Restoring cardiolipin homeostasis mitigates cerebral ischemia–reperfusion injury by suppressing ATG5-mediated neuronal autophagy-dependent ferroptosis

• EV-mediated CL release contributes to CL decrease in peri -infarct brain tissues following cerebral I/R injury. • CL-targeting peptide, SS-31, inhibits CL release, preserves CL content and restores CL homeostasis. • Restoring CL homeostasis suppresses ATG5-mediated autophagy-dependent neuronal ferroptosis. • Plasma CL levels correlate with 3-month outcomes in patients undergoing endovascular treatment. Cardiolipins (CLs) are mitochondria-specific phospholipids critically involved in neurological disorders. However, their role in cerebral ischemia/reperfusion (I/R) injury remains largely underexplored. This study aimed to characterize CL alterations following cerebral I/R injury, identify potential mechanisms underlying CL loss, and investigate the pathophysiological effects of these changes. Ischemic stroke was modeled by using middle cerebral occlusion/reperfusion (MCAO/R) in mice and oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro . CL alterations following cerebral I/R injury were evaluated through lipidomic analysis and fluorescent staining. The role of extracellular vesicles (EVs) in CL release was investigated. The effects of CL-targeting peptide SS-31 on CL release and homeostasis were assessed, and underlying mechanisms were explored by analyzing neuronal autophagy and ferroptosis. Plasma CL levels in stroke patients were also measured to elucidate clinical relevance. Significant CL depletion and acyl chain remodeling were observed in peri -infarct brain tissues. EV-mediated release was identified as one of the mechanisms of CL loss, and inhibiting EV release restored neuronal CL levels. SS-31 inhibits CL release, preserves CL content, and subsequently restores CL homeostasis. Restoring CL homeostasis attenuated cerebral I/R injury by suppressing neuronal ferroptosis. Mechanistically, SS-31 downregulated autophagy-associated genes, with ATG5 identified as the crucial target. Activating autophagy or overexpressing ATG5 reversed the protective effects of SS-31. Clinically, plasma CL levels in patients undergoing endovascular treatment correlated with 90-day functional outcomes. Our findings establish CL preservation as a novel neuroprotective strategy, in which SS-31 mitigates I/R injury by restoring CL homeostasis and ameliorating ATG5-mediated autophagy-dependent neuronal ferroptosis, offering a novel therapeutic avenue for stroke.