Literature context: 6 RRID: AB_2534095 RRID:AB_2576217 RRID: AB_142924 RRID: AB_142540
Morphological and functional alterations of peripheral somatosensory neurons during the aging process lead to a decline of somatosensory perception. Here, we analyze the changes occurring with aging in trigeminal ganglion (TG), TRPM8-expressing cold thermoreceptor neurons innervating the mouse cornea, which participate in the regulation of basal tearing and blinking and have been implicated in the pathogenesis of dry eye disease (DED). TG cell bodies and axonal branches were examined in a mouse line (TRPM8BAC -EYFP) expressing a fluorescent reporter. In 3 months old animals, about 50% of TG cold thermoreceptor neurons were intensely fluorescent, likely providing strongly fluorescent axons and complex corneal nerve terminals with ongoing activity at 34°C and low-threshold, robust responses to cooling. The remaining TRPM8+ corneal axons were weakly fluorescent with nonbeaded axons, sparsely ramified nerve terminals, and exhibited a low-firing rate at 34°C, responding moderately to cooling pulses as do weakly fluorescent TG neurons. In aged (24 months) mice, the number of weakly fluorescent TG neurons was strikingly high while the morphology of TRPM8+ corneal axons changed drastically; 89% were weakly fluorescent, unbranched, and often ending in the basal epithelium. Functionally, 72.5% of aged cold terminals responded as those of young animals, but 27.5% exhibited very low-background activity and abnormal responsiveness to cooling pulses. These morpho-functional changes develop in parallel with an enhancement of tear's basal flow and osmolarity, suggesting that the aberrant sensory inflow to the brain from impaired peripheral cold thermoreceptors contributes to age-induced abnormal tearing and to the high incidence of DED in elderly people.
Literature context: Thermo Fisher: A11034
α-Synuclein (α-syn) is an abundant presynaptic protein that is the primary constituent of inclusions that define Lewy body diseases (LBDs). In these inclusions, α-syn is phosphorylated at the serine-129 residue. Antibodies directed to this phosphorylation site are used to measure inclusion abundance and stage disease progression in preclinical models as well as in postmortem tissues in LBDs. While it is critical to reliably identify inclusions, phospho-specific antibodies often cross-react with nonspecific antigens. Four commercially available monoclonal antibodies, two from rabbits (clones EP1536Y and MJF-R13) and two from mice (81a and pSyn#64), have been the most widely used in detecting pS129-α-syn inclusions. Here, we systematically evaluated these antibodies in brain sections and protein lysates from rats and mice. All antibodies detected pS129-α-syn inclusions in the brain that were induced by preformed α-syn fibrils in wild-type rats and mice. Antibody titrations revealed that clones EP1536Y and 81a comparably labeled inclusions in both the perikarya and neuronal processes in contrast to clones MJF-R13 and pSyn#64 that incompletely labeled inclusions at various antibody concentrations. Except for EP1536Y, the clones produced nonspecific diffuse neuropil labeling in α-syn knockout mice as well as mice and rats injected with monomeric α-syn, with some nonspecific staining titrating with pS129-α-syn inclusions. By immunoblot, all the clones cross-reacted with proteins other than α-syn, warranting caution in interpretations of specificity. Clone EP1536Y uniquely and robustly detected endogenous pS129-α-syn in highly soluble protein fractions from the mouse brain. In summary, EP1536Y had the highest sensitivity and specificity for detecting pS129-α-syn.
Literature context: Fisher Scientific Cat# A-11034; RRID:AB_2576217 Experimental Models: Cell Lines
The ability to convert human somatic cells efficiently to neurons facilitates the utility of patient-derived neurons for studying neurological disorders. As such, ectopic expression of neuronal microRNAs (miRNAs), miR-9/9∗ and miR-124 (miR-9/9∗-124) in adult human fibroblasts has been found to evoke extensive reconfigurations of the chromatin and direct the fate conversion to neurons. However, how miR-9/9∗-124 break the cell fate barrier to activate the neuronal program remains to be defined. Here, we identified an anti-neurogenic function of EZH2 in fibroblasts that acts outside its role as a subunit of Polycomb Repressive Complex 2 to directly methylate and stabilize REST, a transcriptional repressor of neuronal genes. During neuronal conversion, miR-9/9∗-124 induced the repression of the EZH2-REST axis by downregulating USP14, accounting for the opening of chromatin regions harboring REST binding sites. Our findings underscore the interplay between miRNAs and protein stability cascade underlying the activation of neuronal program.
Literature context: Fisher Scientific CAT# A-11034; RRID:AB_2576217 Goat anti-Rabbit Alexa Fluor 56
Dendritic filopodia of developing neurons function as environmental sensors, regulating the spatial organization of dendrites and proper targeting to presynaptic partners. Dendritic filopodia morphology is determined by the balance of F-actin assembled via two major nucleating pathways, the ARP2/3 complex and formins. The inverse-BAR protein MTSS1 is highly expressed in Purkinje cells (PCs) and has been shown to upregulate ARP2/3 activity. PCs in MTSS1 conditional knockout mice showed dendrite hypoplasia due to excessive contact-induced retraction during development. This phenotype was concomitant with elongated dendritic filopodia and was phenocopied by overactivation of the actin nucleator formin DAAM1 localized in the tips of PC dendritic protrusions. Cell biology assays including single-molecule speckle microscopy demonstrated that MTSS1's C terminus binds to DAAM1 and paused DAAM1-mediated F-actin polymerization. Thus, MTSS1 plays a dual role as a formin inhibitor and ARP2/3 activator in dendritic filopodia, determining final neuronal morphology.
Literature context: (RRID:AB_2576217) Dilution 1:2000
The architecture of normal and diseased tissues strongly influences the development and progression of disease as well as responsiveness and resistance to therapy. We describe a tissue-based cyclic immunofluorescence (t-CyCIF) method for highly multiplexed immuno-fluorescence imaging of formalin-fixed, paraffin-embedded (FFPE) specimens mounted on glass slides, the most widely used specimens for histopathological diagnosis of cancer and other diseases. t-CyCIF generates up to 60-plex images using an iterative process (a cycle) in which conventional low-plex fluorescence images are repeatedly collected from the same sample and then assembled into a high dimensional representation. t-CyCIF requires no specialized instruments or reagents and is compatible with super-resolution imaging; we demonstrate its application to quantifying signal transduction cascades, tumor antigens and immune markers in diverse tissues and tumors. The simplicity and adaptability of t-CyCIF makes it an effective method for pre-clinical and clinical research and a natural complement to single-cell genomics.
Literature context: Invitrogen, Carlsbad, CA, USA; RRID:AB_2576217) for 1 h at 37Â°C. Nuclei were c
One characteristic of Alzheimer's disease (AD) is excessive accumulation of amyloid-β (Aβ) in the brain. Aβ brain accumulation is, in part, due to a reduction in Aβ clearance from the brain across the blood-brain barrier. One key element that contributes to Aβ brain clearance is P-glycoprotein (P-gp) that transports Aβ from brain to blood. In AD, P-gp protein expression and transport activity levels are significantly reduced, which impairs Aβ brain clearance. The mechanism responsible for reduced P-gp expression and activity levels is poorly understood. We recently demonstrated that Aβ40 triggers P-gp degradation through the ubiquitin-proteasome pathway. Consistent with these data, we show here that ubiquitinated P-gp levels in brain capillaries isolated from brain samples of AD patients are increased compared to capillaries isolated from brain tissue of cognitive normal individuals. We extended this line of research to in vivo studies using transgenic human amyloid precursor protein (hAPP)-overexpressing mice (Tg2576) that were treated with PYR41, a cell-permeable, irreversible inhibitor of the ubiquitin-activating enzyme E1. Our data show that inhibiting P-gp ubiquitination protects the transporter from degradation, and immunoprecipitation experiments confirmed that PYR41 prevented P-gp ubiquitination. We further found that PYR41 treatment prevented reduction of P-gp protein expression and transport activity levels and substantially lowered Aβ brain levels in hAPP mice. Together, our findings provide in vivo proof that the ubiquitin-proteasome system mediates reduction of blood-brain barrier P-gp in AD and that inhibiting P-gp ubiquitination prevents P-gp degradation and lowers Aβ brain levels. Thus, targeting the ubiquitin-proteasome system may provide a novel therapeutic approach to protect blood-brain barrier P-gp from degradation in AD and other Aβ-based pathologies.
Literature context: 1034, Thermo Fisher Scientific; RRID:AB_2576217) and anti-mouse Alexa Fluor 594
Perineuronal nets (PNNs), composed mainly of chondroitin sulfate proteoglycans, are the extracellular matrix that surrounds cell bodies, proximal dendrites, and axon initial segments of adult CNS neurons. PNNs are known to regulate neuronal plasticity, although their physiological roles in cerebellar functions have yet to be elucidated. Here, we investigated the contribution of PNNs to GABAergic transmission from cerebellar Purkinje cells (PCs) to large glutamatergic neurons in the deep cerebellar nuclei (DCN) in male mice by recording IPSCs from cerebellar slices, in which PNNs were depleted with chondroitinase ABC (ChABC). We found that PNN depletion increased the amplitude of evoked IPSCs and enhanced the paired-pulse depression. ChABC treatment also facilitated spontaneous IPSCs and increased the miniature IPSC frequency without changing not only the amplitude but also the density of PC terminals, suggesting that PNN depletion enhances presynaptic GABA release. We also demonstrated that the enhanced GABAergic transmission facilitated rebound firing in large glutamatergic DCN neurons, which is expected to result in the efficient induction of synaptic plasticity at synapses onto DCN neurons. Furthermore, we tested whether PNN depletion affects cerebellar motor learning. Mice having received the enzyme into the interpositus nuclei, which are responsible for delay eyeblink conditioning, exhibited the conditioned response at a significantly higher rate than control mice. Therefore, our results suggest that PNNs of the DCN suppress GABAergic transmission between PCs and large glutamatergic DCN neurons and restrict synaptic plasticity associated with motor learning in the adult cerebellum.SIGNIFICANCE STATEMENT Perineuronal nets (PNNs) are one of the extracellular matrices of adult CNS neurons and implicated in regulating various brain functions. Here we found that enzymatic PNN depletion in the mouse deep cerebellar nuclei (DCN) reduced the paired-pulse ratio of IPSCs and increased the miniature IPSC frequency without changing the amplitude, suggesting that PNN depletion enhances GABA release from the presynaptic Purkinje cell (PC) terminals. Mice having received the enzyme in the interpositus nuclei exhibited a higher conditioned response rate in delay eyeblink conditioning than control mice. These results suggest that PNNs regulate presynaptic functions of PC terminals in the DCN and functional plasticity of synapses on DCN neurons, which influences the flexibility of adult cerebellar functions.
Literature context: Fisher Scientific Cat #A11034; RRID:AB_2576217 Goat polyclonal anti-Rabbit Ale
The epigenetic landscape is dynamically remodeled during neurogenesis. However, it is not understood how chromatin modifications in neural stem cells instruct the formation of complex structures in the brain. We report that the histone methyltransferase PRDM16 is required in radial glia to regulate lineage-autonomous and stage-specific gene expression programs that control number and position of upper layer cortical projection neurons. PRDM16 regulates the epigenetic state of transcriptional enhancers to activate genes involved in intermediate progenitor cell production and repress genes involved in cell migration. The histone methyltransferase domain of PRDM16 is necessary in radial glia to promote cortical neuron migration through transcriptional silencing. We show that repression of the gene encoding the E3 ubiquitin ligase PDZRN3 by PRDM16 determines the position of upper layer neurons. These findings provide insights into how epigenetic control of transcriptional enhancers in radial glial determines the organization of the mammalian cerebral cortex.
Literature context: (Cat. # A-11034, RRID:AB_2576217 and Cat. # A11030, RRID:AB_1446
Although psychotropic drugs act on neurons and glial cells, how glia respond, and whether glial responses are involved in therapeutic effects are poorly understood. Here, we show that fluoxetine (FLX), an anti-depressant, mediates its anti-depressive effect by increasing the gliotransmission of ATP. FLX increased ATP exocytosis via vesicular nucleotide transporter (VNUT). FLX-induced anti-depressive behavior was decreased in astrocyte-selective VNUT-knockout mice or when VNUT was deleted in mice, but it was increased when astrocyte-selective VNUT was overexpressed in mice. This suggests that VNUT-dependent astrocytic ATP exocytosis has a critical role in the therapeutic effect of FLX. Released ATP and its metabolite adenosine act on P2Y11 and adenosine A2b receptors expressed by astrocytes, causing an increase in brain-derived neurotrophic factor in astrocytes. These findings suggest that in addition to neurons, FLX acts on astrocytes and mediates its therapeutic effects by increasing ATP gliotransmission.
Literature context: conjugate ThermoFisher A-11034; RRID:AB_2576217 Anti-rabbit IgG (H+L) HRP conju
The vertebrate protein STING, an intracellular sensor of cyclic dinucleotides, is critical to the innate immune response and the induction of type I interferon during pathogenic infection. Here, we show that a STING ortholog (dmSTING) exists in Drosophila, which, similar to vertebrate STING, associates with cyclic dinucleotides to initiate an innate immune response. Following infection with Listeria monocytogenes, dmSTING activates an innate immune response via activation of the NF-κB transcription factor Relish, part of the immune deficiency (IMD) pathway. DmSTING-mediated activation of the immune response reduces the levels of Listeria-induced lethality and bacterial load in the host. Of significance, dmSTING triggers an innate immune response in the absence of a known functional cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) ortholog in the fly. Together, our results demonstrate that STING is an evolutionarily conserved antimicrobial effector between flies and mammals, and it comprises a key component of host defense against pathogenic infection in Drosophila.
Literature context: Molecular Probes Cat# A-11034, RRID:AB_2576217 goat anti mouse AlexaFluor 647
The daily light-dark cycles represent a key signal for synchronizing circadian clocks. Both insects and mammals possess dedicated "circadian" photoreceptors but also utilize the visual system for clock resetting. In Drosophila, circadian clock resetting is achieved by the blue-light photoreceptor cryptochrome (CRY), which is expressed within subsets of the brain clock neurons. In addition, rhodopsin-expressing photoreceptor cells contribute to light synchronization. Light resets the molecular clock by CRY-dependent degradation of the clock protein Timeless (TIM), although in specific subsets of key circadian pacemaker neurons, including the small ventral lateral neurons (s-LNvs), TIM and Period (PER) oscillations can be synchronized by light independent of CRY and canonical visual Rhodopsin phototransduction. Here, we show that at least three of the seven Drosophila rhodopsins can utilize an alternative transduction mechanism involving the same α-subunit of the heterotrimeric G protein operating in canonical visual phototransduction (Gq). Surprisingly, in mutants lacking the canonical phospholipase C-β (PLC-β) encoded by the no receptor potential A (norpA) gene, we uncovered a novel transduction pathway using a different PLC-β encoded by the Plc21C gene. This novel pathway is important for behavioral clock resetting to semi-natural light-dark cycles and mediates light-dependent molecular synchronization within the s-LNv clock neurons. The same pathway appears to be responsible for norpA-independent light responses in the compound eye. We show that Rhodopsin 5 (Rh5) and Rh6, present in the R8 subset of retinal photoreceptor cells, drive both the long-term circadian and rapid light responses in the eye.
Literature context: Fisher Scientific Cat# A11034, RRID:AB_2576217 Secondary antibodies Anti-mouse
Rubinstein-Taybi syndrome (RSTS) is a neurodevelopmental disorder characterized by growth retardation, skeletal anomalies and intellectual disability, caused by heterozygous mutation in either the CREBBP (RSTS1) or EP300 (RSTS2) genes. We generated an induced pluripotent stem cell line from an RSTS2 patient's blood mononuclear cells by Sendai virus non integrative reprogramming method. The iPSC line (IAIi001RSTS2-65-A) displayed iPSC morphology, expressed pluripotency markers, possessed trilineage differentiation potential and was stable by karyotyping. Mutation and western blot analyses demonstrated in IAIi001RSTS2-65-A the patient's specific non sense mutation in exon 23 c.3829A > T, p.(Lys 1277*) and showed reduced quantity of wild type p300 protein.
Literature context: t AlexA 488Life TechnologiesCat#A11034Anti-mouse AlexA 647Life Technol
Memory consolidation is a crucial step for long-term memory (LTM) storage. However, we still lack a clear picture of how memory consolidation is regulated at the neuronal circuit level. Here, we took advantage of the well-described anatomy of the Drosophila olfactory memory center, the mushroom body (MB), to address this question in the context of appetitive LTM. The MB lobes, which are made by the fascicled axons of the MB intrinsic neurons, are organized into discrete anatomical modules, each covered by the terminals of a defined type of dopaminergic neuron (DAN) and the dendrites of a corresponding type of MB output neuron (MBON). We previously revealed the essential role of one DAN, the MP1 neuron, in the formation of appetitive LTM. The MP1 neuron is anatomically matched to the GABAergic MBON MVP2, which has been attributed feedforward inhibitory functions recently. Here, we used behavior experiments and in vivo imaging to challenge the existence of MP1-MVP2 synapses and investigate their role in appetitive LTM consolidation. We show that MP1 and MVP2 neurons form an anatomically and functionally recurrent circuit, which features a feedback inhibition that regulates consolidation of appetitive memory. This circuit involves two opposite type 1 and type 2 dopamine receptors in MVP2 neurons and the metabotropic GABAB-R1 receptor in MP1 neurons. We propose that this dual-receptor feedback supports a bidirectional self-regulation of MP1 input to the MB. This mechanism displays striking similarities with the mammalian reward system, in which modulation of the dopaminergic signal is primarily assigned to inhibitory neurons.
Literature context: antibodies (catalog # A-11034, RRID:AB_2576217) were obtained from Thermo Fish
Mouse Cx50D47A and human Cx50D47N are non-functional connexin mutants that cause dominantly-inherited cataracts. In tissue culture expression experiments, they both exhibit impaired cellular trafficking and gap junction plaque formation. Lenses of mice expressing Cx50D47A have cataracts, reduced size, drastically decreased levels of connexin50, and less severely reduced levels of connexin46. The PERK-dependent pathway of the ER response to misfolded proteins is activated, and they have impaired differentiation with retained cellular organelles. Since treatments that enhance protein folding improve trafficking and plaque formation by Cx50D47N and other mutant connexins in vitro, and they are successful therapeutics for some other diseases caused by misfolded proteins, we tested the efficacy of the chemical chaperone, 4-phenylbutyrate (4-PBA) in cultured cells and mice expressing Cx50D47A. 4-PBA treatment increased the formation of Cx50D47A-containing plaques at appositional membranes of transiently transfected HeLa cells. Heterozygous Cx50D47A mice were treated with 4-PBA by addition to the drinking water and parenteral injection of pregnant mice (starting 10 days after pairing of males and females) and their pups. Lenses from 1-month-old mice were examined by darkfield illumination and immunofluorescence microscopy. Protein levels were determined by immunoblotting. Cataract size and density were not detectably different between the control and the 4-PBA-treated groups. Lens size was not increased following treatment. Levels of connexin46 and connexin50 were significantly increased in 4-PBA-treated lenses compared with saline-treated lenses. Immunofluorescence showed an increased abundance of connexin46 immunoreactivity and puncta. The ratio of phosphorylated to total EIF2α was not altered, and levels of organellar proteins were not significantly reduced, suggesting that the ER response to misfolded proteins and differentiation were not changed. Thus, treatment with 4-PBA improved critical pathological issues in these mice (low connexin and gap junction abundance), but the magnitude of this recovery (especially for Cx50) was inadequate to impact the reduced size or the opacification of Cx50D47A lenses.
Literature context: it secondary antibodyThermoCAT# A-11034Alexa546 anti-mouse secondary an
Many cancer-related proteins are controlled by composite post-translational modifications (PTMs), but prevalent strategies only target one type of modification. Here we describe a designed peptide that controls two types of modifications of the p53 tumor suppressor, based on the discovery of a protein complex that suppresses p53 (suppresome). We found that Morn3, a cancer-testis antigen, recruits different PTM enzymes, such as sirtuin deacetylase and ubiquitin ligase, to confer composite modifications on p53. The molecular functions of Morn3 were validated through in vivo assays and chemico-biological intervention. A rationally designed Morn3-targeting peptide (Morncide) successfully activated p53 and suppressed tumor growth. These findings shed light on the regulation of protein PTMs and present a strategy for targeting two modifications with one molecule.
