Glia

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.

Organotypic slice cultures (OSCs) are widely used to study cellular properties in a functional and developmental tissue context. With the recent advent of transgenic mouse lines and viral tools we postulated that OSCs may enable the study of multicellular glial and neuroglial interactions in development, as well homeostatic and pathological conditions. Here, we made mouse cortical OSCs and used markers for oligodendroglial, microglial states and neuronal types between 1 to 28 days in vitro (DIV). The OSC was characterized by in-vivo like cortical layering, including layer 5 pyramidal neurons and produced highly robust synchronized period bursts resembling Up- and Down states. Glial cells showed a strong cortical layer- and time-dependent development pattern: in the first week (DIV 1-7), slicing-related debris clearance and developmentally restricted sparse oligodendroglial myelination created an environment with highly phagocytic, non-homeostatic microglia (assessed with CD68 and purinergic receptor P2Y12, respectively). Between DIV 14 and 21, however, slices showed stereotypical cortical myelin patterns and the emergence of a homeostatic microglia phenotype while exhibiting continued phagocytosis. Furthermore, live two-photon imaging and morphometric analyses revealed highly ramified microglia and myelinated axons with compact myelination, exceeding lamellae count compared to age-matched in vivo axons. Lastly, from DIV 28 and onwards, myelin integrity became impaired and associated with phagocytic microglia. Together, the results indicate that between DIV14 and 21 cortical OSCs are well suited for live imaging of homeostatic and activity-dependent neuron-glia interactions, bridging the gap between in vivo investigations and primary cell cultures.

Phagocytic and immune-like cells have been observed in the satellite envelope of neuronal somata in peripheral sensory ganglia of many species for several decades. These cells likely play an important role in normal function of sensory neurons and they may also play an important role in neuronal dysfunction and neurodegeneration seen with neuropathy. Recent findings have described a satellite macrophage population transcriptomically similar to microglia in peripheral ganglia of some mammalian species. The function of these cells, and the mechanisms by which they may influence neurons in neuropathy are unclear. We sought to understand the phenotype and localization of these cells in the human dorsal root ganglion (hDRG) using large-scale single nucleus and spatial transcriptomic datasets from individuals with and without a history of peripheral diabetic neuropathy. We observed a large population of macrophages that express classical microglia makers such as TMEM119 and P2RY12 in the hDRG, as previously described. Our findings confirm that these microglia-like cells (MLCs) localize to the satellite envelope around neuronal somata, yet are transcriptomically distinct from all glial cell types characterized in the hDRG. These MLCs exhibit changes in abundance and localization with diabetic painful neuropathy (DPN) in both the hDRG and sural nerves suggesting that they are not exclusively localized to the DRG. We conclude that microglia-like cells are likely the resident tissue macrophage (RTM) of the hDRG, and perhaps the peripheral nervous system (PNS) given their localization to the sural nerve and other ganglia, where they are predicted to regulate homeostatic neuronal functions and response to injury. HighlightsO_LIMLCs are likely the RTM of hDRGs C_LIO_LIMLCs localize to the satellite envelope and recede with Nageotte nodule formation C_LIO_LIMLC activation state and signaling shift with diabetic neuropathy C_LIO_LIMLCs are also present in other ganglia and sural nerve C_LI

The effect of sortilin inhibition on acute inner retinal neurodegeneration induced by optic nerve crush was investigated. Pharmacological sortilin inhibition using intravitreal delivery of a polyclonal antibody or a small-molecule inhibitor was evaluated in C57BL/6JRj male mice subjected to unilateral crush. Inner retinal thickness was evaluated by optical coherence tomography, and retinal ganglion cell density was determined in retinal flat mounts. Furthermore, the effect of constitutive sortilin deficiency was examined using Sort1-/- mice. Changes in protein and mRNA levels of sortilin, p75NTR, and associated injury markers were analyzed. Neither pharmacological inhibition or constitutive loss of sortilin protected against inner retinal thinning or retinal ganglion cell loss following optic nerve crush. A transient 1.4-fold increase in p75NTR mRNA was observed early after injury, accompanied by a two-fold increase in protein levels. While sortilin expression remained largely unchanged, sortilin deficiency was associated with an altered baseline retinal state, including increased GFAP, p75NTR, and proBDNF levels. Following optic nerve crush, the induction of p75NTR was significantly attenuated in sortilin-deficient retinas compared with wild type, without affecting the extent of RGC degeneration. In summary, sortilin inhibition does not preserve inner retinal structure following optic nerve crush, but modulates glial activation, inflammatory signaling, and proneurotrophin dynamics. These findings indicate that sortilin-dependent pathways are not key drivers of optic nerve crush-induced neurodegeneration but may be more relevant in disease contexts characterized by chronic stress and neuroinflammation.

