evolutionarily conserved. Together, the data suggest that MET+ neurons in the brainstem vagal motor nuclei are anatomically positioned to regulate distinct portions of the gastrointestinal tract, with implications for the pathophysiology of gastrointestinal comorbidities of ASD.
Literature context: muscle of male C57BL/6 J mice (https://www.jax.org/strain/000664). Animals were supplied by the
Acetazolamide (AZ), a molecule frequently used to treat different neurological syndromes, is an inhibitor of the carbonic anhydrase (CA), an enzyme that regulates pH inside and outside cells. We combined fluorescent FM styryl dyes and electrophysiological techniques at ex vivo levator auris longus neuromuscular junctions (NMJs) from mice to investigate the modulation of synaptic transmission and vesicle recycling by AZ. Transmitter release was minimally affected by AZ, as evidenced by evoked and spontaneous end-plate potential measurements. However, optical evaluation with FM-styryl dyes of vesicle exocytosis elicited by 50 Hz stimuli showed a strong reduction in fluorescence loss in AZ treated NMJ, an effect that was abolished by bathing the NMJ in Hepes. The remaining dye was quenched by bromophenol, a small molecule capable of diffusing inside vesicles. Furthermore, in transgenic mice expressing Synaptophysin-pHluorin (SypHy), the fluorescence responses of motor nerve terminals to a 50 Hz train of stimuli was decrease to a 50% of controls in the presence of AZ. Immunohistochemistry experiments to evaluate the state of the Myosin light chain kinase (MLCK), an enzyme involved in vesicle recycling, demonstrated that MLCK phosphorylation was much stronger in the presence than AZ than in its absence in 50 Hz stimulated NMJs. We postulate that AZ, via cytosol acidification and activation of MLCK, shifts synaptic vesicle recycling to a fast (kiss-and-run) mode, which changes synaptic performance. These changes may contribute to the therapeutic action reported in many neurological syndromes like ataxia, epilepsy, and migraine.
Literature context: kcrossed to C57BL/6J (Stock No. 000664, https://www.jax.org/strain/000
Oligodendrocytes are essential regulators of axonal energy homeostasis and electrical conduction and emerging target cells for restoration of neurological function. Here we investigate the role of protease activated receptor 2 (PAR2), a unique protease activated G protein-coupled receptor, in myelin development and repair using the spinal cord as a model. Results demonstrate that genetic deletion of PAR2 accelerates myelin production, including higher proteolipid protein (PLP) levels in the spinal cord at birth and higher levels of myelin basic protein and thickened myelin sheaths in adulthood. Enhancements in spinal cord myelin with PAR2 loss-of-function were accompanied by increased numbers of Olig2- and CC1-positive oligodendrocytes, as well as in levels of cyclic adenosine monophosphate (cAMP), and extracellular signal related kinase 1/2 (ERK1/2) signaling. Parallel promyelinating effects were observed after blocking PAR2 expression in purified oligodendrocyte cultures, whereas inhibiting adenylate cyclase reversed these effects. Conversely, PAR2 activation reduced PLP expression and this effect was prevented by brain derived neurotrophic factor (BDNF), a promyelinating growth factor that signals through cAMP. PAR2 knockout mice also showed improved myelin resiliency after traumatic spinal cord injury and an accelerated pattern of myelin regeneration after focal demyelination. These findings suggest that PAR2 is an important controller of myelin production and regeneration, both in the developing and adult spinal cord.
Literature context: oratory Cat#000664; RRID:IMSR_JAX:000664 Mouse: C57BL/6-Trim21tm1Hm/J Th
Methods for the targeted disruption of protein function have revolutionized science and greatly expedited the systematic characterization of genes. Two main approaches are currently used to disrupt protein function: DNA knockout and RNA interference, which act at the genome and mRNA level, respectively. A method that directly alters endogenous protein levels is currently not available. Here, we present Trim-Away, a technique to degrade endogenous proteins acutely in mammalian cells without prior modification of the genome or mRNA. Trim-Away harnesses the cellular protein degradation machinery to remove unmodified native proteins within minutes of application. This rapidity minimizes the risk that phenotypes are compensated and that secondary, non-specific defects accumulate over time. Because Trim-Away utilizes antibodies, it can be applied to a wide range of target proteins using off-the-shelf reagents. Trim-Away allows the study of protein function in diverse cell types, including non-dividing primary cells where genome- and RNA-targeting methods are limited.
Literature context: 2) The Jackson Laboratories Cat#JAX:000664; RRID: IMSR_JAX:000664
Granulocyte-monocyte progenitors (GMPs) and monocyte-dendritic cell progenitors (MDPs) produce monocytes during homeostasis and in response to increased demand during infection. Both progenitor populations are thought to derive from common myeloid progenitors (CMPs), and a hierarchical relationship (CMP-GMP-MDP-monocyte) is presumed to underlie monocyte differentiation. Here, however, we demonstrate that mouse MDPs arose from CMPs independently of GMPs, and that GMPs and MDPs produced monocytes via similar but distinct monocyte-committed progenitors. GMPs and MDPs yielded classical (Ly6Chi) monocytes with gene expression signatures that were defined by their origins and impacted their function. GMPs produced a subset of "neutrophil-like" monocytes, whereas MDPs gave rise to a subset of monocytes that yielded monocyte-derived dendritic cells. GMPs and MDPs were also independently mobilized to produce specific combinations of myeloid cell types following the injection of microbial components. Thus, the balance of GMP and MDP differentiation shapes the myeloid cell repertoire during homeostasis and following infection.
Literature context: th wild-type females (C57BL/6J; RRID:IMSR_JAX:000664). Yapflox/flox;Tazflox/flox [RR
Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are functionally redundant transcriptional regulators that are downstream effectors of the Hippo signaling pathway. They act as major regulators of stem cell maintenance, cell growth, and differentiation. To characterize their roles in the adrenal cortex, we generated a mouse model in which Yap and Taz were conditionally deleted in steroidogenic cells (Yapflox/flox;Tazflox/flox;Nr5a1cre/+). Male Yapflox/flox;Tazflox/flox;Nr5a1cre/+ mice were characterized by an age-dependent degeneration of the adrenal cortex associated with an increase in apoptosis and a progressive reduction in the expression levels of steroidogenic genes. Evaluation of the expression levels of stem and progenitor cell population markers in the adrenal glands of Yapflox/flox;Tazflox/flox;Nr5a1cre/+ mice also showed the downregulation of sonic hedgehog (Shh), a marker of the subcapsular progenitor cell population. Gross degenerative changes were not observed in the adrenal glands of Yapflox/flox;Tazflox/flox;Nr5a1cre/+ females, although steroidogenic capacity and Shh expression were reduced, suggesting that mechanisms of adrenocortical maintenance are sex specific. These results define a crucial role for YAP and TAZ in the maintenance of the postnatal adrenal cortex.
Literature context: RRID:IMSR_JAX:000664
Regulation of rod gene expression has emerged as a potential therapeutic strategy to treat retinal degenerative diseases like retinitis pigmentosa (RP). We previously reported on a small molecule modulator of the rod transcription factor Nr2e3, Photoregulin1 (PR1), that regulates the expression of photoreceptor-specific genes. Although PR1 slows the progression of retinal degeneration in models of RP in vitro, in vivo analyses were not possible with PR1. We now report a structurally unrelated compound, Photoregulin3 (PR3) that also inhibits rod photoreceptor gene expression, potentially though Nr2e3 modulation. To determine the effectiveness of PR3 as a potential therapy for RP, we treated RhoP23H mice with PR3 and assessed retinal structure and function. PR3-treated RhoP23H mice showed significant structural and functional photoreceptor rescue compared with vehicle-treated littermate control mice. These results provide further support that pharmacological modulation of rod gene expression provides a potential strategy for the treatment of RP.
Literature context: Mouse (C57BL/6J) Jackson Labs RRID:IMSR_JAX:000664 Mouse (GlyT2-Cre) MMRRC 030730-
Action potentials clustered into high-frequency bursts play distinct roles in neural computations. However, little is known about ionic currents that control the duration and probability of these bursts. We found that, in cartwheel inhibitory interneurons of the dorsal cochlear nucleus, the likelihood of bursts and the interval between their spikelets were controlled by Ca2+ acting across two nanodomains, one between plasma membrane P/Q Ca2+ channels and endoplasmic reticulum (ER) ryanodine receptors and another between ryanodine receptors and large-conductance, voltage- and Ca2+-activated K+ (BK) channels. Each spike triggered Ca2+-induced Ca2+ release (CICR) from the ER immediately beneath somatic, but not axonal or dendritic, plasma membrane. Moreover, immunolabeling demonstrated close apposition of ryanodine receptors and BK channels. Double-nanodomain coupling between somatic plasma membrane and hypolemmal ER cisterns provides a unique mechanism for rapid control of action potentials on the millisecond timescale.
Literature context: Jackson Laboratory (CLEA Japan) RRID:JAX:000664 Mouse: Il1râˆ’/âˆ’: B6.129S7-Il1r1t
Staphylococcus aureus commonly colonizes the epidermis, but the mechanisms by which the host senses virulent, but not commensal, S. aureus to trigger inflammation remain unclear. Using a murine epicutaneous infection model, we found that S. aureus-expressed phenol-soluble modulin (PSM)α, a group of secreted virulence peptides, is required to trigger cutaneous inflammation. PSMα induces the release of keratinocyte IL-1α and IL-36α, and signaling via IL-1R and IL-36R was required for induction of the pro-inflammatory cytokine IL-17. The levels of released IL-1α and IL-36α, as well as IL-17 production by γδ T cells and ILC3 and neutrophil infiltration to the site of infection, were greatly reduced in mice with total or keratinocyte-specific deletion of the IL-1R and IL-36R signaling adaptor Myd88. Further, Il17a-/-f-/- mice showed blunted S. aureus-induced inflammation. Thus, keratinocyte Myd88 signaling in response to S. aureus PSMα drives an IL-17-mediated skin inflammatory response to epicutaneous S. aureus infection.
Literature context: ttee. Adult male C57BL/6J mice (RRID:IMSR_JAX:000664) (8- to 10-week-old; 25-30 g ea
Idiopathic Parkinson's disease and manganese-induced atypical parkinsonism are characterized by movement disorder and nigrostriatal pathology. Although clinical features, brain region involved and responsiveness to levodopa distinguish both, differences at the neuronal level are largely unknown. We studied the morphological, neurophysiological and molecular differences in dopaminergic neurons exposed to the Parkinson's disease toxin 1-methyl-4-phenylpyridinium ion (MPP+ ) and manganese (Mn), followed by validation in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and Mn mouse models. Morphological analysis highlighted loss of neuronal processes in the MPP+ and not the Mn model. Cellular network dynamics of dopaminergic neurons characterized by spike frequency and inter-spike intervals indicated major neuronal population (~ 93%) with slow discharge rates (0-5 Hz). While MPP+ exposure suppressed the firing of these neurons, Mn neither suppressed nor elevated the neuronal activity. High-throughput transcriptomic analysis revealed up-regulation of 694 and 603 genes and down-regulation of 428 and 255 genes in the MPP+ and Mn models respectively. Many differentially expressed genes were unique to either models and contributed to neuroinflammation, metabolic/mitochondrial function, apoptosis and nuclear function, synaptic plasticity, neurotransmission and cytoskeleton. Analysis of the Janus kinase-signal transducer and activator of transcription pathway with implications for neuritogenesis and neuronal proliferation revealed contrasting profile in both models. Genome-wide DNA methylomics revealed differences between both models and substantiated the epigenetic basis of the difference in the Janus kinase-signal transducer and activator of transcription pathway. We conclude that idiopathic Parkinson's disease and atypical parkinsonism have divergent neurotoxicological manifestation at the dopaminergic neuronal level with implications for pathobiology and evolution of novel therapeutics. Cover Image for this issue: doi. 10.1111/jnc.13821.
Literature context: iceThe Jackson LaboratoriesCat# 000664Mouse: TCRÎ³Î´GFP: C57BL/6-Trdctm1
Intestinal intraepithelial lymphocytes (IELs) are located at the critical interface between the intestinal lumen, which is chronically exposed to food and microbes, and the core of the body. Using high-resolution microscopy techniques and intersectional genetic tools, we investigated the nature of IEL responses to luminal microbes. We observed that TCRγδ IELs exhibit unique microbiota-dependent location and movement patterns in the epithelial compartment. This behavioral pattern quickly changes upon exposure to different enteric pathogens, resulting in increased interepithelial cell (EC) scanning, expression of antimicrobial genes, and glycolysis. Both dynamic and metabolic changes to γδ IEL depend on pathogen sensing by ECs. Direct modulation of glycolysis is sufficient to change γδ IEL behavior and susceptibility to early pathogen invasion. Our results uncover a coordinated EC-IEL response to enteric infections that modulates lymphocyte energy utilization and dynamics and supports maintenance of the intestinal epithelial barrier. VIDEO ABSTRACT.
Literature context: ). C57BL/6J mice (stock 000664; RRID:IMSR_JAX:000664) mice, B6;129-Gt(ROSA)26Sortm1(
Developmental morphogenesis, tissue injury, and oncogenic transformation can cause the detachment of epithelial cells. These cells are eliminated by a specialized form of apoptosis (anoikis). While the processes that contribute to this form of cell death have been studied, the underlying mechanisms remain unclear. Here, we tested the role of the cJUN NH2-terminal kinase (JNK) signaling pathway using murine models with compound JNK deficiency in mammary and kidney epithelial cells. These studies demonstrated that JNK is required for efficient anoikis in vitro and in vivo. Moreover, JNK-promoted anoikis required pro-apoptotic members of the BCL2 family of proteins. We show that JNK acts through a BAK/BAX-dependent apoptotic pathway by increasing BIM expression and phosphorylating BMF, leading to death of detached epithelial cells.
Literature context: R_JAX:026033C57BL/6JMus musculusIMSR_JAX:000664OligonucleotidesFATP1CACCGGTCGTT
Elevated reactive oxygen species (ROS) induce the formation of lipids in neurons that are transferred to glia, where they form lipid droplets (LDs). We show that glial and neuronal monocarboxylate transporters (MCTs), fatty acid transport proteins (FATPs), and apolipoproteins are critical for glial LD formation. MCTs enable glia to secrete and neurons to absorb lactate, which is converted to pyruvate and acetyl-CoA in neurons. Lactate metabolites provide a substrate for synthesis of fatty acids, which are processed and transferred to glia by FATP and apolipoproteins. In the presence of high ROS, inhibiting lactate transfer or lowering FATP or apolipoprotein levels decreases glial LD accumulation in flies and in primary mouse glial-neuronal cultures. We show that human APOE can substitute for a fly glial apolipoprotein and that APOE4, an Alzheimer's disease susceptibility allele, is impaired in lipid transport and promotes neurodegeneration, providing insights into disease mechanisms.
Literature context: e (The Jackson Laboratory, Cat# RRID:JAX:000664); PLP-EGFP mice (University of
The oligodendrocyte density is greater and myelin sheaths are thicker in the adult male mouse brain when compared with females. Here, we show that these sex differences emerge during the first 10 postnatal days, precisely at a stage when a late wave of oligodendrocyte progenitor cells arises and starts differentiating. Androgen levels, analyzed by gas chromatography/tandem-mass spectrometry, were higher in males than in females during this period. Treating male pups with flutamide, an androgen receptor (AR) antagonist, or female pups with 5α-dihydrotestosterone (5α-DHT), revealed the importance of postnatal androgens in masculinizing myelin and their persistent effect into adulthood. A key role of the brain AR in establishing the sexual phenotype of myelin was demonstrated by its conditional deletion. Our results uncover a new persistent effect of postnatal AR signaling, with implications for neurodevelopmental disorders and sex differences in multiple sclerosis.
Literature context: on Laboratory Stock No: 000664; RRID:IMSR_JAX:000664 Mouse: BALB/cJ Jackson Laborato
Staphylococcus aureus colonization contributes to skin inflammation in diseases such as atopic dermatitis, but the signaling pathways involved are unclear. Herein, epicutaneous S. aureus exposure to mouse skin promoted MyD88-dependent skin inflammation initiated by IL-36, but not IL-1α/β, IL-18, or IL-33. By contrast, an intradermal S. aureus challenge promoted MyD88-dependent host defense initiated by IL-1β rather than IL-36, suggesting that different IL-1 cytokines trigger MyD88 signaling depending on the anatomical depth of S. aureus cutaneous exposure. The bacterial virulence factor PSMα, but not α-toxin or δ-toxin, contributed to the skin inflammation, which was driven by IL-17-producing γδ and CD4+ T cells via direct IL-36R signaling in the T cells. Finally, adoptive transfer of IL-36R-expressing T cells to IL-36R-deficient mice was sufficient for mediating S. aureus-induced skin inflammation. Together, this study defines a previously unknown pathway by which S. aureus epicutaneous exposure promotes skin inflammation involving IL-36R/MyD88-dependent IL-17 T cell responses.
Literature context: atory RRID:JAX:000664 Mouse: Lrig1CreERT2: Lrig1tm1.1
Modern single-cell technologies allow multiplexed sampling of cellular states within a tissue. However, computational tools that can infer developmental cell-state transitions reproducibly from such single-cell data are lacking. Here, we introduce p-Creode, an unsupervised algorithm that produces multi-branching graphs from single-cell data, compares graphs with differing topologies, and infers a statistically robust hierarchy of cell-state transitions that define developmental trajectories. We have applied p-Creode to mass cytometry, multiplex immunofluorescence, and single-cell RNA-seq data. As a test case, we validate cell-state-transition trajectories predicted by p-Creode for intestinal tuft cells, a rare, chemosensory cell type. We clarify that tuft cells are specified outside of the Atoh1-dependent secretory lineage in the small intestine. However, p-Creode also predicts, and we confirm, that tuft cells arise from an alternative, Atoh1-driven developmental program in the colon. These studies introduce p-Creode as a reliable method for analyzing large datasets that depict branching transition trajectories.
Literature context: C57BL/6J The Jackson Laboratory RRID:IMSR_JAX:000664 Mouse: MOR-KO: B6.129S2-Oprm1tm
Biased agonism has been proposed as a means to separate desirable and adverse drug responses downstream of G protein-coupled receptor (GPCR) targets. Herein, we describe structural features of a series of mu-opioid-receptor (MOR)-selective agonists that preferentially activate receptors to couple to G proteins or to recruit βarrestin proteins. By comparing relative bias for MOR-mediated signaling in each pathway, we demonstrate a strong correlation between the respiratory suppression/antinociception therapeutic window in a series of compounds spanning a wide range of signaling bias. We find that βarrestin-biased compounds, such as fentanyl, are more likely to induce respiratory suppression at weak analgesic doses, while G protein signaling bias broadens the therapeutic window, allowing for antinociception in the absence of respiratory suppression.
Literature context: 12â€¯weeks of age). C57BL/6J (WT; RRID:IMSR_JAX:000664) or B6.129P2-Il10tm1Cgn/J (IL-1
Studies show that spinal (intrathecal; i.t.) interleukin-10 (IL-10) gene therapy reverses neuropathic pain in animal models, and co-administration with the mannose receptor (MR; CD206) ligand d-mannose (DM) greatly improves therapeutic efficacy. However, the actions of endogenous IL-10 may be required for enduring pain control observed following i.t. IL-10 gene therapy, potentially narrowing the application of this non-viral transgene delivery approach. Here, we show that i.t. application of naked plasmid DNA expressing the IL-10 transgene co-injected with DM (DM/pDNA-IL-10) for the treatment of peripheral neuropathic pain in IL-10 deficient (IL-10 KO) mice results in a profound and prolonged bilateral pain suppression. Neuropathic pain is induced by unilateral sciatic chronic constriction injury (CCI), and while enduring relief of light touch sensitivity (mechanical allodynia) in both wild type (WT) and IL-10 KO mice was observed following DM/pDNA-IL-10 co-therapy, transient reversal from allodynia was observed following i.t. DM alone. In stably pain-relieved IL-10 KO mice given DM/pDNA-IL-10, mRNA for the IL-10 transgene is detected in the cauda equina and ipsilateral dorsal root ganglia (DRG), but not the lumbar spinal cord. Further, DM/pDNA-IL-10 application increases anti-inflammatory TGF-β1 and decreases pro-inflammatory TNF mRNA in the ipsilateral DRG compared to allodynic controls. Additionally, DM/pDNA-IL-10 treated mice exhibit decreased spinal pro-inflammatory mRNA expression for TNF, CCL2 (MCP-1), and for the microglial-specific marker TMEM119. Similarly, DM/pDNA-IL-10 treatment decreases immunoreactivity for the astrocyte activation marker GFAP in lumbar spinal cord dorsal horn. Despite transient reversal and early return to allodynia in DM-treated mice, lumbar spinal cord revealed elevated TNF, CCL2 and TMEM119 mRNA levels. Both MR (CD206) and IL-10 receptor mRNAs are increased in the DRG following CCI manipulation independent of injection treatment, suggesting that pathological conditions stimulate upregulation and availability of relevant receptors in critical anatomical regions required for the therapeutic actions of the DM/pDNA-IL-10 co-therapy. Taken together, the current report demonstrates that non-viral DM/pDNA-IL-10 gene therapy does not require endogenous IL-10 for enduring relief of peripheral neuropathic pain and does not require direct contact with the spinal cord dorsal horn for robust and enduring relief of neuropathic pain. Spinal non-viral DM/pDNA-IL-10 co-therapy may offer a framework for the development of non-viral gene therapeutic approaches for other diseases of the central nervous system.
Literature context: 140-NB4). Eight-week-old female C57BL/6 mice were obtained from the Jac
Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of skin and internal organs. Protein tyrosine phosphatases have received little attention in the study of SSc or fibrosis. Here, we show that the tyrosine phosphatase PTP4A1 is highly expressed in fibroblasts from patients with SSc. PTP4A1 and its close homolog PTP4A2 are critical promoters of TGFβ signaling in primary dermal fibroblasts and of bleomycin-induced fibrosis in vivo. PTP4A1 promotes TGFβ signaling in human fibroblasts through enhancement of ERK activity, which stimulates SMAD3 expression and nuclear translocation. Upstream from ERK, we show that PTP4A1 directly interacts with SRC and inhibits SRC basal activation independently of its phosphatase activity. Unexpectedly, PTP4A2 minimally interacts with SRC and does not promote the SRC-ERK-SMAD3 pathway. Thus, in addition to defining PTP4A1 as a molecule of interest for TGFβ-dependent fibrosis, our study provides information regarding the functional specificity of different members of the PTP4A subclass of phosphatases.
Literature context: ild type mice Jackson Labs IMSR_RRID:JAX:000664 genetic reagent (mus musculus)
A leading theory holds that neurodevelopmental brain disorders arise from imbalances in excitatory and inhibitory (E/I) brain circuitry. However, it is unclear whether this one-dimensional model is rich enough to capture the multiple neural circuit alterations underlying brain disorders. Here, we combined computational simulations with analysis of in vivo two-photon Ca2+ imaging data from somatosensory cortex of Fmr1 knock-out (KO) mice, a model of Fragile-X Syndrome, to test the E/I imbalance theory. We found that: (1) The E/I imbalance model cannot account for joint alterations in the observed neural firing rates and correlations; (2) Neural circuit function is vastly more sensitive to changes in some cellular components over others; (3) The direction of circuit alterations in Fmr1 KO mice changes across development. These findings suggest that the basic E/I imbalance model should be updated to higher dimensional models that can better capture the multidimensional computational functions of neural circuits.
Literature context: knockout mice (C57BL/6J based; RRID:IMSR_JAX:000664) were originally supplied by Ch
Serotonergic and glutamatergic neurons of median raphe region (MRR) play a pivotal role in the modulation of affective and cognitive functions. These neurons synapse both onto themselves and remote cortical areas. P2X7 receptors (P2rx7) are ligand gated ion channels expressed by central presynaptic excitatory nerve terminals and involved in the regulation of neurotransmitter release. P2rx7s are implicated in various neuropsychiatric conditions such as schizophrenia and depression. Here we investigated whether 5-HT release released from the hippocampal terminals of MRR is subject to modulation by P2rx7s. To achieve this goal, an optogenetic approach was used to selectively activate subpopulation of serotonergic terminals derived from the MRR locally, and one of its target area, the hippocampus. Optogenetic activation of neurons in the MRR with 20 Hz was correlated with freezing and enhanced locomotor activity of freely moving mice and elevated extracellular levels of 5-HT, glutamate but not GABA in vivo. Similar optical stimulation (OS) significantly increased [3H]5-HT and [3H]glutamate release in acute MRR and hippocampal slices. We examined spatial and temporal patterns of [3H]5-HT release and the interaction between the serotonin and glutamate systems. Whilst [3H]5-HT release from MRR neurons was [Ca2+]o-dependent and sensitive to TTX, CNQX and DL-AP-5, release from hippocampal terminals was not affected by the latter drugs. Hippocampal [3H]5-HT released by electrical but not OS was subject to modulation by 5- HT1B/D receptors agonist sumatriptan (1 μM), whereas the selective 5-HT1A agonist buspirone (0.1 μM) was without effect. [3H]5-HT released by electrical and optical stimulation was decreased in mice genetically deficient in P2rx7s, and after perfusion with selective P2rx7 antagonists, JNJ-47965567 (0.1 μM), and AZ-10606120 (0.1 μM). Optical and electrical stimulation elevated the extracellular level of ATP. Our results demonstrate for the first time the modulation of 5-HT release from hippocampal MRR terminals by the endogenous activation of P2rx7s. P2rx7 mediated modulation of 5-HT release could contribute to various physiological and pathophysiological phenomena, related to hippocampal serotonergic transmission.
Literature context: genomic DNA) were mated to C57 (RRID:IMSR_JAX:000664 or CD1 (RRID:IMSR_CRL:22) mice
The human distal limbs have a high spatial acuity for noxious stimuli but a low density of pain-sensing neurites. To elucidate mechanisms underlying regional differences in processing nociception, we sparsely traced non-peptidergic nociceptors across the body using a newly generated MrgprdCreERT2 mouse line. We found that mouse plantar paw skin is also innervated by a low density of Mrgprd+ nociceptors, while individual arbors in different locations are comparable in size. Surprisingly, the central arbors of plantar paw and trunk innervating nociceptors have distinct morphologies in the spinal cord. This regional difference is well correlated with a heightened signal transmission for plantar paw circuits, as revealed by both spinal cord slice recordings and behavior assays. Taken together, our results elucidate a novel somatotopic functional organization of the mammalian pain system and suggest that regional central arbor structure could facilitate the "enlarged representation" of plantar paw regions in the CNS.
Literature context: BL/6 background. C57BL/6J mice (RRID:IMSR_JAX:000664) were purchased from Jackson La
Aberrant alternative pre-mRNA splicing (AS) events have been associated with several disorders. However, it is unclear whether deregulated AS directly contributes to disease. Here, we reveal a critical role of the AS regulator epithelial splicing regulator protein 1 (ESRP1) for intestinal homeostasis and pathogenesis. In mice, reduced ESRP1 function leads to impaired intestinal barrier integrity, increased susceptibility to colitis and altered colorectal cancer (CRC) development. Mechanistically, these defects are produced in part by modified expression of ESRP1-specific Gpr137 isoforms differently activating the Wnt pathway. In humans, ESRP1 is downregulated in inflamed biopsies from inflammatory bowel disease patients. ESRP1 loss is an adverse prognostic factor in CRC. Furthermore, generation of ESRP1-dependent GPR137 isoforms is altered in CRC and expression of a specific GPR137 isoform predicts CRC patient survival. These findings indicate a central role of ESRP1-regulated AS for intestinal barrier integrity. Alterations in ESRP1 function or expression contribute to intestinal pathology.
Literature context: titute of Pharmacology (Zurich) RRID:IMSR_JAX:000664 â€ƒâ€ƒâ€ƒâ€ƒ129SVJ Institute of Pharmac
Glycoprotein-deleted rabies virus-mediated monosynaptic tracing has become a standard method for neuronal circuit mapping, and is applied to virtually all parts of the rodent nervous system, including the spinal cord and primary sensory neurons. Here we identified two classes of unmyelinated sensory neurons (nonpeptidergic and C-fiber low-threshold mechanoreceptor neurons) resistant to direct and trans-synaptic infection from the spinal cord with rabies viruses that carry glycoproteins in their envelopes and that are routinely used for infection of CNS neurons (SAD-G and N2C-G). However, the same neurons were susceptible to infection with EnvA-pseudotyped rabies virus in tumor virus A receptor transgenic mice, indicating that resistance to retrograde infection was due to impaired virus adsorption rather than to deficits in subsequent steps of infection. These results demonstrate an important limitation of rabies virus-based retrograde tracing of sensory neurons in adult mice, and may help to better understand the molecular machinery required for rabies virus spread in the nervous system. In this study, mice of both sexes were used.SIGNIFICANCE STATEMENT To understand the neuronal bases of behavior, it is important to identify the underlying neural circuitry. Rabies virus-based monosynaptic tracing has been used to identify neuronal circuits in various parts of the nervous system. This has included connections between peripheral sensory neurons and their spinal targets. These connections form the first synapse in the somatosensory pathway. Here we demonstrate that two classes of unmyelinated sensory neurons, which account for >40% of dorsal root ganglia neurons, display resistance to rabies infection. Our results are therefore critical for interpreting monosynaptic rabies-based tracing in the sensory system. In addition, identification of rabies-resistant neurons might provide a means for future studies addressing rabies pathobiology.
Literature context: C57Bl/6j Jackson Lab, RRID:JAX:000664, IMSR_
We previously reported that 1,3-bisbenzylimidazolium (DBZIM) bromide was neuroprotective for the dopaminergic system in Parkinson's disease (PD) models of rodent, however the underlying mechanism was unclear. We currently further confirmed that DBZIM ameliorated the Parkinsonian motor deficit and protected the nigrostriatal tract from the neurotoxicity of 1-methyl-4-(2'-methylphenyl)-1,2,3,6-tetrahydropyridine (2'-CH3-MPTP) in C57Bl/6 mice. The dopaminergic degeneration in the substantia nigra par compacta (SNc) and striatum was analyzed by immunohistochemistry while the monoamine oxidase B (MAO-B) inhibition effect of DBZIM was determined by enzyme kinetics. DBZIM was at least as effective as the clinically approved anti-PD drug, l-deprenyl (Selegiline), for both neuroprotection and correction of motor deficits. Mechanistically, DBZIM inhibited the specific activity of MAO-B in the striatum and C6 cells without affecting the protein expression. DBZIM directly inhibited the enzymatic activity of a purified MAO-B protein with an estimated Ki value from 780 to 940nM, in par with that of l-deprenyl (970nM). The physical interaction between DBZIM and MAO-B was proven by NMR analysis, with Kd around 21.5-46.8μM. Our binding and modelling data further illustrated that DBZIM is a mixed inhibitor with its binding to active site partially hindering the substrate binding. Therefore, inhibiting MAO-B is a major mechanism through which DBZIM confers neuroprotection for the dopaminergic neurons against 2'-CH3-MPTP toxicity. Remarkably, the post-lesion treatment with DBZIM provided greater anti-parkinsonian and neuroprotective effects than the l-deprenyl. The current study, together with our previous findings in a 6-OHDA PD model, demonstrated that DBZIM is a promising neuroprotectant for PD with anti-MAO-B property.
Literature context: on Laboratory, Bar Harbor, ME) (RRID:IMSR_JAX:000664) were group housed on a 12:12 h
Adrenal chromaffin cells (ACCs) are the neuroendocrine arm of the sympathetic nervous system and key mediators of the physiological stress response. Acetylcholine (ACh) released from preganglionic splanchnic nerves activates nicotinic acetylcholine receptors (nAChRs) on chromaffin cells causing membrane depolarization, opening voltage-gated Ca2+ channels (VGCC), and exocytosis of catecholamines and neuropeptides. The serotonin transporter is expressed in ACCs and interacts with 5-HT1A receptors to control secretion. In addition to blocking the serotonin transporter, some selective serotonin reuptake inhibitors (SSRIs) are also agonists at sigma-1 receptors which function as intracellular chaperone proteins and can translocate to the plasma membrane to modulate ion channels. Therefore, we investigated whether SSRIs and other sigma-1 receptor ligands can modulate stimulus-secretion coupling in ACCs. Escitalopram and fluvoxamine (100 nM to 1 μM) reversibly inhibited nAChR currents. The sigma-1 receptor antagonists NE-100 and BD-1047 also blocked nAChR currents (≈ 50% block at 100 nM) as did PRE-084, a sigma-1 receptor agonist. Block of nAChR currents by fluvoxamine and NE-100 was not additive suggesting a common site of action. VGCC currents were unaffected by the drugs. Neither the increase in cytosolic [Ca2+ ] nor the resulting catecholamine secretion evoked by direct membrane depolarization to bypass nAChRs was altered by fluvoxamine or NE-100. However, both Ca2+ entry and catecholamine secretion evoked by the cholinergic agonist carbachol were significantly reduced by fluvoxamine or NE-100. Together, our data suggest that sigma-1 receptors do not acutely regulate catecholamine secretion. Rather, SSRIs and other sigma-1 receptor ligands inhibit secretion evoked by cholinergic stimulation because of direct block of Ca2+ entry via nAChRs.
Literature context: A colony of C57BL/6J mice (RRID:IMSR_JAX:000664) was established at the Nationa
Mutations of SLC26A4 are a common cause of hearing loss associated with enlargement of the endolymphatic sac (EES). Slc26a4 expression in the developing mouse endolymphatic sac is required for acquisition of normal inner ear structure and function. Here, we show that the mouse endolymphatic sac absorbs fluid in an SLC26A4-dependent fashion. Fluid absorption was sensitive to ouabain and gadolinium but insensitive to benzamil, bafilomycin and S3226. Single-cell RNA-seq analysis of pre- and postnatal endolymphatic sacs demonstrates two types of differentiated cells. Early ribosome-rich cells (RRCs) have a transcriptomic signature suggesting expression and secretion of extracellular proteins, while mature RRCs express genes implicated in innate immunity. The transcriptomic signature of mitochondria-rich cells (MRCs) indicates that they mediate vectorial ion transport. We propose a molecular mechanism for resorption of NaCl by MRCs during development, and conclude that disruption of this mechanism is the root cause of hearing loss associated with EES.
Literature context: ained on a C57BL/6J background (RRID:IMSR_JAX:000664). To identify individual mice c
GPR151 is a G-protein coupled receptor for which the endogenous ligand remains unknown. In the nervous system of vertebrates, its expression is enriched in specific diencephalic structures, where the highest levels are observed in the habenular area. The habenula has been implicated in a range of different functions including behavioral flexibility, decision making, inhibitory control, and pain processing, which makes it a promising target for treating psychiatric and neurological disease. This study aimed to further characterize neurons expressing the Gpr151 gene, by tracing the afferent connectivity of this diencephalic cell population. Using pseudotyped rabies virus in a transgenic Gpr151-Cre mouse line, monosynaptic afferents of habenular and thalamic Gpr151-expressing neuronal populations could be visualized. The habenular and thalamic Gpr151 systems displayed both shared and distinct connectivity patterns. The habenular neurons primarily received input from basal forebrain structures, the bed nucleus of stria terminalis, the lateral preoptic area, the entopeduncular nucleus, and the lateral hypothalamic area. The Gpr151-expressing neurons in the paraventricular nucleus of the thalamus was primarily contacted by medial hypothalamic areas as well as the zona incerta and projected to specific forebrain areas such as the prelimbic cortex and the accumbens nucleus. Gpr151 mRNA was also detected at low levels in the lateral posterior thalamic nucleus which received input from areas associated with visual processing, including the superior colliculus, zona incerta, and the visual and retrosplenial cortices. Knowledge about the connectivity of Gpr151-expressing neurons will facilitate the interpretation of future functional studies of this receptor.
Literature context: C57BL/6JThe Jackson Laboratory#000664Mouse: BALB/cThe Jackson Laborat
Humans do not usually develop effective immunity to Staphylococcus aureus reinfection. Using a murine model that mimics human infection, we show that lack of protective immunity to S. aureus systemic reinfection is associated with robust interleukin-10 (IL-10) production and impaired protective Th17 responses. In dendritic cell co-culture assays, priming with S. aureus promotes robust T cell proliferation, but limits Th cells polarization and production of IL-1β and other cytokines important for Th1 and Th17 differentiation. We show that O-acetylation of peptidoglycan, a mechanism utilized by S. aureus to block bacterial cell wall breakdown, limits the induction of pro-inflammatory signals required for optimal Th17 polarization. IL-10 deficiency in mice restores protective immunity to S. aureus infection, and adjuvancy with a staphylococcal peptidoglycan O-acetyltransferase mutant reduces IL-10, increases IL-1β, and promotes development of IL-17-dependent, Th cell-transferable protective immunity. Overall, our study suggests a mechanism whereby S. aureus modulates cytokines critical for induction of protective Th17 immunity.
Literature context: C57BL/6J (RRID:IMSR_JAX:000664), CD80/86âˆ’/âˆ’ (RRID: IMSR_JAX:00
T cell receptor (TCR) signaling without CD28 can elicit primary effector T cells, but memory T cells generated during this process are anergic, failing to respond to secondary antigen exposure. We show that, upon T cell activation, CD28 transiently promotes expression of carnitine palmitoyltransferase 1a (Cpt1a), an enzyme that facilitates mitochondrial fatty acid oxidation (FAO), before the first cell division, coinciding with mitochondrial elongation and enhanced spare respiratory capacity (SRC). microRNA-33 (miR33), a target of thioredoxin-interacting protein (TXNIP), attenuates Cpt1a expression in the absence of CD28, resulting in cells that thereafter are metabolically compromised during reactivation or periods of increased bioenergetic demand. Early CD28-dependent mitochondrial engagement is needed for T cells to remodel cristae, develop SRC, and rapidly produce cytokines upon restimulation-cardinal features of protective memory T cells. Our data show that initial CD28 signals during T cell activation prime mitochondria with latent metabolic capacity that is essential for future T cell responses.
Literature context: : C57BL/6JThe Jackson Laboratory000664Mouse: NCr nudeTaconicNCRNU-FOli
Glioblastoma (GBM) is a devastating malignancy with few therapeutic options. We identify PRMT5 in an in vivo GBM shRNA screen and show that PRMT5 knockdown or inhibition potently suppresses in vivo GBM tumors, including patient-derived xenografts. Pathway analysis implicates splicing in cellular PRMT5 dependency, and we identify a biomarker that predicts sensitivity to PRMT5 inhibition. We find that PRMT5 deficiency primarily disrupts the removal of detained introns (DIs). This impaired DI splicing affects proliferation genes, whose downregulation coincides with cell cycle defects, senescence and/or apoptosis. We further show that DI programs are evolutionarily conserved and operate during neurogenesis, suggesting that they represent a physiological regulatory mechanism. Collectively, these findings reveal a PRMT5-regulated DI-splicing program as an exploitable cancer vulnerability.
Literature context: : C57BL/6JThe Jackson Laboratory000664Recombinant DNAcDNA: Trichoderma
Targeted cancer therapies that use genetics are successful, but principles for selectively targeting tumor metabolism that is also dependent on the environment remain unknown. We now show that differences in rate-controlling enzymes during the Warburg effect (WE), the most prominent hallmark of cancer cell metabolism, can be used to predict a response to targeting glucose metabolism. We establish a natural product, koningic acid (KA), to be a selective inhibitor of GAPDH, an enzyme we characterize to have differential control properties over metabolism during the WE. With machine learning and integrated pharmacogenomics and metabolomics, we demonstrate that KA efficacy is not determined by the status of individual genes, but by the quantitative extent of the WE, leading to a therapeutic window in vivo. Thus, the basis of targeting the WE can be encoded by molecular principles that extend beyond the status of individual genes.
Literature context: T C57BL6/J Jackson Laboratories RRID:JAX:000664 Mouse: miR-155tm1.Rsky/J Jackso
MiRNAs are regulatory molecules that can be packaged into exosomes and secreted from cells. Here, we show that adipose tissue macrophages (ATMs) in obese mice secrete miRNA-containing exosomes (Exos), which cause glucose intolerance and insulin resistance when administered to lean mice. Conversely, ATM Exos obtained from lean mice improve glucose tolerance and insulin sensitivity when administered to obese recipients. miR-155 is one of the miRNAs overexpressed in obese ATM Exos, and earlier studies have shown that PPARγ is a miR-155 target. Our results show that miR-155KO animals are insulin sensitive and glucose tolerant compared to controls. Furthermore, transplantation of WT bone marrow into miR-155KO mice mitigated this phenotype. Taken together, these studies show that ATMs secrete exosomes containing miRNA cargo. These miRNAs can be transferred to insulin target cell types through mechanisms of paracrine or endocrine regulation with robust effects on cellular insulin action, in vivo insulin sensitivity, and overall glucose homeostasis.
