Glia

Astrocytes are the most abundant glial cells in the central nervous system and interact with other cell types, including neurons and microglia, via Gq protein-coupled receptors (Gq GPCRs) present on their surface. Astrocytic Gq GPCR activation induces Ca2+ release from internal stores, leading to intracellular Ca2+ elevations. There is emerging evidence supporting that astrocytic Gq GPCR Ca2+ elevations are upregulated and dysregulated in neurodegenerative diseases and are thought to play an important role in the pathogenesis of such diseases. Furthermore, astrocytic Gq GPCR Ca2+-dependent release of neuroactive or inflammatory molecules from astrocytes may occur in the early steps of the stress/inflammatory process in the diseased brain. In addition, low grade and chronic brain inflammation is involved in the etiology of neurodegenerative diseases. We hypothesized that chronic activation of astrocytic Gq GPCR Ca2+ signaling leads to an altered production of glutamate or pro-inflammatory factors from astrocytes, and consequent deficits in synaptic transmission, long-term potentiation (LTP), and memory formation. To test this hypothesis, we used an AAV-based chemogenetic tool to selectively activate astrocyte Gq GPCR Ca2+ signaling combined with in vivo electrophysiology, immunohistochemistry, and biochemistry. Using the mouse primary visual cortex (V1) as a model system, we found that chronically increased astrocytic Gq GPCR Ca2+ signaling leads to a decrease in LTP of visual-evoked potentials. Such LTP impairment was associated with microglial reactive phenotype - displaying a hyper-ramified and proliferative state - as well as a decrease in the number of interleukin 33 (IL-33)-expressing astrocytes. Our study is the first to have shown that chronic astrocytic Gq GPCR activation is sufficient to alter visual LTP and induce astrocyte-to-microglia communication, possibly through and IL-33 pathway in the adult brain. Because GPCRs are important drug targets, our study could have relevant therapeutic implications in the treatment of some neurodegenerative diseases.

Endocannabinoids (eCB) are lipid-based neurotransmitters that are known to influence synaptic function in the visual system. eCBs are also known to suppress neuroinflammation in different pathological states. However, nothing is known about the roles of the eCB system during reprogramming of Muller glia (MG) into proliferating progenitor-like cells in the retina. Accordingly, we used the chick and mouse model to characterize expression patterns of eCB-related genes and applied pharmacological agents to examine how the eCB system impacts glial reactivity and the capacity of MG to become Muller glia-derived progenitor cells (MGPCs). We probed single cell RNA-seq libraries to identify eCB-related genes and identify cells with dynamic patterns of expression in damaged retinas. MG and inner retinal neurons expressed the eCB receptor CNR1, as well as enzymes involved in eCB metabolism. In the chick, intraocular injections of 2-Arachidonoylglycerol (2-AG) and Anandamide (AEA) potentiated the formation of MGPCs. Consistent with these findings, CNR1-agonists and MGLL-inhibitor promoted reprogramming, whereas CNR1-antagonist and inhibitors of eCB synthesis suppressed reprogramming. Surprisingly, retinal microglia were largely unaffected by increases or decreases in eCB signaling in both chick and mouse models. However, eCB-signaling suppressed the activation of NFkB-reporter in MG in damaged mouse retinas. We conclude that the eCB system in the retina influences the reactivity of MG and is important for regulating glial reactivity and the reprogramming of MG into proliferating MGPCs, but not for regulating the reactivity of immune cells in the retina. Main PointsMuller glia express CNR1 receptor and endocannabinoid synthesis genes. Endocannabinoids after retinal damage promote the formation of Muller glia derived progenitor cells in chick. Endocannabinoids reduce NFkB activity in mouse Muller glia.

