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Goat Anti-Rabbit IgG (H+L) Antibody, Alexa Fluor ?? 568 Conjugated

RRID:AB_143157

Antibody ID

AB_143157

Target Antigen

Rabbit IgG (H+L) rabbit

Proper Citation

(Molecular Probes Cat# A-11011, RRID:AB_143157)

Clonality

unknown

Comments

Discontinued; This product offered by Molecular Probes (Invitrogen), now part of Thermo Fisher:

Host Organism

goat

Vendor

Molecular Probes

Cat Num

A-11011 also A11011, A 11011

MTSS1 Regulation of Actin-Nucleating Formin DAAM1 in Dendritic Filopodia Determines Final Dendritic Configuration of Purkinje Cells.

  • Kawabata Galbraith K
  • Cell Rep
  • 2018 Jul 3

Literature context:


Abstract:

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.

Funding information:
  • Breast Cancer Now - 2012NOVSP024(United Kingdom)

Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency.

  • Stein CS
  • Cell Rep
  • 2018 Jun 26

Literature context:


Abstract:

Mitochondria are composed of many small proteins that control protein synthesis, complex assembly, metabolism, and ion and reactive oxygen species (ROS) handling. We show that a skeletal muscle- and heart-enriched long non-coding RNA, LINC00116, encodes a highly conserved 56-amino-acid microprotein that we named mitoregulin (Mtln). Mtln localizes to the inner mitochondrial membrane, where it binds cardiolipin and influences protein complex assembly. In cultured cells, Mtln overexpression increases mitochondrial membrane potential, respiration rates, and Ca2+ retention capacity while decreasing mitochondrial ROS and matrix-free Ca2+. Mtln-knockout mice display perturbations in mitochondrial respiratory (super)complex formation and activity, fatty acid oxidation, tricarboxylic acid (TCA) cycle enzymes, and Ca2+ retention capacity. Blue-native gel electrophoresis revealed that Mtln co-migrates alongside several complexes, including the complex I assembly module, complex V, and supercomplexes. Under denaturing conditions, Mtln remains in high-molecular-weight complexes, supporting its role as a sticky molecular tether that enhances respiratory efficiency by bolstering protein complex assembly and/or stability.

Funding information:
  • NIGMS NIH HHS - GM034869(United States)

Collision of Expanding Actin Caps with Actomyosin Borders for Cortical Bending and Mitotic Rounding in a Syncytium.

  • Zhang Y
  • Dev. Cell
  • 2018 Jun 4

Literature context:


Abstract:

The early Drosophila embryo is a large syncytial cell that compartmentalizes mitotic spindles with furrows. Before furrow ingression, an Arp2/3 actin cap forms above each nucleus and is encircled by actomyosin. We investigated how these networks transform a flat cortex into a honeycomb-like compartmental array. The growing caps circularize and ingress upon meeting their actomyosin borders, which become the furrow base. Genetic perturbations indicate that the caps physically displace their borders and, reciprocally, that the borders resist and circularize their caps. These interactions create an actomyosin cortex arrayed with circular caps. The Rac-GEF Sponge, Rac-GTP, Arp3, and actin coat the caps as a growing material that can drive cortical bending for initial furrow ingression. Additionally, laser ablations indicate that actomyosin contraction squeezes the cytoplasm, producing counterforces that swell the caps. Thus, Arp2/3 caps form clearances of the actomyosin cortex and control buckling and swelling of these clearances for metaphase compartmentalization.

Funding information:
  • NIGMS NIH HHS - R01 GM099773(United States)

Selective Loss of PARG Restores PARylation and Counteracts PARP Inhibitor-Mediated Synthetic Lethality.

  • Gogola E
  • Cancer Cell
  • 2018 Jun 11

Literature context:


Abstract:

Inhibitors of poly(ADP-ribose) (PAR) polymerase (PARPi) have recently entered the clinic for the treatment of homologous recombination (HR)-deficient cancers. Despite the success of this approach, drug resistance is a clinical hurdle, and we poorly understand how cancer cells escape the deadly effects of PARPi without restoring the HR pathway. By combining genetic screens with multi-omics analysis of matched PARPi-sensitive and -resistant Brca2-mutated mouse mammary tumors, we identified loss of PAR glycohydrolase (PARG) as a major resistance mechanism. We also found the presence of PARG-negative clones in a subset of human serous ovarian and triple-negative breast cancers. PARG depletion restores PAR formation and partially rescues PARP1 signaling. Importantly, PARG inactivation exposes vulnerabilities that can be exploited therapeutically.

Funding information:
  • NIGMS NIH HHS - R01 GM061835(United States)

Recovery of "Lost" Infant Memories in Mice.

  • Guskjolen A
  • Curr. Biol.
  • 2018 Jun 26

Literature context:


Abstract:

Hippocampus-dependent, event-related memories formed in early infancy in human and non-human animals are rapidly forgotten. Recently we found that high levels of hippocampal neurogenesis contribute to accelerated rates of forgetting during infancy. Here, we ask whether these memories formed in infancy are permanently erased (i.e., storage failure) or become progressively inaccessible with time (i.e., retrieval failure). To do this, we developed an optogenetic strategy that allowed us to permanently express channelrhodopsin-2 (ChR2) in neuronal ensembles that were activated during contextual fear encoding in infant mice. We then asked whether reactivation of ChR2-tagged ensembles in the dentate gyrus was sufficient for memory recovery in adulthood. We found that optogenetic stimulation of tagged dentate gyrus neurons recovered "lost" infant memories up to 3 months following training and that memory recovery was associated with broader reactivation of tagged hippocampal and cortical neuronal ensembles.

Funding information:
  • NCI NIH HHS - R01CA79548(United States)

Repurposing High-Throughput Image Assays Enables Biological Activity Prediction for Drug Discovery.

  • Simm J
  • Cell Chem Biol
  • 2018 May 17

Literature context:


Abstract:

In both academia and the pharmaceutical industry, large-scale assays for drug discovery are expensive and often impractical, particularly for the increasingly important physiologically relevant model systems that require primary cells, organoids, whole organisms, or expensive or rare reagents. We hypothesized that data from a single high-throughput imaging assay can be repurposed to predict the biological activity of compounds in other assays, even those targeting alternate pathways or biological processes. Indeed, quantitative information extracted from a three-channel microscopy-based screen for glucocorticoid receptor translocation was able to predict assay-specific biological activity in two ongoing drug discovery projects. In these projects, repurposing increased hit rates by 50- to 250-fold over that of the initial project assays while increasing the chemical structure diversity of the hits. Our results suggest that data from high-content screens are a rich source of information that can be used to predict and replace customized biological assays.

Funding information:
  • NIAID NIH HHS - R01 AI15608(United States)
  • NIGMS NIH HHS - R35 GM122547()

Ejaculation Induced by the Activation of Crz Neurons Is Rewarding to Drosophila Males.

  • Zer-Krispil S
  • Curr. Biol.
  • 2018 May 7

Literature context:


Abstract:

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 [5], 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 [5]. 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 [5], 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) [8] 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.

Funding information:
  • NIH HHS - DP2 OD007417(United States)

Role for Wnt Signaling in Retinal Neuropil Development: Analysis via RNA-Seq and In Vivo Somatic CRISPR Mutagenesis.

  • Sarin S
  • Neuron
  • 2018 Apr 4

Literature context:


Abstract:

Screens for genes that orchestrate neural circuit formation in mammals have been hindered by practical constraints of germline mutagenesis. To overcome these limitations, we combined RNA-seq with somatic CRISPR mutagenesis to study synapse development in the mouse retina. Here synapses occur between cellular layers, forming two multilayered neuropils. The outer neuropil, the outer plexiform layer (OPL), contains synapses made by rod and cone photoreceptor axons on rod and cone bipolar dendrites, respectively. We used RNA-seq to identify selectively expressed genes encoding cell surface and secreted proteins and CRISPR-Cas9 electroporation with cell-specific promoters to assess their roles in OPL development. Among the genes identified in this way are Wnt5a and Wnt5b. They are produced by rod bipolars and activate a non-canonical signaling pathway in rods to regulate early OPL patterning. The approach we use here can be applied to other parts of the brain.

