Ganapathy Kavina, Datta Indrani, Sowmithra Sowmithra, Joshi Preeti, Bhonde Ramesh
School of Regenerative Medicine, Manipal University, Bengaluru, Karnataka, India.
Department of Biophysics, National Institute of Mental Health and Neurosciences, an Institute of National Importance, Bengaluru, Karnataka, India.
J Cell Biochem. 2016 Dec;117(12):2719-2736. doi: 10.1002/jcb.25570. Epub 2016 May 5.
Post mortem studies on familial and sporadic Parkinson's disease patient striatal tissue have shown that nearly 90% of α-synuclein deposited in Lewy-bodies is phosphorylated at serine-129 (pSyn-129) as opposed to only 4% in normal human brain. We aimed to find the influence of endogenous neurotoxin 6-hydroxydopamine (6-OHDA) on α-synuclein phosphorylation, resting vesicles, and vesicular dopamine release. The relative distribution of pSyn-129+ cells in apoptotic and non-apoptotic populations at different 6-OHDA concentrations was assessed along with changes in oxidant-antioxidant system, mitochondrial membrane-potential, and intracellular-Ca . Exposing SH-SY5Y cells to different concentrations of 6-OHDA for 48 h showed cell-death and apoptosis. Immunocytochemical analysis indicated an increase in pSyn-129 with increasing 6-OHDA concentration, and ELISA-estimation showed a significant increase in the pSyn-129 to α-synuclein ratio. FACS analysis also showed a significant increase in pSyn-129; and at sub-lethal 6-OHDA concentrations, pSyn-129+ cells were primarily distributed in the non-apoptotic population, suggesting that phosphorylation of α-synuclein precedes apoptosis. At higher 6-OHDA concentrations, the pSyn-129+ cell count significantly increased in the apoptotic population and decreased in the non-apoptotic population. Cytosolic co-localization of α-synuclein and ubiquitin was noticed at higher doses of 6-OHDA. FACS analysis showed decrease in vesicular monoamine transporter-2 (VMAT2) expression in 6-OHDA-treated cells, confirmed by reduction in functional dopamine-release on KCl and ATP stimulation. Significant decrease in VMAT2 expression and vesicular dopamine-release were observed with the lower 6-OHDA concentration, together with mild occurrence of apoptosis and significant increase in phosphorylated α-synuclein. This suggests that at sub-lethal 6-OHDA concentrations, the decrease in resting vesicles (VMAT2) and vesicular dopamine release are not attributable to apoptotic cell death and occur concomitantly with the phosphorylation of α-synuclein. J. Cell. Biochem. 117: 2719-2736, 2016. © 2016 Wiley Periodicals, Inc.
对家族性和散发性帕金森病患者纹状体组织的尸检研究表明，沉积在路易小体中的α-突触核蛋白近90%在丝氨酸129位点发生磷酸化（pSyn-129），而在正常人类大脑中这一比例仅为4%。我们旨在研究内源性神经毒素6-羟基多巴胺（6-OHDA）对α-突触核蛋白磷酸化、静息囊泡以及囊泡多巴胺释放的影响。评估了不同6-OHDA浓度下凋亡和非凋亡群体中pSyn-129+细胞的相对分布，以及氧化还原系统、线粒体膜电位和细胞内钙的变化。将SH-SY5Y细胞暴露于不同浓度的6-OHDA中48小时，结果显示细胞死亡和凋亡。免疫细胞化学分析表明，随着6-OHDA浓度的增加，pSyn-129增加，酶联免疫吸附测定显示pSyn-129与α-突触核蛋白的比例显著增加。流式细胞术分析也显示pSyn-129显著增加；在亚致死性6-OHDA浓度下，pSyn-129+细胞主要分布在非凋亡群体中，这表明α-突触核蛋白的磷酸化先于细胞凋亡。在较高的6-OHDA浓度下，凋亡群体中pSyn-129+细胞计数显著增加，而非凋亡群体中则减少。在较高剂量的6-OHDA下，观察到α-突触核蛋白和泛素在细胞质中共定位。流式细胞术分析显示，6-OHDA处理的细胞中囊泡单胺转运体2（VMAT2）表达降低，氯化钾和ATP刺激后功能性多巴胺释放减少证实了这一点。在较低的6-OHDA浓度下，观察到VMAT2表达和囊泡多巴胺释放显著降低，同时伴有轻度细胞凋亡和磷酸化α-突触核蛋白显著增加。这表明在亚致死性6-OHDA浓度下，静息囊泡（VMAT2）减少和囊泡多巴胺释放并非归因于凋亡性细胞死亡，而是与α-突触核蛋白的磷酸化同时发生。《细胞生物化学杂志》117: 2719 - 2736, 2016。© 2016威利期刊公司
