M2 microglia-derived extracellular vesicles promote white matter repair and functional recovery via miR-23a-5p after cerebral ischemia in mice

Abstract

Rationale: White matter repair is critical for the cognitive and neurological functional recovery after ischemic stroke. M2 microglia are well-documented to enhance remyelination and their extracellular vesicles (EVs) mediate cellular function after brain injury. However, whether M2 microglia-derived EVs could promote white matter repair after cerebral ischemia and its underlying mechanism are largely unknown. Methods: EVs were isolated from IL-4 treated microglia (M2-EVs) and untreated microglia (M0-EVs). Adult ICR mice subjected to 90-minute transient middle cerebral artery occlusion received intravenous EVs treatment for seven consecutive days. Brain atrophy volume, neurobehavioral tests were examined within 28 days following ischemia. Immunohistochemistry, myelin transmission electron microscope and compound action potential measurement were performed to assess white matter structural remodeling, functional repair and oligodendrogenesis. The effects of M2-EVs on oligodendrocyte precursor cells (OPCs) were also examined in vitro . EVs' miRNA sequencing, specific miR-23a-5p knockdown in M2-EVs and luciferase reporter assay were used to explore the underlying mechanism. Results: M2-EVs reduced brain atrophy volume, promoted functional recovery, oligodendrogenesis and white matter repair in vivo , increased OPC proliferation, survival and differentiation in vitro . miR-23a-5p was enriched in M2-EVs and could promote OPC proliferation, survival and maturation, while knocking down miR-23a-5p in M2-EVs reversed the beneficial effects of M2-EVs both in vitro and in vivo . Luciferase reporter assay showed that miR-23a-5p directly targeted Olig3. Conclusion: Our results demonstrated that M2 microglia could communicate to OPCs through M2-EVs and promote white matter repair via miR-23a-5p possibly by directly targeting Olig3 after ischemic stroke, suggesting M2-EVs is a novel and promising therapeutic strategy for white matter repair in stroke and demyelinating disease.

Dynamics of Microglia Activation in the Ischemic Brain: Implications for Myelin Repair and Functional Recovery

“…These results were corroborated by another independent study, showing that intravenous delivery of pro-regenerative microglial EVs significantly promoted OL maturation and white matter structural remodeling after transient MCAo (Li et al, 2022b). In both cases, microglial EVs were able to successfully promote remyelination and long-term functional recovery after stroke (Raffaele et al, 2021;Li et al, 2022b).…”

“…In this respect, infusion of pro-regenerative microglial EVs in the ipsilateral corpus callosum of ischemic mice was found to significantly increase OPC recruitment at lesion boundaries and enhance their maturation (Raffaele et al, 2021 ). These results were corroborated by another independent study, showing that intravenous delivery of pro-regenerative microglial EVs significantly promoted OL maturation and white matter structural remodeling after transient MCAo (Li et al, 2022b ). In both cases, microglial EVs were able to successfully promote remyelination and long-term functional recovery after stroke (Raffaele et al, 2021 ; Li et al, 2022b ).…”

“…Furthermore, the purified lipid fraction of microglial EVs was found to promote OPC maturation even more efficiently than intact EVs, although the precise vesicular lipids responsible for this effect remain to be elucidated (Lombardi et al, 2019 ; Gualerzi et al, 2021 ). Finally, the pro-differentiating effects of microglial EVs may be also partly mediated by their miRNA cargo, as the knockdown of miRNA-23a-5p in donor microglia was found to abolish the pro-myelinating properties of EVs (Li et al, 2022b ).…”

The multiple faces of extracellular vesicles released by microglia: Where are we 10 years after?

“…Proteomic analysis of microglial EVs has been performed on different samples: EVs from N9 ( Potolicchio et al, 2005 ) and BV2 ( Yang et al, 2018 ) cell lines, primary rat microglia ( Hooper et al, 2012 ; Drago et al, 2017 ; Mukherjee et al, 2020 ), MACS sorted rat microglia ( Murgoci et al, 2020 ), primary leech microglia ( Arab et al, 2019 ; Lemaire et al, 2019 ), microglia-like cells from human iPSCs ( Mallach et al, 2021a , b ; You et al, 2022 ), CD11b + EVs from human brain tissue ( Cohn et al, 2021 ). Their lipid ( Osteikoetxea et al, 2015 ; Cohn et al, 2021 ; Kumar et al, 2021 ) and small non-coding RNA (miRNA) composition ( Cunha et al, 2016 ; Prada et al, 2018 ; Lemaire et al, 2019 ; Tian et al, 2019 ; Cohn et al, 2021 ; Li et al, 2022 ) have also been investigated. A summary of these studies is reported in Table 2 .…”

“…A few studies have analyzed the miRNA cargo of microglial EVs. Sequencing studies identified 41 miRNAs differentially expressed in small EVs isolated from IL-4 polarized BV2 cells compared to unstimulated controls, including some upregulated miRNAs (miR-23a-5p, miR-221-3p, miR-129-5p) involved in oligodendrogenesis ( Li et al, 2022 ). Moreover, an independent study compared the miRNA profiles of small EVs from unpolarized, LPS-polarized and IL-4-polarized BV2 cells, detecting a specific enrichment of the proangiogenic miR-26a in pro-regenerative EVs with respect to the other two conditions ( Tian et al, 2019 ).…”