The purpose of this study was to investigate how Sphingosine-1-phosphate (S1P) signaling regulates glial phenotype, neuroprotection, and reprogramming of Muller glia (MG) into neurogenic MG-derived progenitor cells (MGPCs) in the adult mouse retina. We found that S1P-related genes were dynamically regulated following retinal damage. S1pr1 (encoding S1P receptor 1) and Sphk1 (encoding sphingosine kinase 1) are expressed at low levels by resting MG and are rapidly upregulated following acute damage. Overexpression of the neurogenic bHLH transcription factor Ascl1 in MG downregulates S1pr1, and inhibition of Sphk1 and S1pr1/3 enhances Ascl1-driven differentiation of bipolar-like cells and suppresses glial differentiation. Treatments that activate S1pr1 or increase retinal levels of S1P initiate pro-inflammatory NF{kappa}B-signaling in MG, whereas treatments that inhibit S1pr1 or decreased levels of S1P suppress NF{kappa}B-signaling in MG in damaged retinas. Conditional knock-out of NF{kappa}B-signaling in MG increases glial expression of S1pr1 but decreases levels of S1pr3 and Sphk1. Conditional knock-out (cKO) of S1pr1 in MG, but not Sphk1, enhances the accumulation of immune cells in acutely damaged retinas. cKO of S1pr1 is neuroprotective to ganglion cells, whereas cKO of Sphk1 is neuroprotective to amacrine cells in NMDA-damaged retinas. Consistent with these findings, pharmacological treatments that inhibit S1P receptors or inhibit Sphk1 had protective effects upon inner retinal neurons. We conclude that the S1P-signaling pathway is activated in MG after damage and this pathway acts secondarily to restrict the accumulation of immune cells, impairs neuron survival and suppresses the reprogramming of MG into neurogenic progenitors in the adult mouse retina.

Astrocytes are abundant star-shaped glial cells in the mammalian brain, with essential roles in metabolism, development, homeostasis, response to injury, behavior, and learning. Surprisingly, most regions of the teleost brain are thought to lack astrocytes, based primarily on the use of GFAP (glial fibrillary acidic protein) as a marker1. Here, drawing on recent evidence that astrocytes are molecularly heterogeneous, we propose that astrocytes exist in the teleost brain, albeit of the olig2 subtype2. Highly branched cells are present throughout the zebrafish brain, as shown here in Tg(sox10:EGFP) fish and previously in Tg(olig2:GFP) fish. Transcriptome data indicates the presence of brain cells that are olig2 and sox10 positive, which also express the astrocyte markers sox9b, sparcl1 and slc1a2b but lack gfap and the oligodendrocyte marker mbp. In situ hybridization confirms that stellate sox10:EGFP cells express olig2 and sox9b, while immunofluorescence indicates that they lack HuC/D and GFAP. We suggest that these cells be classified as astrocytes as this may more accurately reflect their functions.

Recent studies have demonstrated the complex coordination of pro-inflammatory signaling and reactive microglia/macrophage on the formation Muller glial-derived progenitor cells (MGPCs) in the retinas of fish, birds and mice. We generated scRNA-seq libraries to identify transcriptional changes in Muller glia (MG) that result from the depletion of microglia from the chick retina. We found significant changes in different networks of genes in MG in normal and damaged retinas when the microglia are ablated. We identified a failure of MG to upregulate Wnt-ligands, Heparin binding epidermal growth factor (HBEGF), Fibroblast growth factor (FGF), retinoic acid receptors and genes related to Notch-signaling. Inhibition of GSK3{beta}, to simulate Wnt-signaling, failed to rescue the deficit in formation of proliferating MGPCs in damaged retinas missing microglia. By comparison, application of HBEGF or FGF2 completely rescued the formation of proliferating MGPCs in microglia-depleted retinas. Similarly, injection of a small molecule inhibitor to Smad3 or agonist to retinoic acid receptors partially rescued the formation of proliferating MGPCs in microglia-depleted damaged retinas. According to scRNA-seq libraries, patterns of expression of ligands, receptors, signal transducers and/or processing enzymes to cell-signaling via HBEGF, FGF, retinoic acid and TGF{beta} are rapidly and transiently upregulated by MG after neuronal damage, consistent with important roles for these cell-signaling pathways in regulating the formation of MGPCs. We conclude that quiescent and activated microglia have a significant impact upon the transcriptomic profile of MG. We conclude that signals produced by reactive microglia in damaged retinas stimulate MG to upregulate cell signaling through HBEGF, FGF and retinoic acid, and downregulate signaling through TGF{beta}/Smad3 to promote the reprogramming on MG into proliferating MGPCs.

