RIPK1/RIPK3 and MLKL differentially control mitophagy and mitochondrial homeostasis in hypoxic-ischaemic encephalopathy

Hypoxic-ischaemic encephalopathy (HIE) is a devastating brain injury in newborns, yet the relationship between necroptotic signalling and mitochondrial quality control remains incompletely understood. Here, we investigated the roles of RIPK1/RIPK3 and MLKL in regulating mitophagy and mitochondrial homoeostasis in ischaemia-reperfusion (I/R) brain injury such as HIE. Using oxygen-glucose deprivation and reperfusion (OGD/Rep) in mouse primary neurons or mouse neuroblastoma Neuro-2a cells, we show that OGD/Rep activates necroptosis with increased phosphorylation of mixed lineage kinase domain-like (MLKL) and mitochondrial translocation of necrosome components.

This is accompanied by induction of mitophagy, including upregulation of Parkin and BNIP3, increased mitochondrial conversion of LC3A/B-II, and induction of the mitochondrial protease caseinolytic peptidase protease (ClpP). Pharmacological inhibition of RIPK1 or RIPK3 attenuates OGD/Rep-induced mitophagy, suggesting that RIPK1/RIPK3 signalling promotes the mitochondrial quality control response. In contrast, MLKL knockdown enhanced mitophagy while similarly suppresses mitochondrial ClpP induction and restores mitochondrial function and morphology.

In a neonatal mouse model of HIE, hypoxia-ischaemia challenge concurrently activates necroptosis and mitophagy pathways in the hippocampus. Spatial transcriptomic analysis further reveals region-specific signatures of necroptosis-related mitochondrial stress and mitophagy in the hippocampus and cortex. Taken together, our findings uncover a previously unrecognised divergence within the necroptosis signalling cascade, whereby RIPK1/RIPK3 promote mitophagy and MLKL contributes to mitochondrial dysfunction.

Our work indicates the complex crosstalk between necroptotic signalling and mitochondrial quality control, providing a mechanistic rationale for targeting distinct components of the pathway in HIE and related I/R injuries. We thank Professor Libing Zhou (Guangdong-Hong Kong-Macau Institute of CNS Regeneration, Jinan University, China) for his support on the project. This work is supported by the British Journal of Anesthesia Project grant (D.

Ma) and Ph.D. studentship grant (L. Wu), London, UK; European Society of Anaesthesiology and Intensive Care Andreas Hoeft’s Grant (ESAIC_GR_2022_DM), Brussels, Belgium; and the Ningbo Top Medical and Health Research Programme (2024010317), Ningbo, Zhejiang, China. These authors contributed equally: Lingzhi Wu, Enqiang Chang.

Division of Anaesthetics, Pain Medicine and Intensive Care, Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, Chelsea and Westminster Hospital, London, UK Lingzhi Wu, Enqiang Chang, Jiaqi Li, Ge Huang, Hailin Zhao, Qian Chen & Daqing Ma Department of Anesthesiology, Guangxi Medical University Cancer Hospital, Guangxi, Clinical Research Center for Anesthesiology, Guangxi, China Department of Anesthesiology, Guangzhou Women and Children’s Medical Center, Guangzhou Medical University, Guangdong Provincial Clinical Research Center for Child Health, Guangzhou, China Perioperative and Systems Medicine Laboratory and Department of Anesthesiology, Children’s Hospital, Zhejiang University School of Medicine, National Clinical Research Center for Children and Adolescents’ Health and Diseases, Hangzhou, Zhejiang, China Department of Anesthesiology, The First Affiliated Hospital of Ningbo University, Ningbo, Zhejiang, China The authors declare no competing interests. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.