An Engineered Cellular Carrier Delivers miR-138-5p to Enhance Mitophagy and Protect Hypoxic-Injured Neurons via the DNMT3A/Rhebl1 Axis

Mitophagy influences the progression and prognosis of ischemic stroke (IS). However, whether DNA methylation in the brain is associated with altered mitophagy in hypoxia-injured neurons remains unclear. Here, miR-138-5p was found to be highly expressed in exosomes secreted by astrocytes stimulated with oxygen and glucose deprivation/re-oxygenation (OGD/R), which could influence the recovery of OGD/R-injured neurons through autophagy. Mechanistically, miR-138-5p promotes the stable expression of Ras homolog enriched in brain like 1(Rhebl1) through DNA-methyltransferase-3a (DNMT3A), thereby enhancing ubiquitin-dependent mitophagy to maintain mitochondrial homeostasis. Furthermore, we employed glycosylation engineering and bioorthogonal click reactions to load mirna onto the surface of microglia and deliver them to injured region utilising the inflammatory chemotactic properties of microglia to achieve drug-targeted delivery to the central nervous system (CNS). Our findings demonstrate miR-138-5p improves mitochondrial function in neurons through the miR-138-5p/DNMT3A/Rhebl1 axis. Additionally, our engineered cell vector-targeted delivery system could be promising for treating IS. STATEMENT OF SIGNIFICANCE: In this study, we demonstrated that miR-138-5p in exosomes secreted by astrocytes under hypoxia plays a critical role in the treatment of hypoxia-injured neurons. And we find a new target of miR-138-5p, DNMT3A, which affects neuronal mitophagy and thus exerts a protective effect by regulating the methylation of Rbebl1. Furthermore, we have developed a carrier delivery system by combining miR-138-5p with the cell membrane of microglia and utilized the inflammatory chemotactic properties of microglia to deliver this system to the brain via intravenous injection. This groundbreaking study not only provides a novel therapeutic approach for ischemia-reperfusion treatment but also establishes a solid theoretical foundation for further research on targeted drug delivery for central nervous system diseases with promising clinical applications.