Literature context: trogen Catalog No: A11034, RRID:AB_2576217 Bacterial and Virus Strains
Intracellular signals triggered by DNA breakage flow through proteins containing BRCT (BRCA1 C-terminal) domains. This family, comprising 23 conserved phosphopeptide-binding modules in man, is inaccessible to small-molecule chemical inhibitors. Here, we develop Bractoppin, a drug-like inhibitor of phosphopeptide recognition by the human BRCA1 tandem (t)BRCT domain, which selectively inhibits substrate binding with nanomolar potency in vitro. Structure-activity exploration suggests that Bractoppin engages BRCA1 tBRCT residues recognizing pSer in the consensus motif, pSer-Pro-Thr-Phe, plus an abutting hydrophobic pocket that is distinct in structurally related BRCT domains, conferring selectivity. In cells, Bractoppin inhibits substrate recognition detected by Förster resonance energy transfer, and diminishes BRCA1 recruitment to DNA breaks, in turn suppressing damage-induced G2 arrest and assembly of the recombinase, RAD51. But damage-induced MDC1 recruitment, single-stranded DNA (ssDNA) generation, and TOPBP1 recruitment remain unaffected. Thus, an inhibitor of phosphopeptide recognition selectively interrupts BRCA1 tBRCT-dependent signals evoked by DNA damage.
Literature context: Fisher Sceintific Cat#A-11034; RRID:AB_2576217 Bacterial and Virus Strains
Cellular homeostasis is coordinated through communication between mitochondria and the nucleus, organelles that each possess their own genomes. Whereas the mitochondrial genome is regulated by factors encoded in the nucleus, the nuclear genome is currently not known to be actively controlled by factors encoded in the mitochondrial DNA. Here, we show that MOTS-c, a peptide encoded in the mitochondrial genome, translocates to the nucleus and regulates nuclear gene expression following metabolic stress in a 5'-adenosine monophosphate-activated protein kinase (AMPK)-dependent manner. In the nucleus, MOTS-c regulated a broad range of genes in response to glucose restriction, including those with antioxidant response elements (ARE), and interacted with ARE-regulating stress-responsive transcription factors, such as nuclear factor erythroid 2-related factor 2 (NFE2L2/NRF2). Our findings indicate that the mitochondrial and nuclear genomes co-evolved to independently encode for factors to cross-regulate each other, suggesting that mitonuclear communication is genetically integrated.
Literature context: Thermo Fisher Cat# A-11034, RRID:AB_2576217 Goat anti-rabbit IgG Alexa 555
The dynamics of extracellular signal-regulated kinase (ERK) signaling underlies its versatile functions in cell differentiation, cell proliferation, and cell motility. Classical studies in Drosophila established that a gradient of epidermal growth factor receptor (EGFR)-ERK signaling is essential for these cellular responses. However, we challenge this view by the real-time monitoring of ERK activation; we show that a switch-like ERK activation is essential for the invagination movement of the Drosophila tracheal placode. This switch-like ERK activation stems from the positive feedback regulation of the EGFR-ERK signaling and a resultant relay of EGFR-ERK signaling among tracheal cells. A key transcription factor Trachealess (Trh) permissively regulates the iteration of the relay, and the ERK activation becomes graded in trh mutant. A mathematical model based on these observations and a molecular link between ERK activation dynamics and myosin shows that the relay mechanism efficiently promotes epithelial invagination while the gradient mechanism does not.
Literature context: orbed Invitrogen Cat#: A-11034; RRID:AB_2576217 Alexa Fluor 594 goat anti-mouse
Recent reports indicate that hypoxia influences the circadian clock through the transcriptional activities of hypoxia-inducible factors (HIFs) at clock genes. Unexpectedly, we uncover a profound disruption of the circadian clock and diurnal transcriptome when hypoxic cells are permitted to acidify to recapitulate the tumor microenvironment. Buffering against acidification or inhibiting lactic acid production fully rescues circadian oscillation. Acidification of several human and murine cell lines, as well as primary murine T cells, suppresses mechanistic target of rapamycin complex 1 (mTORC1) signaling, a key regulator of translation in response to metabolic status. We find that acid drives peripheral redistribution of normally perinuclear lysosomes away from perinuclear RHEB, thereby inhibiting the activity of lysosome-bound mTOR. Restoring mTORC1 signaling and the translation it governs rescues clock oscillation. Our findings thus reveal a model in which acid produced during the cellular metabolic response to hypoxia suppresses the circadian clock through diminished translation of clock constituents.
Literature context: RRID:AB_2576217
In most animals, the brain controls the body via a set of descending neurons (DNs) that traverse the neck. DN activity activates, maintains or modulates locomotion and other behaviors. Individual DNs have been well-studied in species from insects to primates, but little is known about overall connectivity patterns across the DN population. We systematically investigated DN anatomy in Drosophila melanogaster and created over 100 transgenic lines targeting individual cell types. We identified roughly half of all Drosophila DNs and comprehensively map connectivity between sensory and motor neuropils in the brain and nerve cord, respectively. We find the nerve cord is a layered system of neuropils reflecting the fly's capability for two largely independent means of locomotion -- walking and flight -- using distinct sets of appendages. Our results reveal the basic functional map of descending pathways in flies and provide tools for systematic interrogation of neural circuits.
Literature context: gateThermo Fisher ScientificCat#A-11034Anti-mouse IgG, Alexa 568 conjug
A class of long noncoding RNAs (lncRNAs) has architectural functions in nuclear body construction; however, specific RNA domains dictating their architectural functions remain uninvestigated. Here, we identified the domains of the architectural NEAT1 lncRNA that construct paraspeckles. Systematic deletion of NEAT1 portions using CRISPR/Cas9 in haploid cells revealed modular domains of NEAT1 important for RNA stability, isoform switching, and paraspeckle assembly. The middle domain, containing functionally redundant subdomains, was responsible for paraspeckle assembly. Artificial tethering of the NONO protein to a NEAT1_2 mutant lacking the functional subdomains rescued paraspeckle assembly, and this required the NOPS dimerization domain of NONO. Paraspeckles exhibit phase-separated properties including susceptibility to 1,6-hexanediol treatment. RNA fragments of the NEAT1_2 subdomains preferentially bound NONO/SFPQ, leading to phase-separated aggregates in vitro. Thus, we demonstrate that the enrichment of NONO dimers on the redundant NEAT1_2 subdomains initiates construction of phase-separated paraspeckles, providing mechanistic insights into lncRNA-based nuclear body formation.
Literature context: ated Thermo Fisher Cat#A-11034; RRID:AB_2576217 Rabbit polyclonal anti-53BP1 No
DNA polymerase ε (POLE) is a four-subunit complex and the major leading strand polymerase in eukaryotes. Budding yeast orthologs of POLE3 and POLE4 promote Polε processivity in vitro but are dispensable for viability in vivo. Here, we report that POLE4 deficiency in mice destabilizes the entire Polε complex, leading to embryonic lethality in inbred strains and extensive developmental abnormalities, leukopenia, and tumor predisposition in outbred strains. Comparable phenotypes of growth retardation and immunodeficiency are also observed in human patients harboring destabilizing mutations in POLE1. In both Pole4-/- mouse and POLE1 mutant human cells, Polε hypomorphy is associated with replication stress and p53 activation, which we attribute to inefficient replication origin firing. Strikingly, removing p53 is sufficient to rescue embryonic lethality and all developmental abnormalities in Pole4 null mice. However, Pole4-/-p53+/- mice exhibit accelerated tumorigenesis, revealing an important role for controlled CMG and origin activation in normal development and tumor prevention.
Literature context: o Fisher Scientific Cat# A11034 RRID:AB_2576217 Chemicals, Peptides, and Recomb
The reward system is a collection of circuits that reinforce behaviors necessary for survival [1, 2]. Given the importance of reproduction for survival, actions that promote successful mating induce pleasurable feeling and are positively reinforced [3, 4]. This principle is conserved in Drosophila, where successful copulation is naturally rewarding to male flies, induces long-term appetitive memories , increases brain levels of neuropeptide F (NPF, the fly homolog of neuropeptide Y), and prevents ethanol, known otherwise as rewarding to flies [6, 7], from being rewarding . It is not clear which of the multiple sensory and motor responses performed during mating induces perception of reward. Sexual interactions with female flies that do not reach copulation are not sufficient to reduce ethanol consumption , suggesting that only successful mating encounters are rewarding. Here, we uncoupled the initial steps of mating from its final steps and tested the ability of ejaculation to mimic the rewarding value of full copulation. We induced ejaculation by activating neurons that express the neuropeptide corazonin (CRZ)  and subsequently measured different aspects of reward. We show that activating Crz-expressing neurons is rewarding to male flies, as they choose to reside in a zone that triggers optogenetic stimulation of Crz neurons and display conditioned preference for an odor paired with the activation. Reminiscent of successful mating, repeated activation of Crz neurons increases npf levels and reduces ethanol consumption. Our results demonstrate that ejaculation stimulated by Crz/Crz-receptor signaling serves as an essential part of the mating reward mechanism in Drosophila. VIDEO ABSTRACT.
Literature context: jugate Invitrogen Cat# A-11034, RRID:AB_2576217 Anti-CD11b antibody abcam Cat#
Stem cell self-renewal is critical for tissue homeostasis, and its dysregulation can lead to organ failure or tumorigenesis. While obesity can induce varied abnormalities in bone marrow components, it is unclear how diet might affect hematopoietic stem cell (HSC) self-renewal. Here, we show that Spred1, a negative regulator of RAS-MAPK signaling, safeguards HSC homeostasis in animals fed a high-fat diet (HFD). Under steady-state conditions, Spred1 negatively regulates HSC self-renewal and fitness, in part through Rho kinase activity. Spred1 deficiency mitigates HSC failure induced by infection mimetics and prolongs HSC lifespan, but it does not initiate leukemogenesis due to compensatory upregulation of Spred2. In contrast, HFD induces ERK hyperactivation and aberrant self-renewal in Spred1-deficient HSCs, resulting in functional HSC failure, severe anemia, and myeloproliferative neoplasm-like disease. HFD-induced hematopoietic abnormalities are mediated partly through alterations to the gut microbiota. Together, these findings reveal that diet-induced stress disrupts fine-tuning of Spred1-mediated signals to govern HSC homeostasis.
Literature context: RRID:AB_2576217 Alexa 647 anti-rat ThermoFisher
Monoaminergic modulation of cortical and thalamic inputs to the dorsal striatum (DS) is crucial for reward-based learning and action control. While dopamine has been extensively investigated in this context, the synaptic effects of serotonin (5-HT) have been largely unexplored. Here, we investigated how serotonergic signaling affects associative plasticity at glutamatergic synapses on the striatal projection neurons of the direct pathway (dSPNs). Combining chemogenetic and optogenetic approaches reveals that impeding serotonergic signaling preferentially gates spike-timing-dependent long-term depression (t-LTD) at thalamostriatal synapses. This t-LTD requires dampened activity of the 5-HT4 receptor subtype, which we demonstrate controls dendritic Ca2+ signals by regulating BK channel activity, and which preferentially localizes at the dendritic shaft. The synaptic effects of 5-HT signaling at thalamostriatal inputs provide insights into how changes in serotonergic levels associated with behavioral states or pathology affect striatal-dependent processes.
Literature context: (H+L) Molecular Probes A11034; RRID:AB_2576217 Alexa Fluor 488 goat anti-human
The production of haploid gametes requires the maintenance of centromeric cohesion between sister chromatids through the transition between two successive meiotic divisions, meiosis I and meiosis II. One mechanism for the cohesion maintenance is shugoshin-dependent protection of centromeric cohesin at anaphase I onset [1-3]. However, how centromeric cohesion is maintained during late anaphase I and telophase I, when centromeric shugoshin is undetectable [1-3], remains largely unexplored. Here we show that the centromeric small ubiquitin-related modifier (SUMO) pathway is critical for the maintenance of centromeric cohesion during post-anaphase-I periods in mouse oocytes. SUMO2/3 and E3 ligase PIAS are enriched near centromeres during late anaphase I and telophase I. Specific perturbation of the centromeric SUMO pathway results in precocious loss of centromeric cohesin at telophase I, although shugoshin-dependent centromeric protection at anaphase I onset remains largely intact. Prevention of the SUMO perturbation during post-anaphase-I periods restores the maintenance of centromeric cohesion through the meiosis I-II transition. Thus, the post-anaphase-I centromeric SUMO pathway ensures continuous maintenance of centromeric cohesion through the meiosis I-II transition.
Literature context: 42. RRID:AB_2576217.
NT, a 13-amino acid peptide, is predominantly released from enteroendocrine cells of the small bowel in response to fat ingestion. Free fatty acid receptors, FFAR1 and FFAR4, regulate secretion of gut hormones and insulin. Here, we show that docosahexaenoic acid, a long-chain fatty acid, has the most dramatic effect on NT release. FFAR1 agonists slightly whereas FFAR4 agonists dramaticly stimulate and amplify NT secretion. Double knockdown of FFAR1 and FFAR4 decreases, but overexpression of FFAR4 but not FFAR1 increases, NT release. Administration of cpdA, the FFAR4 agonist, but not TAK-875, a selective FFAR1 agonist, increases plasma NT and further increases olive oil-stimulated plasma NT levels. Inhibition of MEK/ERK1/2 decreased FA-stimulated NT release but increased AMPK phosphorylation. In contrast, inhibition of AMPK further increased NT secretion and ERK1/2 phosphorylation mediated by FFAR1 or 4. Our results indicate that FFAR4 plays more critical role vs. FFAR1 in mediation of fat-regulated NT release and an inhibitory crosstalk between MEK/ERK1/2 and AMPK in the control of NT release downstream of FFAR1/4.
Literature context: 034 (Thermo Fisher Scientific); RRID:AB_2576217). This reaction was used either
KEY POINTS: By taking advantage of calcium imaging and electrophysiology, we provide direct pharmacological evidence for the functional expression of TRPV1 channels in hippocampal Cajal-Retzius cells. Application of the TRPV1 activator capsaicin powerfully enhances spontaneous synaptic transmission in the hippocampal layers that are innervated by the axons of Cajal-Retzius cells. Capsaicin-triggered calcium responses and membrane currents in Cajal-Retzius cells, as well as layer-specific modulation of spontaneous synaptic transmission, are absent when the drug is applied to slices prepared from TRPV1- /- animals. We discuss the implications of the functional expression of TRPV1 channels in Cajal-Retzius cells and of the observed TRPV1-dependent layer-specific modulation of synaptic transmission for physiological and pathological network processing. ABSTRACT: The vanilloid receptor TRPV1 forms complex polymodal channels that are expressed by sensory neurons and play a critical role in nociception. Their distribution pattern and functions in cortical circuits are, however, much less understood. Although TRPV1 reporter mice have suggested that, in the hippocampus, TRPV1 is predominantly expressed by Cajal-Retzius cells (CRs), direct functional evidence is missing. As CRs powerfully excite GABAergic interneurons of the molecular layers, TRPV1 could play important roles in the regulation of layer-specific processing. Here, we have taken advantage of calcium imaging with the genetically encoded indicator GCaMP6s and patch-clamp techniques to study the responses of hippocampal CRs to the activation of TRPV1 by capsaicin, and have compared the effect of TRPV1 stimulation on synaptic transmission in layers innervated or non-innervated by CRs. Capsaicin induced both calcium responses and membrane currents in ∼50% of the cell tested. Neither increases of intracellular calcium nor whole-cell currents were observed in the presence of the TRPV1 antagonists capsazepine/Ruthenium Red or in slices prepared from TRPV1 knockout mice. We also report a powerful TRPV1-dependent enhancement of spontaneous synaptic transmission onto interneurons with dendritic trees confined to the layers innervated by CRs. In conclusion, our work establishes that functional TRPV1 is expressed by a significant fraction of CRs and we propose that TRPV1 activity may regulate layer-specific synaptic transmission in the hippocampus. Lastly, as CR density decreases during postnatal development, we also propose that functional TRPV1 receptors may be related to mechanisms involved in CR progressive reduction by calcium-dependent toxicity/apoptosis.
Literature context: Fisher Scientific Cat#A-11034; RRID:AB_2576217 Chemicals, Peptides, and Recomb
Taming cell-to-cell variability in gene expression is critical for precise pattern formation during embryonic development. To investigate the source and buffering mechanism of expression variability, we studied a biological clock, the vertebrate segmentation clock, controlling the precise spatiotemporal patterning of the vertebral column. By counting single transcripts of segmentation clock genes in zebrafish, we show that clock genes have low RNA amplitudes and expression variability is primarily driven by gene extrinsic sources, which is suppressed by Notch signaling. We further show that expression noise surprisingly increases from the posterior progenitor zone to the anterior segmentation and differentiation zone. Our computational model reproduces the spatial noise profile by incorporating spatially increasing time delays in gene expression. Our results, suggesting that expression variability is controlled by the balance of time delays and cell signaling in a vertebrate tissue, will shed light on the accuracy of natural clocks in multi-cellular systems and inspire engineering of robust synthetic oscillators.
Literature context: Fisher Scientific Cat# A-11034, RRID:AB_2576217 Goat anti-Rabbit IgG (H+L) High
The triggering receptor expressed on myeloid cells 2 (TREM2) is a microglial innate immune receptor associated with a lethal form of early, progressive dementia, Nasu-Hakola disease, and with an increased risk of Alzheimer's disease. Microglial defects in phagocytosis of toxic aggregates or apoptotic membranes were proposed to be at the origin of the pathological processes in the presence of Trem2 inactivating mutations. Here, we show that TREM2 is essential for microglia-mediated synaptic refinement during the early stages of brain development. The absence of Trem2 resulted in impaired synapse elimination, accompanied by enhanced excitatory neurotransmission and reduced long-range functional connectivity. Trem2-/- mice displayed repetitive behavior and altered sociability. TREM2 protein levels were also negatively correlated with the severity of symptoms in humans affected by autism. These data unveil the role of TREM2 in neuronal circuit sculpting and provide the evidence for the receptor's involvement in neurodevelopmental diseases.
Literature context: hermo Fisher Scientific A11034; RRID:AB_2576217 Anti-Chicken IgY, Alexa Fluor 6
The ability of dying cells to activate antigen-presenting cells (APCs) is carefully controlled to avoid unwarranted inflammatory responses. Here, we show that engulfed cells containing cytosolic double-stranded DNA species (viral or synthetic) or cyclic di-nucleotides (CDNs) are able to stimulate APCs via extrinsic STING (stimulator of interferon genes) signaling, to promote antigen cross-presentation. In the absence of STING agonists, dying cells were ineffectual in the stimulation of APCs in trans. Cytosolic STING activators, including CDNs, constitute cellular danger-associated molecular patterns (DAMPs) only generated by viral infection or following DNA damage events that rendered tumor cells highly immunogenic. Our data shed insight into the molecular mechanisms that drive appropriate anti-tumor adaptive immune responses, while averting harmful autoinflammatory disease, and provide a therapeutic strategy for cancer treatment.
