Metabolic Reprogramming and Exosome Remodeling in an Ndufs4 ‐Deficient Model of Leigh Syndrome

Leigh syndrome is a severe neurometabolic disorder with an incompletely understood pathogenesis. This study investigated the role of Ndufs4 knockout in Leigh syndrome by exploring its impact on cellular metabolism and exosome biology. Initially, we observed that homozygous knockout ( Ndufs4 −/− ) mice exhibited significantly reduced body size and spleen weight compared to wild‐type ( Ndufs4 +/+ ) controls, although the spleen index remained unchanged. Notably, the brain index was significantly increased. Histological analysis revealed structural abnormalities in brain tissue, accompanied by elevated expression of the astrocytic marker GFAP, both in vivo and in differentiated neural stem cells (NSCs), indicating a neuropathological effect of Ndufs4 knockout. Metabolically, Ndufs4 knockout in NSCs did not reduce ATP levels but increased reactive oxygen species (ROS) levels, enhanced glucose uptake (2‐NBDG), and decreased mitochondrial membrane potential (JC‐1). Concurrent shifts in metabolites—decreased L‐lactate, increased acetyl‐CoA, and a lower NADP + /NADPH ratio—implied a potential redirection of metabolic flux toward the pentose phosphate pathway. Furthermore, although the total exosome concentration was reduced in Ndufs4 −/− mice, the protein content per exosome was significantly elevated, indicating enhanced protein loading per vesicle. Proteomic profiling of brain‐derived exosomes identified increased levels of flotillin‐1 and Slc6a5, proteins associated with the mTOR pathway. Consistent with this, downstream mTOR signaling (Phospho‐S6 Ribosomal Protein (Ser235/236; p‐RPS6) and Phospho‐p70 S6 kinase (Thr389; p‐p70S6K)) was substantially attenuated in Ndufs4 −/− mice, indicating suppression of the mTOR pathway. Together, these results elucidate novel mechanisms through which Ndufs4 knockout drives exosomal remodeling, reprograms cellular metabolism, and suppresses mTOR signaling, providing a conceptual framework for understanding Leigh syndrome pathogenesis and informing therapeutic development.