K+ channels are important for controlling membrane potential and regulating functional properties of microglia. Whereas the inward-rectifying K+ (Kir) channel 2.1 modulates proliferation, voltage-gated K+ channels (Kv) are linked to inflammatory response in mouse microglia (mMG). These channels serve as possible drug targets but little is known regarding their activity in human microglia. We used patch-clamp recording to study membrane currents of primary human microglia (hMG) and human induced pluripotent stem cell-derived microglia-like cells (hiPSC-MGL) and compared them with mMG. Unlike mMG, hMG and hiPSC-MGL exhibited Kir2.1 currents only after LPS+IFN-{gamma} stimulation. Interestingly, Kv currents were not observed in hMG or hiPSC-MGL under any condition. While mMG had a progressively ameboid morphology after stimulation, hMG showed few morphological changes and hiPSC-MGL increased ramification. Overall, the activity of Kir2.1 and Kv channels in hMG and hiPSC-MGL differs fundamentally from mMG. Our findings highlight differences between species and underscore the need for translational approaches.

The diversity of reactive astrocytes is key to understanding complicated pathological processes in the brain. The accumulation of reactive astrocytes expressing the neural stem/precursor cell marker Nestin is common after brain injury, but the pathological implications of this reactive astrocyte subpopulation remain elusive. This study initially aimed to determine the origin and fate of these reactive astrocytes expressing Nestin by characterizing cells labeled with green fluorescent protein (GFP) after closed-head injury, using a Nestin promoter region widely utilized to study neural stem/precursor cells. Unexpectedly, oligodendrocyte progenitor cells (OPCs), rather than astrocytes, were robustly and selectively labeled with GFP. A fraction of these cells showed a subsequent upregulation of astrocyte markers and were incorporated into glial scars. These glial scars are aggregates of reactive astrocytes that form between lesion cores and the perilesional recovering region. Deletion of the Stat3 gene, which is essential for astrocyte activation, using a Nestin promoter reduced glial scars, further confirming that OPCs are involved in glial scar formation. Reactive astrocytes labeled with a glial fibrillary acidic protein promoter differed in morphology and distribution from astrocytes derived from OPCs. This confirms that astrocytes and OPCs produce distinct reactive astrocyte subpopulations. Some GFP-labeled OPCs lacking astrocyte markers were found to distribute in perilesional recovering regions. The reduced expression of Nestin and OPC markers in these non-astrocytic descendants of OPCs, coupled with a significant fraction of these cells remaining olig2-positive, suggests that OPCs give rise to both reactive astrocytes and oligodendrocytes. These findings suggest that OPCs are activated by a novel process after brain injury.

Neuroinflammation comprises biochemical and cellular responses of the nervous system to injury, emerging as a central process in many neurological conditions. Although beneficial in nature, over-activation of the immune response may result in the production of neurotoxic factors that exacerbate the disease state. Due to the relevant role of histone acetylation in transcriptional regulation, inhibition of histone deacetylase (HDAC) activity plays a central role in the pathogenesis of inflammation. Here, we investigated the impact of HDACs inhibition in the regulation of the inflammatory response elicited by lipopolysaccharide (LPS) in glia cells using trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA). We observed that acute and prolonged exposure to low doses of TSA boosts the inflammatory response in glia cells. Conversely, TSA pre-treatment in cells exposed to LPS decreases inflammation. Additionally, we observed that low and high doses of SAHA exert opposite effects on LPS-induced inflammatory response. Low dose (100 nM) potentiates inflammatory response by increasing the production of pro-inflammatory cytokines tumour necrosis factor (TNF)- and interleukin (IL) - 1{beta}; and reducing the anti-inflammatory mediator IL-10. Nevertheless, 5 {micro}M SAHA reverts the pro-towards anti-inflammatory profile. To better characterize SAHA dualistic effects in glia cells, we generated comparative genome-wide gene expression data. Simultaneous exposure to 5 {micro}M SAHA and LPS (10 ng/ml) alters the expression of 1628 genes, while the combination of 100 nM SAHA and LPS regulates the expression of 97 transcripts. Both doses of SAHA differentially regulate important pathways involved in cellular function maintenance, homeostasis, and survival. Likewise, inhibition of the Janus Kinase - signal transducers and activators of the transcription (JAK/STAT) signalling pathway seems to mediate the anti-inflammatory effects of 5 {micro}M SAHA. On the other hand, 100 nM SAHA increases pro-inflammatory cytokine levels possibly via modulation of the underlined feedback mechanisms triggered by IL-10 expression regulation. Together, these results contribute to outline a comprehensive picture of the involvement of HDACs inhibitors (HDACi) in the onset or prevention of neuroinflammation, showing that their effects depend on cell types, HDACi dosage and specificity, and protocol used.

