Brain mitovesicles undergo stage-specific remodelling during the progression of amyloid-β pathology

Mitochondrial dysfunction is an early feature of Alzheimer's disease that shows nonlinear changes as the disease progresses. However, how these changes are reflected in the mitochondrial material released by brain cells remains poorly understood. Mitovesicles are a recently described extracellular vesicle subpopulation of mitochondrial origin that provide a unique insight into mitochondrial alterations in the brain and could potentially show how nonlinear changes evolve across disease stages of amyloid-beta pathology. Here, we isolated forebrain-derived small extracellular vesicles and mitovesicles from wild-type and Amyloid precursor protein (App) knock-in (AppKI) mice, carrying familial AD mutations and progressively accumulating amyloid-beta plaques, at presymptomatic (2-month) and symptomatic (12-month) stages using high-resolution density gradient fractionation. Vesicle identity and composition were assessed by morphological, biophysical and quantitative proteomic analyses, and mitovesicle respiratory function was measured by extracellular flux analysis. We show that mitovesicles carry a distinct, predominantly mitochondrial inner membrane proteome that is remodelled by age and disease in a stage-specific manner. Compared with small extracellular vesicles, mitovesicle proteomic profiles captured disease progression more clearly, with symptomatic AppKI mice forming the most molecularly distinct group. At the presymptomatic stage, AppKI mitovesicles showed reduced mitochondrial protein representation, most notably of fatty acid {beta}-oxidation and lipid-metabolism proteins associated with astroglial and vascular signatures, preceding any detectable respiratory impairment. At the symptomatic stage this reversed: AppKI mitovesicles were enriched for oxidative phosphorylation, mitochondrial membrane and transporter proteins, yet showed reduced Complex IV-linked respiration, revealing a dissociation between mitochondrial cargo abundance and respiratory function. Selected cargo changes were not mirrored in whole-brain tissue, indicating selective remodelling of mitovesicle content. Comparison with mitovesicles from post-mortem human Alzheimer's disease frontotemporal cortex and hippocampus identified a conserved 62-protein mitovesicle core, dominated by oxidative phosphorylation subunits and mitochondrial carriers. In addition, 25 of these proteins were enriched in mitovesicles derived from Alzheimer's and AppKI forebrains, defining a conserved mitovesicular disease signature. Together, these findings establish forebrain-derived mitovesicles as a dynamically remodelled vesicle population that reflects progressive mitochondrial dysfunction in the Alzheimer's disease brain and provides a cross-species resource for future biomarker and mechanistic studies.