Literature context: Jackson Laboratory Cat#000664; RRID:IMSR_JAX:000664 CD45.1 (Ly5.1)- congenic C57BL/
Diet, and specifically dietary metals, can modify the risk of infection. However, the mechanisms by which manganese (Mn), a common dietary supplement, alters infection remain unexplored. We report that dietary Mn levels dictate the outcome of systemic infections caused by Staphylococcus aureus, a leading cause of bacterial endocarditis. Mice fed a high Mn diet display alterations in Mn levels and localization within infected tissues, and S. aureus virulence and infection of the heart are enhanced. Although the canonical mammalian Mn-sequestering protein calprotectin surrounds staphylococcal heart abscesses, calprotectin is not released into the abscess nidus and does not limit Mn in this organ. Consequently, excess Mn is bioavailable to S. aureus in the heart. Bioavailable Mn is utilized by S. aureus to detoxify reactive oxygen species and protect against neutrophil killing, enhancing fitness within the heart. Therefore, a single dietary modification overwhelms vital host antimicrobial strategies, leading to fatal staphylococcal infection.
Literature context: oratory RRID:JAX:000664 Mouse: MLH3tm1Lpkn Cohen Lab, (
Faithful chromosome segregation in meiosis requires crossover (CO) recombination, which is regulated to ensure at least one CO per homolog pair. We investigate the failure to ensure COs in juvenile male mice. By monitoring recombination genome-wide using cytological assays and at hotspots using molecular assays, we show that juvenile mouse spermatocytes have fewer COs relative to adults. Analysis of recombination in the absence of MLH3 provides evidence for greater utilization in juveniles of pathways involving structure-selective nucleases and alternative complexes, which can act upon precursors to generate noncrossovers (NCOs) at the expense of COs. We propose that some designated CO sites fail to mature efficiently in juveniles owing to inappropriate activity of these alternative repair pathways, leading to chromosome mis-segregation. We also find lower MutLγ focus density in juvenile human spermatocytes, suggesting that weaker CO maturation efficiency may explain why younger men have a higher risk of fathering children with Down syndrome.
Literature context: were C57BL/6 (JAX stock 000664, RRID:IMSR_JAX:000664). All mice were fed ad libitum
The lateral hypothalamic area (LHA) lies at the intersection of multiple neural and humoral systems and orchestrates fundamental aspects of behavior. Two neuronal cell types found in the LHA are defined by their expression of hypocretin/orexin (Hcrt/Ox) and melanin-concentrating hormone (MCH) and are both important regulators of arousal, feeding, and metabolism. Conflicting evidence suggests that these cell populations have a more complex signaling repertoire than previously appreciated, particularly in regard to their coexpression of other neuropeptides and the machinery for the synthesis and release of GABA and glutamate. Here, we undertook a single-cell expression profiling approach to decipher the neurochemical phenotype, and heterogeneity therein, of Hcrt/Ox and MCH neurons. In transgenic mouse lines, we used single-cell quantitative polymerase chain reaction (qPCR) to quantify the expression of 48 key genes, which include neuropeptides, fast neurotransmitter components, and other key markers, which revealed unexpected neurochemical diversity. We found that single MCH and Hcrt/Ox neurons express transcripts for multiple neuropeptides and markers of both excitatory and inhibitory fast neurotransmission. Virtually all MCH and approximately half of the Hcrt/Ox neurons sampled express both the machinery for glutamate release and GABA synthesis in the absence of a vesicular GABA release pathway. Furthermore, we found that this profile is characteristic of a subpopulation of LHA glutamatergic neurons but contrasts with a broad population of LHA GABAergic neurons. Identifying the neurochemical diversity of Hcrt/Ox and MCH neurons will further our understanding of how these populations modulate postsynaptic excitability through multiple signaling mechanisms and coordinate diverse behavioral outputs.
Literature context: The Jackson LaboratoryStock No: 000664Mouse: Ifnar1âˆ’/âˆ’(B6.129.Ifnar1tm
Cellular tropism during persistent viral infection is commonly conferred by the interaction of a viral surface protein with a host receptor complex. Norovirus, the leading global cause of gastroenteritis, can be persistently shed during infection, but its in vivo cellular tropism and tropism determinants remain unidentified. Using murine norovirus (MNoV), we determine that a small number of intestinal epithelial cells (IECs) serve as the reservoir for fecal shedding and persistence. The viral non-structural protein NS1, rather than a viral surface protein, determines IEC tropism. Expression of NS1 from a persistent MNoV strain is sufficient for an acute MNoV strain to target IECs and persist. In addition, interferon-lambda (IFN-λ) is a key host determinant blocking MNoV infection in IECs. The inability of acute MNoV to shed and persist is rescued in Ifnlr1-/- mice, suggesting that NS1 evades IFN-λ-mediated antiviral immunity. Thus, NS1 and IFN-λ interactions govern IEC tropism and persistence of MNoV.
Literature context: Jackson Laboratory Cat# 000664, RRID:IMSR_JAX:000664 Software and Algorithms
Single-cell RNA sequencing offers a promising opportunity for probing cell types mediating specific behavioral functions and the underlying molecular programs. However, this has been hampered by a long-standing issue in transcriptional profiling of dissociated cells, specifically the transcriptional perturbations that are artificially induced during conventional whole-cell dissociation procedures. Here, we develop Act-seq, which minimizes artificially induced transcriptional perturbations and allows for faithful detection of both baseline transcriptional profiles and acute transcriptional changes elicited by behavior/experience-driven activity. Using Act-seq, we provide the first detailed molecular taxonomy of distinct cell types in the amygdala. We further show that Act-seq robustly detects seizure-induced acute gene expression changes in multiple cell types, revealing cell-type-specific activation profiles. Furthermore, we find that acute stress preferentially activates neuronal subpopulations that express the neuropeptide gene Cck. Act-seq opens the way for linking physiological stimuli with acute transcriptional dynamics in specific cell types in diverse complex tissues.
Literature context: C57BL/6J The Jackson Laboratory RRID:JAX:000664 Mouse: Ldlrâˆ’/âˆ’ C57BL/6J The Jac
Clearance of apoptotic cells (ACs) by phagocytes (efferocytosis) prevents post-apoptotic necrosis and dampens inflammation. Defective efferocytosis drives important diseases, including atherosclerosis. For efficient efferocytosis, phagocytes must be able to internalize multiple ACs. We show here that uptake of multiple ACs by macrophages requires dynamin-related protein 1 (Drp1)-mediated mitochondrial fission, which is triggered by AC uptake. When mitochondrial fission is disabled, AC-induced increase in cytosolic calcium is blunted owing to mitochondrial calcium sequestration, and calcium-dependent phagosome formation around secondarily encountered ACs is impaired. These defects can be corrected by silencing the mitochondrial calcium uniporter (MCU). Mice lacking myeloid Drp1 showed defective efferocytosis and its pathologic consequences in the thymus after dexamethasone treatment and in advanced atherosclerotic lesions in fat-fed Ldlr-/- mice. Thus, mitochondrial fission in response to AC uptake is a critical process that enables macrophages to clear multiple ACs and to avoid the pathologic consequences of defective efferocytosis in vivo.
Literature context: O: C57bl6j Jackson Laboratories RRID:JAX:000664 Mouse: Glp1râˆ’/âˆ’ Scrocchi et. al
Chronic inflammation has been proposed to contribute to the pathogenesis of diet-induced obesity. However, scarce therapeutic options are available to treat obesity and the associated immunometabolic complications. Glucocorticoids are routinely employed for the management of inflammatory diseases, but their pleiotropic nature leads to detrimental metabolic side effects. We developed a glucagon-like peptide-1 (GLP-1)-dexamethasone co-agonist in which GLP-1 selectively delivers dexamethasone to GLP-1 receptor-expressing cells. GLP-1-dexamethasone lowers body weight up to 25% in obese mice by targeting the hypothalamic control of feeding and by increasing energy expenditure. This strategy reverses hypothalamic and systemic inflammation while improving glucose tolerance and insulin sensitivity. The selective preference for GLP-1 receptor bypasses deleterious effects of dexamethasone on glucose handling, bone integrity, and hypothalamus-pituitary-adrenal axis activity. Thus, GLP-1-directed glucocorticoid pharmacology represents a safe and efficacious therapy option for diet-induced immunometabolic derangements and the resulting obesity.
Literature context: ackson C57Bl/6J miceJackson Labs000664Muc2-/- C57Bl/6J miceVelcich etÂ
Intestinal microbes are recognized for their role in human disease. Enterotoxigenic Bacteroides fragilis (ETBF) has been implicated in inflammatory bowel disease and colorectal cancer; however, colonization alone is insufficient to cause these illnesses. We hypothesized that homeostasis in healthy carriers is maintained by colonic mucus, the major constituent of which is the glycoprotein Muc2. We found that Muc2-deficient mice succumb to lethal disease from ETBF colonization in a B. fragilis toxin (BFT)-dependent manner. We identify a toxin regulator, the two-component system RprXY, which suppresses BFT expression in vitro and in vivo. Overexpression of either component was sufficient to prevent lethal disease in Muc2-deficient mice. Our studies demonstrate that homeostasis in the context of ETBF colonization is dependent on a dynamic interaction between intestinal mucus, a bacterial toxin, and a toxin regulatory system. Regulation of virulence may offer a therapeutic target to maintain intestinal homeostasis in susceptible patients.
Literature context: oratory 000664; RRID:IMSR_JAX:000664 Mouse: B6.FVB(Cg)-Tg(Rbp4-cre)K
Changes in cortical and striatal function underlie the transition from novel actions to refined motor skills. How discrete, anatomically defined corticostriatal projections function in vivo to encode skill learning remains unclear. Using novel fiber photometry approaches to assess real-time activity of associative inputs from medial prefrontal cortex to dorsomedial striatum and sensorimotor inputs from motor cortex to dorsolateral striatum, we show that associative and sensorimotor inputs co-engage early in action learning and disengage in a dissociable manner as actions are refined. Disengagement of associative, but not sensorimotor, inputs predicts individual differences in subsequent skill learning. Divergent somatic and presynaptic engagement in both projections during early action learning suggests potential learning-related in vivo modulation of presynaptic corticostriatal function. These findings reveal parallel processing within associative and sensorimotor circuits that challenges and refines existing views of corticostriatal function and expose neuronal projection- and compartment-specific activity dynamics that encode and predict action learning.
Literature context: 57/BL6The Jackson LaboratoryCat#000664Mus musculus: S1PR2-/- (B6.129S6
Heparan sulfate proteoglycans (HSPGs) critically modulate adhesion-, growth-, and migration-related processes. Here, we show that the transmembrane protein, Nogo-A, inhibits neurite outgrowth and cell spreading in neurons and Nogo-A-responsive cell lines via HSPGs. The extracellular, active 180 amino acid Nogo-A region, named Nogo-A-Δ20, binds to heparin and brain-derived heparan sulfate glycosaminoglycans (GAGs) but not to the closely related chondroitin sulfate GAGs. HSPGs are required for Nogo-A-Δ20-induced inhibition of adhesion, cell spreading, and neurite outgrowth, as well as for RhoA activation. Surprisingly, we show that Nogo-A-Δ20 can act via HSPGs independently of its receptor, Sphingosine-1-Phosphate receptor 2 (S1PR2). We thereby identify the HSPG family members syndecan-3 and syndecan-4 as functional receptors for Nogo-A-Δ20. Finally, we show in explant cultures ex vivo that Nogo-A-Δ20 promotes the migration of neuroblasts via HSPGs but not S1PR2.
Literature context: 7BL/6J; The Jackson Laboratory, RRID:IMSR_JAX:000664), Ccr2âˆ’/âˆ’ (B6.129S4-Ccr2tm1Ifc/
Wallerian degeneration (WD) is considered an essential preparatory stage to the process of axonal regeneration. In the peripheral nervous system, infiltrating monocyte-derived macrophages, which use the chemokine receptor CCR2 to gain entry to injured tissues from the bloodstream, are purportedly necessary for efficient WD. However, our laboratory has previously reported that myelin clearance in the injured sciatic nerve proceeds unhindered in the Ccr2-/- mouse model. Here, we extensively characterize WD in male Ccr2-/- mice and identify a compensatory mechanism of WD that is facilitated primarily by neutrophils. In response to the loss of CCR2, injured Ccr2-/- sciatic nerves demonstrate prolonged expression of neutrophil chemokines, a concomitant extended increase in the accumulation of neutrophils in the nerve, and elevated phagocytosis by neutrophils. Neutrophil depletion substantially inhibits myelin clearance after nerve injury in both male WT and Ccr2-/- mice, highlighting a novel role for these cells in peripheral nerve degeneration that spans genotypes.SIGNIFICANCE STATEMENT The accepted view in the basic and clinical neurosciences is that the clearance of axonal and myelin debris after a nerve injury is directed primarily by inflammatory CCR2+ macrophages. However, we demonstrate that this clearance is nearly identical in WT and Ccr2-/- mice, and that neutrophils replace CCR2+ macrophages as the primary phagocytic cell. We find that neutrophils play a major role in myelin clearance not only in Ccr2-/- mice but also in WT mice, highlighting their necessity during nerve degeneration in the peripheral nervous system. These degeneration studies may propel improvements in nerve regeneration and draw critical parallels to mechanisms of nerve degeneration and regeneration in the CNS and in the context of peripheral neuropathies.
Literature context: C57BL6/J mice (RRID:IMSR_JAX:000664) were purchased from the Animal
The acquisition of motor skills involves implementing action sequences that increase task efficiency while reducing cognitive loads. This learning capacity depends on specific cortico-basal ganglia circuits that are affected by normal ageing. Here, combining a series of novel behavioural tasks with extensive neuronal mapping and targeted cell manipulations in mice, we explored how ageing of cortico-basal ganglia networks alters the microstructure of action throughout sequence learning. We found that, after extended training, aged mice produced shorter actions and displayed squeezed automatic behaviours characterised by ultrafast oligomeric action chunks that correlated with deficient reorganisation of corticostriatal activity. Chemogenetic disruption of a striatal subcircuit in young mice reproduced age-related within-sequence features, and the introduction of an action-related feedback cue temporarily restored normal sequence structure in aged mice. Our results reveal static properties of aged cortico-basal ganglia networks that introduce temporal limits to action automaticity, something that can compromise procedural learning in ageing.
Literature context: n separate lines: C57BL/6J (WT, RRID:IMSR_JAX:000664), B6(Cg)-Rag2tm1.1Cgn/J (RAG-2âˆ’
When facial nerve axotomy (FNA) is performed on immunodeficient recombinase activating gene-2 knockout (RAG-2-/-) mice, there is greater facial motoneuron (FMN) death relative to wild type (WT) mice. Reconstituting RAG-2-/- mice with whole splenocytes rescues FMN survival after FNA, and CD4+ T cells specifically drive immune-mediated neuroprotection. Evidence suggests that immunodysregulation may contribute to motoneuron death in amyotrophic lateral sclerosis (ALS). Immunoreconstitution of RAG-2-/- mice with lymphocytes from the mutant superoxide dismutase (mSOD1) mouse model of ALS revealed that the mSOD1 whole splenocyte environment suppresses mSOD1 CD4+ T cell-mediated neuroprotection after FNA. The objective of the current study was to characterize the effect of CD4+ T cells on the central molecular response to FNA and then identify if mSOD1 whole splenocytes blocked these regulatory pathways. Gene expression profiles of the axotomized facial motor nucleus were assessed from RAG-2-/- mice immunoreconstituted with either CD4+ T cells or whole splenocytes from WT or mSOD1 donors. The findings indicate that immunodeficient mice have suppressed glial activation after axotomy, and cell transfer of WT CD4+ T cells rescues microenvironment responses. Additionally, mSOD1 whole splenocyte recipients exhibit an increased astrocyte activation response to FNA. In RAG-2-/- + mSOD1 whole splenocyte mice, an elevation of motoneuron-specific Fas cell death pathways is also observed. Altogether, these findings suggest that mSOD1 whole splenocytes do not suppress mSOD1 CD4+ T cell regulation of the microenvironment, and instead, mSOD1 whole splenocytes may promote motoneuron death by either promoting a neurotoxic astrocyte phenotype or inducing Fas-mediated cell death pathways. This study demonstrates that peripheral immune status significantly affects central responses to nerve injury. Future studies will elucidate the mechanisms by which mSOD1 whole splenocytes promote cell death and if inhibiting this mechanism can preserve motoneuron survival in injury and disease.
Literature context: g (P1, P5, P10) C57Bl/6J (B6/J; RRID:IMSR_JAX:000664) mice that were originally obta
The present study has taken advantage of publicly available cell type specific mRNA expression databases in order to identify potential genes participating in the development of retinal AII amacrine cells. We profile two such genes, Delta/Notch-like EGF repeat containing (Dner) and nuclear factor I/A (Nfia), that are each heavily expressed in AII amacrine cells in the mature mouse retina, and which conjointly identify this retinal cell population in its entirety when using antibodies to DNER and NFIA. DNER is present on the plasma membrane, while NFIA is confined to the nucleus, consistent with known functions of each of these two proteins. DNER also identifies some other subsets of retinal ganglion and amacrine cell types, along with horizontal cells, while NFIA identifies a subset of bipolar cells as well as Muller glia and astrocytes. During early postnatal development, NFIA labels astrocytes on the day of birth, AII amacrine cells at postnatal (P) day 5, and Muller glia by P10, when horizontal cells also transiently exhibit NFIA immunofluorescence. DNER, by contrast, is present in ganglion and amacrine cells on P1, also labeling the horizontal cells by P10. Developing AII amacrine cells exhibit accumulating DNER labeling at the dendritic stalk, labeling that becomes progressively conspicuous by P10, as it is in maturity. This developmental time course is consistent with a prospective role for each gene in the differentiation of AII amacrine cells.
Literature context: the Jackson Laboratories (JAX; RRID:IMSR_JAX:000664)1 as heterozygote breeders foll
Previous studies have demonstrated immunosuppressive and anti-inflammatory effects of nicotine, including in the experimental autoimmune encephalomyelitis (EAE) model in mice of some forms of multiple sclerosis (MS). Other studies using knock-out (KO) mice have implicated nicotinic acetylcholine (ACh) receptors containing α7, α9, or β2 subunits (α7*-, α9*- or β2*-nAChR) in different, disease-exacerbating or disease-ameliorating processes. These outcomes are in harmony with gene expression analyses showing nAChR subunit mRNA in many classes of immune system cell types. Consistent with influences on disease status, predictable effects of nAChR subunit (and subtype) KO, or of nicotine exposure, are seen on immune cell numbers and distribution and on cytokine levels or other markers of immunity, inflammation, demyelination, and axonal degradation. Providing support for our hypotheses about distinctive roles for nAChR subtypes in EAE, here we have used direct and adoptive EAE induction and a nAChR subunit gene double knock-out (DKO) strategy. Immune cell expression of nAChR α9 subunits as protein is demonstrated by immunostaining of isolated CD4+, CD8+, CD11b+ and CD11c+ cells from wild-type (WT) mice, but not in cells from nAChR α9 subunit KO animals. Nicotine exposure is protective against directly-induced EAE in WT or α7/α9 DKO animals relative to effects seen in WT/vehicle-treated mice, but, remarkably, EAE is exacerbated in vehicle-treated α7/α9 DKO mice. Brain lesion volume and intra-cranial inflammatory activity similarly are higher in DKO/vehicle than in WT/vehicle-treated animals, although nicotine's protective effects are seen in each instance. By contrast, in adoptive transfer studies, disease severity is attenuated and disease onset is delayed in recipients of splenocytes from WT animals treated with nicotine rather than with vehicle. Moreover, protection as seen in nicotine-treated WT animals is the same in recipients of splenocytes from nAChR α7/α9 DKO mice irrespective of their exposure to nicotine or vehicle. When combined with previous observations, these findings are consistent with disease exacerbation (or even induction) being mediated at least in part via α9*-nAChR in peripheral immune cells. They also suggest protective roles of central nervous system (CNS) α7*-nAChR. The results suggest that both α7*- and α9*-nAChR are potential targets of therapeutic ligands to modulate inflammation and autoimmunity.
Literature context: The Jackson Laboratory 000664; RRID:IMSR_JAX:000664 Oligonucleotides
The mammalian target of rapamycin complex 1 (mTORC1), a transducer of local dendritic translation, participates in learning and memory processes as well as in mechanisms underlying alcohol-drinking behaviors. Using an unbiased RNA-seq approach, we identified Prosapip1 as a novel downstream target of mTORC1 whose translation and consequent synaptic protein expression are increased in the nucleus accumbens (NAc) of mice excessively consuming alcohol. We demonstrate that alcohol-dependent increases in Prosapip1 levels promote the formation of actin filaments, leading to changes in dendritic spine morphology of NAc medium spiny neurons (MSNs). We further demonstrate that Prosapip1 is required for alcohol-dependent synaptic localization of GluA2 lacking AMPA receptors in NAc shell MSNs. Finally, we present data implicating Prosapip1 in mechanisms underlying alcohol self-administration and reward. Together, these data suggest that Prosapip1 in the NAc is a molecular transducer of structural and synaptic alterations that drive and/or maintain excessive alcohol use.
Literature context: : C57BL/6J Jackson Laboratories RRID:IMSR_JAX:000664 Mouse: Ifnlr1âˆ’/âˆ’ C57BL/6J (> 99
Although Zika virus (ZIKV)-induced congenital disease occurs more frequently during early stages of pregnancy, its basis remains undefined. Using established type I interferon (IFN)-deficient mouse models of ZIKV transmission in utero, we found that the placenta and fetus were more susceptible to ZIKV infection at earlier gestational stages. Whereas ZIKV infection at embryonic day 6 (E6) resulted in placental insufficiency and fetal demise, infections at midstage (E9) resulted in reduced cranial dimensions, and infection later in pregnancy (E12) caused no apparent fetal disease. In addition, we found that fetuses lacking type III IFN-λ signaling had increased ZIKV replication in the placenta and fetus when infected at E12, and reciprocally, treatment of pregnant mice with IFN-λ2 reduced ZIKV infection. IFN-λ treatment analogously diminished ZIKV infection in human midgestation fetal- and maternal-derived tissue explants. Our data establish a model of gestational stage dependence of ZIKV pathogenesis and IFN-λ-mediated immunity at the maternal-fetal interface.
Literature context: Il18tm1Aki (IL-18KO, Jackson), C57BL/6J [(IL-18KO controls) Jackson], a
The cytokine interleukin (IL)-18 is a crucial amplifier of natural killer (NK) cell function. IL-18 signaling is regulated by the inhibitory effects of IL-18 binding protein (IL-18BP). Using mice deficient in IL-18BP (IL-18BPKO), we investigated the impact of mismanaged IL-18 signaling on NK cells. We found an overall reduced abundance of splenic NK cells in the absence of IL-18BP. Closer examination of NK cell subsets in spleen and bone marrow using CD27 and CD11b expression revealed that immature NK cells were increased in abundance, while the mature population of NK cells was reduced. Also, NK cells were polarized to greater production of TNF-α, while dedicated IFN-γ producers were reduced. A novel subset of IL-18 receptor α- NK cells contributed to the expansion of immature NK cells in IL-18BPKO mice. Splenocytes cultured with IL-18 resulted in alterations similar to those observed in IL-18BP deficiency. NK cell changes were associated with significantly reduced levels of circulating plasma IL-18. However, IL-18BPKO mice exhibited normal weight gain and responded to LPS challenge with a >10-fold increase in IFN-γ compared to wild type. Finally, we identified that the source of splenic IL-18BP was among dendritic cells/macrophage localized to the T cell-rich regions of the spleen. Our results demonstrate that IL-18BP is required for normal NK cell abundance and function and also contributes to maintaining steady-state levels of circulating IL-18. Thus, IL-18BP appears to have functions suggestive of a carrier protein, not just an inhibitor.
Literature context: kson Laboratory Strain: 000664; RRID:IMSR_JAX:000664 Mice: C57BL/6 Charles River/NCI
How tumor-infiltrating T lymphocytes (TILs) adapt to the metabolic constrains within the tumor microenvironment (TME) and to what degree this affects their ability to combat tumor progression remain poorly understood. Using mouse melanoma models, we report that CD8+ TILs enhance peroxisome proliferator-activated receptor (PPAR)-α signaling and catabolism of fatty acids (FAs) when simultaneously subjected to hypoglycemia and hypoxia. This metabolic switch partially preserves CD8+ TILs' effector functions, although co-inhibitor expression increases during tumor progression regardless of CD8+ TILs' antigen specificity. Further promoting FA catabolism improves the CD8+ TILs' ability to slow tumor progression. PD-1 blockade delays tumor growth without changing TIL metabolism or functions. It synergizes with metabolic reprogramming of T cells to achieve superior antitumor efficacy and even complete cures.
Literature context: ) male wild-type C57BL/6J mice (RRID:IMSR_JAX:000664), were obtained from Envigo (Al
The master circadian pacemaker in mammals is located in the suprachiasmatic nuclei (SCN) which regulate physiology and behaviour, as well as coordinating peripheral clocks throughout the body. Investigating the function of the SCN has often focused on the identification of rhythmically expressed genes. However, not all genes critical for SCN function are rhythmically expressed. An alternative strategy is to characterize those genes that are selectively enriched in the SCN. Here, we examined the transcriptome of the SCN and whole brain (WB) of mice using meta-analysis of publicly deposited data across a range of microarray platforms and RNA-Seq data. A total of 79 microarrays were used (24 SCN and 55 WB samples, 4 different microarray platforms), alongside 17 RNA-Seq data files (7 SCN and 10 WB). 31 684 MGI gene symbols had data for at least one platform. Meta-analysis using a random effects model for weighting individual effect sizes (derived from differential expression between relevant SCN and WB samples) reliably detected known SCN markers. SCN-enriched transcripts identified in this study provide novel insights into SCN function, including identifying genes which may play key roles in SCN physiology or provide SCN-specific drivers.
Literature context: 000664, The Jackson Laboratory; RRID:IMSR_JAX:000664) over several generations and m
Histaminergic (HA) neurons, found in the posterior hypothalamic tuberomammillary nucleus (TMN), extend fibers throughout the brain and exert modulatory influence over numerous physiological systems. Multiple lines of evidence suggest that the activity of HA neurons is important in the regulation of vigilance despite the lack of direct, causal evidence demonstrating its requirement for the maintenance of arousal during wakefulness. Given the strong correlation between HA neuron excitability and behavioral arousal, we investigated both the electrophysiological diversity of HA neurons in brain slices and the effect of their acute silencing in vivo in male mice. For this purpose, we first validated a transgenic mouse line expressing cre recombinase in histidine decarboxylase-expressing neurons (Hdc-Cre) followed by a systematic census of the membrane properties of both HA and non-HA neurons in the ventral TMN (TMNv) region. Through unsupervised hierarchical cluster analysis, we found electrophysiological diversity both between TMNv HA and non-HA neurons, and among HA neurons. To directly determine the impact of acute cessation of HA neuron activity on sleep-wake states in awake and behaving mice, we examined the effects of optogenetic silencing of TMNv HA neurons in vivo We found that acute silencing of HA neurons during wakefulness promotes slow-wave sleep, but not rapid eye movement sleep, during a period of low sleep pressure. Together, these data suggest that the tonic firing of HA neurons is necessary for the maintenance of wakefulness, and their silencing not only impairs arousal but is sufficient to rapidly and selectively induce slow-wave sleep.SIGNIFICANCE STATEMENT The function of monoaminergic systems and circuits that regulate sleep and wakefulness is often disrupted as part of the pathophysiology of many neuropsychiatric disorders. One such circuit is the posterior hypothalamic histamine (HA) system, implicated in supporting wakefulness and higher brain function, but has been difficult to selectively manipulate owing to cellular heterogeneity in this region. Here we use a transgenic mouse to interrogate both the characteristic firing properties of HA neurons and their specific role in maintaining wakefulness. Our results demonstrate that the acute, cell type-specific silencing of HA neurons during wakefulness is sufficient to not only impair arousal but to rapidly and selectively induce slow-wave sleep. This work furthers our understanding of HA-mediated mechanisms that regulate behavioral arousal.
Literature context: C57BL/6J The Jackson Laboratory RRID:JAX:000664 Oligonucleotides
Rab small GTPases control membrane trafficking through effectors that recruit downstream mediators such as motor proteins. Subcellular trafficking typically involves multiple Rabs, with each specific step mediated by a distinct Rab protein. We describe a collaboration between two distinct Rab-protein-orchestrated trafficking circuits in bladder epithelial cells (BECs) that expels intracellular uropathogenic Escherichia coli (UPEC) from their intracellular niche. RAB11a and RAB27b and their trafficking circuitry are simultaneously involved in UPEC expulsion. While RAB11a recruits its effector RAB11FIP3 and cytoskeletal motor Dynein, RAB27b mobilizes the effector MyRIP and motor Myosin VIIa to mediate bacterial expulsion. This collaboration is coordinated by deposition of the exocyst complex on bacteria-containing vesicles, an event triggered by the innate receptor Toll-like receptor 4. Both RAB11a and RAB27b are recruited and activated by the exocyst complex components SEC6/SEC15. Thus, the cell autonomous defense system can mobilize and coalesce multiple subcellular trafficking circuitries to combat infections.
Literature context: 57BL/6 WTJackson LaboratoryCat# 000664C57BL/6 Cybbâˆ’/âˆ’Jackson Laborator
The mucosal inflammatory response induced by Salmonella serovar Typhimurium creates a favorable niche for this gut pathogen. Conventional wisdom holds that S. Typhimurium undergoes an incomplete tricarboxylic acid (TCA) cycle in the anaerobic mammalian gut. One change during S. Typhimurium-induced inflammation is the production of oxidized compounds by infiltrating neutrophils. We show that inflammation-derived electron acceptors induce a complete, oxidative TCA cycle in S. Typhimurium, allowing the bacteria to compete with the microbiota for colonization. A complete TCA cycle facilitates utilization of the microbiota-derived fermentation product succinate as a carbon source. S. Typhimurium succinate utilization genes contribute to efficient colonization in conventionally raised mice, but provide no growth advantage in germ-free mice. Mono-association of gnotobiotic mice with Bacteroides, a major succinate producer, restores succinate utilization in S. Typhimurium. Thus, oxidative central metabolism enables S. Typhimurium to utilize a variety of carbon sources, including microbiota-derived succinate.
Literature context: C57BL/6JJackson LaboratoriesCat#000664OligonucleotidesH2A F/Z Fwd: CGG
Mechanisms that control cell-cycle dynamics during tissue regeneration require elucidation. Here we find in zebrafish that regeneration of the epicardium, the mesothelial covering of the heart, is mediated by two phenotypically distinct epicardial cell subpopulations. These include a front of large, multinucleate leader cells, trailed by follower cells that divide to produce small, mononucleate daughters. By using live imaging of cell-cycle dynamics, we show that leader cells form by spatiotemporally regulated endoreplication, caused primarily by cytokinesis failure. Leader cells display greater velocities and mechanical tension within the epicardial tissue sheet, and experimentally induced tension anisotropy stimulates ectopic endoreplication. Unbalancing epicardial cell-cycle dynamics with chemical modulators indicated autonomous regenerative capacity in both leader and follower cells, with leaders displaying an enhanced capacity for surface coverage. Our findings provide evidence that mechanical tension can regulate cell-cycle dynamics in regenerating tissue, stratifying the source cell features to improve repair.
Literature context: B6The Jackson LaboratoryStock# 000664Mouse: CD1Charles RiverStrain# 0
Organogenesis during embryonic development occurs through the differentiation of progenitor cells. This process is extraordinarily accurate, but the mechanisms ensuring high fidelity are poorly understood. Coronary vessels of the mouse heart derive from at least two progenitor pools, the sinus venosus and endocardium. We find that the ELABELA (ELA)-APJ signaling axis is only required for sinus venosus-derived progenitors. Because they do not depend on ELA-APJ, endocardial progenitors are able to expand and compensate for faulty sinus venosus development in Apj mutants, leading to normal adult heart function. An upregulation of endocardial SOX17 accompanied compensation in Apj mutants, which was also seen in Ccbe1 knockouts, indicating that the endocardium is activated in multiple cases where sinus venosus angiogenesis is stunted. Our data demonstrate that by diversifying their responsivity to growth cues, distinct coronary progenitor pools are able to compensate for each other during coronary development, thereby providing robustness to organ development.
Literature context: StrainsC57BL/6 MiceJacksonCat # 00064Recombinant DNAFosmid: BCL11b en
It is now established that Bcl11b specifies T cell fate. Here, we show that in developing T cells, the Bcl11b enhancer repositioned from the lamina to the nuclear interior. Our search for factors that relocalized the Bcl11b enhancer identified a non-coding RNA named ThymoD (thymocyte differentiation factor). ThymoD-deficient mice displayed a block at the onset of T cell development and developed lymphoid malignancies. We found that ThymoD transcription promoted demethylation at CTCF bound sites and activated cohesin-dependent looping to reposition the Bcl11b enhancer from the lamina to the nuclear interior and to juxtapose the Bcl11b enhancer and promoter into a single-loop domain. These large-scale changes in nuclear architecture were associated with the deposition of activating epigenetic marks across the loop domain, plausibly facilitating phase separation. These data indicate how, during developmental progression and tumor suppression, non-coding transcription orchestrates chromatin folding and compartmentalization to direct with high precision enhancer-promoter communication.
Literature context: 620-UCDMouse: C57/Bl6JacksonCat#000664OligonucleotidesPrimers for ifnÎ²
Double-stranded RNA (dsRNA) is a common by-product of viral infections and acts as a potent trigger of antiviral immunity. In the nematode C. elegans, sid-1 encodes a dsRNA transporter that is highly conserved throughout animal evolution, but the physiological role of SID-1 and its orthologs remains unclear. Here, we show that the mammalian SID-1 ortholog, SIDT2, is required to transport internalized extracellular dsRNA from endocytic compartments into the cytoplasm for immune activation. Sidt2-deficient mice exposed to extracellular dsRNA, encephalomyocarditis virus (EMCV), and herpes simplex virus 1 (HSV-1) show impaired production of antiviral cytokines and-in the case of EMCV and HSV-1-reduced survival. Thus, SIDT2 has retained the dsRNA transport activity of its C. elegans ortholog, and this transport is important for antiviral immunity.
Literature context: C57BL/6J The Jackson Laboratory RRID:IMSR_JAX:000664 Mouse: Crl:CD1(ICR) Charles Riv
Hippocampal CA3 neurons form synapses with CA1 neurons in two layers, stratum oriens (SO) and stratum radiatum (SR). Each layer develops unique synaptic properties but molecular mechanisms that mediate these differences are unknown. Here, we show that SO synapses normally have significantly more mushroom spines and higher-magnitude long-term potentiation (LTP) than SR synapses. Further, we discovered that these differences require the Type II classic cadherins, cadherins-6, -9, and -10. Though cadherins typically function via trans-cellular homophilic interactions, our results suggest presynaptic cadherin-9 binds postsynaptic cadherins-6 and -10 to regulate mushroom spine density and high-magnitude LTP in the SO layer. Loss of these cadherins has no effect on the lower-magnitude LTP typically observed in the SR layer, demonstrating that cadherins-6, -9, and -10 are gatekeepers for high-magnitude LTP. Thus, Type II cadherins may uniquely contribute to the specificity and strength of synaptic changes associated with learning and memory.
Literature context: JThe Jackson LaboratoryStock #: 000664Mouse: 3xTgThe Jackson Laborator
Delta-secretase, a lysosomal asparagine endopeptidase (AEP), simultaneously cleaves both APP and tau, controlling the onset of pathogenesis of Alzheimer's disease (AD). However, how this protease is post-translationally regulated remains unclear. Here we report that serine-arginine protein kinase 2 (SRPK2) phosphorylates delta-secretase and enhances its enzymatic activity. SRPK2 phosphorylates serine 226 on delta-secretase and accelerates its autocatalytic cleavage, leading to its cytoplasmic translocation and escalated enzymatic activities. Delta-secretase is highly phosphorylated in human AD brains, tightly correlated with SRPK2 activity. Overexpression of a phosphorylation mimetic (S226D) in young 3xTg mice strongly promotes APP and tau fragmentation and facilitates amyloid plaque deposits and neurofibrillary tangle (NFT) formation, resulting in cognitive impairment. Conversely, viral injection of the non-phosphorylatable mutant (S226A) into 5XFAD mice decreases APP and tau proteolytic cleavage, attenuates AD pathologies, and reverses cognitive defects. Our findings support that delta-secretase phosphorylation by SRPK2 plays a critical role in aggravating AD pathogenesis.
Literature context: rmacology (Zurich, Switzerland) RRID:IMSR_JAX:000664 Plasmids
Recombinant adeno-associated virus (rAAV) vector-mediated gene transfer into genetically defined neuron subtypes has become a powerful tool to study the neuroanatomy of neuronal circuits in the brain and to unravel their functions. More recently, this methodology has also become popular for the analysis of spinal cord circuits. To date, a variety of naturally occurring AAV serotypes and genetically modified capsid variants are available but transduction efficiency in spinal neurons, target selectivity, and the ability for retrograde tracing are only incompletely characterized. Here, we have compared the transduction efficiency of seven commonly used AAV serotypes after intraspinal injection. We specifically analyzed local transduction of different types of dorsal horn neurons, and retrograde transduction of dorsal root ganglia (DRG) neurons and of neurons in the rostral ventromedial medulla (RVM) and the somatosensory cortex (S1). Our results show that most of the tested rAAV vectors have similar transduction efficiency in spinal neurons. All serotypes analyzed were also able to transduce DRG neurons and descending RVM and S1 neurons via their spinal axon terminals. When comparing the commonly used rAAV serotypes to the recently developed serotype 2 capsid variant rAAV2retro, a > 20-fold increase in transduction efficiency of descending supraspinal neurons was observed. Conversely, transgene expression in retrogradely transduced neurons was strongly reduced when the human synapsin 1 (hSyn1) promoter was used instead of the strong ubiquitous hybrid cytomegalovirus enhancer/chicken β-actin promoter (CAG) or cytomegalovirus (CMV) promoter fragments. We conclude that the use of AAV2retro greatly increases transduction of neurons connected to the spinal cord via their axon terminals, while the hSyn1 promoter can be used to minimize transgene expression in retrogradely connected neurons of the DRG or brainstem. Cover Image for this issue: doi. 10.1111/jnc.13813.
Literature context: miceJackson LaboratoryStock No: 000664 Black 6Chemicals, Peptides, and
Recent studies indicate that even a homogeneous population of cells display heterogeneity in gene expression and response to environmental stimuli. Although promoter structure critically influences the cell-to-cell variation of gene expression in bacteria and lower eukaryotes, it remains unclear what controls the gene expression noise in mammals. Here we report that CTCF decreases cell-to-cell variation of expression by stabilizing enhancer-promoter interaction. We show that CTCF binding sites are interwoven with enhancers within topologically associated domains (TADs) and a positive correlation is found between CTCF binding and the activity of the associated enhancers. Deletion of CTCF sites compromises enhancer-promoter interactions. Using single-cell flow cytometry and single-molecule RNA-FISH assays, we demonstrate that knocking down of CTCF or deletion of a CTCF binding site results in increased cell-to-cell variation of gene expression, indicating that long-range promoter-enhancer interaction mediated by CTCF plays important roles in controlling the cell-to-cell variation of gene expression in mammalian cells.
