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

Tongtong Xu; Dongliang Qian; Shiyu Deng; Yiyan Guo; Hanlai Li; Yanqin Geng; Lin Gan; Qianyuan Lian; Jingjing Xu; Huaitong Yao; Jing Ye; Enkhbat Myagmartsend; Wanlu Li; Zhijun Zhang; Guo‐Yuan Yang; Jixian Wang; Yaohui Tang; Qingzhu An

doi:10.1161/JAHA.125.043532

. 2026 Feb 20;15(5):e043532. doi: 10.1161/JAHA.125.043532

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

Tongtong Xu ^1,^2,^#, Dongliang Qian ^1,^#, Shiyu Deng ², Yiyan Guo ², Hanlai Li ², Yanqin Geng ², Lin Gan ², Qianyuan Lian ², Jingjing Xu ², Huaitong Yao ², Jing Ye ², Enkhbat Myagmartsend ², Wanlu Li ², Zhijun Zhang ², Guo‐Yuan Yang ², Jixian Wang ^3,^✉, Yaohui Tang ^2,^✉, Qingzhu An ^1,^✉

PMCID: PMC13055836 PMID: 41717953

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.

Keywords: extracellular vesicles, interferon‐γ–responsive microglia, neurogenesis, stroke

Subject Categories: Neurogenesis

Nonstandard Abbreviations and Acronyms

CCK‐8: Cell Counting Kit‐8
CFSE: carboxyfluorescein succinimidyl ester
CXCL10: C‐X‐C motif chemokine ligand 10
EV: extracellular vesicle
GFAP: glial fibrillary acidic protein
IBA‐1: ionized calcium‐binding adapter molecule 1
ISGs: interferon‐stimulated genes
NSC: neural stem cell
miR‐kd: microRNA‐199a‐5p‐knockdown
OGD: oxygen–glucose deprivation
scRNA‐seq: single‐cell RNA sequencing
SVZ: subventricular zone
tMCAO: transient middle cerebral artery occlusion
WB: western blot

Research Perspective.

What Is New?

Extracellular vesicles derived from interferon‐γ–stimulated microglia inhibited the survival and neuronal differentiation of neural stem cells and suppressed neurobehavioral recovery and neurogenesis in mice via the microRNA‐199a‐5p/SIRT1 axis after ischemic stroke.

What Question Should Be Addressed Next?

Whether targeted regulation of the microRNA‐199a‐5p/SIRT1 axis in extracellular vesicles derived from interferon‐γ–stimulated microglia could promote neurogenesis and functional recovery following stroke.

Stroke is the leading cause of death and disability worldwide. ¹ Following stroke, neural stem cells (NSCs) located in the subventricular zone (SVZ) and the subgranular zone are activated, proliferate, and migrate to the peri‐infarct area, where they contribute to improving neural function recovery through neurogenesis. ² , ³ However, the inflammatory microenvironment in the lesion region significantly inhibits neurogenesis, thereby hindering neurological recovery. ⁴ Understanding the impact and mechanisms of the poststroke microenvironment on neurogenesis is essential for developing strategies to improve neural function recovery.

Microglia, as resident immune cells of the central nervous system, play a pivotal role in regulating neurogenesis under pathological conditions such as ischemic stroke or traumatic brain injury. ⁵ , ⁶ Following central nervous system injury, microglia rapidly activate and produce a plethora of proinflammatory cytokines, including tumor necrosis factor‐α, interleukin‐1β, and interleukin‐6, which impair the function of NSCs, thereby suppressing neurogenesis and functional recovery. ⁷ , ⁸ However, emerging evidence also suggests that microglia can contribute to promoting neural repair and remodeling by clearing cellular debris and secreting anti‐inflammatory factors. ⁹ These seemingly contradictory findings may stem from the heterogeneity of microglia following stroke and their dynamic roles at different stages of stroke. ¹⁰ Recent advances in single‐cell RNA sequencing (scRNA‐seq) have unveiled the existence of multiple microglial subpopulations in stroke and neurodegenerative diseases, including homeostatic microglia, disease‐associated microglia, white matter–associated microglia, and interferon‐responsive microglia. ¹¹ , ¹² Among these, interferon‐responsive microglia have been shown to play a pivotal role in influencing the neurogenic microenvironment. For instance, Roy et al identified type I interferon–associated microglia to engulf synapses via complement C3 in Alzheimer disease mice, suggesting their involvement in Alzheimer disease pathogenesis. ¹³ Recently, Escoubas et al discovered a population of type I interferon‐responsive microglia in the somatosensory cortex of 5‐day‐old neonatal mice, which actively engulf entire neurons to maintain nervous system homeostasis. ¹⁴ Furthermore, Kaya and his colleagues identified a subset of interferon‐responsive microglia in aged and Alzheimer disease mice that damage white matter function by engulfing myelin, leading to cognitive dysfunction. ¹⁵ However, the roles and mechanisms of interferon‐responsive microglia on neurogenesis after stroke remain poorly understood, warranting further investigation.

Extracellular vesicles (EVs) are nanoscale lipid membrane vesicles that transport a diverse array of bioactive molecules, serving as key mediators of intercellular communication among neurons, glial cells, and endothelial cells. ¹⁶ These vesicles play a critical role in regulating the remodeling of the neurovascular unit following ischemic injury. ¹⁷ Research from our group and others have demonstrated that microglia‐derived EVs can modulate the function of NSCs. For instance, our previous study showed that EVs derived from anti‐inflammatory microglia promoted the proliferation of NSCs and their differentiation into neurons via microRNA‐124 (miR‐124) and its downstream target protein adaptor‐associated kinase 1/Notch. ¹⁸ In another study, Fan et al discovered that microglia‐derived EVs enriched with miR‐146a‐5p inhibited the proliferation and differentiation of NSCs through the KLF4/CDKL5 signaling pathway in a rat model of depression, thereby suppressing neurogenesis. ¹⁹ On the basis of these findings, we hypothesize that EVs secreted by interferon‐responsive microglia may play a significant role in regulating neurogenesis following stroke.

In our study, we identified a subpopulation of interferon‐γ–responsive microglia in the chronic phase following stroke. Importantly, we discovered that EVs derived from these microglia inhibited the viability and neuronal differentiation of NSCs via miR‐199a‐5p/SIRT1, ultimately impairing neural function recovery. These findings provide a novel therapeutic target for enhancing neural repair after ischemic stroke.

Methods

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Microglia Stimulation With Interferon‐γ

BV2 microglia were cultured in DMEM (Meilunbio, Dalian, China) supplemented with 10% heat‐inactivated FBS (Gibco, Waltham, MA) and 1% penicillin–streptomycin (Meilunbio). BV2 microglia were seeded at a density of 2.0×10⁶ cells per 10 cm culture dish and allowed to adhere for 12 hours under standard condition (37 °C, 5% CO₂). To induce an interferon‐γ–responsive phenotype, BV2 microglia were treated with 100 ng/mL recombinant mouse interferon‐γ protein (MCE, Princeton, NJ) for 12 hours. Interferon‐γ–responsive microglia were further identified using quantitative real‐time polymerase chain reaction (RT‐PCR), western blot (WB) analysis, and immunostaining.