Funding information:
  • NHGRI NIH HHS - NIH T32 HG002536(United States)

Rbfox1 Regulates Synaptic Transmission through the Inhibitory Neuron-Specific vSNARE Vamp1.

  • Vuong CK
  • Neuron
  • 2018 Apr 4

Literature context:


Abstract:

Dysfunction of the neuronal RNA binding protein RBFOX1 has been linked to epilepsy and autism spectrum disorders. Rbfox1 loss in mice leads to neuronal hyper-excitability and seizures, but the physiological basis for this is unknown. We identify the vSNARE protein Vamp1 as a major Rbfox1 target. Vamp1 is strongly downregulated in Rbfox1 Nes-cKO mice due to loss of 3' UTR binding by RBFOX1. Cytoplasmic Rbfox1 stimulates Vamp1 expression in part by blocking microRNA-9. We find that Vamp1 is specifically expressed in inhibitory neurons, and that both Vamp1 knockdown and Rbfox1 loss lead to decreased inhibitory synaptic transmission and E/I imbalance. Re-expression of Vamp1 selectively within interneurons rescues the electrophysiological changes in the Rbfox1 cKO, indicating that Vamp1 loss is a major contributor to the Rbfox1 Nes-cKO phenotype. The regulation of interneuron-specific Vamp1 by Rbfox1 provides a paradigm for broadly expressed RNA-binding proteins performing specialized functions in defined neuronal subtypes.

Funding information:
  • NIDDK NIH HHS - DK094311(United States)
  • NIGMS NIH HHS - R01 GM114463()
  • NIGMS NIH HHS - T32 GM007185()
  • NIMH NIH HHS - R01 MH060919()
  • NIMH NIH HHS - R21 MH101684()
  • NINDS NIH HHS - F31 NS093923()

Primary Traumatic Axonopathy in Mice Subjected to Impact Acceleration: A Reappraisal of Pathology and Mechanisms with High-Resolution Anatomical Methods.

  • Ziogas NK
  • J. Neurosci.
  • 2018 Apr 18

Literature context:


Abstract:

Traumatic axonal injury (TAI) is a common neuropathology in traumatic brain injury and is featured by primary injury to axons. Here, we generated TAI with impact acceleration of the head in male Thy1-eYFP-H transgenic mice in which specific populations of neurons and their axons are labeled with yellow fluorescent protein. This model results in axonal lesions in multiple axonal tracts along with blood-brain barrier disruption and neuroinflammation. The corticospinal tract, a prototypical long tract, is severely affected and is the focus of this study. Using optimized CLARITY at single-axon resolution, we visualized the entire corticospinal tract volume from the pons to the cervical spinal cord in 3D and counted the total number of axonal lesions and their progression over time. Our results divulged the presence of progressive traumatic axonopathy that was maximal at the pyramidal decussation. The perikarya of injured corticospinal neurons atrophied, but there was no evidence of neuronal cell death. We also used CLARITY at single-axon resolution to explore the role of the NMNAT2-SARM1 axonal self-destruction pathway in traumatic axonopathy. When we interfered with this pathway by genetically ablating SARM1 or by pharmacological strategies designed to increase levels of Nicotinamide (Nam), a feedback inhibitor of SARM1, we found a significant reduction in the number of axonal lesions early after injury. Our findings show that high-resolution neuroanatomical strategies reveal important features of TAI with biological implications, especially the progressive axonopathic nature of TAI and the role of the NMNAT2-SARM1 pathway in the early stages of axonopathy.SIGNIFICANCE STATEMENT In the first systematic application of novel high-resolution neuroanatomical tools in neuropathology, we combined CLARITY with 2-photon microscopy, optimized for detection of single axonal lesions, to reconstruct the injured mouse brainstem in a model of traumatic axonal injury (TAI) that is a common pathology associated with traumatic brain injury. The 3D reconstruction of the corticospinal tract at single-axon resolution allowed for a more advanced level of qualitative and quantitative understanding of TAI. Using this model, we showed that TAI is an axonopathy with a prominent role of the NMNAT2-SARM1 molecular pathway, that is also implicated in peripheral neuropathy. Our results indicate that high-resolution anatomical models of TAI afford a level of detail and sensitivity that is ideal for testing novel molecular and biomechanical hypotheses.

Funding information:
  • NIDDK NIH HHS - R01 DK083402(United States)

Dopamine receptors in the rat entopeduncular nucleus.

  • Lavian H
  • Brain Struct Funct
  • 2018 Mar 24

Literature context:


Abstract:

Dopamine is critical for the normal functioning of the basal ganglia, modulating both input and output nuclei of this system. The distribution and function of each of the five dopamine receptor subtypes have been studied extensively in the striatum. However, the role of extrastriatal dopamine receptors in basal ganglia information processing is less clear. Here, we studied the anatomical distribution of dopamine receptors in one of the output nuclei of the rodent basal ganglia, the entopeduncular nucleus (EP). The presence of all dopamine receptor subtypes was verified in the EP using immunostaining. We detected co-localization of dopamine receptors with VGAT, which suggests presynaptic expression on GABAergic terminals. D1R and D2R were strongly colocalized with VGAT, whereas DR3-5 showed only sparse co-localization. We further labeled striatal or pallidal neurons with GFP and showed that only D1 receptors were co-localized with striatal terminals, while only D2R and D3R were co-localized with pallidal terminals. Dopamine receptors were also strongly co-localized with MAP2, indicating postsynaptic expression. Overall, these findings suggest that the dopaminergic system modulates activity in the EP both directly via postsynaptic receptors, and indirectly via GABAergic synapses stemming from the direct and indirect pathways.

Funding information:
  • Israel Science Foundation - 138/15()
  • NIDDK NIH HHS - R24 DK064400(United States)
  • Paul Feder foundation - N/A()

Drosophila Fezf coordinates laminar-specific connectivity through cell-intrinsic and cell-extrinsic mechanisms.

  • Peng J
  • Elife
  • 2018 Mar 7

Literature context:


Abstract:

Laminar arrangement of neural connections is a fundamental feature of neural circuit organization. Identifying mechanisms that coordinate neural connections within correct layers is thus vital for understanding how neural circuits are assembled. In the medulla of the Drosophila visual system neurons form connections within ten parallel layers. The M3 layer receives input from two neuron types that sequentially innervate M3 during development. Here we show that M3-specific innervation by both neurons is coordinated by Drosophila Fezf (dFezf), a conserved transcription factor that is selectively expressed by the earlier targeting input neuron. In this cell, dFezf instructs layer specificity and activates the expression of a secreted molecule (Netrin) that regulates the layer specificity of the other input neuron. We propose that employment of transcriptional modules that cell-intrinsically target neurons to specific layers, and cell-extrinsically recruit other neurons is a general mechanism for building layered networks of neural connections.

Funding information:
  • Howard Hughes Medical Institute - Gilliam Fellowship for Advanced Study()
  • NIAID NIH HHS - R21 AI073587(United States)

Inflammation-Modulated Metabolic Reprogramming Is Required for DUOX-Dependent Gut Immunity in Drosophila.