Literature context: se: C57BL/6J Jackson Laboratory RRID:IMSR_JAX:000664 Mouse: C57BL/6J-TgN(grm2-IL2RA/
AMPARs mediate the briefest synaptic currents in the brain by virtue of their rapid gating kinetics. However, at the mossy fiber-to-unipolar brush cell synapse in the cerebellum, AMPAR-mediated EPSCs last for hundreds of milliseconds, and it has been proposed that this time course reflects slow diffusion from a complex synaptic space. We show that upon release of glutamate, synaptic AMPARs were desensitized by transmitter by >90%. As glutamate levels subsequently fell, recovery of transmission occurred due to the presence of the AMPAR accessory protein stargazin that enhances the AMPAR response to low levels of transmitter. This gradual increase in receptor activity following desensitization accounted for the majority of synaptic transmission at this synapse. Moreover, the amplitude, duration, and shape of the synaptic response was tightly controlled by plasma membrane glutamate transporters, indicating that clearance of synaptic glutamate during the slow EPSC is dictated by an uptake process.
Literature context: n 8)N/AC57BL/6Jackson Laboratory000664C57BL/6 Nos3-KOJackson Laborator
The biological and clinical behaviors of hematological malignancies can be influenced by the active crosstalk with an altered bone marrow (BM) microenvironment. In the present study, we provide a detailed picture of the BM vasculature in acute myeloid leukemia using intravital two-photon microscopy. We found several abnormalities in the vascular architecture and function in patient-derived xenografts (PDX), such as vascular leakiness and increased hypoxia. Transcriptomic analysis in endothelial cells identified nitric oxide (NO) as major mediator of this phenotype in PDX and in patient-derived biopsies. Moreover, induction chemotherapy failing to restore normal vasculature was associated with a poor prognosis. Inhibition of NO production reduced vascular permeability, preserved normal hematopoietic stem cell function, and improved treatment response in PDX.
Literature context: and CD45.1) Jackson Laboratory RRID:IMSR_JAX:000664 Mouse: B6.Cg-Rptortm1.1Dmsa/J (
Follicular regulatory T (Tfr) cells differentiate from conventional regulatory T (Treg) cells and suppress excessive germinal center (GC) responses by acting on both GC B cells and T follicular helper (Tfh) cells. Here, we examined the impact of mTOR, a serine/threonine protein kinase that senses and integrates diverse environmental cues, on the differentiation and functional competency of Tfr cells in response to protein immunization or viral infection. By genetically deleting Rptor or Rictor, essential components for mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), respectively, we found that mTORC1 but not mTORC2 is essential for Tfr differentiation. Mechanistically, mTORC1-mediated phosphorylation of the transcription factor STAT3 induced the expression of the transcription factor TCF-1 by promoting STAT3 binding to the Tcf7 5'-regulatory region. Subsequently, TCF-1 bound to the Bcl6 promoter to induce Bcl6 expression, which launched the Tfr cell differentiation program. Thus, mTORC1 initiates Tfr cell differentiation by activating the TCF-1-Bcl-6 axis during immunization or infection.
Literature context: C57BL/6J The Jackson Laboratory RRID:JAX:000664 Mouse: B6.SJL-Ptprca Pepcb/BoyJ
Immune-checkpoint blockade is able to achieve durable responses in a subset of patients; however, we lack a satisfying comprehension of the underlying mechanisms of anti-CTLA-4- and anti-PD-1-induced tumor rejection. To address these issues, we utilized mass cytometry to comprehensively profile the effects of checkpoint blockade on tumor immune infiltrates in human melanoma and murine tumor models. These analyses reveal a spectrum of tumor-infiltrating T cell populations that are highly similar between tumor models and indicate that checkpoint blockade targets only specific subsets of tumor-infiltrating T cell populations. Anti-PD-1 predominantly induces the expansion of specific tumor-infiltrating exhausted-like CD8 T cell subsets. In contrast, anti-CTLA-4 induces the expansion of an ICOS+ Th1-like CD4 effector population in addition to engaging specific subsets of exhausted-like CD8 T cells. Thus, our findings indicate that anti-CTLA-4 and anti-PD-1 checkpoint-blockade-induced immune responses are driven by distinct cellular mechanisms.
Literature context: s oldJackson LaboratoriesStock# 000664B6.129X1(FVB)-Hnf4atm1.1Gonz/JJa
Cold-induced thermogenesis is an energy-demanding process that protects endotherms against a reduction in ambient temperature. Using non-targeted liquid chromatography-mass spectrometry-based lipidomics, we identified elevated levels of plasma acylcarnitines in response to the cold. We found that the liver undergoes a metabolic switch to provide fuel for brown fat thermogenesis by producing acylcarnitines. Cold stimulates white adipocytes to release free fatty acids that activate the nuclear receptor HNF4α, which is required for acylcarnitine production in the liver and adaptive thermogenesis. Once in circulation, acylcarnitines are transported to brown adipose tissue, while uptake into white adipose tissue and liver is blocked. Finally, a bolus of L-carnitine or palmitoylcarnitine rescues the cold sensitivity seen with aging. Our data highlight an elegant mechanism whereby white adipose tissue provides long-chain fatty acids for hepatic carnitilation to generate plasma acylcarnitines as a fuel source for peripheral tissues in mice.
Literature context: nd age-matched control C57BL/6 (000664, Jackson Labs) mice were used in these experi
Hyperphosphorylation and aggregation of tau protein is a critical factor in many neurodegenerative diseases. These diseases are increasing in prevalence, and there are currently no cures. Previous work from our group and others has shown that tyrosine kinase inhibitors (TKIs) can stimulate autophagy, decrease pathological proteins, and improve symptoms in models of neurodegeneration. Here we examined the role of pazopanib in mouse models that express either human mutant P301L tau (TauP301L) or triple mutant amyloid precursor protein (3x-AβPP). The TauP301L mouse expresses P301L tau under the control of a prion promoter in both neurons and astrocytes, reminiscent of some human tauopathies. Pazopanib crosses the blood-brain barrier with no detectable peripheral off-side effects, and decreases p-tau in TauP301L mice. Pazopanib reaches a brain concentration sufficient for inhibition of several tyrosine kinases, including vascular endothelial growth factor receptors (VEGFRs). Further, pazopanib does not affect microglia but reduces astrocyte levels toward nontransgenic controls in TauP301L mice. Pazopanib does not alter amyloid beta levels or astrocytes in 3x-AβPP mice but modulates a number of inflammatory markers (IP-10, MIP-1α, MIP-1β, and RANTES). These data suggest that pazopanib may be involved in p-tau clearance and modulation of astrocytic activity in models of tauopathies.
Literature context: Jackson Laboratory Cat# 000664; RRID:IMSR_JAX:000664 MECIII-Cre mice (pOxr1-Cre) Ton
Hippocampal replays have been demonstrated to play a crucial role in memory. Chains of ripples (ripple bursts) in CA1 have been reported to co-occur with long-range place cell sequence replays during the quiet awake state, but roles of neural inputs to CA1 in ripple bursts and replays are unknown. Here we show that ripple bursts in CA1 and medial entorhinal cortex (MEC) are temporally associated. An inhibition of MECIII input to CA1 during quiet awake reduced ripple bursts in CA1 and restricted the spatial coverage of replays to a shorter distance corresponding to single ripple events. The reduction did not occur with MECIII input inhibition during slow-wave sleep. Inhibition of CA3 activity suppressed ripples and replays in CA1 regardless of behavioral state. Thus, MECIII input to CA1 is crucial for ripple bursts and long-range replays specifically in quiet awake, whereas CA3 input is essential for both, regardless of behavioral state.
Literature context: a C57BL/6J background (p75+/+) (RRID:IMSR_JAX:000664) have a targeted deletion of ex
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive deposition of amyloid beta (Aβ) and dysregulation of neurotrophic signaling, causing synaptic dysfunction, loss of memory, and cell death. The expression of p75 neurotrophin receptor is elevated in the brain of AD patients, suggesting its involvement in this disease. However, the exact mechanism of its action is not yet clear. Here, we show that p75 interacts with beta-site amyloid precursor protein cleaving enzyme-1 (BACE1), and this interaction is enhanced in the presence of Aβ. Our results suggest that the colocalization of BACE1 and amyloid precursor protein (APP) is increased in the presence of both Aβ and p75 in cortical neurons. In addition, the localization of APP and BACE1 in early endosomes is increased in the presence of Aβ and p75. An increased phosphorylation of APP-Thr668 and BACE1-Ser498 by c-Jun N-terminal kinase (JNK) in the presence of Aβ and p75 could be responsible for this localization. In conclusion, our study proposes a potential involvement in amyloidogenesis for p75, which may represent a future therapeutic target for AD.
Literature context: Mouse: C57BL/6 RRID:IMSR_JAX:000664 Mouse: B6.SJL RRID:IMSR_JAX:002
MyD88 is the main adaptor molecule for TLR and IL-1R family members. Here, we demonstrated that T-cell intrinsic MyD88 signaling is required for proliferation, protection from apoptosis and expression of activation/memory genes during infection with the intracellular parasite Trypanosoma cruzi, as evidenced by transcriptome and cytometry analyses in mixed bone-marrow (BM) chimeras. The lack of direct IL-18R signaling in T cells, but not of IL-1R, phenocopied the absence of the MyD88 pathway, indicating that IL-18R is a critical MyD88-upstream pathway involved in the establishment of the Th1 response against an in vivo infection, a presently controvert subject. Accordingly, Il18r1-/- mice display lower levels of Th1 cells and are highly susceptible to infection, but can be rescued from mortality by the adoptive transfer of WT CD4+ T cells. Our findings establish the T-cell intrinsic IL-18R/MyD88 pathway as a crucial element for induction of cognate Th1 responses against an important human pathogen.
Literature context: C57BL/6 (RRID:IMSR_JAX:000664), and B6CB
The α-synuclein protein exists in vivo in a variety of covalently modified and aggregated forms associated with Parkinson's disease (PD) pathology. However, the specific proteoform structures involved with neuropathological disease mechanisms are not clearly defined. Since α-synuclein plays a role in presynaptic neurotransmitter release, an in vitro enzyme-based assay was developed to measure glutamate release from mouse forebrain synaptoneurosomes (SNs) enriched in synaptic endings. Glutamate measurements utilizing SNs from various mouse genotypes (WT, over-expressers, knock-outs) suggested a concentration dependence of α-synuclein on calcium/depolarization-dependent presynaptic glutamate release from forebrain terminals. In vitro reconstitution experiments with recombinant human α-synuclein proteoforms including monomers and aggregated forms (fibrils, oligomers) produced further evidence of this functional impact. Notably, brief exogenous applications of fibrillated forms of α-synuclein enhanced SN glutamate release but monomeric forms did not, suggesting preferential membrane penetration and toxicity by the aggregated forms. However, when applied to brain tissue sections just prior to homogenization, both monomeric and fibrillated forms stimulated glutamate release. Immuno-gold and transmission electron microscopy (TEM) detected exogenous fibrillated α-synuclein associated with numerous SN membranous structures including synaptic terminals. Western blots and immuno-gold TEM were consistent with SN internalization of α-synuclein. Additional studies revealed no evidence of gross disruption of SN membrane integrity or glutamate transporter function by exogenous α-synuclein. Overall excitotoxicity, due to enhanced glutamate release in the face of either overexpressed monomeric α-synuclein or extrasynaptic exposure to fibrillated α-synuclein, should be considered as a potential neuropathological pathway during the progression of PD and other synucleinopathies. © 2017 Wiley Periodicals, Inc.
Literature context: /6J mice (RRID:IMSR_JAX:000664) at 8 to 1
Intracerebral hemorrhage (ICH) is associated with diverse sets of neurological symptoms and prognosis, depending on the site of bleeding. Relative rate of hemorrhage occurring in the cerebral cortex (lobar hemorrhage) has been increasing, but there is no report on effective pharmacotherapeutic approaches for cortical hemorrhage either in preclinical or clinical studies. The present study aimed to establish an experimental model of cortical hemorrhage in mice for evaluation of effects of therapeutic drug candidates. Type VII collagenase at 0.015 U, injected into the parietal cortex, induced hemorrhage expanding into the whole layer of the posterior parts of the sensorimotor cortex in male C57BL/6 mice. Mice with ICH under these conditions exhibited significant motor deficits as revealed by beam-walking test. Daily administration of nicotine (1 and 2 mg/kg), with the first injection given at 3 hr after induction of ICH, improved motor performance of mice in a dose-dependent manner, although nicotine did not alter the volume of hematoma. Immunohistochemical examinations revealed that the number of neurons was drastically decreased within the hematoma region. Nicotine at 2 mg/kg partially but significantly increased the number of remaining neurons within the hematoma at 3 days after induction of ICH. ICH also resulted in inflammatory activation of microglia/macrophages in the perihematoma region, and nicotine (1 and 2 mg/kg) significantly attenuated the increase of microglia. These results suggest that nicotine can provide a therapeutic effect on cortical hemorrhage, possibly via its neuroprotective and anti-inflammatory actions. © 2017 Wiley Periodicals, Inc.
Literature context: e The Jackson Lab stock#000664; RRID:IMSR_JAX:000664 Lysmcre/wt The Jackson Lab stoc
Ten-Eleven-Translocation-2 (Tet2) is a DNA methylcytosine dioxygenase that functions as a tumor suppressor in hematopoietic malignancies. We examined the role of Tet2 in tumor-tissue myeloid cells and found that Tet2 sustains the immunosuppressive function of these cells. We found that Tet2 expression is increased in intratumoral myeloid cells both in mouse models of melanoma and in melanoma patients and that this increased expression is dependent on an IL-1R-MyD88 pathway. Ablation of Tet2 in myeloid cells suppressed melanoma growth in vivo and shifted the immunosuppressive gene expression program in tumor-associated macrophages to a proinflammatory one, with a concomitant reduction of the immunosuppressive function. This resulted in increased numbers of effector T cells in the tumor, and T cell depletion abolished the reduced tumor growth observed upon myeloid-specific deletion of Tet2. Our findings reveal a non-cell-intrinsic, tumor-promoting function for Tet2 and suggest that Tet2 may present a therapeutic target for the treatment of non-hematologic malignancies.
Literature context: Jackson Laboratory JAX#000664; RRID:IMSR_JAX:000664 Mouse: Drd1-Cre; Drd1aCre/+ Lab
Dopamine (DA) neurotransmission controls behaviors important for survival, including voluntary movement, reward processing, and detection of salient events, such as food or mate availability. Dopaminergic tone also influences circadian physiology and behavior. Although the evolutionary significance of this input is appreciated, its precise neurophysiological architecture remains unknown. Here, we identify a novel, direct connection between the DA neurons of the ventral tegmental area (VTA) and the suprachiasmatic nucleus (SCN). We demonstrate that D1 dopamine receptor (Drd1) signaling within the SCN is necessary for properly timed resynchronization of activity rhythms to phase-shifted light:dark cycles and that elevation of DA tone through selective activation of VTA DA neurons accelerates photoentrainment. Our findings demonstrate a previously unappreciated role for direct DA input to the master circadian clock and highlight the importance of an evolutionarily significant relationship between the circadian system and the neuromodulatory circuits that govern motivational behaviors.
Literature context: ed from The Jackson Laboratory (RRID:IMSR_JAX:000664). Both male and female 12-week-
The CA1 region of the hippocampus plays a critical role in spatial and contextual memory, and has well-established circuitry, function and plasticity. In contrast, the properties of the flanking CA2 pyramidal neurons (PNs), important for social memory, and lacking CA1-like plasticity, remain relatively understudied. In particular, little is known regarding the expression of voltage-gated K+ (Kv) channels and the contribution of these channels to the distinct properties of intrinsic excitability, action potential (AP) waveform, firing patterns and neurotransmission between CA1 and CA2 PNs. In the present study, we used multiplex fluorescence immunolabeling of mouse brain sections, and whole-cell recordings in acute mouse brain slices, to define the role of heterogeneous expression of Kv2 family Kv channels in CA1 versus CA2 pyramidal cell excitability. Our results show that the somatodendritic delayed rectifier Kv channel subunits Kv2.1, Kv2.2, and their auxiliary subunit AMIGO-1 have region-specific differences in expression in PNs, with the highest expression levels in CA1, a sharp decrease at the CA1-CA2 boundary, and significantly reduced levels in CA2 neurons. PNs in CA1 exhibit a robust contribution of Guangxitoxin-1E-sensitive Kv2-based delayed rectifier current to AP shape and after-hyperpolarization potential (AHP) relative to that seen in CA2 PNs. Our results indicate that robust Kv2 channel expression confers a distinct pattern of intrinsic excitability to CA1 PNs, potentially contributing to their different roles in hippocampal network function.
Literature context: 57BL/6J) The Jackson Laboratory RRID:IMSR_JAX:000664 Mouse: mGluR2âˆ’/âˆ’ (B6;129S-Grm2t
Compartmentalized signaling in dendritic subdomains is critical for the function of many central neurons. In the retina, individual dendritic sectors of a starburst amacrine cell (SAC) are preferentially activated by different directions of linear motion, indicating limited signal propagation between the sectors. However, the mechanism that regulates this propagation is poorly understood. Here, we find that metabotropic glutamate receptor 2 (mGluR2) signaling, which acts on voltage-gated calcium channels in SACs, selectively restricts cross-sector signal propagation in SACs, but does not affect local dendritic computation within individual sectors. mGluR2 signaling ensures sufficient electrotonic isolation of dendritic sectors to prevent their depolarization during non-preferred motion, yet enables controlled multicompartmental signal integration that enhances responses to preferred motion. Furthermore, mGluR2-mediated dendritic compartmentalization in SACs is important for the functional output of direction-selective ganglion cells (DSGCs). Therefore, our results directly link modulation of dendritic compartmentalization to circuit-level encoding of motion direction in the retina.
Literature context: r 000664; RRID:IMSR_JAX:000664 Oligonucle
Activity-dependent neuronal plasticity is a fundamental mechanism through which the nervous system adapts to sensory experience. Several lines of evidence suggest that parvalbumin (PV+) interneurons are essential in this process, but the molecular mechanisms underlying the influence of experience on interneuron plasticity remain poorly understood. Perineuronal nets (PNNs) enwrapping PV+ cells are long-standing candidates for playing such a role, yet their precise contribution has remained elusive. We show that the PNN protein Brevican is a critical regulator of interneuron plasticity. We find that Brevican simultaneously controls cellular and synaptic forms of plasticity in PV+ cells by regulating the localization of potassium channels and AMPA receptors, respectively. By modulating Brevican levels, experience introduces precise molecular and cellular modifications in PV+ cells that are required for learning and memory. These findings uncover a molecular program through which a PNN protein facilitates appropriate behavioral responses to experience by dynamically gating PV+ interneuron function.
Literature context: ackson Laboratory Cat # 000664; RRID:IMSR_JAX:000664 Mouse: 129/SvEvTac Taconic Bios
How environmental and physiological signals interact to influence neural circuits underlying developmentally programmed social interactions such as male territorial aggression is poorly understood. We have tested the influence of sensory cues, social context, and sex hormones on progesterone receptor (PR)-expressing neurons in the ventromedial hypothalamus (VMH) that are critical for male territorial aggression. We find that these neurons can drive aggressive displays in solitary males independent of pheromonal input, gonadal hormones, opponents, or social context. By contrast, these neurons cannot elicit aggression in socially housed males that intrude in another male's territory unless their pheromone-sensing is disabled. This modulation of aggression cannot be accounted for by linear integration of environmental and physiological signals. Together, our studies suggest that fundamentally non-linear computations enable social context to exert a dominant influence on developmentally hard-wired hypothalamus-mediated male territorial aggression.
Literature context: C57BL/6J (RRID:IMSR_JAX:000664), Nfiatm1R
The nuclear factor I (NFI) family of transcription factors plays an important role in the development of the cerebral cortex in humans and mice. Disruption of nuclear factor IA (NFIA), nuclear factor IB (NFIB), or nuclear factor IX (NFIX) results in abnormal development of the corpus callosum, lateral ventricles, and hippocampus. However, the expression or function of these genes has not been examined in detail in the adult brain, and the cell type-specific expression of NFIA, NFIB, and NFIX is currently unknown. Here, we demonstrate that the expression of each NFI protein shows a distinct laminar pattern in the adult mouse neocortex and that their cell type-specific expression differs depending on the family member. NFIA expression was more frequently observed in astrocytes and oligodendroglia, whereas NFIB expression was predominantly localized to astrocytes and neurons. NFIX expression was most commonly observed in neurons. The NFI proteins were equally distributed within microglia, and the ependymal cells lining the ventricles of the brain expressed all three proteins. In the hippocampus, the NFI proteins were expressed during all stages of neural stem cell differentiation in the dentate gyrus, with higher expression intensity in neuroblast cells as compared to quiescent stem cells and mature granule neurons. These findings suggest that the NFI proteins may play distinct roles in cell lineage specification or maintenance, and establish the basis for further investigation of their function in the adult brain and their emerging role in disease.
Literature context: re purchased from Jackson Labs (RRID:IMSR_JAX:000664; Bar Harbor, ME, USA). Upon arr
Gulf War Illness (GWI) is a chronic multi-symptom disorder affecting veterans of the 1991 Gulf War. Among the symptoms of GWI are those associated with sickness behavior, observations suggestive of underlying neuroinflammation. We have shown that exposure of mice to the stress hormone, corticosterone (CORT), and to diisopropyl fluorophosphate (DFP), as a nerve agent mimic, results in marked neuroinflammation, findings consistent with a stress/neuroimmune basis of GWI. Here, we examined the contribution of irreversible and reversible acetylcholinesterase (AChE) inhibitors to neuroinflammation in our mouse model of GWI. Male C57BL/6J mice received 4 days of CORT (400 mg/L) in the drinking water followed by a single dose of chlorpyrifos oxon (CPO; 8 mg/kg, i.p.), DFP (4 mg/kg, i.p.), pyridostigmine bromide (PB; 3 mg/kg, i.p.), or physostigmine (PHY; 0.5 mg/kg, i.p.). CPO and DFP alone caused cortical and hippocampal neuroinflammation assessed by qPCR of tumor necrosis factor-alpha, IL-6, C-C chemokine ligand 2, IL-1β, leukemia inhibitory factor and oncostatin M; CORT pretreatment markedly augmented these effects. Additionally, CORT exposure prior to DFP or CPO enhanced activation of the neuroinflammation signal transducer, signal transducer and activator of transcription 3 (STAT3). In contrast, PHY or PB alone or with CORT pretreatment did not produce neuroinflammation or STAT3 activation. While all of the CNS-acting AChE inhibitors (DFP, CPO, and PHY) decreased brain AChE activity, CORT pretreatment abrogated these effects for the irreversible inhibitors. Taken together, these findings suggest that irreversible AChE inhibitor-induced neuroinflammation and particularly its exacerbation by CORT, result from non-cholinergic effects of these compounds, pointing potentially to organophosphorylation of other neuroimmune targets.
Literature context: : 000664; RRID:IMSR_JAX:000664 B6.Cg-Tg(S
Blocking motor cortical output with lesions or pharmacological inactivation has identified movements that require motor cortex. Yet, when and how motor cortex influences muscle activity during movement execution remains unresolved. We addressed this ambiguity using measurement and perturbation of motor cortical activity together with electromyography in mice during two forelimb movements that differ in their requirement for cortical involvement. Rapid optogenetic silencing and electrical stimulation indicated that short-latency pathways linking motor cortex with spinal motor neurons are selectively activated during one behavior. Analysis of motor cortical activity revealed a dramatic change between behaviors in the coordination of firing patterns across neurons that could account for this differential influence. Thus, our results suggest that changes in motor cortical output patterns enable a behaviorally selective engagement of short-latency effector pathways. The model of motor cortical influence implied by our findings helps reconcile previous observations on the function of motor cortex.
Literature context: on Laboratory Stock No: 000664; RRID:IMSR_JAX:000664 Mouse: ArcCreERT2 Denny et al.,
The hippocampal CA3 region is classically viewed as a homogeneous autoassociative network critical for associative memory and pattern completion. However, recent evidence has demonstrated a striking heterogeneity along the transverse, or proximodistal, axis of CA3 in spatial encoding and memory. Here we report the presence of striking proximodistal gradients in intrinsic membrane properties and synaptic connectivity for dorsal CA3. A decreasing gradient of mossy fiber synaptic strength along the proximodistal axis is mirrored by an increasing gradient of direct synaptic excitation from entorhinal cortex. Furthermore, we uncovered a nonuniform pattern of reactivation of fear memory traces, with the most robust reactivation during memory retrieval occurring in mid-CA3 (CA3b), the region showing the strongest net recurrent excitation. Our results suggest that heterogeneity in both intrinsic properties and synaptic connectivity may contribute to the distinct spatial encoding and behavioral role of CA3 subregions along the proximodistal axis.
Literature context: 6J WTThe Jackson LaboratoryCat# 000664Mouse: 5XFAD/Tg6799The Jackson L
Elevated risk of developing Alzheimer's disease (AD) is associated with hypomorphic variants of TREM2, a surface receptor required for microglial responses to neurodegeneration, including proliferation, survival, clustering, and phagocytosis. How TREM2 promotes such diverse responses is unknown. Here, we find that microglia in AD patients carrying TREM2 risk variants and TREM2-deficient mice with AD-like pathology have abundant autophagic vesicles, as do TREM2-deficient macrophages under growth-factor limitation or endoplasmic reticulum (ER) stress. Combined metabolomics and RNA sequencing (RNA-seq) linked this anomalous autophagy to defective mammalian target of rapamycin (mTOR) signaling, which affects ATP levels and biosynthetic pathways. Metabolic derailment and autophagy were offset in vitro through Dectin-1, a receptor that elicits TREM2-like intracellular signals, and cyclocreatine, a creatine analog that can supply ATP. Dietary cyclocreatine tempered autophagy, restored microglial clustering around plaques, and decreased plaque-adjacent neuronal dystrophy in TREM2-deficient mice with amyloid-β pathology. Thus, TREM2 enables microglial responses during AD by sustaining cellular energetic and biosynthetic metabolism.
Literature context: /6Jackson LaboratoriesStock No: 000664Mouse: UBI-GFP-BL/6 mice. Strain
Lower urinary tract infections are among the most common human bacterial infections, but extension to the kidneys is rare. This has been attributed to mechanical forces, such as urine flow, that prevent the ascent of bladder microbes. Here, we show that the regional hypersalinity, required for the kidney's urine-concentrating function, instructs epithelial cells to produce chemokines that localize monocyte-derived mononuclear phagocytes (MNPs) to the medulla. This hypersaline environment also increases the intrinsic bactericidal and neutrophil chemotactic activities of MNPs to generate a zone of defense. Because MNP positioning and function are dynamically regulated by the renal salt gradient, we find that patients with urinary concentrating defects are susceptible to kidney infection. Our work reveals a critical accessory role for the homeostatic function of a vital organ in optimizing tissue defense.
Literature context: kson Laboratory Stock # 000664; RRID:IMSR_JAX:000664 Fos-CreERT2 (+/âˆ’) mice Jackson
In auditory fear conditioning, experimental subjects learn to associate an auditory conditioned stimulus (CS) with an aversive unconditioned stimulus. With sufficient training, animals fear conditioned to an auditory CS show fear response to the CS, but not to irrelevant auditory stimuli. Although long-term potentiation (LTP) in the lateral amygdala (LA) plays an essential role in auditory fear conditioning, it is unknown whether LTP is induced selectively in the neural pathways conveying specific CS information to the LA in discriminative fear learning. Here, we show that postsynaptically expressed LTP is induced selectively in the CS-specific auditory pathways to the LA in a mouse model of auditory discriminative fear conditioning. Moreover, optogenetically induced depotentiation of the CS-specific auditory pathways to the LA suppressed conditioned fear responses to the CS. Our results suggest that input-specific LTP in the LA contributes to fear memory specificity, enabling adaptive fear responses only to the relevant sensory cue. VIDEO ABSTRACT.
Literature context: ouse: C57BL/6 Harlan UK and JAX RRID:IMSR_JAX:000664 Mouse: GAD67-Gfp Tamamaki et. a
Whole-cell patch-clamp electrophysiological recording is a powerful technique for studying cellular function. While in vivo patch-clamp recording has recently benefited from automation, it is normally performed "blind," meaning that throughput for sampling some genetically or morphologically defined cell types is unacceptably low. One solution to this problem is to use two-photon microscopy to target fluorescently labeled neurons. Combining this with robotic automation is difficult, however, as micropipette penetration induces tissue deformation, moving target cells from their initial location. Here we describe a platform for automated two-photon targeted patch-clamp recording, which solves this problem by making use of a closed loop visual servo algorithm. Our system keeps the target cell in focus while iteratively adjusting the pipette approach trajectory to compensate for tissue motion. We demonstrate platform validation with patch-clamp recordings from a variety of cells in the mouse neocortex and cerebellum.
Literature context: nditions, mice (male, C57BL/6J, RRID:IMSR_JAX:000664, total n = 32, 6-9 week old) we
The world around us is replete with stimuli that unfold over time. When we hear an auditory stream like music or speech or scan a texture with our fingertip, physical features in the stimulus are concatenated in a particular order. This temporal patterning is critical to interpreting the stimulus. To explore the capacity of mice and humans to learn tactile sequences, we developed a task in which subjects had to recognise a continuous modulated noise sequence delivered to whiskers or fingertips, defined by its temporal patterning over hundreds of milliseconds. GO and NO-GO sequences differed only in that the order of their constituent noise modulation segments was temporally scrambled. Both mice and humans efficiently learned tactile sequences. Mouse sequence recognition depended on detecting transitions in noise amplitude; animals could base their decision on the earliest information available. Humans appeared to use additional cues, including the duration of noise modulation segments.
Literature context: ed to as PINK1+/+ or wild-type, RRID:IMSR_JAX:000664) or timed-pregnant female PINK1
Mitochondrial Protein Kinase A (PKA) and PTEN-induced kinase 1 (PINK1), which is linked to Parkinson's disease, are two neuroprotective serine/threonine kinases that regulate dendrite remodeling and mitochondrial function. We have previously shown that PINK1 regulates dendrite morphology by enhancing PKA activity. Here, we show the molecular mechanisms by which PINK1 and PKA in the mitochondrion interact to regulate dendrite remodeling, mitochondrial morphology, content, and trafficking in dendrites. PINK1-deficient cortical neurons exhibit impaired mitochondrial trafficking, reduced mitochondrial content, fragmented mitochondria, and a reduction in dendrite outgrowth compared to wild-type neurons. Transient expression of wild-type, but not a PKA-binding-deficient mutant of the PKA-mitochondrial scaffold dual-specificity A Kinase Anchoring Protein 1 (D-AKAP1), restores mitochondrial trafficking, morphology, and content in dendrites of PINK1-deficient cortical neurons suggesting that recruiting PKA to the mitochondrion reverses mitochondrial pathology in dendrites induced by loss of PINK1. Mechanistically, full-length and cleaved forms of PINK1 increase the binding of the regulatory subunit β of PKA (PKA/RIIβ) to D-AKAP1 to enhance the autocatalytic-mediated phosphorylation of PKA/RIIβ and PKA activity. D-AKAP1/PKA governs mitochondrial trafficking in dendrites via the Miro-2/TRAK2 complex and by increasing the phosphorylation of Miro-2. Our study identifies a new role of D-AKAP1 in regulating mitochondrial trafficking through Miro-2, and supports a model in which PINK1 and mitochondrial PKA participate in a similar neuroprotective signaling pathway to maintain dendrite connectivity.
Literature context: ype C57BL/6J Jackson Laboratory RRID:JAX:000664 Mouse: Rosa26loxp-stop-loxp-eGF
Reward-seeking behavior is fundamental to survival, but suppression of this behavior can be essential as well, even for rewards of high value. In humans and rodents, the medial prefrontal cortex (mPFC) has been implicated in suppressing reward seeking; however, despite vital significance in health and disease, the neural circuitry through which mPFC regulates reward seeking remains incompletely understood. Here, we show that a specific subset of superficial mPFC projections to a subfield of nucleus accumbens (NAc) neurons naturally encodes the decision to initiate or suppress reward seeking when faced with risk of punishment. A highly resolved subpopulation of these top-down projecting neurons, identified by 2-photon Ca2+ imaging and activity-dependent labeling to recruit the relevant neurons, was found capable of suppressing reward seeking. This natural activity-resolved mPFC-to-NAc projection displayed unique molecular-genetic and microcircuit-level features concordant with a conserved role in the regulation of reward-seeking behavior, providing cellular and anatomical identifiers of behavioral and possible therapeutic significance.
Literature context: The Jackson LaboratoryStock No: 000664B6.Cg-Tg (Alb-cre) 21 Mgn/J (Alb
Type 2 cytokines are important signals triggering biogenesis of thermogenic beige adipocytes in white adipose tissue (WAT) during cold acclimation. However, how cold activates type 2 immunity in WAT remains obscure. Here we show that cold-induced type 2 immune responses and beiging in subcutaneous WAT (scWAT) are abrogated in mice with adipose-selective ablation of FGF21 or its co-receptor β-Klotho, whereas such impairments are reversed by replenishment with chemokine CCL11. Mechanistically, FGF21 acts on adipocytes in an autocrine manner to promote the expression and secretion of CCL11 via activation of ERK1/2, which drives recruitment of eosinophils into scWAT, leading to increases in accumulation of M2 macrophages, and proliferation and commitment of adipocyte precursors into beige adipocytes. These FGF21-elicited type 2 immune responses and beiging are blocked by CCL11 neutralization. Thus, the adipose-derived FGF21-CCL11 axis triggers cold-induced beiging and thermogenesis by coupling sympathetic nervous system to activation of type 2 immunity in scWAT.
Literature context: -type (C57BL/6J, Stock# 000664, RRID:IMSR_JAX:000664), the conditional Gabrd (Stock
GABAA receptor (GABAAR) pentamers are assembled from a pool of 19 subunits, and variety in subunit combinations diversifies GABAAR functions to tune brain activity. Pentamers with distinct subunit compositions localize differentially at synaptic and non-synaptic sites to mediate phasic and tonic inhibition, respectively. Despite multitudes of theoretical permutations, limited subunit combinations have been identified in the brain. Currently, no molecular model exists for combinatorial GABAAR assembly in vivo. Here, we reveal assembly rules of native GABAAR complexes that explain GABAAR subunit subcellular distributions using mice and Xenopus laevis oocytes. First, α subunits possess intrinsic signals to segregate into distinct pentamers. Second, γ2 is essential for GABAAR assembly with Neuroligin-2 (NL2) and GARLHs, which localize GABAARs at synapses. Third, δ suppresses α6 synaptic localization by preventing assembly with GARLHs/NL2. These findings establish the first molecular model for combinatorial GABAAR assembly in vivo and reveal an assembly pathway regulating GABAAR synaptic localization.
Literature context: zin Lab; The Jackson Laboratory RRID:JAX:000664 Mouse: BALB/cJ Golovkina Lab; T
Select humans and animals control persistent viral infections via adaptive immune responses that include production of neutralizing antibodies. The precise genetic basis for the control remains enigmatic. Here, we report positional cloning of the gene responsible for production of retrovirus-neutralizing antibodies in mice of the I/LnJ strain. It encodes the beta subunit of the non-classical major histocompatibility complex class II (MHC-II)-like molecule H2-O, a negative regulator of antigen presentation. The recessive and functionally null I/LnJ H2-Ob allele supported the production of virus-neutralizing antibodies independently of the classical MHC haplotype. Subsequent bioinformatics and functional analyses of the human H2-Ob homolog, HLA-DOB, revealed both loss- and gain-of-function alleles, which could affect the ability of their carriers to control infections with human hepatitis B (HBV) and C (HCV) viruses. Thus, understanding of the previously unappreciated role of H2-O (HLA-DO) in immunity to infections may suggest new approaches in achieving neutralizing immunity to viruses.
Literature context: nd (RRID:IMSR_JAX:000664, obtained from Charles River) w
Mobilization of retrotransposons to new genomic locations is a significant driver of mammalian genome evolution, but these mutagenic events can also cause genetic disorders. In humans, retrotransposon mobilization is mediated primarily by proteins encoded by LINE-1 (L1) retrotransposons, which mobilize in pluripotent cells early in development. Here we show that TEX19.1, which is induced by developmentally programmed DNA hypomethylation, can directly interact with the L1-encoded protein L1-ORF1p, stimulate its polyubiquitylation and degradation, and restrict L1 mobilization. We also show that TEX19.1 likely acts, at least in part, through promoting the activity of the E3 ubiquitin ligase UBR2 towards L1-ORF1p. Moreover, loss of Tex19.1 increases L1-ORF1p levels and L1 mobilization in pluripotent mouse embryonic stem cells, implying that Tex19.1 prevents de novo retrotransposition in the pluripotent phase of the germline cycle. These data show that post-translational regulation of L1 retrotransposons plays a key role in maintaining trans-generational genome stability in mammals.
Literature context: Laboratory catalog #JAX:000664, RRID:IMSR_JAX:000664). Experiments were performed on
SCN8A encephalopathy, or early infantile epileptic encephalopathy 13 (EIEE13), is caused predominantly by de novo gain-of-function mutations in the voltage-gated Na channel Nav1.6. Affected individuals suffer from refractory seizures, developmental delay, cognitive disability, and elevated risk of sudden unexpected death in epilepsy (SUDEP). A knock-in mouse model carrying the patient mutation p.Asn1768Asp (N1768D) reproduces many features of the disorder, including spontaneous seizures and SUDEP. We used the mouse model to examine the effects of the mutation on layer II stellate neurons of the medial entorhinal cortex (mEC), which transmit excitatory input to the hippocampus. Heterozygous (Scn8aD/+), homozygous (Scn8aD/D)), and WT (Scn8a+/+) littermates were compared at 3 weeks of age, the time of seizure onset for homozygous mice. Heterozygotes remain seizure free for another month. mEC layer II neurons of heterozygous and homozygous mice were hyperexcitable and generated long-lasting depolarizing potentials with bursts of action potentials after synaptic stimulation. Recording of Na currents revealed proexcitatory increases in persistent and resurgent currents and rightward shifts in inactivation parameters, leading to significant increases in the magnitude of window currents. The proexcitatory changes were more pronounced in homozygous mice than in heterozygotes, consistent with the earlier age of seizure onset in homozygotes. These studies demonstrate that the N1768D mutation increases the excitability of mEC layer II neurons by increasing persistent and resurgent Na currents and disrupting channel inactivation. The aberrant activities of mEC layer II neurons would provide excessive excitatory input to the hippocampus and contribute to hyperexcitability of hippocampal neurons in this model of SCN8A encephalopathy.SIGNIFICANCE STATEMENTSCN8A encephalopathy is a devastating neurological disorder that results from de novo mutations in the Na channel Nav1.6. In addition to seizures, patients suffer from cognitive and developmental delays and are at high risk for sudden unexpected death in epilepsy (SUDEP). A mouse knock-in model expressing the patient mutation N1768D reproduces several pathological phenotypes, including spontaneous seizures and sudden death. We demonstrate that medial entorhinal cortex (mEC) neurons from the mouse model exhibit proexcitatory alterations in Na channel activity, some of which were not seen in hippocampal or cortical neurons, and resulting in neuronal hyperexcitability. Because mEC neurons regulate the activity of the hippocampus, which plays an important role in seizure onset, we propose that these profound changes in mEC neuron excitability associated with the gain-of-function mutation of Nav1.6 may increase excitatory drive into the hippocampus, culminating in seizure activity and SUDEP.
Literature context: : C57BL/6J Jackson Laboratories RRID:JAX:000664 Mouse: BALB/c Jackson Laborator
Inhibition of cytosolic DNA sensing represents a strategy that tumor cells use for immune evasion, but the underlying mechanisms are unclear. Here we have shown that CD47-signal regulatory protein α (SIRPα) axis dictates the fate of ingested DNA in DCs for immune evasion. Although macrophages were more potent in uptaking tumor DNA, increase of DNA sensing by blocking the interaction of SIRPα with CD47 preferentially occurred in dendritic cells (DCs) but not in macrophages. Mechanistically, CD47 blockade enabled the activation of NADPH oxidase NOX2 in DCs, which in turn inhibited phagosomal acidification and reduced the degradation of tumor mitochondrial DNA (mtDNA) in DCs. mtDNA was recognized by cyclic-GMP-AMP synthase (cGAS) in the DC cytosol, contributing to type I interferon (IFN) production and antitumor adaptive immunity. Thus, our findings have demonstrated how tumor cells inhibit innate sensing in DCs and suggested that the CD47-SIRPα axis is critical for DC-driven antitumor immunity.