Isolation, Identification, and Labeling of EVs

Microglia‐derived EVs were isolated from supernatants of untreated microglia (naive EVs) and interferon‐γ–responsive microglia using sequential ultracentrifugation. To eliminate contaminating EVs from FBS, FBS was preprocessed by ultracentrifugation (100 000g, 16 hours, 4 °C; SW32Ti rotor, Optima Ultracentrifuge, Beckman Coulter, Germany). BV2 microglia were washed with PBS (Meilunbio) and incubated for 12 hours in EV‐free medium with or without 100 ng/mL interferon‐γ. Supernatants were then collected and subjected to differential centrifugation: 300g (10 minutes), 2000g (10 minutes), and 10 000g (30 minutes) at 4 °C to remove cells and cell debris, followed by EV pelleting at 100 000g (70 minutes, 4 °C). EVs were washed once in PBS (100 000g, 70 minutes, 4 °C) and resuspended in PBS. Protein concentration was quantified via bicinchoninic acid assay (Meilunbio). The EV marker CD81 and the negative control calnexin (endoplasmic reticulum marker) were analyzed by WB. Morphology was visualized by transmission electron microscopy (Thermo Fisher Scientific, Waltham, MA). Particle size and concentration were determined via nanoparticle tracking analysis (Brookhaven Instruments, Holtsville, NY). For labeling of EVs with carboxyfluorescein succinimidyl ester (CFSE), 100 μg EVs were incubated with 5 mM CFSE (Thermo Fisher Scientific) in 300 μL PBS for 2 hours at 37 °C. Unbound CFSE was removed by ultracentrifugation (100 000g, 70 minutes, 4 °C).

Animals

All procedures of animal studies were approved by the Institutional Animal Care and Use Committee of Fudan University and Shanghai Jiao Tong University and complied with Animal Research: Reporting In Vivo Experiments guidelines. Mice were maintained under specific pathogen‐free conditions in individually ventilated cages with controlled environmental parameters: temperature 22 °C±2 °C, humidity 55%±5%, ≥60 air changes/h, and 12‐hour light/dark cycle. Eight‐week‐old male mice (≤5 mice/cage) were housed in autoclaved individually ventilated cages containing γ‐irradiated corn cob bedding (Slacom, Shanghai, China). Animals received autoclaved water and irradiated standard chow (Slacom) ad libitum. Nestin^creERT2 mice (Gem Pharmatech, Nanjing, China) were crossed with Rosa26‐LsL‐cas9‐tdTomato (Rosa‐tdTomato) mice (Gem Pharmatech) to generate mice with red fluorescently labeled NSCs (Nestin^creERT2–Rosa‐tdTomato). Tamoxifen (MCE) dissolved in 95% corn oil at a final concentration of 10 mg/mL was intraperitoneally injected (0.075 mg/g body weight) once a day for 5 consecutive days. Mice were used at least 7 days after the final dose of tamoxifen.

Transient Middle Cerebral Artery Occlusion Model

Transient middle cerebral artery occlusion (tMCAO) was induced in mice as previously described. ²⁰ , ²¹ , ²² Briefly, mice were anesthetized with 1.5% isoflurane (RWD, Shenzhen, China) and maintained under normothermic conditions. The common carotid artery, external carotid artery, and internal carotid artery were surgically exposed. A 6‐0 silicone‐coated monofilament (Covidien, St. Louis, MO) was introduced into the external carotid artery lumen and advanced retrograde to occlude the origin of the middle cerebral artery for 90 minutes. Reperfusion was initiated by filament withdrawal. Cerebral blood flow was monitored using laser Doppler flowmetry (Moor Instruments, Devon, UK). Successful occlusion was defined as cerebral blood flow reduction to ≤20% of baseline, with reperfusion confirmed by cerebral blood flow recovery ≥80% of baseline.

Animal Experimental Design

Adult male C57BL/6J mice (22–25 g) were randomly divided into 5 groups (120 in total): (1) PBS treatment following tMCAO; (2) naive microglia‐derived EVs treatment post following tMCAO; (3) interferon‐γ EV treatment following tMCAO; (4) control lentivirus‐transduced interferon‐γ EV treatment following tMCAO (miR‐cn EVs); (5) miR‐199a‐5p‐knockdown (miR‐kd) interferon‐γ EV treatment following tMCAO. Brain tissues were collected at days 14 and 28 for analysis. Neurobehavioral assessments (modified neurological severity score, elevated body swing test, rotarod test, hanging wire test, novel object recognition, and T‐maze test) were conducted over 28 days, with terminal end points at days 14 and 28 for cresyl violet staining and immunostaining. Mice that died due to surgical procedures were excluded from the analysis.

Tail Vein Injection

Naive EVs (100 μg/1.2×10¹⁰ particles in 100 μL PBS per mouse), interferon‐γ EVs (100 μg/1.6×10¹⁰ particles in 100 μL PBS per mouse) or PBS were intravenously injected through the tail vein every other day, starting at day 7 post‐tMCAO. Mice were immobilized in a tail vein imaging chamber under infrared illumination to enhance vasodilation, followed by alcohol disinfection for vein visualization. Using a 1‐mL syringe (air bubbles preexpelled, 29G needle), injections targeted distal‐to‐proximal vein segments at a 15‐degree insertion angle. Venous access was confirmed by blood reflux upon gentle aspiration, after which EVs or PBS were infused at a controlled rate to prevent extravasation. Postinjection hemostasis was ensured by 10‐second pressure application with sterile gauze.

Brain Atrophy Volume Assessment

Mice were euthanized at 14 and 28 days after tMCAO for atrophy volume analysis. Following cardiac perfusion with PBS and 4% paraformaldehyde (Macklin, Shanghai, China), brains were postfixed in 4% paraformaldehyde (4–6 hours, 4 °C) and cryoprotected in 30% sucrose (48 hours, 4 °C) until tissue sinking. Coronal sections (25‐μm thickness) spanning the anterior commissure to the hippocampus were collected using a freezing microtome (Leica, Wetzlar, Germany). Every 10th section (250‐μm intervals) was stained with 0.05% cresyl violet (Sigma‐Aldrich, St. Louis, MO), and hemispheric areas were measured using ImageJ (National Institutes of Health, Bethesda, MD). The atrophy area denoted as ∆S was calculated by subtracting ipsilateral area from contralateral area. The thickness between 2 adjacent brain slices was described as height (H), and the atrophy area of 2 adjacent slices was denoted as ∆Sn and ∆Sn+1. Total brain atrophy volume was then calculated according to the formula: V=∑H/3×[∆Sn+(∆Sn×∆Sn+1)^1/2+∆Sn+1]. ²³

NSC Isolation and Culture

Primary NSCs were isolated from embryonic day 14 mouse embryos obtained from pregnant C57BL/6J mice. Pregnant dams were anesthetized, and embryos were aseptically extracted following abdominal disinfection with 75% ethanol. Fetal brains were dissected under a stereomicroscope, and the cerebral cortices were enzymatically digested with 0.25% trypsin–EDTA (Meilunbio) for 7 to 8 minutes at 37 °C. Digestion was terminated by glial complete medium, followed by mechanical trituration and filtration through a 40‐μm cell filter (Absin, Shanghai, China). The single‐cell suspension was centrifuged at 1000g for 5 minutes, washed twice with PBS to eliminate residual FBS, and cultured in NSC proliferation medium (DMEM/F12 [Meilunbio] supplemented with 1% B27 [Gibco], 1% GlutaMAX [Gibco], 1% penicillin/streptomycin [Meilunbio], 20 ng/mL epidermal growth factor [Peprotech, Cranbury, NJ], and 20 ng/mL basic fibroblast growth factor [Peprotech]) in 10‐cm dishes. Neurospheres formed within 3 to 4 days and were passaged using accutase as the following: cells were digested for 10 minutes at 37 °C with gentle agitation every 3 minutes, neutralized with PBS, filtered, centrifuged, and replated at a 1:2 ratio in fresh medium. NSCs from passages 1 to 3 were maintained in proliferation medium or differentiated in neurobasal‐based medium (2% B27, 1% GlutaMAX, 0.5% penicillin–streptomycin, filter‐sterilized). For proliferation assay, NSCs were cultured in proliferation medium and treated with naive EVs at concentrations of 5 μg/mL (6×10⁸ particles/mL), 10 μg/mL (1.2×10⁹ particles/mL) and 20 μg/mL (2.4×10⁹ particles/mL) or interferon‐γ EVs at concentrations of 5 μg/mL (8×10⁸ particles/mL), 10 μg/mL (1.6×10⁹ particles/mL) and 20 μg/mL (3.2×10⁹ particles/mL). After 48 hours of stimulation, cell viability was quantified using the Cell Counting Kit‐8 (CCK‐8) assay (Meilunbio).

