α-Synuclein fate is determined by USP9X-regulated monoubiquitination

alpha-Synuclein is central to the pathogenesis of Parkinson disease (PD). Mutations as well as accumulation of alpha-synuclein promote the death of dopaminergic neurons and the formation of Lewy bodies. alpha-Synuclein is monoubiquitinated by SIAH, but the regulation and roles of monoubiquitination in alpha-synuclein biology are poorly understood. We now report that the deubiquitinase USP9X interacts in vivo with and deubiquitinates alpha-synuclein. USP9X levels are significantly lower in cytosolic fractions of PD substantia nigra and Diffuse Lewy Body disease (DLBD) cortices compared to controls. This was associated to lower deubiquitinase activity toward monoubiquitinated a-synuclein in DLBD cortical extracts. A fraction of USP9X seems to be aggregated in PD and DLBD, as USP9X immunoreactivity is detected in Lewy bodies. Knockdown of USP9X expression promotes accumulation of monoubiquitinated alpha-synuclein species and enhances the formation of toxic alpha-synuclein inclusions upon proteolytic inhibition. On the other hand, by manipulating USP9X expression levels in the absence of proteolytic impairment, we demonstrate that monoubiquitination controls the partition of alpha-synuclein between different protein degradation systems. Deubiquitinated alpha-synuclein is mostly degraded by autophagy, while monoubiquitinated alpha-synuclein is preferentially degraded by the proteasome. Moreover, monoubiquitination promotes the degradation of alpha-synuclein, whereas deubiquitination leads to its accumulation, suggesting that the degradation of deubiquitinated a-synuclein by the autophagy pathway is less efficient than the proteasomal one. Lower levels of cytosolic USP9X and deubiquitinase activity in alpha-synucleinopathies may contribute to the accumulation and aggregation of monoubiquitinated alpha-synuclein in Lewy bodies. Our data indicate that monoubiquitination is a key determinant of alpha-synuclein fate.