Literature context: Fisher Scientific Cat#A-11034; RRID:AB_2576217 Goat anti-mouse secondary antib
The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is the expansion of a hexanucleotide repeat in a non-coding region of the gene C9orf72. We report that loss-of-function mutations in alfa-1, the Caenorhabditis elegans ortholog of C9orf72, cause a novel phenotypic defect: endocytosed yolk is abnormally released into the extra-embryonic space, resulting in refractile "blobs." The alfa-1 blob phenotype is partially rescued by the expression of the human C9orf72 protein, demonstrating that C9orf72 and alfa-1 function similarly. We show that alfa-1 and R144.5, which we identified from a genetic screen for mutants with the blob phenotype and renamed smcr-8, act in the degradation of endolysosomal content and subsequent lysosome reformation. The alfa-1 abnormality in lysosomal reformation results in a general dysregulation in lysosomal homeostasis, leading to defective degradation of phagosomal and autophagosomal contents. We suggest that, like alfa-1, C9orf72 functions in the degradation of endocytosed material and in the maintenance of lysosomal homeostasis. This previously undescribed function of C9orf72 explains a variety of disparate observations concerning the effects of mutations in C9orf72 and its homologs, including the abnormal accumulation of lysosomes and defective fusion of lysosomes to phagosomes. We suggest that aspects of the pathogenic and clinical features of ALS/FTD caused by C9orf72 mutations, such as altered immune responses, aggregation of autophagy targets, and excessive neuronal excitation, result from a reduction in C9orf72 gene function and consequent abnormalities in lysosomal degradation.
Literature context: ies (411000)Secondary antibodiesanti-Rabbit AlexaFluor 488anti-Mouse AlexaFluor 5551:10001
Juvenile Onset Huntington's Disease (JOHD) is a rare variant of HD withage of onset ≤20 years, accounting for 3-10% of all HD patients. The rarity occurrence of JOHD cases, who severely progress towards mental and physical disability with atypical clinical manifestations compared to classical HD, are responsible of general lack of knowledge about this variant. We obtained a fully reprogrammed iPS cell line from fibroblasts of a JOHD patient carrying 65 CAG repeats and age at onset at age 15. At the biopsy time, the patient showed an advanced stage after 10 years of disease.
Literature context: bNIF Antibody RegistryAB_2534069AB_2576217AB_2534074AB_2556545AB_2633282Di
BACKGROUND: Tumor necrosis factor (TNF) is associated with several neurodegenerative disorders including multiple sclerosis (MS). Although TNF-targeted therapies have been largely unsuccessful in MS, recent preclinical data suggests selective soluble TNF inhibition can promote remyelination. This has renewed interest in regulation of TNF signaling in demyelinating disease, especially given the limited treatment options for progressive MS. Using a mouse model of progressive MS, this study evaluates the effects of sustained TNF on oligodendrocyte (OLG) apoptosis and OLG precursor cell (OPC) differentiation. METHODS: Induction of experimental autoimmune encephalomyelitis (EAE) in transgenic mice expressing a dominant-negative interferon-γ receptor under the human glial fibrillary acidic protein promoter (GFAPγR1Δ) causes severe non-remitting disease associated with sustained TNF. Therapeutic effects in GFAPγR1Δ mice treated with anti-TNF compared to control antibody during acute EAE were evaluated by assessing demyelinating lesion size, remyelination, OLG apoptosis, and OPC differentiation. RESULTS: More severe and enlarged demyelinating lesions in GFAPγR1Δ compared to wild-type (WT) mice were associated with increased OLG apoptosis and reduced differentiated CC1+Olig2+ OLG within lesions, as well as impaired upregulation of TNF receptor-2, suggesting impaired OPC differentiation. TNF blockade during acute EAE in GFAPγR1Δ both limited OLG apoptosis and enhanced OPC differentiation consistent with reduced lesion size and clinical recovery. TNF neutralization further limited increasing endothelin-1 (ET-1) expression in astrocytes and myeloid cells noted in lesions during disease progression in GFAPγR1Δ mice, supporting inhibitory effects of ET-1 on OPC maturation. CONCLUSION: Our data implicate that IFNγ signaling to astrocytes is essential to limit a detrimental positive feedback loop of TNF and ET-1 production, which increases OLG apoptosis and impairs OPC differentiation. Interference of this cycle by TNF blockade promotes repair independent of TNFR2 and supports selective TNF targeting to mitigate progressive forms of MS.
Literature context: RRID:AB_2576217 Ki67 (rabbit monoclonal) 1:100
Multiple sclerosis is a demyelinating disease in which neurological deficits result from damage to myelin, axons, and neuron cell bodies. Prolonged or repeated episodes of demyelination impair remyelination. We hypothesized that augmenting Sonic hedgehog (Shh) signaling in chronically demyelinated lesions could enhance oligodendrogenesis and remyelination. Shh regulates oligodendrocyte development during postnatal myelination, and maintains adult neural stem cells. We used genetic approaches to detect Shh expression and Shh responding cells in vivo. ShhCreERT2 or Gli1CreERT2 mice were crossed to reporter mice for genetic fate-labeling of cells actively transcribing Shh or Gli1, an effective readout of canonical Shh signaling. Tamoxifen induction enabled temporal control of recombination at distinct stages of acute and chronic cuprizone demyelination of the corpus callosum. Gli1 fate-labeled cells were rarely found in the corpus callosum with tamoxifen given during acute demyelination stages to examine activated microglia, reactive astrocytes, or remyelinating cells. Gli1 fate-labeled cells, mainly reactive astrocytes, were observed in the corpus callosum with tamoxifen given after chronic demyelination. However, Shh expressing cells were not detected in the corpus callosum during acute or chronic demyelination. Finally, SAG, an agonist of both canonical and type II non-canonical Hedgehog signaling pathways, was microinjected into the corpus callosum after chronic demyelination. Significantly, SAG delivery increased proliferation and enhanced remyelination. SAG did not increase Gli1 fate-labeled cells in the corpus callosum, which may indicate signaling through the non-canonical Hedgehog pathway. These studies demonstrate that Hedgehog pathway interventions may have therapeutic potential to modulate astrogliosis and to promote remyelination after chronic demyelination.
Literature context: clonal IgG (H+L; Cat. # A11034, RRID:AB_2576217, Thermo Fisher Scientific), and
Alcoholism initiates with episodes of excessive alcohol drinking, known as binge drinking, which is one form of excessive drinking (NIAAA Newsletter, 2004) that is related to impulsivity and anxiety (Ducci et al., 2007; Edenberg et al., 2004) and is also predictive of smoking status. The predisposition of non-alcohol exposed subjects to initiate binge drinking is controlled by neuroimmune signaling that includes an innately activated neuronal Toll-like receptor 4 (TLR4) signal. This signal also regulates cognitive impulsivity, a heritable trait that defines drug abuse initiation. However, the mechanism of signal activation, its function in dopaminergic (TH+) neurons within the reward circuitry implicated in drug-seeking behavior [viz. the ventral tegmental area (VTA)], and its contribution to nicotine co-abuse are still poorly understood. We report that the γ-aminobutyric acidA receptor (GABAAR) α2 subunit activates the TLR4 signal in neurons, culminating in the activation (phosphorylation/nuclear translocation) of cyclic AMP response element binding (CREB) but not NF-kB transcription factors and the upregulation of corticotropin-releasing factor (CRF) and tyrosine hydroxylase (TH). The signal is activated through α2/TLR4 interaction, as evidenced by co-immunoprecipitation, and it is present in the VTA from drug-untreated alcohol-preferring P rats. VTA infusion of neurotropic herpes simplex virus (HSV) vectors for α2 (pHSVsiLA2) or TLR4 (pHSVsiTLR4) but not scrambled (pHSVsiNC) siRNA inhibits signal activation and both binge alcohol drinking and nicotine sensitization, suggesting that the α2-activated TLR4 signal contributes to the regulation of both alcohol and nicotine abuse.
Literature context: Fisher Scientific Cat# A-11034; RRID:AB_2576217 Goat anti-guinea pig IgG Cy3 Ja
Active forgetting explains the intrinsic instability of a labile memory lasting for hours. However, how such memory maintains stability against unwanted disruption is not completely understood. Here, we report a learning-activated active protection mechanism that enables labile memory to resist disruptive sensory experiences in Drosophila. Aversive olfactory conditioning activates mitogen-activated protein kinase (MAPK) transiently in the mushroom-body γ lobe, where labile-aversive memory is stored. This increased MAPK activity significantly prolongs labile memory retention and enhances its resistance to disruption induced by heat shock, electric shock, or odor reactivation. Such experience-induced forgetting cannot be prevented by inhibition of Rac1 activity. Instead, protection of Rac1-independent forgetting correlates with non-muscle myosin II activity and persistence of learning-induced presynaptic structural changes. Increased Raf/MAPK activity, together with suppressed Rac1 activity, completely blocks labile memory decay. Thus, learning not only leads to memory formation, but also activates active protection and active forgetting to regulate the formed memory.
Literature context: ies (411000)Secondary antibodiesAnti-rabbit AlexaFluor 488Anti-mouse AlexaFluor 5551:10000
Smith-Magenis syndrome (SMS) is a complex genetic disorder characterized by developmental delay, behavioural problems and circadian rhythm dysregulation. About 90% of SMS cases are due to a 17p11.2 deletion containing retinoic acid induced1 (RAI1) gene, 10% are due to heterozygous mutations affecting RAI1 coding region. Little is known about RAI1 role.
Literature context: RID:RRID:AB_2576217).
Rare neurological diseases shed light onto universal neurobiological processes. However, molecular mechanisms connecting genetic defects to their disease phenotypes are elusive. Here, we obtain mechanistic information by comparing proteomes of cells from individuals with rare disorders with proteomes from their disease-free consanguineous relatives. We use triple-SILAC mass spectrometry to quantify proteomes from human pedigrees affected by mutations in ATP7A, which cause Menkes disease, a rare neurodegenerative and neurodevelopmental disorder stemming from systemic copper depletion. We identified 214 proteins whose expression was altered in ATP7A-/y fibroblasts. Bioinformatic analysis of ATP7A-mutant proteomes identified known phenotypes and processes affected in rare genetic diseases causing copper dyshomeostasis, including altered mitochondrial function. We found connections between copper dyshomeostasis and the UCHL1/PARK5 pathway of Parkinson disease, which we validated with mitochondrial respiration and Drosophila genetics assays. We propose that our genealogical "omics" strategy can be broadly applied to identify mechanisms linking a genomic locus to its phenotypes.
Literature context: (Invitrogen, A11034; 10 Î¼g/ml: RRID:AB_2576217). The ISH signals were converte
Objectives: Bombesin receptor subtype-3 (BRS-3) has been suggested to play a potential role in energy homeostasis. However, the physiological mechanism of BRS-3 on energy homeostasis remains unknown. Thus, we investigated the BRS-3-mediated neuronal pathway involved in food intake and energy expenditure. Materials and Methods: Expression of BRS-3 in the rat brain was histologically examined. The BRS-3 neurons activated by refeeding-induced satiety or a BRS-3 agonist were identified by c-Fos immunostaining. We also analyzed expression changes in feeding-relating peptides in the brain of fasted rats administered with the BRS-3 agonist. Results: In the paraventricular hypothalamic nucleus (PVH), dorsomedial hypothalamic nucleus (DMH), and medial preoptic area (MPA), strong c-Fos induction was observed in the BRS-3 neurons especially in PVH after refeeding. However, the BRS-3 neurons in the PVH did not express feeding-regulating peptides, while the BRS-3 agonist administration induced c-Fos expression in the DMH and MPA, which were not refeeding-sensitive, as well as in the PVH. The BRS-3 agonist administration changed the Pomc and Cart mRNA level in several brain regions of fasted rats. Conclusion: These results suggest that BRS-3 neurons in the PVH are a novel functional subdivision in the PVH that regulates feeding behavior. As the MPA and DMH are reportedly involved in thermoregulation and energy metabolism, the BRS-3 neurons in the MPA/DMH might mediate the energy expenditure control. POMC and CART may contribute to BRS-3 neuron-mediated energy homeostasis regulation. In summary, BRS-3-expressing neurons could regulate energy homeostasis through a novel neuronal pathway.
Literature context: :500) Thermo Fisher Cat#A11034; RRID:AB_2576217 AffiniPure Goat Anti-Armenian H
The generation of new blood vessels via angiogenesis is critical for meeting tissue oxygen demands. A role for adult stem cells in this process remains unclear. Here, we identified CD157 (bst1, bone marrow stromal antigen 1) as a marker of tissue-resident vascular endothelial stem cells (VESCs) in large arteries and veins of numerous mouse organs. Single CD157+ VESCs form colonies in vitro and generate donor-derived portal vein, sinusoids, and central vein endothelial cells upon transplantation in the liver. In response to injury, VESCs expand and regenerate entire vasculature structures, supporting the existence of an endothelial hierarchy within blood vessels. Genetic lineage tracing revealed that VESCs maintain large vessels and sinusoids in the normal liver for more than a year, and transplantation of VESCs rescued bleeding phenotypes in a mouse model of hemophilia. Our findings show that tissue-resident VESCs display self-renewal capacity and that vascular regeneration potential exists in peripheral blood vessels.
Literature context: : 1737902 RRID:AB_2576217 10â€‰Âµg/ml
Alzheimer's disease and cerebral amyloid angiopathy are characterized by accumulation of amyloid-β (Aβ) at the cerebrovasculature due to decreased clearance at the blood-brain barrier (BBB). However, the exact mechanism of Aβ clearance across this barrier has not been fully elucidated. The hCMEC/D3 cell line has been characterized as a valid model for the BBB. In this study we evaluated the use of this model to study Aβ clearance across the BBB, with an emphasis on brain-to-blood directional permeability. Barrier integrity of hCMEC/D3 monolayers was confirmed for large molecules in both the apical to basolateral and the reverse direction. However, permeability for smaller molecules was substantially higher, especially in basolateral to apical direction, and barrier formation for Aβ was completely absent in this direction. In addition, hCMEC/D3 cells failed to develop a high TEER, possibly caused by incomplete formation of tight junctions. We conclude that the hCMEC/D3 model has several limitations to study the cerebral clearance of Aβ. Therefore, the model needs further characterization before this cell system can be generally applied as a model to study cerebral Aβ clearance. © 2016 The Authors Journal of Neuroscience Research Published by Wiley Periodicals, Inc.
Literature context: so A11034 RRID:AB_2576217), Alexa Fl
Tau is a major component of the neurofibrillary tangles (NFT) that represent a pathological hallmark of Alzheimer's disease (AD). Although generally considered an axonal protein, Tau is found in the somato-dendritic compartment of degenerating neurons and this redistribution is thought to be a trigger of neurodegeneration in AD. Here, we show the presence of tau mRNA in a dendritic ribonucleoprotein (RNP) complex that includes Ca2+-calmodulin dependent protein kinase (CaMK)IIα mRNA and that is translated locally in response to glutamate stimulation. Further, we show that Tau mRNA is a component of mRNP granules that contain RNA-binding proteins, and that it interacts with Myosin Va, a postsynaptic motor protein; these findings suggest that tau mRNA is transported into dendritic spines. We also report that tau mRNA localized in the somato-dendritic component of primary hippocampal cells and that a sub-toxic concentration of glutamate enhances local translation and hyperphosphorylation of tau, effects that are blocked by the gluatamatergic antagonists MK801 and NBQX. These data thus demonstrate that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-d-aspartate (NMDA) stimulation redistributes tau to the somato-dendritic region of neurons where it may trigger neurodegeneration.
Literature context: Fisher Scientific Cat# A-11034, RRID:AB_2576217 Alexa Fluor 555 Î±-rabbit IgG Ab
Accumulating evidence indicates that the MDM2 oncoprotein promotes tumorigenesis beyond its canonical negative effects on the p53 tumor suppressor, but these p53-independent functions remain poorly understood. Here, we show that a fraction of endogenous MDM2 is actively imported in mitochondria to control respiration and mitochondrial dynamics independently of p53. Mitochondrial MDM2 represses the transcription of NADH-dehydrogenase 6 (MT-ND6) in vitro and in vivo, impinging on respiratory complex I activity and enhancing mitochondrial ROS production. Recruitment of MDM2 to mitochondria increases during oxidative stress and hypoxia. Accordingly, mice lacking MDM2 in skeletal muscles exhibit higher MT-ND6 levels, enhanced complex I activity, and increased muscular endurance in mild hypoxic conditions. Furthermore, increased mitochondrial MDM2 levels enhance the migratory and invasive properties of cancer cells. Collectively, these data uncover a previously unsuspected function of the MDM2 oncoprotein in mitochondria that play critical roles in skeletal muscle physiology and may contribute to tumor progression.
Literature context: 8) 1:1000 ThermoFisher A-11034, RRID:AB_2576217
Duchenne muscular dystrophy (DMD) is an X-linked disease caused by mutations in the DMD gene, which spans ~2.4Mb of genomic sequence at locus Xp21. This mutation results in the loss of the protein dystrophin. DMD patients die in their second or third decade due to either respiratory failure or cardiomyopathy, as the absence of dystrophin leads to myofiber membrane fragility and necrosis, eventually resulting in muscle atrophy and contractures. Currently, there is no effective treatment for DMD, therefore induced pluripotent stem cells from DMD patients would be a powerful tool for studying disease mechanisms.
Literature context: Rabbit IgG-Alexa Fluor 488 (RRID:AB_2576217) Goat Thermo Fisher Scientific
The blood-testis barrier (BTB), conferred by Sertoli cells in the mammalian testis, is an important ultrastructure that supports spermatogenesis. Studies using animal models have shown that a disruption of the BTB leads to meiotic arrest, causing defects in spermatogenesis and male infertility. To better understand the regulation of BTB dynamics, we report findings herein to understand the role of ribosomal protein S6 (rpS6), a downstream signaling protein of mammalian target of rapamycin complex 1 (mTORC1), in promoting BTB disruption in the testis in vivo, making the barrier "leaky." Overexpression of wild-type rpS6 (rpS6-WT, the full-length cDNA cloned into the mammalian expression vector pCI-neo) and a constitutively active quadruple phosphomimetic mutant cloned into pCI-neo (p-rpS6-MT) vs. control (empty pCI-neo vector) was achieved by transfecting adult rat testes with the corresponding plasmid DNA using a Polyplus in vivo-jetPEI transfection reagent. On the basis of an in vivo functional BTB integrity assay, p-rpS6-MT was found to induce BTB disruption better than rpS6-WT did (and no effects in empty vector control), leading to defects in spermatogenesis, including loss of spermatid polarity and failure in the transport of cells (e.g., spermatids) and organelles (e.g., phagosomes), to be followed by germ exfoliation. More important, rpS6-WT and p-rpS6-MT exert their disruptive effects through changes in the organization of actin- and microtubule (MT)-based cytoskeletons, which are mediated by changes in the spatiotemporal expression of actin- and MT-based binding and regulatory proteins. In short, mTORC1/rpS6 signaling complex is a regulator of spermatogenesis and BTB by modulating the organization of the actin- and MT-based cytoskeletons.
Literature context: t IgG Molecular Probes A11034; RRID:AB_2576217 Alexa Fluor 594 goat anti-mouse
The lateral habenula has been widely studied for its contribution in generating reward-related behaviors [1, 2]. We have found that this nucleus plays an unexpected role in the sedative actions of the general anesthetic propofol. The lateral habenula is a glutamatergic, excitatory hub that projects to multiple targets throughout the brain, including GABAergic and aminergic nuclei that control arousal [3-5]. When glutamate release from the lateral habenula in mice was genetically blocked, the ability of propofol to induce sedation was greatly diminished. In addition to this reduced sensitivity to propofol, blocking output from the lateral habenula caused natural non-rapid eye movement (NREM) sleep to become highly fragmented, especially during the rest ("lights on") period. This fragmentation was largely reversed by the dual orexinergic antagonist almorexant. We conclude that the glutamatergic output from the lateral habenula is permissive for the sedative actions of propofol and is also necessary for the consolidation of natural sleep.
