Glia

The injury responses of tissue-resident macrophages in the CNS (microglia) and blood-derived macrophages (BDMs) play key roles in successful regeneration of the zebrafish spinal cord, but the origins and dynamic behaviours of these immune cells are not well characterized. Here, we find that microglia, labelled by the p2ry12:GFP reporter gene, migrate long-distance through neural tissue from the brain to the spinal lesion site, while BDMs, labelled by the mpeg1:mCherry reporter gene, migrate mainly from the caudal hematopoietic tissue to the lesion and back. Half of p2ry12:GFP-positive microglia co-express mpeg1:mCherry, while mpeg1:mCherry-positive BDMs are mostly p2ry12:GFP-negative. However, a BDM sub-population starts to express p2ry12:GFP in the lesion. This indicates heterogeneous and dynamic gene expression in macrophage populations. Gene expression profiling reveals several microglia-like and BDM-like clusters in the lesion with gene expression profiles related to proliferation, phagocytosis, pro- and anti-inflammatory phenotypes and distinct expression of regeneration-relevant genes. The most abundant cell cluster are densely-packed microglia-like cells in the lesion core, which express the novel marker g0s2, as well as phagocytosis-related genes. Hence, regenerative success of the zebrafish spinal cord is linked to a heterogeneous and dynamic response of microglia and BDM subpopulations.

Bergmann glia-like progenitors (BGLPs) are transient astroglial progenitors in the postnatal cerebellum, but how their lineage potential changes during development remains incompletely understood. Our previous electroporation-based study suggested that P0 BGLPs possess broader lineage potential than P6 BGLPs. Here, we performed recombination-based lineage tracing by cerebellar surface application of tamoxifen to Ai9/+; GlastCreERT2/+ mice and temporally analyzed the progeny of BGLPs labeled at P0, P3, P6, and P8. We found that BGLPs undergo progressive lineage restriction during postnatal development. P0 BGLPs gave rise to Bergmann glial cells (BGs), inner granule cell layer astrocytes (IGL astrocytes), white matter astrocytes (WM astrocytes), and molecular layer inhibitory neurons (ML-INs), confirming our previous electroporation-based findings. In contrast, P3 BGLPs generated BGs, IGL astrocytes, and WM astrocytes, whereas P6 BGLPs generated BGs and IGL astrocytes, and P8 BGLPs generated predominantly BGs. Thus, BGLP lineage output was progressively restricted from four progeny categories at P0 to a predominantly BG-restricted output by P8, suggesting that BGLPs dynamically adjust their cellular output during postnatal cerebellar maturation. Additional temporal analyses suggested that ML-INs are unlikely to be generated directly from P0 BGLPs, but may arise indirectly through astrocyte-like progenitors (AsLPs) and inhibitory neuron progenitors (INPs). These findings identify postnatal BGLPs as a useful in vivo model for studying progressive lineage restriction and stage-specific cellular supply during cerebellar development.

In young animals, oligodendrocyte progenitor cells (OPCs) undergo robust differentiation, progressing through stages to become pre-myelinating oligodendrocytes (Pre-OL) and ultimately myelinating oligodendrocytes (OLs). However, OPCs from aged animals have reduced differentiation ability. This disrupts myelin maintenance in the central nervous system (CNS), leading to a lack of remyelination following demyelinating injury and impaired adaptive myelination as a mechanism of learning. To uncover novel factors essential for restoring resilience in aged OPCs, we employed a data-driven approach involving the development of a computational pipeline, gSWITCH (accessible at: https://altoslabs.shinyapps.io/gSWITCH/), that allows for capture of precise dynamic gene expression patterns to pinpoint potential switch genes during lineage progression. Using gSWITCH to identify potential switch genes crucial for OPCs, we conducted a comparative analysis of gene expression in OPCs isolated from young and aged animals. This revealed a group of transcription factors with decreased expression in aged OPCs. Further analysis of transcription factor binding site enrichment in OPC switches highlighted Bcl11a, a zinc finger transcription factor that could potentially serve as a master regulator of many switch genes. Ectopic overexpression of Bcl11a in aged OPCs did not enhance their proliferation; however, it significantly enhanced their differentiation into OLs. Overexpression of Bcl11a in aged mice, followed by demyelination injury in spinal cord white matter, significantly increased the differentiation of OPCs into OLs within the injury region compared to control aged mice. Furthermore, we found that Bcl11a is absent in invertebrates and has undergone pervasive purifying selection throughout vertebrate evolution, constraining its amino-acid sequence by eliminating deleterious mutations. Our study shows that reversing the age-related decline of this evolutionarily conserved factor in aged OPCs restores their impaired capacity for differentiation.