Microglial activation is a common feature of neurological and inflammatory diseases and may contribute to some associated symptoms. However, methodological limitations have made it challenging to identify the specific symptoms and behavioral consequences of selective microglial activation. In this study, we examined the spectrum of symptoms elicited by acute chemogenetic activation of microglia in mice and compared them to those induced by endotoxin-driven systemic inflammation. Both interventions upregulated inflammatory gene expression in the brain, reduced voluntary wheel running, and decreased self-care. Systemic inflammation additionally caused anorexia, weight loss, reduced motivation to work for palatable food, and impaired motor performance in the rotarod test--effects not observed with chemogenetic microglial activation. By showing that acute microglial activation reproduces certain motivational aspects of the sickness response while sparing other functions, the findings might shed new light on the contribution of microglia to symptoms and behavioral alterations during disease.

We developed a physiologically relevant light damage model in pigmented mice and determine how Muller glial (MG) Dicer1/microRNA (miRNA) loss impacts retinal structure and function after injury. A moderate light damage paradigm (5,000 lux, 4 hours) was developed in pigmented mice carrying the RPE65 Leu450 variant. MG-specific Dicer1 conditional knockout (cKO) mice across three Cre lines (Rlbp1-CreER, Glast-CreER, Ascl1-CreER) were subjected to light damage at different developmental stages. Retinal structure and function were assessed longitudinally using optical coherence tomography (OCT), histology, and electroretinography (ERG). Preconditioning and double-damage paradigms were included as controls. The model induced progressive photoreceptor degeneration with early functional decline preceding structural loss and delayed inner retinal impairment. Across all MG-specific Dicer1-cKO lines, retinas exhibited partial structural preservation and, more prominently, sustained functional preservation following injury. Inner retinal function (Vmax) was consistently maintained despite reduced photoreceptor input. This phenotype was independent of age, timing of MG manipulation, or baseline retinal condition and was not reproduced by preconditioning paradigms. Dicer-deficient MG displayed reduced glial fibrillary acidic protein (GFAP) immunoreactivity, indicating suppression of reactive gliosis; however, reduced GFAP alone was insufficient to confer neuroprotection. MG-specific miRNA depletion induces a neuroprotective retinal state characterized by preserved inner retinal function and reduced secondary degeneration. These results identify MG Dicer/miRNA networks as crucial regulators of injury responses and highlight not only a glia-driven degeneration mechanism but also a potential therapeutic target.

