In vivo Proximity & Spatial Proteomics with CRISPR Screening Identify STXBP1 as a Protective Modifier of a-synuclein Toxicity in Dopamine Neurons.

Parkinsons disease (PD) is a disease of adults involving the loss of dopaminergic neurons after a long, asymptomatic, prodromal period. -synuclein, LRRK2, and VPS35 are linked to familial PD, however, how these mutations predispose dopamine neurons to death during the early prodromal phases remains unclear. Here, we used in vivo native proximity proteomics (iBioID) and dopaminergic neuron-specific subcellular proteomics across multiple PD models to uncover early alterations preceding neuronal loss. Our analyses identified convergent disruptions in synaptic protein abundance, indicating that presynaptic trafficking defects are early events in PD pathogenesis. Using a targeted CRISPR-based genetic screen in dopamine neurons, we demonstrated that mimicking this misregulation of STXBP1 amplifies vulnerability to -synuclein, implicating it as a previously underappreciated toxicity buffering factor. These findings highlight convergent mechanisms that sensitize dopamine neuronal degeneration and that presynaptic vesicle SNARE-complex proteins could serve as key targets for disease-modifying therapies in PD and related neurodegenerative disorders. HighlightsO_LIIn vivo native-BioID mapping of multiple Parkinsons disease (PD) protein interactomes revealed a convergent presynaptic network. C_LIO_LIiBioID analysis on mutant PD proteins (-synuclein A30P, LRRK2 G2019S, VPS35 D620N) uncovered mutation-specific shifts in local proximity networks, notably in endocytic and vesicle recycling pathways. C_LIO_LISpatial proteomics (iBioCoFrac) of dopamine neurons in vivo identified functional modules with reduced levels of key synaptic proteins in PD models. C_LIO_LIComparative proteomics using iBioCoFrac revealed synaptic vesicle regulation as a primary site of molecular convergence and early molecular signatures in dopamine neurons across multiple PD mouse models. C_LIO_LIAn in vivo CRISPR screen pinpointed the presynaptic protein Stxbp1/Munc18-1 as an -synuclein toxicity modifier in dopaminergic neurons. C_LI