Developing neuron-replacement therapies for retinal ganglion cells (RGCs) lost to injury or disease requires a deeper understanding of how restriction to cell identity acquisition may be overcome. Previously, we showed that overexpression of Klf4 in late retinal progenitor cells (late RPCs), which are normally restricted from RGC production, is sufficient to produce cells that display a subset of canonical RGC properties including RGC-associated gene expression and morphological features. In the present study, we investigated the transcriptional and epigenetic mechanisms by which Klf4 overexpression influences the fate of cell types generated from late RPCs. scRNA-seq analysis revealed that Klf4 induces transcriptional changes, with some cells exhibiting gene expression profiles similar to those of resident RGCs. In addition, we observed widespread changes in chromatin accessibility, suggesting that KLF4 remodels the chromatin of late RPCs and influences their transcriptional profile. Our findings show KLF4-driven reprogramming of late RPCs, providing insight into progenitor competence and fate specification to an RGC-like identity. These results suggest that KLF4 could be a component in regenerative therapies due to its ability to reprogram and induce RGC genes outside of the normal RGC developmental window.

Glaucoma is a group of eye diseases that affects 4 million people in the US and is one of the leading causes of vision loss due to damage to the eyes optic nerve (ON) which is composed of axons from retinal ganglion cells (RGCs) that transmit visual information to the brain. Injury to the ON often triggers RGC death and subsequent loss of visual function. Despite its increasing prevalence worldwide, effective therapies for glaucoma remain elusive. Notably, the thirteen-lined ground squirrel (TLGS) exhibits intrinsic neuroprotection during hibernation; however, reproducing this protective state pharmacologically has proven challenging. To elucidate the metabolic mechanisms underlying this resilience, we conducted untargeted metabolomic analyses on TLGS retinas at 6 hours, 3 days, and 7 days following ON crush. Retinas from awake and hibernating animals were compared to identify temporal and state-dependent metabolic signatures. Distinct metabolomic profiles were observed in hibernating animals relative to their awake counterparts. Pathway analyses revealed coordinated regulation of amino acid, lipid, and purine metabolism that likely contributes to hibernation-induced resilience. Furthermore, our findings indicate that hibernating TLGS retinas increase exosome biogenesis, prompting in vitro validation using TLGS-derived exosomes, which demonstrated robust neuroprotective and anti-inflammatory effects. Proteomic and transcriptomic characterization of exosomal cargo identified conserved miRNAs, mRNAs, and proteins implicated in redox balance, cytoskeletal stabilization, and stress-response regulation. Collectively, these data support the hypothesis that metabolic reprogramming and exosome-mediated intercellular signaling underlie hibernation-associated neuroprotection. Modulating these pathways may provide a blueprint for novel therapeutic strategies to mitigate neurodegeneration and promote recovery following optic nerve injury. Graphical AbstractIllustration depicting state-dependent metabolic responses to optic nerve crush (ONC) injury in Thirteen-lined Ground Squirrels (TLGS). In Awake animals, injury triggers enhanced ATP production through the TCA cycle, leading to excessive reactive oxygen species (ROS) generation and subsequent retinal ganglion cell (RGC) death. In contrast, Hibernating animals shift toward lipid metabolism and utilize ATP for the biosynthesis of ceramides and sphingolipids, promoting membrane integrity and exosomal signaling. Additionally, a range of metabolites associated with hibernation-linked neuroprotection are elevated, contributing to enhanced RGC survival. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/733742v1_ufig1.gif" ALT="Figure 1"> View larger version (81K): org.highwire.dtl.DTLVardef@1ef3a7eorg.highwire.dtl.DTLVardef@e9293dorg.highwire.dtl.DTLVardef@192729forg.highwire.dtl.DTLVardef@1a32cb5_HPS_FORMAT_FIGEXP M_FIG C_FIG