BackgroundMicroglial heterogeneity is a fundamental feature of brain homeostasis and pathology. The purpose of this study was to investigate the complexity of microglial plasticity by characterizing specialized oligodendrocyte-like microglial subsets. MethodsThe study was performed utilizing single-cell transcriptomics analyses and immunofluorescence staining to identify and profile microglial subpopulations. Additionally, spatial transferring and morphological analyses were conducted to determine the anatomical distribution and structural features of these specific cells. ResultsWe identified a distinct microglial subset termed dual-phenotype microglia (DPM), which co-expresses microglial and oligodendrocyte markers. DPM consisted of two subtypes with distinct functions: myelin-associated DPM (mDPM) and neuron-associated DPM (nDPM). Spatial and morphological evaluations revealed that mDPMs were sparsely distributed across the whole brain and exhibited a highly ramified architecture, whereas nDPMs were enriched in the hippocampal dentate gyrus. Mechanistically, we found that mDPM function was driven by the Sox10 regulon to modulate myelin maintenance and axonal ensheathment, while nDPM was orchestrated by Glis2, facilitating essential neuron-glia crosstalk and synaptic regulation. Furthermore, we demonstrated that nDPM and mDPM were predicted to undergo significant alterations in multiple sclerosis and Alzheimers disease. Notably, mDPMs were selectively enriched in active multiple sclerosis lesions, revealing that DPM were closely related to neuropsychiatric disorders. ConclusionsBy comprehensively characterizing the morphology, molecular signatures, and spatial logic of these oligodendrocyte-like microglial subsets, our study elucidated the complexity of microglial plasticity. These findings provided new insights into their diverse roles in central nervous system health and disease. Graphical abstractIdentification, Molecular Profiling, and Functional Modeling of Dual-Phenotype Microglia (DPM). (1) Discovery: Identification of the dual-phenotype microglia (DPM) population through single-cell transcriptomics. (2) Molecular Signatures: The transcriptomic identity of DPM subtypes is governed by specific regulatory networks. (3) Distribution & Pathology: Spatial mapping reveals divergent anatomical logic and disease relations for DPM subtypes. (4) Mechanism/Theory: A proposed functional model of mDPMs as "metabolic relay" and support units. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/724239v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@b7db1dorg.highwire.dtl.DTLVardef@9265e7org.highwire.dtl.DTLVardef@1605d82org.highwire.dtl.DTLVardef@19b048f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Multiple sclerosis (MS) is an autoimmune and neurological disorder characterized by myelin disruption and neuronal degeneration. Currently approved therapies focus on symptom relief but do not promote central nervous system (CNS) repair. In contrast, astrocytes proliferate and repopulate MS-related lesions. Moreover, in active lesions, they hinder regenerative processes such as neural progenitor migration. Here, we propose astrocytes as a potential target for myelin repair in the human diseased brain. To achieve this aim, we investigated whether glial fibrillary acidic protein (GFAP)+ astrocytes can be transdifferentiated into oligodendrocyte lineage cells through forced overexpression of transcription factors both in vitro and ex vivo organotypic cultures of human adult cortex. Our results show that overexpression of OLIG2 and SOX10 in human induced pluripotent stem cell-derived astrocytes gives rise to oligodendrocyte progenitor cells 12 days post-induction, as shown by morphological changes and O4 marker expression. Importantly, transdifferentiation of GFAP-expressing endogenous astrocytes in human adult cortical tissue give rise to mature oligodendrocytes, as shown by expression of CC1, after only 12 days of overexpression of OLIG2 and SOX10. To our knowledge, this is the first study to assess direct astrocyte-to-oligodendrocyte reprogramming in a human platform preserving the native three-dimensional architecture of the brain. Further work will be required to determine whether the reprogrammed cells can myelinate axons and to evaluate the potential of this approach for structural and functional repair in the demyelinated human CNS.

Hypoxic-ischemic injury is a major cause of olfactory dysfunction, yet the cellular and morphological mechanisms underlying this sensory loss remain poorly understood. Here, we investigated the structural, cellular, and functional effects of acute hypoxic exposure on the olfactory system of adult zebrafish (Danio rerio) of both sexes, a model organism with remarkable neuroregenerative capacity. Fish were subjected to 15 minutes of acute severe hypoxia (0.8 mg/L dissolved oxygen) and assessed at 1 and 5 days post-hypoxia (dph). We evaluated olfactory function by means of cadaverine-evoked aversive behavioral assays. Structural and morphological integrity and inflammation of the olfactory epithelium (OE) and olfactory bulb (OB) were characterized using immunohistochemistry, histological stainings, and a 2,3,5-triphenyltetrazolium chloride (TTC) colorimetric assay. Acute hypoxic exposure impaired olfactory-mediated behaviors without affecting locomotion or exploratory behavior. In the peripheral OE, hypoxia caused neurodegeneration, disruption of the nasal mucus layer, and robust leukocytic infiltration. We observed reduced mitochondrial dehydrogenase activity in the olfactory bulb (OB) along with reactive astrogliosis. Olfactory function recovered by 5 days, coinciding with full restoration of OE morphology, and supported by a strong proliferative response. These findings reveal a coordinated degenerative and regenerative response to hypoxia across the olfactory axis, with implications for understanding hypoxia-induced sensory loss and neural repair. SIGNIFICANCEThis work addresses an important gap in knowledge regarding the mechanisms linking hypoxic insult and olfactory dysfunction. By using adult zebrafish, an extraordinarily regenerative vertebrate, it also provides insight into neuronal repair and regenerative processes supporting olfactory recovery. The novelty of our study resides in that, to our knowledge, there are no studies that provide a comprehensive characterization of the effects of hypoxia in the olfactory system across molecular, histological, and functional levels. These findings advance our understanding of hypoxia-induced sensory neurodegeneration and regeneration, and highlight the zebrafish olfactory system as a powerful model for investigating neural repair mechanisms relevant to hypoxic-ischemic brain injury.

