Extracellular Vesicle-Mediated Delivery of Mitochondrial Circular RNA MTCO2 Protects against Cerebral Ischemia by Modulating mPTP-Dependent Ferroptosis

Ischemic stroke remains a major cause of mortality and long-term disability, with few effective neuroprotective treatments currently available. Ferroptosis, an iron-dependent form of regulated cell death marked by lipid peroxidation, is increasingly recognized as a driver of neuronal damage. However, the mitochondrial mechanisms linking ischemia to Ferroptosis remain poorly defined. Here, we identify circMTCO2, a mitochondria-encoded circular RNA (circRNA), as a novel endogenous modulator of neuronal Ferroptosis. circMTCO2 expression is dynamically down-regulated following cerebral ischemia/reperfusion in vitro and in vivo. Mechanistically, circMTCO2 interacts with adenine nucleotide translocase 1 (ANT1), a key regulator associated with the mitochondrial permeability transition pore (mPTP), thereby inhibiting mPTP opening and suppressing mitochondrial Reactive Oxygen Species release. Disruption of the binding site abolishes circMTCO2-ANT1 interaction and eliminates the protective effects of circMTCO2. To restore and enhance this intrinsic defense mechanism, we developed a dual-targeting extracellular vesicle system (RVG-EV^mt-RNA) capable of delivering circMTCO2 specifically to neuronal mitochondria. Systemic administration of RVG-EV^mt-RNA decreased infarct volume, attenuated ferroptosis-associated injury, and improved neurological function in a mouse model of ischemic stroke, without inducing systemic toxicity. These findings establish circMTCO2 as a previously unrecognized mitochondrial circRNA that regulates Ferroptosis by modulating mPTP activity and provide a proof of concept that organ-to-organelle circRNA delivery can be leveraged as a precision neuroprotective strategy for ischemic stroke.