Lysosomes are critical for neuronal physiology and synaptic function, but their organization and roles within astrocytes, an integral component of the tripartite synapse, remain unknown. Here, we use a neuron-astrocyte coculture system that promotes stellate astrocyte morphology to investigate the trafficking of late endosomes and lysosomes (LELs) in astrocytes. As astrocyte branches mature, degradative activity becomes concentrated in the soma and LELs in branches undergo bidirectional motility that becomes progressively attenuated. We establish that the lysosomal cation channel TRPML1 drives LEL immobilization. TRPML1-mediated arrest involves myosin-Va tethering to the actin cytoskeleton, which may position LELs near peripheral astrocyte processes (PAPs), fine actin-enriched protrusions that can contact synapses. Strikingly, TRPML1 activity modulates the phosphorylation of ezrin, radixin, and moesin, actin-membrane linkers enriched in PAPs. These effects are rapid and transient, suggesting a role for lysosomal TRPML1 in regulating PAP membrane dynamics. Thus, TRPML1 positions LELs proximal to PAPs which may influence PAP structural plasticity and astrocyte-synapse contacts.

Microglia are essential for sculpting the developing brain, yet the molecular mechanisms that select beneficial overreactive phagocytosis remain incompletely understood. Connexin 43 (Cx43, encoded by GJA1 in humans) is best known as a gap junction and hemichannel protein, although its non canonical, channel independent functions are increasingly recognized. We found that Cx43 is highly expressed in microglia during the perinatal period in human and mouse, whereas proportion of full length multimeric Cx43 unexpectedly localizes to the nucleoplasm. Deletion of microglial Cx43 in mice during development instigates a transient neurotoxic state with microgliosis, upregulated phagocytic and complement pathways, excessive neuronal apoptosis, translating into depressive like and cognitive deficits in the adulthood. Notably, neither microglia-specific deletion of Cx43 in adulthood nor hemichannel blockade recapitulate these changes, indicating a channel independent, developmental stage-specific neuroprotective mechanism. Nucleus targeted Cx43 overexpression suppresses neurotoxic markers and neural apoptosis. Nuclear Cx43 interacts with transcriptional regulators to restrain proinflammatory gene programs, nuclear import of Cx43 is driven by neurogenic niche derived bFGF, which triggers AKT mediated phosphorylation of a C terminal nucleus localization signal (NLS), 14 3 3 binding, and importin dependent nucleus translocation. These findings reveal a developmentally restricted nuclear Cx43 function that restrains microglial neurotoxicity while promoting microglial physiological functions thus expanding connexin biology to transcriptional co regulation and pointing to a potential avenue for therapeutic intervention.