Nerve injury causes an imbalance of glutamatergic excitation over GABAergic inhibition, contributing thereby to lasting neuropathic pain. Transgenic GAD67-GFP knock-in reporter mice were developed to visualize GABAergic interneurons. The knock-in into glutamate decarboxylase (GAD67) causes haploinsufficiency that manifest in low GABA levels. In this model, we studied if diminished GABA exacerbates neuropathic pain after nerve injury. Adolescent male and female GAD67-GFP knock-in mice were subjected to Spared Sciatic Nerve Injury (SNI). At baseline, nociception and thermal preferences were equal but after SNI, GAD67-GFP mice developed thermal allodynia which was not detected in wildtype littermates. At the electrophysiology level, SNI caused a partial decrease in the excitability in layer 2/3 pyramidal neurons in the projection-hemisphere in wildtype mice. This effect was exacerbated in GAD67-GFP, affecting both sides, and was accompanied with imbalance of field-potential (FP) amplitudes between projection and non-projection hemisphere, which did not occur in wildtype mice. The results suggest that GABA deficiency can be compensated to protect from hyperexcitability at baseline, but it cannot be further upscaled, ultimately leading to network hyperactivity after injury. Metabolomic studies confirmed the moderate loss of GABA in ipsi- and contralateral cortex and lumbar spinal cord of GAD67-GFP mice and failure to raise GABA in the ipsilateral dorsal horn after injury. Carnosine, cystathionine, and glutathione, three important anti-oxidative metabolites, were co-reduced with GABA suggesting that GABAergic activity and anti-oxidative capacity are interconnected and failure of this axis contributes to neuropathic "pain".

Newborns are especially susceptible to bacterial meningitis, primarily caused by Group B Streptococcus (GBS), due to incomplete maturation of immune and barrier systems. While meningitis is well known to break down the blood-brain barrier (BBB), how the meningeal arachnoid barrier, a critical component of the blood-cerebrospinal fluid barrier (B-CSFB), responds to infection is poorly understood. Using a neonatal mouse model of bacterial meningitis, we demonstrate that GBS infection significantly increases arachnoid barrier permeability, coinciding with alterations in Claudin-11 tight junction distribution and elevated meningeal production of proinflammatory cytokines (IL-6, TNF-, CXCL1). CD206+/Lyve1+ border-associated macrophages (BAMs) undergo significant morphological and molecular activation post-infection, but their depletion prior to GBS infection did not attenuate arachnoid barrier leakage or inflammatory cytokine levels during infection. We show that meningeal fibroblasts are a main source of proinflammatory cytokines in response to GBS infection and that exposure to the inflammatory cytokine TNF- alone is sufficient to induce neonatal arachnoid barrier breakdown. These results support neonatal arachnoid barrier is vulnerable to cytokine-induced breakdown in bacterial infection and highlight the role of non-immune meningeal cells like fibroblasts during bacterial infection.

Astrocytic homeostatic programs, many of which are regulated by the circadian clock, are disrupted early in neurodegenerative disease. The core clock transcription factor (TF) BMAL1 is required for normal astrocyte function, but its role during disease remains unclear. This is partly because methods for identifying cell type-specific TF binding sites are limited. Here, we developed MACS-Calling Cards (MACS-CC), a strategy for mapping astrocyte-specific TF occupancy in vivo. We used MACS-CC to define BMAL1 binding in the Cln3{Delta}ex7/8 mouse model of CLN3 disease, a fatal neurodegenerative disorder marked by early astrocyte dysfunction and circadian disruption. BMAL1 binding was extensively redistributed in Cln3{Delta}ex7/8 astrocytes: wild-type-specific binding sites enriched near glial differentiation genes, whereas Cln3{Delta}ex7/8-specific sites lacked functional enrichment. Consistent with these changes, Cln3{Delta}ex7/8 astrocytes decreased expression of mature astrocyte markers. To define mechanisms underlying BMAL1 retargeting, we tested whether altered chromatin accessibility explained the changes in BMAL1 binding. Although chromatin accessibility was broadly remodeled, differential accessibility did not predict BMAL1 redistribution. Instead, motif analysis suggested that loss of cooperative TF interactions drives BMAL1 retargeting. These findings demonstrate that MACS-CC enables astrocyte-specific TF occupancy mapping and reveals mechanisms behind early rewiring of circadian regulatory programs within a model of a neurodegenerative disease. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/721783v2_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1ada239org.highwire.dtl.DTLVardef@7564a3org.highwire.dtl.DTLVardef@122222forg.highwire.dtl.DTLVardef@1f2729c_HPS_FORMAT_FIGEXP M_FIG C_FIG