Literature context: Rabbit IgG-Alexa Fluor 488 (RRID:AB_2576217) Goat Invitrogen A-11034 1:250
Germ cell differentiation during the epithelial cycle of spermatogenesis is accompanied by extensive remodeling at the Sertoli cell-cell and Sertoli cell-spermatid interface to accommodate the transport of preleptotene spermatocytes and developing spermatids across the blood-testis barrier (BTB) and the adluminal compartment of the seminiferous epithelium, respectively. The unique cell junction in the testis is the actin-rich ectoplasmic specialization (ES) designated basal ES at the Sertoli cell-cell interface, and the apical ES at the Sertoli-spermatid interface. Since ES dynamics (i.e., disassembly, reassembly and stabilization) are supported by actin microfilaments, which rapidly converts between their bundled and unbundled/branched configuration to confer plasticity to the ES, it is logical to speculate that actin nucleation proteins play a crucial role to ES dynamics. Herein, we reported findings that Spire 1, an actin nucleator known to polymerize actins into long stretches of linear microfilaments in cells, is an important regulator of ES dynamics. Its knockdown by RNAi in Sertoli cells cultured in vitro was found to impede the Sertoli cell tight junction (TJ)-permeability barrier through changes in the organization of F-actin across Sertoli cell cytosol. Unexpectedly, Spire 1 knockdown also perturbed microtubule (MT) organization in Sertoli cells cultured in vitro. Biochemical studies using cultured Sertoli cells and specific F-actin vs. MT polymerization assays supported the notion that a transient loss of Spire 1 by RNAi disrupted Sertoli cell actin and MT polymerization and bundling activities. These findings in vitro were reproduced in studies in vivo by RNAi using Spire 1-specific siRNA duplexes to transfect testes with Polyplus in vivo-jetPEI as a transfection medium with high transfection efficiency. Spire 1 knockdown in the testis led to gross disruption of F-actin and MT organization across the seminiferous epithelium, thereby impeding the transport of spermatids and phagosomes across the epithelium and perturbing spermatogenesis. In summary, Spire 1 is an ES regulator to support germ cell development during spermatogenesis.
Literature context: Fisher Scientific Cat# A11034, RRID:AB_2576217;
Becker muscular dystrophy (BMD) is a dystrophinopathy caused by mutations in the dystrophin gene on chromosome Xp21. BMD mutations result in truncated semi-functional dystrophin isoforms. Consequently, less severe clinical symptoms become apparent later in life compared to Duchenne muscular dystrophy. Dermal fibroblasts from a BMD patient were electroporated with episomal plasmids containing reprogramming factors to create the induced pluripotent stem cell line: CCMi002BMD-A-9 that showed pluripotent markers, were karyotypically normal and capable of trilineage differentiation. MLPA analyses performed on DNA extracted from CCMi002BMD-A-9 showed an in-frame deletion of exons 45 to 55 (CCMi002BMD-A-9 Δ45-55).
Literature context: Fisher Scientific Cat# A-11034; RRID:AB_2576217 Goat anti-Rabbit IgG (H+L) Seco
Correlative evidence suggests that polyploidization of heart muscle, which occurs naturally in post-natal mammals, creates a barrier to heart regeneration. Here, we move beyond a correlation by demonstrating that experimental polyploidization of zebrafish cardiomyocytes is sufficient to suppress their proliferative potential during regeneration. Initially, we determined that zebrafish myocardium becomes susceptible to polyploidization upon transient cytokinesis inhibition mediated by dominant-negative Ect2. Using a transgenic strategy, we generated adult animals containing mosaic hearts composed of differentially labeled diploid and polyploid-enriched cardiomyocyte populations. Diploid cardiomyocytes outcompeted their polyploid neighbors in producing regenerated heart muscle. Moreover, hearts composed of equivalent proportions of diploid and polyploid cardiomyocytes failed to regenerate altogether, demonstrating that a critical percentage of diploid cardiomyocytes is required to achieve heart regeneration. Our data identify cardiomyocyte polyploidization as a barrier to heart regeneration and suggest that mobilizing rare diploid cardiomyocytes in the human heart will improve its regenerative capacity.
Literature context: or 5551:100001:10000Invitrogen (A11034)Invitrogen (A21422)PrimersTarge
Huntington's disease (HD) is an incurable, autosomal dominant, hereditary neurodegenerative disorder that typically manifests itself in midlife. This pathology is linked to the deregulation of multiple, as yet unknown, cellular processes starting before HD onset. A human iPS cell line was generated from skin fibroblasts of a subject at the presymptomatic life stage, carrying a polyglutamine expansion in HTT gene codifying Huntingtin protein. The iPSC line contained the expected CAG expansion, expressed the expected pluripotency markers, displayed in vivo differentiation potential to the three germ layers and had a normal karyotype.
Literature context: 488 Thermo Fisher Cat# A11034; RRID:AB_2576217 goat anti-rat polyclonal, Alexa
Individual reports suggest that the central nervous system (CNS) contains multiple immune cell types with diverse roles in tissue homeostasis, immune defense, and neurological diseases. It has been challenging to map leukocytes across the entire brain, and in particular in pathology, where phenotypic changes and influx of blood-derived cells prevent a clear distinction between reactive leukocyte populations. Here, we applied high-dimensional single-cell mass and fluorescence cytometry, in parallel with genetic fate mapping systems, to identify, locate, and characterize multiple distinct immune populations within the mammalian CNS. Using this approach, we revealed that microglia, several subsets of border-associated macrophages and dendritic cells coexist in the CNS at steady state and exhibit disease-specific transformations in the immune microenvironment during aging and in models of Alzheimer's disease and multiple sclerosis. Together, these data and the described framework provide a resource for the study of disease mechanisms, potential biomarkers, and therapeutic targets in CNS disease.
Literature context: Thermo Fisher Scientific A11034 RRID:AB_2576217 Goat anti-mouse IgG_AlexaFluor
Injury to the peripheral axons of sensory neurons strongly enhances the regeneration of their central axons in the spinal cord. It remains unclear on what molecules that initiate such conditioning effect. Because ATP is released extracellularly by nerve and other tissue injury, we hypothesize that injection of ATP into a peripheral nerve might mimic the stimulatory effect of nerve injury on the regenerative state of the primary sensory neurons. We found that a single injection of 6 μl of 150 μm ATP into female rat sciatic nerve quadrupled the number of axons growing into a lesion epicenter in spinal cord after a concomitant dorsal column transection. A second boost ATP injection 1 week after the first one markedly reinforced the stimulatory effect of a single injection. Single ATP injection increased expression of phospho-STAT3 and GAP43, two markers of regenerative activity, in sensory neurons. Double ATP injections sustained the activation of phospho-STAT3 and GAP43, which may account for the marked axonal growth across the lesion epicenter. Similar studies performed on P2X7 or P2Y2 receptor knock-out mice indicate P2Y2 receptors are involved in the activation of STAT3 after ATP injection or conditioning lesion, whereas P2X7 receptors are not. Injection of ATP at 150 μm caused little Wallerian degeneration and behavioral tests showed no significant long-term adverse effects on sciatic nerve functions. The results in this study reveal possible mechanisms underlying the stimulation of regenerative programs and suggest a practical strategy for stimulating axonal regeneration following spinal cord injury.SIGNIFICANCE STATEMENT Injury of peripheral axons of sensory neurons has been known to strongly enhance the regeneration of their central axons in the spinal cord. In this study, we found that injection of ATP into a peripheral nerve can mimic the effect of peripheral nerve injury and significantly increase the number of sensory axons growing across lesion epicenter in the spinal cord. ATP injection increased expression of several markers for regenerative activity in sensory neurons, including phospho-STAT3 and GAP43. ATP injection did not cause significant long-term adverse effects on the functions of the injected nerve. These results may lead to clinically applicable strategies for enhancing neuronal responses that support regeneration of injured axons.
Literature context: mo Fisher, Cat# A11034, RRID:AB_2576217 Goat Anti-Mouse488 1:1000 Therm
Fibroblasts were isolated from a skin biopsy of a clinically diagnosed 51-year-old female attention-deficit/hyperactivity disorder (ADHD) patient carrying a duplication of SLC2A3, a gene encoding neuronal glucose transporter-3 (GLUT3). Patient fibroblasts were infected with Sendai virus, a single-stranded RNA virus, to generate transgene-free human induced pluripotent stem cells (iPSCs). SLC2A3-D2-iPSCs showed expression of pluripotency-associated markers, were able to differentiate into cells of the three germ layers in vitro and had a normal female karyotype. This in vitro cellular model can be used to study the role of risk genes in the pathogenesis of ADHD, in a patient-specific manner.
Literature context: Fisher Scientific Cat#A-11034; RRID:AB_2576217 Alexa Fluor 555 donkey anti-Rab
Endothelial-to-mesenchymal transition (EndoMT) is a cellular process often initiated by the transforming growth factor β (TGF-β) family of ligands. Although required for normal heart valve development, deregulated EndoMT is linked to a wide range of pathological conditions. Here, we demonstrate that endothelial fatty acid oxidation (FAO) is a critical in vitro and in vivo regulator of EndoMT. We further show that this FAO-dependent metabolic regulation of EndoMT occurs through alterations in intracellular acetyl-CoA levels. Disruption of FAO via conditional deletion of endothelial carnitine palmitoyltransferase II (Cpt2E-KO) augments the magnitude of embryonic EndoMT, resulting in thickening of cardiac valves. Consistent with the known pathological effects of EndoMT, adult Cpt2E-KO mice demonstrate increased permeability in multiple vascular beds. Taken together, these results demonstrate that endothelial FAO is required to maintain endothelial cell fate and that therapeutic manipulation of endothelial metabolism could provide the basis for treating a growing number of EndoMT-linked pathological conditions.
Literature context: ies (411000)Secondary antibodiesAnti-rabbit AlexaFluor 4881:10000Invitrogen (A11034)Anti-mouse AlexaFluor 5551:10000
Huntington Disease (HD) is an autosomal dominant disorder characterized by motor, cognitive and behavioral features caused by a CAG expansion in the HTT gene beyond 35 repeats. The juvenile form (JHD) may begin before the age of 20years and is associated with expanded alleles as long as 60 or more CAG repeats. In this study, induced pluripotent stem cells were generated from skin fibroblasts of a 8-year-old child carrying a large size mutation of 84 CAG repeats in the HTT gene. HD appeared at age 3 with mixed psychiatric (i.e. autistic spectrum disorder) and motor (i.e. dystonia) manifestations.
Literature context: (Thermo Fisher catalog #A11034, RRID:AB_2576217) and 1:500 anti-rabbit Cy3 (Jac
CCCTC-binding factor (CTCF) is an 11 zinc finger DNA-binding domain protein that regulates gene expression by modifying 3D chromatin structure. Human mutations in CTCF cause intellectual disability and autistic features. Knocking out Ctcf in mouse embryonic neurons is lethal by neonatal age, but the effects of CTCF deficiency in postnatal neurons are less well studied. We knocked out Ctcf postnatally in glutamatergic forebrain neurons under the control of Camk2a-Cre. CtcfloxP/loxP;Camk2a-Cre+ (Ctcf CKO) mice of both sexes were viable and exhibited profound deficits in spatial learning/memory, impaired motor coordination, and decreased sociability by 4 months of age. Ctcf CKO mice also had reduced dendritic spine density in the hippocampus and cerebral cortex. Microarray analysis of mRNA from Ctcf CKO mouse hippocampus identified increased transcription of inflammation-related genes linked to microglia. Separate microarray analysis of mRNA isolated specifically from Ctcf CKO mouse hippocampal neurons by ribosomal affinity purification identified upregulation of chemokine signaling genes, suggesting crosstalk between neurons and microglia in Ctcf CKO hippocampus. Finally, we found that microglia in Ctcf CKO mouse hippocampus had abnormal morphology by Sholl analysis and increased immunostaining for CD68, a marker of microglial activation. Our findings confirm that Ctcf KO in postnatal neurons causes a neurobehavioral phenotype in mice and provide novel evidence that CTCF depletion leads to overexpression of inflammation-related genes and microglial dysfunction.SIGNIFICANCE STATEMENT CCCTC-binding factor (CTCF) is a DNA-binding protein that organizes nuclear chromatin topology. Mutations in CTCF cause intellectual disability and autistic features in humans. CTCF deficiency in embryonic neurons is lethal in mice, but mice with postnatal CTCF depletion are less well studied. We find that mice lacking Ctcf in Camk2a-expressing neurons (Ctcf CKO mice) have spatial learning/memory deficits, impaired fine motor skills, subtly altered social interactions, and decreased dendritic spine density. We demonstrate that Ctcf CKO mice overexpress inflammation-related genes in the brain and have microglia with abnormal morphology that label positive for CD68, a marker of microglial activation. Our findings suggest that inflammation and dysfunctional neuron-microglia interactions are factors in the pathology of CTCF deficiency.
Literature context: Fluor 488 goat anti-rabbit IgG (RRID:AB_2576217; Thermo Fisher Scientific) dilu
There is an ongoing debate on the contribution of the neuronal glutamate transporter EAAC1 to the onset of compulsive behaviors. Here, we used behavioral, electrophysiological, molecular, and viral approaches in male and female mice to identify the molecular and cellular mechanisms by which EAAC1 controls the execution of repeated motor behaviors. Our findings show that, in the striatum, a brain region implicated with movement execution, EAAC1 limits group I metabotropic glutamate receptor (mGluRI) activation, facilitates D1 dopamine receptor (D1R) expression, and ensures long-term synaptic plasticity. Blocking mGluRI in slices from mice lacking EAAC1 restores D1R expression and synaptic plasticity. Conversely, activation of intracellular signaling pathways coupled to mGluRI in D1R-containing striatal neurons of mice expressing EAAC1 leads to reduced D1R protein level and increased stereotyped movement execution. These findings identify new molecular mechanisms by which EAAC1 can shape glutamatergic and dopaminergic signals and control repeated movement execution.SIGNIFICANCE STATEMENT Genetic studies implicate Slc1a1, a gene encoding the neuronal glutamate transporter EAAC1, with obsessive-compulsive disorder (OCD). EAAC1 is abundantly expressed in the striatum, a brain region that is hyperactive in OCD. What remains unknown is how EAAC1 shapes synaptic function in the striatum. Our findings show that EAAC1 limits activation of metabotropic glutamate receptors (mGluRIs) in the striatum and, by doing so, promotes D1 dopamine receptor (D1R) expression. Targeted activation of signaling cascades coupled to mGluRIs in mice expressing EAAC1 reduces D1R expression and triggers repeated motor behaviors. These findings provide new information on the molecular basis of OCD and suggest new avenues for its treatment.
Literature context: her Cat #A-11034; RRID:AB_2576217 Anti-Mouse-Alexa594 Thermo Fish
Maintenance of endoplasmic reticulum (ER) proteostasis is controlled by a dynamic signaling network known as the unfolded protein response (UPR). IRE1α is a major UPR transducer, determining cell fate under ER stress. We used an interactome screening to unveil several regulators of the UPR, highlighting the ER chaperone Hsp47 as the major hit. Cellular and biochemical analysis indicated that Hsp47 instigates IRE1α signaling through a physical interaction. Hsp47 directly binds to the ER luminal domain of IRE1α with high affinity, displacing the negative regulator BiP from the complex to facilitate IRE1α oligomerization. The regulation of IRE1α signaling by Hsp47 is evolutionarily conserved as validated using fly and mouse models of ER stress. Hsp47 deficiency sensitized cells and animals to experimental ER stress, revealing the significance of Hsp47 to global proteostasis maintenance. We conclude that Hsp47 adjusts IRE1α signaling by fine-tuning the threshold to engage an adaptive UPR.
Literature context: Fisher Scientific Cat# A-11034; RRID:AB_2576217 Goat anti-rabbit Alexa Fluor 55
Genetics and neuropathology strongly link α-synuclein aggregation and neurotoxicity to the pathogenesis of Parkinson's disease and related α-synucleinopathies. Here we describe a new Drosophila model of α-synucleinopathy based on widespread expression of wild-type human α-synuclein, which shows robust neurodegeneration, early-onset locomotor deficits, and abundant α-synuclein aggregation. We use results of forward genetic screening and genetic analysis in our new model to demonstrate that α-synuclein expression promotes reorganization of the actin filament network and consequent mitochondrial dysfunction through altered Drp1 localization. Similar changes are present in a mouse α-synucleinopathy model and in postmortem brain tissue from patients with α-synucleinopathy. Importantly, we provide evidence that the interaction of α-synuclein with spectrin initiates pathological alteration of the actin cytoskeleton and downstream neurotoxicity. These findings suggest new therapeutic approaches for α-synuclein induced neurodegeneration.
Literature context: ies (411000)Secondary antibodiesanti-Rabbit AlexaFluor 4881:10000Invitrogen (A11034)anti-Mouse AlexaFluor 5551:10000
Joubert Syndrome (JS) is a rare autosomal recessive or X-linked condition characterized by a peculiar cerebellar malformation, known as the molar tooth sign (MTS), associated with other neurological phenotypes and multiorgan involvement. JS is a ciliopathy, a spectrum of disorders whose causative genes encode proteins involved in the primary cilium apparatus. In order to elucidate ciliopathy-associated molecular mechanisms, human induced pluripotent stem cells (hiPSCs) were derived from a patient affected by JS carrying a homozygous missense mutation in the AHI1 gene (p.H896R) that encodes a protein named Jouberin.
Literature context: ologies, cat. no. A11034, RRID; AB_2576217). VGluT2 labeling was used to f
Cortical circuits are profoundly shaped by experience during postnatal development. The consequences of altered vision during the critical period for ocular dominance plasticity have been extensively studied in rodent primary visual cortex (V1). However, little is known about how eye opening, a naturally occurring event, influences the maturation of cortical microcircuits. Here we used a combination of slice electrophysiology and immunohistochemistry in rat V1 to ask whether manipulating the time of eye opening for 3 or 7 d affects cortical excitatory and inhibitory synaptic transmission onto excitatory neurons uniformly across layers or induces laminar-specific effects. We report that binocular delayed eye opening for 3 d showed similar reductions of excitatory and inhibitory synaptic transmission in layers 2/3, 4, and 5. Synaptic transmission recovered to age-matched control levels if the delay was prolonged to 7 d, suggesting that these changes were dependent on binocular delay duration. Conversely, laminar-specific and long-lasting effects were observed if eye opening was delayed unilaterally. Our data indicate that pyramidal neurons located in different cortical laminae have distinct sensitivity to altered sensory drive; our data also strongly suggest that experience plays a fundamental role in not only the maturation of synaptic transmission, but also its coordination across cortical layers.
Literature context: Fisher Scientific Cat# A-11034; RRID:AB_2576217 Goat anti-rat IgG Cy3 Jackson I
At the optic chiasm choice point, ipsilateral retinal ganglion cells (RGCs) are repelled away from the midline by guidance cues, including Ephrin-B2 and Sonic Hedgehog (Shh). Although guidance cues are normally produced by cells residing at the choice point, the mRNA for Shh is not found at the optic chiasm. Here we show that Shh protein is instead produced by contralateral RGCs at the retina, transported anterogradely along the axon, and accumulates at the optic chiasm to repel ipsilateral RGCs. In vitro, contralateral RGC axons, which secrete Shh, repel ipsilateral RGCs in a Boc- and Smo-dependent manner. Finally, knockdown of Shh in the contralateral retina causes a decrease in the proportion of ipsilateral RGCs in a non-cell-autonomous manner. These findings reveal a role for axon-axon interactions in ipsilateral RGC guidance, and they establish that remotely produced cues can act at axon guidance midline choice points.
Literature context: ThermoFisher Scientific A11034; RRID:AB_2576217 Goat anti-rabbit IgG H+L AlexaF
Mechanisms of selective autophagy of the ER, known as ER-phagy, require molecular delineation, particularly in vivo. It is unclear how these events control ER proteostasis and cellular health. Here, we identify cell-cycle progression gene 1 (CCPG1), an ER-resident protein with no known physiological role, as a non-canonical cargo receptor that directly binds to core autophagy proteins via an LIR motif to mammalian ATG8 proteins and, independently and via a discrete motif, to FIP200. These interactions facilitate ER-phagy. The CCPG1 gene is inducible by the unfolded protein response and thus directly links ER stress to ER-phagy. In vivo, CCPG1 protects against ER luminal protein aggregation and consequent unfolded protein response hyperactivation and tissue injury of the exocrine pancreas. Thus, via identification of this autophagy protein, we describe an unexpected molecular mechanism of ER-phagy and provide evidence that this may be physiologically relevant in ER luminal proteostasis.
