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The major challenge for progressive multiple sclerosis therapy is the promotion of remyelination from inflammation-induced demyelination. A switch from an M1- to an M2-dominant polarization of microglia is critical in these repair processes. In this study, we identified the homeobox gene msh-like homeobox-3 (Msx3) as a new pivotal regulator for microglial polarization. MSX3 was induced during microglia M2 polarization and repressed in M1 cells. The expression of MSX3 in microglia was dynamically regulated during experimental autoimmune encephalomyelitis (EAE), which is an animal model of multiple sclerosis. The overexpression of MSX3 in microglia promoted M2 but impeded M1 polarization. Interrupting MSX3 expression in microglia accelerated inflammation-induced demyelination and neurodegeneration. The conditioned medium from MSX3-transduced microglia promoted oligodendrocyte progenitor survival, differentiation, and neurite outgrowth. The adoptive transfer of MSX3-transduced microglia suppressed EAE and facilitated remyelination within the murine CNS in EAE and the LPC model. Mechanically, chromatin immunoprecipitation assays also indicated that MSX3 directly regulated three key genes associated with microglia M2 polarization, includingPparg,Stat6, andJak3. Importantly, we found that overexpression of MSX3 in human-derived microglia represents the M2 phenotype and ameliorated EAE after intraventricular injection. Our findings suggest a new homeobox protein-dependent mechanism for driving microglia M2 polarization and identify MSX3 as an attractive therapeutic approach for preventing inflammation-induced demyelination and promoting remyelination.

Blood–brain barrier (BBB) impairment is an early prevalent feature of multiple sclerosis (MS), and remains vital for MS progression. Microglial activation precedes BBB disruption and cellular infiltrates in the brain of MS patients. However, little is known about the function of microglia in BBB impairment. Here, microglia acts as an important modulator of BBB integrity in inflammatory demyelination. Microglial depletion profoundly ameliorates BBB impairment in experimental autoimmune encephalomyelitis (EAE). Specifically, miR‐126a‐5p in microglia is positively correlated with BBB integrity in four types of MS plaques. Mechanistically, microglial deletion of miR‐126a‐5p exacerbates BBB leakage and EAE severity. The protective effect of miR‐126a‐5p is mimicked and restored by specific inhibition of MMP9 in microglia. Importantly, Auranofin, an FDA‐approved drug, is identified to protect BBB integrity and mitigate EAE progression via a microglial miR‐126a‐5p dependent mechanism. Taken together, microglia can be manipulated to protect BBB integrity and ameliorate inflammatory demyelination. Targeting microglia to regulate BBB permeability merits consideration in therapeutic interventions in MS.

T-helper 17 (Th17) cells play an important role in the pathogenesis of multiple sclerosis (MS), an autoimmune demyelinating disease that affects the CNS. In the present study, MicroRNA sequencing (miRNA-seq) was performed in mouse Th0 and Th17 cells to determine the critical miRNAs that are related to Th17 differentiation. We found that miR-30a was significantly downregulated during mouse Th17 differentiation. In addition, the level of miR-30a in CD4⁺ T cells from peripheral blood of MS patients and experimental autoimmune encephalomyelitis (EAE) animal models was also decreased and inversely correlated with the expression of interleukin 17a, the canonical cytokine of Th17 cells. Moreover, overexpression of miR-30a inhibited Th17 differentiation and prevented the full development of EAE, whereas interference of miR-30a promoted Th17 differentiation. Mechanism studies showed that miR-30a reduced IRF4 expression by specifically binding with the 3′-untranslated region. Through screening of 640 different Food and Drug Administration (FDA)-approved drugs, we found that disulfiram and diphenhydramine hydrochloride were effective candidates for inhibiting Th17 differentiation and ameliorating EAE development through upregulating miR-30a. To our knowledge, the present work is not only the first miRNA-seq study focusing on Th17 differentiation, but also the first chemical screening for FDA-approved drugs that inhibit Th17 differentiation through regulating miRNA expression. SIGNIFICANCE STATEMENT The present work is the first miRNA sequencing (miRNA-seq) study focusing on T-helper 17 (Th17) differentiation. By miRNA deep sequencing, we found that miR-30a was downregulated during Th17 differentiation. miR-30a was also decreased in CD4⁺ T cells from multiple sclerosis patients and experimental autoimmune encephalomyelitis (EAE) mice. miR-30a reduced IRF4 expression by specific binding with the 3′-untranslated region and thus suppressed Th17 differentiation and prevented the full development of EAE. Interestingly, by performing a chemical screen with Food and Drug Administration-approved small-molecule drugs, we found that disulfiram and diphenhydramine upregulated miR-30a and suppressed Th17-associated autoimmune demyelination.

Myelin is the key structure for high‐speed information transmission and is formed by oligodendrocytes (OLs) which are differentiated from oligodendrocyte precursor cells (OPCs) in the central nervous system. Lipid is the main component of myelin and the role of lipid metabolism‐related molecules in myelination attach increasing attention. Lysophosphatidylcholine acyltransferase 1 (LPCAT1) mediates the conversion of lysophosphatidylcholine (LPC) to phosphatidylcholine (PC), and its role in myelination draws our interest as LPC is a classical demyelination inducer and PC is a major component of myelin. In this work, LPCAT1 is found expressed in the oligodendrocyte lineage cells during myelination. In vitro experiments showed that the expression level of LPCAT1 gradually increased along with the differentiation process from OPCs to OLs, and over‐expression and interference experiments showed that LPCAT1 promoted OPCs differentiation without affecting their proliferation or apoptosis. Mechanistically, the undertaker of LPCAT1's pro‐differentiation role is not PC, but the phosphorylated mTOR which is a key regulator in OPCs differentiation. RNA sequencing analysis showed LPCAT1 promoted the expression of ZBTB20 which is an important transcription factor related to lipid metabolism and regulates mTOR phosphorylation. In vivo, complex myelin tomacula involving multiple axons was formed after conditionally knocking out LPCAT1 in oligodendrocyte lineage cells, but no obvious myelin thickness abnormalities were observed. Our results indicate that LPCAT1 is an important regulator of myelination, and lipid metabolism‐related molecules may be new valuable targets for the treatment of diseases with myelin abnormalities.

Insufficient maturation of oligodendrocyte progenitor cells (OPCs) contributes to the failure of endogenous remyelination in multiple sclerosis (MS). It remains unclear whether dysregulated oligodendroglial microRNAs (miRNAs) impede remyelination in chronic MS lesions. In this study, we demonstrated that miR-126a-3p was enriched in oligodendroglia from chronic inactive MS plaques and chronic lesions in mice with experimental autoimmune encephalomyelitis (EAE). Functional analyses revealed that miR-126a-3p inhibited OPC differentiation in vitro and that the specific deletion of miR-126a-3p in oligodendroglia accelerated the remyelination process in the EAE and lysolecithin-induced demyelination models. Mechanistically, miR-126a-3p exerted an inhibitory effect on OPC differentiation and remyelination by directly targeting peroxin-5 ( Pex5 ) transcripts. A screening of a US Food and Drug Administration–approved drug library based on Pex5 levels led to the identification of ganciclovir, an antiviral agent, as a potent proremyelinating agent after in vivo demyelinating events. These results identify the aberrant miR-126a-3p– Pex5 axis in oligodendroglia as a potential therapeutic target to facilitate remyelination in chronic MS lesions.