Traumatic brain injury (TBI) induces rapid neuroinflammatory responses not only in the brain but also in anatomically and immunologically connected central nervous system (CNS) compartments, including the retina. In our study, we investigated retinal microglial activation, retinal ganglion cell (RGC) calcium dynamics, and caspase-3 activation in adult mice subjected to severe traumatic brain injury using the Marmarou impact-acceleration model at 24 and 48 h post-injury. Carrying out Ca{superscript 2}-imaging, immunohistochemistry, and ex vivo time-lapse microscopy, we found robust microglial activation in both the superficial and deep retinal layers following TBI, accompanied by increased microglial motility. RGCs exhibited a transient surge in degeneration-induced spontaneous activity at 24 h, followed by a marked reduction below control levels at 48 h, consistent with early degenerative changes. Activated caspase-3 levels were significantly elevated in both microglia and other retinal cell types at both time points, indicating ongoing apoptotic effects. Together, these findings demonstrate that TBI rapidly triggers inflammatory and apoptotic mechanisms in the retina, which are detectable within the first 48 hours. Our results highlight the retina as a sensitive indicator of early CNS pathology after traumatic injury and underscore the potential of retinal analysis for monitoring TBI-induced neurodegeneration for future clinical implementation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/734783v1_ufig1.gif" ALT="Figure 1"> View larger version (14K): org.highwire.dtl.DTLVardef@5bc694org.highwire.dtl.DTLVardef@14a4ce4org.highwire.dtl.DTLVardef@fe2d32org.highwire.dtl.DTLVardef@149419d_HPS_FORMAT_FIGEXP M_FIG C_FIG

After spinal injury, zebrafish, in contrast to mammals, show regenerative neurogenesis, characterized by enhanced injury-induced proliferation of ependymo-radial glial cells (ERGs) and an increase in injury-induced generation of neurons from these progenitors. It is unclear whether regenerative neurogenesis simply recapitulates development or uses regeneration-specific mechanisms. Using scRNA-seq and in vivo validation we find a spinal injury-induced state in ERGs (iiERGs) in larval zebrafish. This cell state emerges mostly without proliferation and has stem cell characteristics, including weak expression of neurogenic genes and strong expression of stemness factors, such as lin28a. Expression of lin28a is not detectable during ongoing developmental neurogenesis. Following spinal cord lesion, lin28a disruption increases the numbers of ERGs undergoing neuronal differentiation and of newly-generated neurons, at the expense of proliferating ERGs and iiERGs. This supports a stemness-preserving role of lin28a in iiERGs. Importantly, iiERGs secrete growth factors, including the regeneration-specific cytokine clcf1, which depends in part on lin28a expression. Disruption of clcf1 signalling impairs spinal progenitor proliferation and injury-induced generation of new neurons, but does not affect the emergence of iiERGs. Over-expression of clcf1 is sufficient to augment neurogenesis in unlesioned animals without inducing the iiERG state, indicating that clcf1 acts as a generic growth factor. Hence, we describe an injury-specific stem cell-like ERG population that regulates regenerative neurogenesis by attenuating neuronal differentiation via lin28a and promoting progenitor proliferation via clcf1.

Long non-coding RNAs (lncRNAs) are increasingly recognized as regulators of cellular identity and disease associated gene expression programs, yet their role in astrocyte reactivity remains poorly understood. Here, we profiled lncRNA expression in primary mouse astrocytes exposed to inflammatory activation paradigms that model microglia driven signaling. This identified a conserved set of activation responsive lncRNAs, among which Gm16685 emerged as one of the most strongly induced candidates. Gm16685 and its human homolog MITA1 were enriched in the nucleus, and MITA1 expression was increased in selected human datasets from Alzheimer's disease, Parkinson's disease and frontotemporal dementia patients. Functional depletion of Gm16685 attenuated inflammatory gene expression and several activation associated astrocyte phenotypes, including reactive oxygen species production, glutamate handling, phagocytic activity and proliferation. Time-resolved transcriptomic analysis indicated that Gm16685 is required for the timely induction of inflammatory response genes. Mechanistically, Gm16685/MITA1 interacted with the RNA binding protein PCBP2, and Gm16685 depletion was associated with reduced PCBP2 protein abundance, altered splicing of Inhibitor of NF-{kappa}B Kinase Subunit Beta (IKK{beta}) and a shift in downstream inflammatory signaling. Together, our findings identify Gm16685/MITA1 as a conserved lncRNA regulator of astrocyte reactivity and suggest that non-coding RNA dependent control of RNA binding proteins contributes to inflammatory signaling in neurodegenerative disease relevant contexts.

