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The foam cell-derived exosomal miRNA Novel-3 drives neuroinflammation and ferroptosis during ischemic stroke

Nature Aging volume 4, pages 1845–1861 (2024)Cite this article

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Abstract

Large artery atherosclerosis (LAA) is a prevalent cause of acute ischemic stroke (AIS). Understanding the mechanisms linking atherosclerosis to stroke is essential for developing appropriate intervention strategies. Here, we found that the exosomal miRNA Novel-3 is selectively upregulated in the plasma of patients with LAA–AIS. Notably, Novel-3 was predominantly expressed in macrophage-derived foam cells, and its expression correlated with atherosclerotic plaque vulnerability in patients undergoing carotid endarterectomy. Exploring the function of Novel-3 in a mouse model of cerebral ischemia, we found that Novel-3 exacerbated ischemic injury and targeted microglia and macrophages expressing ionized calcium-binding adapter molecule 1 in peri-infarct regions. Mechanistically, Novel-3 increased ferroptosis and neuroinflammation by interacting with striatin (STRN) and downregulating the phosphoinositide 3-kinase–AKT–mechanistic target of rapamycin signaling pathway. Blocking Novel-3 activity or overexpressing STRN provided neuroprotection under ischemic conditions. Our findings suggest that exosomal Novel-3, which is primarily derived from macrophage-derived foam cells, targets microglia and macrophages in the brain to induce neuroinflammation and could serve as a potential therapeutic target for patients with stroke who have atherosclerosis.

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Fig. 1: Exosomal Novel-3 serves as a predictor of LAA in patients with stroke.
Fig. 2: Novel-3 originates from foam cells and correlates with plaque vulnerability.
Fig. 3: AS-exos target Iba1+ MMs and aggravate ischemic brain injury.
Fig. 4: AS-exos exacerbate microglial ferroptosis-driven neuroinflammation.
Fig. 5: Novel-3 regulates ferroptosis and peroxidation in microglia.
Fig. 6: Anti-Novel-3 shows neuroprotection against ischemic stroke.
Fig. 7: Novel-3 targets STRN and effectively inhibits its expression.
Fig. 8: Overexpression of STRN attenuates Novel-3-induced ischemic injury.

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Data availability

High-throughput sequencing data have been deposited in the Genome Sequence Archive-Human database (HRA005998), the Gene Expression Omnibus database (GSE248793) and figshare (https://doi.org/10.6084/m9.figshare.26719024.v1)40. All data and methods supporting the findings of the study are available in the article and supplementary materials and are available from the corresponding authors upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank Jiangsu Simcere Diagnostics for its technical support for the transcriptional analysis. We also thank S. Wang and R. Chen (Department of Neurosurgery, Tongji Hospital) for providing carotid endarterectomy samples. In addition, we are greatly indebted to all the patients who participated in the study, without whom this study would never have been accomplished. This study was funded by the Ministry of Science and Technology China Brain Initiative Grant (STI2030-Major Projects 2022ZD0204700 to W.W.), the National Natural Science Foundation of China (grants 82371404 to D.-S.T., 82271341 to C.Q. and 81873743 to D.-S.T.) and the Knowledge Innovation Program of Wuhan Shuguang Project (2022020801020454 to C.Q.).

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  1. These authors contributed equally: Chuan Qin, Ming-Hao Dong, Yue Tang, Yun-Hui Chu.

Authors and Affiliations

  1. Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Chuan Qin, Ming-Hao Dong, Yue Tang, Yun-Hui Chu, Luo-Qi Zhou, Hang Zhang, Sheng Yang, Lu-Yang Zhang, Xiao-Wei Pang, Li-Fang Zhu, Wei Wang & Dai-Shi Tian

  2. Hubei Key Laboratory of Neural Injury and Functional Reconstruction, Huazhong University of Science and Technology, Wuhan, China

    Chuan Qin, Ming-Hao Dong, Yue Tang, Yun-Hui Chu, Luo-Qi Zhou, Hang Zhang, Sheng Yang, Lu-Yang Zhang, Xiao-Wei Pang, Li-Fang Zhu, Wei Wang & Dai-Shi Tian

  3. Key Laboratory of Vascular Aging, Ministry of Education, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Chuan Qin, Ming-Hao Dong, Yue Tang, Yun-Hui Chu, Luo-Qi Zhou, Hang Zhang, Sheng Yang, Lu-Yang Zhang, Xiao-Wei Pang, Li-Fang Zhu, Wei Wang & Dai-Shi Tian

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Contributions

D.-S.T. is the lead contact for this article. D.-S.T., W.W., C.Q., M.-H.D., Y.T. and L.-Q.Z. conceptualized and designed the study. C.Q., M.-H.D., Y.T., Y.-H.C., L.-Q.Z., H.Z. and S.Y. analyzed the data. W.W., D.S.T., C.Q., M.H.D., L.Q.Z., L.Y.Z. and X.W.P. interpreted the data. D.S.T., C.Q., M.H.D. and L.F.Z. drafted the manuscript. W.W., D.-S.T., C.Q., M.-H.D., Y.T., Y.-H.C., L.-Q.Z., H.Z., S.Y., L.-Y.Z., X.-W.P. and L.-F.Z. made critical revisions to the manuscript. All authors were involved in the collection and critical review of data. All authors approved the final version of the manuscript.

Corresponding authors

Correspondence to Chuan Qin, Wei Wang or Dai-Shi Tian.

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Nature Aging thanks Marco Bacigaluppi, Lidia Garcia Bonilla and Zhaolong Zhang for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Additional data of small RNA sequencing.

Volcano plot and heatmap depicting differentially expressed plasma ex-miRNAs in LAA patients versus HD, SVO and CES, with Novel-3 highlighted.