Literature context: RRID:AB_2576217 1:500 (IF)
Damaged mitochondria are selectively eliminated by mitophagy. Parkin and PINK1, gene products mutated in familial Parkinson's disease, play essential roles in mitophagy through ubiquitination of mitochondria. Cargo ubiquitination by E3 ubiquitin ligase Parkin is important to trigger selective autophagy. Although autophagy receptors recruit LC3-labeled autophagic membranes onto damaged mitochondria, how other essential autophagy units such as ATG9A-integrated vesicles are recruited remains unclear. Here, using mammalian cultured cells, we demonstrate that RABGEF1, the upstream factor of the endosomal Rab GTPase cascade, is recruited to damaged mitochondria via ubiquitin binding downstream of Parkin. RABGEF1 directs the downstream Rab proteins, RAB5 and RAB7A, to damaged mitochondria, whose associations are further regulated by mitochondrial Rab-GAPs. Furthermore, depletion of RAB7A inhibited ATG9A vesicle assembly and subsequent encapsulation of the mitochondria by autophagic membranes. These results strongly suggest that endosomal Rab cycles on damaged mitochondria are a crucial regulator of mitophagy through assembling ATG9A vesicles.
Literature context: 1:500) Invitrogen Cat# A11034; RRID:AB_2576217 Goat Anti-mouse IgG-Alexa Fluor
The cohesin complex is built upon the SMC1/SMC3 heterodimer, and mammalian meiocytes feature two variants of SMC1 named SMC1α and SMC1β. It is unclear why these two SMC1 variants have evolved. To determine unique versus redundant functions of SMC1β, we asked which of the known functions of SMC1β can be fulfilled by SMC1α. Smc1α was expressed under control of the Smc1β promoter in either wild-type or SMC1β-deficient mice. No effect was seen in the former. However, several major phenotypes of SMC1β-deficient spermatocytes were rescued by SMC1α. We observed extended development before apoptosis and restoration of axial element and synaptonemal complex lengths, chromosome synapsis, sex body formation, processing of DNA double-strand breaks, and formation of MLH1 recombination foci. This supports the concept that the quantity rather than the specific quality of cohesin complexes is decisive for meiotic chromosome architecture. It also suggests plasticity in complex composition, because to replace SMC1β in many functions, SMC1α has to more extensively associate with other cohesins. The cells did not complete meiosis but died to the latest at the pachytene-to-diplotene transition. Telomere aberrations known from Smc1β-/- mice persisted, and DNA damage response and repair proteins accumulated there regardless of expression of SMC1α. Thus, whereas SMC1α can substitute for SMC1β in many functions, the protection of telomere integrity requires SMC1β.
Literature context: trogen Molecular Probes; 1:300; RRID:AB_2576217 or AB_10563288)
Animals adaptively respond to a tactile stimulus by choosing an ethologically relevant behavior depending on the location of the stimuli. Here, we investigate how somatosensory inputs on different body segments are linked to distinct motor outputs in Drosophila larvae. Larvae escape by backward locomotion when touched on the head, while they crawl forward when touched on the tail. We identify a class of segmentally repeated second-order somatosensory interneurons, that we named Wave, whose activation in anterior and posterior segments elicit backward and forward locomotion, respectively. Anterior and posterior Wave neurons extend their dendrites in opposite directions to receive somatosensory inputs from the head and tail, respectively. Downstream of anterior Wave neurons, we identify premotor circuits including the neuron A03a5, which together with Wave, is necessary for the backward locomotion touch response. Thus, Wave neurons match their receptive field to appropriate motor programs by participating in different circuits in different segments.
Literature context: Fisher Scientific Cat# A-11034, RRID:AB_2576217 Goat anti-Rat IgG (H+L), Alexa
Human in vitro generated monocyte-derived dendritic cells (moDCs) and macrophages are used clinically, e.g., to induce immunity against cancer. However, their physiological counterparts, ontogeny, transcriptional regulation, and heterogeneity remains largely unknown, hampering their clinical use. High-dimensional techniques were used to elucidate transcriptional, phenotypic, and functional differences between human in vivo and in vitro generated mononuclear phagocytes to facilitate their full potential in the clinic. We demonstrate that monocytes differentiated by macrophage colony-stimulating factor (M-CSF) or granulocyte macrophage colony-stimulating factor (GM-CSF) resembled in vivo inflammatory macrophages, while moDCs resembled in vivo inflammatory DCs. Moreover, differentiated monocytes presented with profound transcriptomic, phenotypic, and functional differences. Monocytes integrated GM-CSF and IL-4 stimulation combinatorically and temporally, resulting in a mode- and time-dependent differentiation relying on NCOR2. Finally, moDCs are phenotypically heterogeneous and therefore necessitate the use of high-dimensional phenotyping to open new possibilities for better clinical tailoring of these cellular therapies.
Literature context: A-11034, RRID:AB_2576217). Nuclei w
An upregulation of Egr-1 expression has been reported in models of atherosclerosis and intimal hyperplasia and, various vasoactive peptides and growth promoting stimuli have been shown to induce the expression of Egr-1 in vascular smooth muscle cells (VSMC). Angiotensin-II (Ang-II) is a key vasoactive peptide that has been implicated in the pathogenesis of vascular diseases. Ang-II elevates intracellular Ca2+ through activation of the store-operated calcium entry (SOCE) involving an inositol-3-phosphate receptor (IP3R)-coupled depletion of endoplasmic reticular Ca2+ and a subsequent activation of the stromal interaction molecule 1 (STIM-1)/Orai-1 complex. However, the involvement of IP3R/STIM-1/Orai-1-Ca2+ -dependent signaling in Egr-1 expression in VSMC remains unexplored. Therefore, in the present studies, we have examined the role of Ca2+ signaling in Ang-II-induced Egr-1 expression in VSMC and investigated the contribution of STIM-1 or Orai-1 in mediating this response. 2-aminoethoxydiphenyl borate (2-APB), a dual non-competitive antagonist of IP3R and inhibitor of SOCE, decreased Ang-II-induced Ca2+ release and attenuated Ang-II-induced enhanced expression of Egr-1 protein and mRNA levels. Egr-1 upregulation was also suppressed following blockade of calmodulin and CaMKII. Furthermore, RNA interference-mediated depletion of STIM-1 or Orai-1 attenuated Ang-II-induced Egr-1 expression as well as Ang-II-induced phosphorylation of ERK1/2 and CREB. In addition, siRNA-induced silencing of CREB resulted in a reduction in the expression of Egr-1 stimulated by Ang-II. In summary, our data demonstrate that Ang-II-induced Egr-1 expression is mediated by STIM-1/Orai-1/Ca2+ -dependent signaling pathways in A-10 VSMC.
Literature context: 8 ThermoFisher Cat#: A11034; RRID:AB_2576217 Goat anti-rabbit Alexa Fluor 54
Autophagy and autophagy-related genes (Atg) have been attributed prominent roles in tumorigenesis, tumor growth, and metastasis. Extracellular vesicles called exosomes are also implicated in cancer metastasis. Here, we demonstrate that exosome production is strongly reduced in cells lacking Atg5 and Atg16L1, but this is independent of Atg7 and canonical autophagy. Atg5 specifically decreases acidification of late endosomes where exosomes are produced, disrupting the acidifying V1V0-ATPase by removing a regulatory component, ATP6V1E1, into exosomes. The effect of Atg5 on exosome production promotes the migration and in vivo metastasis of orthotopic breast cancer cells. These findings uncover mechanisms controlling exosome release and identify means by which autophagy-related genes can contribute to metastasis in autophagy-independent pathways.
Literature context: Watham, MA, USA, Cat# A-11034, RRID:AB_2576217) and donkey anti-rat Alexa Fluo
Neuron-secreted factors induce astrocytic expression of the glutamate transporter, GLT-1 (excitatory amino acid transporter 2). In addition to their elaborate anatomic relationships with neurons, astrocytes also have processes that extend to and envelop the vasculature. Although previous studies have demonstrated that brain endothelia contribute to astrocyte differentiation and maturation, the effects of brain endothelia on astrocytic expression of GLT-1 have not been examined. In this study, we tested the hypothesis that endothelia induce expression of GLT-1 by co-culturing astrocytes from mice that utilize non-coding elements of the GLT-1 gene to control expression of reporter proteins with the mouse endothelial cell line, bEND.3. We found that endothelia increased steady state levels of reporter and GLT-1 mRNA/protein. Co-culturing with primary rat brain endothelia also increases reporter protein, GLT-1 protein, and GLT-1-mediated glutamate uptake. The Janus kinase/signal transducer and activator of transcription 3, bone morphogenic protein/transforming growth factor β, and nitric oxide pathways have been implicated in endothelia-to-astrocyte signaling; we provide multiple lines of evidence that none of these pathways mediate the effects of endothelia on astrocytic GLT-1 expression. Using transwells with a semi-permeable membrane, we demonstrate that the effects of the bEND.3 cell line are dependent upon contact. Notch has also been implicated in endothelia-astrocyte signaling in vitro and in vivo. The first step of Notch signaling requires cleavage of Notch intracellular domain by γ-secretase. We demonstrate that the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester blocks endothelia-induced increases in GLT-1. We show that the levels of Notch intracellular domain are higher in nuclei of astrocytes co-cultured with endothelia, an effect also blocked by N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. Finally, infection of co-cultures with shRNA directed against recombination signal binding protein for immunoglobulin kappa J, a Notch effector, also reduces endothelia-dependent increases in enhanced green fluorescent protein and GLT-1. Together, these studies support a novel role for Notch in endothelia-dependent induction of GLT-1 expression. Cover Image for this issue: doi. 10.1111/jnc.13825.
Literature context: or 488 ThermoFisher Cat#A11034; RRID:AB_2576217 Goat anti-Rabbit IgG (H+L) High
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: RRID:AB_2576217 2.4. Cell proliferation in vivo
Sonic hedgehog (Shh) regulates a wave of oligodendrocyte production for extensive myelination during postnatal development. During this postnatal period of oligodendrogenesis, we fate-labeled cells exhibiting active Shh signaling to examine their contribution to the regenerative response during remyelination. Bitransgenic mouse lines were generated for induced genetic fate-labeling of cells actively transcribing Shh or Gli1. Gli1 transcription is an effective readout for canonical Shh signaling. ShhCreERT2 mice and Gli1CreERT2 mice were crossed to either R26tdTomato mice to label cells with red fluorescence, or, R26IAP mice to label membranes with alkaline phosphatase. When tamoxifen (TMX) was given on postnatal days 6-9 (P6-9), Shh ligand synthesis was prevalent in neurons of ShhCreERT2; R26tdTomato mice and ShhCreERT2;R26IAP mice. In Gli1CreERT2 crosses, TMX from P6-9 detected Gli1 transcription in cells that populated the corpus callosum (CC) during postnatal myelination. Delaying TMX to P14-17, after the peak of oligodendrogenesis, significantly reduced labeling of Shh synthesizing neurons and Gli1 expressing cells in the CC. Importantly, Gli1CreERT2;R26tdTomato mice given TMX from P6-9 showed Gli1 fate-labeled cells in the adult (P56) CC, including cycling progenitor cells identified by EdU incorporation and NG2 immunolabeling. Furthermore, after cuprizone demyelination of the adult CC, Gli1 fate-labeled cells incorporated EdU and were immunolabeled by NG2 early during remyelination while forming myelin-like membranes after longer periods for remyelination to progress. These studies reveal a postnatal cell population with transient Shh signaling that contributes to oligodendrogenesis during CC myelination, and gives rise to cells that continue to proliferate in adulthood and contribute to CC remyelination.
Literature context: Fisher Scientific Cat# A11034, RRID:AB_2576217 Anti-Mouse IgG2a Cross-Adsorbed
Duchenne muscular dystrophy (DMD) is caused by abnormalities in the dystrophin gene and is clinically characterised by childhood muscle degeneration and cardiomyopathy. We produced an induced pluripotent stem cell line from a DMD patient's dermal fibroblasts by electroporation with episomal vectors containing: hL-MYC, hLIN28, hSOX2, hKLF4, hOCT3/4. The resultant DMD iPSC line (CCMi001DMD-A-3) displayed iPSC morphology, expressed pluripotency markers, possessed trilineage differentiation potential and was karyotypically normal. MLPA analyses performed on DNA extracted from CCMi001DMD-A-3 showed a deletion of exons 49 and 50 (CCMi001DMD-A-3, ∆49, ∆50).
Literature context: 1034, Thermo Fisher Scientific; RRID:AB_2576217) and an Alexa Fluor 555 goat an
Female honeybees use the "waggle dance" to communicate the location of nectar sources to their hive mates. Distance information is encoded in the duration of the waggle phase (von Frisch, 1967). During the waggle phase, the dancer produces trains of vibration pulses, which are detected by the follower bees via Johnston's organ located on the antennae. To uncover the neural mechanisms underlying the encoding of distance information in the waggle dance follower, we investigated morphology, physiology, and immunohistochemistry of interneurons arborizing in the primary auditory center of the honeybee (Apis mellifera). We identified major interneuron types, named DL-Int-1, DL-Int-2, and bilateral DL-dSEG-LP, that responded with different spiking patterns to vibration pulses applied to the antennae. Experimental and computational analyses suggest that inhibitory connection plays a role in encoding and processing the duration of vibration pulse trains in the primary auditory center of the honeybee.SIGNIFICANCE STATEMENT The waggle dance represents a form of symbolic communication used by honeybees to convey the location of food sources via species-specific sound. The brain mechanisms used to decipher this symbolic information are unknown. We examined interneurons in the honeybee primary auditory center and identified different neuron types with specific properties. The results of our computational analyses suggest that inhibitory connection plays a role in encoding waggle dance signals. Our results are critical for understanding how the honeybee deciphers information from the sound produced by the waggle dance and provide new insights regarding how common neural mechanisms are used by different species to achieve communication.
Literature context: goat IgG (Hâ€¯+â€¯L; Cat. # A11034, RRID:AB_2576217 or Cat. # A11055, RRID: AB_1426
Cognitive impulsivity is a heritable trait believed to represent the behavior that defines the volition to initiate alcohol drinking. We have previously shown that a neuronal Toll-like receptor 4 (TLR4) signal located in the central amygdala (CeA) and ventral tegmental area (VTA) controls the initiation of binge drinking in alcohol-preferring P rats, and TLR4 expression is upregulated by alcohol-induced corticotropin-releasing factor (CRF) at these sites. However, the function of the TLR4 signal in the nucleus accumbens shell (NAc-shell), a site implicated in the control of reward, drug-seeking behavior and impulsivity and the contribution of other signal-associated genes, are still poorly understood. Here we report that P rats have an innately activated TLR4 signal in NAc-shell neurons that co-express the α2 GABAA receptor subunit and CRF prior to alcohol exposure. This signal is not present in non-alcohol drinking NP rats. The TLR4 signal is sustained by a CRF amplification loop, which includes TLR4-mediated CRF upregulation through PKA/CREB activation and CRF-mediated TLR4 upregulation through the CRF type 1 receptor (CRFR1) and the MAPK/ERK pathway. NAc-shell Infusion of a neurotropic, non-replicating herpes simplex virus vector for TLR4-specific small interfering RNA (pHSVsiTLR4) inhibits TLR4 expression and cognitive impulsivity, implicating the CRF-amplified TLR4 signal in impulsivity regulation.
Literature context: A-11034, RRID:not available
Although mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of genetic Parkinson's disease, their function is largely unknown. LRRK2 is pleiotropic in nature, shown to be involved in neurodegeneration and in more peripheral processes, including kidney functions, in rats and mice. Recent studies in zebrafish have shown conflicting evidence that removal of the LRRK2 WD40 domain may or may not affect dopaminergic neurons and/or locomotion. This study shows that ∼50% LRRK2 knockdown in zebrafish causes not only neuronal loss but also developmental perturbations such as axis curvature defects, ocular abnormalities, and edema in the eyes, lens, and otic vesicles. We further show that LRRK2 knockdown results in significant neuronal loss, including a reduction of dopaminergic neurons. Immunofluorescence demonstrates that endogenous LRRK2 is expressed in the lens, brain, heart, spinal cord, and kidney (pronephros), which mirror the LRRK2 morphant phenotypes observed. LRRK2 knockdown results further in the concomitant upregulation of β-synuclein, PARK13, and SOD1 and causes β-synuclein aggregation in the diencephalon, midbrain, hindbrain, and postoptic commissure. LRRK2 knockdown causes mislocalization of the Na(+) /K(+) ATPase protein in the pronephric ducts, suggesting that the edema might be linked to renal malfunction and that LRRK2 might be associated with pronephric duct epithelial cell differentiation. Combined, our study shows that LRRK2 has multifaceted roles in zebrafish and that zebrafish represent a complementary model to further our understanding of this central protein. © 2016 Wiley Periodicals, Inc.
Literature context: Rabbit IgG-Alexa Fluor 488 (RRID:AB_2576217) Goat Thermo Fisher Scientific
PFOS induces Sertoli cell injury using testicular cells isolated from rodent testes, but it remains unknown if PFOS has similar effects in humans. Herein, we maintained human Sertoli cells in a mitotically active state in vitro, thus enabling transfection experiments that altered gene expression to explore the molecular mechanism(s) underlying toxicant-induced cell injury. Human Sertoli cells obtained from men at ages 15, 23, 36 and 40 were cultured in vitro. These differentiated Sertoli cells remained mitotically active when cultured in the presence of 10% FBS (fetal bovine serum), with a replication time of ~1-3 weeks. At ~80% confluency, they were used for studies including toxicant exposure, immunoblotting, immunofluorescence analysis, tight junction (TJ)-permeability assessment, and overexpression of BTB (blood-testis barrier) regulatory genes such as FAK and its phosphomimetic mutants. PFOS was found to induce Sertoli cell injury through disruptive effects on actin microfilaments and microtubule (MT) organization across the cell cytosol. As a consequence, these cytoskeletal networks failed to support cell adhesion at the BTB. Overexpression of a FAK phosphomimetic and constitutively active mutant p-FAK-Y407E in these cells was capable of rescuing the PFOS-induced injury through corrective cellular organization of cytoskeletal elements. SUMMARY: PFOS induces human Sertoli cell injury which can be rescued by overexpressing p-FAK-Y407E mutant.
Literature context: IF) Thermo Fisher Cat# A-11034; RRID:AB_2576217 FITC, goat anti-rabbit IgG for
Little is known about the cellular events promoting metastasis. We show that knockout of phospholipase D2 (PLD2), which generates the signaling lipid phosphatidic acid (PA), inhibits lung metastases in the mammary tumor virus (MMTV)-Neu transgenic mouse breast cancer model. PLD2 promotes local invasion through the regulation of the plasma membrane targeting of MT1-MMP and its associated invadopodia. A liposome pull-down screen identifies KIF5B, the heavy chain of the motor protein kinesin-1, as a new PA-binding protein. In vitro assays reveal that PA specifically and directly binds to the C terminus of KIF5B. The binding between PLD2-generated PA and KIF5B is required for the vesicular association of KIF5B, surface localization of MT1-MMP, invadopodia, and invasion in cancer cells. Taken together, these results identify a role of PLD2-generated PA in the regulation of kinesin-1 motor functions and breast cancer metastasis and suggest PLD2 as a potential therapeutic target for metastatic breast cancer.
Literature context: Fisher Scientific Cat# A-11034, RRID:AB_2576217 goat anti-Rat IgG (H+L) Seconda
Many infections and stress signals can rapidly activate the NLRP3 inflammasome to elicit robust inflammatory responses. This activation requires a priming step, which is thought to be mainly for upregulating NLRP3 transcription. However, recent studies report that the NLRP3 inflammasome can be activated independently of transcription, suggesting that the priming process has unknown essential regulatory steps. Here, we report that JNK1-mediated NLRP3 phosphorylation at S194 is a critical priming event and is essential for NLRP3 inflammasome activation. We show that NLRP3 inflammasome activation is disrupted in NLRP3-S194A knockin mice. JNK1-mediated NLRP3 S194 phosphorylation is critical for NLRP3 deubiquitination and facilitates its self-association and the subsequent inflammasome assembly. Importantly, we demonstrate that blocking S194 phosphorylation prevents NLRP3 inflammasome activation in cryopyrin-associated periodic syndromes (CAPS). Thus, our study reveals a key priming molecular event that is a prerequisite for NLRP3 inflammasome activation. Inhibiting NLRP3 phosphorylation could be an effective treatment for NLRP3-related diseases.
