Aggregation of alpha-synuclein (α-Syn) drives Parkinson’s disease (PD), although the initial stages of self-assembly and structural conversion have not been directly observed inside neurons. In this study, we tracked the intracellular conformational states of α-Syn using a single-molecule Förster resonance energy transfer (smFRET) biosensor, and we show here that α-Syn converts from a monomeric state into two distinct oligomeric states in neurons in a concentration-dependent and sequence-specific manner. Three-dimensional FRET-correlative light and electron microscopy (FRET-CLEM) revealed that intracellular seeding events occur preferentially on membrane surfaces, especially at mitochondrial membranes. The mitochondrial lipid cardiolipin triggers rapid oligomerization of A53T α-Syn, and cardiolipin is sequestered within aggregating lipid–protein complexes. Mitochondrial aggregates impair complex I activity and increase mitochondrial reactive oxygen species (ROS) generation, which accelerates the oligomerization of A53T α-Syn and causes permeabilization of mitochondrial membranes and cell death. These processes were also observed in induced pluripotent stem cell (iPSC)–derived neurons harboring A53T mutations from patients with PD. Our study highlights a mechanism of de novo α-Syn oligomerization at mitochondrial membranes and subsequent neuronal toxicity.

The human genome contains thousands of natural antisense transcripts (NAT) that can regulate epigenetic state, transcription, RNA stability, or translation of their overlapping genes 1,2 . We describe MAPT-AS1, a primate-conserved, brain-enriched NAT containing an embedded mammalian-wide interspersed repeat (MIR), which represses tau translation by competing with rRNA pairing to MAPT mRNA internal ribosome entry site (IRES) 3 . Tau, a neuronal intrinsically disordered protein (IDP), stabilises axonal microtubules. Hyperphosphorylated, aggregation-prone tau forms the hallmark inclusions of tauopathies 4 . MAPT mutations cause familial frontotemporal dementia (FTLD-tau), and common variation forming the MAPT H1 haplotype is a significant risk factor in many tauopathies 5 , and Parkinson's disease. Notably, expression of MAPT-AS1 or its minimal essential sequences including MIR reduces, whereas silenced MAPT-AS1 increases neuronal tau, and is correlated with tau pathology in human brain. Moreover, we identified hundreds additional NATs with embedded MIRs (MIR-NATs), which are overrepresented at coding genes linked to neurodegeneration, and/or encoding IDPs, and confirmed MIR-NAT-mediated translational control of one such gene, PLCG1. Collectively, we present the importance of MAPT-AS1 for tauopathies, while also uncovering a potentially broad contribution of MIR-NATs to the tightly controlled translation of IDPs 6 , with particular relevance for proteostasis in neurodegeneration.