Extended Data Fig. 2 Additional data of transcriptomic analysis of microglia both in vivo and in vitro.

a, Volcano plot depicting differentially expressed genes in microglia sorted from MCAO mice treated with Ctrl-exos or AS-exos. Dashed lines reveal fold change and significance thresholds. Up-regulated and down-regulated genes are shown in orange and blue, respectively. b,c, GSEA pathway analysis of differentially expressed genes, highlighting changes to ferroptosis-related, mitochondrion-related, and PI3K-AKT-mTOR pathways. The P value was estimated using an empirical phenotype-based permutation test. d, Heatmap of hierarchically differentially expressed genes in the pathways related to PI3K-AKT-mTOR signaling. e, Volcano plot depicting differentially expressed genes in HMC3 cells treated with LAA-exos or HD-exos. Dashed lines reveal fold change and significance thresholds. Up-regulated and down-regulated genes are shown in purple and blue, respectively. f,g, GSEA pathway analysis of differentially expressed genes, highlighting changes to mitochondrion-related, and PI3K-AKT-mTOR pathways. The P value was estimated using an empirical phenotype-based permutation test. h, Heatmap of hierarchically differentially expressed genes in the pathways related to inflammatory, ROS metabolic, and PI3K-AKT signaling.

Extended Data Fig. 3 Additional data of Novel-3-mediated ferroptosis and mitochondrial peroxidation of recipient microglia.

a, Representative images and quantitative analysis of intracellular Fe2+ levels (FerroOrange) (N = 6), mitochondrial superoxide (MitoSOX), and oxidized lipid (C11-BODIPY581/591). One-way ANOVA and Tukey’s multiple comparison tests. Data are shown as mean ± SD. b, Illustration of the experimental design of microglia sorting and quantitative analysis.

Extended Data Fig. 4 Coculture experiment of neurons and microglia.

a, Illustration of the in vitro experimental design of Coculture experiment of primary neurons and microglia. b, Representative images and quantitative analysis of MAP2+ neurons with proliferation marker (Ki67) (N = 6). Quantitative analysis of cell viability of MAP2+ neurons. One-way ANOVA and Tukey’s multiple comparison tests. Data are shown as mean ± SD.

Extended Data Fig. 5 Additional data of the in vivo treatment of AAV-Novel-3Sponge.

a, Representative immunostaining images and quantitative analysis showing that CreER protein were more distributed in the nucleus one week after tamoxifen injection than without tamoxifen (N = 6). The yellow arrows indicated cells without CreER protein distributed in the nucleus. The red arrows indicated cells with CreER protein distributed in the nucleus. Two-tailed unpaired t-test. Data are presented as mean ± SD. b, Representative images and quantitative analysis of STRN expression in Iba-1+ MM at 3days after MCAO treated with AAV-NC or AAV-Novel-3Sponge (N = 6). Two-tailed unpaired t-test. Data are presented as mean ± SD.

Extended Data Fig. 6 Novel-3 antagomiR showed neuroprotection against ischemic stroke.

a, Illustration of the in vivo experimental design injecting Novel-3 antagomir. b, Representative images and quantitative analysis of Cy3 labeled antagomiR-NC and antagomiR-Novel-3 colocalization with Iba1+ MM at 3 days after MCAO (N = 6). Two-tailed unpaired t-test. Data are shown as mean ± SD. c, Representative images of immunostaining showed Iba-1+ cells, 3D construction and sholl analysis in the peri-infarct areas of MCAO mice with daily administration of AS-exos and treatment of NC antagomiR or Novel-3 antagomiR (N = 6). Quantitative analysis of microglial density, and morphological changes including solidity, round, branch numbers and lengths, and end-point voxels. Two-tailed unpaired t-test. Data are shown as mean ± SD. d, Representative images and quantitative analysis of Iba-1+ microglia with lipid peroxidation marker (4-Hydroxynonenal, 4-HNE) and oxidized DNA marker (8-OHdG) at 3days after MCAO (N = 6). Two-tailed unpaired t-test. Data are shown as mean ± SD. e, Quantitative analysis of behavioral tests as measured by the mNSS, foot fault rate and rotarod test (N = 6). Two-tailed Mann-Whitney U tests and Two-way ANOVA tests for mNSS. Two-way ANOVA tests for foot fault rate and rotarod test. Data are presented as mean ± SD. f, Representative images and quantitative analysis of Nissl staining of MCAO mice with daily administration of AS-exos and treatment of NC antagomiR or Novel-3 antagomiR (N = 6). Black dashed lines indicate the border of infarct area. Dot plots indicated percentage of infarct area of individual mouse in each group. Two-tailed unpaired t-test. Data are presented as mean ± SD.

Extended Data Fig. 7 Additional data of attenuated Novel-3 induced microglial dysfunction and ischemic injury by overexpression of STRN.

a, RT-qPCR analysis and heatmap of genes in the pathways related to inflammatory responses, mitochondrial function, and ferroptosis. b, Representative images and quantitative analysis of intracellular Fe2+ levels (FerroOrange), and oxidized lipid (C11-BODIPY581/591) (N = 6). One-way ANOVA and Tukey’s multiple comparison tests. Data are shown as mean ± SD.

Extended Data Fig. 8 Graphic abstract.

Exosomal Novel-3, which originated predominantly from macrophage-derived foam cells, mainly targeted microglia to induce ferroptosis and neuroinflammation during ischemic stroke.

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Qin, C., Dong, MH., Tang, Y. et al. The foam cell-derived exosomal miRNA Novel-3 drives neuroinflammation and ferroptosis during ischemic stroke. Nat Aging 4, 1845–1861 (2024). https://doi.org/10.1038/s43587-024-00727-8

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