title: Influence of 6-Hydroxydopamine Toxicity on α-Synuclein Phosphorylation, Resting Vesicle Expression, and Vesicular Dopamine Release.
abstract: 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-Ca2+. 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.
authors.affiliation: School of Regenerative Medicine, Manipal University, Bengaluru, Karnataka, India.
authors.forename: Kavina
authors.initials: K
authors.lastname: Ganapathy
authors.affiliation: Department of Biophysics, National Institute of Mental Health and Neurosciences, an Institute of National Importance, Bengaluru, Karnataka, India. indranidatta.nimhans@gmail.com.
authors.forename: Indrani
authors.initials: I
authors.lastname: Datta
authors.affiliation: Department of Biophysics, National Institute of Mental Health and Neurosciences, an Institute of National Importance, Bengaluru, Karnataka, India.
authors.forename: Sowmithra
authors.initials: S
authors.lastname: Sowmithra
authors.affiliation: Department of Biophysics, National Institute of Mental Health and Neurosciences, an Institute of National Importance, Bengaluru, Karnataka, India.
authors.forename: Preeti
authors.initials: P
authors.lastname: Joshi
authors.affiliation: School of Regenerative Medicine, Manipal University, Bengaluru, Karnataka, India.
authors.forename: Ramesh
authors.initials: R
authors.lastname: Bhonde
mesh_terms: D018663:Adrenergic Agents / Q000009:adverse effects; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D049109:Cell Proliferation; D004298:Dopamine / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D009154:Mutation / Q000235:genetics; D009447:Neuroblastoma / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D018384:Oxidative Stress / Q000187:drug effects; D016627:Oxidopamine / Q000009:adverse effects*; D010766:Phosphorylation / Q000187:drug effects; D013572:Synaptic Vesicles / Q000187:drug effects / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D051844:alpha-Synuclein / Q000235:genetics / Q000378:metabolism*
chemical_list: D018663:Adrenergic Agents; D051844:alpha-Synuclein; D016627:Oxidopamine; D004298:Dopamine
keywords: APOPTOSIS; IN VITRO PD MODEL; MITOCHONDRIAL MEMBRANE POTENTIAL; PHOSPHO SERINE129 α-SYNUCLEIN; SUB-LETHAL CONCENTRATION OF 6-OHDA; VESICULAR MONOAMINE TRANSPORTER