Reactive astrogliosis, a hallmark of central nervous system pathologies, involves cellular responses, including morphological remodelling and upregulation of intermediate filaments such as vimentin. These changes are driven by cytoskeletal dynamics and are mediated by focal adhesions (FAs). Our study identifies plectin, a versatile cytoskeletal linker protein, as a critical modulator of FA-associated processes in mouse astrocytes. We demonstrate that plectin localizes to astrocyte FAs, where it regulates their number, maturation, turnover, and the mobility of FA components. Plectin also polarizes within FAs, depending on their maturation state, and controls the recruitment of key cytoskeletal elements particularly vimentin. In plectin-deficient astrocytes, the vimentin network exhibits impaired connectivity, accompanied by altered viscoelastic properties of the cells. In a model of reactive astrocytes FA number and size were elevated along with the expression of plectin, highlighting involvement of plectin in pathological conditions.

The cerebellum is involved in the coordination of movement. Its cellular composition is dominated by GABAergic neuronal types, and glial cells are known to express functional receptors. GABAergic signaling regulates cell proliferation, differentiation, and migration during neurodevelopment. However, little is known about the functional expression of GABA receptors in the cerebellar white matter (WM). Thus, the aim of this study was to test whether glial cells express functional GABA receptors during postnatal development (P7-P9) of cerebellar WM. Immunofluorescence studies showed that half of the astrocytes express GAD67, suggesting that GABA is synthetized by glial cells. Calcium imaging in cerebellar slices revealed that GABA and the GABAA agonist muscimol evoked calcium transients in sulforhodamine B (SRB) negative cells, whereas the GABAB agonist baclofen failed to evoke responses in cerebellar WM. Whole-cell patch-clamp recordings of GFAP+ cells showed dye coupling and a passive current-voltage relation typical of astrocytes. Surprisingly, these cells did not respond to muscimol. Two additional populations were identified as GFAP- cells. The first population showed dye coupling, slow decaying inward and outward currents with no voltage dependence and did not respond to GABAA agonists. The second population showed an outward-rectifying current-voltage relationship and responded to muscimol, but dye coupling was absent. These cells received synaptic input and were NG2+, but evoked calcium waves failed to modulate the frequency of sPSCs or signal directly to NG2 glia. We conclude that GABAA receptor-mediated signaling is selective for NG2 glia in the WM of the cerebellum.

Astrocytes exhibit dual roles in central nervous system (CNS) recovery, offering both beneficial and detrimental effects. Following CNS injury, a subset of astrocytes undergoes proliferation, de-differentiation, and acquires self-renewal and neurosphere-forming capabilities in vitro. This subset of astrocytes represents a promising target for initiating brain repair processes and holds potential for neural recovery. However, studying these rare plastic astrocytes is challenging due to the absence of distinct markers. In our study, we characterized these astrocytic subpopulations using comparative single-cell transcriptome analysis. By leveraging the regenerative properties observed in radial glia of zebrafish, we identified and characterized injury-induced plastic astrocytes in mice. These injury-induced astrocytic subpopulations were predominantly proliferative and demonstrated the capacity for self-renewal and neurosphere formation, ultimately differentiating exclusively into astrocytes. Integration with scRNAseq data of the subependymal zone (SEZ) allowed us to trace the origins of these injury-induced plastic astrocytic subpopulations to parenchymal astrocytes. Our analysis revealed that a subset of these injury-induced astrocytes shares transcriptional similarities with endogenous transient amplifying progenitors (TAPs) within the SEZ, rather than with neural stem cells (NSCs). Notably, these injury-induced TAP-like cells exhibit distinct differentiation trajectories, favoring gliogenic over neurogenic differentiation. In summary, our study identifies a rare subset of injury-induced, proliferative plastic astrocytes with neurosphere-forming capacities. These cells originate from reactive astrocytes and resemble TAPs in their transcriptional profile. This study enhances our understanding of astrocyte plasticity post-injury. HighlightsO_LISingle-cell transcriptomics and cross-species comparisons reveal proliferative and de-differentiated plastic astrocytes following CNS injury. C_LIO_LIInjury-induced de-differentiated astrocytes exhibit remarkable in vitro self-renewal and neurosphere formation but favor glial differentiation. C_LIO_LIDe-differentiated astrocytes exhibit transcriptional similarities to transit-amplifying progenitors (TAPs) over neural stem cells (NSCs) C_LIO_LIInjury-induced TAP-like progenitors exhibit limited spontaneous neuronal differentiation. C_LI