Literature context: oratory Jax: 000664; RRID:IMSR_JAX:000664 Mouse: Slc17a6tm1Lowl/J The Jac
The ability of presynaptic dopamine terminals to tune neurotransmitter release to meet the demands of neuronal activity is critical to neurotransmission. Although vesicle content has been assumed to be static, in vitro data increasingly suggest that cell activity modulates vesicle content. Here, we use a coordinated genetic, pharmacological, and imaging approach in Drosophila to study the presynaptic machinery responsible for these vesicular processes in vivo. We show that cell depolarization increases synaptic vesicle dopamine content prior to release via vesicular hyperacidification. This depolarization-induced hyperacidification is mediated by the vesicular glutamate transporter (VGLUT). Remarkably, both depolarization-induced dopamine vesicle hyperacidification and its dependence on VGLUT2 are seen in ventral midbrain dopamine neurons in the mouse. Together, these data suggest that in response to depolarization, dopamine vesicles utilize a cascade of vesicular transporters to dynamically increase the vesicular pH gradient, thereby increasing dopamine vesicle content.
Literature context: erations. C57BL/6J (JAX 000664; RRID:IMSR_JAX:000664), LfngTm1Grid/J (Lfngâˆ’/+, LfngÎ²
Hippocampal neural stem cells (NSCs) integrate inputs from multiple sources to balance quiescence and activation. Notch signaling plays a key role during this process. Here, we report that Lunatic fringe (Lfng), a key modifier of the Notch receptor, is selectively expressed in NSCs. Further, Lfng in NSCs and Notch ligands Delta1 and Jagged1, expressed by their progeny, together influence NSC recruitment, cell cycle duration, and terminal fate. We propose a new model in which Lfng-mediated Notch signaling enables direct communication between a NSC and its descendants, so that progeny can send feedback signals to the 'mother' cell to modify its cell cycle status. Lfng-mediated Notch signaling appears to be a key factor governing NSC quiescence, division, and fate.
Literature context: aboratory RRID:IMSR_JAX:000664 Mouse: 129
Activation of the pseudokinase mixed lineage kinase domain-like (MLKL) upon its phosphorylation by the protein kinase RIPK3 triggers necroptosis, a form of programmed cell death in which rupture of cellular membranes yields release of intracellular components. We report that MLKL also associated with endosomes and controlled the transport of endocytosed proteins, thereby enhancing degradation of receptors and ligands, modulating their induced signaling and facilitating the generation of extracellular vesicles. This role was exerted on two quantitative grades: a constitutive one independent of RIPK3, and an enhanced one, triggered by RIPK3, where the association of MLKL with the endosomes was enhanced, and it was found to bind endosomal sorting complexes required for transport (ESCRT) proteins and the flotillins and to be excluded, together with them, from cells within vesicles. We suggest that release of phosphorylated MLKL within extracellular vesicles serves as a mechanism for self-restricting the necroptotic activity of this protein.
Literature context: ed with wild-type C57BL/6 mice (RRID:IMSR_JAX:000664) to generate Î”GLS1 HETs.
Dopamine neurons in the ventral tegmental area use glutamate as a cotransmitter. To elucidate the behavioral role of the cotransmission, we targeted the glutamate-recycling enzyme glutaminase (gene Gls1). In mice with a dopamine transporter (Slc6a3)-driven conditional heterozygous (cHET) reduction of Gls1 in their dopamine neurons, dopamine neuron survival and transmission were unaffected, while glutamate cotransmission at phasic firing frequencies was reduced, enabling a selective focus on the cotransmission. The mice showed normal emotional and motor behaviors, and an unaffected response to acute amphetamine. Strikingly, amphetamine sensitization was reduced and latent inhibition potentiated. These behavioral effects, also seen in global GLS1 HETs with a schizophrenia resilience phenotype, were not seen in mice with an Emx1-driven forebrain reduction affecting most brain glutamatergic neurons. Thus, a reduction in dopamine neuron glutamate cotransmission appears to mediate significant components of the GLS1 HET schizophrenia resilience phenotype, and glutamate cotransmission appears to be important in attribution of motivational salience.
Literature context: t#000664; RRID:IMSR_JAX:000664 Mouse: B6.
The release of dopamine (DA) regulates rewarding behavior and motor actions through striatum-targeting efferents from ventral tegmental area (VTA) and substantia nigra pars compacta (SNc). Here, we map and functionally characterize axonal projections from oxytocin neurons in the hypothalamic paraventricular nucleus to midbrain DA regions. Electrophysiological recordings of DA neurons reveal that both the application of oxytocin and optogenetic stimulation of oxytocinergic terminals suffice to increase DA neuron activity in the VTA but downregulate it in SNc. This biased modulation is mediated by oxytocin and vasopressin G-protein-coupled receptors. Oxytocin release directly activates DA neurons and indirectly inhibits them through local GABA neurons, but the relative magnitudes of the two mechanisms differ in VTA and SNc. Oxytocin-modulated DA neurons give rise to canonical striatal projections. Since hypothalamic oxytocinergic projections also target the striatum, oxytocin is poised to bias the balance of DA tone through multiple sites in vertebrate reward circuits.
Literature context: ines and regulations. C57BL/6J (RRID:IMSR_JAX:000664) and App-KO (Jackson Lab B6.129
The concurrent application of subtoxic doses of soluble oligomeric forms of human amyloid-beta (oAβ) and Tau (oTau) proteins impairs memory and its electrophysiological surrogate long-term potentiation (LTP), effects that may be mediated by intra-neuronal oligomers uptake. Intrigued by these findings, we investigated whether oAβ and oTau share a common mechanism when they impair memory and LTP in mice. We found that as already shown for oAβ, also oTau can bind to amyloid precursor protein (APP). Moreover, efficient intra-neuronal uptake of oAβ and oTau requires expression of APP. Finally, the toxic effect of both extracellular oAβ and oTau on memory and LTP is dependent upon APP since APP-KO mice were resistant to oAβ- and oTau-induced defects in spatial/associative memory and LTP. Thus, APP might serve as a common therapeutic target against Alzheimer's Disease (AD) and a host of other neurodegenerative diseases characterized by abnormal levels of Aβ and/or Tau.
Literature context: mental Models: Organisms/StrainsBl6 wildtype mouse, C57BL/6JJax labsStock No:000664BDNFflox/flox mouseProf. Michael
Autophagy is crucial for neuronal integrity. Loss of key autophagic components leads to progressive neurodegeneration and structural defects in pre- and postsynaptic morphologies. However, the molecular mechanisms regulating autophagy in the brain remain elusive. Similarly, while it is widely accepted that protein turnover is required for synaptic plasticity, the contribution of autophagy to the degradation of synaptic proteins is unknown. Here, we report that BDNF signaling via the tropomyosin receptor kinase B (TrkB) and the phosphatidylinositol-3' kinase (PI3K)/Akt pathway suppresses autophagy in vivo. In addition, we demonstrate that suppression of autophagy is required for BDNF-induced synaptic plasticity and for memory enhancement under conditions of nutritional stress. Finally, we identify three key remodelers of postsynaptic densities as cargo of autophagy. Our results establish autophagy as a pivotal component of BDNF signaling, which is essential for BDNF-induced synaptic plasticity. This molecular mechanism underlies behavioral adaptations that increase fitness in times of scarcity.
Literature context: /6J mice (RRID:IMSR_JAX:000664; Jackson L
Taste buds contain multiple cell types with each type expressing receptors and transduction components for a subset of taste qualities. The sour sensing cells, Type III cells, release serotonin (5-HT) in response to the presence of sour (acidic) tastants and this released 5-HT activates 5-HT3 receptors on the gustatory nerves. We show here, using 5-HT3A GFP mice, that 5-HT3 -expressing nerve fibers preferentially contact and receive synaptic contact from Type III taste cells. Further, these 5-HT3 -expressing nerve fibers terminate in a restricted central-lateral portion of the nucleus of the solitary tract (nTS)-the same area that shows increased c-Fos expression upon presentation of a sour tastant (30 mM citric acid). This acid stimulation also evokes c-Fos in the laterally adjacent mediodorsal spinal trigeminal nucleus (DMSp5), but this trigeminal activation is not associated with the presence of 5-HT3 -expressing nerve fibers as it is in the nTS. Rather, the neuronal activation in the trigeminal complex likely is attributable to direct depolarization of acid-sensitive trigeminal nerve fibers, for example, polymodal nociceptors, rather than through taste buds. Taken together, these findings suggest that transmission of sour taste information involves communication between Type III taste cells and 5-HT3 -expressing afferent nerve fibers that project to a restricted portion of the nTS consistent with a crude mapping of taste quality information in the primary gustatory nucleus.
Literature context: l/6 mice (RRID:IMSR_JAX:000664) were purc
Neurogenesis is a well-characterized phenomenon within the dentate gyrus (DG) of the adult hippocampus. Aging and chronic degenerative disorders have been shown to impair hippocampal neurogenesis, but the consequence of chronic inflammation remains controversial. In this study the chronic experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis was used to investigate the long-term effects of T cell-mediated central nervous system inflammation on hippocampal neurogenesis. 5-Bromodeoxyuridine (BrdU)-labeled subpopulations of hippocampal cells in EAE and control mice (coexpressing GFAP, doublecortin, NeuN, calretinin, and S100) were quantified at the recovery phase, 21 days after BrdU administration, to estimate alterations on the rate and differentiation pattern of the neurogenesis process. The core features of EAE mice DG are (i) elevated number of newborn (BrdU+) cells indicating vigorous proliferation, which in the long term subsided; (ii) enhanced migration of newborn cells into the granule cell layer; (iii) increased level of immature neuronal markers (including calretinin and doublecortin); (iv) trending decrease in the percentage of newborn mature neurons; and (v) augmented gliogenesis and differentiation of newborn neural precursor cells (NPCs) to mature astrocytes (BrdU+/S100+). Although the inflammatory environment in the brain of EAE mice enhances the proliferation of hippocampal NPCs, in the long term neurogenesis is progressively depleted, giving prominence to gliogenesis. The discrepancy between the high number of immature cells and the low number of mature newborn cells could be the result of a caused defect in the maturation pathway. © 2016 Wiley Periodicals, Inc.
Literature context: # 000651C57BL/6JJackson labCat# 000664OligonucleotidesHSV1Â UL1 forward
Clearance of pathogens or tumor cells by antibodies traditionally requires both Fab and Fc domains of IgG. Here, we show the Fc domain of IgG alone mediates recognition and clearance of herpes simplex virus (HSV1)-infected cells. The human natural killer (NK) cell surface is naturally coated with IgG bound by its Fc domain to the Fcγ receptor CD16a. NK cells utilize the Fc domain of bound IgG to recognize gE, an HSV1-encoded glycoprotein that also binds the Fc domain of IgG but at a site distinct from CD16a. The bridge formed by the Fc domain between the HSV1-infected cell and the NK cell results in NK cell activation and lysis of the HSV1-infected cell in the absence of HSV1-specific antibody in vitro and prevents fatal HSV1 infection in vivo. This mechanism also explains how bacterial IgG-binding proteins regulate NK cell function and may be broadly applicable to Fcγ-receptor-bearing cells.
Literature context: nimal Care Committee. C57BL/6J (Jackson, 000664) and STOCK Mecp2tm1.1Jae/Mmcd (
Literature context: Adult (25-35 g) male C57BL/6J (RRID:IMSR_JAX:000664) and ThIRES-Cre (RRID:IMSR_EM:0
Increased tonic activity of locus coeruleus noradrenergic (LC-NE) neurons induces anxiety-like and aversive behavior. While some information is known about the afferent circuitry that endogenously drives this neural activity and behavior, the downstream receptors and anatomical projections that mediate these acute risk aversive behavioral states via the LC-NE system remain unresolved. Here we use a combination of retrograde tracing, fast-scan cyclic voltammetry, electrophysiology, and in vivo optogenetics with localized pharmacology to identify neural substrates downstream of increased tonic LC-NE activity in mice. We demonstrate that photostimulation of LC-NE fibers in the BLA evokes norepinephrine release in the basolateral amygdala (BLA), alters BLA neuronal activity, conditions aversion, and increases anxiety-like behavior. Additionally, we report that β-adrenergic receptors mediate the anxiety-like phenotype of increased NE release in the BLA. These studies begin to illustrate how the complex efferent system of the LC-NE system selectively mediates behavior through distinct receptor and projection-selective mechanisms.
Literature context: Wild-type mice (C57BL/6; RRID:IMSR_JAX:000664; postnatal days 12-18; from Har
Spike timing-dependent plasticity (STDP) is under neuromodulatory control, which is correlated with distinct behavioral states. Previously, we reported that dopamine, a reward signal, broadens the time window for synaptic potentiation and modulates the outcome of hippocampal STDP even when applied after the plasticity induction protocol (Brzosko et al., 2015). Here, we demonstrate that sequential neuromodulation of STDP by acetylcholine and dopamine offers an efficacious model of reward-based navigation. Specifically, our experimental data in mouse hippocampal slices show that acetylcholine biases STDP toward synaptic depression, whilst subsequent application of dopamine converts this depression into potentiation. Incorporating this bidirectional neuromodulation-enabled correlational synaptic learning rule into a computational model yields effective navigation toward changing reward locations, as in natural foraging behavior. Thus, temporally sequenced neuromodulation of STDP enables associations to be made between actions and outcomes and also provides a possible mechanism for aligning the time scales of cellular and behavioral learning.
Literature context: mental Models: Organisms/StrainsC57BL/6 miceJackson Laboratory000664A129 miceRossi LaboratoryCD-1Cha
The emergence of Zika virus (ZIKV) and its association with congenital malformations has prompted the rapid development of vaccines. Although efficacy with multiple viral vaccine platforms has been established in animals, no study has addressed protection during pregnancy. We tested in mice two vaccine platforms, a lipid nanoparticle-encapsulated modified mRNA vaccine encoding ZIKV prM and E genes and a live-attenuated ZIKV strain encoding an NS1 protein without glycosylation, for their ability to protect against transmission to the fetus. Vaccinated dams challenged with a heterologous ZIKV strain at embryo day 6 (E6) and evaluated at E13 showed markedly diminished levels of viral RNA in maternal, placental, and fetal tissues, which resulted in protection against placental damage and fetal demise. As modified mRNA and live-attenuated vaccine platforms can restrict in utero transmission of ZIKV in mice, their further development in humans to prevent congenital ZIKV syndrome is warranted.
Literature context: g/strain/013636.html; RRID:IMSR JAX:000664) line that we routinely use in
The whisker system is an important sensory organ with extensive neural representations in the brain of the mouse. Patterned neural modules (barrelettes) in the ipsilateral principal sensory nucleus of the trigeminal nerve (PrV) correspond to the whiskers. Axons of the PrV barrelette neurons cross the midline and confer the whisker-related patterning to the contralateral ventroposteromedial nucleus of the thalamus, and subsequently to the cortex. In this way, specific neural modules called barreloids and barrels in the contralateral thalamus and cortex represent each whisker. Partial midline crossing of the PrV axons, in a conditional Robo3 mutant (Robo3R3-5cKO) mouse line, leads to the formation of bilateral whisker maps in the ventroposteromedial, as well as the barrel cortex. We used voltage-sensitive dye optical imaging and somatosensory and motor behavioral tests to characterize the consequences of bifacial maps in the thalamocortical system. Voltage-sensitive dye optical imaging verified functional, bilateral whisker representation in the barrel cortex and activation of distinct cortical loci following ipsilateral and contralateral stimulation of the specific whiskers. The mutant animals were comparable with the control animals in sensorimotor tests. However, they showed noticeable deficits in all of the whisker-dependent or -related tests, including Y-maze exploration, horizontal surface approach, bridge crossing, gap crossing, texture discrimination, floating in water, and whisking laterality. Our results indicate that bifacial maps along the thalamocortical system do not offer a functional advantage. Instead, they lead to impairments, possibly due to the smaller size of the whisker-related modules and interference between the ipsilateral and contralateral whisker representations in the same thalamus and cortex.SIGNIFICANCE STATEMENT The whisker sensory system plays a quintessentially important role in exploratory behavior of mice and other nocturnal rodents. Here, we studied a novel mutant mouse line, in which the projections from the brainstem to the thalamus are disrupted. This led to formation of bilateral whisker maps in both the thalamus and the cortex. The two whisker maps crowd in a space normally devoted to the contralateral map alone and in a nonoverlapping fashion. Stimulation of the whiskers on either side activates the corresponding region of the map. Mice with bilateral whisker maps perform well in general sensorimotor tasks but show poor performance in specific tests that require whisker-dependent tactile discrimination. These observations indicate that contralateral, instead of bilateral, representation of the sensory space plays a critical role in acuity and fine discrimination during somesthesis.
Literature context: isolation. Male C57BL/6J mice (RRID:IMSR_JAX:000664) obtained from The Jackson Labo
Previous research demonstrates that Slc6a15, a neutral amino acid transporter, is associated with depression susceptibility. However, no study examined Slc6a15 in the ventral striatum [nucleus accumbens (NAc)] in depression. Given our previous characterization of Slc6a15 as a striatal dopamine receptor 2 (D2)-neuron-enriched gene, we examined the role of Slc6a15 in NAc D2-neurons in mediating susceptibility to stress in male mice. First, we showed that Slc6a15 mRNA was reduced in NAc of mice susceptible to chronic social defeat stress (CSDS), a paradigm that produces behavioral and molecular adaptations that resemble clinical depression. Consistent with our preclinical data, we observed Slc6a15 mRNA reduction in NAc of individuals with major depressive disorder (MDD). The Slc6a15 reduction in NAc occurred selectively in D2-neurons. Next, we used Cre-inducible viruses combined with D2-Cre mice to reduce or overexpress Slc6a15 in NAc D2-neurons. Slc6a15 reduction in D2-neurons caused enhanced susceptibility to a subthreshold social defeat stress (SSDS) as observed by reduced social interaction, while a reduction in social interaction following CSDS was not observed when Slc6a15 expression in D2-neurons was restored. Finally, since both D2-medium spiny neurons (MSNs) and D2-expressing choline acetyltransferase (ChAT) interneurons express Slc6a15, we examined Slc6a15 protein in these interneurons after CSDS. Slc6a15 protein was unaltered in ChAT interneurons. Consistent with this, reducing Slc5a15 selectively in NAc D2-MSNs, using A2A-Cre mice that express Cre selectively in D2-MSNs, caused enhanced susceptibility to SSDS. Collectively, our data demonstrate that reduced Slc6a15 in NAc occurs in MDD individuals and that Slc6a15 reduction in NAc D2-neurons underlies stress susceptibility.SIGNIFICANCE STATEMENT Our study demonstrates a role for reduced Slc6a15, a neutral amino acid transporter, in nucleus accumbens (NAc) in depression and stress susceptibility. The reduction of Slc6a15 occurs selectively in the NAc D2-neurons. Genetic reduction of Slc6a15 induces susceptibility to a subthreshold stress, while genetic overexpression in D2-neurons prevents social avoidance after chronic social defeat stress.
Literature context: mental Models: Organisms/StrainsMouse: C57BL/6JJackson laboratories000664OligonucleotidespIRES::CrkI-GFP(
Apoptosis has been implicated in compensatory proliferation signaling (CPS), whereby dying cells induce proliferation in neighboring cells as a means to restore homeostasis. The nature of signaling between apoptotic cells and their neighboring cells remains largely unknown. Here we show that a fraction of apoptotic cells produce and release CrkI-containing microvesicles (distinct from exosomes and apoptotic bodies), which induce proliferation in neighboring cells upon contact. We provide visual evidence of CPS by videomicroscopy. We show that purified vesicles in vitro and in vivo are sufficient to stimulate proliferation in other cells. Our data demonstrate that CrkI inactivation by ExoT bacterial toxin or by mutagenesis blocks vesicle formation in apoptotic cells and inhibits CPS, thus uncoupling apoptosis from CPS. We further show that c-Jun amino-terminal kinase (JNK) plays a pivotal role in mediating vesicle-induced CPS in recipient cells. CPS could have important ramifications in diseases that involve apoptotic cell death.
Literature context: 57BL/6Jackson LaboratoryStock # 000664Thy1.1 (CD90.1), aka B6.PL-Thy1a
Immune cells function in diverse metabolic environments. Tissues with low glucose and high lactate concentrations, such as the intestinal tract or ischemic tissues, frequently require immune responses to be more pro-tolerant, avoiding unwanted reactions against self-antigens or commensal bacteria. T-regulatory cells (Tregs) maintain peripheral tolerance, but how Tregs function in low-glucose, lactate-rich environments is unknown. We report that the Treg transcription factor Foxp3 reprograms T cell metabolism by suppressing Myc and glycolysis, enhancing oxidative phosphorylation, and increasing nicotinamide adenine dinucleotide oxidation. These adaptations allow Tregs a metabolic advantage in low-glucose, lactate-rich environments; they resist lactate-mediated suppression of T cell function and proliferation. This metabolic phenotype may explain how Tregs promote peripheral immune tolerance during tissue injury but also how cancer cells evade immune destruction in the tumor microenvironment. Understanding Treg metabolism may therefore lead to novel approaches for selective immune modulation in cancer and autoimmune diseases.
Literature context: C57BL/6J The Jackson Laboratory RRID:JAX:000664 Mouse: R6/2: B6(Cg)-Tyrc-2J/J T
Aging and obesity induce ectopic adipocyte accumulation in bone marrow cavities. This process is thought to impair osteogenic and hematopoietic regeneration. Here we specify the cellular identities of the adipogenic and osteogenic lineages of the bone. While aging impairs the osteogenic lineage, high-fat diet feeding activates expansion of the adipogenic lineage, an effect that is significantly enhanced in aged animals. We further describe a mesenchymal sub-population with stem cell-like characteristics that gives rise to both lineages and, at the same time, acts as a principal component of the hematopoietic niche by promoting competitive repopulation following lethal irradiation. Conversely, bone-resident cells committed to the adipocytic lineage inhibit hematopoiesis and bone healing, potentially by producing excessive amounts of Dipeptidyl peptidase-4, a protease that is a target of diabetes therapies. These studies delineate the molecular identity of the bone-resident adipocytic lineage, and they establish its involvement in age-dependent dysfunction of bone and hematopoietic regeneration.
Literature context: ; Research Resource Identifier (RRID): IMSR_JAX:000664] were housed in a 12:12 light:d
Many of the best-studied neural sex differences relate to differences in cell number and are due to the hormonal control of developmental cell death. However, several prominent neural sex differences persist even if cell death is eliminated. We hypothesized that these may reflect cell phenotype "decisions" that depend on epigenetic mechanisms, such as DNA methylation. To test this, we treated newborn mice with the DNA methyltransferase (DNMT) inhibitor zebularine, or vehicle, and examined two sexually dimorphic markers at weaning. As expected, control males had more cells immunoreactive for calbindin-D28k (CALB) in the medial preoptic area (mPOA) and fewer cells immunoreactive for estrogen receptor α (ERα) in the ventrolateral portion of the ventromedial nucleus of the hypothalamus (VMHvl) and the mPOA than did females. Neonatal DNMT inhibition markedly increased CALB cell number in both sexes and ERα cell density in males; as a result, the sex differences in ERα in the VMHvl and mPOA were completely eliminated in zebularine-treated animals. Zebularine treatment did not affect developmental cell death or the total density of Nissl-stained cells at weaning. Thus, a neonatal disruption of DNA methylation apparently has long-term effects on the proportion of cells expressing CALB and ERα, and some of these effects are sex specific. We also found that sex differences in CALB in the mPOA and ERα in the VMHvl persist in mice with a neuron-specific depletion of either Dnmt1 or Dnmt3b, indicating that neither DNMT alone is likely to be required for the sexually dimorphic expression of these markers.
Literature context: e Jackson LaboratoryStock 026179Wildtype C57BL/6J miceThe Jackson LaboratoryStock 000664OligonucleotidesGenotyping prime
Dual leucine zipper kinase (DLK) has been implicated in cell death signaling secondary to axonal damage in retinal ganglion cells (RGCs) and other neurons. To better understand the pathway through which DLK acts, we developed enhanced functional genomic screens in primary RGCs, including use of arrayed, whole-genome, small interfering RNA libraries. Explaining why DLK inhibition is only partially protective, we identify leucine zipper kinase (LZK) as cooperating with DLK to activate downstream signaling and cell death in RGCs, including in a mouse model of optic nerve injury, and show that the same pathway is active in human stem cell-derived RGCs. Moreover, we identify four transcription factors, JUN, activating transcription factor 2 (ATF2), myocyte-specific enhancer factor 2A (MEF2A), and SRY-Box 11 (SOX11), as being the major downstream mediators through which DLK/LZK activation leads to RGC cell death. Increased understanding of the DLK pathway has implications for understanding and treating neurodegenerative diseases.
Literature context: ed from The Jackson Laboratory (RRID:IMSR_JAX:000664) were housed in groups of four
Dentate gyrus (DG) is widely thought to provide a teaching signal that enables hippocampal encoding of memories, but its role during retrieval is poorly understood. Some data and models suggest that DG plays no role in retrieval; others encourage the opposite conclusion. To resolve this controversy, we evaluated the effects of optogenetic inhibition of dorsal DG during context fear conditioning, recall, generalization, and extinction in male mice. We found that (1) inhibition during training impaired context fear acquisition; (2) inhibition during recall did not impair fear expression in the training context, unless mice had to distinguish between similar feared and neutral contexts; (3) inhibition increased generalization of fear to an unfamiliar context that was similar to a feared one and impaired fear expression in the conditioned context when it was similar to a neutral one; and (4) inhibition impaired fear extinction. These effects, as well as several seemingly contradictory published findings, could be reproduced by BACON (Bayesian Context Fear Algorithm), a physiologically realistic hippocampal model positing that acquisition and retrieval both involve coordinated activity in DG and CA3. Our findings thus suggest that DG contributes to retrieval and extinction, as well as to the initial establishment of context fear.SIGNIFICANCE STATEMENT Despite abundant evidence that the hippocampal dentate gyrus (DG) plays a critical role in memory, it remains unclear whether the role of DG relates to memory acquisition or retrieval. Using contextual fear conditioning and optogenetic inhibition, we show that DG contributes to both of these processes. Using computational simulations, we identify specific mechanisms through which the suppression of DG affects memory performance. Finally, we show that DG contributes to fear extinction learning, a process in which learned fear is attenuated through exposures to a fearful context in the absence of threat. Our data resolve a long-standing question about the role of DG in memory and provide insight into how disorders affecting DG, including aging, stress, and depression, influence cognitive processes.
Literature context: e C57BL/6JThe Jackson LaboratoryJAX: 000664Mouse: ob/ob obese, leptin-defic
Malonyl-coenzyme A (malonyl-CoA) is a central metabolite in mammalian fatty acid biochemistry generated and utilized in the cytoplasm; however, little is known about noncanonical organelle-specific malonyl-CoA metabolism. Intramitochondrial malonyl-CoA is generated by a malonyl-CoA synthetase, ACSF3, which produces malonyl-CoA from malonate, an endogenous competitive inhibitor of succinate dehydrogenase. To determine the metabolic requirement for mitochondrial malonyl-CoA, ACSF3 knockout (KO) cells were generated by CRISPR/Cas-mediated genome editing. ACSF3 KO cells exhibited elevated malonate and impaired mitochondrial metabolism. Unbiased and targeted metabolomics analysis of KO and control cells in the presence or absence of exogenous malonate revealed metabolic changes dependent on either malonate or malonyl-CoA. While ACSF3 was required for the metabolism and therefore detoxification of malonate, ACSF3-derived malonyl-CoA was specifically required for lysine malonylation of mitochondrial proteins. Together, these data describe an essential role for ACSF3 in dictating the metabolic fate of mitochondrial malonate and malonyl-CoA in mammalian metabolism.
Literature context: trainsMouse C57Bl6/JJackson LabsJAX000664Mouse Albumin-CreLaboratory of D
Decreasing glucagon action lowers the blood glucose and may be useful therapeutically for diabetes. However, interrupted glucagon signaling leads to α cell proliferation. To identify postulated hepatic-derived circulating factor(s) responsible for α cell proliferation, we used transcriptomics/proteomics/metabolomics in three models of interrupted glucagon signaling and found that proliferation of mouse, zebrafish, and human α cells was mTOR and FoxP transcription factor dependent. Changes in hepatic amino acid (AA) catabolism gene expression predicted the observed increase in circulating AAs. Mimicking these AA levels stimulated α cell proliferation in a newly developed in vitro assay with L-glutamine being a critical AA. α cell expression of the AA transporter Slc38a5 was markedly increased in mice with interrupted glucagon signaling and played a role in α cell proliferation. These results indicate a hepatic α islet cell axis where glucagon regulates serum AA availability and AAs, especially L-glutamine, regulate α cell proliferation and mass via mTOR-dependent nutrient sensing.
Literature context: Organisms/StrainsMouse: C57BL6/JThe Jackson LaboratoryJAX: 000664Mouse: B1-8iThe Jackson Laborato
During antibody affinity maturation, germinal center (GC) B cells cycle between affinity-driven selection in the light zone (LZ) and proliferation and somatic hypermutation in the dark zone (DZ). Although selection of GC B cells is triggered by antigen-dependent signals delivered in the LZ, DZ proliferation occurs in the absence of such signals. We show that positive selection triggered by T cell help activates the mechanistic target of rapamycin complex 1 (mTORC1), which promotes the anabolic program that supports DZ proliferation. Blocking mTORC1 prior to growth prevented clonal expansion, whereas blockade after cells reached peak size had little to no effect. Conversely, constitutively active mTORC1 led to DZ enrichment but loss of competitiveness and impaired affinity maturation. Thus, mTORC1 activation is required for fueling B cells prior to DZ proliferation rather than for allowing cell-cycle progression itself and must be regulated dynamically during cyclic re-entry to ensure efficient affinity-based selection.
Literature context: t#000664; RRID:IMSR_JAX:000664 Mouse: B6(
The mammalian pupillary light reflex (PLR) involves a bilateral brain circuit whereby afferent light signals in the optic nerve ultimately drive iris-sphincter-muscle contraction via excitatory cholinergic parasympathetic innervation [1, 2]. Additionally, the PLR in nocturnal and crepuscular sub-primate mammals has a "local" component in the isolated sphincter muscle [3-5], as in amphibians, fish, and bird [6-10]. In mouse, this local PLR requires the pigment melanopsin , originally found in intrinsically photosensitive retinal ganglion cells (ipRGCs) [11-19]. However, melanopsin's presence and effector pathway locally in the iris remain uncertain. The sphincter muscle itself may express melanopsin , or its cholinergic parasympathetic innervation may be modulated by suggested intraocular axonal collaterals of ipRGCs traveling to the eye's ciliary body or even to the iris [20-22]. Here, we show that the muscarinic receptor antagonist, atropine, eliminated the effect of acetylcholine (ACh), but not of light, on isolated mouse sphincter muscle. Conversely, selective genetic deletion of melanopsin in smooth muscle mostly removed the light-induced, but not the ACh-triggered, increase in isolated sphincter muscle's tension and largely suppressed the local PLR in vivo. Thus, sphincter muscle cells are bona fide, albeit unconventional, photoreceptors. We found melanopsin expression in a small subset of mouse iris sphincter muscle cells, with the light-induced contractile signal apparently spreading through gap junctions into neighboring muscle cells. Light and ACh share a common signaling pathway in sphincter muscle. In summary, our experiments have provided details of a photosignaling process in the eye occurring entirely outside the retina.
Literature context: mental Models: Organisms/StrainsMouse: C57BL/6J male miceThe Jackson LaboratoryJAX:000664OligonucleotidesEif2ak3-dluc for
Besides circadian rhythms, oscillations cycling with a 12 hr period exist. However, the prevalence, origin, regulation, and function of mammalian 12 hr rhythms remain elusive. Utilizing an unbiased mathematical approach identifying all superimposed oscillations, we uncovered prevalent 12 hr gene expression and metabolic rhythms in mouse liver, coupled with a physiological 12 hr unfolded protein response oscillation. The mammalian 12 hr rhythm is cell autonomous, driven by a dedicated 12 hr pacemaker distinct from the circadian clock, and can be entrained in vitro by metabolic and ER stress cues. Mechanistically, we identified XBP1s as a transcriptional regulator of the mammalian 12 hr clock. Downregulation of the 12 hr gene expression strongly correlates with human hepatic steatosis and steatohepatitis, implying its importance in maintaining metabolic homeostasis. The mammalian 12 hr rhythm of gene expression also is conserved in nematodes and crustaceans, indicating an ancient origin of the 12 hr clock. Our work sheds new light on how perturbed biological rhythms contribute to human disease.
Literature context: 57BL/6Â J (RRID:IMSR_JAX:000664), B6.129S2
Dengue is the most prevalent arthropod-borne viral disease worldwide and is caused by the four dengue virus serotypes (DENV-1-4). Sequential heterologous DENV infections can be associated with severe disease manifestations. Here, we present an immunocompetent mouse model of secondary DENV infection using non mouse-adapted DENV strains to investigate the pathogenesis of severe dengue disease. C57BL/6 mice infected sequentially with DENV-1 (strain Puerto Rico/94) and DENV-2 (strain Tonga/74) developed low platelet counts, internal hemorrhages, and increase of liver enzymes. Cross-reactive CD8+ T lymphocytes were found to be necessary and sufficient for signs of severe disease by adoptively transferring of DENV-1-immune CD8+T lymphocytes before DENV-2 challenge. Disease signs were associated with production of tumor necrosis factor (TNF)-α and elevated cytotoxicity displayed by heterotypic anti-DENV-1 CD8+ T lymphocytes. These findings highlight the critical role of heterotypic anti-DENV CD8+ T lymphocytes in manifestations of severe dengue disease.
Literature context: d from Jackson Laboratory (JAX #000664). Generation of Acp1 gene-trapp
Obesity-associated insulin resistance plays a central role in type 2 diabetes. As such, tyrosine phosphatases that dephosphorylate the insulin receptor (IR) are potential therapeutic targets. The low-molecular-weight protein tyrosine phosphatase (LMPTP) is a proposed IR phosphatase, yet its role in insulin signaling in vivo has not been defined. Here we show that global and liver-specific LMPTP deletion protects mice from high-fat diet-induced diabetes without affecting body weight. To examine the role of the catalytic activity of LMPTP, we developed a small-molecule inhibitor with a novel uncompetitive mechanism, a unique binding site at the opening of the catalytic pocket, and an exquisite selectivity over other phosphatases. This inhibitor is orally bioavailable, and it increases liver IR phosphorylation in vivo and reverses high-fat diet-induced diabetes. Our findings suggest that LMPTP is a key promoter of insulin resistance and that LMPTP inhibitors would be beneficial for treating type 2 diabetes.
Literature context: StrainsC57BL/6 miceCharles RiverJAX000664Ccr2âˆ’/âˆ’ miceDr. Frank Tacke Enge
Neutrophils play a crucial role in defense against systemic candidiasis, a disease associated with a high mortality rate in patients receiving immunosuppressive therapy, although the early immune mechanisms that boost the candidacidal activity of neutrophils remain to be defined in depth. Here, we used a murine model of systemic candidiasis to explore the role of inflammatory Ly6Chigh monocytes in NK cell-mediated neutrophil activation during the innate immune response against C. albicans. We found that efficient anti-Candida immunity required a collaborative response between the spleen and kidney, which relied on type I interferon-dependent IL-15 production by spleen inflammatory Ly6Chigh monocytes to drive efficient activation and GM-CSF release by spleen NK cells; this in turn was necessary to boost the Candida killing potential of kidney neutrophils. Our findings unveil a role for IL-15 as a critical mediator in defense against systemic candidiasis and hold promise for the design of IL-15-based antifungal immunotherapies.
Literature context: /6J mice (RRID:IMSR_JAX:000664) and selec
Attenuating the strength of fearful memories could benefit people disabled by memories of past trauma. Pavlovian conditioning experiments indicate that a retrieval cue can return a conditioned aversive memory to a labile state. However, means to enhance retrieval and render a memory more labile are unknown. We hypothesized that augmenting synaptic signaling during retrieval would increase memory lability. To enhance synaptic transmission, mice inhaled CO2 to induce an acidosis and activate acid sensing ion channels. Transient acidification increased the retrieval-induced lability of an aversive memory. The labile memory could then be weakened by an extinction protocol or strengthened by reconditioning. Coupling CO2 inhalation to retrieval increased activation of amygdala neurons bearing the memory trace and increased the synaptic exchange from Ca2+-impermeable to Ca2+-permeable AMPA receptors. The results suggest that transient acidosis during retrieval renders the memory of an aversive event more labile and suggest a strategy to modify debilitating memories.
Literature context: vier Labs RRID:IMSR_JAX:000664 Mouse: WT
The liver plays a pivotal role in metabolism and xenobiotic detoxification, processes that must be particularly efficient when animals are active and feed. A major question is how the liver adapts to these diurnal changes in physiology. Here, we show that, in mice, liver mass, hepatocyte size, and protein levels follow a daily rhythm, whose amplitude depends on both feeding-fasting and light-dark cycles. Correlative evidence suggests that the daily oscillation in global protein accumulation depends on a similar fluctuation in ribosome number. Whereas rRNA genes are transcribed at similar rates throughout the day, some newly synthesized rRNAs are polyadenylated and degraded in the nucleus in a robustly diurnal fashion with a phase opposite to that of ribosomal protein synthesis. Based on studies with cultured fibroblasts, we propose that rRNAs not packaged into complete ribosomal subunits are polyadenylated by the poly(A) polymerase PAPD5 and degraded by the nuclear exosome.
Literature context: nsMouse: C57BL/6JThe Jackson LabStock No: 000664Mouse: B6.CB17-Prkdcscid/SzJThe
Hallmarks of aging that negatively impact health include weight gain and reduced physical fitness, which can increase insulin resistance and risk for many diseases, including type 2 diabetes. The underlying mechanism(s) for these phenomena is poorly understood. Here we report that aging increases DNA breaks and activates DNA-dependent protein kinase (DNA-PK) in skeletal muscle, which suppresses mitochondrial function, energy metabolism, and physical fitness. DNA-PK phosphorylates threonines 5 and 7 of HSP90α, decreasing its chaperone function for clients such as AMP-activated protein kinase (AMPK), which is critical for mitochondrial biogenesis and energy metabolism. Decreasing DNA-PK activity increases AMPK activity and prevents weight gain, decline of mitochondrial function, and decline of physical fitness in middle-aged mice and protects against type 2 diabetes. In conclusion, DNA-PK is one of the drivers of the metabolic and fitness decline during aging, and therefore DNA-PK inhibitors may have therapeutic potential in obesity and low exercise capacity.
Literature context: aboratory RRID:IMSR_JAX:000664 Mouse: DBA
Vocalizations play a significant role in social communication across species. Analyses in rodents have used a limited number of spectro-temporal measures to compare ultrasonic vocalizations (USVs), which limits the ability to address repertoire complexity in the context of behavioral states. Using an automated and unsupervised signal processing approach, we report the development of MUPET (Mouse Ultrasonic Profile ExTraction) software, an open-access MATLAB tool that provides data-driven, high-throughput analyses of USVs. MUPET measures, learns, and compares syllable types and provides an automated time stamp of syllable events. Using USV data from a large mouse genetic reference panel and open-source datasets produced in different social contexts, MUPET analyzes the fine details of syllable production and repertoire use. MUPET thus serves as a new tool for USV repertoire analyses, with the capability to be adapted for use with other species.
Literature context: : 000664; RRID:IMSR_JAX:000664 Mouse: MyD
Antibiotic-mediated microbiota destruction and the consequent loss of colonization resistance can result in intestinal domination with vancomycin-resistant Enterococcus (VRE), leading to bloodstream infection in hospitalized patients. Clearance of VRE remains a challenging goal that, if achieved, would reduce systemic VRE infections and patient-to-patient transmission. Although obligate anaerobic commensal bacteria have been associated with colonization resistance to VRE, the specific bacterial species involved remain undefined. Herein, we demonstrate that a precisely defined consortium of commensal bacteria containing the Clostridium cluster XIVa species Blautia producta and Clostridium bolteae restores colonization resistance against VRE and clears VRE from the intestines of mice. While C. bolteae did not directly mediate VRE clearance, it enabled intestinal colonization with B. producta, which directly inhibited VRE growth. These findings suggest that therapeutic or prophylactic administration of defined bacterial consortia to individuals with compromised microbiota composition may reduce inter-patient transmission and intra-patient dissemination of highly antibiotic-resistant pathogens.
