It is well established that inflammation contributes to the ocular complications caused by diabetes; however, the specific molecular events that drive diabetes-induced pro-inflammatory signaling in the retina remain to be fully elucidated. This study investigated the role of Müller glial spleen tyrosine kinase (SYK) in diabetes-induced retinal complications. Hyperglycemic culture conditions increased mitochondrial membrane permeability and cytosolic mitochondrial DNA content in human MIO-M1 Müller cells and enhanced cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling, nuclear factor-κB (NF-κB) activation, and inflammatory cytokine expression. STING inhibition reduced inflammatory cytokine expression in cells exposed to hyperglycemic conditions by acting downstream of the increase in cytosolic mitochondrial DNA levels. In cells exposed to either hyperglycemic conditions or the STING agonist diABZI, SYK signaling was necessary for cGAS-STING pathway activation. Inhibition of SYK-dependent cGAS-STING signaling reduced the expression of inflammatory cytokines, including IL1β, CCL2, and CCL5, under hyperglycemic conditions. In the retina of diabetic mice, Müller glia-specific SYK deletion reduced glial activation and attenuated inflammatory cytokine expression. Müller glia-specific SYK deletion also prevented diabetes-induced retinal thinning and visual function deficits in spatial frequency threshold and contrast sensitivity. The data support an essential role for Müller glial SYK in diabetes-induced retinal inflammation and the development of functional deficits in vision.

ABSTRACTOvercoming remyelination failure is one of the main targets in therapeutic strategies for multiple sclerosis. This process requires the differentiation of oligodendrocyte precursor cells (OPCs) to mature myelinating oligodendrocytes (OLs), a process known to be controlled by thyroid hormone, nuclear receptors, and sonic hedgehog (SHH). Retinoid X receptor gamma (RXRg) is one of the nuclear receptors acting as a positive regulator of remyelination, but little is known about its mechanisms of function. Using transcriptomic and pharmacological analysis of primary neural stem cell‐derived OPCs, we show that RXRg is involved in the induction of the thyroid hormone‐driven differentiation process and in refining it toward an oligodendrogenic cell fate. RXRg also emerged as an important negative modulator of SHH expression and signaling, as Shh and additional genes from this pathway were found to be strongly upregulated in Rxrg−/− OPCs. An inhibition of SHH signaling by cyclopamine or GANT61 entirely normalized the differentiation deficit of Rxrg−/− OPCs, but also myelination of newly generated Rxrg−/− OLs. Such data indicate a key role of SHH hyperactivity in the oligodendrogenesis block associated with the absence of RXRg. Importantly, hyperactivation of the SHH pathway by purmorphamine or SAG inhibited the oligodendrogenesis and myelination potential of wild‐type OPCs, indicating that SHH hyperactivity can also be a sufficient factor to block OPC differentiation. These results point to RXRg as an important regulator of SHH pathway signaling and underline the need of an optimal, fine‐tuning of SHH signaling to assure successful oligodendrogenesis.

Neonatal hypoxic-ischemic encephalopathy (HIE) may induce substantial neuronal damage. In particular, an overactivation of microglia following HIE represents a pathogenically important process. Previous studies have shown that microglial inhibitors can exert neuroprotective effects in HIE; however, the specific mechanisms underlying these effects have not yet been elucidated. Ligation of the left common carotid artery and exposure to 5% O2 were utilized to produce an HIE model in rats. A number of experimental approaches were then used to determine the effect of a microglial inhibition, achieved via the administration of GW2580, a Csf1r inhibitor, and investigate the mechanisms involved. Our HIE models exhibited substantial brain infarction and were significantly impaired in motor functions (p < 0.01-0.001, in all tests examined). In the infarction areas, the number of microglia, macrophages, and neural stem cells (NSCs) was all dramatically increased over that in sham-injured rats, respectively (p < 0.05-0.001). The administration of GW2580 significantly reduced the numbers of microglia and macrophages, but increased the number of NSCs when compared to those in vehicle-treated HIE models (p < 0.05-0.001). Furthermore, GW2580 significantly ameliorated both the histological and behavioral phenotypes in HIE rats and increased STAT phosphorylation (p < 0.05-0.001). Finally, the inhibition or activation of STAT3 respectively decreased or increased the neuroprotective effects of GW2580 (p < 0.05-0.001). Collectively, our findings demonstrate that the STAT3 signaling pathway plays a critical role in the neuroprotective effects of microglial inhibition and may facilitate the development of novel therapeutic strategies to treat stroke.

