Synucleinopathies, including Parkinson's disease, Dementia with Lewy bodies, and Multiple System Atrophy, are characterised by intracellular accumulation of misfolded α-synuclein (αSyn). Although αSyn aggregation is central to disease pathogenesis, the mechanisms linking αSyn to progressive neurodegeneration remain incompletely understood. Mitochondrial dysfunction is recognised as a key pathological process, particularly in experimental models using exogenously applied αSyn species, including preformed fibrils (PFFs). This systematic review synthesises evidence from mammalian in vitro studies that examined the effects of disease-associated αSyn conformations, including monomers, oligomers and PFFs, on mitochondrial structure and function. Following PRISMA 2020 guidelines, EMBASE, MEDLINE, and PsycInfo were searched to identify eligible peer-reviewed studies published up to March 2026, yielding 33 included studies. Models encompassed neuronal, astrocytic, microglial, endothelial, fibroblast, and induced pluripotent stem cell-derived systems, enabling comparison across cell types, αSyn conformations, and genetic backgrounds. Across included studies, oligomeric and fibrillar αSyn consistently induced mitochondrial depolarisation, impaired oxidative phosphorylation, reduced ATP production, complex-I dysfunction and increased reactive oxygen species generation. Additional mitochondrial pathological changes included fragmentation, cristae disruption, impaired TOM20-mediated protein import, calcium dysregulation, defective mitophagy and increased heteroplasmy i.e. accumulation of damaged mitochondria. These alterations frequently preceded cell death and were amplified in dopaminergic and genetically vulnerable models, including αSyn A53T, SNCA triplication, and PARK2-associated transgenic systems. Our review highlights mitochondrial dysfunction as a mechanism underlying αSyn toxicity, whilst suggesting methodological heterogeneity can limit cross-study translation. Priorities for future research using 'in vitro' models of PFF synucleinopathies include standardised fibril preparation protocols, improved mitochondrial phenotyping, and use of human multicellular and organoid models.