Literature context: : TRE-shBRD4Scott W. Lowe LabN/AMouse: C57BL/6JThe Jackson LaboratoryStock #: 000664OligonucleotidessiRNAs, see the
Autophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis. We show that BRD4 knockdown induces autophagy in vitro and in vivo in response to some, but not all, situations. In the case of starvation, a signaling cascade involving AMPK and histone deacetylase SIRT1 displaces chromatin-bound BRD4, instigating autophagy gene activation and cell survival. Importantly, this program is directed independently and also reciprocally to the growth-promoting properties of BRD4 and is potently repressed by BRD4-NUT, a driver of NUT midline carcinoma. These findings therefore identify a distinct and selective mechanism of autophagy regulation.
Literature context: BL6 mice (RRID:IMSR_JAX:000664) of either
A major challenge in experimental epilepsy research is to reconcile the effects of anti-epileptic drugs (AEDs) on individual neurons with their network-level actions. Highlighting this difficulty, it is unclear why carbamazepine (CBZ), a frontline AED with a known molecular mechanism, has been reported to increase epileptiform activity in several clinical and experimental studies. We confirmed in an in vitro mouse model (in both sexes) that the frequency of interictal bursts increased after CBZ perfusion. To address the underlying mechanisms, we developed a method, activity clamp, to distinguish the response of individual neurons from network-level actions of CBZ. We first recorded barrages of synaptic conductances from neurons during epileptiform activity and then replayed them in pharmacologically isolated neurons under control conditions and in the presence of CBZ. CBZ consistently decreased the reliability of the second action potential in each burst of activity. Conventional current-clamp recordings using excitatory ramp or square-step current injections failed to reveal this effect. Network modeling showed that a CBZ-induced decrease of neuron recruitment during epileptic bursts can lead to an increase in burst frequency at the network level by reducing the refractoriness of excitatory transmission. By combining activity clamp with computer simulations, the present study provides a potential explanation for the paradoxical effects of CBZ on epileptiform activity.SIGNIFICANCE STATEMENT The effects of anti-epileptic drugs on individual neurons are difficult to separate from their network-level actions. Although carbamazepine (CBZ) has a known anti-epileptic mechanism, paradoxically, it has also been reported to increase epileptiform activity in clinical and experimental studies. To investigate this paradox during realistic neuronal epileptiform activity, we developed a method, activity clamp, to distinguish the effects of CBZ on individual neurons from network-level actions. We demonstrate that CBZ consistently decreases the reliability of the second action potential in each burst of epileptiform activity. Network modeling shows that this effect on individual neuronal responses could explain the paradoxical effect of CBZ at the network level.
Literature context: aboratory RRID:IMSR_JAX:000664 Mouse: B6.
Lambda interferons (IFNλs) or type III IFNs share homology, expression patterns, signaling cascades, and antiviral functions with type I IFNs. This has complicated the unwinding of their unique non-redundant roles. Through the systematic study of influenza virus infection in mice, we herein show that IFNλs are the first IFNs produced that act at the epithelial barrier to suppress initial viral spread without activating inflammation. If infection progresses, type I IFNs come into play to enhance viral resistance and induce pro-inflammatory responses essential for confronting infection but causing immunopathology. Central to this are neutrophils which respond to both cytokines to upregulate antimicrobial functions but exhibit pro-inflammatory activation only to type I IFNs. Accordingly, Ifnlr1-/- mice display enhanced type I IFN production, neutrophilia, lung injury, and lethality, while therapeutic administration of PEG-IFNλ potently suppresses these effects. IFNλs therefore constitute the front line of antiviral defense in the lung without compromising host fitness.
Literature context: 82C57BL/6JThe Jackson LaboratoryJAX: 000664OligonucleotidesNheI-GFP: CGTCAG
Chronic mitochondrial stress is a central problem associated with neurodegenerative diseases. Early removal of defective mitochondria from axons constitutes a critical step of mitochondrial quality control. Here we investigate axonal mitochondrial response to mild stress in wild-type neurons and chronic mitochondrial defects in Amytrophic Lateral Sclerosis (ALS)- and Alzheimer's disease (AD)-linked neurons. We show that stressed mitochondria are removed from axons triggered by the bulk release of mitochondrial anchoring protein syntaphilin via a new class of mitochondria-derived cargos independent of Parkin, Drp1, and autophagy. Immuno-electron microscopy and super-resolution imaging show the budding of syntaphilin cargos, which then share a ride on late endosomes for transport toward the soma. Releasing syntaphilin is also activated in the early pathological stages of ALS- and AD-linked mutant neurons. Our study provides new mechanistic insights into the maintenance of axonal mitochondrial quality through SNPH-mediated coordination of mitochondrial stress and motility before activation of Parkin-mediated mitophagy. VIDEO ABSTRACT.
Literature context: e C57BL/6JThe Jackson LaboratoryStock No: 000664Mouse: B6(Cg)-Gt(ROSA)26Sortm4(I
Central regulation of metabolic physiology is mediated critically through neuronal functions; however, whether astrocytes are also essential remains unclear. Here we show that the high-order processes of astrocytes in the mediobasal hypothalamus displayed shortening in fasting and elongation in fed status. Chronic overnutrition and astrocytic IKKβ/NF-κB upregulation similarly impaired astrocytic plasticity, leading to sustained shortening of high-order processes. In physiology, astrocytic IKKβ/NF-κB upregulation resulted in early-onset effects, including glucose intolerance and blood pressure rise, and late-onset effects, including body weight and fat gain. Appropriate inhibition in astrocytic IKKβ/NF-κB protected against chronic overnutrition impairing astrocytic plasticity and these physiological functions. Mechanistically, astrocytic regulation of hypothalamic extracellular GABA level and therefore BDNF expression were found partly accountable. Hence, astrocytic process plasticity and IKKβ/NF-κB play significant roles in central control of blood glucose, blood pressure, and body weight as well as the central induction of these physiological disorders leading to disease.
Literature context: atopoietic Biorepository CoreN/AC57BL/6J miceJackson Laboratory000664B6.129S-
Neutrophils are the first line of defense against bacterial infections, and the generation of reactive oxygen species is a key part of their arsenal. Pathogens use detoxification systems to avoid the bactericidal effects of reactive oxygen species. Here we demonstrate that the Gram-negative pathogen Pseudomonas aeruginosa is susceptible to reactive oxygen species but actively blocks the reactive oxygen species burst using two type III secreted effector proteins, ExoS and ExoT. ExoS ADP-ribosylates Ras and prevents it from interacting with and activating phosphoinositol-3-kinase (PI3K), which is required to stimulate the phagocytic NADPH-oxidase that generates reactive oxygen species. ExoT also affects PI3K signaling via its ADP-ribosyltransferase activity but does not act directly on Ras. A non-ribosylatable version of Ras restores reactive oxygen species production and results in increased bacterial killing. These findings demonstrate that subversion of the host innate immune response requires ExoS-mediated ADP-ribosylation of Ras in neutrophils.
Literature context: ERT2)AmcJackson Laboratory009600Mouse: C57BL/6JJackson Laboratory000664Mouse: Sox2-Cretg/tg; Tg(Sox2-cr
The kidney contains the functional units, the nephrons, surrounded by the renal interstitium. Previously we discovered that, once Six2-expressing nephron progenitor cells and Foxd1-expressing renal interstitial progenitor cells form at the onset of kidney development, descendant cells from these populations contribute exclusively to the main body of nephrons and renal interstitial tissues, respectively, indicating a lineage boundary between the nephron and renal interstitial compartments. Currently it is unclear how lineages are regulated during kidney organogenesis. We demonstrate that nephron progenitor cells lacking Pax2 fail to differentiate into nephron cells but can switch fates into renal interstitium-like cell types. These data suggest that Pax2 function maintains nephron progenitor cells by repressing a renal interstitial cell program. Thus, the lineage boundary between the nephron and renal interstitial compartments is maintained by the Pax2 activity in nephron progenitor cells during kidney organogenesis.
Literature context: IMSR_JAX:000691, C57BL/6J (C57) RRID:IMSR_JAX:000664, and FVB/NJ (FVB) RRID:IMSR_JAX
Inbred genetic background significantly influences the expression of phenotypes associated with known genetic perturbations and can underlie variation in disease severity between individuals with the same mutation. However, the effect of epistatic interactions on the development of complex traits, such as craniofacial morphology, is poorly understood. Here, we investigated the effect of three inbred backgrounds (129X1/SvJ, C57BL/6J, and FVB/NJ) on the expression of craniofacial dysmorphology in mice (Mus musculus) with loss of function in three members of the Sprouty family of growth factor negative regulators (Spry1, Spry2, or Spry4) in order to explore the impact of epistatic interactions on skull morphology. We found that the interaction of inbred background and the Sprouty genotype explains as much craniofacial shape variation as the Sprouty genotype alone. The most severely affected genotypes display a relatively short and wide skull, a rounded cranial vault, and a more highly angled inferior profile. Our results suggest that the FVB background is more resilient to Sprouty loss of function than either C57 or 129, and that Spry4 loss is generally less severe than loss of Spry1 or Spry2 While the specific modifier genes responsible for these significant background effects remain unknown, our results highlight the value of intercrossing mice of multiple inbred backgrounds to identify the genes and developmental interactions that modulate the severity of craniofacial dysmorphology. Our quantitative results represent an important first step toward elucidating genetic interactions underlying variation in robustness to known genetic perturbations in mice.
Literature context: C57BL/6 (RRID:IMSR_JAX:000664) mice, and
Hair cells of the cochlea are mechanosensors for the perception of sound. Mutations in the LRTOMT gene, which encodes a protein with homology to the catecholamine methyltransferase COMT that is linked to schizophrenia, cause deafness. Here, we show that Tomt/Comt2, the murine ortholog of LRTOMT, has an unexpected function in the regulation of mechanotransduction by hair cells. The role of mTOMT in hair cells is independent of mTOMT methyltransferase function and mCOMT cannot substitute for mTOMT function. Instead, mTOMT binds to putative components of the mechanotransduction channel in hair cells and is essential for the transport of some of these components into the mechanically sensitive stereocilia of hair cells. Our studies thus suggest functional diversification between mCOMT and mTOMT, where mTOMT is critical for the assembly of the mechanotransduction machinery of hair cells. Defects in this process are likely mechanistically linked to deafness caused by mutations in LRTOMT/Tomt.
Literature context: 6/J mice (RRID:IMSR_JAX:000664) were isol
The complement system, typically associated with innate immunity, is emerging as a key controller of nonimmune systems including in development, with recent studies linking complement mutations with neurodevelopmental disease. A key effector of the complement response is the activation fragment C5a, which, through its receptor C5aR1, is a potent driver of inflammation. Surprisingly, C5aR1 is also expressed during early mammalian embryogenesis; however, no clearly defined function is ascribed to C5aR1 in development. Here we demonstrate polarized expression of C5aR1 on the apical surface of mouse embryonic neural progenitor cells in vivo and on human embryonic stem cell-derived neural progenitors. We also show that signaling of endogenous C5a during mouse embryogenesis drives proliferation of neural progenitor cells within the ventricular zone and is required for normal brain histogenesis. C5aR1 signaling in neural progenitors was dependent on atypical protein kinase C ζ, a mediator of stem cell polarity, with C5aR1 inhibition reducing proliferation and symmetric division of apical neural progenitors in human and mouse models. C5aR1 signaling was shown to promote the maintenance of cell polarity, with exogenous C5a increasing the retention of polarized rosette architecture in human neural progenitors after physical or chemical disruption. Transient inhibition of C5aR1 during neurogenesis in developing mice led to behavioral abnormalities in both sexes and MRI-detected brain microstructural alterations, in studied males, demonstrating a requirement of C5aR1 signaling for appropriate brain development. This study thus identifies a functional role for C5a-C5aR1 signaling in mammalian neurogenesis and provides mechanistic insight into recently identified complement gene mutations and brain disorders.SIGNIFICANCE STATEMENT The complement system, traditionally known as a controller of innate immunity, now stands as a multifaceted signaling family with a broad range of physiological actions. These include roles in the brain, where complement activation is associated with diseases, including epilepsy and schizophrenia. This study has explored complement regulation of neurogenesis, identifying a novel relationship between the complement activation peptide C5a and the neural progenitor proliferation underpinning formation of the mammalian brain. C5a was identified as a regulator of cell polarity, with inhibition of C5a receptors during embryogenesis leading to abnormal brain development and behavioral deficits. This work demonstrates mechanisms through which dysregulation of complement causes developmental disease and highlights the potential risk of complement inhibition for therapeutic purposes in pregnancy.
Literature context: s: Organisms/StrainsC57BL/6 miceJackson LaboratoryStock# 000664Wistar ratCharles RiverStock# 00
A decline in proteasome function is causally connected to neuronal aging and aging-associated neuropathologies. By using hippocampal neurons in culture and in vivo, we show that aging triggers a reduction and a cytoplasm-to-nucleus redistribution of the E3 ubiquitin ligase mahogunin (MGRN1). Proteasome impairment induces MGRN1 monoubiquitination, the key post-translational modification for its nuclear entry. One potential mechanism for MGRN1 monoubiquitination is via progressive deubiquitination at the proteasome of polyubiquitinated MGRN1. Once in the nucleus, MGRN1 potentiates the transcriptional cellular response to proteotoxic stress. Inhibition of MGRN1 impairs ATF3-mediated neuronal responsiveness to proteosomal stress and increases neuronal stress, while increasing MGRN1 ameliorates signs of neuronal aging, including cognitive performance in old animals. Our results imply that, among others, the strength of neuronal survival in a proteasomal deterioration background, like during aging, depends on the fine-tuning of ubiquitination-deubiquitination.
Literature context: he Jackson LaboratoryJAX: 006000Mouse: C57BL/6The Jackson LaboratoryJAX: 000664Mouse: C57BL/6 pupsCharles River
Organ-specific functions of tissue-resident macrophages in the steady-state heart are unknown. Here, we show that cardiac macrophages facilitate electrical conduction through the distal atrioventricular node, where conducting cells densely intersperse with elongated macrophages expressing connexin 43. When coupled to spontaneously beating cardiomyocytes via connexin-43-containing gap junctions, cardiac macrophages have a negative resting membrane potential and depolarize in synchrony with cardiomyocytes. Conversely, macrophages render the resting membrane potential of cardiomyocytes more positive and, according to computational modeling, accelerate their repolarization. Photostimulation of channelrhodopsin-2-expressing macrophages improves atrioventricular conduction, whereas conditional deletion of connexin 43 in macrophages and congenital lack of macrophages delay atrioventricular conduction. In the Cd11bDTR mouse, macrophage ablation induces progressive atrioventricular block. These observations implicate macrophages in normal and aberrant cardiac conduction.
Literature context: L/6 mice (RRID:IMSR_JAX:000664) were purc
Plastic changes in the CNS in response to peripheral sensory nerve injury are a series of complex processes, ranging from local circuit remodeling to somatotopic reorganization. However, the link between circuit remodeling and somatotopic reorganization remains unclear. We have previously reported that transection of the primary whisker sensory nerve causes the abnormal rewiring of lemniscal fibers (sensory afferents) on a neuron in the mouse whisker sensory thalamus (V2 VPM). In the present study, using transgenic mice whose lemniscal fibers originate from the whisker sensory principle trigeminal nucleus (PrV2) are specifically labeled, we identified that the transection induced retraction of PrV2-originating lemniscal fibers and invasion of those not originating from PrV2 in the V2 VPM. This anatomical remodeling with somatotopic reorganization was highly correlated with the rewiring of lemniscal fibers. Origins of the non-PrV2-origin lemniscal fibers in the V2 VPM included the mandibular subregion of trigeminal nuclei and the dorsal column nuclei (DCNs), which normally represent body parts other than whiskers. The transection also resulted in ectopic receptive fields of V2 VPM neurons and extraterritorial pain behavior on the uninjured mandibular region of the face. The anatomical remodeling, emergence of ectopic receptive fields, and extraterritorial pain behavior all concomitantly developed within a week and lasted more than three months after the transection. Our findings, thus, indicate a strong linkage between these plastic changes after peripheral sensory nerve injury, which may provide a neural circuit basis underlying large-scale reorganization of somatotopic representation and abnormal ectopic sensations.
Literature context: aboratory RRID:IMSR_JAX:000664 Mouse: Hpr
Respiration is a rhythmic activity as well as one that requires responsiveness to internal and external circumstances; both the rhythm and neuromodulatory responses of breathing are controlled by brainstem neurons in the preBötzinger complex (preBötC) and the retrotrapezoid nucleus (RTN), but the specific ion channels essential to these activities remain to be identified. Because deficiency of sodium leak channel, non-selective (Nalcn) causes lethal apnea in humans and mice, we investigated Nalcn function in these neuronal groups. We found that one-third of mice lacking Nalcn in excitatory preBötC neurons died soon after birth; surviving mice developed apneas in adulthood. Interestingly, in both preBötC and RTN neurons, the Nalcn current influences the resting membrane potential, contributes to maintenance of stable network activity, and mediates modulatory responses to the neuropeptide substance P. These findings reveal Nalcn's specific role in both rhythmic stability and responsiveness to neuropeptides within the respiratory network.
Literature context: # 000664; RRID:IMSR_JAX:000664 Recombinan
Genome-wide association studies have struggled to identify functional genes and variants underlying complex phenotypes. We recruited a multi-ethnic cohort of healthy volunteers (n = 91) and used their tissue to generate induced pluripotent stem cells (iPSCs) and hepatocyte-like cells (HLCs) for genome-wide mapping of expression quantitative trait loci (eQTLs) and allele-specific expression (ASE). We identified many eQTL genes (eGenes) not observed in the comparably sized Genotype-Tissue Expression project's human liver cohort (n = 96). Focusing on blood lipid-associated loci, we performed massively parallel reporter assays to screen candidate functional variants and used genome-edited stem cells, CRISPR interference, and mouse modeling to establish rs2277862-CPNE1, rs10889356-DOCK7, rs10889356-ANGPTL3, and rs10872142-FRK as functional SNP-gene sets. We demonstrated HLC eGenes CPNE1, VKORC1, UBE2L3, and ANGPTL3 and HLC ASE gene ACAA2 to be lipid-functional genes in mouse models. These findings endorse an iPSC-based experimental framework to discover functional variants and genes contributing to complex human traits.
Literature context: : C57BL/6JThe Jackson LaboratoryJAX:000664Mouse: B6.129-Ucp1tm1Kz/JThe Jac
Cold and other environmental factors induce "browning" of white fat depots-development of beige adipocytes with morphological and functional resemblance to brown fat. Similar to brown fat, beige adipocytes are assumed to express mitochondrial uncoupling protein 1 (UCP1) and are thermogenic due to the UCP1-mediated H+ leak across the inner mitochondrial membrane. However, this assumption has never been tested directly. Herein we patch clamped the inner mitochondrial membrane of beige and brown fat to provide a direct comparison of their thermogenic H+ leak (IH). All inguinal beige adipocytes had robust UCP1-dependent IH comparable to brown fat, but it was about three times less sensitive to purine nucleotide inhibition. Strikingly, only ∼15% of epididymal beige adipocytes had IH, while in the rest UCP1-dependent IH was undetectable. Despite the absence of UCP1 in the majority of epididymal beige adipocytes, these cells employ prominent creatine cycling as a UCP1-independent thermogenic mechanism.
Literature context: o, Italy; RRID:IMSR_JAX:000664) weighing
Light-operated drugs constitute a major target in drug discovery, since they may provide spatiotemporal resolution for the treatment of complex diseases (i.e. chronic pain). JF-NP-26 is an inactive photocaged derivative of the metabotropic glutamate type 5 (mGlu5) receptor negative allosteric modulator raseglurant. Violet light illumination of JF-NP-26 induces a photochemical reaction prompting the active-drug's release, which effectively controls mGlu5 receptor activity both in ectopic expressing systems and in striatal primary neurons. Systemic administration in mice followed by local light-emitting diode (LED)-based illumination, either of the thalamus or the peripheral tissues, induced JF-NP-26-mediated light-dependent analgesia both in neuropathic and in acute/tonic inflammatory pain models. These data offer the first example of optical control of analgesia in vivo using a photocaged mGlu5 receptor negative allosteric modulator. This approach shows potential for precisely targeting, in time and space, endogenous receptors, which may allow a better management of difficult-to-treat disorders.
Literature context: nimal Co. RRID:IMSR_JAX:000664 Mouse:Fxr-
Cholestasis is a common complication of sepsis, and the increased plasma levels of bile acids are predictive of sepsis-associated mortality. However, the exact mechanism by which cholestasis aggravates sepsis development remains elusive. Here, we show that bile acids are danger-associated molecular patterns (DAMPs) that can activate both signal 1 and 2 of the NLRP3 inflammasome in inflammatory macrophages. Mechanistically, bile acids induce a prolonged calcium influx and activate the NLRP3 inflammasome synergistically with ATP. Experimental cholestasis sensitizes, while cholestyramine, a bile acid sequestrant, protects mice from LPS-induced sepsis. FXR negatively regulates the NLRP3 inflammasome via physical interaction with NLRP3 and caspase 1. Fxr-null mice are more sensitive, while FXR-overexpressing mice are more resistant, to endoxemia shock. These findings suggest that bile acids and FXR play pivotal roles in sepsis via controlling the NLRP3 inflammasome, and that targeting FXR may represent a therapeutic strategy for cholestasis-associated sepsis.
Literature context: C57BL/6J (RRID:IMSR_JAX:000664) genome (G
The mouse olfactory sensory neuron (OSN) repertoire is composed of 10 million cells and each expresses one olfactory receptor (OR) gene from a pool of over 1000. Thus, the nose is sub-stratified into more than a thousand OSN subtypes. Here, we employ and validate an RNA-sequencing-based method to quantify the abundance of all OSN subtypes in parallel, and investigate the genetic and environmental factors that contribute to neuronal diversity. We find that the OSN subtype distribution is stereotyped in genetically identical mice, but varies extensively between different strains. Further, we identify cis-acting genetic variation as the greatest component influencing OSN composition and demonstrate independence from OR function. However, we show that olfactory stimulation with particular odorants results in modulation of dozens of OSN subtypes in a subtle but reproducible, specific and time-dependent manner. Together, these mechanisms generate a highly individualized olfactory sensory system by promoting neuronal diversity.
Literature context: C57BL/6J, RRID:IMSR_JAX:000664) were obta
The δ opioid receptor (δR) is a promising alternate target for pain management because δR agonists show decreased abuse potential compared with current opioid analgesics that target the μ opioid receptor. A critical limitation in developing δR as an analgesic target, however, is that δR agonists show relatively low efficacy in vivo, requiring the use of high doses that often cause adverse effects, such as convulsions. Here we tested whether intracellular retention of δR in sensory neurons contributes to this low δR agonist efficacy in vivo by limiting surface δR expression. Using direct visualization of δR trafficking and localization, we define a phosphatase and tensin homolog (PTEN)-regulated checkpoint that retains δR in the Golgi and decreases surface delivery in rat and mice sensory neurons. PTEN inhibition releases δR from this checkpoint and stimulates delivery of exogenous and endogenous δR to the neuronal surface both in vitro and in vivo PTEN inhibition in vivo increases the percentage of TG neurons expressing δR on the surface and allows efficient δR-mediated antihyperalgesia in mice. Together, we define a critical role for PTEN in regulating the surface delivery and bioavailability of the δR, explain the low efficacy of δR agonists in vivo, and provide evidence that active δR relocation is a viable strategy to increase δR antinociception.SIGNIFICANCE STATEMENT Opioid analgesics, such as morphine, which target the μ opioid receptor (μR), have been the mainstay of pain management, but their use is highly limited by adverse effects and their variable efficacy in chronic pain. Identifying alternate analgesic targets is therefore of great significance. Although the δ opioid receptor (δR) is an attractive option, a critical limiting factor in developing δR as a target has been the low efficacy of δR agonists. Why δR agonists show low efficacy is still under debate. This study provides mechanistic and functional data that intracellular localization of δR in neurons is a key factor that contributes to low agonist efficacy, and presents a proof of mechanism that relocating δR improves efficacy.
Literature context: o:000664; RRID:IMSR_JAX:000664 Recombinan
In cells experiencing unrelieved endoplasmic reticulum (ER) stress, the ER transmembrane kinase/endoribonuclease (RNase)-IRE1α-endonucleolytically degrades ER-localized mRNAs to promote apoptosis. Here we find that the ABL family of tyrosine kinases rheostatically enhances IRE1α's enzymatic activities, thereby potentiating ER stress-induced apoptosis. During ER stress, cytosolic ABL kinases localize to the ER membrane, where they bind, scaffold, and hyperactivate IRE1α's RNase. Imatinib-an anti-cancer tyrosine kinase inhibitor-antagonizes the ABL-IRE1α interaction, blunts IRE1α RNase hyperactivity, reduces pancreatic β cell apoptosis, and reverses type 1 diabetes (T1D) in the non-obese diabetic (NOD) mouse model. A mono-selective kinase inhibitor that allosterically attenuates IRE1α's RNase-KIRA8-also efficaciously reverses established diabetes in NOD mice by sparing β cells and preserving their physiological function. Our data support a model wherein ER-stressed β cells contribute to their own demise during T1D pathogenesis and implicate the ABL-IRE1α axis as a drug target for the treatment of an autoimmune disease.
Literature context: AC57BL/6JJackson LaboratoryCat# 000664NOD.Cg-PrkdcscidÂ Il2rgtm1Wjl/SzJ
The transcription factor Meis1 drives myeloid leukemogenesis in the context of Hox gene overexpression but is currently considered undruggable. We therefore investigated whether myeloid progenitor cells transformed by Hoxa9 and Meis1 become addicted to targetable signaling pathways. A comprehensive (phospho)proteomic analysis revealed that Meis1 increased Syk protein expression and activity. Syk upregulation occurs through a Meis1-dependent feedback loop. By dissecting this loop, we show that Syk is a direct target of miR-146a, whose expression is indirectly regulated by Meis1 through the transcription factor PU.1. In the context of Hoxa9 overexpression, Syk signaling induces Meis1, recapitulating several leukemogenic features of Hoxa9/Meis1-driven leukemia. Finally, Syk inhibition disrupts the identified regulatory loop, prolonging survival of mice with Hoxa9/Meis1-driven leukemia.
Literature context: trainsSPF miceJackson LaboratoryC57BL/6J Cat#000664GF miceGerm-free C57BL/6J from c
Regulatory T cells (Tregs) are required to establish immune tolerance to commensal microbes. Tregs accumulate abruptly in the skin during a defined window of postnatal tissue development. However, the mechanisms mediating Treg migration to neonatal skin are unknown. Here we show that hair follicle (HF) development facilitates the accumulation of Tregs in neonatal skin and that upon skin entry these cells localize to HFs, a primary reservoir for skin commensals. Further, germ-free neonates had reduced skin Tregs indicating that commensal microbes augment Treg accumulation. We identified Ccl20 as a HF-derived, microbiota-dependent chemokine and found its receptor, Ccr6, to be preferentially expressed by Tregs in neonatal skin. The Ccl20-Ccr6 pathway mediated Treg migration in vitro and in vivo. Thus, HF morphogenesis, commensal microbe colonization, and local chemokine production work in concert to recruit Tregs into neonatal skin, thereby establishing this tissue Treg niche early in life.
Literature context: germ-free conditionsJackson labs000664Mouse: B6.129S-Tnftm1Gkl/J 10-14
Levels of inflammatory mediators in circulation are known to increase with age, but the underlying cause of this age-associated inflammation is debated. We find that, when maintained under germ-free conditions, mice do not display an age-related increase in circulating pro-inflammatory cytokine levels. A higher proportion of germ-free mice live to 600 days than their conventional counterparts, and macrophages derived from aged germ-free mice maintain anti-microbial activity. Co-housing germ-free mice with old, but not young, conventionally raised mice increases pro-inflammatory cytokines in the blood. In tumor necrosis factor (TNF)-deficient mice, which are protected from age-associated inflammation, age-related microbiota changes are not observed. Furthermore, age-associated microbiota changes can be reversed by reducing TNF using anti-TNF therapy. These data suggest that aging-associated microbiota promote inflammation and that reversing these age-related microbiota changes represents a potential strategy for reducing age-associated inflammation and the accompanying morbidity.
Literature context: embryos and wild-type C57BL/6J (RRID:IMSR_JAX:000664) controls, and immortalised by
Proteolytic cleavage and release from the cell surface of membrane-tethered ligands is an important mechanism of regulating intercellular signalling. TACE is a major shedding protease, responsible for the liberation of the inflammatory cytokine TNFα and ligands of the epidermal growth factor receptor. iRhoms, catalytically inactive members of the rhomboid-like superfamily, have been shown to control the ER-to-Golgi transport and maturation of TACE. Here, we reveal that iRhom2 remains associated with TACE throughout the secretory pathway, and is stabilised at the cell surface by this interaction. At the plasma membrane, ERK1/2-mediated phosphorylation and 14-3-3 protein binding of the cytoplasmic amino-terminus of iRhom2 alter its interaction with mature TACE, thereby licensing its proteolytic activity. We show that this molecular mechanism is responsible for triggering inflammatory responses in primary mouse macrophages. Overall, iRhom2 binds to TACE throughout its lifecycle, implying that iRhom2 is a primary regulator of stimulated cytokine and growth factor signalling.
Literature context: aboratory RRID:IMSR_JAX:000664 129S4/SvJa
RNA-binding proteins (RBPs), in addition to their functions in cellular homeostasis, play important roles in lineage specification and maintaining cellular identity. Despite their diverse and essential functions, which touch on nearly all aspects of RNA metabolism, the roles of RBPs in somatic cell reprogramming are poorly understood. Here we show that the DEAD-box RBP DDX5 inhibits reprogramming by repressing the expression and function of the non-canonical polycomb complex 1 (PRC1) subunit RYBP. Disrupting Ddx5 expression improves the efficiency of iPSC generation and impedes processing of miR-125b, leading to Rybp upregulation and suppression of lineage-specific genes via RYBP-dependent ubiquitination of H2AK119. Furthermore, RYBP is required for PRC1-independent recruitment of OCT4 to the promoter of Kdm2b, a histone demethylase gene that promotes reprogramming by reactivating endogenous pluripotency genes. Together, these results reveal important functions of DDX5 in regulating reprogramming and highlight the importance of a Ddx5-miR125b-Rybp axis in controlling cell fate.
Literature context: ouse: C57BL/6JJackson LaboratoryJAX: 000664OligonucleotidesVPS35 shRNA targ
Alterations in the function of the retromer, a multisubunit protein complex that plays a specialized role in endosomal sorting, have been linked to Alzheimer's and Parkinson's diseases, yet little is known about the retromer's role in the mature brain. Using in vivo knockdown of the critical retromer component VPS35, we demonstrate a specific role for this endosomal sorting complex in the trafficking of AMPA receptors during NMDA-receptor-dependent LTP at mature hippocampal synapses. The impairment of LTP due to VPS35 knockdown was mechanistically independent of any role of the retromer in the production of Aβ from APP. Finally, we find surprising differences between Alzheimer's- and Parkinson's-disease-linked VPS35 mutations in supporting this pathway. These findings demonstrate a key role for the retromer in LTP and provide insights into how retromer malfunction in the mature brain may contribute to symptoms of common neurodegenerative diseases. VIDEO ABSTRACT.
Literature context: /6J mice (RRID:IMSR_JAX:000664) were obta
The brain shows various sex differences in its structures. Various mammalian species exhibit sex differences in the sexually dimorphic nucleus of the preoptic area (SDN-POA) and parts of the extended amygdala such as the principal nucleus of the bed nucleus of the stria terminalis (BNSTpr) and posterodorsal part of the medial amygdala (MePD). The SDN-POA and BNSTpr are male-biased sexually dimorphic nuclei, and characterized by the expression of calbindin D-28K (calbindin 1). However, calbindin-immunoreactive cells are not restricted to the SDN-POA, but widely distributed outside of the SDN-POA. To find genes that are more specific to sexually dimorphic nuclei, we selected candidate genes by searching the Allen brain atlas and examined the detailed expressions of the candidate genes using in situ hybridization. We found that the strong expression of monooxygenase DBH-like 1 (Moxd1) was restricted to the SDN-POA, BNSTpr and MePD. The numbers of Moxd1-positive cells in the SDN-POA, BNSTpr and MePD in male mice were larger than those in female mice. Most of the Moxd1-positive cells in the SDN-POA and BNSTpr expressed calbindin. Neonatal castration of male mice reduced the number of Moxd1-positive cells in the SDN-POA, whereas gonadectomy in adulthood did not change the expression of the Moxd1 gene in the SDN-POA in both sexes. These results suggest that the Moxd1 gene is a suitable marker for sexual dimorphic nuclei in the POA, BNST and amygdala, which enables us to manipulate sexually dimorphic neurons to examine their roles in sex-biased physiology and behaviors.
Literature context: ouse: C57BL/6JJackson Laboratory000664Mouse: C57BL/6NJJackson Laborato
Sirtuins are NAD+-dependent protein deacylases that regulate several aspects of metabolism and aging. In contrast to the other mammalian sirtuins, the primary enzymatic activity of mitochondrial sirtuin 4 (SIRT4) and its overall role in metabolic control have remained enigmatic. Using a combination of phylogenetics, structural biology, and enzymology, we show that SIRT4 removes three acyl moieties from lysine residues: methylglutaryl (MG)-, hydroxymethylglutaryl (HMG)-, and 3-methylglutaconyl (MGc)-lysine. The metabolites leading to these post-translational modifications are intermediates in leucine oxidation, and we show a primary role for SIRT4 in controlling this pathway in mice. Furthermore, we find that dysregulated leucine metabolism in SIRT4KO mice leads to elevated basal and stimulated insulin secretion, which progressively develops into glucose intolerance and insulin resistance. These findings identify a robust enzymatic activity for SIRT4, uncover a mechanism controlling branched-chain amino acid flux, and position SIRT4 as a crucial player maintaining insulin secretion and glucose homeostasis during aging.
Literature context: L/6 mice (RRID:IMSR_JAX:000664) were prov
Cognitive behavioral therapy, such as environmental enrichment combined with voluntary exercise (EE-VEx), is under active investigation as an adjunct to pharmaceutical treatment for chronic pain. However, the effectiveness and underlying mechanisms of EE-VEx remain unclear. In mice with intraplantar injection of complete Freund's adjuvant, our results revealed that EE-VEx alleviated perceptual, affective, and cognitive dimensions of chronic inflammatory pain. These effects of EE-VEx on chronic pain were contingent on the occurrence of adult neurogenesis in the dentate gyrus in a functionally dissociated manner along the dorsoventral axis: neurogenesis in the ventral dentate gyrus participated in alleviating perceptual and affective components of chronic pain by EE-VEx, whereas neurogenesis in the dorsal dentate gyrus was involved in EE-VEx's cognitive-enhancing effects. Chronic inflammatory pain was accompanied by decreased levels of brain-derived neurotrophic factor (BDNF) in the dentate gyrus, which were reversed by EE-VEx. Overexpression of BDNF in the dentate gyrus mimicked the effects of EE-VEx. Our results demonstrate distinct contribution of adult hippocampal neurogenesis along the dorsoventral axis to EE-VEx's beneficial effects on different dimensions of chronic pain.SIGNIFICANCE STATEMENT Environmental enrichment combined with voluntary exercise (EE-VEx) is under active investigation as an adjunct to pharmaceutical treatment for chronic pain, but its effectiveness and underlying mechanisms remain unclear. In a mouse model of inflammatory pain, the present study demonstrates that the beneficial effects of EE-VEx on chronic pain depend on adult neurogenesis with a dorsoventral dissociation along the hippocampal axis. Adult neurogenesis in the ventral dentate gyrus participates in alleviating perceptual and affective components of chronic pain by EE-VEx, whereas that in the dorsal pole is involved in EE-VEx's cognitive-enhancing effects in chronic pain.
Literature context: : C57BL/6JThe Jackson LaboratoryStock No: 000664Mouse:B6;129S2-Nr4a1tm1Jmi/JThe
Mitochondria play an integral role in cell death, autophagy, immunity, and inflammation. We previously showed that Nur77, an orphan nuclear receptor, induces apoptosis by targeting mitochondria. Here, we report that celastrol, a potent anti-inflammatory pentacyclic triterpene, binds Nur77 to inhibit inflammation and induce autophagy in a Nur77-dependent manner. Celastrol promotes Nur77 translocation from the nucleus to mitochondria, where it interacts with tumor necrosis factor receptor-associated factor 2 (TRAF2), a scaffold protein and E3 ubiquitin ligase important for inflammatory signaling. The interaction is mediated by an LxxLL motif in TRAF2 and results not only in the inhibition of TRAF2 ubiquitination but also in Lys63-linked Nur77 ubiquitination. Under inflammatory conditions, ubiquitinated Nur77 resides at mitochondria, rendering them sensitive to autophagy, an event involving Nur77 interaction with p62/SQSTM1. Together, our results identify Nur77 as a critical intracellular target for celastrol and unravel a mechanism of Nur77-dependent clearance of inflamed mitochondria to alleviate inflammation.
Literature context: Mice, 6Â weeksJackson LabsStock#00664OligonucleotidesPlease refer to
The intricate connection between the circadian clock and metabolism remains poorly understood. We used high temporal resolution metabolite profiling to explore clock regulation of mouse liver and cell-autonomous metabolism. In liver, ∼50% of metabolites were circadian, with enrichment of nucleotide, amino acid, and methylation pathways. In U2 OS cells, 28% were circadian, including amino acids and NAD biosynthesis metabolites. Eighteen metabolites oscillated in both systems and a subset of these in primary hepatocytes. These 18 metabolites were enriched in methylation and amino acid pathways. To assess clock dependence of these rhythms, we used genetic perturbation. BMAL1 knockdown diminished metabolite rhythms, while CRY1 or CRY2 perturbation generally shortened or lengthened rhythms, respectively. Surprisingly, CRY1 knockdown induced 8 hr rhythms in amino acid, methylation, and vitamin metabolites, decoupling metabolite from transcriptional rhythms, with potential impact on nutrient sensing in vivo. These results provide the first comprehensive views of circadian liver and cell-autonomous metabolism.
Literature context: _002865; IMSR_JAX:000664; RRID:SCR_
Little is known about the function of the cholinergic efferents innervating peripheral vestibular hair cells. We measured vestibular sensory evoked potentials (VsEPs) in α9 knockout (KO) mice, α10 KO mice, α7 KO mice, α9/10 and α7/9 double KO mice, and wild-type (WT) controls. We also studied the morphology and ultrastructure of efferent terminals on vestibular hair cells in α9, α10, and α9/10 KOs. Both type I and type ll vestibular hair cells express the α9 and α10 subunits. The efferent boutons on vestibular cells in α9, α10, and α9/10 KOs appeared normal, but a quantitative analysis was not performed. Mean VsEP thresholds were significantly elevated in α9 and α9/10 KO animals. Some α9 and α9/10 KO animals, however, had normal or near-normal thresholds, whereas others were greatly affected. Despite individual variability in threshold responses, latencies were consistently shortened. The double α7/9 KO resulted in decreased variance by normalizing waveforms and latencies. The phenotypes of the α7 and α10 single KOs were identical. Both α7 and α10 KO mice evidenced normal thresholds, decreased activation latencies, and larger amplitudes compared with WT mice. The data suggest a complex interaction of nicotinic acetylcholine receptors (nAChRs) in regulating vestibular afferent gain and activation timing. Although the α9/10 heteromeric nAChR is an important component of vestibular efferent activity, other peripheral or central nAChRs involving the α7 subunit or α10 subunit and α9 homomeric receptors are also important. J. Comp. Neurol. 525:1216-1233, 2017. © 2016 Wiley Periodicals, Inc.