Oxygen–Glucose Deprivation Model

NSCs were exposed to oxygen–glucose deprivation (OGD) using a modified protocol. NSCs were washed with PBS, then cultured in glucose‐free DMEM, and transferred to a hypoxia chamber preequilibrated with oxygen concentration below 0.1% at 37 °C for 10 hours. Control NSCs were maintained under normoxic conditions with complete medium throughout the experiment. After OGD, NSCs were reoxygenated (5% CO₂/95% air) in fresh proliferation medium and treated with naive EVs at concentrations of 5 μg/mL (6×10⁸ particles/mL), 10 μg/mL (1.2×10⁹ particles/mL), 20 μg/mL (2.4×10⁹ particles/mL) and 40 μg/mL (4.8×10⁹ particles/mL) or interferon‐γ EVs at concentrations of 5 μg/mL (8×10⁸ particles/mL), 10 μg/mL (1.6×10⁹ particles/mL), 20 μg/mL (3.2×10⁹ particles/mL), and 40 μg/mL (6.4×10⁹ particles/mL). After 38 hours of treatment, cell viability was quantified using the Cell Counting Kit‐8 assay.

NSC Differentiation Assay

NSCs were seeded on poly‐D‐lysine (Sigma‐Aldrich)‐coated glass coverslips (24‐well plate, 1×10⁵ cells/well) or poly‐D‐lysine–coated 6‐well plates (5×10⁵ cells/well) for immunostaining and WB analyses, respectively. After 48‐hour culture in proliferation medium, cells were differentiated for 7 days in differentiation medium containing 20 μg/mL EVs with medium refreshed once every 3 days. Differentiation outcomes were assessed by dual immunostaining for neuronal marker βIII‐tubulin (Tuj‐1, Millipore), and astrocytic marker GFAP (glial fibrillary acidic protein; Oasis, Zhejiang, China). For miRNA mimic experiments, NSCs were transfected with mimics for 6 hours, allowed to recover in proliferation medium for 18 hours, then differentiated for 6 days before Tuj‐1/GFAP costaining.

RNA Extraction and RT‐PCR

Total RNA was isolated from ischemic brain or cultured cells using TRIzol Reagent (Invitrogen, Carlsbad, CA), followed by reverse transcription with the ZymoScript II First Strand cDNA Synthesis Kit (Zymo Research, Irvine, CA). Quantitative real‐time polymerase chain reaction (RT‐PCR) amplification was performed on a QuantStudio 6 Pro System (Applied Biosystems, Foster City, CA) under the following conditions: initial denaturation at 95 °C for 5 minutes and 40 cycles of 95 °C for 10 seconds and 60 °C for 30 seconds. Gene expression was normalized to GAPDH. Primer sequences of mRNA are provided in Table S1.

For EV‐derived miRNA analysis, total miRNA was extracted using the miRNeasy Serum/Plasma Kit (Qiagen, Hilden, Germany), reverse‐transcribed with the miRCURY LNA RT Kit (Qiagen), and quantified via SYBR Green‐based RT‐PCR (miRCURY SYBR Green PCR Kit, Qiagen). All reactions complied with Minimum Information for Publication of Quantitative Real‐Time PCR Experiments guidelines, with U6 serving as the endogenous control. Primer sequences of miRNA are provided in Table S2.

Immunostaining

Paraformaldehyde ‐fixed brain sections or cultured cells were rinsed 3 times with PBS (5 min/wash), permeabilized with 0.3% Triton X‐100 for 10 minutes at room temperature, and blocked with 3% BSA for 1 hour at room temperature. Samples were incubated overnight at 4 °C with primary antibodies of interferon‐responsive proteins IFIT3 (1:100; Santa Cruz, Texas, TX), interferon‐stimulated gene (ISG) 15 (1:100, Santa Cruz), CXCL10 (C‐X‐C motif chemokine ligand 10; 1:100, Santa Cruz), and IBA‐1 (ionized calcium‐binding adapter molecule 1; 1:200, Oasis), Nestin (1:100, Millipore, Boston, MA), Tuj‐1 (1:200), GFAP (1:200), doublecortin (1:200; Abcam, Cambridge, UK), neuronal nuclei (1:200, Huabio, China), c‐Fos (1:200, Huabio) and Ki67 (1:200; Abcam). After washing 3 times with PBS (10 min/wash), samples were incubated with species‐matched Alexa Fluor–conjugated secondary antibodies (1:500; Invitrogen) for 1 hour at room temperature, followed by nuclear counterstaining with DAPI (1:1000; Beyotime, Jiangsu, China). Images were captured by confocal (Leica). For quantitative analysis, images were acquired from 10 randomly selected fields at 20× magnification for each cell coverslip, and 3 to 4 representative sections were obtained from each mouse.

scRNA‐Seq of Mice Brain

Fresh mice brain tissues were dissociated into single‐cell suspensions using a tissue dissociation kit (Miltenyi, Cologne, Germany), followed by live cell sorting (viability >85%). Single cells were captured using the 10× Genomics Chromium platform with a target of 8000 to 12 000 cells per sample to account for brain cellular heterogeneity. Cell suspensions were filtered through a 40‐μm strainer before loading to prevent microfluidic clogging. Postlysis cDNA synthesis and library construction followed the manufacturer’s protocol. Sequencing was conducted on a NovaSeq 6000 (Illumina, San Diego, CA) with a depth of 50 000 to 100 000 reads per cell to enhance transcriptome coverage of low‐abundance neuronal RNAs. Raw data were processed using Cell Ranger (version 7.1.0, mm10/GRCm39 reference genome), and downstream analysis in Seurat (version 5.0.1) included adjusted quality control thresholds (genes/cell >800, mitochondrial reads <15% for neuronal cells) and doublet removal by DoubletFinder (version 2.0.3). Cell clusters were annotated using mouse brain‐specific marker genes from the Allen Brain Atlas.

miRNA Sequencing of Microglia‐Derived EVs

miRNA sequencing of naive EVs and interferon‐γ EVs was performed by OE Biotech Co., Ltd. (Shanghai, China). Total RNA was extracted from EVs. Libraries were prepared using 5 μg RNA through 3′/5′ adapter ligation, reverse transcription, and PCR amplification. Products (140–160 bp) were size selected and sequenced on a HiSeq 2500 platform (Illumina) to generate single‐end 50 bp reads. Raw data were processed by removing reads lacking 3′ adapters or insert tags, and those outside 15 to 41 nt length thresholds. Clean reads were mapped to the Rfam version 10.1 (https://rfam.org) and GenBank (https://www.ncbi.nlm.nih.gov/genbank/) databases for annotation. Differential miRNA expression was identified using DESeq2 with significance thresholds set at |log2(fold change)| >1 and adjusted P value <0.05.

