Search

SummaryIn the developing central nervous system, oligodendrocyte precursor cells (OPCs) differentiate into oligodendrocytes, which form myelin around axons. Oligodendrocytes and myelin are essential for the function of the central nervous system, as evidenced by the severe neurological symptoms that arise in demyelinating diseases such as multiple sclerosis and leukodystrophy. Although many cell-intrinsic mechanisms that regulate oligodendrocyte development and myelination have been reported, it remains unclear whether interactions among oligodendrocyte-lineage cells (OPCs and oligodendrocytes) affect oligodendrocyte development and myelination. Here, we show that blocking vesicle-associated membrane protein (VAMP) 1/2/3-dependent exocytosis from oligodendrocyte-lineage cells impairs oligodendrocyte development, myelination, and motor behavior in mice. Adding oligodendrocyte-lineage cell-secreted molecules to secretion-deficient OPC cultures partially restores the morphological maturation of oligodendrocytes. Moreover, we identified L-type prostaglandin D synthase as an oligodendrocyte-lineage cell-secreted protein that promotes oligodendrocyte development and myelination in vivo. These findings reveal a novel autocrine/paracrine loop model for the regulation of oligodendrocyte and myelin development.

SUMMARYOligodendrocytes are the sole myelin producing cells in the central nervous system. Oligodendrocyte numbers are tightly controlled across diverse brain regions to match local axon type and number, but the underlying mechanisms and functional significance remain unclear. Here, we show that autophagy, an evolutionarily conserved cellular process that promotes cell survival under canonical settings, elicits premyelinating oligodendrocyte apoptosis during development and regulates critical aspects of nerve pulse propagation. Autophagy flux is increased in premyelinating oligodendrocytes, and its genetic blockage causes ectopic oligodendrocyte survival throughout the entire brain. Autophagy acts in the TFEB-Bax/Bak pathway and elevatesPUMAmRNA levels to trigger premyelinating oligodendrocyte apoptosis cell-autonomously. Autophagy continuously functions in the myelinating oligodendrocytes to limit myelin sheath numbers and fine-tune nerve pulse propagation. Our results providein vivoevidence showing that autophagy promotes apoptosis in mammalian cells under physiological conditions and reveal key intrinsic mechanisms governing oligodendrocyte number.HIGHLIGHTSAutophagy flux increases in the premyelinating and myelinating oligodendrocytesAutophagy promotes premyelinating oligodendrocyte (pre-OL) apoptosis to control myelination location and timingAutophagy acts in the TFEB-PUMA-Bax/Bak pathway and elevatesPUMAmRNA levels to determine pre-OL fateAutophagy continuously functions in the myelinating oligodendrocytes to limit myelin sheath thickness and finetune nerve pulse propagation

SummaryMicroglia are increasingly recognized for their major contributions during brain development and neurodegenerative disease. It is currently unknown if these functions are carried out by subsets of microglia during different stages of development and adulthood or within specific brain regions. Here, we performed deep single-cell RNA sequencing (scRNA-seq) of microglia and related myeloid cells sorted from various regions of embryonic, postnatal, and adult mouse brains. We found that the majority of adult microglia with homeostatic signatures are remarkably similar in transcriptomes, regardless of brain region. By contrast, postnatal microglia represent a more heterogeneous population. We discovered that postnatal white matter-associated microglia (WAM) are strikingly different from microglia in other regions and express genes enriched in degenerative disease-associated microglia. These postnatal WAM have distinct amoeboid morphology, are metabolically active, and phagocytose newly formed oligodendrocytes. This scRNA-seq atlas will be a valuable resource for dissecting innate immune functions in health and disease.HighlightsMyeloid scRNA-seq atlas across brain regions and developmental stagesLimited transcriptomic heterogeneity of homeostatic microglia in the adult brainPhase-specific gene sets of proliferating microglia along cell cycle pseudotimePhagocytic postnatal white matter-associated microglia sharing DAM gene signatures

Nervous system function depends on proper myelination for insulation and critical trophic support for axons. Myelination is tightly regulated spatially and temporally, but how it is controlled molecularly remains largely unknown. Here, we identified key molecular mechanisms governing the regional and temporal specificity of central nervous system (CNS) myelination. We show that transcription factor EB (TFEB) is highly expressed by differentiating oligodendrocytes and that its loss causes precocious and ectopic myelination in many parts of the murine brain. TFEB functions cell-autonomously through PUMA induction and Bax/Bak activation to promote programmed cell death of a subset of premyelinating oligodendrocytes, allowing for selective elimination of oligodendrocytes in normally unmyelinated brain regions. This pathway is conserved across diverse brain areas and is critical for myelination timing. Our findings define an oligodendrocyte-intrinsic mechanism underlying the spatiotemporal specificity of CNS myelination, shedding light on how myelinating glia sculpt the nervous system during development.

