RESEARCH ARTICLE Soluble α-synuclein facilitates priming and fusion by releasing Ca 2+ from the thapsigargin-sensitive Ca 2+ pool in PC12 cells Chien-Chang Huang 1,2 , Tai-Yu Chiu 2 , Tzu-Ying Lee 2 , Hsin-Jui Hsieh 2 , Chung-Chih Lin 1,2,3, * and Lung-Sen Kao 1,2, * ABSTRACT α-Synuclein is associated with Parkinson’s disease, and is mainly localized in presynaptic terminals and regulates exocytosis, but its physiological roles remain controversial. Here, we studied the effects of soluble and aggregated α-synuclein on exocytosis, and explored the molecular mechanism by which α-synuclein interacts with regulatory proteins, including Rab3A, Munc13-1 (also known as Unc13a) and Munc18-1 (also known as STXBP1), in order to regulate exocytosis. Through fluorescence recovery after photobleaching experiments, overexpressed α-synuclein in PC12 cells was found to be in a monomeric form, which promotes exocytosis. In contrast, aggregated α-synuclein induced by lactacystin treatment inhibits exocytosis. Our results show that α-synuclein is involved in vesicle priming and fusion. α-Synuclein and phorbol 12-myristate 13-acetate (PMA), which is known to enhance vesicle priming mediated by Rab3A, Munc13-1 and Munc18-1, act on the same population of vesicles, but regulate priming independently. Furthermore, the results show a novel effects of α-synuclein on mobilizing Ca 2+ release from thapsigargin-sensitive Ca 2+ pools to enhance the ATP-induced [Ca 2+ ] i increase, which enhances vesicle fusion. Our results provide a detailed understanding of the action of α-synuclein during the final steps of exocytosis. KEY WORDS: α-Synuclein, Rab3A, Exocytosis, Priming, Dilation of fusion pore, TG-sensitive pools INTRODUCTION α-Synuclein is a small soluble protein that is highly abundant in nerve terminals (Maroteaux et al., 1988). It is the major component of Lewy bodies and is present in the polymerized fibrils found in the brains of patients who suffer from Parkinson’s disease (Spillantini et al., 1998). The familial A30P and A53T mutations, and the expression of the abnormal α-synuclein, have been suggested to play key roles in the neurodegenerative process associated with Parkinson’s disease (Narhi et al., 1999). α-Synuclein has also been shown to associate with the plasma membrane and is found as a soluble protein in human and mouse brain extracts (Fortin et al., 2004; Kahle et al., 2000) and in rat hippocampal neurons (Fortin et al., 2005). Overexpression of human α-synuclein leads to abnormal aggregation and neuronal degeneration both at the cellular level (Outeiro et al., 2008) and in a mouse model (Masliah et al., 2000). Studies on transgenic mice overexpressing α-synuclein, show that α-synuclein has at least three distinct subcellular forms in presynaptic terminals. These consist of unbound soluble protein, proteins bound to presynaptic vesicles and proteins in the form of microaggregates (Spinelli et al., 2014). Several lines of evidence have shown that α-synuclein is involved in the regulation of exocytosis. Firstly, α-synuclein is mainly localized within the presynaptic terminals and has been reported to regulate the size of the presynaptic vesicular pool, vesicular turnover, synaptic plasticity and the assembly of the soluble NSF attachment protein receptor (SNARE) complex (Abeliovich et al., 2000; Cabin et al., 2002; Chandra et al., 2005; Larsen et al., 2006; Murphy et al., 2000). Secondly, α-synuclein is able to rescue neurodegeneration caused by knockout of the synaptic co-chaperone protein cysteine-string protein-α (CSPα; also known as DNAJC5) (Chandra et al., 2005). Thirdly, α-synuclein has been shown to perturb Ca 2+ homeostasis in SH-SY5Y cells (Hettiarachchi et al., 2009). Ca 2+ plays important roles in the regulation of exocytosis, including the activation of phospholipase C and the assembly of the SNARE complex (Giraudo et al., 2006). According to previous biochemical investigations, α-synuclein directly interacts with several synaptic proteins, including Rab3A, rabphilin, septin-4 and synapsin 1 (Dalfó et al., 2004a; Ihara et al., 2007; Nemani et al., 2010). However, it remains unclear as to how α-synuclein interacts with molecules that are involved in exocytotic process within living cells. It has been shown that Rab3A associates with α-synuclein in Lewy bodies (Dalfó et al., 2004a). Rab3A is a small G-protein of Rab family that is involved in the late steps of exocytosis. Rab3A acts as a gate keeper and plays multiple roles in the secretory process, including in docking, priming and fusion (Dulubova et al., 2005; Huang et al., 2011; Leenders et al., 2001; Lin et al., 2007; Wang et al., 2008). It has been shown that α-synuclein is present in the homogenate obtained from the entorhinal cortex from Lewy body disease patients and can be pulled down with Rab3A by immunoprecipitation (Dalfó et al., 2004a,b). We have previously found that the rate of Rab3A dissociation is involved in regulating fusion pore dilation and that Rab3A also involved in modulating Munc13-1 (also known as Unc13a)- and Munc18-1 (also known as STXBP1)-dependent vesicle priming (Huang et al., 2011; Lin et al., 2007). In this study, we focus on the effects of soluble α-synuclein and its aggregates on exocytosis, and explored the molecular mechanism by which α-synuclein interacts with regulatory proteins, including Rab3A, Munc13-1 and Munc18-1, to regulate exocytosis. This was carried out by using total internal reflection fluorescence (TIRF) microscopy to examine the process of exocytosis in living PC12 cells. Our results suggest that α-synuclein acts at multiple steps during exocytosis including Received 28 November 2017; Accepted 12 October 2018 1 Brain Research Center, National Yang-Ming University, Taipei 112, Taiwan, Republic of China. 2 Department of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei 112, Taiwan, Republic of China. 3 Biophotonics Interdisciplinary Research Center, National Yang-Ming University, Taipei 112, Taiwan, Republic of China. *Authors for correspondence (lskao@ym.edu.tw; cclin2@ym.edu.tw) C.-C.H., 0000-0001-8089-251X; T.-Y.L., 0000-0001-6275-0550; C.-C.L., 0000- 0002-3390-882X; L.-S.K., 0000-0001-6712-8306 1 © 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs213017. doi:10.1242/jcs.213017 Journal of Cell Science