The protracted timing required for oligodendrocyte differentiation from human pluripotent stem cells (hPSCs) has limited their use in disease modeling, drug screening, and cell therapy. In particular, the signals that drive oligodendrocyte specification and maturation after neural induction and ventral patterning remain poorly understood. Here, we present a protocol to derive human oligodendrocytes from hPSCs that is based solely on extrinsic cues, and we identify dual inhibition of BMP and Notch signaling as critical drivers of oligodendrocyte commitment and maturation. By day 42 of differentiation, up to 70% of the cells are positive for the oligodendrocyte marker O4, with minimal astrocyte contamination, and show robust expression of mature myelin markers including MBP, MOG, and MAG. These hPSC-derived oligodendrocytes closely match the molecular identity of primary fetal human oligodendrocytes as assessed by single-cell RNA sequencing and are functional as shown by in vitro myelination assays. In addition to the rapid generation of myelinating oligodendrocytes, the new protocol can be modularly adapted for the efficient production of PDGFR+ oligodendrocyte precursors or mixed glial populations containing AQP4+ astrocytes, thereby providing a cellular toolbox for the study of human glial lineages in translational applications.

Systemic inflammation drives neurodegeneration, yet its differential effects across neural tissues and genetic backgrounds remain poorly understood. We performed RNA-sequencing on brain, optic nerve head (ONH), and retina from four genetically diverse mouse strains (B6, CAST, NZO, WSB) following lipopolysaccharide (LPS)-induced systemic inflammation. The ONH mounted the largest response to LPS (9510 DEGs), followed by retina (5152) and brain (4586). A conserved core of 1444 DEGs across all tissues was enriched for innate immune and acute-phase pathways. Tissue-specific responses were apparent; the retina downregulated phototransduction and visual perception genes; ONH exhibited bidirectional remodeling with upregulated proteasome and ribosome biogenesis and suppressed lipid metabolism and lysosomal function; yet the brain displayed no significant pathway level enrichment. Genetic background strongly modulated the LPS response across the three tissues; the retina exhibited the greatest strain-dependent divergence. Interestingly, differing genetic context affected the ONH response to LPS the least despite its markedly larger response to LPS overall. In totality, both genetic and physical context dictate the neuroinflammatory response to LPS.

Photoreceptors are among the most metabolically active cells in the retina and are therefore highly sensitive to fluctuations in oxygen availability. Age-related tissue changes in the eye affect oxygen delivery to the outer retina, which may result in hypoxic stress within photoreceptors and can contribute to disease development and retinal degeneration. To investigate how chronic hypoxic signalling affects photoreceptor metabolism, we examined a rod-pecific Vhl knockout mouse (RodVhl), in which constitutive HIF activation mimics the molecular response to hypoxia. Combining a cell-type-enriched multi-omics approach with metabolic flux analysis, we identified an early metabolic response in the retina of Rod{Delta}Vhl mice prior to degeneration. This response was characterized by a shift towards an oxidative redox environment indicated by a decrease in nucleotide precursors and an increased antioxidant response. While steady-state glycolytic flux remained unchanged, the dynamic 13C-glucose tracing revealed accelerated carbon flow through the three-carbon glycolytic intermediates, indicating a carbon rerouting. Outer segment lipidomics revealed selective remodelling of phosphatidylcholine and phosphatidylethanolamine species toward more oxidation-resistant and elongated acyl chains, supported by early gene upregulation of essential enzymes involved in fatty acid elongation, desaturation and oxidation. Together, these findings indicate a coordinated shift in metabolic and lipid pathways in photoreceptors under chronic hypoxic stress, consistent with an adaptive response that may help preserve outer segment integrity and improve stress resilience.