Transfection of miRNA Mimics in NSCs

Primary NSCs were transfected with miR‐199a‐5p, miR‐206‐3p, or miR‐100‐5p miRNA mimics (Synbio, Suzhou, China), or negative control oligonucleotides (miR‐NC mimic) using Lipofectamine RNAiMAX (Thermo Fisher) at 48 hours after seeding, according to the manufacturer’s protocol. The final concentration of miRNA mimic was 100 nM. Following 6 hours of transfection, cell viability was measured by CCK‐8, and differentiation was assessed by immunostaining. Sequences of miRNA mimics were provided in Table S3.

Statistical Analysis

Data were expressed as mean±SD for normally distributed variables, and median (interquartile range) for nonnormally distributed variables. The Shapiro–Wilk test was used to confirm normality. Intergroup comparisons used unpaired 2‐tailed t tests for 2‐group analyses and 1‐way ANOVA with Tukey’s post hoc tests for multigroup comparisons, implemented in Prism version 9.0 (GraphPad Software, San Diego, CA). Data that failed the Shapiro–Wilk normality test (P<0.05) were analyzed using nonparametric methods. The Mann–Whitney U test was used for 2‐group comparisons, and the Kruskal–Wallis test followed by Dunn’s post hoc test was used for multiple‐group comparisons. A 2‐tailed P value <0.05 defined statistical significance. Raw data are provided in Table S4.

ELISA, WB analysis, neurobehavioral assessments, lentiviral transduction of BV2 cCells, Golgi staining, dual‐luciferase reporter assay, and pharmacological activation of SIRT1 in NSCs are described in Data S1.

Results

Increase of Interferon‐γ–Responsive Microglia in Mouse Brain After Stroke

We first used scRNA‐seq to decipher different phenotypes of microglia after stroke. Our scRNA‐seq data demonstrated 12 clusters of cells in sham and tMCAO mice brains, including microglia, endothelial cells, oligodendrocytes, astrocytes, and the like (Figure 1A) on the basis of their marker genes (Figure S1A). Microglia were further divided into 3 subclusters including homeostatic microglia, disease‐associated microglia, and interferon‐responsive microglia, which expressed interferon‐responsive genes (ISGs) such as IFIT3, ISG15, CXCL10, IFIT2, and CCL12 (Figure 1B and 1C; Figure S1B). The number of disease‐associated microglia and interferon‐responsive microglia was increased after stroke, while the number of homeostatic microglia was reduced (Figure 1B). Our immunostaining and RT‐PCR validated the presence of interferon‐responsive microglia in the lesioned brain at 14 days after stroke, and ISGs were upregulated in stroke mice brain (Figure 1D and 1E). In addition, our ELISA data showed that interferon‐γ was significantly increased after stroke, while interferon‐α and interferon‐β showed no significant changes, suggesting these interferon‐responsive microglia were mainly interferon‐γ–responsive microglia (Figure 1F).

A, UMAP plot of single cells from striatum in sham and tMCAO mice on day 14. B, UMAP plot and quantification of microglia subclusters in sham and tMCAO mice. C, Feature plots of ISGs (*IFIT3*, *ISG15*, *CXCL10*, *IFIT2*, and *CCL12*) expressed in interferon microglia after stroke. D, Representative coimmunostaining images and quantitative analysis showed the expression of ISGs in IBA‐1–positive microglia in sham and tMCAO mice. Scale bar=50 μm. n=3 to 4 mice/group. E, RT‐PCR showed the expression of ISGs in sham and tMCAO mice. n=5 mice/group. F, ELISA data exhibited the concentrations of interferon‐α, interferon‐β, and interferon‐γ in sham and tMCAO mice brains. n=4 mice/group. Data are mean±SD and median (interquartile range) (D [*ISG15*] and F [interferon‐α all subtype]), *P<0.05, **P<0.01, ***P<0.001, using t‐test (D, E, F) and Mann–Whitney U test (D [*ISG15*] and F [interferon‐α all subtype]). DAM indicates disease associated micorglia; IBA‐1, ionized calcium‐binding adapter molecule 1; ISG, interferon‐stimulated gene; RT‐PCR, quantitative real‐time polymerase chain reaction; tMCAO, transient middle cerebral artery occlusion; and UMAP, Uniform Manifold Approximation and Projection.

Identification of Interferon‐γ–Responsive Microglia and Microglia‐Derived EVs

To test the function of interferon‐γ–responsive microglia, BV2 microglia were first stimulated with interferon‐γ at different concentrations and times to generate interferon‐γ–responsive microglia. We found that interferon‐γ treatment upregulated ISGs including ISG15, CXCL10, and IFIT2 in microglia in a dose‐dependent manner (Figure 2A). Microglia treated with 100 ng/mL interferon‐γ for 12 hours achieved the highest expression of ISGs (Figure 2A). Therefore, stimulation of microglia with 100 ng/mL interferon‐γ for 12 hours was used for our subsequent studies. Bright field analysis revealed that 80% of interferon‐γ–treated microglia exhibited amoeboid morphology, with enlarged cell body and stubby processes (Figure 2B), suggesting they were activated. The immunostaining and WB results also indicated that interferon‐γ treatment remarkably upregulated ISG15, CXCL10, and IFIT2 expression in microglia (Figure 2C and 2D). EVs were then collected from the medium of interferon‐γ treated and untreated microglia (interferon‐γ EVs and naive EVs) by ultracentrifugation. WB images showed that EV marker CD81 was expressed in interferon‐γ EVs and naive EVs, and endoplasmic reticulum marker calnexin was expressed only in microglia but not EVs (Figure 2E). Transmission electron microscopy images revealed the typical cup‐shaped morphology of EVs, and nanoparticle tracking analysis data showed that the diameter of EVs was around 50 to 200 nm (Figure 2F).

A, RT‐PCR showed the expression of *ISG15*, *CXCL10*, and *IFIT2* in BV2 microglia stimulated with different concentrations of interferon‐γ for 12 hours, and 100 ng/mL interferon‐γ for different times. n=3 to 4/group. B, Bright field images and quantitative analysis of activated microglia after stimulation with vehicle or 100 ng/mL interferon‐γ for 12 hours. n=4/group. Scale bar=25 μm. C, Representative coimmunostaining images of IBA‐1–positive BV2 microglia with *IFIT3*, *ISG15*, and *CXCL10* after vehicle or 100 ng/mL interferon‐γ treatment for 12 hours. Scale bar=100 μm. D, WB analysis of *IFIT3* and *ISG15* expression in BV2 treated with vehicle or interferon‐γ. n=4/group. E, Representative images of CD81, calnexin, and β‐actin in cell lysis and EVs detected by WB. F, Transmission electron microscopy and nanoparticle tracking analysis showed the morphology and size distribution of EVs. Scale bar=100 nm. Data are mean±SD, *P<0.05, **P<0.01, ***P<0.001, using t test (B, D) and 1‐way ANOVA with Tukey’s post hoc test (A). EV indicates extracellular vesicle; IBA‐1, ionized calcium‐binding adapter molecule 1; IFN, interferon; RT‐PCR, quantitative real‐time polymerase chain reaction; and WB, western blot

Interferon‐γ EVs Inhibited the Survival and Differentiation of NSCs but Not Proliferation In Vitro

To investigate the effects of interferon‐γ EVs on NSCs, we first assessed the uptake capacity of NSCs. As shown in Figure 3A, CFSE‐labeled EVs were internalized by NSCs in a dose‐dependent manner. After being treated with different concentrations of EVs (5, 10, 20 μg/mL), we found that interferon‐γ EV treatment did not affect the viability of NSCs (Figure 3B), while 40 μg/mL interferon‐γ EVs reduced the viability of NSCs subjected to OGD (Figure 3C). Moreover, our coimmunostaining revealed that interferon‐γ EV treatment reduced NSC differentiation into Tuj‐1–positive neurons and increased differentiation into GFAP⁺ astrocytes (Figure 3D and 3E). These results indicated that interferon‐γ EVs inhibited the survival and differentiation of NSCs into neurons in vitro.

