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.