Interferon‐γ–Responsive Microglia‐Derived Extracellular Vesicles Inhibited Neurogenesis After Stroke via MicroRNA‐199a‐5p/ SIRT1 Axis

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Abstract
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.

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