Literature context: -tdTomato)Hze/J), and wild-type C57BL/6J mice were obtained from The Jac
Mutations in the Pejvakin (PJVK) gene are thought to cause auditory neuropathy and hearing loss of cochlear origin by affecting noise-induced peroxisome proliferation in auditory hair cells and neurons. Here we demonstrate that loss of pejvakin in hair cells, but not in neurons, causes profound hearing loss and outer hair cell degeneration in mice. Pejvakin binds to and colocalizes with the rootlet component TRIOBP at the base of stereocilia in injectoporated hair cells, a pattern that is disrupted by deafness-associated PJVK mutations. Hair cells of pejvakin-deficient mice develop normal rootlets, but hair bundle morphology and mechanotransduction are affected before the onset of hearing. Some mechanotransducing shorter row stereocilia are missing, whereas the remaining ones exhibit overextended tips and a greater variability in height and width. Unlike previous studies of Pjvk alleles with neuronal dysfunction, our findings reveal a cell-autonomous role of pejvakin in maintaining stereocilia architecture that is critical for hair cell function.SIGNIFICANCE STATEMENT Two missense mutations in the Pejvakin (PJVK or DFNB59) gene were first identified in patients with audiological hallmarks of auditory neuropathy spectrum disorder, whereas all other PJVK alleles cause hearing loss of cochlear origin. These findings suggest that complex pathogenetic mechanisms underlie human deafness DFNB59. In contrast to recent studies, we demonstrate that pejvakin in auditory neurons is not essential for normal hearing in mice. Moreover, pejvakin localizes to stereociliary rootlets in hair cells and is required for stereocilia maintenance and mechanosensory function of the hair bundle. Delineating the site of the lesion and the mechanisms underlying DFNB59 will allow clinicians to predict the efficacy of different therapeutic approaches, such as determining compatibility for cochlear implants.
Literature context: 6 miceJackson LaboratoryStock#: 000664AG129 miceDiamond LaboratoryN/AO
The emergence of ZIKV infection has prompted a global effort to develop safe and effective vaccines. We engineered a lipid nanoparticle (LNP) encapsulated modified mRNA vaccine encoding wild-type or variant ZIKV structural genes and tested immunogenicity and protection in mice. Two doses of modified mRNA LNPs encoding prM-E genes that produced virus-like particles resulted in high neutralizing antibody titers (∼1/100,000) that protected against ZIKV infection and conferred sterilizing immunity. To offset a theoretical concern of ZIKV vaccines inducing antibodies that cross-react with the related dengue virus (DENV), we designed modified prM-E RNA encoding mutations destroying the conserved fusion-loop epitope in the E protein. This variant protected against ZIKV and diminished production of antibodies enhancing DENV infection in cells or mice. A modified mRNA vaccine can prevent ZIKV disease and be adapted to reduce the risk of sensitizing individuals to subsequent exposure to DENV, should this become a clinically relevant concern.
Literature context: C57BL/6J (RRID:IMSR_JAX:000664), Thy1:GCaMP6 (Dana et al., 201
Rhythmic neuronal activity of multiple frequency bands has been described in many brain areas and attributed to numerous brain functions. Among these, little is known about the mechanism and role of infra-slow oscillations, which have been demonstrated recently in the mouse accessory olfactory bulb (AOB). Along with prolonged responses to stimuli and distinct network connectivity, they inexplicably affect the AOB processing of social relevant stimuli. Here, we show that assemblies of AOB mitral cells are synchronized by lateral interactions through chemical and electrical synapses. Using a network model, we demonstrate that the synchronous oscillations in these assemblies emerge from interplay between intrinsic membrane properties and network connectivity. As a consequence, the AOB network topology, in which each mitral cell receives input from multiple glomeruli, enables integration of chemosensory stimuli over extended time scales by interglomerular synchrony of infra-slow bursting. These results provide a possible functional significance for the distinct AOB physiology and topology. Beyond the AOB, this study presents a general model for synchronous infra-slow bursting in neuronal networks.SIGNIFICANCE STATEMENT Infra-slow rhythmic neuronal activity with a very long (>10 s) duration has been described in many brain areas, but little is known about the role of this activity and the mechanisms that produce it. Here, we combine experimental and computational methods to show that synchronous infra-slow bursting activity in mitral cells of the mouse accessory olfactory bulb (AOB) emerges from interplay between intracellular dynamics and network connectivity. In this novel mechanism, slow intracellular Na+ dynamics endow AOB mitral cells with a weak tendency to burst, which is further enhanced and stabilized by chemical and electrical synapses between them. Combined with the unique topology of the AOB network, infra-slow bursting enables integration and binding of multiple chemosensory stimuli over a prolonged time scale.
Literature context: (C57BL/6J)The Jackson LaboratoryStock#: 000664Mouse: Cre-dependent Cas9 knocki
Ionotropic neurotransmitter receptors mediate fast synaptic transmission by functioning as ligand-gated ion channels. Fast inhibitory transmission in the brain is mediated mostly by ionotropic GABAA receptors (GABAARs), but their essential components for synaptic localization remain unknown. Here, we identify putative auxiliary subunits of GABAARs, which we term GARLHs, consisting of LH4 and LH3 proteins. LH4 forms a stable tripartite complex with GABAARs and neuroligin-2 in the brain. Moreover, LH4 is required for the synaptic localization of GABAARs and inhibitory synaptic transmission in the hippocampus. Our findings propose GARLHs as the first identified auxiliary subunits for anion channels. These findings provide new insights into the regulation of inhibitory transmission and the molecular constituents of native anion channels in vivo.
Literature context: ainsC57BL/6JJackson Laboratories000664IL2rÎ³âˆ’/âˆ’ (B6.129S4-Il2rgtm1Wjl/J
Loss of photoreceptors is a common endpoint in degenerative retinal diseases. Human pluripotent stem cells provide a potential source for photoreceptor replacement, but, even in mouse models, the efficiency and efficacy of transplantation-based repair remains poor. In this study, we examined the degree to which immune rejection contributes to these disappointing outcomes using an immunodeficient IL2 receptor γ (IL2rγ)-null mouse model. Our results show that prevention of cell rejection in the normal and degenerating retinal environment significantly improves long-term survival and integration of hESC-derived donor retinal cells. Transplanted cells are able to differentiate into mature photoreceptors expressing various opsins and can functionally integrate into congenitally blind mice. Our work suggests that even though the retina is often considered immune-privileged, suppression of host immune-mediated cell rejection may well be a useful approach for improving long-term integration of transplanted cells with a view to successful clinical outcomes.
Literature context: /6J mice (RRID:IMSR_JAX:000664; C57BL/6J)
BACKGROUND: A variety of neurological disorders, including Alzheimer's disease, Parkinson's disease, major depressive disorder, dyslexia and autism, are differentially prevalent between females and males. To better understand the possible molecular basis for the sex-biased nature of neurological disorders, we used a developmental series of female and male mice at 1, 2, and 4 months of age to assess both mRNA and protein in the hippocampus with RNA-sequencing and mass-spectrometry, respectively. RESULTS: The transcriptomic analysis identifies 2699 genes that are differentially expressed between animals of different ages. The bulk of these differentially expressed genes are changed in both sexes at one or more ages, but a total of 198 transcripts are differentially expressed between females and males at one or more ages. The number of transcripts that are differentially expressed between females and males is greater in adult animals than in younger animals. Additionally, we identify 69 transcripts that show complex and sex-specific patterns of temporal regulation through postnatal development, 8 of which are heat-shock proteins. We also find a modest correlation between levels of mRNA and protein in the mouse hippocampus (Rho = 0.53). CONCLUSION: This study adds to the substantial body of evidence for transcriptomic regulation in the hippocampus during postnatal development. Additionally, this analysis reveals sex differences in the transcriptome of the developing mouse hippocampus, and further clarifies the need to include both female and male mice in longitudinal studies involving molecular changes in the hippocampus.
Literature context: ier Labs; RRID:IMSR_JAX:000664) aged 6-8
Calibration of the vestibulo-ocular reflex (VOR) depends on the presence of visual feedback. However, the cellular mechanisms associated with VOR modifications at the level of the brainstem remain largely unknown. A new protocol was designed to expose freely behaving mice to a visuo-vestibular mismatch during a 2-week period. This protocol induced a 50% reduction of the VOR. In vivo pharmacological experiments demonstrated that the VOR reduction depends on changes located outside the flocculus/paraflocculus complex. The cellular mechanisms associated with the VOR reduction were then studied in vitro on brainstem slices through a combination of vestibular afferent stimulation and patch-clamp recordings of central vestibular neurons. The evoked synaptic activity demonstrated that the efficacy of the synapses between vestibular afferents and central vestibular neurons was decreased. In addition, a long-term depression protocol failed to further decrease the synapse efficacy, suggesting that the VOR reduction might have occurred through depression-like mechanisms. Analysis of the intrinsic membrane properties of central vestibular neurons revealed that the synaptic changes were supplemented by a decrease in the spontaneous discharge and excitability of a subpopulation of neurons. Our results provide evidence that a long-lasting visuo-vestibular mismatch leads to changes in synaptic transmission and intrinsic properties of central vestibular neurons in the direct VOR pathway. Overall, these results open new avenues for future studies on visual and vestibular interactions conducted in vivo and in vitro.
Literature context: C57BL/6 (RRID:IMSR_JAX:000664, Jackson L
Mechanotransducer channels at the tips of sensory stereocilia of inner ear hair cells are gated by the tension of 'tip links' interconnecting stereocilia. To ensure maximal sensitivity, tip links are tensioned at rest, resulting in a continuous influx of Ca2+ into the cell. Here, we show that this constitutive Ca2+ influx, usually considered as potentially deleterious for hair cells, is in fact essential for stereocilia stability. In the auditory hair cells of young postnatal mice and rats, a reduction in mechanotransducer current, via pharmacological channel blockers or disruption of tip links, leads to stereocilia shape changes and shortening. These effects occur only in stereocilia that harbor mechanotransducer channels, recover upon blocker washout or tip link regeneration and can be replicated by manipulations of extracellular Ca2+ or intracellular Ca2+ buffering. Thus, our data provide the first experimental evidence for the dynamic control of stereocilia morphology by the mechanotransduction current.
Literature context: T: C57BL/6The Jackson LaboratoryStrain: #000664Recombinant DNARNA probe: Plexin
Transcription factors control cell identity by regulating diverse developmental steps such as differentiation and axon guidance. The mammalian binocular visual circuit is comprised of projections of retinal ganglion cells (RGCs) to ipsilateral and contralateral targets in the brain. A transcriptional code for ipsilateral RGC identity has been identified, but less is known about the transcriptional regulation of contralateral RGC development. Here we demonstrate that SoxC genes (Sox4, 11, and 12) act on the progenitor-to-postmitotic transition to implement contralateral, but not ipsilateral, RGC differentiation, by binding to Hes5 and thus repressing Notch signaling. When SoxC genes are deleted in postmitotic RGCs, contralateral RGC axons grow poorly on chiasm cells in vitro and project ipsilaterally at the chiasm midline in vivo, and Plexin-A1 and Nr-CAM expression in RGCs is downregulated. These data implicate SoxC transcription factors in the regulation of contralateral RGC differentiation and axon guidance.
Literature context: : 000664, RRID:IMSR_JAX:000664 Mouse: CD-
The Reelin signaling pathway plays a crucial role in regulating neocortical development. However, little is known about how Reelin controls the cytoskeleton during neuronal migration. Here, we identify CLASP2 as a key cytoskeletal effector in the Reelin signaling pathway. We demonstrate that CLASP2 has distinct roles during neocortical development regulating neuron production and controlling neuron migration, polarity, and morphogenesis. We found downregulation of CLASP2 in migrating neurons leads to mislocalized cells in deeper cortical layers, abnormal positioning of the centrosome-Golgi complex, and aberrant length/orientation of the leading process. We discovered that Reelin regulates several phosphorylation sites within the positively charged serine/arginine-rich region that constitute consensus GSK3β phosphorylation motifs of CLASP2. Furthermore, phosphorylation of CLASP2 regulates its interaction with the Reelin adaptor Dab1 and this association is required for CLASP2 effects on neurite extension and motility. Together, our data reveal that CLASP2 is an essential Reelin effector orchestrating cytoskeleton dynamics during brain development.
Literature context: : 000664; RRID:IMSR_JAX:000664 129(Cg)-Fo
Voltage-gated sodium channel (VGSC) mutations cause severe epilepsies marked by intermittent, pathological hypersynchronous brain states. Here we present two mechanisms that help to explain how mutations in one VGSC gene, Scn8a, contribute to two distinct seizure phenotypes: (1) hypoexcitation of cortical circuits leading to convulsive seizure resistance, and (2) hyperexcitation of thalamocortical circuits leading to non-convulsive absence epilepsy. We found that loss of Scn8a leads to altered RT cell intrinsic excitability and a failure in recurrent RT synaptic inhibition. We propose that these deficits cooperate to enhance thalamocortical network synchrony and generate pathological oscillations. To our knowledge, this finding is the first clear demonstration of a pathological state tied to disruption of the RT-RT synapse. Our observation that loss of a single gene in the thalamus of an adult wild-type animal is sufficient to cause spike-wave discharges is striking and represents an example of absence epilepsy of thalamic origin.
Literature context: e:C57BL/6JThe Jackson Laboratory000664Mouse:C57BL/6J - DIOThe Jackson
Type 2 diabetes (T2D) is a worldwide epidemic with a medical need for additional targeted therapies. Suppression of hepatic glucose production (HGP) effectively ameliorates diabetes and can be exploited for its treatment. We hypothesized that targeting PGC-1α acetylation in the liver, a chemical modification known to inhibit hepatic gluconeogenesis, could be potentially used for treatment of T2D. Thus, we designed a high-throughput chemical screen platform to quantify PGC-1α acetylation in cells and identified small molecules that increase PGC-1α acetylation, suppress gluconeogenic gene expression, and reduce glucose production in hepatocytes. On the basis of potency and bioavailability, we selected a small molecule, SR-18292, that reduces blood glucose, strongly increases hepatic insulin sensitivity, and improves glucose homeostasis in dietary and genetic mouse models of T2D. These studies have important implications for understanding the regulatory mechanisms of glucose metabolism and treatment of T2D.
Literature context: mental Models: Organisms/StrainsC57BL/6 miceCharles River Lab. (CRL, Saint-G
Primary immune responses generate short-term effectors and long-term protective memory cells. The delineation of the genealogy linking naive, effector, and memory cells has been complicated by the lack of phenotypes discriminating effector from memory differentiation stages. Using transcriptomics and phenotypic analyses, we identify Bcl2 and Mki67 as a marker combination that enables the tracking of nascent memory cells within the effector phase. We then use a formal approach based on mathematical models describing the dynamics of population size evolution to test potential progeny links and demonstrate that most cells follow a linear naive→early effector→late effector→memory pathway. Moreover, our mathematical model allows long-term prediction of memory cell numbers from a few early experimental measurements. Our work thus provides a phenotypic means to identify effector and memory cells, as well as a mathematical framework to investigate their genealogy and to predict the outcome of immunization regimens in terms of memory cell numbers generated.
Literature context: : 000644; RRID:IMSR_JAX:000664) mice as a
The spatial receptive fields of neurons in medial entorhinal cortex layer II (MECII) and in the hippocampus suggest general and environment-specific maps of space, respectively. However, the relationship between these receptive fields remains unclear. We reversibly manipulated the activity of MECII neurons via chemogenetic receptors and compared the changes in downstream hippocampal place cells to those of neurons in MEC. Depolarization of MECII impaired spatial memory and elicited drastic changes in CA1 place cells in a familiar environment, similar to those seen during remapping between distinct environments, while hyperpolarization did not. In contrast, both manipulations altered the firing rate of MEC neurons without changing their firing locations. Interestingly, only depolarization caused significant changes in the relative firing rates of individual grid fields, reconfiguring the spatial input from MEC. This suggests a novel mechanism of hippocampal remapping whereby rate changes in MEC neurons lead to locational changes of hippocampal place fields.
Literature context: e; Envigo RRID:IMSR_JAX:000664) were used
Circadian rhythms optimize physiology and behavior to the varying demands of the 24 h day. The master circadian clock is located in the suprachiasmatic nuclei (SCN) of the hypothalamus and it regulates circadian oscillators in tissues throughout the body to prevent internal desynchrony. Here, we demonstrate for the first time that, under standard 12 h:12 h light/dark (LD) cycles, object, visuospatial, and olfactory recognition performance in C57BL/6J mice is consistently better at midday relative to midnight. However, under repeated exposure to constant light (rLL), recognition performance becomes desynchronized, with object and visuospatial performance better at subjective midday and olfactory performance better at subjective midnight. This desynchrony in behavioral performance is mirrored by changes in expression of the canonical clock genes Period1 and Period2 (Per1 and Per2), as well as the immediate-early gene Fos in the SCN, dorsal hippocampus, and olfactory bulb. Under rLL, rhythmic Per1 and Fos expression is attenuated in the SCN. In contrast, hippocampal gene expression remains rhythmic, mirroring object and visuospatial performance. Strikingly, Per1 and Fos expression in the olfactory bulb is reversed, mirroring the inverted olfactory performance. Temporal desynchrony among these regions does not result in arrhythmicity because core body temperature and exploratory activity rhythms persist under rLL. Our data provide the first demonstration that abnormal lighting conditions can give rise to temporal desynchrony between autonomous circadian oscillators in different regions, with different consequences for performance across different sensory domains. Such a dispersed network of dissociable circadian oscillators may provide greater flexibility when faced with conflicting environmental signals.SIGNIFICANCE STATEMENT A master circadian clock in the suprachiasmatic nuclei (SCN) of the hypothalamus regulates physiology and behavior across the 24 h day by synchronizing peripheral clocks throughout the brain and body. Without the SCN, these peripheral clocks rapidly become desynchronized. Here, we provide a unique demonstration that, under lighting conditions in which the central clock in the SCN is dampened, peripheral oscillators in the hippocampus and olfactory bulb become desynchronized, along with the behavioral processes mediated by these clocks. Multiple clocks that adopt different phase relationships may enable processes occurring in different brain regions to be optimized to specific phases of the 24 h day. Moreover, such a dispersed network of dissociable circadian clocks may provide greater flexibility when faced with conflicting environmental signals (e.g., seasonal changes in photoperiod).
Literature context: C57BL/6J (RRID:IMSR_JAX:000664) wild-type
The responses of neurons in the visual cortex (V1) of adult mammals have long been thought to be stable over long periods. Here, we investigated whether repeated exposure to specific stimuli would enhance V1 visual responses in mice using intrinsic signal imaging through the intact skull and two-photon imaging of calcium signals in single neurons. Mice ran on Styrofoam balls floating on air while viewing one of three different, high-contrast visual stimuli. V1 responses to the stimuli that were viewed by the animal were specifically enhanced, while responses to other stimuli were unaffected. Similar exposure in stationary mice or in mice in which NMDA receptors were partially blocked did not significantly enhance responses. These findings indicate that stimulus-specific plasticity in the adult visual cortex depends on concurrent locomotion, presumably as a result of the high-gain state of the visual cortex induced by locomotion.SIGNIFICANCE STATEMENT We report a rapid and persistent increase in visual cortical responses to visual stimuli presented during locomotion in intact mice. We first used a method that is completely noninvasive to image intrinsic signals through the intact skull. We then measured the same effects on single neurons using two-photon calcium imaging and found that the increase in response to a particular stimulus produced by locomotion depends on how well the neuron is initially driven by the stimulus. To our knowledge, this is the first time such enhancement has been described in single neurons or using noninvasive measurements.
Literature context: oratories RRID:IMSR_JAX:000664 Mouse: FVB
Natural killer (NK) cells play a key role in innate immunity by detecting alterations in self and non-self ligands via paired NK cell receptors (NKRs). Despite identification of numerous NKR-ligand interactions, physiological ligands for the prototypical NK1.1 orphan receptor remain elusive. Here, we identify a viral ligand for the inhibitory and activating NKR-P1 (NK1.1) receptors. This murine cytomegalovirus (MCMV)-encoded protein, m12, restrains NK cell effector function by directly engaging the inhibitory NKR-P1B receptor. However, m12 also interacts with the activating NKR-P1A/C receptors to counterbalance m12 decoy function. Structural analyses reveal that m12 sequesters a large NKR-P1 surface area via a "polar claw" mechanism. Polymorphisms in, and ablation of, the viral m12 protein and host NKR-P1B/C alleles impact NK cell responses in vivo. Thus, we identify the long-sought foreign ligand for this key immunoregulatory NKR family and reveal how it controls the evolutionary balance of immune recognition during host-pathogen interplay.
Literature context: WT mice (RRID:IMSR_JAX:000664) were purc
Across many studies, animals with enhanced synaptic plasticity exhibit either enhanced or impaired learning, raising a conceptual puzzle: how enhanced plasticity can yield opposite learning outcomes? Here, we show that the recent history of experience can determine whether mice with enhanced plasticity exhibit enhanced or impaired learning in response to the same training. Mice with enhanced cerebellar LTD, due to double knockout (DKO) of MHCI H2-Kb/H2-Db (KbDb-/-), exhibited oculomotor learning deficits. However, the same mice exhibited enhanced learning after appropriate pre-training. Theoretical analysis revealed that synapses with history-dependent learning rules could recapitulate the data, and suggested that saturation may be a key factor limiting the ability of enhanced plasticity to enhance learning. Optogenetic stimulation designed to saturate LTD produced the same impairment in WT as observed in DKO mice. Overall, our results suggest that the recent history of activity and the threshold for synaptic plasticity conspire to effect divergent learning outcomes.
Literature context: rN/AC57BL6/JJackson Laboratories000664Mx1-CreJackson Laboratories00355
Literature context: ild-type C57BL/6J mice (strain #664, Jackson Laboratories, Bar Harb
Purpose: How retinal bipolar cell interneurons are specified and assigned to specialized subtypes is only partially understood. In part, this is due to a lack of early pan- and subtype-specific bipolar cell markers. To discover these factors, we identified genes that were upregulated in Blimp1 (Prdm1) mutant retinas, which exhibit precocious bipolar cell development. Methods: Postnatal day (P)2 retinas from Blimp1 conditional knock-out (CKO) mice and controls were processed for RNA sequencing. Genes that increased at least 45% and were statistically different between conditions were considered candidate bipolar-specific factors. Candidates were further evaluated by RT-PCR, in situ hybridization, and immunohistochemistry. Knock-in Tmem215-LacZ mice were used to better trace retinal expression. Results: A comparison between Blimp1 CKO and control RNA-seq datasets revealed approximately 40 significantly upregulated genes. We characterized the expression of three genes that have no known function in the retina, Gsg1 (germ cell associated gene), Trnp1 (TMF-regulated nuclear protein), and Tmem215 (a predicted transmembrane protein). Germ cell associated gene appeared restricted to a small subset of cone bipolars while Trnp1 was seen in all ON type bipolar cells. Using Tmem215-LacZ heterozygous knock-in mice, we observed that β-galactosidase expression started early in bipolar cell development. In adults, Tmem215 was expressed by a subset of ON and OFF cone bipolar cells. Conclusions: We have identified Gsg1, Tmem215, and Trnp1 as novel bipolar subtype-specific genes. The spatial and temporal pattern of their expression is consistent with a role in controlling bipolar subtype fate choice, differentiation, or physiology.
Literature context: ME, USA, RRID:IMSR_JAX:000664)] were use
The basolateral amygdala (BLA) integrates sensory input from cortical and subcortical regions, a function that requires marked synaptic plasticity. Here we provide evidence that cytochrome P450 aromatase (AROM), the enzyme converting testosterone to 17β-estradiol (E2), contributes to the regulation of this plasticity in a sex-specific manner. We show that AROM is expressed in the BLA, particularly in the basolateral nucleus (BL), in male and female rodents. Systemic administration of the AROM inhibitor letrozole reduced spine synapse density in the BL of adult female mice but not in the BL of male mice. Similarly, in organotypic corticoamygdalar slice cultures from immature rats, treatment with letrozole significantly reduced spine synapses in the BL only in cultures derived from females. In addition, letrozole sex-specifically altered synaptic properties in the BL: in acute slices from juvenile (prepubertal) female rats, wash-in of letrozole virtually abolished long-term potentiation (LTP), whereas it did not prevent the generation of LTP in the slices from males. Together, these data indicate that neuron-derived E2 modulates synaptic plasticity in rodent BLA sex-dependently. As protein expression levels of AROM, estrogen and androgen receptors did not differ between males and females and were not sex-specifically altered by letrozole, the findings suggest sex-specific mechanisms of E2 signaling.SIGNIFICANCE STATEMENT The basolateral amygdala (BLA) is a key structure of the fear circuit. This research reveals a sexually dimorphic regulation of synaptic plasticity in the BLA involving neuronal aromatase, which produces the neurosteroid 17β-estradiol (E2). As male and female neurons in rodent BLA responded differently to aromatase inhibition both in vivo and in vitro, our findings suggest that E2 signaling in BLA neurons is regulated sex-dependently, presumably via mechanisms that have been established during sexual determination. These findings could be relevant for the understanding of sex differences in mood disorders and of the side effects of cytochrome P450 aromatase inhibitors, which are frequently used for breast cancer therapy.
Literature context: ouse: C57Bl6Jackson Laboratories000664Recombinant DNArAAV2-retro.CAG.C
Mossy cells in the hilus of the dentate gyrus constitute a major excitatory principal cell type in the mammalian hippocampus; however, it remains unknown how these cells behave in vivo. Here, we have used two-photon Ca2+ imaging to monitor the activity of mossy cells in awake, behaving mice. We find that mossy cells are significantly more active than dentate granule cells in vivo, exhibit spatial tuning during head-fixed spatial navigation, and undergo robust remapping of their spatial representations in response to contextual manipulation. Our results provide a functional characterization of mossy cells in the behaving animal and demonstrate their active participation in spatial coding and contextual representation.
Literature context: #000664; RRID:IMSR_JAX:000664 Mouse / Ca
When conflicts occur during integration of visual and auditory information, one modality often dominates the other, but the underlying neural circuit mechanism remains unclear. Using auditory-visual discrimination tasks for head-fixed mice, we found that audition dominates vision in a process mediated by interaction between inputs from the primary visual (VC) and auditory (AC) cortices in the posterior parietal cortex (PTLp). Co-activation of the VC and AC suppresses VC-induced PTLp responses, leaving AC-induced responses. Furthermore, parvalbumin-positive (PV+) interneurons in the PTLp mainly receive AC inputs, and muscimol inactivation of the PTLp or optogenetic inhibition of its PV+ neurons abolishes auditory dominance in the resolution of cross-modal sensory conflicts without affecting either sensory perception. Conversely, optogenetic activation of PV+ neurons in the PTLp enhances the auditory dominance. Thus, our results demonstrate that AC input-specific feedforward inhibition of VC inputs in the PTLp is responsible for the auditory dominance during cross-modal integration.
Literature context: les River RRID:IMSR_JAX:000664 Mouse: AP-
Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Recent data suggest that at physiological temperature SVs are internalized via clathrin-independent ultrafast endocytosis (UFE) within hundreds of milliseconds, while other studies have postulated a key role for clathrin-mediated endocytosis (CME) of SV proteins on a timescale of seconds to tens of seconds. Here we demonstrate using cultured hippocampal neurons as a model that at physiological temperature SV endocytosis occurs on several timescales from less than a second to several seconds, yet, is largely independent of clathrin. Clathrin-independent endocytosis (CIE) of SV membranes is mediated by actin-nucleating formins such as mDia1, which are required for the formation of presynaptic endosome-like vacuoles from which SVs reform. Our results resolve previous discrepancies in the field and suggest that SV membranes are predominantly retrieved via CIE mediated by formin-dependent actin assembly.
Literature context: X:000664; RRID:IMSR_JAX:000664; 6-8 weeks
In the adult mammalian central nervous system (CNS), axonal damage often triggers neuronal cell death and glial activation, with very limited spontaneous axon regeneration. In this study, we performed optic nerve injury in adult naked mole-rats, the longest living rodent, with a maximum life span exceeding 30 years, and found that injury responses in this species are quite distinct from those in other mammalian species. In contrast to what is seen in other mammals, the majority of injured retinal ganglion cells (RGCs) survive with relatively high spontaneous axon regeneration. Furthermore, injured RGCs display activated signal transducer and activator of transcription-3 (STAT3), whereas astrocytes in the optic nerve robustly occupy and fill the lesion area days after injury. These neuron-intrinsic and -extrinsic injury responses are reminiscent of those in "cold-blooded" animals, such as fish and amphibians, suggesting that the naked mole-rat is a powerful model for exploring the mechanisms of neuronal injury responses and axon regeneration in mammals. J. Comp. Neurol. 525:380-388, 2017. © 2016 Wiley Periodicals, Inc.
Literature context: /6J mice (RRID:IMSR_JAX:000664), three we
Paternal environmental conditions can influence phenotypes in future generations, but it is unclear whether offspring phenotypes represent specific responses to particular aspects of the paternal exposure history, or a generic response to paternal 'quality of life'. Here, we establish a paternal effect model based on nicotine exposure in mice, enabling pharmacological interrogation of the specificity of the offspring response. Paternal exposure to nicotine prior to reproduction induced a broad protective response to multiple xenobiotics in male offspring. This effect manifested as increased survival following injection of toxic levels of either nicotine or cocaine, accompanied by hepatic upregulation of xenobiotic processing genes, and enhanced drug clearance. Surprisingly, this protective effect could also be induced by a nicotinic receptor antagonist, suggesting that xenobiotic exposure, rather than nicotinic receptor signaling, is responsible for programming offspring drug resistance. Thus, paternal drug exposure induces a protective phenotype in offspring by enhancing metabolic tolerance to xenobiotics.
Literature context: ype mice (RRID:IMSR_JAX:000664) were used
The mammalian inner ear (IE) subserves auditory and vestibular sensations via highly specialized cells and proteins. Sensory receptor hair cells (HCs) are necessary for transducing mechanical inputs and stimulating sensory neurons by using a host of known and as yet unknown protein machinery. To understand the protein composition of these unique postmitotic cells, in which irreversible protein degradation or damage can lead to impaired hearing and balance, we analyzed IE samples by tandem mass spectrometry to generate an unbiased, shotgun-proteomics view of protein identities and abundances. By using Pou4f3/eGFP-transgenic mice in which HCs express GFP driven by Pou4f3, we FACS purified a population of HCs to analyze and compare the HC proteome with other IE subproteomes from sensory epithelia and whole IE. We show that the mammalian HC proteome comprises hundreds of uniquely or highly expressed proteins. Our global proteomic analysis of purified HCs extends the existing HC transcriptome, revealing previously undetected gene products and isoform-specific protein expression. Comparison of our proteomic data with mouse and human databases of genetic auditory/vestibular impairments confirms the critical role of the HC proteome for normal IE function, providing a cell-specific pool of candidates for novel, important HC genes. Several proteins identified exclusively in HCs by proteomics and verified by immunohistochemistry map to human genetic deafness loci, potentially representing new deafness genes. SIGNIFICANCE STATEMENT: Hearing and balance rely on specialized sensory hair cells (HCs) in the inner ear (IE) to convey information about sound, acceleration, and orientation to the brain. Genetically and environmentally induced perturbations to HC proteins can result in deafness and severe imbalance. We used transgenic mice with GFP-expressing HCs, coupled with FACS sorting and tandem mass spectrometry, to define the most complete HC and IE proteome to date. We show that hundreds of proteins are uniquely identified or enriched in HCs, extending previous gene expression analyses to reveal novel HC proteins and isoforms. Importantly, deafness-linked proteins were significantly enriched in HCs, suggesting that this in-depth proteomic analysis of IE sensory cells may hold potential for deafness gene discovery.
Literature context: 9Mouse: B6The Jackson Laboratory000664Mouse: Col1a2-CreERT:R26RtdTomat
Mammalian tissues calcify with age and injury. Analogous to bone formation, osteogenic cells are thought to be recruited to the affected tissue and induce mineralization. In the heart, calcification of cardiac muscle leads to conduction system disturbances and is one of the most common pathologies underlying heart blocks. However the cell identity and mechanisms contributing to pathological heart muscle calcification remain unknown. Using lineage tracing, murine models of heart calcification and in vivo transplantation assays, we show that cardiac fibroblasts (CFs) adopt an osteoblast cell-like fate and contribute directly to heart muscle calcification. Small-molecule inhibition of ENPP1, an enzyme that is induced upon injury and regulates bone mineralization, significantly attenuated cardiac calcification. Inhibitors of bone mineralization completely prevented ectopic cardiac calcification and improved post injury heart function. Taken together, these findings highlight the plasticity of fibroblasts in contributing to ectopic calcification and identify pharmacological targets for therapeutic development.
Literature context: Both C57BL/6J (RRID:IMSR_JAX:000664) (Doucette and Restrepo, 2008;
The firing rate of the mitral/tufted cells in the olfactory bulb is known to undergo significant trial-to-trial variability and is affected by anesthesia. Here we ask whether odorant-elicited changes in firing rate depend on the rate before application of the stimulus in the awake and anesthetized mouse. We find that prestimulus firing rate varies widely on a trial-to-trial basis and that the stimulus-induced change in firing rate decreases with increasing prestimulus firing rate. Interestingly, this prestimulus firing rate dependence was different when the behavioral task did not involve detecting the valence of the stimulus. Finally, when the animal was learning to associate the odor with reward, the prestimulus firing rate was smaller for false alarms compared with correct rejections, suggesting that intrinsic activity reflects the anticipatory status of the animal. Thus, in this sensory modality, changes in behavioral status alter the intrinsic prestimulus activity, leading to a change in the responsiveness of the second-order neurons. We speculate that this trial-to-trial variability in odorant responses reflects sampling of the massive parallel input by subsets of mitral cells.SIGNIFICANCE STATEMENT The olfactory bulb must deal with processing massive parallel input from ∼1200 distinct olfactory receptors. In contrast, the visual system receives input from a small number of photoreceptors and achieves recognition of complex stimuli by allocating processing for distinct spatial locations to different brain areas. Here we find that the change in firing rate elicited by the odorant in second-order mitral cells depends on the intrinsic activity leading to a change of magnitude in the responsiveness of these neurons relative to this prestimulus activity. Further, we find that prestimulus firing rate is influenced by behavioral status. This suggests that there is top-down modulation allowing downstream brain processing areas to perform dynamic readout of olfactory information.
Literature context: ratories; RRID:IMSR_JAX:000664) using aÂ s
Diverse cellular signaling events, including B cell receptor (BCR) activation, are hypothesized to be facilitated by domains enriched in specific plasma membrane lipids and proteins that resemble liquid-ordered phase-separated domains in model membranes. This concept remains controversial and lacks direct experimental support in intact cells. Here, we visualize ordered and disordered domains in mouse B lymphoma cell membranes using super-resolution fluorescence localization microscopy, demonstrate that clustered BCR resides within ordered phase-like domains capable of sorting key regulators of BCR activation, and present a minimal, predictive model where clustering receptors leads to their collective activation by stabilizing an extended ordered domain. These results provide evidence for the role of membrane domains in BCR signaling and a plausible mechanism of BCR activation via receptor clustering that could be generalized to other signaling pathways. Overall, these studies demonstrate that lipid mediated forces can bias biochemical networks in ways that broadly impact signal transduction.
Literature context: Jackson LaboratoryStock number: 000664Mouse: APCmin/+The Jackson Labor
Centrosome amplification is a common feature of human tumors, but whether this is a cause or a consequence of cancer remains unclear. Here, we test the consequence of centrosome amplification by creating mice in which centrosome number can be chronically increased in the absence of additional genetic defects. We show that increasing centrosome number elevated tumor initiation in a mouse model of intestinal neoplasia. Most importantly, we demonstrate that supernumerary centrosomes are sufficient to drive aneuploidy and the development of spontaneous tumors in multiple tissues. Tumors arising from centrosome amplification exhibit frequent mitotic errors and possess complex karyotypes, recapitulating a common feature of human cancer. Together, our data support a direct causal relationship among centrosome amplification, genomic instability, and tumor development.
Literature context: e Committee. Male C57BL/6 mice (RRID:IMSR_JAX:000664) between the ages of 8 and 30 w
In both dichromats and trichromats, cone opsin signals are maintained independently in cones and combined at the bipolar and retinal ganglion cell level, creating parallel color opponent pathways to the central visual system. Like other dichromats, the mouse retina expresses a short-wavelength (S) and a medium-wavelength (M) opsin, with the S-opsin shifted to peak sensitivity in the ultraviolet (UV) range. Unlike in primates, nonuniform opsin expression across the retina and coexpression in single cones creates a mostly mixed chromatic signal. Here, we describe the visuotopic and chromatic organization of spiking responses in the dorsal lateral geniculate and of the local field potentials in their recipient zone in primary visual cortex (V1). We used an immersive visual stimulus dome that allowed us to present spatiotemporally modulated UV and green luminance in any region of the visual field of an awake, head-fixed mouse. Consistent with retinal expression of opsins, we observed graded UV-to-green dominated responses from the upper to lower visual fields, with a smaller difference across azimuth. In addition, we identified a subpopulation of cells (<10%) that exhibited spectrally opponent responses along the S-M axis. Luminance signals of each wavelength and color signals project to the middle layers of V1. SIGNIFICANCE STATEMENT: In natural environments, color information is useful for guiding behavior. How small terrestrial mammals such as mice use graded expression of cone opsins to extract visual information from their environments is not clear, even as the use of mice for studying visually guided behavior grows. In this study, we examined the color signals that the retina sends to the visual cortex via the lateral geniculate nucleus of the thalamus. We found that green dominated responses in the lower and nasal visual field and ultraviolet dominated responses in the upper visual field. We describe a subset of cells that exhibit color opponent responses.
Literature context: les River RRID:IMSR_JAX:000664 Recombinan
The thalamus receives sensory input from different circuits in the periphery. How these sensory channels are integrated at the level of single thalamic cells is not well understood. We performed targeted single-cell-initiated transsynaptic tracing to label the retinal ganglion cells that provide input to individual principal cells in the mouse lateral geniculate nucleus (LGN). We identified three modes of sensory integration by single LGN cells. In the first, 1-5 ganglion cells of mostly the same type converged from one eye, indicating a relay mode. In the second, 6-36 ganglion cells of different types converged from one eye, revealing a combination mode. In the third, up to 91 ganglion cells converged from both eyes, revealing a binocular combination mode in which functionally specialized ipsilateral inputs joined broadly distributed contralateral inputs. Thus, the LGN employs at least three modes of visual input integration, each exhibiting different degrees of specialization.
Literature context: aboratory RRID:IMSR_JAX:000664 Recombinan
The hippocampal dentate gyrus is often viewed as a segregator of upstream information. Physiological support for such function has been hampered by a lack of well-defined characteristics that can identify granule cells and mossy cells. We developed an electrophysiology-based classification of dentate granule cells and mossy cells in mice that we validated by optogenetic tagging of mossy cells. Granule cells exhibited sparse firing, had a single place field, and showed only modest changes when the mouse was tested in different mazes in the same room. In contrast, mossy cells were more active, had multiple place fields and showed stronger remapping of place fields under the same conditions. Although the granule cell-mossy cell synapse was strong and facilitating, mossy cells rarely "inherited" place fields from single granule cells. Our findings suggest that the granule cells and mossy cells could be modulated separately and their joint action may be critical for pattern separation.
Literature context: C57BL/6J (RRID:IMSR_JAX:000664) backgroun
The study was designed to determine the role of long noncoding RNA (lncRNA), metastasis-associated lung adenocarcinoma transcript 1 (Malat1), in ischemic stroke outcome. Primary mouse brain microvascular endothelial cells (BMECs) were cultured and treated with Malat1 GapmeR before 16 h oxygen and glucose depravation (OGD). Cell death was assayed by LDH and MTT methods. Malat1 knock-out and wild-type mice were subjected to 1 h of middle cerebral artery occlusion (MCAO) and 24-72 h of reperfusion. To explore the underlying mechanism, apoptotic and inflammatory factors were measured by qPCR, ELISA, and Western blotting. The physical interaction between Malat1 and apoptotic or inflammatory factors was measured by RNA immunoprecipitation. Increased Malat1 levels were found in cultured mouse BMECs after OGD as well as in isolated cerebral microvessels in mice after MCAO. Silencing of Malat1 by Malat1 GapmeR significantly increased OGD-induced cell death and Caspase 3 activity in BMECs. Silencing of Malat1 also significantly aggravated OGD-induced expression of the proapoptotic factor Bim and proinflammatory cytokines MCP-1, IL-6, and E-selectin. Moreover, Malat1 KO mice presented larger brain infarct size, worsened neurological scores, and reduced sensorimotor functions. Consistent with in vitro findings, significantly increased expression of proapoptotic and proinflammatory factors was also found in the cerebral cortex of Malat1 KO mice after ischemic stroke compared with WT controls. Finally, we demonstrated that Malat1 binds to Bim and E-selectin both in vitro and in vivo Our study suggests that Malat1 plays critical protective roles in ischemic stroke.SIGNIFICANCE STATEMENT Accumulative studies have demonstrated the important regulatory roles of microRNAs in vascular and neural damage after ischemic stroke. However, the functional significance and mechanisms of other classes of noncoding RNAs in cerebrovascular pathophysiology after stroke are less studied. Here we demonstrate a novel role of Malat1, a long noncoding RNA that has been originally identified as a prognostic marker for non-small cell lung cancer, in cerebrovascular pathogenesis of ischemic stroke. Our experiments have provided the first evidence that Malat1 plays anti-apoptotic and anti-inflammatory roles in brain microvasculature to reduce ischemic cerebral vascular and parenchymal damages. Our studies also suggest that lncRNAs can be therapeutically targeted to minimize poststroke brain damage.