Injury to the central nervous system (CNS) such as spinal cord results in multifaceted cellular responses, including the glial scars, a structural formation of reactive glia around an area of severe tissue damage. However, the functional heterogeneity within the cells in glial scars remains unclear. In this study, we found Aldh1L1 lineage cells exhibited reactive astrocyte distribution, culminating in glial scar formation via proliferation and differentiation after SCI. Interestingly, the Aldh1L1+ glial scars were distinct from the GFAP+ glial scars, encircled an area closer to the injury center and harbored scattered Aldh1L1 lineage cells within the central zone. At day 7 after SCI, Aldh1L1 lineage cells reached peak activation; some of them exhibited an injury responsive intermediate state with neural stem cell-like properties, and a significant portion of the Aldh1L1+ glial scar arose from the differentiation and migration of central canal ependymal cells, not derived from invading meningeal cell lineages or pial astroglia. Some of the Aldh1L1+ cells in glial scar exhibited expression of oligodendrocyte progenitor cell markers. Mechanistically, YAP was highly expressed and activated in Aldh1L1+ cells after SCI, and YAP deletion in Aldh1L1+ cells inhibited glial scar formation and differentiation into astrocytes from Aldh1L1+ ependymal cells, exacerbated neuronal loss, and inhibited the functional recovery of mice after SCI. These results suggest that Aldh1L1 lineage cells contribute to the functional heterogeneity within the cells in glial scars after SCI and uncover the pivotal role of YAP signaling in the formation of glial scars derived from Aldh1L1 lineage cells.

ABSTRACTCortical demyelination is a critical contributor to progressive disease in multiple sclerosis (MS). The barriers to cortical remyelination following demyelination are not fully understood, and there are no remyelinating treatments for MS. We previously took advantage of the spatial and temporal resolution of longitudinal in vivo imaging to study cortical oligodendrocyte regeneration following cuprizone‐induced demyelination and found that oligodendrocyte regeneration was impaired. In this study, we investigated whether cortical reactive microglia disrupt oligodendrocyte regeneration. To do so, we used a combination of in situ RNA and immunofluorescence labeling to characterize cortical microglia reactive states following cuprizone‐mediated demyelination. We then depleted cortical microglia by administering a Csf1r inhibitor during the recovery period from cuprizone and quantified oligodendrocyte recovery. We found that following cortical demyelination, deep cortical microglia change morphology, downregulate homeostatic markers (P2RY12, TMEM119), and upregulate a marker (CD68) associated with activated macrophages. These reactive changes persisted through early recovery post‐cuprizone but resolved by late recovery. Depleting cortical microglia post‐cuprizone restored the baseline density of deep cortical ASPA+ oligodendrocytes at early and late recovery. There were also more deep cortical BCAS1+ differentiating oligodendrocytes at early recovery when microglia were depleted, suggesting that transient deep cortical reactive microglia impair oligodendrocyte differentiation following demyelinating injury. Together, we found that cortical microglia adopt spatially restricted reactive functions after demyelination and deep cortical reactive microglia transiently reduce differentiating oligodendrocytes. A potential therapeutic strategy for progressive MS could involve targeting transiently reactive microglia at the right time and place in cortical lesions to promote oligodendrocyte regeneration.

ABSTRACTOligodendrocytes, traditionally recognized for their role in central nervous system myelination, have emerged during the last decades as key participants maintaining brain homeostasis in response to metabolic demands and stress. In addition, injury to myelin prompts a regenerative response that leads to the formation of new myelin sheaths. However, the signals regulating effective remyelination by oligodendrocytes are still not completely understood. Here, we report that oligodendrocytes can internalize exogenous myelin both in vitro and in vivo, which leads to an increase in oligodendroglial lineage progression. RNA sequencing reveals that myelin debris alters the oligodendrocyte transcriptional profile, leading to the suppression of immune‐related pathways and de novo cholesterol and fatty acid biosynthesis, while promoting lipid droplet formation for the storage and processing internalized myelin particles. In primary cultures, myelin exposure increases oligodendrocyte progenitor (OPC) proliferation and overall oligodendroglia lineage progression, accompanied by greater cellular complexity and a larger myelinated area per cell, without altering the relative OPC‐to‐mature oligodendrocyte ratio. Stereotaxic injection of fluorescent myelin into mouse cortex and zebrafish ventricles shows internalization by microglia and, to a lesser extent, by oligodendroglia. Notably, in the zebrafish model, ventricular injections of myelin also increase the number of ventral oligodendrocytes in the spinal cord, further supporting that myelin can promote lineage progression. These findings challenge the classical view that myelin debris intrinsically inhibits oligodendrocyte proliferation, suggesting instead that oligodendrocytes can use myelin to support self‐renewal and maturation across vertebrate species, acting as a trophic factor in the absence of pathological cues.