  • Lee KA
  • Cell Host Microbe
  • 2018 Mar 14

Literature context:


Abstract:

DUOX, a member of the NADPH oxidase family, acts as the first line of defense against enteric pathogens by producing microbicidal reactive oxygen species. DUOX is activated upon enteric infection, but the mechanisms regulating DUOX activity remain incompletely understood. Using Drosophila genetic tools, we show that enteric infection results in "pro-catabolic" signaling that initiates metabolic reprogramming of enterocytes toward lipid catabolism, which ultimately governs DUOX homeostasis. Infection induces signaling cascades involving TRAF3 and kinases AMPK and WTS, which regulate TOR kinase to control the balance of lipogenesis versus lipolysis. Enhancing lipogenesis blocks DUOX activity, whereas stimulating lipolysis via ATG1-dependent lipophagy is required for DUOX activation. Drosophila with altered activity in TRAF3-AMPK/WTS-ATG1 pathway components exhibit abolished infection-induced lipolysis, reduced DUOX activation, and enhanced susceptibility to enteric infection. Thus, this work uncovers signaling cascades governing inflammation-induced metabolic reprogramming and provides insight into the pathophysiology of immune-metabolic interactions in the microbe-laden gut epithelia.

Funding information:
  • NIDCD NIH HHS - F31-DC010519(United States)

Juxtanodin in retinal pigment epithelial cells: Expression and biological activities in regulating cell morphology and actin cytoskeleton organization.

  • Liang F
  • J. Comp. Neurol.
  • 2018 Feb 1

Literature context:


Abstract:

Juxtanodin (JN, also known as ermin) was initially identified as an actin cytoskeleton-related oligodendroglial protein in the rat central nervous system. It was subsequently also found in the rat olfactory neuroepithelium, especially at the apical junctional belt of the sustentacular cells. We further examined JN expression and functional roles in the retina using fluorescence histochemistry, confocal microscopy, immuno-electron microscopy, molecular biology, and cell culture. Prominent JN expression was found in the photoreceptor-supporting retinal pigment epithelium (RPE), especially in a zone corresponding to the apices of RPE cells, at the roots of the RPE microvilli, and at the base of RPE cells next to the Bruch's membrane. Partial co-localization of JN immunoreactivity with F-actin (labeled with phalloidin) was observed at the apices and bases of RPE cells. No JN was detected in other cell types of the retina. In cultured human RPE cell line ARPE-19, expression of extrinsic JN up-regulated formation of actin cytoskeleton stress fibers, caused redistribution of more F-actin fibers to the cell periphery, and promoted spreading/enlargement of transfected cells. These findings suggest possible roles of JN in RPE molecular transport, phagocytosis and formation of outer blood-retinal barrier, or possible involvement of JN expression perturbations in pathogenesis of such retinal disorders as proliferative vitreoretinopathy and age-related macular degeneration.

Engineered Tissue Folding by Mechanical Compaction of the Mesenchyme.

  • Hughes AJ
  • Dev. Cell
  • 2018 Jan 22

Literature context:


Abstract:

Many tissues fold into complex shapes during development. Controlling this process in vitro would represent an important advance for tissue engineering. We use embryonic tissue explants, finite element modeling, and 3D cell-patterning techniques to show that mechanical compaction of the extracellular matrix during mesenchymal condensation is sufficient to drive tissue folding along programmed trajectories. The process requires cell contractility, generates strains at tissue interfaces, and causes patterns of collagen alignment around and between condensates. Aligned collagen fibers support elevated tensions that promote the folding of interfaces along paths that can be predicted by modeling. We demonstrate the robustness and versatility of this strategy for sculpting tissue interfaces by directing the morphogenesis of a variety of folded tissue forms from patterns of mesenchymal condensates. These studies provide insight into the active mechanical properties of the embryonic mesenchyme and establish engineering strategies for more robustly directing tissue morphogenesis ex vivo.

Funding information:
  • Cancer Research UK - 11008(United Kingdom)
  • NICHD NIH HHS - DP2 HD080351()
  • NIDCR NIH HHS - R01 DE016402()
  • NIDCR NIH HHS - R01 DE025668()
  • NIH HHS - S10 OD021664()

Orexin-A/hypocretin-1 Immunoreactivity in the Lateral Hypothalamus is Reduced in Genetically Obese but not in Diet-induced Obese Mice.

  • González JA
  • Neuroscience
  • 2018 Jan 15

Literature context:


Abstract:

The mechanisms that link diet and body weight are not fully understood. A diet high in fat often leads to obesity, and this in part is the consequence of diet-induced injury to specific hypothalamic nuclei. It has been suggested that a diet high in fat leads to cell loss in the lateral hypothalamus, which contains specific populations of neurons that are essential for regulating energy homoeostasis; however, we do not know which cell types are affected by the diet. We studied the possibility that high-fat diet leads to a reduction in orexin-A/hypocretin-1 (Hcrt1) and/or melanin-concentrating hormone (MCH) immunoreactivity in the lateral hypothalamus. We quantified immuno-labeled Hcrt1 and MCH cells in brain sections of mice fed a diet high in fat for up to 12 weeks starting at 4 weeks of age and found that this diet did not modify the number of Hcrt1- or MCH-immunoreactive neurons. By contrast, there were fewer Hcrt1- (but not MCH-) immunoreactive cells in genetically obese db/db mice compared to wild-type mice. Non-obese, heterozygous db/+ mice also had fewer Hcrt1-immunoreactive cells. Differences in the number of Hcrt1-immunoreactive cells were only a function of the db genotype but not of diet or body weight. Our findings show that the lateral hypothalamus is affected differently in the db genotype and in diet-induced obesity, and support the idea that not all hypothalamic neurons involved in energy balance regulation are sensitive to the effects of diet.

Neuropeptide Y Regulates Sleep by Modulating Noradrenergic Signaling.

  • Singh C
  • Curr. Biol.
  • 2017 Dec 18

Literature context:


Abstract:

Sleep is an essential and evolutionarily conserved behavioral state whose regulation remains poorly understood. To identify genes that regulate vertebrate sleep, we recently performed a genetic screen in zebrafish, and here we report the identification of neuropeptide Y (NPY) as both necessary for normal daytime sleep duration and sufficient to promote sleep. We show that overexpression of NPY increases sleep, whereas mutation of npy or ablation of npy-expressing neurons decreases sleep. By analyzing sleep architecture, we show that NPY regulates sleep primarily by modulating the length of wake bouts. To determine how NPY regulates sleep, we tested for interactions with several systems known to regulate sleep, and provide anatomical, molecular, genetic, and pharmacological evidence that NPY promotes sleep by inhibiting noradrenergic signaling. These data establish NPY as an important vertebrate sleep/wake regulator and link NPY signaling to an established arousal-promoting system.

Funding information:
  • NIGMS NIH HHS - GM 080571(United States)
  • NINDS NIH HHS - R01 NS070911()
  • NINDS NIH HHS - R01 NS095824()
  • NINDS NIH HHS - R01 NS101158()
  • NINDS NIH HHS - R01 NS101665()

Methylglyoxal Requires AC1 and TRPA1 to Produce Pain and Spinal Neuron Activation.