Literature context: les River RRID:IMSR_JAX:000664 Recombinan
The developmental mechanisms regulating the number of adult neural stem cells (aNSCs) are largely unknown. Here we show that the cleavage plane orientation in murine embryonic radial glia cells (RGCs) regulates the number of aNSCs in the lateral ganglionic eminence (LGE). Randomizing spindle orientation in RGCs by overexpression of Insc or a dominant-negative form of Lgn (dnLgn) reduces the frequency of self-renewing asymmetric divisions while favoring symmetric divisions generating two SNPs. Importantly, these changes during embryonic development result in reduced seeding of aNSCs. Interestingly, no effects on aNSC numbers were observed when Insc was overexpressed in postnatal RGCs or aNSCs. These data suggest a new mechanism for controlling aNSC numbers and show that the role of spindle orientation during brain development is highly time and region dependent.
Literature context: /6J mice (RRID:IMSR_JAX:000664) at postna
Addictive drugs usurp neural plasticity mechanisms that normally serve reward-related learning and memory, primarily by evoking changes in glutamatergic synaptic strength in the mesocorticolimbic dopamine circuitry. Here, we show that repeated cocaine exposure in vivo does not alter synaptic strength in the mouse prefrontal cortex during an early period of withdrawal, but instead modifies a Hebbian quantitative synaptic learning rule by broadening the temporal window and lowers the induction threshold for spike-timing-dependent LTP (t-LTP). After repeated, but not single, daily cocaine injections, t-LTP in layer V pyramidal neurons is induced at +30 ms, a normally ineffective timing interval for t-LTP induction in saline-exposed mice. This cocaine-induced, extended-timing t-LTP lasts for ∼1 week after terminating cocaine and is accompanied by an increased susceptibility to potentiation by fewer pre-post spike pairs, indicating a reduced t-LTP induction threshold. Basal synaptic strength and the maximal attainable t-LTP magnitude remain unchanged after cocaine exposure. We further show that the cocaine facilitation of t-LTP induction is caused by sensitized D1-cAMP/protein kinase A dopamine signaling in pyramidal neurons, which then pathologically recruits voltage-gated l-type Ca2+ channels that synergize with GluN2A-containing NMDA receptors to drive t-LTP at extended timing. Our results illustrate a mechanism by which cocaine, acting on a key neuromodulation pathway, modifies the coincidence detection window during Hebbian plasticity to facilitate associative synaptic potentiation in prefrontal excitatory circuits. By modifying rules that govern activity-dependent synaptic plasticity, addictive drugs can derail the experience-driven neural circuit remodeling process important for executive control of reward and addiction. SIGNIFICANCE STATEMENT: It is believed that addictive drugs often render an addict's brain reward system hypersensitive, leaving the individual more susceptible to relapse. We found that repeated cocaine exposure alters a Hebbian associative synaptic learning rule that governs activity-dependent synaptic plasticity in the mouse prefrontal cortex, characterized by a broader temporal window and a lower threshold for spike-timing-dependent LTP (t-LTP), a cellular form of learning and memory. This rule change is caused by cocaine-exacerbated D1-cAMP/protein kinase A dopamine signaling in pyramidal neurons that in turn pathologically recruits l-type Ca2+ channels to facilitate coincidence detection during t-LTP induction. Our study provides novel insights on how cocaine, even with only brief exposure, may prime neural circuits for subsequent experience-dependent remodeling that may underlie certain addictive behavior.
Literature context: Brd/BrdCrCrlMouse: C57BL6/J miceJackson Laboratory (Stock #000664)C57BL6/JMouse: Mice possessing
Bone mineral density (BMD) is a highly heritable predictor of osteoporotic fracture. Genome-wide association studies (GWAS) for BMD have identified dozens of associations; yet, the genes responsible for most associations remain elusive. Here, we used a bone co-expression network to predict causal genes at BMD GWAS loci based on the premise that genes underlying a disease are often functionally related and functionally related genes are often co-expressed. By mapping genes implicated by BMD GWAS onto a bone co-expression network, we predicted and inferred the function of causal genes for 30 of 64 GWAS loci. We experimentally confirmed that two of the genes predicted to be causal, SPTBN1 and MARK3, are potentially responsible for the effects of GWAS loci on chromosomes 2p16.2 and 14q32.32, respectively. This approach provides a roadmap for the dissection of additional BMD GWAS associations. Furthermore, it should be applicable to GWAS data for a wide range of diseases.
Literature context: Male 8â€“9-wk-old C57BL/6J mice (000664; The Jackson Laboratory) and IF
The brain has a tightly regulated environment that protects neurons and limits inflammation, designated "immune privilege." However, there is not an absolute lack of an immune response. We tested the ability of the brain to initiate an innate immune response to a virus, which was directly injected into the brain parenchyma, and to determine whether this response could limit viral spread. We injected vesicular stomatitis virus (VSV), a transsynaptic tracer, or naturally occurring VSV-derived defective interfering particles (DIPs), into the caudate-putamen (CP) and scored for an innate immune response and inhibition of virus spread. We found that the brain parenchyma has a functional type I interferon (IFN) response that can limit VSV spread at both the inoculation site and among synaptically connected neurons. Furthermore, we characterized the response of microglia to VSV infection and found that infected microglia produced type I IFN and uninfected microglia induced an innate immune response following virus injection.
Literature context: : C57BL/6JThe Jackson LaboratoryStock No: 000664Mouse: B6D2F1/JThe Jackson Labor
Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naïve hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.
Literature context: : C57BL/6JThe Jackson LaboratoryJAX: 000664Mouse: Slc32a1tm2(cre)Lowl/JThe
Superior predatory skills led to the evolutionary triumph of jawed vertebrates. However, the mechanisms by which the vertebrate brain controls predation remain largely unknown. Here, we reveal a critical role for the central nucleus of the amygdala in predatory hunting. Both optogenetic and chemogenetic stimulation of central amygdala of mice elicited predatory-like attacks upon both insect and artificial prey. Coordinated control of cervical and mandibular musculatures, which is necessary for accurately positioning lethal bites on prey, was mediated by a central amygdala projection to the reticular formation in the brainstem. In contrast, prey pursuit was mediated by projections to the midbrain periaqueductal gray matter. Targeted lesions to these two pathways separately disrupted biting attacks upon prey versus the initiation of prey pursuit. Our findings delineate a neural network that integrates distinct behavioral modules and suggest that central amygdala neurons instruct predatory hunting across jawed vertebrates.
Literature context: sed either C57BL/6J mice (RRID: IMSR_JAX:000664) or NPY-hrGFP mice (B6.FVB-Tg(N
Neuropeptide Y (NPY) has robust anxiolytic properties and is reduced in patients with anxiety disorders. However, the mechanisms by which NPY modulates circuit function to reduce anxiety behavior are not known. Anxiolytic effects of NPY are mediated in the CA1 region of hippocampus, and NPY injection into hippocampus alleviates anxiety symptoms in the predator scent stress model of stress-induced anxiety. The mechanisms that regulate NPY release, and its effects on CA1 synaptic function, are not fully understood. Here we show in acute hippocampal slices from mice that endogenous NPY, released in response to optogenetic stimulation or synaptically evoked spiking of NPY+ cells, suppresses both of the feedforward pathways to CA1. Stimulation of temporoammonic synapses with a physiologically derived spike train causes NPY release that reduces short-term facilitation, whereas the release of NPY that modulates Schaffer collateral synapses requires integration of both the Schaffer collateral and temporoammonic pathways. Pathway specificity of NPY release is conferred by three functionally distinct NPY+ cell types, with differences in intrinsic excitability and short-term plasticity of their inputs. Predator scent stress abolishes the release of endogenous NPY onto temporoammonic synapses, a stress-sensitive pathway, thereby causing enhanced short-term facilitation. Our results demonstrate how stress alters CA1 circuit function through the impairment of endogenous NPY release, potentially contributing to heightened anxiety. SIGNIFICANCE STATEMENT: Neuropeptide Y (NPY) has robust anxiolytic properties, and its levels are reduced in patients with post-traumatic stress disorder. The effects of endogenously released NPY during physiologically relevant stimulation, and the impact of stress-induced reductions in NPY on circuit function, are unknown. By demonstrating that NPY release modulates hippocampal synaptic plasticity and is impaired by predator scent stress, our results provide a novel mechanism by which stress-induced anxiety alters circuit function. These studies fill an important gap in knowledge between the molecular and behavioral effects of NPY. This article also advances the understanding of NPY+ cells and the factors that regulate their spiking, which could pave the way for new therapeutic targets to increase endogenous NPY release in patients in a spatially and temporally appropriate manner.
Literature context: The Jackson LaboratoryStock No: 000664Recombinant DNApFENHK-Halo-MLLPr
Chromosomal translocations of the mixed-lineage leukemia (MLL) gene with various partner genes result in aggressive leukemia with dismal outcomes. Despite similar expression at the mRNA level from the wild-type and chimeric MLL alleles, the chimeric protein is more stable. We report that UBE2O functions in regulating the stability of wild-type MLL in response to interleukin-1 signaling. Targeting wild-type MLL degradation impedes MLL leukemia cell proliferation, and it downregulates a specific group of target genes of the MLL chimeras and their oncogenic cofactor, the super elongation complex. Pharmacologically inhibiting this pathway substantially delays progression, and it improves survival of murine leukemia through stabilizing wild-type MLL protein, which displaces the MLL chimera from some of its target genes and, therefore, relieves the cellular oncogenic addiction to MLL chimeras. Stabilization of MLL provides us with a paradigm in the development of therapies for aggressive MLL leukemia and perhaps for other cancers caused by translocations.
Literature context: C57BL/6J RRID:IMSR_JAX:000664 Â C57BL/6N
Deficits in the domains of attention and response inhibition are central to many psychiatric disorders. As such, animal models of disorders purporting to replicate these behavioral deficits first require tests that can accurately assess the behaviors with high fidelity. The gold-standard clinical test of attention and response inhibition is the continuous performance test (CPT). Although there are a number of CPTs, all share the premise of responding to target stimuli and inhibiting from responding to non-target stimuli. The recently developed rodent five-choice CPT (5C-CPT) requires similar behavioral responses, enabling signal detection parameter calculations. With demonstrable feasibility for rodent testing, the 5C-CPT permits/facilitates: (1) delineation of neural mechanisms underlying these behaviors; (2) multifactorial analyses of the complex interplay between genetic and environmental manipulations relevant to psychiatric disorders; and hence (3) development of novel targeted treatments. All data to date indicate that the rodent 5C-CPT described here has direct translatability to clinical CPTs, producing equivalent measures of behavior in experimental animals to those assessed in humans. The 5C-CPT task provides an important tool toward delineating these mechanisms and developing treatments. However, it is also complex, with long training times and nuances requiring a thorough understanding before utilization. This unit will enable researchers to avoid potential missteps, greatly increasing the likelihood of success. © 2017 by John Wiley & Sons, Inc.
Literature context: BL/6J miceThe Jackson LaboratoryJAX: 000664Recombinant DNApMXs-Oct3/4Addgen
Aging is the major risk factor for many human diseases. In vitro studies have demonstrated that cellular reprogramming to pluripotency reverses cellular age, but alteration of the aging process through reprogramming has not been directly demonstrated in vivo. Here, we report that partial reprogramming by short-term cyclic expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) ameliorates cellular and physiological hallmarks of aging and prolongs lifespan in a mouse model of premature aging. Similarly, expression of OSKM in vivo improves recovery from metabolic disease and muscle injury in older wild-type mice. The amelioration of age-associated phenotypes by epigenetic remodeling during cellular reprogramming highlights the role of epigenetic dysregulation as a driver of mammalian aging. Establishing in vivo platforms to modulate age-associated epigenetic marks may provide further insights into the biology of aging.
Literature context: AX:000664 RRID:IMSR_JAX:000664), 129S-Par
After traumatic damage of the brain or spinal cord, many surviving neurons are disconnected, and recovery of function is limited by poor axon regeneration. Recent data have suggested that poly ADP-ribosylation plays a role in limiting axonal regrowth such that inhibition of poly (ADP-ribose) polymerase (PARP) may have therapeutic efficacy for neurological recovery after trauma. Here, we tested systemic administration of the PARP inhibitor, veliparib, and showed effective suppression of PARylation in the mouse CNS. After optic nerve crush injury or dorsal hemisection of the thoracic spinal cord in mice, treatment with veliparib at doses with pharmacodynamic action had no benefit for axonal regeneration or functional recovery. We considered whether PARP gene family specificity might play a role. In vitro mouse cerebral cortex axon regeneration experiments revealed that short hairpin RNA (shRNA)-mediated suppression of PARP1 promoted axonal regeneration, whereas suppression of other PARP isoforms either had no effect or decreased regeneration. Therefore, we examined recovery from neurological trauma in mice lacking PARP1. No increase of axonal regeneration was observed in Parp1-/- mice after optic nerve crush injury or dorsal hemisection of the thoracic spinal cord, and there was no improvement in motor function recovery. Thus, comprehensive in vivo analysis reveals no indication that clinical PARP inhibitors will on their own provide benefit for recovery from CNS trauma.
Literature context: /6J mice (RRID:IMSR_JAX:000664), were obt
Background Disruption of rhythms in activity and rest occur in many diseases, and provide an important indicator of healthy physiology and behaviour. However, outside the field of sleep and circadian rhythm research, these rhythmic processes are rarely measured due to the requirement for specialised resources and expertise. Until recently, the primary approach to measuring activity in laboratory rodents has been based on voluntary running wheel activity. By contrast, measuring sleep requires the use of electroencephalography (EEG), which involves invasive surgical procedures and time-consuming data analysis. Methods Here we describe a simple, non-invasive system to measure home cage activity in mice based upon passive infrared (PIR) motion sensors. Careful calibration of this system will allow users to simultaneously assess sleep status in mice. The use of open-source tools and simple sensors keeps the cost and the size of data-files down, in order to increase ease of use and uptake. Results In addition to providing accurate data on circadian activity parameters, here we show that extended immobility of >40 seconds provides a reliable indicator of sleep, correlating well with EEG-defined sleep (Pearson's r >0.95, 4 mice). Conclusions Whilst any detailed analysis of sleep patterns in mice will require EEG, behaviourally-defined sleep provides a valuable non-invasive means of simultaneously phenotyping both circadian rhythms and sleep. Whilst previous approaches have relied upon analysis of video data, here we show that simple motion sensors provide a cheap and effective alternative, enabling real-time analysis and longitudinal studies extending over weeks or even months. The data files produced are small, enabling easy deposition and sharing. We have named this system COMPASS - Continuous Open Mouse Phenotyping of Activity and Sleep Status. This simple approach is of particular value in phenotyping screens as well as providing an ideal tool to assess activity and rest cycles for non-specialists.
Literature context: J strain (RRID:IMSR_JAX:000664) forÂ >10 g
Endoplasmic reticulum (ER) stress is implicated in many chronic diseases, but very little is known about how the unfolded protein response (UPR) responds to persistent ER stress in vivo. Here, we experimentally reconstituted chronic ER stress in the mouse liver, using repeated injection of a low dose of the ER stressor tunicamycin. Paradoxically, this treatment led to feedback-mediated suppression of a select group of mRNAs, including those encoding the ER chaperones BiP and GRP94. This suppression was due to both silencing of the ATF6α pathway of UPR-dependent transcription and enhancement of mRNA degradation, possibly via regulated IRE1-dependent decay (RIDD). The suppression of mRNA encoding BiP was phenocopied by ectopic overexpression of BiP protein, and was also observed in obese mice. Our findings suggest that persistent cycles of UPR activation and deactivation create an altered, quasi-stable setpoint for UPR-dependent transcriptional regulation-an outcome that could be relevant to conditions such as metabolic syndrome.
Literature context: /6JThe Jackson LaboratoryStock#:000664Mouse: B6.129S7-Ifngtm1Ts/JThe J
Therapeutic blocking of the PD1 pathway results in significant tumor responses, but resistance is common. We demonstrate that prolonged interferon signaling orchestrates PDL1-dependent and PDL1-independent resistance to immune checkpoint blockade (ICB) and to combinations such as radiation plus anti-CTLA4. Persistent type II interferon signaling allows tumors to acquire STAT1-related epigenomic changes and augments expression of interferon-stimulated genes and ligands for multiple T cell inhibitory receptors. Both type I and II interferons maintain this resistance program. Crippling the program genetically or pharmacologically interferes with multiple inhibitory pathways and expands distinct T cell populations with improved function despite expressing markers of severe exhaustion. Consequently, tumors resistant to multi-agent ICB are rendered responsive to ICB monotherapy. Finally, we observe that biomarkers for interferon-driven resistance associate with clinical progression after anti-PD1 therapy. Thus, the duration of tumor interferon signaling augments adaptive resistance and inhibition of the interferon response bypasses requirements for combinatorial ICB therapies.
Literature context: e: C57BL/6The Jackson LaboratoryJAX: 000664Mouse: C3H/HeOuThe Jackson Labor
Poorly immunogenic tumor cells evade host immunity and grow even in the presence of an intact immune system, but the complex mechanisms regulating tumor immunogenicity have not been elucidated. Here, we discovered an unexpected role of the Hippo pathway in suppressing anti-tumor immunity. We demonstrate that, in three different murine syngeneic tumor models (B16, SCC7, and 4T1), loss of the Hippo pathway kinases LATS1/2 (large tumor suppressor 1 and 2) in tumor cells inhibits tumor growth. Tumor regression by LATS1/2 deletion requires adaptive immune responses, and LATS1/2 deficiency enhances tumor vaccine efficacy. Mechanistically, LATS1/2-null tumor cells secrete nucleic-acid-rich extracellular vesicles, which induce a type I interferon response via the Toll-like receptors-MYD88/TRIF pathway. LATS1/2 deletion in tumors thus improves tumor immunogenicity, leading to tumor destruction by enhancing anti-tumor immune responses. Our observations uncover a key role of the Hippo pathway in modulating tumor immunogenicity and demonstrate a proof of concept for targeting LATS1/2 in cancer immunotherapy.
Literature context: C57BL/6J (RRID:IMSR_JAX:000664) with at l
Mechanisms underlying the vein development remain largely unknown. Tie2 signaling mediates endothelial cell (EC) survival and vascular maturation and its activating mutations are linked to venous malformations. Here we show that vein formation are disrupted in mouse skin and mesentery when Tie2 signals are diminished by targeted deletion of Tek either ubiquitously or specifically in embryonic ECs. Postnatal Tie2 attenuation resulted in the degeneration of newly formed veins followed by the formation of haemangioma-like vascular tufts in retina and venous tortuosity. Mechanistically, Tie2 insufficiency compromised venous EC identity, as indicated by a significant decrease of COUP-TFII protein level, a key regulator in venogenesis. Consistently, angiopoietin-1 stimulation increased COUP-TFII in cultured ECs, while Tie2 knockdown or blockade of Tie2 downstream PI3K/Akt pathway reduced COUP-TFII which could be reverted by the proteasome inhibition. Together, our results imply that Tie2 is essential for venous specification and maintenance via Akt mediated stabilization of COUP-TFII.
Literature context: X:007905, RRID:IMSR_JAX:000664), species
The hippocampal theta rhythm plays important roles in information processing; however, the mechanisms of its generation are not well understood. We developed a data-driven, supercomputer-based, full-scale (1:1) model of the rodent CA1 area and studied its interneurons during theta oscillations. Theta rhythm with phase-locked gamma oscillations and phase-preferential discharges of distinct interneuronal types spontaneously emerged from the isolated CA1 circuit without rhythmic inputs. Perturbation experiments identified parvalbumin-expressing interneurons and neurogliaform cells, as well as interneuronal diversity itself, as important factors in theta generation. These simulations reveal new insights into the spatiotemporal organization of the CA1 circuit during theta oscillations.
Literature context: /6J mice (RRID:IMSR_JAX:000664), Boy/J CD
IgE can trigger potent allergic responses, yet the mechanisms regulating IgE production are poorly understood. Here we reveal that IgE+ B cells are constrained by chronic activity of the IgE B cell receptor (BCR). In the absence of cognate antigen, the IgE BCR promoted terminal differentiation of B cells into plasma cells (PCs) under cell culture conditions mimicking T cell help. This antigen-independent PC differentiation involved multiple IgE domains and Syk, CD19, BLNK, Btk, and IRF4. Disruption of BCR signaling in mice led to consistently exaggerated IgE+ germinal center (GC) B cell but variably increased PC responses. We were unable to confirm reports that the IgE BCR directly promoted intrinsic apoptosis. Instead, IgE+ GC B cells exhibited poor antigen presentation and prolonged cell cycles, suggesting reduced competition for T cell help. We propose that chronic BCR activity and access to T cell help play critical roles in regulating IgE responses.
Literature context: C57BL/6J (RRID:IMSR_JAX:000664) mice obta
While the aging process is central to the pathogenesis of age-dependent diseases, it is poorly understood at the molecular level. We identified a mouse mutant with accelerated aging in the retina as well as pathologies observed in age-dependent retinal diseases, suggesting that the responsible gene regulates retinal aging, and its impairment results in age-dependent disease. We determined that a mutation in the transmembrane 135 (Tmem135) is responsible for these phenotypes. We observed localization of TMEM135 on mitochondria, and imbalance of mitochondrial fission and fusion in mutant Tmem135 as well as Tmem135 overexpressing cells, indicating that TMEM135 is involved in the regulation of mitochondrial dynamics. Additionally, mutant retina showed higher sensitivity to oxidative stress. These results suggest that the regulation of mitochondrial dynamics through TMEM135 is critical for protection from environmental stress and controlling the progression of retinal aging. Our study identified TMEM135 as a critical link between aging and age-dependent diseases.
Literature context: (000664, RRID:IMSR_JAX:000664) were from
Toll-like receptor 2 (TLR2) is a pattern recognition receptor that recognizes many types of PAMPs that originate from gram-positive bacteria. Here we describe a novel mechanism regulating TLR2 protein expression and subsequent cytokine release through the ubiquitination and degradation of the receptor in response to ligand stimulation. We show a new mechanism in which an uncharacterized RING finger E3 ligase, PPP1R11, directly ubiquitinates TLR2 both in vitro and in vivo, which leads to TLR2 degradation and disruption of the signaling cascade. Lentiviral gene transfer or knockdown of PPP1R11 in mouse lungs significantly affects lung inflammation and the clearance of Staphylococcus aureus. There is a negative correlation between PPP1R11 and TLR2 levels in white blood cell samples isolated from patients with Staphylococcus aureus infections. These results suggest that PPP1R11 plays an important role in regulating innate immunity and gram-positive bacterial clearance by functioning, in part, through the ubiquitination and degradation of TLR2.
Literature context: oratories RRID:IMSR_JAX:000664 BMT WT and
In obesity, macrophages and other immune cells accumulate in insulin target tissues, promoting a chronic inflammatory state and insulin resistance. Galectin-3 (Gal3), a lectin mainly secreted by macrophages, is elevated in both obese subjects and mice. Administration of Gal3 to mice causes insulin resistance and glucose intolerance, whereas inhibition of Gal3, through either genetic or pharmacologic loss of function, improved insulin sensitivity in obese mice. In vitro treatment with Gal3 directly enhanced macrophage chemotaxis, reduced insulin-stimulated glucose uptake in myocytes and 3T3-L1 adipocytes and impaired insulin-mediated suppression of glucose output in primary mouse hepatocytes. Importantly, we found that Gal3 can bind directly to the insulin receptor (IR) and inhibit downstream IR signaling. These observations elucidate a novel role for Gal3 in hepatocyte, adipocyte, and myocyte insulin resistance, suggesting that Gal3 can link inflammation to decreased insulin sensitivity. Inhibition of Gal3 could be a new approach to treat insulin resistance.
Literature context: rN/AC57BL6/JJackson Laboratories000664Mx1-CreJackson Laboratories00355
Stem cells determine homeostasis and repair of many tissues and are increasingly recognized as functionally heterogeneous. To define the extent of-and molecular basis for-heterogeneity, we overlaid functional, transcriptional, and epigenetic attributes of hematopoietic stem cells (HSCs) at a clonal level using endogenous fluorescent tagging. Endogenous HSC had clone-specific functional attributes over time in vivo. The intra-clonal behaviors were highly stereotypic, conserved under the stress of transplantation, inflammation, and genotoxic injury, and associated with distinctive transcriptional, DNA methylation, and chromatin accessibility patterns. Further, HSC function corresponded to epigenetic configuration but not always to transcriptional state. Therefore, hematopoiesis under homeostatic and stress conditions represents the integrated action of highly heterogeneous clones of HSC with epigenetically scripted behaviors. This high degree of epigenetically driven cell autonomy among HSCs implies that refinement of the concepts of stem cell plasticity and of the stem cell niche is warranted.
Literature context: , males); RRID:IMSR_JAX:000664.
The cerebral cortex of the mouse has become one of the most important systems for studying information processing and the neural correlates of behavior. Multiple studies have examined the first stages of visual cortical processing: primary visual cortex (V1) and its thalamic inputs from the dorsal lateral geniculate nucleus (dLGN), but more rarely in the lateral posterior nucleus (LP) in mice. Multiple single-unit surveys of dLGN and V1, both with electrophysiology and two-photon calcium imaging, have described receptive fields in anesthetized animals. Increasingly, awake animals are being used in physiological studies, so it is important to compare neuronal responses between awake and anesthetized state. We have performed a comprehensive survey of spatial and temporal response properties in V1, dLGN, and lateral posterior nucleus of both anesthetized and awake animals, using a common set of stimuli: drifting sine-wave gratings spanning a broad range of spatial and temporal parameters, and sparse noise stimuli consisting of flashed light and dark squares. Most qualitative receptive field parameters were found to be unchanged between the two states, such as most aspects of spatial processing, but there were significant differences in several parameters, most notably in temporal processing. Compared with anesthetized animals, the temporal frequency that evoked the peak response was shifted toward higher values in the dLGN of awake mice and responses were more sustained. Further, the peak response to a flashed stimulus was earlier in all three areas. Overall, however, receptive field properties in the anesthetized animal remain a good model for those in the awake animal. SIGNIFICANCE STATEMENT: The primary visual cortex (V1) of the mouse and its inputs from visual thalamus (dLGN), have become a dominant model for studying information processing in the brain. Early surveys of visual response properties (receptive fields) were performed in anesthetized animals. Although most recent studies of V1 have been performed in awake animals to examine links between vision and behavior, there have been few comprehensive studies of receptive field properties in the awake mouse, especially in dLGN and lateral posterior nucleus. We have performed a comparative survey of receptive fields in dLGN, lateral posterior nucleus, and V1 in anesthetized and awake mice. We found multiple differences in processing of time-varying stimuli, whereas the spatial aspects of receptive fields remain comparatively unchanged.
Literature context: ckground (RRID:IMSR_JAX:000664) of either
Misfolded α-synuclein is a key factor in the pathogenesis of Parkinson's disease (PD). However, knowledge about a physiological role for the native, unfolded α-synuclein is limited. Using brains of mice lacking α-, β-, and γ-synuclein, we report that extracellular monomeric α-synuclein enters neurons and localizes to mitochondria, interacts with ATP synthase subunit α, and modulates ATP synthase function. Using a combination of biochemical, live-cell imaging and mitochondrial respiration analysis, we found that brain mitochondria of α-, β-, and γ-synuclein knock-out mice are uncoupled, as characterized by increased mitochondrial respiration and reduced mitochondrial membrane potential. Furthermore, synuclein deficiency results in reduced ATP synthase efficiency and lower ATP levels. Exogenous application of low unfolded α-synuclein concentrations is able to increase the ATP synthase activity that rescues the mitochondrial phenotypes observed in synuclein deficiency. Overall, the data suggest that α-synuclein is a previously unrecognized physiological regulator of mitochondrial bioenergetics through its ability to interact with ATP synthase and increase its efficiency. This may be of particular importance in times of stress or PD mutations leading to energy depletion and neuronal cell toxicity. SIGNIFICANCE STATEMENT: Misfolded α-synuclein aggregations in the form of Lewy bodies have been shown to be a pathological hallmark in histological staining of Parkinson's disease (PD) patient brains. It is known that misfolded α-synuclein is a key driver in PD pathogenesis, but the physiological role of unfolded monomeric α-synuclein remains unclear. Using neuronal cocultures and isolated brain mitochondria of α-, β-, and γ-synuclein knock-out mice and monomeric α-synuclein, this current study shows that α-synuclein in its unfolded monomeric form improves ATP synthase efficiency and mitochondrial function. The ability of monomeric α-synuclein to enhance ATP synthase efficiency under physiological conditions may be of importance when α-synuclein undergoes the misfolding and aggregation reported in PD.
Literature context: # 000664; RRID:IMSR_JAX:000664 Recombinan
Antibody blockade of the inhibitory CTLA-4 pathway has led to clinical benefit in a subset of patients with metastatic melanoma. Anti-CTLA-4 enhances T cell responses, including production of IFN-γ, which is a critical cytokine for host immune responses. However, the role of IFN-γ signaling in tumor cells in the setting of anti-CTLA-4 therapy remains unknown. Here, we demonstrate that patients identified as non-responders to anti-CTLA-4 (ipilimumab) have tumors with genomic defects in IFN-γ pathway genes. Furthermore, mice bearing melanoma tumors with knockdown of IFN-γ receptor 1 (IFNGR1) have impaired tumor rejection upon anti-CTLA-4 therapy. These data highlight that loss of the IFN-γ signaling pathway is associated with primary resistance to anti-CTLA-4 therapy. Our findings demonstrate the importance of tumor genomic data, especially IFN-γ related genes, as prognostic information for patients selected to receive treatment with immune checkpoint therapy.
Literature context: mental Models: Organisms/StrainsMouse: DIO: C57bl6jJackson LaboratoriesN/AMouse: Ld
Glucagon and thyroid hormone (T3) exhibit therapeutic potential for metabolic disease but also exhibit undesired effects. We achieved synergistic effects of these two hormones and mitigation of their adverse effects by engineering chemical conjugates enabling delivery of both activities within one precisely targeted molecule. Coordinated glucagon and T3 actions synergize to correct hyperlipidemia, steatohepatitis, atherosclerosis, glucose intolerance, and obesity in metabolically compromised mice. We demonstrate that each hormonal constituent mutually enriches cellular processes in hepatocytes and adipocytes via enhanced hepatic cholesterol metabolism and white fat browning. Synchronized signaling driven by glucagon and T3 reciprocally minimizes the inherent harmful effects of each hormone. Liver-directed T3 action offsets the diabetogenic liability of glucagon, and glucagon-mediated delivery spares the cardiovascular system from adverse T3 action. Our findings support the therapeutic utility of integrating these hormones into a single molecular entity that offers unique potential for treatment of obesity, type 2 diabetes, and cardiovascular disease.
Literature context: aboratory RRID:IMSR_JAX:000664 Mouse: Atm
Heritability and genome stability are shaped by meiotic recombination, which is initiated via hundreds of DNA double-strand breaks (DSBs). The distribution of DSBs throughout the genome is not random, but mechanisms molding this landscape remain poorly understood. Here, we exploit genome-wide maps of mouse DSBs at unprecedented nucleotide resolution to uncover previously invisible spatial features of recombination. At fine scale, we reveal a stereotyped hotspot structure-DSBs occur within narrow zones between methylated nucleosomes-and identify relationships between SPO11, chromatin, and the histone methyltransferase PRDM9. At large scale, DSB formation is suppressed on non-homologous portions of the sex chromosomes via the DSB-responsive kinase ATM, which also shapes the autosomal DSB landscape at multiple size scales. We also provide a genome-wide analysis of exonucleolytic DSB resection lengths and elucidate spatial relationships between DSBs and recombination products. Our results paint a comprehensive picture of features governing successive steps in mammalian meiotic recombination.
Literature context: use: C57BL/6Jackson LaboratoriesCat# 000664Mouse: CBySmn.CB17-Prkdc SCID/JJ
Monkeypox (MPXV) and cowpox (CPXV) are emerging agents that cause severe human infections on an intermittent basis, and variola virus (VARV) has potential for use as an agent of bioterror. Vaccinia immune globulin (VIG) has been used therapeutically to treat severe orthopoxvirus infections but is in short supply. We generated a large panel of orthopoxvirus-specific human monoclonal antibodies (Abs) from immune subjects to investigate the molecular basis of broadly neutralizing antibody responses for diverse orthopoxviruses. Detailed analysis revealed the principal neutralizing antibody specificities that are cross-reactive for VACV, CPXV, MPXV, and VARV and that are determinants of protection in murine challenge models. Optimal protection following respiratory or systemic infection required a mixture of Abs that targeted several membrane proteins, including proteins on enveloped and mature virion forms of virus. This work reveals orthopoxvirus targets for human Abs that mediate cross-protective immunity and identifies new candidate Ab therapeutic mixtures to replace VIG.
Literature context: #000664, RRID:IMSR_JAX:000664) mice. All
Many animals display morning and evening bimodal activities in the day/night cycle. However, little is known regarding the potential components involved in the regulation of bimodal behavioral rhythms in mammals. Here, we identified that the zinc finger protein gene Zbtb20 plays a crucial role in the regulation of bimodal activities in mice. Depletion of Zbtb20 in nerve system resulted in the loss of early evening activity, but the increase of morning activity. We found that Zbtb20-deficient mice exhibited a pronounced decrease in the expression of Prokr2 and resembled phenotypes of Prok2 and Prokr2-knockout mice. Injection of adeno-associated virus-double-floxed Prokr2 in suprachiasmatic nucleus could partly restore evening activity in Nestin-Cre; Zbtb20fl/fl (NS-ZB20KO) mice. Furthermore, loss of Zbtb20 in Foxg1 loci, but intact in the suprachiasmatic nucleus, was not responsible for the unimodal activity of NS-ZB20KO mice. Our study provides evidence that ZBTB20-mediated PROKR2 signaling is critical for the evening behavioral rhythms.
Literature context: oratories RRID:IMSR_JAX:000664 Mouse: GLH
A vaccine that elicits broadly neutralizing antibodies (bNAbs) against HIV-1 is likely to be protective, but this has not been achieved. To explore immunization regimens that might elicit bNAbs, we produced and immunized mice expressing the predicted germline PGT121, a bNAb specific for the V3-loop and surrounding glycans on the HIV-1 spike. Priming with an epitope-modified immunogen designed to activate germline antibody-expressing B cells, followed by ELISA-guided boosting with a sequence of directional immunogens, native-like trimers with decreasing epitope modification, elicited heterologous tier-2-neutralizing responses. In contrast, repeated immunization with the priming immunogen did not. Antibody cloning confirmed elicitation of high levels of somatic mutation and tier-2-neutralizing antibodies resembling the authentic human bNAb. Our data establish that sequential immunization with specifically designed immunogens can induce high levels of somatic mutation and shepherd antibody maturation to produce bNAbs from their inferred germline precursors.
Literature context: ainsMouse: C57BL/6JCharles RiverJAX: 000664Mouse: Lgr5-EGFP-Ires-CreERT2Jac
The murine epidermis with its hair follicles represents an invaluable model system for tissue regeneration and stem cell research. Here we used single-cell RNA-sequencing to reveal how cellular heterogeneity of murine telogen epidermis is tuned at the transcriptional level. Unbiased clustering of 1,422 single-cell transcriptomes revealed 25 distinct populations of interfollicular and follicular epidermal cells. Our data allowed the reconstruction of gene expression programs during epidermal differentiation and along the proximal-distal axis of the hair follicle at unprecedented resolution. Moreover, transcriptional heterogeneity of the epidermis can essentially be explained along these two axes, and we show that heterogeneity in stem cell compartments generally reflects this model: stem cell populations are segregated by spatial signatures but share a common basal-epidermal gene module. This study provides an unbiased and systematic view of transcriptional organization of adult epidermis and highlights how cellular heterogeneity can be orchestrated in vivo to assure tissue homeostasis.
Literature context: e strain (RRID:IMSR_JAX:000664, Jackson L
BACKGROUND: Epidemiological and animal studies have reported that prenatal nicotine exposure (PNE) leads to obesity and type-2 diabetes in offspring. Central leptin-melanocortin signaling via hypothalamic arcuate proopiomelanocortin (POMC) neurons is crucial for the regulation of energy and glucose balance. Furthermore, hypothalamic POMC neurons were recently found to mediate the anorectic effects of nicotine through activation of acetylcholine receptors. Here, we hypothesized that PNE impairs leptin-melanocortinergic regulation of energy balance in first-generation offspring by altering expression of long non-coding RNAs (lncRNAs) putatively regulating development and/or function of hypothalamic POMC neurons. METHODS: C57BL/6J females were exposed ad libitum to nicotine through drinking water and crossed with C57BL/6J males. Nicotine exposure was sustained during pregnancy and discontinued at parturition. Offspring development was monitored from birth into adulthood. From the age of 8 weeks, central leptin-melanocortin signaling, diabetes, and obesity susceptibility were assessed in male offspring fed a low-fat or high-fat diet for 16 weeks. Nicotine-exposed and non-exposed C57BL/6J females were also crossed with C57BL/6J males expressing the enhanced green fluorescent protein specifically in POMC neurons. Transgenic male offspring were subjected to laser microdissections and RNA sequencing (RNA-seq) analysis of POMC neurons for determination of nicotine-induced gene expression changes and regulatory lncRNA/protein-coding gene interactions. RESULTS: Contrary to expectation based on previous studies, PNE did not impair but rather enhanced leptin-melanocortinergic regulation of energy and glucose balance via POMC neurons in offspring. RNA-seq of laser microdissected POMC neurons revealed only one consistent change, upregulation of Gm15851, a lncRNA of yet unidentified function, in nicotine-exposed offspring. RNA-seq further suggested 82 cis-regulatory lncRNA/protein-coding gene interactions, 19 of which involved coding genes regulating neural development and/or function, and revealed expression of several previously unidentified metabolic, neuroendocrine, and neurodevelopment pathways in POMC neurons. CONCLUSIONS: PNE does not result in obesity and type 2 diabetes but instead enhances leptin-melanocortinergic feeding and body weight regulation via POMC neurons in adult offspring. PNE leads to selective upregulation of Gm15851, a lncRNA, in adult offspring POMC neurons. POMC neurons express several lncRNAs and pathways possibly regulating POMC neuronal development and/or function.
Literature context: Wildtype (RRID:IMSR_JAX:000664) and rd10
In retinal degenerative disease (RD), the diminished light signal from dying photoreceptors has been considered the sole cause of visual impairment. Recent studies show a 10-fold increase in spontaneous activity in the RD network, challenging this paradigm. This aberrant activity forms a new barrier for the light signal, and not only exacerbates the loss of vision, but also may stand in the way of visual restoration. This activity originates in AII amacrine cells and relies on excessive activation of gap junctions. However, it remains unclear whether aberrant activity affects central visual processing and what mechanisms lead to this excessive activation of gap junctions. By combining genetic manipulation with electrophysiological recordings of light-induced activity in both living mice and isolated wholemount retina, we demonstrate that aberrant activity extends along retinotectal projections to alter activity in higher brain centers. Next, to selectively eliminate Cx36-containing gap junctions, which are the primary type expressed by AII amacrine cells, we crossed rd10 mice, a slow-degenerating model of RD, with Cx36 knockout mice. We found that retinal aberrant activity was reduced in the rd10/Cx36KO mice compared to rd10 controls, a direct evidence for involvement of Cx36-containing gap junctions in generating aberrant activity in RD. These data provide an essential support for future experiments to determine if selectively targeting these gap junctions could be a valid strategy for reducing aberrant activity and restoring light responses in RD.
