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Demyelinating diseases such as multiple sclerosis (MS) are situations with the core feature of primary or secondary damage to myelin and oligodendrocytes (OLs) due to various insults including autoimmune attack and inflammation which ultimately lead to axonal injury and neurological dysfunction. Currently, immunomodulatory therapies for MS have very limited effects on disease progression and long-term prognosis. Therefore, pro-myelinating strategy has been attracting more and more attention. Astrocyte reactivation has been reported in many demyelination conditions and is supposed to play a duplex effect during demyelination and remyelination, while the detailed underlying mechanism remains unknown. Here, we report that reactive astrocytes in cuprizone-induced demyelination mice showed sustained upregulation of the expression of P2 purinergic receptor X1 (P2X1). Pharmacologic blockade of P2X1 receptor signaling by receptor-specific antagonist NF449 significantly enhanced/accelerated remyelination and altered new OL production dynamics. Furthermore, astrocyte-specific conditional knockout of the P2X1 gene promoted remyelination and led to early onset of new OL production. In addition, conditioned medium (CM) from ATP treated astrocyte culture that overexpressed P2X1 significantly hindered the differentiation of oligodendrocyte precursor cells (OPCs) in vitro compared to CM from empty vector virus-infected astrocytes with the same treatment, suggesting the secretion of certain inhibitory factors for OPC maturation and myelination by astrocytes with upregulated P2X1 receptor expression. Taken together, our study indicates that abnormal P2X1 receptor signaling in reactive astrocytes may be an important endogenous inhibitory factor for remyelination, and blockade of this pathway might be a potential target for pro-myelination therapy strategy for demyelinating diseases.

Regulatory mechanisms underlying microglia-dependent restorative effects during the early stages following spinal cord injury (SCI) remain uncertain. In adult mice, microglia depletion exacerbates injury and impairs functional recovery, suggesting that microglia play a protective role after SCI. We performed RNA sequencing on four spinal cord conditions (uninjured, injured, microglia-depleted uninjured, and microglia-depleted injured) to identify the core transcriptional signature of microglia-dependent restorative functions after SCI. Bioinformatics analysis identified 16 genes as critical microglia-dependent reparative regulators. Furthermore, our findings demonstrated that manipulating select genes and restoring core microglia-dependent signaling pathways resulted in a significant reduction in lesion size, increased neuronal survival, enhanced axonal regeneration, and promoted substantial locomotor recovery in a SCI model with microglia depletion. Our findings identify key transcriptional networks that regulate microglia reparative properties in the acute phases after SCI and suggest novel microglia-dependent strategies for SCI treatment.

Activation of satellite glial cells (SGCs) within the dorsal root ganglia and trigeminal ganglia (TG) has been reported to be involved in pain associated with peripheral neuropathy. One of the activation mechanisms of SGCs is that adenosine triphosphate (ATP) released from ganglion neurons transmits information via the purinergic receptor 7 (P2X₇-R) expressed on SGCs. However, it remains unknown whether primary nociceptive neurons change their excitability following activation of the ATP-P2X₇-R pathway. In the present study, excitatory changes in TG neurons following P2X₇-R-mediated SGCs activation were analyzed using whole-cell patch-clamp recordings with whole-mounted or sliced TG preparations from Sprague–Dawley rats. When the P2X₇-R agonist BzATP was applied instead of ATP, the minimum current amplitude required to induce an action potential in TG neurons was significantly reduced compared with that in the vehicle-treated group. No neuronal excitability changes were induced by BzATP in the presence of the selective P2X₇-R antagonist A740003, the metabotropic glutamate receptor 5 (mGluR5) inhibitor MTEP, or the hemichannel inhibitor carbenoxolone. Immunofluorescence examination revealed that P2X₇-R was localized in SGCs, and mGluR5 was localized in TG neurons. We confirmed that primary cultured SGCs released glutamate following BzATP stimulation. These results indicate that glutamate released from SGCs via hemichannels in a paracrine manner may alter the excitability of neighboring TG neurons. Following activation of SGCs, we analyzed the excitatory changes occurring in neurons using patch-clamp recording in intact TG preparations, and, for the first time, identified that glutamate released from SGCs activates mGluR5 on neurons, contributing to these excitatory changes.