A, Representative confocal images illustrated the uptake of different concentrations of CFSE‐labeled EVs (green) by Nestin‐positive NSCs (red). Scale bar=25 μm. B, CCK‐8 examined the cell viability of NSCs treated with different concentrations of naive EVs and interferon‐γ EVs for 48 hours under proliferation medium. n=4/group. C, CCK‐8 data showed the cell viability of NSCs treated with different concentrations of naive EVs and interferon‐γ EVs under OGD. n=3/group. D, Representative coimmunostaining images of Tuj‐1–positive neurons and GFAP‐positive astrocytes after naive EVs and interferon‐γ EV treatment. Scale bar=50 μm. E, Statistical analysis showed the percentage of Tuj‐1–positive neurons and GFAP‐positive astrocytes. n=6/group. Data are mean±SD, *P<0.05, **P<0.01, ***P<0.001, using 1‐way ANOVA with Tukey’s post hoc test (B, C, E). CCK‐8 indicates Cell Counting Kit‐8; CFSE, carboxyfluorescein succinimidyl ester; EV, extracellular vesicle; GFAP, glial fibrillary acidic protein; NSC, neural stem cell; and OGD, oxygen–glucose deprivation.

Interferon‐γ EV Treatment Inhibited Neural Function Recovery of Mice After Stroke

To test the effects of interferon‐γ EVs on stroke mice, 100 μg interferon‐γ EVs in 100 μL PBS were intravenously injected into mice at 7, 9, and 11 days after tMCAO (Figure 4A). We noticed that CFSE‐labeled EVs were taken up by NSCs in SVZ (11.3%), microglia (12.3%), astrocytes (6.2%), and endothelial cells (6.6%) (Figure S2). The modified neurological severity score, elevated body swing test, rotarod test, and hanging wire test demonstrated that injection of interferon‐γ EVs inhibited neural function recovery, as evidenced by increased neurological deficit scores, worse swing asymmetry, reduced motor coordination, and decreased grip strength (Figure 4B through 4E). We also performed the long‐term memory tests using novel object recognition and T‐maze test (Figure S3A). Results demonstrated that interferon‐γ EVs impaired neurological memory recovery of tMCAO mice at days 14 and 28 (Figure S3B through S3I). Cresyl violet staining showed that administration of interferon‐γ EVs increased brain atrophy volume at 14 and 28 days after stroke (Figure 4F), suggesting that interferon‐γ EVs treatment exacerbated neurological dysfunction and suppressed functional recovery in mice following stroke.

A, Schematic diagram of the experiment. Neurobehavioral tests including modified neurological severity score (B), elevated body swing test (C), rotarod test (D), and hanging wire (E) were conducted in stroke mice treated with PBS, naive EVs, and interferon‐γ EVs. n=12 mice/group. F, Cresyl violet staining showed brain atrophy at 14 and 28 d after stroke. n=6 mice/group. Data are mean±SD and median (interquartile range) (B, C, D, E), *P<0.05, **P<0.01, ***P<0.001, using 1‐way ANOVA with Tukey’s post hoc test (F) and Kruskal–Wallis test with Dunn’s post hoc test (B, C, D, E). EBST indicates elevated body swing test; EV, extracellular vesicle; mNSS, modified neurological severity score; and tMCAO, transient middle cerebral artery occlusion.

Interferon‐γ EV Treatment Inhibited Neurogenesis of Mice After Stroke

Doublecortin, a microtubule‐associated protein specifically expressed in migrating and immature neurons, ²⁴ was stained to test the effects of interferon‐γ EVs on neurogenesis of mice after stroke. Our results showed that administration of interferon‐γ EVs significantly reduced doublecortin expression at both 14 and 28 days after tMCAO (Figure 5A and 5B). Additionally, coimmunostaining of Nestin with Ki67, a marker of cell proliferation, further confirmed the suppressive effect of interferon‐γ EVs on neurogenesis, in line with the observed neurobehavioral impairments (Figure 5C and 5D). We used Nestin^creERT2–Rosa‐tdTomato transgenic mice for the fate‐mapping study. Tamoxifen was injected into mice to induce the expression of tdTomato in Nestin‐positive NSCs, and tMCAO was performed. Then, PBS, naive EVs, and interferon‐γ EVs were injected intravenously at days 7, 9, and 11 after tMCAO, and the mice were euthanized at day 14. Cell differentiation was detected through immunostaining of doublecortin and GFAP. Results showed that interferon‐γ EVs suppressed the differentiation of tdTomato‐labeled NSCs into doublecortin‐positive neuroblast, indicating the inhibition of neuronal differentiation (Figure S4). Furthermore, Golgi staining was used to evaluate dendritic spine density, revealing a marked reduction in neural spine density in mice treated with interferon‐γ EVs (Figure 5E and 5F). Together, these findings indicated that interferon‐γ EV treatment remarkably impaired neurogenesis of stroke mice.

A, B, Representative immunostaining images and quantitative analysis of doublecortin intensity in the SVZ of stroke mice after PBS, naive EV, and interferon‐γ EV treatment at days 14 and 28. Scale bar=50 μm. n=4 to 5 mice/group. C, D, Representative coimmunostaining images and corresponding quantification of Nestin and Ki67 in SVZ. Scale bar=50 μm. n=4 to 5 mice/group. E, F, Golgi staining images and quantitative analysis of dendritic spine density (spines per 100 μm). Scale bar=20 μm. n=4 to 5 mice/group. Data are mean±SD, *P<0.05, **P<0.01, ***P<0.001, using 1‐way ANOVA with Tukey’s post hoc test (B, D, F). EV indicates extracellular vesicle; SVZ, subventricular zone; and tMCAO, transient middle cerebral artery occlusion.

Interferon‐γ EVs Suppressed Neurogenesis and Worsened Stroke Outcome via miR‐199a‐5p

To decipher the mechanism of interferon‐γ EVs in inhibiting neurogenesis, miRNA array was performed to compare interferon‐γ EVs with naive EVs. The data revealed upregulation of several miRNAs in interferon‐γ EVs, including miR‐206‐3p, miR‐100‐5p, miR‐199a‐5p, miR‐92b‐5p, miR‐9‐5p, miR‐18a‐5p, and so on (Figure 6A). Our RT‐PCR results further verified the upregulation of miR‐206‐3p, miR‐100‐5p, and miR‐199a‐5p in interferon‐γ EVs (Figure 6B). To determine the functional relevance, NSCs were treated with miRNA mimics of miR‐199a‐5p, miR‐206‐3p, and miR‐100‐5p (Figure S5). Among the candidate miRNAs, only miR‐199a‐5p significantly suppressed NSC survival and neuronal differentiation without affecting cell proliferation, mirroring the effects observed with interferon‐γ EVs (Figure 6C through 6F). These results suggested that miR‐199a‐5p played a pivotal role in mediating the inhibitory effects of interferon‐γ EVs on neurogenesis.

