Proximity interactome mapping and cryo-ET visualization define the neuronal uptake and mitochondrial engagement of α-syn fibrils

ABSTRACT: Parkinson’s disease (PD) is driven by the prion-like propagation of α-synuclein (α-syn) fibrils, but the molecular and structural mechanisms governing their neuronal uptake, intracellular trafficking, and mitochondrial engagement remain incompletely defined. Here, we integrated two core cutting-edge approaches—time-resolved proximity labeling (PL) proteomics and cryogenic electron tomography (cryo-ET)—along with functional analyses to resolve these critical processes. To dissect the dynamic protein interactions that mediate α-syn fibrils neuronal uptake and trafficking, we developed a novel Rose Bengal-conjugated α-syn fibrils (RB-PFFs) proximity labeling strategy, a powerful tool that enabled the capture of stage-specific interactomes throughout α-syn fibrils intracellular transit. A key insight from this time-resolved proximity proteomics approach was the progressive enrichment of mitochondrial proteins, which directly guided our subsequent investigation of α-syn fibrils-mitochondrial interactions. Complementing these proteomic analyses, cryo-ET—validated by correlative light and electron microscopy (CLEM)—provided definitive near-atomic resolution visualization of α-syn fibrils interacting with receptor-rich densities on the axonal plasma membrane prior to internalization, being sequestered within lysosomal compartments following intracellular trafficking, and subsequently escaping lysosomes to make direct contact with the outer mitochondrial membrane (OMM) in naive neurons, resolving the structural basis of this critical pathogenic interaction. Functional analyses further confirmed that α-syn fibrils engagement with mitochondria induces mitochondrial DNA (mtDNA) release, with Voltage-Dependent Anion Channel 1 (VDAC1) implicated as a mediator of this process. Together, our findings, anchored in proximity proteomics and cryo-ET, establish a comprehensive molecular and structural framework for α-syn fibrils neuronal uptake and mitochondrial engagement, identifying potential therapeutic targets for mitigating α-syn-related pathogenesis in PD.

ORGANISM(S): Mus Musculus

SUBMITTER: Lihua Zhang  

PROVIDER: PXD078571 | iProX | Tue May 19 00:00:00 BST 2026

REPOSITORIES: iProX