  • Griggs RB
  • Front Neurosci
  • 2017 Dec 23

Literature context:


Abstract:

Methylglyoxal (MG) is a metabolite of glucose that may contribute to peripheral neuropathy and pain in diabetic patients. MG increases intracellular calcium in sensory neurons and produces behavioral nociception via the cation channel transient receptor potential ankyrin 1 (TRPA1). However, rigorous characterization of an animal model of methylglyoxal-evoked pain is needed, including testing whether methylglyoxal promotes negative pain affect. Furthermore, it remains unknown whether methylglyoxal is sufficient to activate neurons in the spinal cord dorsal horn, whether this requires TRPA1, and if the calcium-sensitive adenylyl cyclase 1 isoform (AC1) contributes to MG-evoked pain. We administered intraplantar methylglyoxal and then evaluated immunohistochemical phosphorylation of extracellular signal-regulated kinase (p-ERK) and multiple pain-like behaviors in wild-type rats and mice and after disruption of either TRPA1 or AC1. Methylglyoxal produced conditioned place avoidance (CPA) (a measure of affective pain), dose-dependent licking and lifting nociceptive behaviors, hyperalgesia to heat and mechanical stimulation, and p-ERK in the spinal cord dorsal horn. TRPA1 knockout or intrathecal administration of a TRPA1 antagonist (HC030031) attenuated methylglyoxal-evoked p-ERK, nociception, and hyperalgesia. AC1 knockout abolished hyperalgesia but not nociceptive behaviors. These results indicate that intraplantar administration of methylglyoxal recapitulates multiple signs of painful diabetic neuropathy found in animal models of or patients with diabetes, including the activation of spinal nociresponsive neurons and the potential involvement of a TRPA1-AC1 sensitization mechanism. We conclude that administration of MG is a valuable model for investigating both peripheral and central components of a MG-TRPA1-AC1 pathway that contribute to painful diabetic neuropathy.

Funding information:
  • NIDA NIH HHS - R01 DA037621()
  • NIGMS NIH HHS - R01 GM101180(United States)
  • NINDS NIH HHS - F31 NS083292()
  • NINDS NIH HHS - R01 NS045954()
  • NINDS NIH HHS - R01 NS062306()
  • NINDS NIH HHS - T32 NS077889()

NOTCH2 Hajdu-Cheney Mutations Escape SCFFBW7-Dependent Proteolysis to Promote Osteoporosis.

  • Fukushima H
  • Mol. Cell
  • 2017 Nov 16

Literature context:


Abstract:

Hajdu-Cheney syndrome (HCS), a rare autosomal disorder caused by heterozygous mutations in NOTCH2, is clinically characterized by acro-osteolysis, severe osteoporosis, short stature, neurological symptoms, cardiovascular defects, and polycystic kidneys. Recent studies identified that aberrant NOTCH2 signaling and consequent osteoclast hyperactivity are closely associated with the bone-related disorder pathogenesis, but the exact molecular mechanisms remain unclear. Here, we demonstrate that sustained osteoclast activity is largely due to accumulation of NOTCH2 carrying a truncated C terminus that escapes FBW7-mediated ubiquitination and degradation. Mice with osteoclast-specific Fbw7 ablation revealed osteoporotic phenotypes reminiscent of HCS, due to elevated Notch2 signaling. Importantly, administration of Notch inhibitors in Fbw7 conditional knockout mice alleviated progressive bone resorption. These findings highlight the molecular basis of HCS pathogenesis and provide clinical insights into potential targeted therapeutic strategies for skeletal disorders associated with the aberrant FBW7/NOTCH2 pathway as observed in patients with HCS.

Funding information:
  • NIMH NIH HHS - R01 MH086920(United States)

Dmrt5, a Novel Neurogenic Factor, Reciprocally Regulates Lhx2 to Control the Neuron-Glia Cell-Fate Switch in the Developing Hippocampus.

  • Muralidharan B
  • J. Neurosci.
  • 2017 Nov 15

Literature context:


Abstract:

Regulation of the neuron-glia cell-fate switch is a critical step in the development of the CNS. Previously, we demonstrated that Lhx2 is a necessary and sufficient regulator of this process in the mouse hippocampal primordium, such that Lhx2 overexpression promotes neurogenesis and suppresses gliogenesis, whereas loss of Lhx2 has the opposite effect. We tested a series of transcription factors for their ability to mimic Lhx2 overexpression and suppress baseline gliogenesis, and also to compensate for loss of Lhx2 and suppress the resulting enhanced level of gliogenesis in the hippocampus. Here, we demonstrate a novel function of Dmrt5/Dmrta2 as a neurogenic factor in the developing hippocampus. We show that Dmrt5, as well as known neurogenic factors Neurog2 and Pax6, can each not only mimic Lhx2 overexpression, but also can compensate for loss of Lhx2 to different extents. We further uncover a reciprocal regulatory relationship between Dmrt5 and Lhx2, such that each can compensate for loss of the other. Dmrt5 and Lhx2 also have opposing regulatory control on Pax6 and Neurog2, indicating a complex bidirectionally regulated network that controls the neuron-glia cell-fate switch.SIGNIFICANCE STATEMENT We identify Dmrt5 as a novel regulator of the neuron-glia cell-fate switch in the developing hippocampus. We demonstrate Dmrt5 to be neurogenic, and reciprocally regulated by Lhx2: loss of either factor promotes gliogenesis; overexpression of either factor suppresses gliogenesis and promotes neurogenesis; each can substitute for loss of the other. Furthermore, each factor has opposing effects on established neurogenic genes Neurog2 and Pax6 Dmrt5 is known to suppress their expression, and we show that Lhx2 is required to maintain it. Our study reveals a complex regulatory network with bidirectional control of a fundamental feature of CNS development, the control of the production of neurons versus astroglia in the developing hippocampus.Finally, we confirm that Lhx2 binds a highly conserved putative enhancer of Dmrt5, suggesting an evolutionarily conserved regulatory relationship between these factors. Our findings uncover a complex network that involves Lhx2, Dmrt5, Neurog2, and Pax6, and that ensures the appropriate amount and timing of neurogenesis and gliogenesis in the developing hippocampus.

Funding information:
  • Howard Hughes Medical Institute - (United States)

Nanos promotes epigenetic reprograming of the germline by down-regulation of the THAP transcription factor LIN-15B.

  • Lee CS
  • Elife
  • 2017 Nov 7

Literature context:


Abstract:

Nanos RNA-binding proteins are required for germline development in metazoans, but the underlying mechanisms remain poorly understood. We have profiled the transcriptome of primordial germ cells (PGCs) lacking the nanos homologs nos-1 and nos-2 in C. elegans. nos-1nos-2 PGCs fail to silence hundreds of transcripts normally expressed in oocytes. We find that this misregulation is due to both delayed turnover of maternal transcripts and inappropriate transcriptional activation. The latter appears to be an indirect consequence of delayed turnover of the maternally-inherited transcription factor LIN-15B, a synMuvB class transcription factor known to antagonize PRC2 activity. PRC2 is required for chromatin reprogramming in the germline, and the transcriptome of PGCs lacking PRC2 resembles that of nos-1nos-2 PGCs. Loss of maternal LIN-15B restores fertility to nos-1nos-2 mutants. These findings suggest that Nanos promotes germ cell fate by downregulating maternal RNAs and proteins that would otherwise interfere with PRC2-dependent reprogramming of PGC chromatin.

KCa2 channel localization and regulation in the axon initial segment.

  • Abiraman K
  • FASEB J.
  • 2017 Nov 29

Literature context:


Abstract:

Small conductance calcium-activated potassium (KCa2) channels are expressed throughout the CNS and play a critical role in synaptic and neuronal excitability. KCa2 channels have a somatodendritic distribution with their highest expression in distal dendrites. It is unclear whether KCa2 channels are specifically present on the axon initial segment (AIS), the site at which action potentials are initiated in neurons. Through a powerful combination of toxin pharmacology, single-molecule atomic force microscopy, and dual-color fluorescence microscopy, we report here that KCa2 channels-predominantly the KCa2.3 subtype-are indeed present on the AIS. We also report that cAMP-PKA controls the axonal KCa2 channel surface expression. Surprisingly, and in contrast to KCa2 channels that were observed in the soma and dendrites, the inhibition of cAMP-PKA increased the surface expression of KCa2 channels without promoting nanoclustering. Lastly, we found that axonal KCa2 channels seem to undergo endocytosis in a dynamin-independent manner, unlike KCa2 channels in the soma and dendrites. Together, these novel results demonstrate that the distribution and membrane recycling of KCa2 channels differs among various neuronal subcompartments.-Abiraman, K., Tzingounis, A. V., Lykotrafitis, G. KCa2 channel localization and regulation in the axon initial segment.