The intestinal mesentery has been recently classified as a 'new' organ and contains various cell types including adipocytes, preadipocytes, endothelial cells, and immune cells. In addition, neuronal cell bodies are found in the small intestinal mesentery and are situated either individually or clustered together with glial cells in small ganglion structures close to the gut wall. However, little is known about the origin or function of these extra-intestinal mesenteric neurons. The aim of this study was to better these characterize mesenteric neurons and to examine their connectivity with the ENS using calcium imaging in adult mouse ileum with the mesentery attached. Here we show that neurons in the mesentery express typical ENS neurochemical markers, respond to 5-HT, ATP and the nicotinic agonist DMPP, and receive nicotinic synaptic inputs. Furthermore, using labeling with the neuronal tracer DiI, some mesenteric neurons were found to project into the gut wall and can provide functional excitatory inputs to myenteric neurons. By contrast, we did not find evidence for mesenteric neurons providing inputs to other extrinsic neuronal targets, suggesting that they preferentially interact with the ENS. We also demonstrate that mesenteric neurons can be activated by intestinal distension and that the mesentery provides a source of inhibition to the myenteric plexus. Taken together, we show that the ENS not only interacts with vagal and spinal afferents, and sympathetic and parasympathetic nerves, but also neurons situated in the mesentery. Finally, our data suggest that these neurons may provide a form of negative feedback to the myenteric plexus such as in the event of intestinal distension. These findings have important implications for the regulation of intestinal motility in physiological and pathophysiological conditions.

Microbes residing in the gastrointestinal tract exert immunomodulatory impacts on the brain through the gut-brain axis. Short-chain fatty acids (SCFAs) produced by bacterial fermentation of dietary fiber can enter the brain parenchyma and are implicated in microglia-mediated inflammation. While the gut microbiome is required to maintain microglial homeostasis, the mechanisms by which microbiota-derived metabolites affect microglia remains unknown. We examined the roles of SCFAs, specifically butyrate, propionate and acetate, on microglial function in response to SCFAs both in vitro using BV2 cells and in vivo in mice. We observed in vivo that SCFAs impact microglial transcriptional responses to LPS in a sex- and metabolite-specific manner with butyrate having the strongest effect. Enriched gene sets included signatures associated with LPS responsive microglia, Arg1 positive microglia, microglial cell cycle related genes and genes affiliated with changes in microglial morphology. We observed a similar effect in vitro, where metabolite administration enhanced phagocytosis, blunted proliferation and nitric oxide production. We then evaluated global histone modification levels following metabolite treatment and detected an enhancement of H3K9ac, H3K27ac, and H3K4me3 both in vivo and in BV2 cells treated with butyrate. Finally, we showed that butyrate is a potent HDAC inhibitor possibly contributing to enhanced acetylation. Hence, our findings suggest that SCFAs impact microglial function in a metabolite- and sex-specific manner, and that butyrate blunts inflammation by regulating microglial histone acetylation. Our results provide a more in-depth understanding of gut microbiome-microglia crosstalk, opening the door for new microbiome- and microglia-targeted therapies.