A, Heatmap shows the expression profiles of miRNAs in interferon‐γ EVs and naive EVs, as determined by miRNA array. B, RT‐PCR showed the expression of miRNAs in interferon‐γ EVs and naive EVs. n=4/group. C, CCK‐8 detected the cell viability of NSCs transfected with control mimics (miR‐NC mimic), miR‐199a‐5p, miR‐206‐3p, and miR‐100‐5p mimics for 48 hours under proliferation medium. n=4 to 5/group. D, CCK‐8 detected the cell viability of NSCs transfected with miRNAs mimics under OGD. n=3 to 4/group. E, Representative coimmunostaining images of Tuj‐1–positive neurons and GFAP‐positive astrocytes after treatment with different miRNA mimics. Scale bar=100 μm. F, Statistical analysis of the proportion of Tuj‐1–positive neurons and GFAP‐positive astrocytes. n=4/group. Data are mean±SD, *P<0.05, **P<0.01, ***P<0.001, using t test (B) and 1‐way ANOVA with Tukey’s post hoc test (C, D, F). CCK‐8 indicates Cell Counting Kit‐8; EV, extracellular vesicle; GFAP, glial fibrillary acidic protein; NC, negative control; NSC, neural stem cell; OGD, oxygen–glucose deprivation; and RT‐PCR, quantitative real‐time polymerase chain reaction.

To explore whether miR‐199a‐5p mediated the inhibition effects of interferon‐γ EVs on neurogenesis and poststroke recovery, EVs were isolated from miR‐kd interferon‐γ‐responsive microglia. EVs derived from microglia transfected with Zs‐Green–fused nontargeting miRNA sequence were used as control (Figure S6). We found that mice treated with miR‐kd EVs exhibited significantly improved neural function recovery compared with Zs‐Green–fused nontargeting miRNA sequence EVs (Figure 7A and 7B). Moreover, miR‐kd EV treatment reduced brain atrophy volume (Figure 7C), as well as increased dendritic spine density (Figure 7D), suggesting enhanced synaptic remodeling and neuronal connectivity. In addition, immunostaining revealed that injection of miR‐kd EVs promoted neurogenesis as evidenced by increased doublecortin‐positive neuroblasts and a higher proportion of proliferative Nestin‐positive/Ki67‐positive NSCs in SVZ (Figure 7E and 7F). c‐Fos is a well‐recognized immediate early gene expressed in activated neurons and has been widely used to reflect neuronal functional activity in response to physiological and pathological stimuli. ²⁵ We assessed neuronal activity using neuronal nuclei and c‐Fos double staining. Results demonstrated that interferon‐γ EVs alleviated neural activity, while the knockdown of miR‐199a‐5p in interferon‐γ EVs reversed the inhibitory effect (Figure S7). These data collectively indicated that miR‐199a‐5p was a key effector in interferon‐γ EV‐mediated inhibition of neurogenesis, and its suppression could partially reverse the antineurogenesis of interferon‐γ EVs and neurobehavioral recovery.

Neurobehavioral tests including (A) modified neurological severity score and elevated body swing test, (B) rotarod test and hanging wire were performed in stroke mice treated with PBS, miR‐cn EVs, and miR‐kd EVs. n=8 to 12 mice. C, Cresyl violet staining images and analysis of tMCAO mice after miR‐kd EVs treatment. n=5 mice/group. D, Golgi staining images and quantitative analysis of tMCAO mice after miR‐kd EV treatment. n=5 mice/group. E, Representative immunostaining images and quantitation of doublecortin intensity in SVZ of stroke mice treated with miR‐kd EVs at day 28. Scale bar=50 μm. n=4 to 5 mice/group. F, Representative coimmunostaining images and quantitative analysis of Nestin and Ki67 in SVZ of stroke mice treated with miR‐kd EV treatment at day 28. Scale bar=50 μm. n=5 mice/group. Data are mean±SD and median±IQR (A, B), *P<0.05, **P<0.01, ***P<0.001, using 1‐way ANOVA with Tukey’s post hoc test (C, D, E, F) and Kruskal–Wallis test with Dunn’s post hoc test (A, B). DCX indicates doublecortin; EV, extracellular vesicle; miR‐cn, Zs‐Green–fused nontargeting microRNA sequence; miR‐kd, microRNA‐199a‐5p‐knockdown; SVZ, subventricular zone; and tMCAO, transient middle cerebral artery occlusion.

Interferon‐γ EV‐Derived miR‐199a‐5p Inhibited NSC Survival and Neuronal Differentiation by Downregulating SIRT1

We used 3 prediction databases including miRDB, TarBase, and TargetScan to predict potential downstream targets of miR‐199a‐5p derived from interferon‐γ EVs. Analysis across these databases consistently predicted that SRGAP3, HIF‐1α, PDE4D, SIRT1, and TGF‐β2 may serve as downstream targets of miR‐199a‐5p (Figure 8A). We then transfected NSCs with miR‐199a‐5p mimic and examined mRNA expression using RT‐PCR. Notably, only SIRT1 expression was significantly downregulated (Figure 8B). To confirm the interaction between miR‐199a‐5p and SIRT1, a dual luciferase reporter gene assay was conducted. The results demonstrated that miR‐199a‐5p directly bound to the 3′ untranslated region of SIRT1, resulting in reduced luciferase activity, thereby verifying SIRT1 as a target of miR‐199a‐5p (Figure S8; Figure 8C). SRT1720, an agonist of SIRT1, was used to elevate SIRT1 levels. ²⁶ We found that 2 μM SRT1720 significantly upregulated the expression of SIRT1 in NSCs (Figure 8D). Importantly, pretreatment with SRT1720 reversed the inhibitory effects of miR‐199a‐5p mimic on NSCs survival and neuronal differentiation, as shown by CCK‐8 and immunostaining (Figure 8E and 8F). These findings demonstrated that SIRT1 acted as a functional downstream effector of miR‐199a‐5p–inhibited NSCs survival and neuronal differentiation, and pharmacological activation of SIRT1 may offer a potential therapeutic strategy for ischemic stroke.

A, microRNA databases predicted the targeted downstream genes of miR‐199a‐5p. B, RT‐PCR verified the expression of predicted genes in NSCs treated with miR‐199a‐5p mimic. n=4 to 7/group. C, Dual luciferase reporter gene assay showed the interaction of miR‐199a‐5p with *SIRT1*. n=5/group. D, RT‐PCR detected *SIRT1* expression of NSCs after miR‐199a‐5p mimic treatment and its activator SRT with different concentrations for 48 hours. n=4 to 5/group. E, CCK‐8 detected the cell viability of NSCs with miR‐199a‐5p mimic and SRT for 48 hours under proliferation medium and OGD. n=5/group. F, Representative coimmunostaining images and quantitative analysis of Tuj‐1–positive neurons and GFAP‐positive astrocytes after miR‐199a‐5p mimic and SRT treatment. Scale bar=100 μm. n=6/group. Data are mean±SD and median (interquartile range) (E [right]), *P<0.05, **P<0.01, ***P<0.001, using t test (B, C), 1‐way ANOVA with Tukey’s post hoc test (D, E, F), and Kruskal–Wallis test with Dunn’s post hoc test (E [right]). CCK‐8 indicates Cell Counting Kit‐8; EV, extracellular vesicle; GFAP, glial fibrillary acidic protein; NC, negative control; NSC, neural stem cell; OGD, oxygen–glucose deprivation; and RT‐PCR, quantitative real‐time polymerase chain reaction.

Discussion

In this study, we identified a population of interferon‐γ–responsive microglia at 14 days after stroke. These microglia secrete EVs that are taken up by NSCs, resulting in reduced neuronal differentiation and neurogenesis via the miR‐199a‐5p/SIRT1 signaling axis, thereby impairing neurological recovery following stroke. Our study not only deepens our understanding of the mechanisms underlying neural repair after stroke but also provides critical scientific insights for developing targeted therapeutic strategies to enhance neurogenesis and functional recovery. These findings hold significant potential for clinical applications.

