Identification of a highly neurotoxic alpha-synuclein species inducing mitochondrial damage and mitophagy in Parkinson's disease

Exposure of cultured primary neurons to preformed alpha-synuclein fibrils (PFFs) leads to the recruitment of endogenous alpha-synuclein and its templated conversion into fibrillar phosphorylated alpha-synuclein (p alpha-synF) aggregates resembling those involved in Parkinson's disease (PD) pathogenesis. P alpha-synF was described previously as inclusions morphologically similar to Lewy bodies and Lewy neurites in PD patients. We discovered the existence of a conformationally distinct, nonfibrillar, phosphorylated alpha-syn species that we named p alpha-syn*. We uniquely describe the existence of p alpha-syn* in PFF-seeded primary neurons, mice brains, and PD patients' brains. Through immunofluorescence and pharmacological manipulation we showed that p alpha-syn* results from incomplete autophagic degradation of p alpha-synF. P alpha-synF was decorated with autophagic markers, but p alpha-syn* was not. Western blots revealed that p alpha-syn* was N- and C-terminally trimmed, resulting in a 12.5-kDa fragment and a SDS-resistant dimer. After lysosomal release, p alpha-syn* aggregates associated with mitochondria, inducing mitochondrial membrane depolarization, cytochrome C release, and mitochondrial fragmentation visualized by confocal and stimulated emission depletion nanoscopy. P alpha-syn* recruited phosphorylated acetyl-CoA carboxylase 1 (ACC1) with which it remarkably colocalized. ACC1 phosphorylation indicates low ATP levels, AMPK activation, and oxidative stress and induces mitochondrial fragmentation via reduced lipoylation. P alpha-syn* also colocalized with BiP, a master regulator of the unfolded protein response and a resident protein of mitochondria-associated endoplasmic reticulum membranes that are sites of mitochondrial fission and mitophagy. P alpha-syn* aggregates were found in Parkin-positive mitophagic vacuoles and imaged by electron microscopy. Collectively, we showed that p alpha-syn* induces mitochondrial toxicity and fission, energetic stress, and mitophagy, implicating p alpha-syn* as a key neurotoxic alpha-syn species and a therapeutic target.