Neurodegeneration is a major determinant of disability progression in multiple sclerosis (MS), yet the pathophysiological mechanisms associating inflammation to neuronal insult remain poorly understood. We recently identified Dendritic Syntaphilin Intrusion (DSI), a novel excitoxicity pathway in which the axonal mitochondrial anchor syntaphilin (SNPH) aberrantly translocates into dendrites, causing neurodegeneration in a non-inflammatory model of MS. However, whether this protein intrudes abruptly into dendrites in inflammatory MS pathology is still not clear. Here, we investigated the role of synaptic and extrasynaptic NMDA receptors (NMDAR) in regulating the intrusion of Syntaphilin into dendrites. Using primary hippocampal neuronal cultures, we examined how the balance between synaptic GluN2A-containing and extrasynaptic GluN2B-containing NMDARs influences DSI under inflammatory conditions. Pharmacological and viral-mediated approaches were employed to manipulate NMDAR subtype activity and evaluate their impact on DSI. Inflammatory cytokines discernibly sensitized neurons to DSI. Our results revealed that blockade of synaptic NMDARs significantly increased DSI, whereas inhibition of extrasynaptic NMDARs reduced DSI. These findings demonstrate opposing roles of NMDAR subtypes, with GluN2A-containing synaptic receptors inhibiting DSI and fostering neuronal survival, while GluN2B-containing extrasynaptic receptors enhancing DSI and neurodegenerative signaling. Manipulation of the GluN2A/GluN2B balance showed opposite effect on DSI, suggesting a relationship between NMDAR subtype signaling and SNPH mislocalization. Overall, our findings extend the relevance of DSI from non-inflammatory MS to inflammatory MS and identify DSI as a downstream convergence point linking inflammatory cytokines and excitotoxic NMDAR signaling to neuronal insult. These results reveal DSI as a potential mechanistic link between inflammatory signaling and excitotoxic neuronal injury and indicate that modulation of GluN2B-dependent pathways warrants further investigation in inflammatory neurodegenerative disorders.

Myelin formation in peripheral nerves is orchestrated by axon-derived signals and the ability of Schwann cells to expand lipid-rich membrane. Axonal Neuregulin-1 type III (NRG1t3) is known to promote thicker myelin and is associated with increased lipid abundance in peripheral nerves. NRG1t3 also strongly upregulates peripheral myelin protein 2 (FABP8/PMP2), a fatty acid binding protein in myelinating Schwann cells. Here, we directly tested whether elevating PMP2 in Schwann cells is sufficient to enhance myelin growth in nerves. We generated transgenic mice with Schwann cell-specific PMP2 overexpression and showed that PMP2 overexpression drove significant myelin thickening in peripheral nerves, without altering myelin ultrastructure, impairing nerve conduction, or causing detectable adverse effects despite sustained expression. Strikingly, this hypermyelination occurred without activating canonical promyelinating signaling pathways. Instead, elevated PMP2 selectively enhanced fatty acid uptake in Schwann cells, identifying PMP2 as a dosage sensitive enhancer of myelin growth. These findings suggest that in the absence of axonal signaling-driven upregulation, enhanced PMP2 expression itself promotes myelin membrane expansion and enhanced fatty acid availability that could favor complex lipid synthesis. These findings reveal new role for PMP2 as a regulator for myelin expansion in vivo, highlighting a key link between lipid trafficking and myelin growth.

Progressive and chronic neuroinflammation is associated with numerous neurodegenerative diseases, including primary tauopathies such as frontotemporal dementia and progressive supranuclear palsy. Unlike Alzheimer's disease, there is no clear genetic association implicating microglial dysfunction as a primary driver of tauopathy. As such, the contributions of microglia to tauopathy pathogenesis have been less well defined. Here, we explore the cell autonomous effects of the pathogenic MAPT-S305N variant on microglial function, across two distinct iPSC-microglia protocols, followed by examination of the non-cell autonomous effects of microglial MAPT genotype on neuronal health and function. We find that different protocols produce cells of equivalent microglial identity, but result in microglia in different functional states, thereby influencing reactivity and detectable phenotypes. Regardless, across both protocols we find that MAPT-S305N induces microglial hypoactivity, evidenced by impaired phagocytosis, reduced cytokine release and diminished regulation of synaptic function. We conclude that microglial hypoactivity may be an early event in disease pathogenesis, where MAPT mutation microglia fail to adequately respond to pathogenic stimuli, thereby contributing to subsequent neuronal vulnerability and susceptibility. Further studies are required to understand how and when this initial hypoactive state may switch to a toxic pro-inflammatory state, and whether early detection and correction may be of therapeutic value.