Oligodendrocyte precursor cells (OPCs) are unique glial cells that communicate bidirectionally with neurons. Neuronal inputs drive various OPC behaviors, including proliferation and differentiation, immunomodulation, blood brain barrier regulation, synapse engulfment and axonal remodeling. OPCs are implicated in numerous stress and pain conditions, where their involvement is likely driven by neuronal activity (ie. neurotransmitter and neuropeptide signaling). One neuropeptide causally involved in chronic pain and stress conditions is calcitonin gene-related peptide (CGRP). Here, we tested the hypothesis that OPCs receive direct inputs from CGRP-containing neurons in the adult brain. Using RNAscope, immunofluorescence and analysis of single-cell datasets, we find that OPCs express receptors for CGRP and we identify close spatial contacts between CGRP and OPCs, with nearly half of CGRP puncta occurring within 1 {micro}m of an OPC. Some of these contacts appear to be synaptic, with CGRP-OPC contacts colocalizing with the presynaptic protein Bassoon and the postsynaptic protein PSD-95. This work suggests the presence of both diffuse and more direct forms of CGRP signaling to OPCs, raising the importance of future experiments to identify both the mode of CGRP release onto OPCs and the functional effects of these different contact types.

Microglia are the brains resident immune cells that exhibit complex signaling behavior, including phagocytic activity in response to threats and prolonged neuronal activity. Adenosine triphosphate (ATP) is a chemoattractant for microglia. In the nucleus accumbens (NAc), ATP is co-packaged and released with DA, and microglia express dopamine (DA) receptors and ATP receptors. The present work examines microglia chemotactic motility for these transmitters using iontophoresis and multiphoton microscopy approaches in NAc brain slices from GFP-monocyte labeled transgenic mice. ATP chemoattraction was more regularly observed than DA chemoattraction, and DA chemoattraction occurred in only a small subset of microglia. The DA chemoattraction of this subset was blocked by DA D1 antagonism. Microglia are reactive oxygen species (ROS) scavengers. Application of glucose oxidase produces mild but consistent increases in ROS and induced inflammatory-related changes in microglial morphology and motility. Glucose oxidase application decreased DA release but had variable effects on ATP release. The toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS) transitioned microglia from ramified to amoeboid morphology over a period of 4 hours, and increased DA and ATP release across this same period. These studies highlight the complex relationship between local immune activation and DA terminal functionality.

Communication between gut microbiota and immune cells within the brain is essential for neurotypical development. Specifically, microglia are known to play a key role in regulating and supporting neural progenitor stem cell production during brain development, and are sensitive to changes in the maternal gut microbial composition during perinatal development. Here, we employed a germ-free (GF) porcine paradigm to examine how the absence of the microbiome affects microglial dynamics during a key epoch of brain development. We utilized automated software to evaluate microglial density and morphology across three developmentally significant regions: the ventricular/subventricular zone (VZ/SVZ), the prefrontal subcortical white matter (PFCSWM), and layers II/III of the prefrontal cortex (PFCII-III). We found no significant differences in microglial morphology or density in the VZ/SVZ or PFCSWM. In contrast, the PFCII-III of P16 piglets exhibited an increase in microglia density paired with morphologies indicative of an activated/reactive functional state. Notably, these effects were identified with no overall changes in microglial density in any of the regions assessed. Transcriptomics on RNA isolated from the PFCII-III revealed a significant upregulation of genes related to neuroinflammation, in agreement with a region-specific microglial and immune response in the absence of microbial colonization during postnatal development. Together, these findings build on the limited knowledge available on how microbiota influence brain development in large animal model organisms with high similarities to human brain anatomy and developmental trajectories. Significance StatementThe prefrontal cortex of porcine display unique, ramified microglia which are sensitive to germ-free conditions whereby they display alterations in morphology with a more transcriptionally reactive signature. These findings indicate that microglia are regionally sensitive to stimuli in the periphery, and studies in lissencephalic mammalian models may not be directly correlative to other higher-order species. The neuroanatomical heterogeneity of microglia across species is informative and understudied, but necessary, to draw conclusions on the array of perturbations spanning neurodevelopmental trajectories in health and disease.