Literature context: /6J (RRID:IMSR_JAX:000664; B6), DBA/
Identifying sex differences in gene expression within the brain is critical for determining why multiple neurological and behavioral disorders differentially affect males and females. Several disorders are more common or severe in males (e.g., autism and schizophrenia) or in females (e.g., Alzheimer's disease and depression). We analyzed transcriptomic data from the mouse hippocampus of six inbred strains (129S1/SvImJ, A/J, C57BL/6J, DBA/1J, DBA/2J, and PWD/Ph) to provide a perspective on differences between male and female gene expression. Our data show that 1) gene expression differences in males vs. females varies substantially across the strains, 2) only a few genes are differentially expressed across all of the strains (termed core genes), and 3) >2,600 genes differ in the individual strain comparisons (termed noncore genes). We found that DBA/2J uniquely has a substantial majority (89%) of differentially expressed genes (DEGs) that are more highly expressed in females than in males (female-biased); 129/SvImJ has a majority (69%) of DEGs that are more highly expressed in males. To gain insight into the function of the DEGs, we examined gene ontology and pathway and phenotype enrichment and found significant enrichment in phenotypes related to abnormal nervous system morphology and physiology, among others. In addition, several pathways are enriched significantly, including Alzheimer's disease (AD), with 32 genes implicated in AD, eight of which are male-biased. Three of the male-biased genes have been implicated in a neuroprotective role in AD. Our transcriptomic data provide new insight into the possible genetic bases for sex-specific susceptibility and severity of brain disorders. J. Comp. Neurol. 524:2696-2710, 2016. © 2016 Wiley Periodicals, Inc.
Literature context: nsC57BL/6JThe Jackson Laboratory000664Oxtr-cre ON82Laboratory of Natha
The frequency of human social and emotional disorders varies significantly between males and females. We have recently reported that oxytocin receptor interneurons (OxtrINs) modulate female sociosexual behavior. Here, we show that, in male mice, OxtrINs regulate anxiety-related behaviors. We demonstrate that corticotropin-releasing-hormone-binding protein (CRHBP), an antagonist of the stress hormone CRH, is specifically expressed in OxtrINs. Production of CRHBP blocks the CRH-induced potentiation of postsynaptic layer 2/3 pyramidal cell activity of male, but not female, mice, thus producing an anxiolytic effect. Our data identify OxtrINs as critical for modulation of social and emotional behaviors in both females and males and reveal a molecular mechanism that acts on local medial prefrontal cortex (mPFC) circuits to coordinate responses to OXT and CRH. They suggest that additional studies of the impact of the OXT/OXTR and CRHBP/CRH pathways in males and females will be important in development of gender-specific therapies.
Literature context: oratories RRID:IMSR_JAX:000664 Mouse: B6;
Mammalian DNA methylation is a critical epigenetic mechanism orchestrating gene expression networks in many biological processes. However, investigation of the functions of specific methylation events remains challenging. Here, we demonstrate that fusion of Tet1 or Dnmt3a with a catalytically inactive Cas9 (dCas9) enables targeted DNA methylation editing. Targeting of the dCas9-Tet1 or -Dnmt3a fusion protein to methylated or unmethylated promoter sequences caused activation or silencing, respectively, of an endogenous reporter. Targeted demethylation of the BDNF promoter IV or the MyoD distal enhancer by dCas9-Tet1 induced BDNF expression in post-mitotic neurons or activated MyoD facilitating reprogramming of fibroblasts into myoblasts, respectively. Targeted de novo methylation of a CTCF loop anchor site by dCas9-Dnmt3a blocked CTCF binding and interfered with DNA looping, causing altered gene expression in the neighboring loop. Finally, we show that these tools can edit DNA methylation in mice, demonstrating their wide utility for functional studies of epigenetic regulation.
Literature context: /6J mice (RRID:IMSR_JAX:000664), B6.Cg-Le
Alternative pre-mRNA splicing expands the complexity of the transcriptome and controls isoform-specific gene expression. Whether alternative splicing contributes to metabolic regulation is largely unknown. Here we investigated the contribution of alternative splicing to the development of diet-induced obesity. We found that obesity-induced changes in adipocyte gene expression include alternative pre-mRNA splicing. Bioinformatics analysis associated part of this alternative splicing program with sequence specific NOVA splicing factors. This conclusion was confirmed by studies of mice with NOVA deficiency in adipocytes. Phenotypic analysis of the NOVA-deficient mice demonstrated increased adipose tissue thermogenesis and improved glycemia. We show that NOVA proteins mediate a splicing program that suppresses adipose tissue thermogenesis. Together, these data provide quantitative analysis of gene expression at exon-level resolution in obesity and identify a novel mechanism that contributes to the regulation of adipose tissue function and the maintenance of normal glycemia.
Literature context: t#000664; RRID:IMSR_JAX:000664 PMEL Jacks
Reversing the dysfunctional T cell state that arises in cancer and chronic viral infections is the focus of therapeutic interventions; however, current therapies are effective in only some patients and some tumor types. To gain a deeper molecular understanding of the dysfunctional T cell state, we analyzed population and single-cell RNA profiles of CD8(+) tumor-infiltrating lymphocytes (TILs) and used genetic perturbations to identify a distinct gene module for T cell dysfunction that can be uncoupled from T cell activation. This distinct dysfunction module is downstream of intracellular metallothioneins that regulate zinc metabolism and can be identified at single-cell resolution. We further identify Gata-3, a zinc-finger transcription factor in the dysfunctional module, as a regulator of dysfunction, and we use CRISPR-Cas9 genome editing to show that it drives a dysfunctional phenotype in CD8(+) TILs. Our results open novel avenues for targeting dysfunctional T cell states while leaving activation programs intact.
Literature context: C57BL/6 (RRID:IMSR_JAX:000664), as well
While more than 70 genes have been linked to deafness, most of which are expressed in mechanosensory hair cells of the inner ear, a challenge has been to link these genes into molecular pathways. One example is Myo7a (myosin VIIA), in which deafness mutations affect the development and function of the mechanically sensitive stereocilia of hair cells. We describe here a procedure for the isolation of low-abundance protein complexes from stereocilia membrane fractions. Using this procedure, combined with identification and quantitation of proteins with mass spectrometry, we demonstrate that MYO7A forms a complex with PDZD7, a paralog of USH1C and DFNB31. MYO7A and PDZD7 interact in tissue-culture cells, and co-localize to the ankle-link region of stereocilia in wild-type but not Myo7a mutant mice. Our data thus describe a new paradigm for the interrogation of low-abundance protein complexes in hair cell stereocilia and establish an unanticipated link between MYO7A and PDZD7.
Literature context: oratories RRID:IMSR_JAX:000664 CD200 KO m
Cellular compartments that cannot be biochemically isolated are challenging to characterize. Here we demonstrate the proteomic characterization of the synaptic clefts that exist at both excitatory and inhibitory synapses. Normal brain function relies on the careful balance of these opposing neural connections, and understanding how this balance is achieved relies on knowledge of their protein compositions. Using a spatially restricted enzymatic tagging strategy, we mapped the proteomes of two of the most common excitatory and inhibitory synaptic clefts in living neurons. These proteomes reveal dozens of synaptic candidates and assign numerous known synaptic proteins to a specific cleft type. The molecular differentiation of each cleft allowed us to identify Mdga2 as a potential specificity factor influencing Neuroligin-2's recruitment of presynaptic neurotransmitters at inhibitory synapses.
Literature context: gy.Wild-type male C57BL/6 mice (RRID:IMSR_JAX:000664) 8 weeks of age were purchased
Over the last decade, striatal cholinergic interneurons (ChIs) have reemerged as key actors in the pathophysiology of basal-ganglia-related movement disorders. However, the mechanisms involved are still unclear. In this study, we address the role of ChI activity in the expression of parkinsonian-like motor deficits in a unilateral nigrostriatal 6-hydroxydopamine (6-OHDA) lesion model using optogenetic and pharmacological approaches. Dorsal striatal photoinhibition of ChIs in lesioned ChAT(cre/cre) mice expressing halorhodopsin in ChIs reduces akinesia, bradykinesia, and sensorimotor neglect. Muscarinic acetylcholine receptor (mAChR) blockade by scopolamine produces similar anti-parkinsonian effects. To decipher which of the mAChR subtypes provides these beneficial effects, systemic and intrastriatal administration of the selective M1 and M4 mAChR antagonists telenzepine and tropicamide, respectively, were tested in the same model of Parkinson's disease. The two compounds alleviate 6-OHDA lesion-induced motor deficits. Telenzepine produces its beneficial effects by blocking postsynaptic M1 mAChRs expressed on medium spiny neurons (MSNs) at the origin of the indirect striatopallidal and direct striatonigral pathways. The anti-parkinsonian effects of tropicamide were almost completely abolished in mutant lesioned mice that lack M4 mAChRs specifically in dopamine D1-receptor-expressing neurons, suggesting that postsynaptic M4 mAChRs expressed on direct MSNs mediate the antiakinetic action of tropicamide. The present results show that altered cholinergic transmission via M1 and M4 mAChRs of the dorsal striatum plays a pivotal role in the occurrence of motor symptoms in Parkinson's disease. SIGNIFICANCE STATEMENT: The striatum, where dopaminergic and cholinergic systems interact, is the pivotal structure of basal ganglia involved in pathophysiological changes underlying Parkinson's disease. Here, using optogenetic and pharmacological approaches, we investigated the involvement of striatal cholinergic interneurons (ChIs) and muscarinic receptor subtypes (mAChRs) in the occurrence of a wide range of motor deficits such as akinesia, bradykinesia, motor coordination, and sensorimotor neglect after unilateral nigrostriatal 6-hydroxydopamine lesion in mice. Our results show that photoinhibition of ChIs in the dorsal striatum and pharmacological blockade of muscarinic receptors, specifically postsynaptic M1 and M4 mAChRs, alleviate lesion-induced motor deficits. The present study points to these receptor subtypes as potential targets for the symptomatic treatment of parkinsonian-like motor symptoms.
Literature context: C57Bl/6J (RRID:IMSR_JAX:000664) male (nâ€‰=
The posterodorsal aspect of the medial amygdala (MePD) in rats is sexually dimorphic, being larger and containing more and larger neurons in males than in females. It is also highly lateralized, with the right MePD larger than the left in both sexes, but with the smaller left MePD actually containing more and larger neurons than the larger right. Astrocytes are also strikingly sexually differentiated, with male-biased numbers and lateralized favoring the right in the rat MePD. However, comparable information is scant for mice where genetic tools offer greater experimental power. Hence, we examined the MePD from adult male and female C57Bl/6(J) mice. We now report that the MePD is larger in males than in females, with the MePD in males containing more astrocytes and neurons than in females. However, we did not find sex differences in astrocyte complexity or overall glial number nor effects of laterality in either measure. While the mouse MePD is generally less lateralized than in rats, we did find that the sex difference in astrocyte number is only on the right because of a significant lateralization in females, with significantly fewer astrocytes on the right than the left but only in females. A sex difference in neuronal soma size favoring males was also evident, but only on the left. Sex differences in the number of neurons and astrocytes common to both rodent species may represent core morphological features that critically underlie the expression of sex-specific behaviors that depend on the MePD. J. Comp. Neurol. 524:2492-2502, 2016. © 2016 Wiley Periodicals, Inc.
Literature context: /6J mice (RRID:IMSR_JAX:000664), B6.SJL-P
Arterial occlusive diseases are major causes of morbidity and mortality. Blood flow to the affected tissue must be restored quickly if viability and function are to be preserved. We report that disruption of the mixed-lineage protein kinase (MLK) - cJun NH2-terminal kinase (JNK) signaling pathway in endothelial cells causes severe blockade of blood flow and failure to recover in the murine femoral artery ligation model of hindlimb ischemia. We show that the MLK-JNK pathway is required for the formation of native collateral arteries that can restore circulation following arterial occlusion. Disruption of the MLK-JNK pathway causes decreased Dll4/Notch signaling, excessive sprouting angiogenesis, and defects in developmental vascular morphogenesis. Our analysis demonstrates that the MLK-JNK signaling pathway is a key regulatory mechanism that protects against ischemia in arterial occlusive disease.
Literature context: ild-type (RRID:IMSR_JAX:000664; n = 12) a
Mapping specific sensory features to future motor actions is a crucial capability of mammalian nervous systems. We investigated the role of visual (V1), posterior parietal (PPC), and frontal motor (fMC) cortices for sensorimotor mapping in mice during performance of a memory-guided visual discrimination task. Large-scale calcium imaging revealed that V1, PPC, and fMC neurons exhibited heterogeneous responses spanning all task epochs (stimulus, delay, response). Population analyses demonstrated unique encoding of stimulus identity and behavioral choice information across regions, with V1 encoding stimulus, fMC encoding choice even early in the trial, and PPC multiplexing the two variables. Optogenetic inhibition during behavior revealed that all regions were necessary during the stimulus epoch, but only fMC was required during the delay and response epochs. Stimulus identity can thus be rapidly transformed into behavioral choice, requiring V1, PPC, and fMC during the transformation period, but only fMC for maintaining the choice in memory prior to execution.
Literature context: J male mice (2â€“3 months of age; IMSR_JAX:000664) were obtained from Jackson lab
Brief periods of sleep loss have long-lasting consequences such as impaired memory consolidation. Structural changes in synaptic connectivity have been proposed as a substrate of memory storage. Here, we examine the impact of brief periods of sleep deprivation on dendritic structure. In mice, we find that five hours of sleep deprivation decreases dendritic spine numbers selectively in hippocampal area CA1 and increased activity of the filamentous actin severing protein cofilin. Recovery sleep normalizes these structural alterations. Suppression of cofilin function prevents spine loss, deficits in hippocampal synaptic plasticity, and impairments in long-term memory caused by sleep deprivation. The elevated cofilin activity is caused by cAMP-degrading phosphodiesterase-4A5 (PDE4A5), which hampers cAMP-PKA-LIMK signaling. Attenuating PDE4A5 function prevents changes in cAMP-PKA-LIMK-cofilin signaling and cognitive deficits associated with sleep deprivation. Our work demonstrates the necessity of an intact cAMP-PDE4-PKA-LIMK-cofilin activation-signaling pathway for sleep deprivation-induced memory disruption and reduction in hippocampal spine density.
Literature context: C56Bl/6J (RRID:IMSR_JAX:000664), PV-IRES-
Stimulus processing in fear conditioning is constrained by parvalbumin interneurons (PV-INs) through inhibition of principal excitatory neurons. However, the contributions of PV-IN microcircuits to input gating and long-term plasticity in the fear system remain unknown. Here we interrogate synaptic connections between afferent pathways, PV-INs, and principal excitatory neurons in the basolateral amygdala. We find that subnuclei of this region are populated two functionally distinct PV-IN networks. PV-INs in the lateral (LA), but not the basal (BA), amygdala possess complex dendritic arborizations, receive potent excitatory drive, and mediate feedforward inhibition onto principal neurons. After fear conditioning, PV-INs exhibit nucleus- and target-selective plasticity, resulting in persistent reduction of their excitatory input and inhibitory output in LA but not BA. These data reveal previously overlooked specializations of amygdala PV-INs and indicate specific circuit mechanisms for inhibitory plasticity during the encoding of associative fear memories.
Literature context: ted to C57BL/6J (B6, JAX stock #000664) mice to generate both B6.APBTg
Studies have assessed individual components of a western diet, but no study has assessed the long-term, cumulative effects of a western diet on aging and Alzheimer's disease (AD). Therefore, we have formulated the first western-style diet that mimics the fat, carbohydrate, protein, vitamin and mineral levels of western diets. This diet was fed to aging C57BL/6J (B6) mice to identify phenotypes that may increase susceptibility to AD, and to APP/PS1 mice, a mouse model of AD, to determine the effects of the diet in AD. Astrocytosis and microglia/monocyte activation were dramatically increased in response to diet and was further increased in APP/PS1 mice fed the western diet. This increase in glial responses was associated with increased plaque burden in the hippocampus. Interestingly, given recent studies highlighting the importance of TREM2 in microglia/monocytes in AD susceptibility and progression, B6 and APP/PS1 mice fed the western diet showed significant increases TREM2+ microglia/monocytes. Therefore, an increase in TREM2+ microglia/monocytes may underlie the increased risk from a western diet to age-related neurodegenerative diseases such as Alzheimer's disease. This study lays the foundation to fully investigate the impact of a western diet on glial responses in aging and Alzheimer's disease.
Literature context: son Labs; RRID:IMSR_JAX:000664) were used
The ventral hippocampus (vHPC), medial prefrontal cortex (mPFC), and basolateral amygdala (BLA) are each required for the expression of anxiety-like behavior. Yet the role of each individual element of the circuit is unclear. The projection from the vHPC to the mPFC has been implicated in anxiety-related neural synchrony and spatial representations of aversion. The role of this projection was examined using multi-site neural recordings combined with optogenetic terminal inhibition. Inhibition of vHPC input to the mPFC disrupted anxiety and mPFC representations of aversion, and reduced theta synchrony in a pathway-, frequency- and task-specific manner. Moreover, bilateral, but not unilateral, inhibition altered physiological correlates of anxiety in the BLA, mimicking a safety-like state. These results reveal a specific role for the vHPC-mPFC projection in anxiety-related behavior and the spatial representation of aversive information within the mPFC.
Literature context: arrival; RRID:IMSR_JAX:000664; The Jacks
Changes in executive function are at the root of most cognitive problems associated with Parkinson's disease. Because dopaminergic treatment does not necessarily alleviate deficits in executive function, it has been hypothesized that dysfunction of neurotransmitters/systems other than dopamine (DA) may be associated with this decrease in cognitive function. We have reported decreases in motor function and dopaminergic/glutamatergic biomarkers in a progressive 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Parkinson's mouse model. Assessment of executive function and dopaminergic/glutamatergic biomarkers within the limbic circuit has not previously been explored in our model. Our results show progressive behavioral decline in a cued response task (a rodent model for frontal cortex cognitive function) with increasing weekly doses of MPTP. Although within the dorsolateral (DL) striatum mice that had been given MPTP showed a 63% and 83% loss of tyrosine hydroxylase and dopamine transporter expression, respectively, there were no changes in the nucleus accumbens or medial prefrontal cortex (mPFC). Furthermore, dopamine-1 receptor and vesicular glutamate transporter (VGLUT)-1 expression increased in the mPFC following DA loss. There were significant MPTP-induced decreases and increases in VGLUT-1 and VGLUT-2 expression, respectively, within the DL striatum. We propose that the behavioral decline following MPTP treatment may be associated with a change not only in cortical-cortical (VGLUT-1) glutamate function but also in striatal DA and glutamate (VGLUT-1/VGLUT-2) input.
Literature context: . 000664; RRID:IMSR_JAX:000664), and a co
It has been shown that vitamin C (VC) is transported at synaptic boutons, but how this occurs has not been elucidated. This study investigates the role of the sodium-dependent vitamin C transporter-2 (SVCT2) in transporting VC at the cortical nerve terminal. Immunostaining of cultured mouse superior cervical ganglion cells showed the SVCT2 to be expressed in presynaptic boutons, colocalizing with the vesicular monoamine transporter-2 and the norepinephrine transporter. Immunoblotting of enriched cortical synaptosomes demonstrated that the SVCT2 was enriched in presynaptic fractions, confirming a predominantly presynaptic location. In crude synaptosomes, known inhibitors of SVCT2 inhibited uptake of VC. Furthermore, the kinetic features of VC uptake were consistent with SVCT2-mediated function. VC was also found to efflux from synaptosomes by a mechanism not involving the SVCT2. Indeed, VC efflux was substantially offset by reuptake of VC on the SVCT2. The presence and function of the SVCT2 at the presynaptic nerve terminal suggest that it is the transporter responsible for recovery of VC released into the synaptic cleft.
Literature context: BL/6J WT (RRID:IMSR_JAX:000664) controls.
Activation of taste buds triggers the release of several neurotransmitters, including ATP and serotonin (5-hydroxytryptamine; 5-HT). Type III taste cells release 5-HT directly in response to acidic (sour) stimuli and indirectly in response to bitter and sweet tasting stimuli. Although ATP is necessary for activation of nerve fibers for all taste stimuli, the role of 5-HT is unclear. We investigated whether gustatory afferents express functional 5-HT3 receptors and, if so, whether these receptors play a role in transmission of taste information from taste buds to nerves. In mice expressing GFP under the control of the 5-HT(3A) promoter, a subset of cells in the geniculate ganglion and nerve fibers in taste buds are GFP-positive. RT-PCR and in situ hybridization confirmed the presence of 5-HT(3A) mRNA in the geniculate ganglion. Functional studies show that only those geniculate ganglion cells expressing 5-HT3A-driven GFP respond to 10 μM 5-HT and this response is blocked by 1 μM ondansetron, a 5-HT3 antagonist, and mimicked by application of 10 μM m-chlorophenylbiguanide, a 5-HT3 agonist. Pharmacological blockade of 5-HT3 receptors in vivo or genetic deletion of the 5-HT3 receptors reduces taste nerve responses to acids and other taste stimuli compared with controls, but only when urethane was used as the anesthetic. We find that anesthetic levels of pentobarbital reduce taste nerve responses apparently by blocking the 5-HT3 receptors. Our results suggest that 5-HT released from type III cells activates gustatory nerve fibers via 5-HT3 receptors, accounting for a significant proportion of the neural taste response.
Literature context: ckground (RRID:IMSR_JAX:000664) such that
The Kv2 family of voltage-gated potassium channel α subunits, comprising Kv2.1 and Kv2.2, mediate the bulk of the neuronal delayed rectifier K(+) current in many mammalian central neurons. Kv2.1 exhibits robust expression across many neuron types and is unique in its conditional role in modulating intrinsic excitability through changes in its phosphorylation state, which affect Kv2.1 expression, localization, and function. Much less is known of the highly related Kv2.2 subunit, especially in forebrain neurons. Here, through combined use of cortical layer markers and transgenic mouse lines, we show that Kv2.1 and Kv2.2 are localized to functionally distinct cortical cell types. Kv2.1 expression is consistently high throughout all cortical layers, especially in layer (L) 5b pyramidal neurons, whereas Kv2.2 expression is primarily limited to neurons in L2 and L5a. In addition, L4 of primary somatosensory cortex is strikingly devoid of Kv2.2 immunolabeling. The restricted pattern of Kv2.2 expression persists in Kv2.1-KO mice, suggesting distinct cell- and layer-specific functions for these two highly related Kv2 subunits. Analyses of endogenous Kv2.2 in cortical neurons in situ and recombinant Kv2.2 expressed in heterologous cells reveal that Kv2.2 is largely refractory to stimuli that trigger robust, phosphorylation-dependent changes in Kv2.1 clustering and function. Immunocytochemistry and voltage-clamp recordings from outside-out macropatches reveal distinct cellular expression patterns for Kv2.1 and Kv2.2 in intratelencephalic and pyramidal tract neurons of L5, indicating circuit-specific requirements for these Kv2 paralogs. Together, these results support distinct roles for these two Kv2 channel family members in mammalian cortex. SIGNIFICANCE STATEMENT: Neurons within the neocortex are arranged in a laminar architecture and contribute to the input, processing, and/or output of sensory and motor signals in a cell- and layer-specific manner. Neurons of different cortical layers express diverse populations of ion channels and possess distinct intrinsic membrane properties. Here, we show that the Kv2 family members Kv2.1 and Kv2.2 are expressed in distinct cortical layers and pyramidal cell types associated with specific corticostriatal pathways. We find that Kv2.1 and Kv2.2 exhibit distinct responses to acute phosphorylation-dependent regulation in brain neurons in situ and in heterologous cells in vitro. These results identify a molecular mechanism that contributes to heterogeneity in cortical neuron ion channel function and regulation.
Literature context: /6J mice (RRID:IMSR_JAX:000664; 7-8 weeks
MicroRNAs (miRNAs) are modulators of gene expression that play key regulatory roles in distinct cellular processes. Methamphetamine (METH) induces various aberrant changes in the limbic system by affecting a complex gene regulatory mechanism, yet the involvement of miRNAs in the effects of METH exposure remains unclear. This study identifies METH-responsive miRNAs and their potential effects in the nucleus accumbens (NAc) of mice. Using miRNA sequencing, we examined the expression of miRNAs in the NAc of saline- and METH-treated mice and identified 45 known miRNAs to be METH responsive. Additionally, we identified two novel miRNA candidates that were METH responsive (novel-m002C and novel-m009C). Our target prediction analysis suggested that the known METH-regulated miRNAs might target genes that are involved in cellular autophagy, cellular metabolism, and immune responses and that the novel METH-regulated miRNA candidates might target genes that are related to drug addiction. We also matched the predicted targets of METH-regulated miRNAs with the NAc messenger RNA expression profile, revealing eight putative METH-regulated target genes (Arc, Capn9, Gbp5, Lefty1, Patl2, Pde4c, Strc, and Vmn1r58). Thus, METH triggers an alteration in NAc miRNA expression, which could contribute to METH-induced changes in neuron autophagy, metabolism, and immune responses. The differential expression of putative target genes suggests their involvement following exposure to METH.
Literature context: osylation;IMSR_JAX:000664;RRID: IMSR
Nitric oxide (NO) synthesis in the retina is triggered by light stimulation. NO has been shown to modulate visual signal processing at multiple sites in the vertebrate retina, via activation of the most sensitive target of NO signaling, soluble guanylate cyclase. NO can also alter protein structure and function and exert biological effects directly by binding to free thiol groups of cysteine residues in a chemical reaction called S-nitrosylation. However, in the central nervous system, including the retina, this reaction has not been considered to be significant under physiological conditions. Here we provide immunohistochemical evidence for extensive S-nitrosylation that takes place in the goldfish and mouse retinas under physiologically relevant light intensities, in an intensity-dependent manner, with a strikingly similar pattern in both species. Pretreatment with N-ethylmaleimide (NEM), which occludes S-nitrosylation, or with 1-(2-trifluromethylphenyl)imidazole (TRIM), an inhibitor of neuronal NO synthase, eliminated the light-evoked increase in S-nitrosylated protein immunofluorescence (SNI) in the retinas of both species. Similarly, light did not increase SNI, above basal levels, in retinas of transgenic mice lacking neuronal NO synthase. Qualitative analysis of the light-adapted mouse retina with mass spectrometry revealed more than 300 proteins that were S-nitrosylated upon illumination, many of which are known to participate directly in retinal signal processing. Our data strongly suggest that in the retina light-evoked NO production leads to extensive S-nitrosylation and that this process is a significant posttranslational modification affecting a wide range of proteins under physiological conditions.
Literature context: B6) mice (RRID:IMSR_JAX:000664), to reduc
Mice with high numbers of the Cu/Zn superoxide dismutase-1 G93A transgene (SOD1(G93A) G1H) have become the most commonly used animal model to study amyotrophic lateral sclerosis. This study investigated changes in size, numbers, and cell stress/death markers of motor neuron numbers in G1H mice that re-express the common p75 neurotrophin receptor (p75NTR). SOD1(G93A) G1H mice and age-matched C57BL/6J controls at 60, 80, 100, 120 days and end stage/140 days were analyzed for p75NTR, choline acetyltransferase (ChAT), activating transcription factor 3 (ATF3), and cleaved caspase-3. In addition, motor neuron counts and soma sizes were recorded. Motor neurons re-expressing p75NTR in SOD1(G93A) G1H mice were first observed at 80 days, and this continued to 140 days, peaking at 100-120 days at ∼5%. The soma area of motor neurons re-expressing p75NTR was always 600-800 µm(2) , suggesting that these are alpha motor neurons, which was confirmed after examination of somas post injection of a retrogradely transported antibody to p75NTR in 110-day-old SOD1(G93A) G1H mice. In motor neurons not re-expressing p75NTR, the frequency of small soma 200-400 µm2 motor neurons increased, whereas the larger 600-900 µm2 motor neurons decreased with progression, indicating that large motor neurons were dying off and shrinking in the process. There was minimal coexpression of p75NTR with ATF3, a marker for cell stress, but 85% coexpressed the apoptotic marker cleaved caspase-3. These findings indicate that in SOD1(G93A) G1H mice, p75NTR re-expression is detectable from 80 days in a small population of large motor neurons that represent 5% of the total motor neurons. Furthermore, p75NTR re-expression occurs in larger alpha motor neurons that express cleaved caspsase-3 and are destined to die.
Literature context: rbor, ME; RRID:IMSR_JAX:000664) of both s
High-voltage-activated calcium channels are hetero-oligomeric protein complexes that mediate multiple cellular processes, including the influx of extracellular Ca(2+), neurotransmitter release, gene transcription, and synaptic plasticity. These channels consist of a primary α(1) pore-forming subunit, which is associated with an extracellular α(2)δ subunit and an intracellular β auxiliary subunit, which alter the gating properties and trafficking of the calcium channel. The cellular localization of the α(2)δ(3) subunit in the mouse and rat retina is unknown. In this study using RT-PCR, a single band at ∼ 305 bp corresponding to the predicted size of the α(2)δ(3) subunit fragment was found in mouse and rat retina and brain homogenates. Western blotting of rodent retina and brain homogenates showed a single 123-kDa band. Immunohistochemistry with an affinity-purified antibody to the α(2)δ(3) subunit revealed immunoreactive cell bodies in the ganglion cell layer and inner nuclear layer and immunoreactive processes in the inner plexiform layer and the outer plexiform layer. α(2)δ(3) immunoreactivity was localized to multiple cell types, including ganglion, amacrine, and bipolar cells and photoreceptors, but not horizontal cells. The expression of the α(2)δ(3) calcium channel subunit to multiple cell types suggests that this subunit participates widely in Ca-channel-mediated signaling in the retina.
Literature context: ratories; RRID:IMSR_JAX:000664), 2D2 T-ce
Stroke-induced immunodepression (SIDS) is an essential cause of poststroke infections. Pharmacological inhibition of SIDS appears promising in preventing life-threatening infections in stroke patients. However, SIDS might represent an adaptive mechanism preventing autoreactive immune responses after stroke. To address this, we used myelin oligodendrocyte glycoprotein (MOG) T-cell receptor transgenic (2D2) mice where >80% of peripheral CD4(+) T cells express a functional receptor for MOG. We investigated in a murine model of middle cerebral artery occlusion the effect of blocking SIDS by inhibiting body's main stress axes, the sympathetic nervous system (SNS) with propranolol and the hypothalamic-pituitary-adrenal axis (HPA) with mifepristone. Blockade of both stress axes robustly reduced infarct volumes, decreased infection rate, and increased long-term survival of 2D2 and C57BL/6J wild-type mice. Despite these protective effects, blockade of SIDS increased CNS antigen-specific Type1 T helper cell (Th1) responses in the brains of 2D2 mice 14 d after middle cerebral artery occlusion. One month after experimental stroke, 2D2 mice developed signs of polyradiculitis, which were diminished by SIDS blockade. Adoptive transfer of CD4(+) T cells, isolated from 2D2 mice, into lymphocyte-deficient Rag-1KO mice did not reveal differences between SIDS blockade and vehicle treatment in functional long-term outcome after stroke. In conclusion, inhibiting SIDS by pharmacological blockade of body's stress axes increases autoreactive CNS antigen-specific T-cell responses in the brain but does not worsen functional long-term outcome after experimental stroke, even in a mouse model where CNS antigen-specific autoreactive T-cell responses are boosted.
Literature context: rbor, ME; RRID:IMSR_JAX:000664), three Gp
The habenula is a phylogenetically conserved brain structure in the epithalamus. It is a major node in the information flow between fronto-limbic brain regions and monoaminergic brainstem nuclei, and is thus anatomically and functionally ideally positioned to regulate emotional, motivational, and cognitive behaviors. Consequently, the habenula may be critically important in the pathophysiology of psychiatric disorders such as addiction and depression. Here we investigated the expression pattern of GPR151, a G protein-coupled receptor (GPCR), whose mRNA has been identified as highly and specifically enriched in habenular neurons by in situ hybridization and translating ribosome affinity purification (TRAP). In the present immunohistochemical study we demonstrate a pronounced and highly specific expression of the GPR151 protein in the medial and lateral habenula of rodent brain. Specific expression was also seen in efferent habenular fibers projecting to the interpeduncular nucleus, the rostromedial tegmental area, the rhabdoid nucleus, the mesencephalic raphe nuclei, and the dorsal tegmental nucleus. Using confocal microscopy and quantitative colocalization analysis, we found that GPR151-expressing axons and terminals overlap with cholinergic, substance P-ergic, and glutamatergic markers. Virtually identical expression patterns were observed in rat, mouse, and zebrafish brains. Our data demonstrate that GPR151 is highly conserved, specific for a subdivision of the habenular neurocircuitry, and constitutes a promising novel target for psychiatric drug development.
Literature context: 8127Original brand not specifiedC57/BL6J (H-2b)Mouse lineJackson LaboratoryStra
The Kang et al. (2011), published in Nature in 2011. The experiments that will be replicated are those reported in Figures 3B, 3C, 3E, and 4A. In these experiments, Kang et al. (2011) demonstrate the phenomenon of oncogene-induced cellular senescence and immune-mediated clearance of senescent cells after intrahepatic injection of NRAS (Figures 2I, 3B, 3C, and 3E). Additionally, Kang et al. (2011) show the specific necessity of CD4+ T cells for immunoclearance of senescent cells (Figure 4A). The Reproducibility Project: Cancer Biology is a collaboration between the eLife.
Literature context: son Labs, RRID:IMSR_JAX:000664), were use
Neocortical interactions with the dorsal striatum support many motor and executive functions, and such underlying functional networks are particularly vulnerable to a variety of developmental, neurological, and psychiatric brain disorders, including autism spectrum disorders, Parkinson's disease, and Huntington's disease. Relatively little is known about the development of functional corticostriatal interactions, and in particular, virtually nothing is known of the molecular mechanisms that control generation of prefrontal cortex-striatal circuits. Here, we used regional and cellular in situ hybridization techniques coupled with neuronal tract tracing to show that Cadherin-8 (Cdh8), a homophilic adhesion protein encoded by a gene associated with autism spectrum disorders and learning disability susceptibility, is enriched within striatal projection neurons in the medial prefrontal cortex and in striatal medium spiny neurons forming the direct or indirect pathways. Developmental analysis of quantitative real-time polymerase chain reaction and western blot data show that Cdh8 expression peaks in the prefrontal cortex and striatum at P10, when cortical projections start to form synapses in the striatum. High-resolution immunoelectron microscopy shows that Cdh8 is concentrated at excitatory synapses in the dorsal striatum, and Cdh8 knockdown in cortical neurons impairs dendritic arborization and dendrite self-avoidance. Taken together, our findings indicate that Cdh8 delineates developing corticostriatal circuits where it is a strong candidate for regulating the generation of normal cortical projections, neuronal morphology, and corticostriatal synapses.
Literature context: 6JOlaHsd (RRID:IMSR_JAX:000664) 8- to 10-
The medial septum (MS) is required for theta rhythmic oscillations and grid cell firing in the medial entorhinal cortex (MEC). While GABAergic, glutamatergic, and cholinergic neurons project from the MS to the MEC, their synaptic targets are unknown. To investigate whether MS neurons innervate specific layers and cell types in the MEC, we expressed channelrhodopsin-2 in mouse MS neurons and used patch-clamp recording in brain slices to determine the response to light activation of identified cells in the MEC. Following activation of MS axons, we observed fast monosynaptic GABAergic IPSPs in the majority (>60%) of fast-spiking (FS) and low-threshold-spiking (LTS) interneurons in all layers of the MEC, but in only 1.5% of nonstellate principal cells (NSPCs) and in no stellate cells. We also observed fast glutamatergic responses to MS activation in a minority (<5%) of NSPCs, FS, and LTS interneurons. During stimulation of MS inputs at theta frequency (10 Hz), the amplitude of GABAergic IPSPs was maintained, and spike output from LTS and FS interneurons was entrained at low (25-60 Hz) and high (60-180 Hz) gamma frequencies, respectively. By demonstrating cell type-specific targeting of the GABAergic projection from the MS to the MEC, our results support the idea that the MS controls theta frequency activity in the MEC through coordination of inhibitory circuits.
Literature context: Wild-type (WT; C57BL/6J) mice, rd10 mice, and Cx3cr1GFP
Retinitis pigmentosa (RP) is a photoreceptor-degenerative disease caused by various mutations and is characterized by death of rod photoreceptor cell followed by gradual death of cone photoreceptors. The molecular mechanisms that lead to rod and cone death are not yet fully understood. Neuroinflammation contributes to the progression of many chronic neurodegenerative disorders. However, it remains to be determined how microglia contribute to photoreceptor disruption in RP. In this study, we explored the role of microglia as a contributor to photoreceptor degeneration in the rd10 mouse model of RP. First, we demonstrated that microglia activation was an early alteration in RP retinas. Inhibition of microglia activation by minocycline reduced photoreceptor apoptosis and significantly improved retinal structure and function and visual behavior in rd10 mice. Second, we identified that minocycline exerted its neuroprotective effects through both anti-inflammatory and anti-apoptotic mechanisms. Third, we found that Cx3cr1 deficiency dysregulated microglia activation and subsequently resulted in increased photoreceptor vulnerability in rd10 mice, suggesting that the Cx3cl1/Cx3cr1 signaling pathway might protect against microglia neurotoxicity. We concluded that suppression of neuroinflammatory responses could be a potential treatment strategy aimed at improving photoreceptor survival in human RP.
Literature context: 010], and C57BL/6J mice (stock #000664) were purchased from the Mutant
Ataxia, episodic dyskinesia, and thalamocortical seizures are associated with an inherited loss of P/Q-type voltage-gated Ca(2+) channel function. P/Q-type channels are widely expressed throughout the neuraxis, obscuring identification of the critical networks underlying these complex neurological disorders. We showed recently that the conditional postnatal loss of P/Q-type channels in cerebellar Purkinje cells (PCs) in mice (purky) leads to these aberrant phenotypes, suggesting that intrinsic alteration in PC output is a sufficient pathogenic factor for disease initiation. The question arises whether P/Q-type channel deletion confined to a single upstream cerebellar synapse might induce the pathophysiological abnormality of genomically inherited P/Q-type channel disorders. PCs integrate two excitatory inputs, climbing fibers from inferior olive and parallel fibers (PFs) from granule cells (GCs) that receive mossy fiber (MF) input derived from precerebellar nuclei. In this study, we introduce a new mouse model with a selective knock-out of P/Q-type channels in rhombic-lip-derived neurons including the PF and MF pathways (quirky). We found that in quirky mice, PF-PC synaptic transmission is reduced during low-frequency stimulation. Using focal light stimulation of GCs that express optogenetic light-sensitive channels, channelrhodopsin-2, we found that modulation of PC firing via GC input is reduced in quirky mice. Phenotypic analysis revealed that quirky mice display ataxia, dyskinesia, and absence epilepsy. These results suggest that developmental alteration of patterned input confined to only one of the main afferent cerebellar excitatory synaptic pathways has a significant role in generating the neurological phenotype associated with the global genomic loss of P/Q-type channel function.
Literature context: Review Form-approved. C57BL/6 (Jax 000664), mdx (dystrophin-deficient, Ja
Mutations in fukutin-related protein (FKRP) give rise to mild and more severe forms of muscular dystrophy. FKRP patients have reduced glycosylation of the extracellular protein dystroglycan, and FKRP itself shows sequence similarity to glycosyltransferases, implicating FKRP in the processing of dystroglycan. However, FKRP localization is controversial, and no FKRP complexes are known, so any FKRP-dystroglycan link remains elusive. Here, we demonstrate a novel FKRP localization in vivo; in mouse, both endogenous and recombinant FKRP are present at the sarcolemma. Biochemical analyses revealed that mouse muscle FKRP and dystroglycan co-enrich and co-fractionate, indicating that FKRP coexists with dystroglycan in the native dystrophin-glycoprotein complex. Furthermore, FKRP sedimentation shifts with dystroglycan in disease models involving the dystrophin-glycoprotein complex, and sarcolemmal FKRP immunofluorescence mirrors that of dystroglycan in muscular dystrophy mice, suggesting that FKRP localization may be mediated by dystroglycan. These data offer the first evidence of an FKRP complex in muscle and suggest that FKRP may influence the glycosylation status of dystroglycan from within the sarcolemmal dystrophin-glycoprotein complex.