Dopamine Cytotoxicity Involves Both Oxidative and Nonoxidative Pathways in SH-SY5Y Cells: Potential Role of Alpha-Synuclein Overexpression and Proteasomal Inhibition in the Etiopathogenesis of Parkinson's Disease

Abstract

Background. The cytotoxic effects of dopamine (DA) on several catecholaminergic cell lines involve DA oxidation products like reactive oxygen species (ROS) and toxic quinones and have implications in the pathogenesis of sporadic Parkinson's disease (PD). However, many molecular details are yet to be elucidated, and the possible nonoxidative mechanism of dopamine cytotoxicity has not been studied in great detail. Results. Cultured SH-SY5Y cells treated with DA (up to 400 μM) or lactacystin (5 μM) or DA (400 μM) plus N-acetylcysteine (NAC, 2.5 mM) for 24 h are processed accordingly to observe the cell viability, mitochondrial dysfunctions, oxidative stress parameters, proteasomal activity, expression of alpha-synuclein gene, and intracellular accumulation of the protein. DA causes mitochondrial dysfunction and extensive loss of cell viability partially inhibited by NAC, potent inhibition of proteasomal activity marginally prevented by NAC, and overexpression with accumulation of intracellular alpha-synuclein partially preventable by NAC. Under similar conditions of incubation, NAC completely prevents enhanced production of ROS and increased formation of quinoprotein adducts in DA-treated SH-SY5Y cells. Separately, proteasomal inhibitor lactacystin causes accumulation of alpha-synuclein as well as mitochondrial dysfunction and cell death. Conclusions. DA cytotoxicity includes both oxidative and nonoxidative modes and may involve overexpression and accumulation of alpha-synuclein as well as proteasomal inhibition.

Mild mitochondrial metabolic deficits by α-ketoglutarate dehydrogenase inhibition cause prominent changes in intracellular autophagic signaling: Potential role in the pathobiology of Alzheimer's disease

“…On reaching 70–80% confluence, the cells were trypsinized in 2 ml of 0.05% trypsin-EDTA for 2 min, followed by centrifugation at 200g for 10 min and suspension of the resulting cell-pellet in fresh medium. For subsequent maintenance, the cells were split and 0.5 x 10 6 viable cells, counted by trypan blue method (Banerjee et al, 2014), were seeded in freshly obtained sterile 100 mm tissue culture plates. The culture medium was changed every 2–3 days.…”

“…Typically, 100–200 cells were counted in each group with a Neubauer counting chamber by an observer blind to the experimental history. The cell death was expressed as the percentage of trypan blue positive cells in the total population of both stained and unstained cells as dead and viable, respectively (Banerjee et al, 2014). …”

A Comprehensive View of the Neurotoxicity Mechanisms of Cocaine and Ethanol

“…DA toxicity is believed to be due to the formation of reactive oxygen species (ROS) resulting from its metabolism (by autooxidation or MAO action), leading to oxidative stress (Banerjee et al 2014) and posteriorly to apoptosis. As referred above, MAO catalyses the conversion of DA to DOPAC and hydrogen peroxide (H 2 O 2 ), which, via Haber-Weiss/Fenton reaction, can react with transition metal ions, to give the highly toxic hydroxyl radical (ÁOH) (Caudle et al 2007;González-Hernández et al 2004).…”

“…As referred above, MAO catalyses the conversion of DA to DOPAC and hydrogen peroxide (H 2 O 2 ), which, via Haber-Weiss/Fenton reaction, can react with transition metal ions, to give the highly toxic hydroxyl radical (ÁOH) (Caudle et al 2007;González-Hernández et al 2004). On the other hand, DA auto-oxidation may occur in the extracellular medium, leading to the generation of superoxide (O 2 Á-) and toxic quinones (Banerjee et al 2014). Furthermore, O 2 Á-reacts with nitric oxide, a product of arginine oxidation by neuronal nitric oxide synthase (nNOS), forming the highly toxic peroxynitrite (ONOO -) (Cadet and Brannock 1998).…”

Effect of lysosomal and ubiquitin-proteasome system dysfunction on the abnormal aggregation of α-synuclein in PC12 cells

“…It has subsequently been found that, whether in familial or sporadic PD, the aggregation of abnormal α-synuclein forms LBs and that this abnormal α-synuclein aggregation in the neurons of the substantia nigra constitutes a pathological morphological marker for PD (14). In normal conditions, α-synuclein has an unordered and unfolded structure; however, under high concentrations or conditions conducive to abnormal modifications the protein undergoes a conformational change into fibers rich in β-sheets, which form oligomers and exhibit cytotoxicity (15,16). Since it is believed that α-synuclein aggregation could play an important role in the occurrence and development of PD, it is crucial to elucidate the mechanism underling the α-synuclein aggregation in cells in order to facilitate the understanding of PD pathogenesis.…”