Literature context: 8 Life Technologies Cat#A11034; RRID:AB_2576217 Experimental Models: Organisms/
The Bruce effect refers to pregnancy termination in recently pregnant female rodents upon exposure to unfamiliar males . This event occurs in specific combinations of laboratory mouse strains via the vomeronasal system [2, 3]; however, the responsible chemosensory signals have not been fully identified. Here we demonstrate that the male pheromone exocrine gland-secreting peptide 1 (ESP1) is one of the key factors that causes pregnancy block. Female mice exhibited high pregnancy failure rates upon encountering males that secreted different levels of ESP1 compared to the mated male. The effect was not observed in mice that lacked the ESP1 receptor, V2Rp5, which is expressed in vomeronasal sensory neurons. Prolactin surges in the blood after mating, which are essential for maintaining luteal function, were suppressed by ESP1 exposure, suggesting that a neuroendocrine mechanism underlies ESP1-mediated pregnancy failure. The single peptide pheromone ESP1 conveys not only maleness to promote female receptivity but also the males' characteristics to facilitate memorization of the mating partner.
Literature context: ti-rabbit (AF488)InvitrogenCat# A11034Goat anti-rabbit (AF555)Invitrog
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: fe Technologies Cat# A11034; RRID:AB_2576217 488 Donkey anti-rat (1:200) Lif
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: abbit IgG, Antibody Registry ID RRID:AB_2576217 used at 1/200 dilution, and mon
The shapes of homologous skeletal elements in the vertebrate forelimb and hindlimb are distinct, with each element exquisitely adapted to their divergent functions. Many of the signals and signalling pathways responsible for patterning the developing limb bud are common to both forelimb and hindlimb. How disparate morphologies are generated from common signalling inputs during limb development remains poorly understood. We show that, similar to what has been shown in the chick, characteristic differences in mouse forelimb and hindlimb cartilage morphology are maintained when chondrogenesis proceeds in vitro away from the endogenous limb bud environment. Chondrogenic nodules that form in high-density micromass cultures derived from forelimb and hindlimb buds are consistently different in size and shape. We described analytical tools we have developed to quantify these differences in nodule morphology and demonstrate that characteristic hindlimb nodule morphology is lost in the absence of the hindlimb-restricted limb modifier gene Pitx1. Furthermore, we show that ectopic expression of Pitx1 in the forelimb is sufficient to generate nodule patterns characteristic of the hindlimb. We also demonstrate that hindlimb cells are less adhesive to the tissue culture substrate and, within the limb environment, to the extracellular matrix and to each other. These results reveal autonomously programmed differences in forelimb and hindlimb cartilage precursors of the limb skeleton are controlled, at least in part, by Pitx1 and suggest this has an important role in generating distinct limb-type morphologies. Our results demonstrate that the micromass culture system is ideally suited to study cues governing morphogenesis of limb skeletal elements in a simple and experimentally tractable in vitro system that reflects in vivo potential.
Literature context: t-IgG Invitrogen Cat # A11034, RRID:AB_2576217 Alexa Fluor 546 Goat anti-rabbi
Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating side effect of many cancer treatments. The hallmark of CIPN is degeneration of long axons required for transmission of sensory information; axonal degeneration causes impaired tactile sensation and persistent pain. Currently the molecular mechanisms of CIPN are not understood, and there are no available treatments. Here we show that the chemotherapeutic agent paclitaxel triggers CIPN by altering IP3 receptor phosphorylation and intracellular calcium flux, and activating calcium-dependent calpain proteases. Concomitantly paclitaxel impairs axonal trafficking of RNA-granules and reduces synthesis of Bclw (bcl2l2), a Bcl2 family member that binds IP3R1 and restrains axon degeneration. Surprisingly, Bclw or a stapled peptide corresponding to the Bclw BH4 domain interact with axonal IP3R1 and prevent paclitaxel-induced degeneration, while Bcl2 and BclxL cannot do so. Together these data identify a Bclw-IP3R1-dependent cascade that causes axon degeneration and suggest that Bclw-mimetics could provide effective therapy to prevent CIPN.
Literature context: rabbit488 (ThermoFisher A11034, RRID:AB_2576217), goat anti-mouse555 (ThermoFis
The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al., 2014, 2015). Here, we show that by using specific intracellular immunolabelling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabelled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, termed 'early risers'. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide the first a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase.
Literature context: cular Probes CAT# A11034; RRID:AB_2576217 Goat anti-Rabbit Alexa 594 Mole
The generation of precise synaptic connections between developing neurons is critical to the formation of functional neural circuits. Astrocyte-secreted glypican 4 induces formation of active excitatory synapses by recruiting AMPA glutamate receptors to the postsynaptic cell surface. We now identify the molecular mechanism of how glypican 4 exerts its effect. Glypican 4 induces release of the AMPA receptor clustering factor neuronal pentraxin 1 from presynaptic terminals by signaling through presynaptic protein tyrosine phosphatase receptor δ. Pentraxin then accumulates AMPA receptors on the postsynaptic terminal forming functional synapses. Our findings reveal a signaling pathway that regulates synaptic activity during central nervous system development and demonstrates a role for astrocytes as organizers of active synaptic connections by coordinating both pre and post synaptic neurons. As mutations in glypicans are associated with neurological disorders, such as autism and schizophrenia, this signaling cascade offers new avenues to modulate synaptic function in disease.
Literature context: y Ab ThermoFisher Cat#: A11034; RRID:AB_2576217 IF: Goat anti-Rat IgG, Texas Re
Mitochondrial antiviral signaling platform protein (MAVS) acts as a central hub for RIG-I receptor proximal signal propagation. However, key components in the assembly of the MAVS mitochondrial platform that promote RIG-I mitochondrial localization and optimal activation are still largely undefined. Employing pooled RNAi and yeast two-hybrid screenings, we report that the mitochondrial adaptor protein tripartite motif (TRIM)14 provides a docking platform for the assembly of the mitochondrial signaling complex required for maximal activation of RIG-I-mediated signaling, consisting of WHIP and protein phosphatase PPP6C. Following viral infection, the ubiquitin-binding domain in WHIP bridges RIG-I with MAVS by binding to polyUb chains of RIG-I at lysine 164. The ATPase domain in WHIP contributes to stabilization of the RIG-I-dsRNA interaction. Moreover, phosphatase PPP6C is responsible for RIG-I dephosphorylation. Together, our findings define the WHIP-TRIM14-PPP6C mitochondrial signalosome required for RIG-I-mediated innate antiviral immunity.
Literature context: 488 Thermo Fisher Cat# A-11034; RRID:AB_2576217 Goat anti-Rabbit IgG Alexa Fluo
We define how chronic cigarette smoke-induced time-dependent epigenetic alterations can sensitize human bronchial epithelial cells for transformation by a single oncogene. The smoke-induced chromatin changes include initial repressive polycomb marking of genes, later manifesting abnormal DNA methylation by 10 months. At this time, cells exhibit epithelial-to-mesenchymal changes, anchorage-independent growth, and upregulated RAS/MAPK signaling with silencing of hypermethylated genes, which normally inhibit these pathways and are associated with smoking-related non-small cell lung cancer. These cells, in the absence of any driver gene mutations, now transform by introducing a single KRAS mutation and form adenosquamous lung carcinomas in mice. Thus, epigenetic abnormalities may prime for changing oncogene senescence to addiction for a single key oncogene involved in lung cancer initiation.
Literature context: :1000)Invitrogen (Thermo Fisher)A-11034, A-11037,A-21245Donkey anti-Rab
Dural cerebral veins (CV) are required for cerebrospinal fluid reabsorption and brain homeostasis, but mechanisms that regulate their growth and remodeling are unknown. We report molecular and cellular processes that regulate dural CV development in mammals and describe venous malformations in humans with craniosynostosis and TWIST1 mutations that are recapitulated in mouse models. Surprisingly, Twist1 is dispensable in endothelial cells but required for specification of osteoprogenitor cells that differentiate into preosteoblasts that produce bone morphogenetic proteins (BMPs). Inactivation of Bmp2 and Bmp4 in preosteoblasts and periosteal dura causes skull and CV malformations, similar to humans harboring TWIST1 mutations. Notably, arterial development appears normal, suggesting that morphogens from the skull and dura establish optimal venous networks independent from arterial influences. Collectively, our work establishes a paradigm whereby CV malformations result from primary or secondary loss of paracrine BMP signaling from preosteoblasts and dura, highlighting unique cellular interactions that influence tissue-specific angiogenesis in mammals.
Literature context: 488Thermo Fisher ScientificCat#A11034Chemicals, Peptides, and Recombi
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: conjugateInvitrogenCat#A-11034; RRID: AB_2576217Goat polyclonal anti-mouse, Alex
Astrocytes produce and supply metabolic substrates to neurons through gap junction-mediated astroglial networks. However, the role of astroglial metabolic networks in behavior is unclear. Here, we demonstrate that perturbation of astroglial networks impairs the sleep-wake cycle. Using a conditional Cre-Lox system in mice, we show that knockout of the gap junction subunit connexin 43 in astrocytes throughout the brain causes excessive sleepiness and fragmented wakefulness during the nocturnal active phase. This astrocyte-specific genetic manipulation silenced the wake-promoting orexin neurons located in the lateral hypothalamic area (LHA) by impairing glucose and lactate trafficking through astrocytic networks. This global wakefulness instability was mimicked with viral delivery of Cre recombinase to astrocytes in the LHA and rescued by in vivo injections of lactate. Our findings propose a novel regulatory mechanism critical for maintaining normal daily cycle of wakefulness and involving astrocyte-neuron metabolic interactions.
Literature context: Fisher Scientific Cat# A-11034; RRID:AB_2576217 Goat anti-Rabbit IgG (H+L) High
A genome-wide association study identified LMO1, which encodes an LIM-domain-only transcriptional cofactor, as a neuroblastoma susceptibility gene that functions as an oncogene in high-risk neuroblastoma. Here we show that dβh promoter-mediated expression of LMO1 in zebrafish synergizes with MYCN to increase the proliferation of hyperplastic sympathoadrenal precursor cells, leading to a reduced latency and increased penetrance of neuroblastomagenesis. The transgenic expression of LMO1 also promoted hematogenous dissemination and distant metastasis, which was linked to neuroblastoma cell invasion and migration, and elevated expression levels of genes affecting tumor cell-extracellular matrix interaction, including loxl3, itga2b, itga3, and itga5. Our results provide in vivo validation of LMO1 as an important oncogene that promotes neuroblastoma initiation, progression, and widespread metastatic dissemination.
Literature context: Life Technologies Cat#A11034; RRID:AB_2576217 N-MYC Santa Cruz Biotechnology
While many tumors initially respond to chemotherapy, regrowth of surviving cells compromises treatment efficacy in the long term. The cell-biological basis of this regrowth is not understood. Here, we characterize the response of individual, patient-derived neuroblastoma cells driven by the prominent oncogene MYC to the first-line chemotherapy, doxorubicin. Combining live-cell imaging, cell-cycle-resolved transcriptomics, and mathematical modeling, we demonstrate that a cell's treatment response is dictated by its expression level of MYC and its cell-cycle position prior to treatment. All low-MYC cells enter therapy-induced senescence. High-MYC cells, by contrast, disable their cell-cycle checkpoints, forcing renewed proliferation despite treatment-induced DNA damage. After treatment, the viability of high-MYC cells depends on their cell-cycle position during treatment: newborn cells promptly halt in G1 phase, repair DNA damage, and form re-growing clones; all other cells show protracted DNA repair and ultimately die. These findings demonstrate that fast-proliferating tumor cells may resist cytotoxic treatment non-genetically, by arresting within a favorable window of the cell cycle.
Literature context: nd goat anti-rabbit AF488 (cat# A-11034) were from Molecular Probes (In
Activation of antigen-presenting dendritic cells (DCs) and the complement system are essential early events in the immune defense against invading pathogens. Recently, we and others demonstrated immunological crosstalk between signaling from receptors recognizing complement activation products and PAMPs on DCs. This affects DC effector function, as demonstrated by the finding that C5a prevents induction of pro-inflammatory cytokines by toll-like receptor (TLR) ligands in human monocyte-derived DCs (moDCs). Here, we demonstrate that this regulatory crosstalk is specifically important in 6-sulfo LacNAc dendritic cells (slanDCs), the most pro-inflammatory DC subset found in human. C5aR and TLR signaling show profound interference in the ERK/p38/CREB1 signaling pathways. C5aR signaling accelerates TLR-induced CREB1 phosphorylation both in moDC and slanDC. This is key in the regulatory effect of C5a on pro-inflammatory DC maturation by mediating induction of IL-10, which subsequently inhibits pro-inflammatory cytokine production via negative feedback signaling. Importantly, the regulatory effect of C5a affects T-cell immunity by decreasing Th1 and cytotoxic CD8 T-cell responses. The finding that the pro-inflammatory effector function of slanDC can be down modulated by activation products of the complement system highlights the existence of intricate regulatory interactions between various arms of the immune system. Intensive immune monitoring of patients suffering from complement-mediated diseases or patients receiving complement modulating compounds can give more inside in the contribution of complement receptor and TLR crosstalk in APCs in disease.
Literature context: Alexa488/594/647Molecular ProbesRRID: AB_2576217 / RRID: AB_2534095 / RRID: AB_2
The conserved polarity effector proteins PAR-3, PAR-6, CDC-42, and atypical protein kinase C (aPKC) form a core unit of the PAR protein network, which plays a central role in polarizing a broad range of animal cell types. To functionally polarize cells, these proteins must activate aPKC within a spatially defined membrane domain on one side of the cell in response to symmetry-breaking cues. Using the Caenorhabditis elegans zygote as a model, we find that the localization and activation of aPKC involve distinct, specialized aPKC-containing assemblies: a PAR-3-dependent assembly that responds to polarity cues and promotes efficient segregation of aPKC toward the anterior but holds aPKC in an inactive state, and a CDC-42-dependent assembly in which aPKC is active but poorly segregated. Cycling of aPKC between these distinct functional assemblies, which appears to depend on aPKC activity, effectively links cue-sensing and effector roles within the PAR network to ensure robust establishment of polarity.
Literature context: r Scientific Cat#A-11034/11037; RRID:AB_2576217, AB_2534095
GABAergic interneurons play important roles in cortical circuit development. However, there are multiple populations of interneurons and their respective developmental contributions remain poorly explored. Neuregulin 1 (NRG1) and its interneuron-specific receptor ERBB4 are critical genes for interneuron maturation. Using a conditional ErbB4 deletion, we tested the role of vasoactive intestinal peptide (VIP)-expressing interneurons in the postnatal maturation of cortical circuits in vivo. ErbB4 removal from VIP interneurons during development leads to changes in their activity, along with severe dysregulation of cortical temporal organization and state dependence. These alterations emerge during adolescence, and mature animals in which VIP interneurons lack ErbB4 exhibit reduced cortical responses to sensory stimuli and impaired sensory learning. Our data support a key role for VIP interneurons in cortical circuit development and suggest a possible contribution to pathophysiology in neurodevelopmental disorders. These findings provide a new perspective on the role of GABAergic interneuron diversity in cortical development. VIDEO ABSTRACT.
Literature context: so A11034 RRID:AB_2576217; Goat anti
Sonic hedgehog (SHH) is a glycoprotein associated with development that is also expressed in the adult CNS and released after brain injury. Since the SHH receptors patched homolog-1 and Smoothened are highly expressed on astrocytes, we hypothesized that SHH regulates astrocyte function. Primary mouse cortical astrocytes derived from embryonic Swiss mouse cortices, were treated with two chemically distinct agonists of the SHH pathway, which caused astrocytes to elongate and proliferate. These changes are accompanied by decreases in the major astrocyte glutamate transporter-1 and the astrocyte intermediate filament protein glial fibrillary acidic protein. Multisite electrophysiological recordings revealed that the SHH agonist, smoothened agonist suppressed neuronal firing in astrocyte-neuron co-cultures and this was abolished by the astrocyte metabolic inhibitor ethylfluoroacetate, revealing that SHH stimulation of metabolically active astrocytes influences neuronal firing. Using three-dimensional co-culture, MAP2 western blotting and immunohistochemistry, we show that SHH-stimulated astrocytes protect neurons from kainate-induced cell death. Altogether the results show that SHH regulation of astrocyte function represents an endogenous neuroprotective mechanism.
Literature context: a Fluor 488 Invitrogen A-11034; RRID:AB_2576217 Goat polyclonal anti-guinea pig
The formation and retrieval of a memory is thought to be accomplished by activation and reactivation, respectively, of the memory-holding cells (engram cells) by a common set of neural circuits, but this hypothesis has not been established. The medial temporal-lobe system is essential for the formation and retrieval of episodic memory for which individual hippocampal subfields and entorhinal cortex layers contribute by carrying out specific functions. One subfield whose function is poorly known is the subiculum. Here, we show that dorsal subiculum and the circuit, CA1 to dorsal subiculum to medial entorhinal cortex layer 5, play a crucial role selectively in the retrieval of episodic memories. Conversely, the direct CA1 to medial entorhinal cortex layer 5 circuit is essential specifically for memory formation. Our data suggest that the subiculum-containing detour loop is dedicated to meet the requirements associated with recall such as rapid memory updating and retrieval-driven instinctive fear responses.
Literature context: t antibody Invitrogen A11034; RRID:AB_2576217 Mcm2 (BM28) primary antibody BD
In late mitosis and G1, origins of DNA replication must be "licensed" for use in the upcoming S phase by being encircled by double hexamers of the minichromosome maintenance proteins MCM2-7. A "licensing checkpoint" delays cells in G1 until sufficient origins have been licensed, but this checkpoint is lost in cancer cells. Inhibition of licensing can therefore kill cancer cells while only delaying normal cells in G1. In a high-throughput cell-based screen for licensing inhibitors we identified a family of 2-arylquinolin-4-amines, the most potent of which we call RL5a. The binding of the origin recognition complex (ORC) to origin DNA is the first step of the licensing reaction. We show that RL5a prevents ORC forming a tight complex with DNA that is required for MCM2-7 loading. Formation of this ORC-DNA complex requires ATP, and we show that RL5a inhibits ORC allosterically to mimic a lack of ATP.
Literature context: #A-11034; RRID:AB_2576217 Goat anti-
In this study, we investigated whether intrinsic glial dysfunction contributes to the pathogenesis of schizophrenia (SCZ). Our approach was to establish humanized glial chimeric mice using glial progenitor cells (GPCs) produced from induced pluripotent stem cells derived from patients with childhood-onset SCZ. After neonatal implantation into myelin-deficient shiverer mice, SCZ GPCs showed premature migration into the cortex, leading to reduced white matter expansion and hypomyelination relative to controls. The SCZ glial chimeras also showed delayed astrocytic differentiation and abnormal astrocytic morphologies. When established in myelin wild-type hosts, SCZ glial mice showed reduced prepulse inhibition and abnormal behavior, including excessive anxiety, antisocial traits, and disturbed sleep. RNA-seq of cultured SCZ human glial progenitor cells (hGPCs) revealed disrupted glial differentiation-associated and synaptic gene expression, indicating that glial pathology was cell autonomous. Our data therefore suggest a causal role for impaired glial maturation in the development of schizophrenia and provide a humanized model for its in vivo assessment.
Literature context: bit IgG (H + L) (RRID:AB_2576217), Alexa Fluor 546 goat anti-rab
Impact of stress on diseases including gastrointestinal failure is well-known, but molecular mechanism is not understood. Here we show underlying molecular mechanism using EAE mice. Under stress conditions, EAE caused severe gastrointestinal failure with high-mortality. Mechanistically, autoreactive-pathogenic CD4+ T cells accumulated at specific vessels of boundary area of third-ventricle, thalamus, and dentate-gyrus to establish brain micro-inflammation via stress-gateway reflex. Importantly, induction of brain micro-inflammation at specific vessels by cytokine injection was sufficient to establish fatal gastrointestinal failure. Resulting micro-inflammation activated new neural pathway including neurons in paraventricular-nucleus, dorsomedial-nucleus-of-hypothalamus, and also vagal neurons to cause fatal gastrointestinal failure. Suppression of the brain micro-inflammation or blockage of these neural pathways inhibited the gastrointestinal failure. These results demonstrate direct link between brain micro-inflammation and fatal gastrointestinal disease via establishment of a new neural pathway under stress. They further suggest that brain micro-inflammation around specific vessels could be switch to activate new neural pathway(s) to regulate organ homeostasis.