Funding information:
  • NCI NIH HHS - P50CA-58183(United States)
  • NIMH NIH HHS - R21 MH110887()

Optimized method for isolating highly purified and functional porcine aortic endothelial and smooth muscle cells.

  • Beigi F
  • J. Cell. Physiol.
  • 2017 Nov 6

Literature context:


Abstract:

Numerous protocols exist for isolating aortic endothelial and smooth muscle cells from small animals. However, establishing a protocol for isolating pure cell populations from large animal vessels that are more elastic has been challenging. We developed a simple sequential enzymatic approach to isolate highly purified populations of porcine aortic endothelial and smooth muscle cells. The lumen of a porcine aorta was filled with 25 U/ml dispase solution and incubated at 37°C to dissociate the endothelial cells. The smooth muscle cells were isolated by mincing the tunica media of the treated aorta and incubating the pieces in 0.2% and then 0.1% collagenase type I solution. The isolated endothelial cells stained positive for von Willebrand factor, and 97.2% of them expressed CD31. Early and late passage endothelial cells had a population doubling time of 38 hr and maintained a capacity to take up DiI-Ac-LDL and form tubes in Matrigel®. The isolated smooth muscle cells stained highly positive for alpha-smooth muscle actin, and an impurities assessment showed that only 1.8% were endothelial cells. Population doubling time for the smooth muscle cells was ∼70 hr at passages 3 and 7; and the cells positively responded to endothelin-1, as shown by a 66% increase in the intracellular calcium level. This simple protocol allows for the isolation of highly pure populations of endothelial and smooth muscle cells from porcine aorta that can survive continued passage in culture without losing functionality or becoming overgrown by fibroblasts.

A Coincidence Detection Mechanism Controls PX-BAR Domain-Mediated Endocytic Membrane Remodeling via an Allosteric Structural Switch.

  • Lo WT
  • Dev. Cell
  • 2017 Nov 20

Literature context:


Abstract:

Clathrin-mediated endocytosis occurs by bending and remodeling of the membrane underneath the coat. Bin-amphiphysin-rvs (BAR) domain proteins are crucial for endocytic membrane remodeling, but how their activity is spatiotemporally controlled is largely unknown. We demonstrate that the membrane remodeling activity of sorting nexin 9 (SNX9), a late-acting endocytic PX-BAR domain protein required for constriction of U-shaped endocytic intermediates, is controlled by an allosteric structural switch involving coincident detection of the clathrin adaptor AP2 and phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2) at endocytic sites. Structural, biochemical, and cell biological data show that SNX9 is autoinhibited in solution. Binding to PI(3,4)P2 via its PX-BAR domain, and concomitant association with AP2 via sequences in the linker region, releases SNX9 autoinhibitory contacts to enable membrane constriction. Our results reveal a mechanism for restricting the latent membrane remodeling activity of BAR domain proteins to allow spatiotemporal coupling of membrane constriction to the progression of the endocytic pathway.

Funding information:
  • NINDS NIH HHS - R01 NS031718(United States)

Age-related alterations in histone deacetylase expression in Purkinje neurons of ethanol-fed rats.

  • Khurana A
  • Brain Res.
  • 2017 Nov 15

Literature context:


Abstract:

Ethanol and age-induced pathologies of the Purkinje neuron (PN) may result from histone deacetylases (HDACs), enzymes which repress transcription through coiling of the DNA. The purposes of this study were to investigate expression patterns of Class 1 and IIa HDACs in PN and the effects of aging and alcohol on the density of HDACs and histone acetylation in PN. Ninety, eight month old rats (30/diet) were fed a liquid ethanol, liquid control, or rat chow diet for 10, 20, or 40weeks (30/treatment duration). Double immunocytochemical labeling on tissue sections from these rats used antibodies against HDAC isoforms or acetylated histones, and calbindin, a marker for PN. Fluorescent intensities were also measured. Results showed a significant age but not an alcohol-related decrease in the densities of HDACs 2, 3, and 7. In contrast, there were age related-increases in the densities of phosphorylated form of HDAC (4, 5, 7) PN and in PN nuclei expressing HDAC 7. There were also a trend towards ethanol-induced inhibition of acetylation as the density of AH2b PN nuclei and AH3 and AH2b fluorescent intensity was significantly lower in the EF compared to the PF rats. This study presents unique data concerning which HDACs are commonly expressed in PN and indicates that aging rather than lengthy alcohol expression alters expression of the HDACs studied here. These results also suggest that lengthy ethanol consumption may inhibit histone deacetylation in PN.

Channel Nucleoporins Recruit PLK-1 to Nuclear Pore Complexes to Direct Nuclear Envelope Breakdown in C. elegans.

  • Martino L
  • Dev. Cell
  • 2017 Oct 23

Literature context:


Abstract:

In animal cells, nuclear envelope breakdown (NEBD) is required for proper chromosome segregation. Whereas mitotic kinases have been implicated in NEBD, how they coordinate their activity to trigger this event is unclear. Here, we show that both in human cells and Caenorhabditis elegans, the Polo-like kinase 1 (PLK-1) is recruited to the nuclear pore complexes, just prior to NEBD, through its Polo-box domain (PBD). We provide evidence that PLK-1 localization to the nuclear envelope (NE) is required for efficient NEBD. We identify the central channel nucleoporins NPP-1/Nup58, NPP-4/Nup54, and NPP-11/Nup62 as the critical factors anchoring PLK-1 to the NE in C. elegans. In particular, NPP-1, NPP-4, and NPP-11 primed at multiple Polo-docking sites by Cdk1 and PLK-1 itself physically interact with the PLK-1 PBD. We conclude that nucleoporins play an unanticipated regulatory role in NEBD, by recruiting PLK-1 to the NE thereby facilitating phosphorylation of critical downstream targets.

A Circuit Node that Integrates Convergent Input from Neuromodulatory and Social Behavior-Promoting Neurons to Control Aggression in Drosophila.

  • Watanabe K
  • Neuron
  • 2017 Aug 30

Literature context:


Abstract:

Diffuse neuromodulatory systems such as norepinephrine (NE) control brain-wide states such as arousal, but whether they control complex social behaviors more specifically is not clear. Octopamine (OA), the insect homolog of NE, is known to promote both arousal and aggression. We have performed a systematic, unbiased screen to identify OA receptor-expressing neurons (OARNs) that control aggression in Drosophila. Our results uncover a tiny population of male-specific aSP2 neurons that mediate a specific influence of OA on aggression, independent of any effect on arousal. Unexpectedly, these neurons receive convergent input from OA neurons and P1 neurons, a population of FruM+ neurons that promotes male courtship behavior. Behavioral epistasis experiments suggest that aSP2 neurons may constitute an integration node at which OAergic neuromodulation can bias the output of P1 neurons to favor aggression over inter-male courtship. These results have potential implications for thinking about the role of related neuromodulatory systems in mammals.

Funding information:
  • NIMH NIH HHS - MH084020(United States)

The Helicase Aquarius/EMB-4 Is Required to Overcome Intronic Barriers to Allow Nuclear RNAi Pathways to Heritably Silence Transcription.

