Monitoring α-synuclein ubiquitination dynamics reveals key endosomal effectors mediating its trafficking and degradation

While defective & alpha;-synuclein homeostasis is central to Parkinson's pathogenesis, fundamental questions about its degradation remain unresolved. We have developed a bimolecular fluorescence complementation assay in living cells to monitor de novo ubiquitination of & alpha;-synuclein and identified lysine residues 45, 58, and 60 as critical ubiquitination sites for its degradation. This is mediated by NBR1 binding and entry into endosomes in a process that involves ESCRT I-III for subsequent lysosomal degradation. Autophagy or the autophagic chaperone Hsc70 is dispensable for this pathway. Antibodies against diglycine-modified & alpha;-synuclein peptides confirmed that endogenous & alpha;-synuclein is similarly ubiquitinated in the brain and targeted to lysosomes in primary and iPSC-derived neurons. Ubiquitinated & alpha;-synuclein was detected in Lewy bodies and cellular models of aggregation, suggesting that it may be entrapped with endo/lysosomes in inclusions. Our data elucidate the intracellular trafficking of de novo ubiquitinated & alpha;-synuclein and provide tools for investigating the rapidly turned-over fraction of this disease-causing protein.

Automated summary

We developed a bimolecular fluorescence complementation assay to monitor de novo ubiquitination of alpha-synuclein, and identified lysine residues 45, 58, and 60 as critical sites for its degradation. The degradation involves NBR1 binding, entry into endosomes and subsequent lysosomal degradation. Our data also show the presence of ubiquitinated alpha-synuclein in Lewy bodies and cellular models of aggregation.