Literature context: Fisher Scientific Cat# A11034; RRID:AB_2576217 AlexaFluor goat anti-rabbit 594
Appropriate growth and synaptic integration of GABAergic inhibitory interneurons are essential for functional neural circuits in the brain. Here, we demonstrate that disruption of primary cilia function following the selective loss of ciliary GTPase Arl13b in interneurons impairs interneuronal morphology and synaptic connectivity, leading to altered excitatory/inhibitory activity balance. The altered morphology and connectivity of cilia mutant interneurons and the functional deficits are rescued by either chemogenetic activation of ciliary G-protein-coupled receptor (GPCR) signaling or the selective induction of Sstr3, a ciliary GPCR, in Arl13b-deficient cilia. Our results thus define a specific requirement for primary cilia-mediated GPCR signaling in interneuronal connectivity and inhibitory circuit formation.
Literature context: A-11034; RRID:AB_2576217 Alexa Fluo
Assigning behavioral functions to neural structures has long been a central goal in neuroscience and is a necessary first step toward a circuit-level understanding of how the brain generates behavior. Here, we map the neural substrates of locomotion and social behaviors for Drosophila melanogaster using automated machine-vision and machine-learning techniques. From videos of 400,000 flies, we quantified the behavioral effects of activating 2,204 genetically targeted populations of neurons. We combined a novel quantification of anatomy with our behavioral analysis to create brain-behavior correlation maps, which are shared as browsable web pages and interactive software. Based on these maps, we generated hypotheses of regions of the brain causally related to sensory processing, locomotor control, courtship, aggression, and sleep. Our maps directly specify genetic tools to target these regions, which we used to identify a small population of neurons with a role in the control of walking.
Literature context: 37/21244; AB_2576217, RRID: AB_
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: tÂ A-11034Â Goat; polyclonalÂ 1:10Â AB_2576217Â Peptide/Protein TargetÂ Antigen
The cytokine leukemia inhibitory factor (LIF) is essential for rendering the uterus receptive for blastocyst implantation. In mice, LIF receptor expression (LIFR) is largely restricted to the uterine luminal epithelium (LE). LIF, secreted from the endometrial glands (GEs), binds to the LIFR, activating the Janus kinase-signal transducer and activation of transcription (STAT) 3 (Jak-Stat3) signaling pathway in the LE. JAK-STAT activation converts the LE to a receptive state so that juxtaposed blastocysts begin to implant. To specifically delete the LIFR in the LE, we derived a line of mice in which Cre recombinase was inserted into the endogenous lactoferrin gene (Ltf-Cre). Lactoferrin expression in the LE is induced by E2, and we demonstrate that Cre recombinase activity is restricted to the LE and GE. To determine the requirement of the LIFR in implantation, we derived an additional mouse line carrying a conditional (floxed) Lifrflx/flx gene. Crossing Ltf-Cre mice with Lifrflx/flx mice generated Lifrflx/Δ:LtfCre/+ females that were overtly normal but infertile. Many of these females, despite repeated matings, did not become pregnant. Unimplanted blastocysts were recovered from the Lifrflx/Δ:LtfCre/+ uteri and, when transferred to wild-type recipients, implanted normally, indicating that uterine receptivity rather than the embryo's competency is compromised. The loss of Lifr results in both the failure for STAT3 to translocate to the LE nuclei and a reduction in the expression of the LIF regulated gene Msx1 that regulates uterine receptivity. These results reveal that uterine expression of the LIFR is essential for embryo implantation and further define the components of the LIF signaling pathway necessary for effective implantation.
Formin 1 confers actin nucleation by generating long stretches of actin microfilaments to support cell movement, cell shape, and intracellular protein trafficking. Formin 1 is likely involved in microtubule (MT) dynamics due to the presence of a MT binding domain near its N terminus. Here, formin 1 was shown to structurally interact with α-tubulin, the building block of MT, and also end-binding protein 1 (a MT plus [+]-end-binding protein that stabilizes MT) in the testis. Knockdown of formin 1 in Sertoli cells with an established tight junction barrier was found to induce down-regulation of detyrosinated MT (a stabilized form of MT), and disorganization of MTs, in which MTs were retracted from the cell cortical zone, mediated through a loss of MT polymerization and down-regulation of Akt1/2 signaling kinase. An efficient knockdown of formin 1 in the testis reduced the number of track-like structures conferred by MTs and F-actin considerably, causing defects in spermatid and phagosome transport across the seminiferous epithelium. In summary, formin1 maintains MT and F-actin track-like structures to support spermatid and phagosome transport across the seminiferous epithelium during spermatogenesis.
Biologically active vitamin D (1,25-dihydroxycholecalciferol or 1,25(OH)2D) is synthetized from inactive prohormone 25-hydroxycholecalciferol (25(OH)D) by the enzyme CYP27B1 1-α-hydroxylase in kidney and several extrarenal tissues including breast. Although the development of breast cancer has been linked to inadequate vitamin D status, the importance of bioactive vitamin D production within tumors themselves is not fully understood. To investigate the role of tumoral vitamin D production in mammary epithelial cell progression to breast cancer, we conducted a Cre-loxP-mediated Cyp27b1 gene ablation in the mammary epithelium of the polyoma middle T antigen-mouse mammary tumor virus (PyMT-MMTV) mouse breast cancer model. Targeted ablation of Cyp27b1 was accompanied by significant acceleration in initiation of spontaneous mammary tumorigenesis. In vivo, cell proliferation, angiogenesis, cell cycle progression, and survival markers were up-regulated in tumors by Cyp27b1 ablation, and apoptosis was decreased. AK thymoma (AKT) phosphorylation and expression of several components of nuclear factor κB (NF-κB), integrin, and signal transducer and activator of transcription 3 (STAT3) signaling pathways were increased in Cyp27b1-ablated tumors compared with nonablated controls. In vitro, 1,25(OH)2D treatment induced a strong antiproliferative action on tumor cells from both ablated and nonablated mice, accompanied by rapid disappearance of NF-κB p65 from the nucleus and segregation in the cytoplasm. In contrast, treatment with the metabolic precursor 25(OH)D was only effective against cells from nonablated mice. 25(OH)D did not inhibit growth of Cyp27b1-ablated cells, and their nuclear NF-κB p65 remained abundant. Our findings demonstrate that in-tumor CYP27B1 1-α-hydroxylase activity plays a crucial role in controlling early oncogene-mediated mammary carcinogenesis events, at least in part by modulating tumoral cell NF-κB p65 nuclear translocation.
Literature context: A-11034; RRID:AB_2576217 Alexa Fluo
The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent on its Ser727 phosphorylation by CDK1. Upon replication stress, RECQ5 associates with CFSs in early mitosis through its physical interaction with MUS81 and promotes MUS81-dependent mitotic DNA synthesis. RECQ5 depletion or mutational inactivation of its ATP-binding site, RAD51-interacting domain, or phosphorylation site causes excessive binding of RAD51 to CFS loci and impairs CFS expression. This leads to defective chromosome segregation and accumulation of CFS-associated DNA damage in G1 cells. Biochemically, RECQ5 alleviates the inhibitory effect of RAD51 on 3'-flap DNA cleavage by MUS81-EME1 through its RAD51 filament disruption activity. These data suggest that RECQ5 removes RAD51 filaments stabilizing stalled replication forks at CFSs and hence facilitates CFS cleavage by MUS81-EME1.
Literature context: A-11034; RRID:AB_2576217 Alexa 488
Animals rely on dedicated sensory circuits to extract and encode environmental features. How individual neurons integrate and translate these features into behavioral responses remains a major question. Here, we identify a visual projection neuron type that conveys predator approach information to the Drosophila giant fiber (GF) escape circuit. Genetic removal of this input during looming stimuli reveals that it encodes angular expansion velocity, whereas other input cell type(s) encode angular size. Motor program selection and timing emerge from linear integration of these two features within the GF. Linear integration improves size detection invariance over prior models and appropriately biases motor selection to rapid, GF-mediated escapes during fast looms. Our findings suggest feature integration, and motor control may occur as simultaneous operations within the same neuron and establish the Drosophila escape circuit as a model system in which these computations may be further dissected at the circuit level. VIDEO ABSTRACT.
Literature context: #ab18197; RRID:AB_2576217 Mouse mono
Mutations truncating a single copy of the tumor suppressor, BRCA2, cause cancer susceptibility. In cells bearing such heterozygous mutations, we find that a cellular metabolite and ubiquitous environmental toxin, formaldehyde, stalls and destabilizes DNA replication forks, engendering structural chromosomal aberrations. Formaldehyde selectively depletes BRCA2 via proteasomal degradation, a mechanism of toxicity that affects very few additional cellular proteins. Heterozygous BRCA2 truncations, by lowering pre-existing BRCA2 expression, sensitize to BRCA2 haploinsufficiency induced by transient exposure to natural concentrations of formaldehyde. Acetaldehyde, an alcohol catabolite detoxified by ALDH2, precipitates similar effects. Ribonuclease H1 ameliorates replication fork instability and chromosomal aberrations provoked by aldehyde-induced BRCA2 haploinsufficiency, suggesting that BRCA2 inactivation triggers spontaneous mutagenesis during DNA replication via aberrant RNA-DNA hybrids (R-loops). These findings suggest a model wherein carcinogenesis in BRCA2 mutation carriers can be incited by compounds found pervasively in the environment and generated endogenously in certain tissues with implications for public health.
Literature context: n A11034; RRID:AB_2576217 Alexa 546
Mixed-lineage leukemia (MLL), along with multisubunit (WDR5, RbBP5, ASH2L, and DPY30) complex catalyzes the trimethylation of H3K4, leading to gene activation. Here, we characterize a chromatin-independent role for MLL during mitosis. MLL and WDR5 localize to the mitotic spindle apparatus, and loss of function of MLL complex by RNAi results in defects in chromosome congression and compromised spindle formation. We report interaction of MLL complex with several kinesin and dynein motors. We further show that the MLL complex associates with Kif2A, a member of the Kinesin-13 family of microtubule depolymerase, and regulates the spindle localization of Kif2A during mitosis. We have identified a conserved WDR5 interaction (Win) motif, so far unique to the MLL family, in Kif2A. The Win motif of Kif2A engages in direct interactions with WDR5 for its spindle localization. Our findings highlight a non-canonical mitotic function of MLL complex, which may have a direct impact on chromosomal stability, frequently compromised in cancer.
Literature context: s used at a dilution of 1:1000 (RRID:AB_2576217, ThermoFisher A11034).
Monoaminergic neurotransmission is greatly dependent on the function of the vesicular monoamine transporter VMAT2, which is responsible for loading monoamines into secretory vesicles. The role of VMAT2 in histaminergic neurotransmission is poorly understood. We studied the structure and function of the histaminergic system in larval zebrafish following inhibition of VMAT2 function by reserpine. We found that reserpine treatment greatly reduced histamine immunoreactivity in neurons and an almost total disappearance of histamine-containing nerve fibers in the dorsal telencephalon and habenula, the most densely innervated targets of the hypothalamic histamine neurons. The reserpine treated larvae had an impaired histamine-dependent dark-induced flash response seen during the first second after onset of darkness, implying that function of the histaminergic network is VMAT2 dependent. Levels of histamine and other monoamines were decreased in reserpine treated animals. This study provides conclusive evidence of the relevance of VMAT2 in histaminergic neurotransmission, further implying that the storage and release mechanism of neural histamine is comparable to that of other monoamines. Our results also reveal potential new insights about the roles of monoaminergic neurotransmitters in the regulation of locomotion increase during adaptation to darkness.
Literature context: -rabbit AlexaFluor 488 (A11034, RRID:AB_2576217), goat anti-mouse AlexaFluor 55
Resolving the spatial distribution of the human proteome at a subcellular level can greatly increase our understanding of human biology and disease. Here we present a comprehensive image-based map of subcellular protein distribution, the Cell Atlas, built by integrating transcriptomics and antibody-based immunofluorescence microscopy with validation by mass spectrometry. Mapping the in situ localization of 12,003 human proteins at a single-cell level to 30 subcellular structures enabled the definition of the proteomes of 13 major organelles. Exploration of the proteomes revealed single-cell variations in abundance or spatial distribution and localization of about half of the proteins to multiple compartments. This subcellular map can be used to refine existing protein-protein interaction networks and provides an important resource to deconvolute the highly complex architecture of the human cell.
Literature context: bbit 488 (RRID:AB_2576217, 1:800, In
Large scale transitions between active (up) and silent (down) states during quiet wakefulness or NREM sleep regulate fundamental cortical functions and are known to involve both excitatory and inhibitory cells. However, if and how inhibition regulates these activity transitions is unclear. Using fluorescence-targeted electrophysiological recording and cell-specific optogenetic manipulation in both anesthetized and non-anesthetized mice, we found that two major classes of interneurons, the parvalbumin and the somatostatin positive cells, tightly control both up-to-down and down-to-up state transitions. Inhibitory regulation of state transition was observed under both natural and optogenetically-evoked conditions. Moreover, perturbative optogenetic experiments revealed that the inhibitory control of state transition was interneuron-type specific. Finally, local manipulation of small ensembles of interneurons affected cortical populations millimetres away from the modulated region. Together, these results demonstrate that inhibition potently gates transitions between cortical activity states, and reveal the cellular mechanisms by which local inhibitory microcircuits regulate state transitions at the mesoscale.
Literature context: : AB_2576217 (antibody_GTÎ±RBaf488) RRID: AB_
Dopamine- and tyrosine hydroxylase-immunopositive cells (TH cells) modulate visually driven signals as they flow through retinal photoreceptor, bipolar, and ganglion cells. Previous studies suggested that TH cells release dopamine from varicose axons arborizing in the inner and outer plexiform layers after glutamatergic synapses depolarize TH cell dendrites in the inner plexiform layer and these depolarizations propagate to the varicosities. Although it has been proposed that these excitatory synapses are formed onto appendages resembling dendritic spines, spines have not been found on TH cells of most species examined to date or on TH cell somata that release dopamine when exposed to glutamate receptor agonists. By use of protocols that preserve proximal retinal neuron morphology, we have examined the shape, distribution, and synapse-related immunoreactivity of adult rat TH cells. We report here that TH cell somata, tapering and varicose inner plexiform layer neurites, and varicose outer plexiform layer neurites all bear spines, that some of these spines are immunopositive for glutamate receptor and postsynaptic density proteins (viz., GluR1, GluR4, NR1, PSD-95, and PSD-93), that TH cell somata and tapering neurites are also immunopositive for a γ-aminobutyric acid (GABA) receptor subunit (GABAA Rα1 ), and that a synaptic ribbon-specific protein (RIBEYE) is found adjacent to some colocalizations of GluR1 and TH in the inner plexiform layer. These results identify previously undescribed sites at which glutamatergic and GABAergic inputs may stimulate and inhibit dopamine release, especially at somata and along varicose neurites that emerge from these somata and arborize in various levels of the retina. J. Comp. Neurol. 525:1707-1730, 2017. © 2016 Wiley Periodicals, Inc.
Literature context: echnologies, A-11034Â GoatÂ 1:400Â AB_2576217Â Antigen Sequence (if Known)Â Nam
Impaired neurodevelopment in preterm infants is caused by prematurity itself; however, hypoxia/ischemia, inflammation, and hyperoxia contribute to the extent of impairment. Because preterm birth is accompanied by a dramatic decrease in 17β-estradiol (E2) and progesterone, preliminary clinical studies have been carried out to substitute these steroids in preterm infants; however, they failed to confirm significantly improved neurologic outcomes. We therefore hypothesized that the persistently high postnatal production of fetal zone steroids [mainly dehydroepiandrosterone (DHEA)] until term could interfere with E2-mediated protection. We investigated whether E2 could reduce hyperoxia-mediated apoptosis in three immature glial cell types and detected the involved receptors. Thereafter, we investigated protection by the fetal zone steroids DHEA, 16α-hydroxy-DHEA, and androstenediol. For DHEA, the involved receptors were evaluated. We examined aromatases, which convert fetal zone steroids into more estrogenic compounds. Finally, cotreatment was compared against single hormone treatment to investigate synergism. In all cell types, E2 and fetal zone steroids resulted in significant dose-dependent protection, whereas the mediating receptors differed. The neuroprotection by fetal zone steroids highly depended on the cell type-specific expression of aromatases, the receptor repertoire, and the potency of the fetal zone steroids toward these receptors. No synergism in fetal zone steroid and E2 cotreatment was detected in two of three cell types. Therefore, E2 supplementation may not be beneficial with respect to neuroprotection because fetal zone steroids circulate in persistently high concentrations until term in preterm infants. Hence, a refined experimental model for preterm infants is required to investigate potential treatments.
Literature context: s A11034; RRID:AB_2576217 Alexa Fluo
Chromosome segregation during meiosis in oocytes is error prone. The uniquely large cytoplasmic size of oocytes, which provides support for embryogenesis after fertilization, might be a predisposing factor for meiotic errors. However, this hypothesis remains unproven. Here, we show that cytoplasmic size affects the functionality of the acentrosomal spindle. Artificially decreasing the cytoplasmic size in mouse oocytes allows the acentrosomal spindle poles to have a better-focused distribution of microtubule-organizing centers and to biorient chromosomes more efficiently, whereas enlargement of the cytoplasmic size has the opposite effects. Moreover, we found that the cytoplasmic size-dependent dilution of nuclear factors, including anaphase inhibitors that are preformed at the nuclear membrane, limits the spindle's capacity to prevent anaphase entry with misaligned chromosomes. The present study defines a large cytoplasmic volume as a cell-intrinsic feature linked to the error-prone nature of oocytes. This may represent a trade-off between meiotic fidelity and post-fertilization developmental competence.
Literature context: A-11034; RRID:AB_2576217 Goat anti-
Myriad experiences produce transient memory, yet, contingent on the internal state of the organism and the saliency of the experience, only some memories persist over time. How experience and internal state influence the duration of memory at the molecular level remains unknown. A self-assembled aggregated state of Drosophila Orb2A protein is required specifically for long-lasting memory. We report that in the adult fly brain the mRNA encoding Orb2A protein exists in an unspliced non-protein-coding form. The convergence of experience and internal drive transiently increases the spliced protein-coding Orb2A mRNA. A screen identified pasilla, the fly ortholog of mammalian Nova-1/2, as a mediator of Orb2A mRNA processing. A single-nucleotide substitution in the intronic region that reduces Pasilla binding and intron removal selectively impairs long-term memory. We posit that pasilla-mediated processing of unspliced Orb2A mRNA integrates experience and internal state to control Orb2A protein abundance and long-term memory formation.
Literature context: #A-11034; RRID:AB_2576217 Rabbit pol
Skeletal muscle is a major site of postprandial glucose disposal. Inadequate insulin action in skeletal myocytes contributes to hyperglycemia in diabetes. Although glucose is known to stimulate insulin secretion by β cells, whether it directly engages nutrient signaling pathways in skeletal muscle to maintain systemic glucose homeostasis remains largely unexplored. Here we identified the Baf60c-Deptor-AKT pathway as a target of muscle glucose sensing that augments insulin action in skeletal myocytes. Genetic activation of this pathway improved postprandial glucose disposal in mice, whereas its muscle-specific ablation impaired insulin action and led to postprandial glucose intolerance. Mechanistically, glucose triggers KATP channel-dependent calcium signaling, which promotes HDAC5 phosphorylation and nuclear exclusion, leading to Baf60c induction and insulin-independent AKT activation. This pathway is engaged by the anti-diabetic sulfonylurea drugs to exert their full glucose-lowering effects. These findings uncover an unexpected mechanism of glucose sensing in skeletal myocytes that contributes to homeostasis and therapeutic action.