Astrocytes have long been considered to be a largely homogeneous cell population. Recent studies however suggest that astrocytes are highly adapted to the local neuronal circuitry. Glucose utilization in the retinorecipient superior colliculus (SC) is one of the highest in the brain. Since metabolic support to neurons is a major function of astrocytes, they could be of particular relevance in this region and display specific features. However, little is known about astrocytes and their interactions with neurons in this multisensory brain area. We thus here investigated region-specific cellular and structural properties of astrocytes in the visual layer of the SC. Using morphological reconstructions, fluorescent recovery after photobleaching (FRAP) and superresolution imaging, we found that astrocytes from the visual layers of the SC are highly distinct with a higher cellular density, a more complex morphology and a stronger proximity to synapses compared to astrocytes from the primary visual cortex and the hippocampus. These data point to astroglial diversity and specialization within neural circuits integrating sensory information in the adult brain.

The severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), causing human coronavirus disease 2019 (COVID-19), not only affects the respiratory tract, but also impacts other organs including the brain. A considerable number of COVID-19 patients develop neuropsychiatric symptoms that may linger for weeks and months and contribute to "long-COVID". While the neurological symptoms of COVID-19 are well described, the cellular mechanisms of neurologic disorders attributed to the infection are still enigmatic. Here, we studied the effect of an infection with SARS-CoV-2 on the structure and expression of marker proteins of astrocytes and microglial cells in the frontal cortex of patients who died from COVID-19 in comparison to non-COVID-19 controls. Most of COVID-19 patients had microglial cells with retracted processes and rounded and enlarged cell bodies in both gray and white matter, as visualized by anti-Iba1 staining and confocal fluorescence microscopy. In addition, gray matter astrocytes in COVID-19 patients were frequently labeled by intense anti-GFAP staining, whereas in non-COVID-19 controls, most gray matter astrocytes expressed little GFAP. The most striking difference between astrocytes in COVID-19 patients and controls was found by anti-aquaporin-4 (AQP4) staining. In COVID-19 patients, a large number of gray matter astrocytes showed an increase in AQP4. In addition, AQP4 polarity was lost and AQP4 covered the entire cell, including the cell body and all cell processes, while in controls, AQP4 immunostaining was mainly detected in endfeet around blood vessels and did not visualize the cell body. In summary, our data suggest neuroinflammation upon SARS-CoV-2 infection including microgliosis and astrogliosis, including loss of AQP4 polarity.

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/587565v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1899db6org.highwire.dtl.DTLVardef@1d152fcorg.highwire.dtl.DTLVardef@19f4754org.highwire.dtl.DTLVardef@3f853_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG Brain damage triggers diverse cellular and molecular events, with astrocytes playing a crucial role in activating local neuroprotective and reparative signaling within damaged neuronal circuits. Here, we investigated reactive astrocytes using a multidimensional approach to categorize their responses into different subtypes based on morphology using the StarTrack lineage tracer, single-cell imaging reconstruction and multivariate data analysis. Our findings revealed three profiles of reactive astrocyte responses affecting cell size- and shape-related morphological parameters: "moderate," "strong," and "very strong". We also explored the heterogeneity in astrocyte reactivity, with a particular emphasis in the spatial and clonal distribution. Our research highlights the importance of the relationships between the different astrocyte subpopulations with their reactive responses, showing an enrichment of protoplasmic and fibrous astrocytes within the "strong" and "very strong" subtypes. Overall, our study contributes to a better understanding of astrocyte heterogeneity in response to an injury. By elucidating the diverse reactive responses among astrocyte subpopulations, we pave the way for future research aimed at uncovering novel therapeutic targets for mitigating the effects of brain damage and promoting neural repair.

