p21-activated kinase 4 controls the aggregation of alpha-synuclein by reducing the monomeric and aggregated forms of alpha-synuclein: involvement of the E3 ubiquitin ligase NEDD4-1

Aggregation of misfolded alpha-synuclein (alpha-synuclein) is a central player in the pathogenesis of neurodegenerative diseases. Therefore, the regulatory mechanism underlying alpha-synuclein aggregation has been intensively studied in Parkinson's disease (PD) but remains poorly understood. Here, we report p21-activated kinase 4 (PAK4) as a key regulator of alpha-synuclein aggregation. Immunohistochemical analysis of human PD brain tissues revealed an inverse correlation between PAK4 activity and alpha-synuclein aggregation. To investigate their causal relationship, we performed loss-of-function and gain-of-function studies using conditional PAK4 depletion in nigral dopaminergic neurons and the introduction of lentivirus expressing a constitutively active form of PAK4 (caPAK4; PAK4(S445N/S474E)), respectively. For therapeutic relevance in the latter setup, we injected lentivirus into the striatum following the development of motor impairment and analyzed the effects 6 weeks later. In the loss-of-function study, Cre-driven PAK4 depletion in dopaminergic neurons enhanced alpha-synuclein aggregation, intracytoplasmic Lewy body-like inclusions and Lewy-like neurites, and reduced dopamine levels in PAK4(DAT-CreER) mice compared to controls. Conversely, caPAK4 reduced alpha-synuclein aggregation, as assessed by a marked decrease in both proteinase K-resistant and Triton X100-insoluble forms of alpha-synuclein in the AAV-alpha-synuclein-induced PD model. Mechanistically, PAK4 specifically interacted with the NEDD4-1 E3 ligase, whose pharmacological inhibition and knockdown suppressed the PAK4-mediated downregulation of alpha-synuclein. Collectively, these results provide new insights into the pathogenesis of PD and suggest PAK4-based gene therapy as a potential disease-modifying therapy in PD.