Objective: To study whether ubiquitination in Lewy bodies is directly relevant to α-synuclein turnover and Parkinson’s pathogenesis.

Background: In Parkinson’s disease, misfolded α-synuclein accumulates, often in a ubiquitinated form, in neuronal inclusions termed Lewy bodies. It is currently unknown whether ubiquitin conjugation in Lewy bodies is due to a defect in enzymes that regulate α-synuclein degradation. This is important as identification and pharmacological modulation of such enzymes could be targeted for therapies.

Methods: Comparative immunohistochemistry and immunoblotting between three brain regions was used to determine the type and abundance of ubiquitin conjugates as well as relevant interacting proteins in neurons with Lewy bodies (n=20 cases). The co-localisation of Usp8 and α-synuclein was investigated using bi-fluorescence complementation assays and in human iPSc-derived neurons. Transient overexpression and shRNA mediated knockdown of Usp8 was used in human cell lines to investigate the functional interaction between the two proteins. The deubiquitination of α-synuclein was investigated in cells and with purified proteins. Drosophila genetics were used to study the effect of Usp8 and other deubiquitinases against α-synuclein toxicity in vivo.

Results: By comparative analysis in human post-mortem brains, we found that ubiquitin immunoreactivity in Lewy bodies is largely due to K63-linked ubiquitin chains and markedly reduced in the substantia nigra compared to the neocortex. The ubiquitin staining in cells with Lewy bodies inversely correlated with the content and pathological localization of the deubiquitinase Usp8. Usp8 interacted and partly co-localized with α-synuclein in endosomal membranes and both in cells and after purification, it deubiquitinated K63-linked chains on α-synuclein. Knockdown of Usp8 in the Drosophila eye reduced α-synuclein levels and α-synuclein-induced eye toxicity. Accordingly, in human cells Usp8 knockdown decreased α-synuclein levels. In the dopaminergic neurons of the Drosophila model, unlike knockdown of other deubiquitinases, Usp8 protected from α-synuclein-induced locomotor deficits and cell loss.

Conclusions: These findings strongly suggest that removal of K63-linked ubiquitin chains on α-synuclein by Usp8 is a critical mechanism in slowing α-synuclein degradation in dopaminergic neurons that may contribute to α-synuclein accumulation in Lewy body disease.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/identification-of-usp8-as-a-toxicity-modifying-deubiquitinase-for-synuclein/