  • Akay A
  • Dev. Cell
  • 2017 Aug 7

Literature context:


Abstract:

Small RNAs play a crucial role in genome defense against transposable elements and guide Argonaute proteins to nascent RNA transcripts to induce co-transcriptional gene silencing. However, the molecular basis of this process remains unknown. Here, we identify the conserved RNA helicase Aquarius/EMB-4 as a direct and essential link between small RNA pathways and the transcriptional machinery in Caenorhabditis elegans. Aquarius physically interacts with the germline Argonaute HRDE-1. Aquarius is required to initiate small-RNA-induced heritable gene silencing. HRDE-1 and Aquarius silence overlapping sets of genes and transposable elements. Surprisingly, removal of introns from a target gene abolishes the requirement for Aquarius, but not HRDE-1, for small RNA-dependent gene silencing. We conclude that Aquarius allows small RNA pathways to compete for access to nascent transcripts undergoing co-transcriptional splicing in order to detect and silence transposable elements. Thus, Aquarius and HRDE-1 act as gatekeepers coordinating gene expression and genome defense.

Light-Dependent Regulation of Sleep and Wake States by Prokineticin 2 in Zebrafish.

  • Chen S
  • Neuron
  • 2017 Jul 5

Literature context:


Abstract:

Light affects sleep and wake behaviors by providing an indirect cue that entrains circadian rhythms and also by inducing a direct and rapid regulation of behavior. While circadian entrainment by light is well characterized at the molecular level, mechanisms that underlie the direct effect of light on behavior are largely unknown. In zebrafish, a diurnal vertebrate, we found that both overexpression and mutation of the neuropeptide prokineticin 2 (Prok2) affect sleep and wake behaviors in a light-dependent but circadian-independent manner. In light, Prok2 overexpression increases sleep and induces expression of galanin (galn), a hypothalamic sleep-inducing peptide. We also found that light-dependent, Prok2-induced sedation requires prokineticin receptor 2 (prokr2) and is strongly suppressed in galn mutants. These results suggest that Prok2 antagonizes the direct wake-promoting effect of light in zebrafish, in part through the induction of galn expression in the hypothalamus.

Lysosomal Degradation Is Required for Sustained Phagocytosis of Bacteria by Macrophages.

  • Wong CO
  • Cell Host Microbe
  • 2017 Jun 14

Literature context:


Abstract:

Clearance of bacteria by macrophages involves internalization of the microorganisms into phagosomes, which are then delivered to endolysosomes for enzymatic degradation. These spatiotemporally segregated processes are not known to be functionally coupled. Here, we show that lysosomal degradation of bacteria sustains phagocytic uptake. In Drosophila and mammalian macrophages, lysosomal dysfunction due to loss of the endolysosomal Cl- transporter ClC-b/CLCN7 delayed degradation of internalized bacteria. Unexpectedly, defective lysosomal degradation of bacteria also attenuated further phagocytosis, resulting in elevated bacterial load. Exogenous application of bacterial peptidoglycans restored phagocytic uptake in the lysosomal degradation-defective mutants via a pathway requiring cytosolic pattern recognition receptors and NF-κB. Mammalian macrophages that are unable to degrade internalized bacteria also exhibit compromised NF-κB activation. Our findings reveal a role for phagolysosomal degradation in activating an evolutionarily conserved signaling cascade, which ensures that continuous uptake of bacteria is preceded by lysosomal degradation of microbes.

Funding information:
  • NIH HHS - P40 OD018537()
  • NINDS NIH HHS - R01 NS081301()
  • NINDS NIH HHS - R21 NS094860()

Homeostatic Plasticity Shapes Cell-Type-Specific Wiring in the Retina.

  • Tien NW
  • Neuron
  • 2017 May 3

Literature context:


Abstract:

Convergent input from different presynaptic partners shapes the responses of postsynaptic neurons. Whether developing postsynaptic neurons establish connections with each presynaptic partner independently or balance inputs to attain specific responses is unclear. Retinal ganglion cells (RGCs) receive convergent input from bipolar cell types with different contrast responses and temporal tuning. Here, using optogenetic activation and pharmacogenetic silencing, we found that type 6 bipolar (B6) cells dominate excitatory input to ONα-RGCs. We generated mice in which B6 cells were selectively removed from developing circuits (B6-DTA). In B6-DTA mice, ONα-RGCs adjusted connectivity with other bipolar cells in a cell-type-specific manner. They recruited new partners, increased synapses with some existing partners, and maintained constant input from others. Patch-clamp recordings revealed that anatomical rewiring precisely preserved contrast and temporal frequency response functions of ONα-RGCs, indicating that homeostatic plasticity shapes cell-type-specific wiring in the developing retina to stabilize visual information sent to the brain.

Funding information:
  • NEI NIH HHS - R01 EY023341()
  • NEI NIH HHS - R01 EY026978()
  • NEI NIH HHS - R01 EY027411()

A PTEN-Regulated Checkpoint Controls Surface Delivery of δ Opioid Receptors.

  • Shiwarski DJ
  • J. Neurosci.
  • 2017 Apr 5

Literature context:


Abstract:

The δ opioid receptor (δR) is a promising alternate target for pain management because δR agonists show decreased abuse potential compared with current opioid analgesics that target the μ opioid receptor. A critical limitation in developing δR as an analgesic target, however, is that δR agonists show relatively low efficacy in vivo, requiring the use of high doses that often cause adverse effects, such as convulsions. Here we tested whether intracellular retention of δR in sensory neurons contributes to this low δR agonist efficacy in vivo by limiting surface δR expression. Using direct visualization of δR trafficking and localization, we define a phosphatase and tensin homolog (PTEN)-regulated checkpoint that retains δR in the Golgi and decreases surface delivery in rat and mice sensory neurons. PTEN inhibition releases δR from this checkpoint and stimulates delivery of exogenous and endogenous δR to the neuronal surface both in vitro and in vivo PTEN inhibition in vivo increases the percentage of TG neurons expressing δR on the surface and allows efficient δR-mediated antihyperalgesia in mice. Together, we define a critical role for PTEN in regulating the surface delivery and bioavailability of the δR, explain the low efficacy of δR agonists in vivo, and provide evidence that active δR relocation is a viable strategy to increase δR antinociception.SIGNIFICANCE STATEMENT Opioid analgesics, such as morphine, which target the μ opioid receptor (μR), have been the mainstay of pain management, but their use is highly limited by adverse effects and their variable efficacy in chronic pain. Identifying alternate analgesic targets is therefore of great significance. Although the δ opioid receptor (δR) is an attractive option, a critical limiting factor in developing δR as a target has been the low efficacy of δR agonists. Why δR agonists show low efficacy is still under debate. This study provides mechanistic and functional data that intracellular localization of δR in neurons is a key factor that contributes to low agonist efficacy, and presents a proof of mechanism that relocating δR improves efficacy.

Funding information:
  • NIDA NIH HHS - R00 DA031243()

Fatty Acids Regulate Germline Sex Determination through ACS-4-Dependent Myristoylation.

  • Tang H
  • Cell
  • 2017 Apr 20

Literature context:


Abstract:

Fat metabolism has been linked to fertility and reproductive adaptation in animals and humans, and environmental sex determination potentially plays a role in the process. To investigate the impact of fatty acids (FA) on sex determination and reproductive development, we examined and observed an impact of FA synthesis and mobilization by lipolysis in somatic tissues on oocyte fate in Caenorhabditis elegans. The subsequent genetic analysis identified ACS-4, an acyl-CoA synthetase and its FA-CoA product, as key germline factors that mediate the role of FA in promoting oocyte fate through protein myristoylation. Further tests indicated that ACS-4-dependent protein myristoylation perceives and translates the FA level into regulatory cues that modulate the activities of MPK-1/MAPK and key factors in the germline sex-determination pathway. These findings, including a similar role of ACS-4 in a male/female species, uncover a likely conserved mechanism by which FA, an environmental factor, regulates sex determination and reproductive development.

The Drosophila Postsynaptic DEG/ENaC Channel ppk29 Contributes to Excitatory Neurotransmission.