Cerebral small vessel disease (cSVD) is a major contributor to stroke and cognitive decline, ultimately leading to vascular dementia (VaD). Genetic factors play a key role in the disease susceptibility and progression, and variants in COL4A1 cause one of the most common genetic cSVD. COL4A1 encodes the 1 subunit of type IV collagen, the principle extracellular matrix (ECM) protein in the basement membrane of vasculature. In the central nervous system (CNS), the neurovascular unit (NVU) has the unique astrocyte-derived parenchymal basement membrane (pBM), in addition to the vascular basement membrane (vBM), which together contributing to the regulation of the blood-brain barrier (BBB) function. However, the role of pBM in cSVD remains under investigated and poorly understood. The lack of relevant human models has limited our ability to dissect specific cell-cell and cell-matrix interactions, hindering the identification of effective therapeutic targets. In this study, we hypothesised that astrocyte-mediated ECM remodelling contributes to BBB dysfunction in COL4A1-associated cSVD. To investigate this, human induced pluripotent stem cells (hiPSCs) derived from a patient carrying the COL4A1G755R variant and its isogenic control line were differentiated into astrocytes and brain microvascular endothelial cells (BMECs). Comparing to isogenic controls, the COL4A1G755R astrocytes significantly reduced the expression of ECM-related genes and abnormally increased glutamate uptake. ECM preparations from COL4A1G755R astrocytes significantly damaged the tight junction (TJ) structure formed by control iPSC-derived BMECs and failed to rescue the compromised TJ integrity in COL4A1G755R BMECs. The secretome from COL4A1G755R astrocytes exaggerated the ECM abnormality in COL4A1G755R BMECs. Most importantly, reduced expression of HTRA1, a crucial serine protease known to regulate both ECM turnover and homeostasis, and increased TGF-{beta} signalling was observed in COL4A1G755R astrocytes. Functional rescue by recombinant human HTRA1 protein restored the disrupted TJ continuity in COL4A1G755R BMECs and normalized TGF-{beta} signalling and glutamate uptake in astrocytes. Together, these findings defined a previously unrecognised astrocyte-driven pBM mechanism in COL4A1-associated cSVD and highlight HTRA1 in ECM remodelling as a therapeutic target.

Porcine organotypic retinal explant cultures are widely used to study retinal neurodegeneration under controlled conditions, but the biological process that occurs in the retinal explant over time due to preparation-induced injury and culture are not well understood. Here, we generated a time-resolved transcriptomic reference for porcine neural retinal explants-maintained ex vivo for 10 days. Global expression profiles are strongly separated by culture time, with Day 0 clearly distinct from cultured samples and at Day 7 and Day 10 showing the highest similarity, indicating a transition toward a later stabilized state. Across the time course, 3,187 genes were differentially expressed relative to Day 0, with the largest shifts occurring at an early stage of culture (Day 1-Day 3). Pathway-level analyses revealed coordinated remodeling involving inflammatory signaling, and metabolic/bioenergetic changes, including reduced mitochondrial and oxidative phosphorylation-related programs at later time points. Here, we provide a time-resolved transcriptomics reference dataset for cultured porcine retinal explants. These data can build a foundation to interpret data generated in this model, differentiate changes inherent to the explant culture from treatment-specific effects and to select appropriate experimental windows for mechanistic studies of retinal degeneration.

