Shiyu Deng

Promoting white matter repair is crucial for functional recovery after ischemic stroke. Our previous study has identified an IFN‐ γ ‐responsive microglia phenotype in the brain after stroke. However, its role in white matter repair remains poorly understood. Herein, we find that extracellular vesicles derived from IFN‐ γ ‐responsive microglia (IFN‐ γ EVs) are internalized by oligodendrocyte precursor cells (OPCs), leading to impaired OPCs proliferation, survival, and differentiation and reduced white matter repair in a murine transient middle cerebral artery occlusion (tMCAO) model. Mechanistically, miR‐9‐5p enrichment in IFN‐ γ EVs suppresses NEDD4 expression, a key regulator of myelination, leading to reduced OPCs maturation and white matter repair. Genetic interventions including miR‐9‐5p knockdown in IFN‐ γ EVs or NEDD4 overexpression in OPCs restore remyelination and functional recovery. Our findings demonstrate that IFN‐ γ EVs act as negative regulators of OPCs differentiation and remyelination following ischemic stroke via miR‐9‐5p/NEDD4 pathway. Targeting this signaling axis may represent a novel therapeutic strategy to enhance remyelination and functional recovery after stroke.

Background: Previous studies have reported the presence of interferon‐responsive microglia in the brain after central nervous system injury. However, their roles and the underlying mechanisms in neurological function recovery remain poorly understood. Methods: Adult male mice were subjected to 90‐minute transient middle cerebral artery occlusion, and brain tissues were analyzed using single‐cell RNA sequencing (scRNA‐seq) at 14 days after stroke. Immunostaining, quantitative real‐time polymerase chain reaction and ELISA were conducted to validate the presence of interferon‐γ–responsive microglia in stroke mice brains. Extracellular vesicles (EVs) were isolated from interferon‐γ–treated BV2 microglia via ultracentrifugation. Interferon‐γ EVs were then used to treat neural stem cells (NSCs) in vitro or administered intravenously to mice every other day, starting at 7 days after transient middle cerebral artery occlusion. Neurobehavioral tests, cresyl violet staining, Golgi staining, and immunostaining were performed to evaluate NSC differentiation, neurogenesis, and neurobehavioral recovery. Micro RNA (miR) sequencing and bioinformatic analysis were conducted to explore targeted genes and signaling pathways underlying interferon‐γ EV‐mediated inhibition of neurogenesis. Results: Single‐cell RNA sequencing, immunostaining, quantitative real‐time polymerase chain reaction, and ELISA showed the presence of interferon‐γ–responsive microglia in stroke mice brains. Interferon‐γ EVs were internalized by NSCs, leading to reduced NSC survival and neuronal differentiation. Administration of interferon‐γ EVs increased brain atrophy volume, inhibited neurobehavioral recovery and neurogenesis in mice after stroke. miRNA array revealed 12 upregulated microRNAs, and treatment with miR‐199a‐5p mimic inhibited the survival and neuronal differentiation of NSCs, and knockdown of miR‐199a‐5p in interferon‐γ EVs increased neurogenesis in stroke mice. miRNA database analysis and luciferase reporter assay identified SIRT1 as a downstream target gene of miR‐199a‐5p. Treatment with SIRT1 agonist promoted the survival and neuronal differentiation of NSCs, confirming that interferon‐γ EVs inhibited neurogenesis via miR‐199a‐5p/ SIRT1 . Conclusions: Our study demonstrated that interferon‐γ EVs inhibited the survival and neuronal differentiation of NSCs, exacerbating brain injury via the miR‐199a‐5p/ SIRT1 axis after ischemic stroke, providing a novel target for treating ischemic stroke.