Cleavage of normal and pathological forms of α-synuclein by neurosin in vitro

Abstract Neurosin is one of the serine proteases predominantly expressed in the central nervous system. Neurosin is presumed to play an important role in the degradation of α-synuclein (α-syn), since a previous study showed that neurosin degrades α-syn, inhibits polymerization of α-syn in vitro, and exists in Lewy bodies. However, the details of α-syn degradation by neurosin are little known. We investigated neurosin-mediated cleavage of α-syn by immunoblotting and liquid chromatography-ion trap mass spectrometry (LC/MS/MS). We also compared α-syn degradation by neurosin between phosphorylated and non-phosphorylated forms of α-syn, and between mutant and wild-type α-syn. Neurosin cleaved α-syn at specific sites. The major cleavage site was localized between Lys80 and Thr81 within the NAC region (E61 to V95), which is important for α-syn aggregation, and accordingly may preclude α-syn polymerization. Meanwhile, alternative, minor forms of processing also occur. They conserve the NAC region with truncation of the C-terminal region, and accordingly may contribute to α-syn polymerization. Phosphorylated α-syn was more resistant to degradation by neurosin than non-phosphorylated α-syn. The A30P mutant was more resistant to degradation than the wild-type and other α-syn mutants. This resistance to neurosin-mediated degradation of phosphorylated α-syn and the A30P mutant, which are, respectively, posttranslational and genetic factors related to the development of Parkinson's disease (PD), provides supporting evidence that neurosin is involved in the pathogenesis of PD.