ABSTRACTMicroglia, the brain's innate immune cells, possess complex, highly motile branched processes. These act independently, enabling individual processes to carry out entirely distinct functions in parallel. Intracellular Ca2+ signaling is implicated in many of these distinct microglial functions. However, it has been difficult to quantify how such Ca2+ activity is compartmentalized in space and time to prevent unwanted cross‐talk between signaling pathways. Previous studies have typically relied on manually drawn regions‐of‐interest (ROIs), which averages fluorescence within predefined compartments and therefore cannot resolve the fine‐scale spatio‐temporal propagation patterns that may be functionally relevant. To address this, we adopt an unbiased non‐ROI‐based analytical approach to comprehensively characterize the temporal, spatial and spatio‐temporal dimensions of microglial Ca2+ activity in vivo. We find that microglial Ca2+ activity predominantly occurs in processes, tends to remain localized at its site of origin, and, when it propagates, often follows a well‐defined direction (either toward or away from the soma) rather than spreading isotropically as would be expected under purely passive diffusion. The tendency of microglial Ca2+ activity to spread between intracellular regions does not correlate with peak amplitude, but appears to be limited by the branching points of the microglial processes. Finally, we show that Ca2+ activity can differ between the microglial soma and its processes in response to various pharmacological stimuli. These results suggest that Ca2+ signals are actively compartmentalized within microglia in a context dependent manner, rather than being synchronized across the entire cell.

ABSTRACTMultiple sclerosis (MS) affects women more frequently than men, but the disease progresses more aggressively in men. We have demonstrated that acute intermittent hypoxia (AIH), a noninvasive therapy, promotes repair and remyelination and alters disease course in the female MOG35‐55 experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Given the importance of understanding sex‐specific responses to potential MS therapies, we investigated whether AIH exerts similar therapeutic effects in male EAE mice. EAE was induced by MOG35‐55 immunization in C57BL/6 male mice. Male EAE mice received either AIH (10 cycles‐5 min 11% oxygen alternating with 5 min 21% oxygen) or Normoxia (21% oxygen for same duration) once daily for 7d beginning at near peak EAE disease clinical score of 2.5. Mice were followed post‐last treatment for an additional 7d or 14d before assessing histopathology. Clinical scores, inflammation, myelination, and neurorepair were evaluated. Compared to Normoxia, AIH significantly improved clinical scores in male EAE mice with mice exhibiting reduced inflammation and increased myelination/remyelination within inflamed regions. Further, AIH polarized remaining immune cells toward a pro‐repair phenotype, promoted OPC recruitment to demyelinated regions, and increased the presence of mature, myelinating oligodendrocytes, and myelination. An axon protective phenotype was also significantly improved with AIH, supporting enhanced neuroprotection. Our findings reveal that AIH has comparable, albeit slightly less robust beneficial therapeutic effects in male as was previously shown in female EAE mice. Altogether, this study highlights the potential of AIH as a therapy for MS, capable of addressing the disease's differential impacts in both sexes.

Glutamate-mediated neurotoxicity is a key factor in the pathogenesis of Alzheimer's disease, yet its regulatory mechanism and connection to the amyloid β (Aβ) cascade remain unclear. This study examined the role of the astrocytic Aβ receptor, PirB, on glutamate reuptake, excitotoxic neuronal death, and cognition. Invitro, primary astrocytes were treated with soluble PirB extracellular peptide (PEP), the PirB inhibitor Fluspirilene, or lentiviral vectors encoding PirB or shPirB. We measured excitatory amino acid transporter (EAAT) expression, intracellular glutamate levels, and calcium influx in astrocytes. We also evaluated neuronal apoptosis in neuron-astrocyte co-cultures. PirB inhibition (via PEP or Fluspirilene) significantly increased EAAT1 and EAAT2 expression and activated the mTOR signaling. This was accompanied by elevated astrocytic glutamate levels, decreased intracellular calcium, as well as reduced neuronal apoptosis, whereas PirB overexpression had the opposite effects. Invivo, mice with astrocyte-specific PirB conditional knockout (cKO) were injected with Aβ oligomers into the hippocampus and subjected to cognitive behavioral assays. Hippocampal neuronal loss and apoptosis were evaluated through Nissl and TUNEL staining. PirB cKO mice showed enhanced exploratory behavior, improved working memory, and higher EAAT expression, along with higher Bcl-2/Bax ratios, mTOR activation, and reduced neuronal apoptosis compared with Aβ-injected PirBflox/flox mice. Overall, our findings identify astrocytic PirB as a key driver of Aβ-triggered glutamate-mediated neurotoxicity and suggest that suppressing the PirB-mTOR-EAAT pathway could be a promising therapeutic strategy for mitigating Aβ-related cognitive impairment.