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Updated: Jun 29, 2026

Sequential Extraction of Soluble and Insoluble Alpha-Synuclein from Parkinsonian Brains
Published on: January 5, 2016
Monitoring α-synuclein ubiquitination dynamics reveals key endosomal effectors mediating its trafficking and
Dmitry Zenko1,2, Jade Marsh1,2, Andrew R Castle1,2
1Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK.
Abstract:
While defective α-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 α-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 α-synuclein peptides confirmed that endogenous α-synuclein is similarly ubiquitinated in the brain and targeted to lysosomes in primary and iPSC-derived neurons. Ubiquitinated α-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 α-synuclein and provide tools for investigating the rapidly turned-over fraction of this disease-causing protein.
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