  • Hill A
  • J. Neurosci.
  • 2017 Mar 22

Literature context:


Abstract:

The protein family of degenerin/epithelial sodium channels (DEG/ENaCs) is composed of diverse animal-specific, non-voltage-gated ion channels that play important roles in regulating cationic gradients across epithelial barriers. Some family members are also enriched in neural tissues in both vertebrates and invertebrates. However, the specific neurophysiological functions of most DEG/ENaC-encoding genes remain poorly understood. The fruit fly Drosophila melanogaster is an excellent model for deciphering the functions of DEG/ENaC genes because its genome encodes an exceptionally large number of DEG/ENaC subunits termed pickpocket (ppk) 1-31 Here we demonstrate that ppk29 contributes specifically to the postsynaptic modulation of excitatory synaptic transmission at the larval neuromuscular junction. Electrophysiological data indicate that the function of ppk29 in muscle is necessary for normal postsynaptic responsivity to neurotransmitter release and for normal coordinated larval movement. The ppk29 mutation does not affect gross synaptic morphology and ultrastructure, which indicates that the observed phenotypes are likely due to defects in glutamate receptor function. Together, our data indicate that DEG/ENaC ion channels play a fundamental role in the postsynaptic regulation of excitatory neurotransmission.SIGNIFICANCE STATEMENT Members of the degenerin/epithelial sodium channel (DEG/ENaC) family are broadly expressed in epithelial and neuronal tissues. To date, the neurophysiological functions of most family members remain unknown. Here, by using the power of Drosophila genetics in combination with electrophysiological and behavioral approaches, we demonstrate that the DEG/ENaC-encoding gene pickpocket 29 contributes to baseline neurotransmission, possibly via the modulation of postsynaptic glutamate receptor functionality.

Funding information:
  • Medical Research Council - (United Kingdom)
  • NIAMS NIH HHS - P30 AR057235()
  • NIH HHS - S10 OD021694()
  • NINDS NIH HHS - R01 NS091546()
  • NINDS NIH HHS - R21 NS089834()
  • NINDS NIH HHS - R24 NS086741()

Hallmarks of Alzheimer's Disease in Stem-Cell-Derived Human Neurons Transplanted into Mouse Brain.

  • Espuny-Camacho I
  • Neuron
  • 2017 Mar 8

Literature context:


Abstract:

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.

Transcription factor Emx2 controls stereociliary bundle orientation of sensory hair cells.

  • Jiang T
  • Elife
  • 2017 Mar 7

Literature context:


Abstract:

The asymmetric location of stereociliary bundle (hair bundle) on the apical surface of mechanosensory hair cells (HCs) dictates the direction in which a given HC can respond to cues such as sound, head movements, and water pressure. Notably, vestibular sensory organs of the inner ear, the maculae, exhibit a line of polarity reversal (LPR) across which, hair bundles are polarized in a mirror-image pattern. Similarly, HCs in neuromasts of the zebrafish lateral line system are generated as pairs, and two sibling HCs develop opposite hair bundle orientations. Within these sensory organs, expression of the transcription factor Emx2 is restricted to only one side of the LPR in the maculae or one of the two sibling HCs in neuromasts. Emx2 mediates hair bundle polarity reversal in these restricted subsets of HCs and generates the mirror-image pattern of the sensory organs. Downstream effectors of Emx2 control bundle polarity cell-autonomously via heterotrimeric G proteins.

Small-Molecule Stabilization of 14-3-3 Protein-Protein Interactions Stimulates Axon Regeneration.

  • Kaplan A
  • Neuron
  • 2017 Mar 8

Literature context:


Abstract:

Damaged central nervous system (CNS) neurons have a poor ability to spontaneously regenerate, causing persistent functional deficits after injury. Therapies that stimulate axon growth are needed to repair CNS damage. 14-3-3 adaptors are hub proteins that are attractive targets to manipulate cell signaling. We identify a positive role for 14-3-3s in axon growth and uncover a developmental regulation of the phosphorylation and function of 14-3-3s. We show that fusicoccin-A (FC-A), a small-molecule stabilizer of 14-3-3 protein-protein interactions, stimulates axon growth in vitro and regeneration in vivo. We show that FC-A stabilizes a complex between 14-3-3 and the stress response regulator GCN1, inducing GCN1 turnover and neurite outgrowth. These findings show that 14-3-3 adaptor protein complexes are druggable targets and identify a new class of small molecules that may be further optimized for the repair of CNS damage.

Sex-Dependent Regulation of Aromatase-Mediated Synaptic Plasticity in the Basolateral Amygdala.

  • Bender RA
  • J. Neurosci.
  • 2017 Feb 8

Literature context:


Abstract:

The basolateral amygdala (BLA) integrates sensory input from cortical and subcortical regions, a function that requires marked synaptic plasticity. Here we provide evidence that cytochrome P450 aromatase (AROM), the enzyme converting testosterone to 17β-estradiol (E2), contributes to the regulation of this plasticity in a sex-specific manner. We show that AROM is expressed in the BLA, particularly in the basolateral nucleus (BL), in male and female rodents. Systemic administration of the AROM inhibitor letrozole reduced spine synapse density in the BL of adult female mice but not in the BL of male mice. Similarly, in organotypic corticoamygdalar slice cultures from immature rats, treatment with letrozole significantly reduced spine synapses in the BL only in cultures derived from females. In addition, letrozole sex-specifically altered synaptic properties in the BL: in acute slices from juvenile (prepubertal) female rats, wash-in of letrozole virtually abolished long-term potentiation (LTP), whereas it did not prevent the generation of LTP in the slices from males. Together, these data indicate that neuron-derived E2 modulates synaptic plasticity in rodent BLA sex-dependently. As protein expression levels of AROM, estrogen and androgen receptors did not differ between males and females and were not sex-specifically altered by letrozole, the findings suggest sex-specific mechanisms of E2 signaling.SIGNIFICANCE STATEMENT The basolateral amygdala (BLA) is a key structure of the fear circuit. This research reveals a sexually dimorphic regulation of synaptic plasticity in the BLA involving neuronal aromatase, which produces the neurosteroid 17β-estradiol (E2). As male and female neurons in rodent BLA responded differently to aromatase inhibition both in vivo and in vitro, our findings suggest that E2 signaling in BLA neurons is regulated sex-dependently, presumably via mechanisms that have been established during sexual determination. These findings could be relevant for the understanding of sex differences in mood disorders and of the side effects of cytochrome P450 aromatase inhibitors, which are frequently used for breast cancer therapy.

Synaptic Vesicle Endocytosis Occurs on Multiple Timescales and Is Mediated by Formin-Dependent Actin Assembly.

  • Soykan T
  • Neuron
  • 2017 Feb 22

Literature context:


Abstract:

Neurotransmission is based on the exocytic fusion of synaptic vesicles (SVs) followed by endocytic membrane retrieval and the reformation of SVs. Recent data suggest that at physiological temperature SVs are internalized via clathrin-independent ultrafast endocytosis (UFE) within hundreds of milliseconds, while other studies have postulated a key role for clathrin-mediated endocytosis (CME) of SV proteins on a timescale of seconds to tens of seconds. Here we demonstrate using cultured hippocampal neurons as a model that at physiological temperature SV endocytosis occurs on several timescales from less than a second to several seconds, yet, is largely independent of clathrin. Clathrin-independent endocytosis (CIE) of SV membranes is mediated by actin-nucleating formins such as mDia1, which are required for the formation of presynaptic endosome-like vacuoles from which SVs reform. Our results resolve previous discrepancies in the field and suggest that SV membranes are predominantly retrieved via CIE mediated by formin-dependent actin assembly.

Angiotensin-II-induced Muscle Wasting is Mediated by 25-Hydroxycholesterol via GSK3β Signaling Pathway.