Most excitatory synapses in the mammalian brain are formed on dendritic spines, and spine density has a profound impact on synaptic transmission, integration, and plasticity. Membrane-associated guanylate kinase (MAGUK) proteins are intracellular scaffolding proteins with well established roles in synapse function. However, whether MAGUK proteins are required for the formation of dendritic spines in vivo is unclear. We isolated a novel disc large-5 (Dlg5) allele in mice, Dlg5(LP), which harbors a missense mutation in the DLG5 SH3 domain, greatly attenuating its ability to interact with the DLG5 GUK domain. We show here that DLG5 is a MAGUK protein that regulates spine formation, synaptogenesis, and synaptic transmission in cortical neurons. DLG5 regulates synaptogenesis by enhancing the cell surface localization of N-cadherin, revealing a key molecular mechanism for regulating the subcellular localization of this cell adhesion molecule during synaptogenesis.

Stratification of retinal neuronal cell bodies and lamination of their processes provide a scaffold upon which neural circuits can be built. However, the molecular mechanisms that direct retinal ganglion cells (RGCs) to resolve into a single-cell retinal ganglion cell layer (GCL) are not well understood. The extracellular matrix protein laminin conveys spatial information that instructs the migration, process outgrowth, and reorganization of GCL cells. Here, we show that the β1-Integrin laminin receptor is required for RGC positioning and reorganization into a single-cell GCL layer. β1-Integrin signaling within migrating GCL cells requires Cas signaling-adaptor proteins, and in the absence of β1-Integrin or Cas function retinal neurons form ectopic cell clusters beyond the inner limiting membrane (ILM), phenocopying laminin mutants. These data reveal a novel and essential role for Cas adaptor proteins in β1-Integrin-mediated signaling events critical for the formation of the single-cell GCL in the mammalian retina.

Neurons receive input from the outside world or from other neurons through neuronal receptive endings (NREs). Glia envelop NREs to create specialized microenvironments; however, glial functions at these sites are poorly understood. Here we report a molecular mechanism by which glia control NRE shape and associated animal behavior. The C. elegans AMsh glial cell ensheathes NREs of twelve neurons, including the thermosensory neuron AFD. KCC-3, a K/Cl transporter, localizes specifically to a glial microdomain surrounding AFD receptive-ending microvilli, where it regulates K+ and Cl− levels. We find that Cl− ions function as direct inhibitors of an NRE-localized receptor-guanylyl-cyclase, GCY-8, which synthesizes cGMP. High cGMP mediates the effects of glial KCC-3 on AFD shape by antagonizing the actin-regulator WSP-1/NWASP. Components of this pathway are broadly expressed throughout the nervous system, suggesting that ionic regulation of the NRE microenvironment may be a conserved mechanism by which glia control neuron shape and function.

In the vertebrate retina, neuronal circuitry required for visual perception is organized within specific laminae. Photoreceptors convey external visual information to bipolar and horizontal cells at triad ribbon synapses established within the outer plexiform layer (OPL), initiating retinal visual processing. However, the molecular mechanisms that organize these three classes of neuronal processes within the OPL, thereby ensuring appropriate ribbon synapse formation, remain largely unknown. Here we show that mice with null mutations inSema6AorPlexinA4(PlexA4) exhibit a pronounced defect in OPL stratification of horizontal cell axons without any apparent deficits in bipolar cell dendrite or photoreceptor axon targeting. Furthermore, these mutant horizontal cells exhibit aberrant dendritic arborization and reduced dendritic self-avoidance within the OPL. Ultrastructural analysis shows that the horizontal cell contribution to rod ribbon synapse formation inPlexA4−/−retinas is disrupted. These findings define molecular components required for outer retina lamination and ribbon synapse formation.

Tyrosine phosphorylation mediates multiple signal transduction pathways that play key roles in developmental processes and behavioral plasticity. The level of tyrosine phosphorylation is regulated by protein tyrosine kinases and protein tyrosine phosphatases (PTPs). Extensive studies have investigated the roles of tyrosine kinases in memory formation. However, there were few studies on PTPs. To date, learning has been shown to be defective only for mouse knock-outs of PTPα, leukocyte common antigen-related, or PTPδ. A major limitation of these studies arises from their inability to distinguish an acute (biochemical) impairment of memory formation from a more chronic abnormality in neurodevelopment. From a behavioral screen for defective long-term memory, we foundchimutants to disrupt expression of the PTP10D protein tyrosine phosphatase gene. We show thatchimutants are normal for learning, early memory, and anesthesia-resistant memory, whereas long-term memory specifically is abolished. Significantly, induction of a heat shock-PTP10D+transgene before training fully rescues the memory defect ofchimutants, thereby demonstrating an acute role for PTP10D in behavioral plasticity. We show that PTP10D is widely expressed in the embryonic CNS and in the adult brain. Transgenic expression of upstream activating sequence-PTP10D+in mushroom bodies is sufficient to rescue the memory defect ofchimutants. Our data clearly demonstrate that signaling through PTP10D in mushroom bodies is critical for the formation of long-term memory.

