The role of α-synuclein in exocytosis - relationship between SNAP-25 and α-synuclein

• Main Authors: Kai-Ning Shih, 施凱寧
• Other Authors: Lung-Sen Kao
• Format: Others
• Language: en_US
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/83467363348511932090

碩士 === 國立陽明大學 === 生命科學系暨基因體科學研究所 === 101 === α-Synuclein, a presynaptic protein, has been proposed as a key player in the pathogenesis of Parkinson’s disease. In previous investigations, CSPα-knockout mice have abnormal degradation of SNAP-25A to reduce SNARE complex assembly and result in neurodegeneration, and overexpression of α-synuclein help more SNARE complex formation. Previous studies show that SNAP-25A functions in vesicle docking and priming. Our previous studies show that overexpression of α-synuclein promote vesicle priming and accelerate fusion pore dilation in PC12 cells. The goal of my study is to investigate the relationship between α-synuclein and SNAP-25A in regulated exocytosis. PC12 cells co-expressing NPY-EGFP with α-synuclein and SNAP-25A or mutants were stimulated by ATP for exocytosis. The individual exocytotic events were followed by the fluorescence of NPY-EGFP using a total internal reflection fluorescence microscopy (TIRFM). Overexpression of wt SNAP-25A, PML38 (mutant lacking of interaction with VAMP2), or α-synuclein alone promotes vesicle secretion. In combination of α-synuclein with SNAP-25A or PML38, exocytotic activity was similar to that of overexpression of α-synuclein. Overexpressed PML446 (lacking of interaction with syntaxin1A) or PML39 (palmitoylation sites only) with or without α-synuclein exhibited similar level of vesicle secretion as the cells overexpressing α-synuclein only and the control cells. Overexpression of PML444 (lacking the ability of membrane binding) inhibited secretion, and co-expression of α-synuclein did not rescue the secretion. In the presence of α-synuclein, PC12 cells expressed SNAP-25A WT, PML446, PML38, or PML39 exhibited similar level of exocytosis activity to cells expressed α-synuclein alone. Moreover, overexpression of α-synuclein has no effect on subcellular localization of SNAP-25A and its mutants. These results suggested that α-synuclein plays a role in downstream of SNAP-25A to regulate exocytosis.