Repetitive opioid treatment promotes analgesic tolerance through mechanisms involving opioid-induced neuroinflammation. Here, we report that Wnt signaling activation can rescue opioid analgesic effects and suppress neuroinflammation by regulating microglial cells in the spinal cord. We used prolonged repetitive morphine treatment (10 mg/kg, daily) to induce morphine analgesic tolerance in adult C57BL/6J mice. Experiments were performed in mice and in the BV-2 microglial cell line to determine the potential cellular mechanisms underlying the effects of recombinant Wnt3a on microglial reactive states. Our in vivo studies showed that repetitive morphine treatment suppressed the level of Wnt ligands and β-catenin activity in the spinal cord. After 4 days of repetitive morphine treatment, intrathecal administration of Wnt3a rescued the analgesic effect of morphine. Ablation of the microglial population by PLX5622 revealed that microglial cells were required for Wnt3a to restore the analgesic efficacy of morphine. In vitro results indicated that exogenous Wnt3a inhibited neuroinflammation and regulated the reactive states of microglial cells by suppressing the activity of NF-κB and increasing the nuclear expression of STAT3. These findings demonstrate that Wnt3a signaling activation can rescue the analgesic effect of morphine and suppress morphine tolerance-induced neuroinflammation by regulating microglial reactivation in the spinal cord. This study provides mechanistic insights into the pathophysiology of morphine tolerance and potential therapeutic targets to control morphine-induced neuroinflammation.

Microglia make important contributions to central nervous system (CNS) development, but the breadth of their distinct developmental functions remains poorly understood. The mouse retina has been a key model system for understanding fundamental mechanisms controlling the assembly of the CNS. To gain insight into where and how microglia might influence retinal development, here we identified molecularly unique myeloid cell populations that are selectively present during development and characterized their anatomical locations. Development-specific transcriptional states were identified using single-cell (sc) and single-nucleus RNA-sequencing (RNA-seq) across multiple timepoints. Transcriptional states were validated in vivo by histological staining for key RNA and/or protein markers. Several of these development-specific myeloid populations have been described before in brain scRNA-seq atlases but not validated in vivo, while others are unique to our retinal dataset. We identify two closely related microglial populations, labeled by the Spp1 and Hmox1 genes, that are distinguished mainly by transcriptional targets of the NRF2 transcription factor. Both types are present selectively within the developing retinal nerve fiber layer where they engulf neurons and astrocytes undergoing developmental cell death. Hmox1⁺ microglia were also localized selectively at the wavefront of developing vasculature during retinal angiogenesis, suggesting that developmental events associated with angiogenesis modulate NRF2 activity and thereby induce microglia to switch between the Spp1⁺ and Hmox1⁺ states. Overall, our results identify transcriptional profiles that define specific populations of retinal microglia, opening the way to future investigations of how these programs support microglial functions during development.

Exposure to hypoxic environments leads to neurological dysfunction, with recent studies implicating microglia-derived neuroinflammation involved in hypoxia-induced neuronal impairment. However, the underlying pathological mechanisms remain largely unclear. Lipid-droplet-accumulating microglia (LDAM) have been linked to age-related and genetic forms of neurodegeneration, prompting the investigation of their role in hypoxia-induced neuronal impairment. In this study, we observed that hypoxia induced lipid droplets accumulation in microglia, accompanied by increased levels of RETSAT, an enzyme involved in lipid metabolism regulation. Conditional knockout of RETSAT in microglia decreased lipid droplets accumulation and alleviates hypoxia-induced microglial-derived neuroinflammation and oxidative stress, both in vitro and in vivo. Our biological studies indicate that the beneficial effects of RETSAT knockout on lipid droplets degradation are primarily mediated through enhanced activity of hormone-sensitive lipase (HSL). Furthermore, we found that the hypoxic adaptation-related RETSAT mutation Q247R promotes microglia lipolysis under hypoxic conditions. These findings suggest that RetSat is a potential therapeutic target for the prevention and treatment of hypoxia-induced microglial activation.