Promoting neurogenesis has profound implications for the recovery and survival of stroke patients, significantly improving neurobehavioral recovery, accelerating functional repair, and enhancing quality of life while reducing the risk of long‐term disability. ²⁷ Neurogenesis facilitates the reconstruction of damaged neural networks, restoring connections between the brain and other parts of the body, thereby enhancing patients’ self‐care abilities and independence. Thus, promoting neurogenesis is a critical strategy in stroke treatment. However, pathological changes triggered by stroke, such as ischemia, inflammation, oxidative stress, and glial scar formation, inhibit neuronal survival, axonal growth, and synaptic reconstruction. ²⁸ Additionally, the poststroke microenvironment is characterized by a reduction in neurotrophic factors, an increase in inhibitory molecules (eg, Nogo‐A, myelin‐associated proteins), and abnormalities in the vascular and immune systems, which further impede the process of neurogenesis. ²⁹ , ³⁰ Consequently, although the brain possesses a certain capacity for self‐repair, the endogenous neurogenesis following stroke is often insufficient to achieve functional recovery.

Microglia, as the primary immune cells of central nervous system, play a dual regulatory role in neurogenesis following stroke, which is closely associated with the distinct functions of different microglial phenotypes activated following stroke. ³¹ , ³² Using scRNA‐seq, we characterized the subpopulations of microglia in the brains of stroke mice and identified homeostatic microglia, disease‐associated microglia, major histocompatibility complex–associated microglia, and interferon‐responsive microglia. Among these, the interferon‐responsive microglia exhibited high expression of ISGs, including IFIT2, IFIT3, ISG15, and CXCL10 (Figure 1A). Combining scRNA‐seq with ELISA assays, we found that the poststroke brain primarily showed an increase in type II interferon‐responsive microglia (Figure 1E). After stroke, T cells and natural killer cells accumulate significantly and serve as the main producers of interferon‐γ. ³³ These cells can bind to the interferon‐γ receptor on microglia, activating the Janus kinase/signal transducer and activator of transcription 1 signaling pathway and inducing the expression of a series of ISGs, thereby transforming microglia into an interferon‐γ–responsive state. ³⁴ , ³⁵ In future studies, we aim to investigate the interactions between T cells, natural killer cells, and microglia to elucidate the mechanisms driving the generation of interferon‐γ–responsive microglia.

Type II interferon (interferon‐γ) plays a critical role in the “priming” of microglia and other tissue‐resident macrophages, a process accompanied by various cellular adaptive changes, including morphological alterations, proliferation, and the moderate release of nitric oxide and interleukin ‐6. ³⁶ , ³⁷ In hippocampal slice cultures, continuous exposure to interferon‐γ and lipopolysaccharide (a Toll‐like receptor 4 ligand) significantly induces the high‐level release of interleukin‐6, tumor necrosis factor‐α, and nitric oxide. ³⁸ , ³⁹ This is accompanied by the loss of neuronal network activity and the onset of neurodegeneration. In mice, microglia primed by interferon‐γ injection suppress the proliferation of neural stem and precursor cells and induce apoptosis of immature neurons, ultimately impairing adult hippocampal neurogenesis. ⁴⁰ Existing studies reveal that acute cerebral ischemia triggers excessive activation of interferon signaling in the injured brain, particularly in “primed” microglia. ⁴¹ Recently, the presence of interferon‐responsive microglia subpopulations has also been identified in mouse models of ischemic brain injury, especially in aged mice, suggesting that interferon‐γ has considerable effects in central nervous system. ¹⁵

EVs serve as critical mediators of intercellular communication and play pivotal roles in neurodevelopment, synaptic plasticity, neuroinflammation, and neural repair. ⁴² , ⁴³ Our previous studies have demonstrated that EVs secreted by anti‐inflammatory microglia exhibit neuroprotective effects, promote white matter repair, and enhance neurogenesis. ¹⁸ , ²³ , ⁴⁴ Arvanitaki et al reported that the deletion of xeroderma pigmentosum F‐excision repair cross complementation group in microglia triggers a type I interferon response, leading to neuronal cell death and progressive neurodegeneration. ⁴⁵ In contrast, the impact and mechanisms of EVs secreted by interferon‐γ–responsive microglia on neural function remain poorly understood. In our study, we stimulated microglia in vitro to transform into an interferon‐γ–responsive state and isolated EVs. Even though in vivo immunostaining data indicated that interferon‐γ–stimulated EVs were taken up by microglia, astrocytes, and endothelial cells, our fate‐mapping experiments and in vitro functional study collectively provided the strongest support for an NSC‐centric mechanism of interferon‐γ EVs. We found that interferon‐γ EVs inhibited poststroke neurogenesis. Multiple stimuli, including cytokines, lipopolysaccharide, capsaicin, serotonin, amyloid plaques, and ATP, have been shown to activate EVs production in microglia. ⁴⁶ It is now evident that microglia dynamically modify EVs composition in response to environmental stimuli, resulting in a heterogeneous EVs population. We did not observe significant changes in EV morphology or secretion levels upon interferon‐γ stimulation. Instead, the primary alterations were in the internal components of the EVs, particularly in miRNA profiles. Additionally, EVs are enriched with proteomes, lipids, and other bioactive molecules, and whether these components are altered in interferon‐γ–induced EVs requires further investigation.

According to our miRNA array results, a number of proinflammatory or stress‐related miRNAs including miR‐122‐5p, miR‐16‐5p, miR‐143‐3p, and miR‐155‐5p, were highly enriched in naive EVs, which may contribute to a minor deleterious effect on tMCAO mice (Figure 4C). miR‐122‐5p has been reported to aggravate neuronal apoptosis via targeting semaphorin 3A following ischemic stroke. ⁴⁷ Similarly, miR‐16‐5p contributed to neuronal death by targeting antiapoptotic genes such as Bcl‐2 and HSP70, thereby enhancing mitochondrial‐mediated apoptosis. ⁴⁸ , ⁴⁹ miR‐143‐3p has been shown to negatively regulate angiogenesis and neurorepair, partly through suppression of insulin‐like growth factor 1 receptor and vascular endothelial growth factor–related pathways. ⁵⁰ , ⁵¹ miR‐155‐5p is a well‐established proinflammatory miRNA, which activates Nuclear factor κ‐light‐chain‐enhancer of activated B cells signaling and promotes the release of cytokines such as tumor necrosis factor‐α ‐α and interleukin‐1β, exacerbating poststroke inflammation. ⁵² , ⁵³ We also noticed that miR‐199a‐5p mimic treatment had minor effects on NSCs viability and differentiation (Figure 6), especially when compared with the dramatic differences in interferon EVs from miR‐kd BV2 microglia (Figure 7). The concern could be addressed from the relatively high level of miR‐199a‐5p of NSCs themselves ⁵⁴ and the broad transcriptional and translational changes within microglia after miRNA knockdown. ⁵⁵ , ⁵⁶

Our study revealed that miR‐199a‐5p inhibited neural regeneration in ischemic stroke by downregulating the target gene, SIRT1, a key regulator of NSC survival and neuronal differentiation. ⁵⁷ The regulatory interaction of miR‐199a‐5p and SIRT1 has been previously validated. ⁵⁸ , ⁵⁹ Here, we reported that miR‐199a‐5p was enriched in interferon‐γ–derived EVs and functionally contributed to the suppressive effects of EVs on NSCs by reducing SIRT1 expression. miR‐199a‐5p has also been reported to target several components of the PI3K/Akt and mammalian target of rapamycin signaling pathways, both of which play critical roles in neuronal survival and differentiation. ⁶⁰ , ⁶¹ However, several studies have reported contrasting findings, suggesting that miR‐199a‐5p may exert neuroprotective effects. We propose that the disparate findings are context dependent rather than mutually exclusive, for the following reasons. First, the functional outcome is dictated by the specific cell types and primary target genes. In our study, miR‐199a‐5p enrichment in NSCs silences SIRT1, thereby impairing cell viability and differentiation. In contrast, other studies report protective effects through targeting CAV1 in vascular cells, DDR1 in neurons, or Hif‐1α in cardiomyocytes. ⁶² , ⁶³ , ⁶⁴ Second, temporal dynamics are critical. The neurotoxic effect we observed, which was linked to impaired neurogenesis, is most relevant during the recovery phase, whereas the neuroprotection reported by others pertains mainly to early injury phases. Thus, miR‐199a‐5p may be beneficial in the acute phase but detrimental later by inhibiting neural repair. Finally, species‐ and model‐specific differences cannot be ruled out. Variations in ischemic lesion size, location (cortex or striatum), and the intrinsic neurogenic response between rats and mice may influence the relative abundance of miR‐199a‐5p across different cell types, thereby altering its primary function. Thus, comprehensive understanding of the roles of miR‐199a‐5p on ischemic stroke is critical for determining its therapeutic potential.

