LOSS AND PROTEOLYTIC REMODELING OF EBP1 ALTERS MITOCHONDRIAL DYNAMICS IN AGING AND ALZHEIMER’S DISEASE MODELS

Mitochondrial quality control relies on the tight coordination between mitochondrial fusion and selective removal of damaged mitochondria by mitophagy, yet the molecular mechanisms coupling these processes remain incompletely understood. EBP1, a known substrate of Parkin, has been implicated in promoting mitophagy during ischemia/reperfusion injury; however, how EBP1 integrates mitochondrial dynamics with mitophagic signaling, and the fate of mitochondria that evade degradation in its absence, remain unclear. Here, we identify EBP1 as a critical molecular link between MFN2-dependent mitochondrial fusion and Parkin-mediated mitophagy. Ebp1-null mouse embryonic fibroblasts exhibited pronounced mitochondrial fragmentation, indicating impaired fusion. Biochemical analyses revealed that EBP1 selectively interacts with the mitochondrial fusion protein MFN2, and this interaction was significantly attenuated by a K376A mutation, identifying Lys376 as a key determinant of MFN2 binding. Under Alzheimer’s disease–like conditions, EBP1 underwent proteolytic cleavage by asparagine endopeptidase at residues N84 and N204, generating a C-terminal 205–394 fragment with enhanced mitochondrial localization. This fragment displayed increased association with MFN2 and, when co-expressed with mitochondria-targeted amyloid-β, robustly activated mitophagy and facilitated amyloid-β clearance. In aging 5xFAD mice, total EBP1 levels were reduced, leading to diminished EBP1-dependent mitophagy; notably, the remaining EBP1 exhibited enhanced association with MFN2. Together, these findings establish EBP1 as a stress-responsive regulator that coordinates mitochondrial fusion and mitophagy, thereby maintaining mitochondrial homeostasis under pathological conditions such as neurodegenerative disease.