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

  • J Adv Res. 2026 Mar 12:S2090-1232(26)00234-1. doi: 10.1016/j.jare.2026.03.014.
Weichen Dong  1 ,  Wenxin Zhang  2 ,  Li Huang  1 ,  Lulu Xiao  1 ,  Yuanfei Luo  3 ,  Linying Yuan  1 ,  Yike Jiang  1 ,  Anyu Liao  1 ,  Hongting Zhao  4 ,  Zhihui Liu  1 ,  Jia Wang  1 ,  Ying Zhao  1 ,  Yulong Cai  1 ,  Mengna Peng  1 ,  Dong Yang  1 ,  Yi Xie  5 ,  Kuanyu Li  6 ,  Wusheng Zhu  7
Affiliations
  • 1. State Key Laboratory of Pharmaceutical Biotechnology, Department of Neurology, Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210002, China.
  • 2. State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Key Laboratory of Molecular Medicine, Medical School, Nanjing University, Nanjing 210093, China; Gulbenkian Institute for Molecular Medicine, Lisboa 2780-156, Portugal.
  • 3. Department of Neurology, Third Affiliated Hospital, Soochow University, Changzhou 213003, China.
  • 4. Affiliated Hospital of Nanjing University of Chinese Medicine, Nanjing 210029, China; the Chinese Clinical Medicine Innovation Center of Obstetrics, Gynecology, and Reproduction in Jiangsu Province, Nanjing 210029, China.
  • 5. Department of Neurology, Nanjing First Hospital, Nanjing Medical University, Nanjing 210006, China. Electronic address: [email protected].
  • 6. State Key Laboratory of Pharmaceutical Biotechnology, Jiangsu Key Laboratory of Molecular Medicine, Medical School, Nanjing University, Nanjing 210093, China. Electronic address: [email protected].
  • 7. State Key Laboratory of Pharmaceutical Biotechnology, Department of Neurology, Jinling Hospital, Affiliated Hospital of Medical School, Nanjing University, Nanjing 210002, China. Electronic address: [email protected].
PMID: 41831680 DOI: 10.1016/j.jare.2026.03.014
Abstract

Introduction: Cardiolipins (CLs) are mitochondria-specific Phospholipids critically involved in neurological disorders. However, their roles in cerebral ischemia/reperfusion (I/R) injury remains largely underexplored.

Objectives: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Keywords
Autophagy; Autophagy-related gene 5 (ATG5); Cardiolipin; Cerebral ischemia/reperfusion injury; Extracellular vesicle; Ferroptosis; SS-31.
Products