Literature context: , A11034, RRID:AB_2576217), anti-rat
Microtubule-associated proteins (MAPs) are main candidates to stabilize neuronal microtubules, playing an important role in establishing axon-dendrite polarity. However, how MAPs are selectively targeted to specific neuronal compartments remains poorly understood. Here, we show specific localization of microtubule-associated protein 6 (MAP6)/stable tubule-only polypeptide (STOP) throughout neuronal maturation and its role in axonal development. In unpolarized neurons, MAP6 is present at the Golgi complex and in secretory vesicles. As neurons mature, MAP6 is translocated to the proximal axon, where it binds and stabilizes microtubules. Further, we demonstrate that dynamic palmitoylation, mediated by the family of α/β Hydrolase domain-containing protein 17 (ABHD17A-C) depalmitoylating enzymes, controls shuttling of MAP6 between membranes and microtubules and is required for MAP6 retention in axons. We propose a model in which MAP6's palmitoylation mediates microtubule stabilization, allows efficient organelle trafficking, and controls axon maturation in vitro and in situ.
Literature context: ientific; RRID:AB_2576217]. For BrdU
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: # A11034; RRID:AB_2576217 Alexa Fluo
Synaptic refinement is a critical step in nervous system maturation, requiring a carefully timed reorganization and refinement of neuronal connections. We have identified myrf-1 and myrf-2, two C. elegans homologs of Myrf family transcription factors, as key regulators of synaptic rewiring. MYRF-1 and its paralog MYRF-2 are functionally redundant specifically in synaptic rewiring. They co-exist in the same protein complex and act cooperatively to regulate synaptic rewiring. We find that the MYRF proteins localize to the ER membrane and that they are cleaved into active N-terminal fragments, which then translocate into the nucleus to drive synaptic rewiring. Overexpression of active forms of MYRF is sufficient to accelerate synaptic rewiring. MYRF-1 and MYRF-2 are the first genes identified to be indispensable for promoting synaptic rewiring in C. elegans. These findings reveal a molecular mechanism underlying synaptic rewiring and developmental circuit plasticity.
Literature context: so A11034 RRID:AB_2576217) or Alexa
Regeneration of skeletal muscle in adults is mediated by satellite stem cells. Accumulation of misfolded proteins triggers endoplasmic reticulum stress that leads to unfolded protein response (UPR). The UPR is relayed to the cell through the activation of PERK, IRE1/XBP1, and ATF6. Here, we demonstrate that levels of PERK and IRE1 are increased in satellite cells upon muscle injury. Inhibition of PERK, but not the IRE1 arm of the UPR in satellite cells inhibits myofiber regeneration in adult mice. PERK is essential for the survival and differentiation of activated satellite cells into the myogenic lineage. Deletion of PERK causes hyper-activation of p38 MAPK during myogenesis. Blocking p38 MAPK activity improves the survival and differentiation of PERK-deficient satellite cells in vitro and muscle formation in vivo. Collectively, our results suggest that the PERK arm of the UPR plays a pivotal role in the regulation of satellite cell homeostasis during regenerative myogenesis.
Literature context: A-11034, RRID:AB_2534092; 1:500), A
Blast traumatic brain injury (bTBI) affects civilians, soldiers, and veterans worldwide and presents significant health concerns. The mechanisms of neurodegeneration following bTBI remain elusive and current therapies are largely ineffective. It is important to better characterize blast-evoked cellular changes and underlying mechanisms in order to develop more effective therapies. In the present study, our group utilized rat organotypic hippocampal slice cultures (OHCs) as an in vitro system to model bTBI. OHCs were exposed to either 138 ± 22 kPa (low) or 273 ± 23 kPa (high) overpressures using an open-ended helium-driven shock tube, or were assigned to sham control group. At 2 hours (h) following injury, we have characterized the astrocytic response to a blast overpressure. Immunostaining against the astrocytic marker glial fibrillary acidic protein (GFAP) revealed acute shearing and morphological changes in astrocytes, including clasmatodendrosis. Moreover, overlap of GFAP immunostaining and propidium iodide (PI) indicated astrocytic death. Quantification of the number of dead astrocytes per counting area in the hippocampal cornu Ammonis 1 region (CA1), demonstrated a significant increase in dead astrocytes in the low- and high-blast, compared to sham control OHCs. However only a small number of GFAP-expressing astrocytes were co-labeled with the apoptotic marker Annexin V, suggesting necrosis as the primary type of cell death in the acute phase following blast exposure. Moreover, western blot analyses revealed calpain mediated breakdown of GFAP. The dextran exclusion additionally indicated membrane disruption as a potential mechanism of acute astrocytic death. Furthermore, although blast exposure did not evoke significant changes in glutamate transporter 1 (GLT-1) expression, loss of GLT-1-expressing astrocytes suggests dysregulation of glutamate uptake following injury. Our data illustrate the profound effect of blast overpressure on astrocytes in OHCs at 2 h following injury and suggest increased calpain activity and membrane disruption as potential underlying mechanisms.
Literature context: so A11034 RRID:AB_2576217 Anti-rabbi
Human pluripotent stem cells (PSCs) provide a unique entry to study species-specific aspects of human disorders such as Alzheimer's disease (AD). However, in vitro culture of neurons deprives them of their natural environment. Here we transplanted human PSC-derived cortical neuronal precursors into the brain of a murine AD model. Human neurons differentiate and integrate into the brain, express 3R/4R Tau splice forms, show abnormal phosphorylation and conformational Tau changes, and undergo neurodegeneration. Remarkably, cell death was dissociated from tangle formation in this natural 3D model of AD. Using genome-wide expression analysis, we observed upregulation of genes involved in myelination and downregulation of genes related to memory and cognition, synaptic transmission, and neuron projection. This novel chimeric model for AD displays human-specific pathological features and allows the analysis of different genetic backgrounds and mutations during the course of the disease.
Literature context: #A-11034; RRID:AB_2576217), Alexa Fl
Microglia are the intrinsic immune sentinels of the central nervous system. Their activation restricts tissue injury and pathogen spread, but in some settings, including viral infection, this response can contribute to cell death and disease. Identifying mechanisms that control microglial responses is therefore an important objective. Using replication-incompetent adenovirus 5 (Ad5)-based vectors as a model, we investigated the mechanisms through which microglia recognize and respond to viral uptake. Transgenic, immunohistochemical, molecular-genetic, and fluorescence imaging approaches revealed that phosphatidylserine (PtdSer) exposure on the outer leaflet of transduced cells triggers their engulfment by microglia through TAM receptor-dependent mechanisms. We show that inhibition of phospholipid scramblase 1 (PLSCR1) activity reduces intracellular calcium dysregulation, prevents PtdSer externalization, and enables months-long protection of vector-transduced, transgene-expressing cells from microglial phagocytosis. Our study identifies PLSCR1 as a potent target through which the innate immune response to viral vectors, and potentially other stimuli, may be controlled.
Literature context: Alexa Fluor 488Life TechnologiesA11034Goat anti-mouse Alexa Fluor 594L
Chromatin regulation is a principal mechanism governing animal development, yet it is unclear to what extent structural changes in chromatin underlie tissue regeneration. Non-mammalian vertebrates such as zebrafish activate cardiomyocyte (CM) division after tissue damage to regenerate lost heart muscle. Here, we generated transgenic zebrafish expressing a biotinylatable H3.3 histone variant in CMs and derived cell-type-specific profiles of histone replacement. We identified an emerging program of putative enhancers that revise H3.3 occupancy during regeneration, overlaid upon a genome-wide reduction of H3.3 from promoters. In transgenic reporter lines, H3.3-enriched elements directed gene expression in subpopulations of CMs. Other elements increased H3.3 enrichment and displayed enhancer activity in settings of injury- and/or Neuregulin1-elicited CM proliferation. Dozens of consensus sequence motifs containing predicted transcription factor binding sites were enriched in genomic regions with regeneration-responsive H3.3 occupancy. Thus, cell-type-specific regulatory programs of tissue regeneration can be revealed by genome-wide H3.3 profiling.
Literature context: A-11034, RRID:AB_2576217 Alexa Fluo
Mechanisms regulating the surveillance and clearance of synaptic proteins are not well understood. Intriguingly, the loss of the presynaptic active zone proteins Piccolo and Bassoon triggers the loss of synaptic vesicles (SVs) and compromises synaptic integrity. Here we report that the destruction of SVs in boutons lacking Piccolo and Bassoon was associated with the induction of presynaptic autophagy, a process that depended on poly-ubiquitination, but not the E3 ubiquitin ligase Siah1. Surprisingly, gain or loss of function (LOF) of Bassoon alone suppressed or enhanced presynaptic autophagy, respectively, implying a fundamental role for Bassoon in the local regulation of presynaptic autophagy. Mechanistically, Bassoon was found to interact with Atg5, an E3-like ligase essential for autophagy, and to inhibit the induction of autophagy in heterologous cells. Importantly, Atg5 LOF as well as targeting an Atg5-binding peptide derived from Bassoon inhibited presynaptic autophagy in boutons lacking Piccolo and Bassoon, providing insights into the molecular mechanisms regulating presynaptic autophagy.
Literature context: (anti-rabbit, catalog #A-11034 RRID:AB_2576217), AlexaFluor-488 (anti-mouse, c
Long-term potentiation (LTP) is an activity-dependent and persistent increase in synaptic transmission. Currently available techniques to measure LTP are time-intensive and require highly specialized expertise and equipment, and thus are not well suited for screening of multiple candidate treatments, even in animal models. To expand and facilitate the analysis of LTP, here we use a flow cytometry-based method to track chemically induced LTP by detecting surface AMPA receptors in isolated synaptosomes: fluorescence analysis of single-synapse long-term potentiation (FASS-LTP). First, we demonstrate that FASS-LTP is simple, sensitive, and models electrically induced LTP recorded in intact circuitries. Second, we conducted FASS-LTP analysis in two well-characterized Alzheimer's disease (AD) mouse models (3xTg and Tg2576) and, importantly, in cryopreserved human AD brain samples. By profiling hundreds of synaptosomes, our data provide the first direct evidence to support the idea that synapses from AD brain are intrinsically defective in LTP. Third, we used FASS-LTP for drug evaluation in human synaptosomes. Testing a panel of modulators of cAMP and cGMP signaling pathways, FASS-LTP identified vardenafil and Bay-73-6691 (phosphodiesterase-5 and -9 inhibitors, respectively) as potent enhancers of LTP in synaptosomes from AD cases. These results indicate that our approach could provide the basis for protocols to study LTP in both healthy and diseased human brains, a previously unattainable goal. SIGNIFICANCE STATEMENT: Learning and memory depend on the ability of synapses to strengthen in response to activity. Long-term potentiation (LTP) is a rapid and persistent increase in synaptic transmission that is thought to be affected in Alzheimer's disease (AD). However, direct evidence of LTP deficits in human AD brain has been elusive, primarily due to methodological limitations. Here, we analyze LTP in isolated synapses from AD brain using a novel approach that allows testing LTP in cryopreserved brain. Our analysis of hundreds of synapses supports the idea that AD-diseased synapses are intrinsically defective in LTP. Further, we identified pharmacological agents that rescue LTP in AD, thus opening up a new avenue for drug screening and evaluation of strategies for alleviating memory impairments.
Literature context: -rabbit AlexaFluor 488InvitrogenA11034Donkey anti-rabbit AlexaFluor 59
The removal of unwanted or damaged mitochondria by autophagy, a process called mitophagy, is essential for key events in development, cellular homeostasis, tumor suppression, and prevention of neurodegeneration and aging. However, the precise mechanisms of mitophagy remain uncertain. Here, we identify the inner mitochondrial membrane protein, prohibitin 2 (PHB2), as a crucial mitophagy receptor involved in targeting mitochondria for autophagic degradation. PHB2 binds the autophagosomal membrane-associated protein LC3 through an LC3-interaction region (LIR) domain upon mitochondrial depolarization and proteasome-dependent outer membrane rupture. PHB2 is required for Parkin-induced mitophagy in mammalian cells and for the clearance of paternal mitochondria after embryonic fertilization in C. elegans. Our findings pinpoint a conserved mechanism of eukaryotic mitophagy and demonstrate a function of prohibitin 2 that may underlie its roles in physiology, aging, and disease.
Literature context: #A-11034; RRID:AB_2576217 Goat anti-
There is growing evidence that stress-coping mechanisms represent tumor cell vulnerabilities that may function as therapeutically beneficial targets. Recent work has delineated an integrated stress adaptation mechanism that is characterized by the formation of cytoplasmic mRNA and protein foci, termed stress granules (SGs). Here, we demonstrate that SGs are markedly elevated in mutant KRAS cells following exposure to stress-inducing stimuli. The upregulation of SGs by mutant KRAS is dependent on the production of the signaling lipid molecule 15-deoxy-delta 12,14 prostaglandin J2 (15-d-PGJ2) and confers cytoprotection against stress stimuli and chemotherapeutic agents. The secretion of 15-d-PGJ2 by mutant KRAS cells is sufficient to enhance SG formation and stress resistance in cancer cells that are wild-type for KRAS. Our findings identify a mutant KRAS-dependent cell non-autonomous mechanism that may afford the establishment of a stress-resistant niche that encompasses different tumor subclones. These results should inform the design of strategies to eradicate tumor cell communities.
Literature context: (RRID:AB_2576217)
When unperturbed, somatic stem cells are poised to affect immediate tissue restoration upon trauma. Yet, little is known regarding the mechanistic basis controlling initial and homeostatic 'scaling' of stem cell pool sizes relative to their target tissues for effective regeneration. Here, we show that TEAD1-expressing skeletal muscle of transgenic mice features a dramatic hyperplasia of muscle stem cells (i.e. satellite cells, SCs) but surprisingly without affecting muscle tissue size. Super-numeral SCs attain a 'normal' quiescent state, accelerate regeneration, and maintain regenerative capacity over several injury-induced regeneration bouts. In dystrophic muscle, the TEAD1 transgene also ameliorated the pathology. We further demonstrate that hyperplastic SCs accumulate non-cell-autonomously via signal(s) from the TEAD1-expressing myofiber, suggesting that myofiber-specific TEAD1 overexpression activates a physiological signaling pathway(s) that determines initial and homeostatic SC pool size. We propose that TEAD1 and its downstream effectors are medically relevant targets for enhancing muscle regeneration and ameliorating muscle pathology.
Adults born preterm are at increased risk of impaired glucose tolerance and diabetes. Late gestation fetuses exposed to high blood glucose concentration also are at increased risk of impaired glucose tolerance as adults. Preterm babies commonly become hyperglycemic and are thus exposed to high blood glucose concentration at an equivalent stage of pancreatic maturation. It is not known whether preterm birth itself, or complications of prematurity, such as hyperglycemia, alter later pancreatic function. To distinguish these, we made singleton preterm lambs hyperglycemic (HYPER) for 12 days after birth with a dextrose infusion and compared them with vehicle-treated preterm and term controls and with HYPER lambs made normoglycemic with an insulin infusion. Preterm birth reduced β-cell mass, apparent by 4 weeks after term and persisting to adulthood (12 mo), and was associated with reduced insulin secretion at 4 months (juvenile) and reduced insulin mRNA expression in adulthood. Hyperglycemia in preterm lambs further down-regulated key pancreatic gene expression in adulthood. These findings indicate that reduced β-cell mass after preterm birth may be an important factor in increased risk of diabetes after preterm birth and may be exacerbated by postnatal hyperglycemia.
Literature context: ntific Cat# A-11034 also A11034 RRID:AB_2576217). For rat neuron immunostaining
The authors discovered some errors regarding reference group labels in Table 2. The corrected table is attached. The authors regret these errors.
During spermatogenesis, the transport of spermatids and the release of sperms at spermiation and the remodeling of the blood-testis barrier (BTB) in the seminiferous epithelium of rat testes require rapid reorganization of the actin-based cytoskeleton. However, the mechanism(s) and the regulatory molecule(s) remain unexplored. Herein we report findings that unfold the functional significance of ezrin in the organization of the testis-specific adherens junction at the spermatid-Sertoli cell interface called apical ectoplasmic specialization (ES) in the adluminal compartment and the Sertoli cell-cell interface known as basal ES at the BTB. Ezrin is expressed at the basal ES/BTB in all stages, except from late VIII to IX, of the epithelial cycle. Its knockdown by RNA interference (RNAi) in vitro perturbs the Sertoli cell tight junction-permeability barrier via a disruption of the actin microfilaments in Sertoli cells, which in turn impeded basal ES protein (eg, N-cadherin) distribution, perturbing the BTB function. These findings were confirmed by a knockdown study in vivo. However, the expression of ezrin at the apical ES is restricted to stage VIII of the cycle and limited only between step 19 spermatids and Sertoli cells. A knockdown of ezrin in vivo by RNAi was found to impede spermatid transport, causing defects in spermiation in which spermatids were embedded deep inside the epithelium, and associated with a loss of spermatid polarity. Also, ezrin was associated with residual bodies and phagosomes, and its knockdown by RNAi in the testis also impeded the transport of residual bodies/phagosomes from the apical to the basal compartment. In summary, ezrin is involved in regulating actin microfilament organization at the ES in rat testes.
Environmental toxicants such as perfluorooctanesulfonate (PFOS) have been implicated in male reproductive dysfunction, including reduced sperm count and semen quality, in humans. However, the underlying mechanism(s) remains unknown. Herein PFOS at 10-20 μM (∼5-10 μg/mL) was found to be more potent than bisphenol A (100 μM) in perturbing the blood-testis barrier (BTB) function by disrupting the Sertoli cell tight junction-permeability barrier without detectable cytotoxicity. We also delineated the underlying molecular mechanism by which PFOS perturbed Sertoli cell BTB function using an in vitro model that mimics the BTB in vivo. First, PFOS perturbed F-actin organization in Sertoli cells, causing truncation of actin filaments at the BTB. Thus, the actin-based cytoskeleton was no longer capable of supporting the distribution and/or localization of actin-regulatory and adhesion proteins at the cell-cell interface necessary to maintain BTB integrity. Second, PFOS was found to perturb inter-Sertoli cell gap junction (GJ) communication based on a dye-transfer assay by down-regulating the expression of connexin-43, a GJ integral membrane protein. Third, phosphorylated focal adhesion kinase (FAK)-Tyr(407) was found to protect the BTB from the destructive effects of PFOS as shown in a study via an overexpression of an FAK Y407E phosphomimetic mutant. Also, transfection of Sertoli cells with an FAK-specific microRNA, miR-135b, to knock down the expression of phosphorylated FAK-Tyr(407) was found to worsen PFOS-mediated Sertoli cell tight junction disruption. In summary, PFOS-induced BTB disruption is mediated by down-regulating phosphorylated FAK-Tyr(407) and connexin-43, which in turn perturbed F-actin organization and GJ-based intercellular communication, leading to mislocalization of actin-regulatory and adhesion proteins at the BTB.
GH deficiency is thought to be involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, therapy with GH and/or IGF-I has not shown benefit. To gain a better understanding of the role of GH secretion in ALS pathogenesis, we assessed endogenous GH secretion in wild-type and hSOD1(G93A) mice throughout the course of ALS disease. Male wild-type and hSOD1(G93A) mice were studied at the presymptomatic, onset, and end stages of disease. To assess the pathological features of disease, we measured motor neuron number and neuromuscular innervation. We report that GH secretion profile varies at different stages of disease progression in hSOD1(G93A) mice; compared with age-matched controls, GH secretion is unchanged prior to the onset of disease symptoms, elevated at the onset of disease symptoms, and reduced at the end stage of disease. In hSOD1(G93A) mice at the onset of disease, GH secretion is positively correlated with the percentage of neuromuscular innervation but not with motor neuron number. Moreover, this occurs in parallel with an elevation in the expression of muscle IGF-I relative to controls. Our data imply that increased GH secretion at symptom onset may be an endogenous endocrine response to increase the local production of muscle IGF-I to stimulate reinnervation of muscle, but that in the latter stages of disease this response no longer occurs.