Purpose: Photoreceptor Kv2.1/Kv8.2 voltage-gated potassium channels carry an outward potassium current, helping to set the resting membrane potential and to shape dim light responses. Because potassium flux in the outer retina influences extracellular osmolarity and fluid distribution, we hypothesized that Kv2.1/Kv8.2 channels also contribute to fluid homeostasis in this region of the retina. Methods: OCT imaging was performed in Kv8.2 heterozygous (Het) and knockout (KO) mice aged 4-7 weeks under dark- and light-adapted conditions. Light-dark differences in the distance between the external limiting membrane (ELM) and retinal pigment epithelium (RPE) ({Delta}ELM-RPE) were calculated to quantify light-evoked expansion of the subretinal space (SRS). As a secondary outcome, outer nuclear layer (ONL) thickness was also measured under both lighting conditions. Retinal gene expression was assessed by RNA-seq and droplet digital RT-PCR. Retinal protein expression was determined by western blotting and immunolabeling. Results: {Delta}ELM-RPE was significantly reduced in Kv8.2 KO mice compared with Het controls, indicating reduced SRS hydration. ONL thickness exhibited a small but significant light-dark change that was different between genotypes. Transcriptomic analyses revealed upregulation of osmosensitive genes, including osmolyte transporters and aquaporins. AQP1 protein expression in photoreceptors increased. Conclusions: These findings reveal a previously unrecognized role for Kv2.1/Kv8.2 channels in outer retinal fluid homeostasis and support a model in which photoreceptor potassium efflux contributes to osmotic water movement into the subretinal space.

BackgroundExtracellular matrix (ECM) remodeling is increasingly recognized as an important component of neuroinflammatory pathology in multiple sclerosis (MS), yet the mechanisms by which CNS cells sense and respond to alterations in their mechanical environment and the spatial across which mechanical changes can influence cellular behavior remain poorly understood. Piezo1 is a mechanosensitive ion channel that regulates cellular responses to mechanical stimuli and has recently emerged as a potential modulator of neuroinflammation. MethodsExperimental autoimmune encephalomyelitis (EAE) was induced in C57BL/6 wildtype mice using myelin oligodendrocyte glycoprotein (MOG):35-55. Immunohistochemical analyses were performed in spinal cord gray matter (GM), normal-appearing white matter (NAWM), and white matter lesion (LES) regions to assess ECM remodeling, total Piezo1 expression, and astrocyte-specific Piezo1 expression during acute and chronic EAE stages. Correlations with clinical EAE severity were determined. In parallel, mixed primary murine glial cultures were exposed to substrates of different stiffness and analyzed by transcriptomic profiling to investigate mechanobiological responses in vitro. ResultsECM-associated proteins, including glial fibrillary acidic protein (GFAP), fibronectin-1 and matrix metalloproteinase-3 (MMP3), were regionally upregulated during EAE, indicating widespread tissue remodeling beyond focal inflammatory lesions. Total Piezo1 expression was increased within lesions and transiently elevated in GM, whereas astrocyte-specific Piezo1 remained persistently upregulated during both acute and chronic EAE. Astrocytic Piezo1 expression correlated closely with ECM remodeling and clinical EAE severity, particularly in GM and NAWM. Notably, both total and astrocyte-specific Piezo1 showed stronger associations with clinical disability than classical inflammatory markers. Transcriptomic analysis revealed pronounced stiffness-dependent responses in glial cells, including alterations in extracellular matrix organization, cytokine signaling, cell adhesion, and proliferative pathways. ConclusionsOur findings identify astrocytic Piezo1 as a prominent component of neuroinflammatory tissue remodeling during EAE. The close association of Piezo1 with ECM alterations, clinical disease severity, and stiffness-dependent glial responses supports a link between neuroinflammation and mechanosensory signaling. These results highlight mechanosensation as a potentially important contributor to CNS pathology and establish Piezo1 alteration as a candidate biomarker for neuroinflammatory disease.