Proteomic and lipidomic profiling of microglia‐derived EVs are crucial for elucidating their underlying mechanisms. Several published studies have characterized the molecular composition of EVs released by microglia under pathological stimulation. For instance, proteomic analysis of EVs from lipopolysaccharide‐stimulated microglia identified more than 200 previously unreported proteins, which were capable of inducing proinflammatory changes. ⁶⁵ , ⁶⁶ These alterations included an enrichment of proteins related to proteasome activity and a reduction in those associated with chromosomal organization. Lipidomic profiling of EVs from postmortem brains showed significant enrichment of specific lipids, particularly glycerophosphoinositol bisphosphate, which is linked to acute phase response signaling and neuroinflammatory pathways. ⁶⁷

There are some limitations in our study. First, we used BV2 microglia cell line instead of primary microglia, which may be closer to the in vivo condition. However, cell lines maintain the capability to respond to EV release stimuli such as ATP and lipopolysaccharide and are widely used due to their workability. ¹⁶ Second, only male mice were used to avoid variability from female hormonal cycles in this study. Estrogen is known to exert significant neuroprotective effects in cerebrovascular diseases, which could confound EV treatment outcomes. ⁶⁸ , ⁶⁹ , ⁷⁰ Future studies will include female mice to explore potential sex‐related differences and improve clinical relevance. Besides, we assessed the impact of EVs on neurogenesis primarily through immunostaining, which provided insights into cellular and structural changes. Nevertheless, we did not evaluate functional aspects of neuronal activity, such as electrophysiological properties, which are critical for understanding the comprehensive effects of EVs on neural function. Future studies incorporating electrophysiological techniques, such as patch‐clamp recordings or multielectrode array analysis, would provide a more holistic understanding of how EVs influence neuronal network activity and overall functional recovery following stroke.

Overall, we demonstrated that interferon‐γ EVs derived from microglia after stroke were internalized by NSCs, leading to impaired survival and neuronal differentiation, inhibited neurogenesis, and hindered functional recovery through the miR‐199a‐5p/SIRT1 signaling pathway, highlighting a novel therapeutic target for ischemic stroke.

Sources of Funding

This study was supported by grants from the National Natural Science Foundation of China (82171312 [Q.A.], 82172529 [J.W.], 82472590 [J.W.], 82371307 [Y.T.], 82271320 [Z.Z.], and 32301146 [W.L.]), the National Key R&D Program of China [No. 2022YFA1603600 [Z.Z.]), the Young Leading Scientists Cultivation Plan supported by Shanghai Municipal Education Commission (ZXWH1082101 [Y.T.]), the Fundamental Research Funds for the Central Universities (YG2023ZD02 [Y.T.] and YG2023ZD18 [Y.T.]), and the Shanghai Sailing Program (23YF1420700 [W.L.]).

Disclosures

None.

Supporting information

Data S1

Tables S1–S4

Figures S1–S8

Unedited Gels

ARRIVE Checklist

JAH3-15-e043532-s001.zip^{(1.4MB, zip)}

This manuscript was sent to Neel Singhal, MD, PhD, Associate Editor, for review by expert referees, editorial decision, and final disposition.

Supplemental Material is available at https://www.ahajournals.org/doi/suppl/10.1161/JAHA.125.043532

For Sources of Funding and Disclosures, see page 19.

Contributor Information

Jixian Wang, Email: wangjixian6@163.com.

Yaohui Tang, Email: yaohuitang@sjtu.edu.cn.

Qingzhu An, Email: anqingzhu@fudan.edu.cn.

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PERMALINK

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

Tongtong Xu, PhD

Dongliang Qian, MD

Shiyu Deng, PhD

Yiyan Guo, PhD

Hanlai Li, PhD

Yanqin Geng, MD, PhD

Lin Gan, PhD

Qianyuan Lian, PhD

Jingjing Xu, MS

Huaitong Yao, PhD

Jing Ye, PhD

Enkhbat Myagmartsend, PhD

Wanlu Li, PhD

Zhijun Zhang, PhD

Guo‐Yuan Yang, MD, PhD

Jixian Wang, MD, PhD

Yaohui Tang, PhD

Qingzhu An, MD, PhD

Abstract

Background

Methods

Results

Conclusions

Nonstandard Abbreviations and Acronyms

Research Perspective.

What Is New?

What Question Should Be Addressed Next?

Methods

Microglia Stimulation With Interferon‐γ

Isolation, Identification, and Labeling of EVs

Animals

Transient Middle Cerebral Artery Occlusion Model

Animal Experimental Design

Tail Vein Injection

Brain Atrophy Volume Assessment

NSC Isolation and Culture

Oxygen–Glucose Deprivation Model

NSC Differentiation Assay

RNA Extraction and RT‐PCR

Immunostaining

scRNA‐Seq of Mice Brain

miRNA Sequencing of Microglia‐Derived EVs

Transfection of miRNA Mimics in NSCs

Statistical Analysis

Results

Increase of Interferon‐γ–Responsive Microglia in Mouse Brain After Stroke

Figure 1. Verification of interferon‐γ–responsive microglia in mice brains after stroke.

Identification of Interferon‐γ–Responsive Microglia and Microglia‐Derived EVs

Figure 2. Identification of interferon‐γ–responsive microglia and microglia‐derived EVs.

Interferon‐γ EVs Inhibited the Survival and Differentiation of NSCs but Not Proliferation In Vitro

Figure 3. Interferon‐γ EV treatment reduced the survival and neuronal differentiation of NSCs.

Interferon‐γ EV Treatment Inhibited Neural Function Recovery of Mice After Stroke

Figure 4. Interferon‐γ EV treatment inhibited neural function recovery of mice following tMCAO.

Interferon‐γ EV Treatment Inhibited Neurogenesis of Mice After Stroke

Figure 5. Interferon‐γ EV treatment inhibited neurogenesis of mice following tMCAO.

Interferon‐γ EVs Suppressed Neurogenesis and Worsened Stroke Outcome via miR‐199a‐5p

Figure 6. Interferon‐γ EVs suppressed neurogenesis via miR‐199a‐5p.

Figure 7. Interferon‐γ EVs suppressed neurogenesis and worsen stroke outcome via miR‐199a‐5p.

Interferon‐γ EV‐Derived miR‐199a‐5p Inhibited NSC Survival and Neuronal Differentiation by Downregulating SIRT1

Figure 8. Interferon‐γ EVs inhibited NSC survival and neuronal differentiation via miR‐199a‐5p/SIRT1 axis.

Discussion

Sources of Funding

Disclosures

Supporting information

Contributor Information

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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