  • Shen C
  • EBioMedicine
  • 2017 Feb 5

Literature context:


Abstract:

While angiotensin II (ang II) has been implicated in the pathogenesis of cardiac cachexia (CC), the molecules that mediate ang II's wasting effect have not been identified. It is known TNF-α level is increased in patients with CC, and TNF-α release is triggered by ang II. We therefore hypothesized that ang II induced muscle wasting is mediated by TNF-α. Ang II infusion led to skeletal muscle wasting in wild type (WT) but not in TNF alpha type 1 receptor knockout (TNFR1KO) mice, suggesting that ang II induced muscle loss is mediated by TNF-α through its type 1 receptor. Microarray analysis identified cholesterol 25-hydroxylase (Ch25h) as the down stream target of TNF-α. Intraperitoneal injection of 25-hydroxycholesterol (25-OHC), the product of Ch25h, resulted in muscle loss in C57BL/6 mice, accompanied by increased expression of atrogin-1, MuRF1 and suppression of IGF-1/Akt signaling pathway. The identification of 25-OHC as an inducer of muscle wasting has implications for the development of specific treatment strategies in preventing muscle loss.

A Surveillance Mechanism Ensures Repair of DNA Lesions during Zygotic Reprogramming.

  • Ladstätter S
  • Cell
  • 2016 Dec 15

Literature context:


Abstract:

Sexual reproduction culminates in a totipotent zygote with the potential to produce a whole organism. Sperm chromatin reorganization and epigenetic reprogramming that alter DNA and histone modifications generate a totipotent embryo. Active DNA demethylation of the paternal genome has been proposed to involve base excision and DNA repair-based mechanisms. The nature and consequence of DNA lesions generated during reprogramming are not known. Using mouse genetics and chemical biology, we discovered that Tet3-dependent zygotic reprogramming generates paternal DNA lesions that are monitored by a surveillance mechanism. In vivo structure-function rescue assays revealed that cohesin-dependent repair of paternal DNA lesions prevents activation of a Chk1-dependent checkpoint that delays mitotic entry. Culturing conditions affect checkpoint stringency, which has implications for human in vitro fertilization. We propose the zygotic checkpoint senses DNA lesions generated during paternal DNA demethylation and ensures reprogrammed loci are repaired before mitosis to prevent chromosome fragmentation, embryo loss, and infertility.

Funding information:
  • NHLBI NIH HHS - R01 HL070029(United States)

Distinctive Features of the Human Marginal Zone and Cajal-Retzius Cells: Comparison of Morphological and Immunocytochemical Features at Midgestation.

  • Tkachenko LA
  • Front Neuroanat
  • 2016 Apr 6

Literature context:


Abstract:

Despite a long history of research of cortical marginal zone (MZ) organization and development, a number of issues remain unresolved. One particular issue is the problem of Cajal-Retzius cells (C-R) identification. It is currently based on morphology and Reelin expression. The aim of this research is to investigate MZ cytoarchitectonics and Reelin-producing cells morphotypes in the superior temporal, pre- and postcentral cortex at GW24-26. We used Reelin (Reln) as the marker for C-R cells and microtubule-associated protein 2 (MAP2) and neurofilament heavy chain protein (N200) as markers of neuronal maturation. The MZ of all of the investigated areas had the distinct cytoarchitectonic of alternating cell sparse (MZP, SR) and cell dense (SGL, DGL) layers. The distribution of the neuromarkers across the MZ also showed layer specificity. MAP2-positive cells were only found in the SGL. N200 and Reelin-positive neurons in the MZP. N200-positive processes were forming a plexus at the DGL level. All of the N200-positive neurons found were in the MZP and had distinctive morphological features of C-R cells. All of the N200-positive neurons in MZ were also positive for Reelin, whereas MAP2-positive cells lack Reelin. Thus, the joint use of two immunomarkers allowed us to discern the C-R cells based on their morphotype and neurochemistry and indicate that the Reelin-positive cells of MZ at 24-26 GW were morphologically C-R cells. In the current study, we identified three C-R cells morphotypes. Using a 3D reconstruction, we made sure that all of them belonged to the single morphotype of triangular C-R cells. This approach will allow future studies to separate C-R cells from other Reelin-producing neurons which appear at later corticogenesis stages. In addition, our findings support the assumption that a plexus could be formed not only with C-R cells processes but also possibly by other cell processes by the poorly researched DGL, which is only allocated as a part of the human MZ.

Blocking stroke-induced immunodeficiency increases CNS antigen-specific autoreactivity but does not worsen functional outcome after experimental stroke.

  • Römer C
  • J. Neurosci.
  • 2015 May 20

Literature context:


Abstract:

Stroke-induced immunodepression (SIDS) is an essential cause of poststroke infections. Pharmacological inhibition of SIDS appears promising in preventing life-threatening infections in stroke patients. However, SIDS might represent an adaptive mechanism preventing autoreactive immune responses after stroke. To address this, we used myelin oligodendrocyte glycoprotein (MOG) T-cell receptor transgenic (2D2) mice where >80% of peripheral CD4(+) T cells express a functional receptor for MOG. We investigated in a murine model of middle cerebral artery occlusion the effect of blocking SIDS by inhibiting body's main stress axes, the sympathetic nervous system (SNS) with propranolol and the hypothalamic-pituitary-adrenal axis (HPA) with mifepristone. Blockade of both stress axes robustly reduced infarct volumes, decreased infection rate, and increased long-term survival of 2D2 and C57BL/6J wild-type mice. Despite these protective effects, blockade of SIDS increased CNS antigen-specific Type1 T helper cell (Th1) responses in the brains of 2D2 mice 14 d after middle cerebral artery occlusion. One month after experimental stroke, 2D2 mice developed signs of polyradiculitis, which were diminished by SIDS blockade. Adoptive transfer of CD4(+) T cells, isolated from 2D2 mice, into lymphocyte-deficient Rag-1KO mice did not reveal differences between SIDS blockade and vehicle treatment in functional long-term outcome after stroke. In conclusion, inhibiting SIDS by pharmacological blockade of body's stress axes increases autoreactive CNS antigen-specific T-cell responses in the brain but does not worsen functional long-term outcome after experimental stroke, even in a mouse model where CNS antigen-specific autoreactive T-cell responses are boosted.

Specialized pathways from the primate amygdala to posterior orbitofrontal cortex.

  • Timbie C
  • J. Neurosci.
  • 2014 Jun 11

Literature context:


Abstract:

The primate amygdala sends dense projections to posterior orbitofrontal cortex (pOFC) in pathways that are critical for processing emotional content, but the synaptic mechanisms are not understood. We addressed this issue by investigating pathways in rhesus monkeys (Macaca mulatta) from the amygdala to pOFC at the level of the system and synapse. Terminations from the amygdala were denser and larger in pOFC compared with the anterior cingulate cortex, which is also strongly connected with the amygdala. Axons from the amygdala terminated most densely in the upper layers of pOFC through large terminals. Most of these terminals innervated spines of presumed excitatory neurons and many were frequently multisynaptic and perforated, suggesting high synaptic efficacy. These amygdalar synapses in pOFC exceeded in size and specialization even thalamocortical terminals from the prefrontal-related thalamic mediodorsal nucleus to the middle cortical layers, which are thought to be highly efficient drivers of cortical neurons. Pathway terminals in the upper layers impinge on the apical dendrites of neurons in other layers, suggesting that the robust amygdalar projections may also activate neurons in layer 5 that project back to the amygdala and beyond to autonomic structures. Among inhibitory neurons, the amygdalar pathway innervated preferentially the neurochemical classes of calbindin and calretinin neurons in the upper layers of pOFC, which are synaptically suited to suppress noise and enhance signals. These features provide a circuit mechanism for flexibly shifting focus and adjusting emotional drive in processes disrupted in psychiatric disorders, such as phobias and obsessive-compulsive disorder.

Funding information:
  • NIDCD NIH HHS - R01DC011582(United States)