Astrocyte morphology in vivo is heterogeneous across different subtypes and dynamically changes in response to various stimuli. However, several questions on the mechanistic links between shape and function remain unanswered. Here, we developed an efficient protocol to generate pure populations of morphologically distinct human astrocytes in vitro, which we used for a systematic analysis of shape-function relationships. We performed a structural, molecular, and functional characterization of these populations and highlighted how their distinct morphologies mirror distinct functional and transcriptional patterns at the population level. We were also able to both correlate gene expression profiles of these morphologically distinct astrocyte subtypes with in vivo astrocytes in the human brain, and to validate our findings with primary isolated murine astrocytes in vitro. Moreover, we show that the observed morphological differences are correlated with changes in key cytoskeletal proteins, which offers a potential link to the observed functional differences. Finally, we demonstrated that different morphological subtypes of astrocytes have distinct reactivity responses to a common stimulus. This study offers a glimpse into the shape-function dynamics of human astrocytes, highlighting potential mechanistic links between cytoskeletal usage and astrocyte function, while also providing tools and datasets that will be useful for further studies into human glial biology in health and disease.

Astrocytes are key players in CNS neuroinflammation and neuroregeneration that may help or hinder recovery, depending on the context of the injury. Although pro-inflammatory factors that promote astrocyte-mediated neurotoxicity have been shown to be secreted by reactive microglia, anti-inflammatory factors that suppress astrocyte activation are not well-characterized. Olfactory ensheathing cells (OECs), glial cells that wrap axons of olfactory sensory neurons, have been shown to moderate astrocyte reactivity, creating an environment conducive to regeneration. Similarly, astrocytes cultured in medium conditioned by cultured OECs (OEC-CM) show reduced nuclear translocation of Nuclear Factor kappa-B (NF{kappa}B), a pro-inflammatory protein that induces neurotoxic reactivity in astrocytes. In this study, we screened primary and immortalized OEC lines to identify these factors and discovered that Alpha B-crystallin (CryAB), an antiinflammatory protein, is secreted by OECs via exosomes, coordinating an intercellular immune response. Our results showed: 1) OEC exosomes block nuclear NF{kappa}B translocation in astrocytes while exosomes from CryAB-null OECs could not; 2) OEC exosomes could be taken up by astrocytes and 3) CryAB treatment suppressed multiple neurotoxicity-associated astrocyte transcripts. Our results indicate that OEC-secreted factors are potential agents that can ameliorate, or even reverse, the growth-inhibitory environment created by neurotoxic reactive astrocytes following CNS injuries. Main PointsO_LIAstrocytes uptake OEC-secreted exosomes. C_LIO_LIWT OEC-exosomes, but not CryAB-null OEC-exosomes, block nuclear NF{kappa}B translocation in astrocytes. C_LIO_LICryAB, and other factors secreted by OECs, suppresses multiple neurotoxicity-associated astrocyte transcripts. C_LI

Satellite glial cells (SGCs) envelop the somata, axon hillock, and initial axon segment of sensory neurons in the dorsal root ganglia (DRG), playing a critical role in regulating the neuronal microenvironment. While DRG neurons have been extensively studied and classified based on size, molecular markers, and functional characteristics, very little is still known about SGC heterogeneity and its potential implications on sensory processing in the DRG. Single cell transcriptional analyses have proposed the existence of SGC subtypes, yet in situ validation, spatial distribution, and potential functional implications of such subtypes are still largely unexplored. Here, we present the first comprehensive in situ characterization of SGC heterogeneity within the mouse DRG. By integrating single-cell RNA sequencing with immunohistochemistry, in situ hybridization, and advanced imaging techniques, distinct SGC subclusters were identified, validated, and spatially mapped within their native anatomical context. We visually identify four distinct subpopulations: 1) a predominant population of perisomatic SGC sheaths defined by the expression of marker proteins traditionally used to characterize the entire SGC population, including FABP7, KIR4.1, GS, and CX43. 2) OCT6+ SGCs occasionally being found in mosaic perisomatic sheaths, and consistently associated with axonal glomeruli, primarily ensheathing initial segment axon. 3) SCN7A+ SGCs, exhibiting no/low expression of traditional SGC markers and forming specialized homogenous sheaths around non-peptidergic neuron subtypes, implicating their potential role in pruritic (itch-related) conditions. 4) Interferon response gene-expressing SGCs, responding to Herpes Simplex Virus infection, suggesting potential involvement in antiviral protection. Finally, we investigate human DRG and find an inner perisomatic SGC layer surrounded by an outer SGC layer, with traditional and novel markers distinctively distributed between the two layers. Our results provide novel insight into SGC heterogeneity in the DRG and suggests distinct functional properties for such subtypes of relevance for the neuronal microenvironment.