Astrocytes directly influence neuronal survival and increasingly are understood to contribute to the progression of neurodegenerative diseases including Parkinsons disease (PD). Mitochondrial damage is a hallmark of PD pathology in both neurons and astrocytes. Damaged mitochondria are cleared by PINK1/Parkin-mediated mitophagy; loss-of-function mutations in either PINK1 or Parkin are sufficient to cause PD. Neuronal mitophagy is well-studied, but far less is known about how mitochondrial dysfunction in astrocytes affects neural health. While microglial release of pro-inflammatory cytokines has been shown to induce astrocytes to mount their own inflammatory response, we hypothesize that a more direct pathway is involved, and that mitochondrial damage to astrocytes directly triggers release of proinflammatory cytokines. To address these questions, we treated primary murine cortical astrocytes with oxidative phosphorylation (OXPHOS) inhibitors antimycin A (AA) and oligomycin A (OA) and observed the PINK1-dependent accumulation of Parkin on damaged mitochondria, leading to phospho-ubiquitination of proteins in the outer mitochondrial membrane and the recruitment of the autophagy receptor SQSTM1/p62. To identify transcriptional changes caused by mitochondrial damage and the resulting activation of mitophagic machinery, we performed bulk RNA-sequencing on astrocytes isolated from WT, PINK1-/-, or Parkin-/- mice treated with AA/OA or a vehicle control. In WT astrocytes, TNF- signaling via NF-{kappa}B was the most significantly upregulated pathway following OXPHOS inhibition. OXPHOS inhibitor treatment also stimulated p62 expression, while NF-{kappa}B inhibition prevented this upregulation. Astrocytic secretion of cytokines, including TNF-, was increased following mitochondrial damage; this secretion was dependent on NF-{kappa}B activation and occurred at levels sufficient to induce mitochondrial depolarization in hippocampal neurons. Compared to WT astrocytes, PINK1-/- astrocytes showed a significant reduction in transcriptional signatures associated with TNF- signaling following mitochondrial damage, while Parkin-/- astrocytes exhibited upregulation of both IFN-{gamma} and IFN- signaling. These findings indicate altered inflammatory responses to mitochondrial damage in the absence of functional PINK1 or Parkin. Finally, we analyzed scRNA-sequencing data from substantia nigra astrocytes harvested from human brain tissue from PD-positive or control samples. Distinct clusters comprised predominantly of PD-positive or control astrocytes emerged. Astrocytes in the PD-positive cluster were enriched for NF-{kappa}B, IFN- and IFN-{gamma} responses, consistent with the signaling observed in vitro post-OXPHOS inhibition. Together, these findings identify inflammatory signatures activated by mitochondrial damage in astrocytes, and establish this pathway as a potential contributor to neuroinflammation in PD.

The central role of microglia in CNS function in health and disease has resulted in large interest in targeting microglial as treatments for neurodegenerative disease; understanding the factors that regulate microglial gene expression will be crucial to this goal. microRNAs (miRNAs) are among the most abundant post transcriptional regulators of gene expression. miRNAs suggests miRNA were likely key to significant evolutionary events as regulators of gene expression. The miRNAome of microglia is critical to their correct functioning but the miRNA that define microglia identity and regulate key functions have not been fully defined. In this study we performed a detailed analysis of the microglial miRNAome to identify miRNA enriched in microglia that are conserved across species (human, mouse, and xenopus). We further characterised the expression of these conserved miRNAs during demyelination and remyelination and identified conserved function of a microglial-enriched miRNA across species. These findings reveal evolutionary conservation of specific miRNAs, suggesting an important role in establishing and maintaining microglial identity. They also highlight miRNAs that may be critical for microglial function in the central nervous system in both health and disease. Overall, this work advances our understanding of the factors that regulate microglial gene expression.

Nerve growth factor (NGF) exerts neuroprotective effects in the retina, and accumulating evidence indicates that microglia represent a key cellular target of NGF/TrkA signaling. However, evidence showing that the NGF/TrkA signaling in microglia is required for downstream neuroprotective actions remains unresolved. Here, we directly addressed this question by pharmacologically depleting microglia and assessing the impact on NGF pathway activity and retinal integrity. Adult C57BL/6J mice were treated with the CSF1R inhibitor PLX5622 for three weeks, resulting in a robust ([~]77%) depletion of retinal microglia. Microglial ablation induced marked structural and cellular alterations, including significant loss of retinal ganglion cells (RGCs) and thinning of retinal layers, in the absence of any other lesion or insult. Residual microglia exhibited layer-specific phenotypic changes, with a phagocytic profile in the ganglion cell layer and a more ramified morphology in the outer plexiform layer. Strikingly, microglial depletion led to a profound decrease of NGF signaling, with a strong reduction in total and phosphorylated TrkA, and decreased p75NTR levels, in retinal extracts. The amount of TrkA expression is strongly correlated with microglial levels, supporting a primary role of microglia in sustaining NGF signaling in the retina. Together, these findings demonstrate that microglia are required for NGF/TrkA signaling and identify these cells as essential mediators of NGF-dependent neuroprotection in the retina.