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-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.

Modification of α-Synuclein by Phosphorylation: A Pivotal Event in the Cellular Pathogenesis of Parkinson’s Disease

“…Perfeito et al [118] showed through an in vitro model that exposure to ferrous iron and rotenone resulted in increase in pSyn. A similar increase was reported by Ganapathy et al [112] in the presence of the endogenous toxin 6-hydroxydopamine. Proteosomal inhibition by epoxomycin and increased oxidative stress by paraquat treatment has led to increases in pSyn [97].…”

“…In studies using PD rat models, the phospho-resistant S129A was found to be localized in the nucleus at higher levels than the S129D form, and was found to correlate with enhanced toxicity [110,111]. Our group too demonstrated the nuclear localization of pSyn in SH-SY5Y cells under 6-hydroxydopamine toxicity [112]. Gonçalves and Outeiro [113] showed that S129 phosphorylation modulates the shuttling of α-Syn between nucleus and cytoplasm in human neuroglioma cells, using photo-activatable green fluorescent protein as a reporter.…”

“…The work of Perfeito et al [118] suggest that stimuli that promote ROS formation and mitochondrial alterations highly correlate with mutant α-Syn phosphorylation at serine129, which may precede cell degeneration in PD. Similarly, we have shown in our recent work that at sub-lethal 6-hydroxydopamine 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-Syn [112]. The evidence of pSyn accumulation in the brain has been collected largely from post mortem analysis and it fails to answer if this accumulation occurs during the early or late stages of synucleinopathies.…”

“…Although the function of α-Syn in the nucleus is still unclear, it appears to be related to pathological insults. In particular, nuclear localization of α-Syn increases under oxidative stress conditions [112,114,115]. Nuclear α-Syn interacts with histones, inhibits acetylation, and promotes neurotoxicity [116,117].…”

Deferred Administration of Afobazole Induces Sigma1R-Dependent Restoration of Striatal Dopamine Content in a Mouse Model of Parkinson’s Disease

“…Nevertheless, there is evidence that α-synuclein is involved in the lesions caused by 6-OHDA [ 67 ]. 6-OHDA increased, in a concentration-dependent manner, the content of monomeric and oligomeric α-synuclein in SH-SY5Y cells in vitro and enhanced the phosphorylation of α-synuclein (pSyn-129) characteristic for PD [ 68 ], which is considered to be an LB-like feature [ 65 ]. These properties of the 6-OHDA model are consistent with the changes in the brains of Sigmar1 knockout mice [ 23 ].…”