Oligodendrocyte lineage cells (OLCs) are glia that arise as oligodendrocyte progenitor cells (OPCs) in the central nervous system (CNS) and may persist as progenitors or differentiate into myelin-producing oligodendrocytes (OLs). OLCs are sensitive to neuronal activity, which can influence myelin formation via activity-dependent myelination. Calcium influx in OLCs regulates many developmental processes, including stabilizing newly formed sheaths. OLCs possess P/Q-type voltage-gated calcium channels (VGCC) that contribute to calcium influx and mutations in these channels are implicated in a spectrum of neurological disorders, yet the functional significance of P/Q-type channels in OLCs is not well understood. In this study, we employ zebrafish to investigate the role of P/Q-type channels in OLCs in vivo during development. We use global and cell-type specific CRISPR/Cas9-mediated genome editing approaches in conjunction with live imaging and physiology to characterize the morphology and signaling properties of OLCs with mutated P/Q-type channel genes. P/Q-type channels are required for normal myelination in the developing CNS and mutants present with decreased amplitude sheath calcium transients, reduced myelin production, shorter myelin sheaths and blebbing membrane structures during development. These findings provide new insight into the role of P/Q-type calcium channels in regulating OLC development and myelination.

Glial cells in the peripheral nerve wrap axons to insulate them and ensure efficient conduction of neuronal signals. In the myelin sheath, it is proposed that the autotypic tight junctions and adherens junctions form glia-glia complexes that stabilize the glia sheath in myelinating glia. Yet the role of adhesion junctions in non-myelinating glia of vertebrates or invertebrates has not been clearly established. Many components of adhering junctions contain PDZ (PSD-95, Dlg, ZO1) domains or are recruited to these junctions by PDZ binding motifs. To test for the role of PDZ domain proteins in glial sheath formation, we carried out an RNAi screen using Drosophila melanogaster to knockdown each of the 66 predicted PDZ domain proteins in the peripheral glia. We identified six PDZ genes with potential roles in glial morphology, and further investigated Discs-large 5 (Dlg5), a scaffolding protein with no previously known function in glia. Knockdown of Dlg5 disrupts subperineurial glia (SPG) morphology, including gaps in the membrane that coincide with disruption of septate junction proteins. To further our investigation of Dlg5, we focused on cadherins and found both N-Cadherin and E-Cadherin are expressed throughout peripheral glia. Knockdown of E-Cadherin phenocopied the loss of Dlg5 leading to gaps in the SPG and septate junctions while only simultaneous loss of both N-Cadherins (NCad, and CadN2) had the same effect. The loss of all three Cadherins enhanced these phenotypes as did loss of Dlg5 when paired with cadherin knockdown. This leads to a model where Dlg5 plays a role in conjunction with cadherins in glial membrane stabilization and septate junction formation in the subperineurial glia.

NG2 glia represent a distinct type of macroglial cells in the CNS and are unique among glia because they receive synaptic input from neurons. They are abundantly present in white and grey matter. While the majority of white matter NG2 glia differentiates into oligodendrocytes, the physiological impact of grey matter NG2 glia and their synaptic input are ill defined yet. Here we asked whether dysfunctional NG2 glia affect neuronal signaling and behavior. We generated mice with inducible deletion of the K+ channel Kir4.1 in NG2 glia and performed comparative electrophysiological, immunohistochemical, molecular and behavioral analyses. Focussing on the hippocampus, we found that loss of the Kir4.1 potentiated synaptic depolarizations of NG2 glia and enhanced the expression of myelin basic protein. Notably, while mice with targeted deletion of the K+ channel in NG2 glia showed impaired long term potentiation at CA3-CA1 synapses, they demonstrated improved spatial memory as revealed by testing new object location recognition. Our data demonstrate that proper NG2 glia function is critical for normal brain function and behavior.