Introduction Direction-selective responses to visual cues depend upon precise connectivity between inhibitory starburst amacrine cells (SACs) and direction-selective ganglion cells (DSGCs). Motion is detected by SAC responses to illumination onset (On) or cessation (Off). On and Off SACs costratify in the inner plexiform layer of the vertebrate retina with distinct DSGC dendritic arborizations that mediate On or Off directional responses. Here, we study the molecular mechanisms that specify On versus Off SACs and the signaling pathways governing the functional assembly of murine retinal direction-selective circuitry. We show that signaling between the transmembrane guidance cue semaphorin 6A (Sema6A) and its receptor plexinA2 (PlexA2) regulates dendritic morphology of On but not Off SACs, thereby controlling direction-selective responses to visual stimuli. Development of direction-selective circuitry. On and Off mouse SACs normally stratify in discrete layers (top left) and exhibit radial dendrite morphology (top right). Sema6A and its PlexA2 receptor are expressed in On SACs, but only PlexA2 is expressed in Off SACs. In Sema6A mutants, SACs fail to stratify (bottom left) and On SACs are misshapen (bottom right), compromising responses to “light on” directional cues. Methods We analyzed SAC stratification in the inner plexiform layer, and dendritic morphology of individual On and Off SACs, throughout retinal development in Sema6A and PlexA2 mutant mice. We used dissociated retinal cultures to determine how neurites from genetically labeled SACs respond to exogenous Sema6A. We determined the light-evoked excitatory and inhibitory responses of Sema6A–/– On SACs. Finally, we analyzed direction-selective responses in isolated retinas by On-Off direction-selective ganglion cells. Results Sema6A is expressed in On, but not Off, SACs, whereas PlexA2 is expressed in all SACs. In vitro, exogenous Sema6A repels neurites only from PlexA2+, Sema6A– SACs, the in vivoexpression profile of Off SACs.In PlexA2–/– or Sema6A–/–retinas, SAC dendritic stratifications fail to completely segregate from each other; therefore, in vitro and in vivo observations suggest that repulsive interactions between Sema6A and PlexA2 mediate SAC dendritic stratification. Analysis of dendritic morphology in individual SACs in the x-y plane reveals that On SACs in PlexA2–/– and Sema6A–/– mutants are missing extensive portions of their dendritic fields, have asymmetric dendritic arbors, and exhibit self-avoidance defects; Off SACs in these mutants have normal x-y plane dendritic arbors. Specific On-Off bistratified direction-selective ganglion cells in Sema6A–/–mutant retinas exhibit decreased tuning of On-directional motion responses, whereas Off responses in these same cells are unaffected, correlating the elaboration of symmetric SAC dendritic morphology and asymmetric responses to motion. Discussion Our findings show that, in addition to contributing to the separation between On and Off SAC dendritic stratifications into distinct inner plexiform layer laminae, Sema6A-PlexA2 signaling selectively regulates the elaboration of symmetric On SAC dendritic fields. Disruption of Sema6A-PlexA2 signaling ultimately results in compromised On, but not Off, directional tuning in a subclass of On-Off direction-selective ganglion cells. Our elucidation of molecular events critical for functional assembly of retinal direction-selective circuitry may have general implications for understanding the establishment of circuitry in which individual neurons participate in multiple distinct pathways.

In the vertebrate retina, the formation of neural circuits within discrete laminae is critical for the establishment of retinal visual function. Precise formation of retinal circuits requires the coordinated actions of adhesive and repulsive molecules, including repulsive transmembrane semaphorins (Sema6A, Sema5A, and Sema5B). These semaphorins signal through different Plexin A (PlexA) receptors, thereby regulating distinct aspects of retinal circuit assembly. Here, we investigate the physiological roles of three Class 6 transmembrane semaphorins (Sema6B, Sema6C, and Sema6D), previously identified as PlexA receptor ligands in non-retinal tissues, in mammalian retinal development. We performed expression analysis and also phenotypic analyses of mice that carry null mutations in each of genes encoding these proteins using a broad range of inner and outer retinal markers. We find that these Class 6 semaphorins are uniquely expressed throughout postnatal retinal development in specific domains and cell types of the developing retina. However, we do not observe defects in stereotypical lamina-specific neurite stratification of retinal neuron subtypes in Sema6B-/- or Sema6C-/-; Sema6D-/- retinas. These findings indicate these Class 6 transmembrane semaphorins are unlikely to serve as major PlexA receptor ligands for the assembly of murine retinal circuit laminar organization.