Interaction of α-synuclein and Parkin in iron toxicity on SH-SY5Y cells: implications in the pathogenesis of Parkinson's disease.

The toxicity of accumulated α-synuclein plays a key role in the neurodegeneration of Parkinson's disease (PD). This study has demonstrated that iron in varying concentrations (up to 400 µM) causes an increase in α-synuclein content in SH-SY5Y cells associated with mitochondrial depolarization, decreased cellular ATP content and loss of cell viability during incubation up to 96 h. Knocking-down α-synuclein expression prevents cytotoxic actions of iron, which can also be prevented by cyclosporine A (a blocker of mitochondrial permeability transition pore). These results indicate that iron cytotoxicity is mediated by α-synuclein acting on mitochondria. Likewise siRNA mediated knock-down of Parkin causes an accumulation of α-synuclein accompanied by mitochondrial dysfunction and cell death during 48 h incubation under basal conditions, but these changes are not further aggravated by co-incubation with iron (400 µM). We have also analyzed mitochondrial dysfunction and cell viability in SH-SY5Y cells under double knock-down (α-synuclein and Parkin concurrently) conditions during incubation for 48 h with or without iron. Our results tend to suggest that iron inactivates Parkin in SH-SY5Y cells and thereby inhibits the proteasomal degradation of α-synuclein, and the accumulated α-synuclein causes mitochondrial dysfunction and cell death. These results have implications in the pathogenesis of sporadic PD and also familial type with Parkin mutations.

Dopamine Cytotoxicity on SH-SY5Y Cells: Involvement of α-Synuclein and Relevance in the Neurodegeneration of Sporadic Parkinson's Disease.

The cytotoxicity of dopamine on cultured cells of neural origin has been used as a tool to explore the mechanisms of dopaminergic neurodegeneration in Parkinson's disease. In the current study, we have shown that dopamine induces a dose-dependent (10-40 µM) and time-dependent (up to 96 h) loss of cell viability associated with mitochondrial dysfunction and increased intra-cellular accumulation of α-synuclein in cultured SH-SY5Y cells. Dopamine-induced mitochondrial dysfunction and the loss of cell viability under our experimental conditions could be prevented by cyclosporine, a blocker of mitochondrial permeability transition pore, as well as the antioxidant N-acetylcysteine. Interestingly, the dopamine effects on cell viability and mitochondrial functions were significantly prevented by knocking down α-synuclein expression by specific siRNA. Our results suggest that dopamine cytotoxicity is mediated by α-synuclein acting on the mitochondria and impairing its bioenergetic functions.

α-Synuclein-induced mitochondrial dysfunction in isolated preparation and intact cells: implications in the pathogenesis of Parkinson's disease.

This study has shown that purified recombinant human α-synuclein (20 µM) causes membrane depolarization and loss of phosphorylation capacity of isolated purified rat brain mitochondria by activating permeability transition pore complex. In intact SHSY5Y (human neuroblastoma cell line) cells, lactacystin (5 µM), a proteasomal inhibitor, causes an accumulation of α-synuclein with concomitant mitochondrial dysfunction and cell death. The effects of lactacystin on intact SHSY5Y cells are, however, prevented by knocking down α-synuclein expression by specific siRNA. Furthermore, in wild-type (non-transfected) SHSY5Y cells, the effects of lactacystin on mitochondrial function and cell viability are also prevented by cyclosporin A (1 µM) which blocks the activity of the mitochondrial permeability transition pore. Likewise, in wild-type SHSY5Y cells, typical mitochondrial poison like antimycin A (50 nM) produces loss of cell viability comparable to that of lactacystin (5 µM). These data, in combination with those from isolated brain mitochondria, strongly suggest that intracellularly accumulated α-synuclein can interact with mitochondria in intact SHSY5Y cells causing dysfunction of the organelle which drives the cell death under our experimental conditions. The results have clear implications in the pathogenesis of sporadic Parkinson's disease. α-Synuclein is shown to cause mitochondrial impairment through interaction with permeability transition pore complex in isolated preparations. Intracellular accumulation of α-synuclein in SHSY5Y cells following proteasomal inhibition leads to mitochondrial impairment and cell death which could be prevented by knocking down α-synuclein gene. The results link mitochondrial dysfunction and α-synuclein accumulation, two key pathogenic mechanisms of Parkinson's disease, in a common damage pathway.

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.

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.

Raised serum adenosine deaminase level in nonobese type 2 diabetes mellitus.

The role of inflammation being minimal in the pathogenesis of type 2 diabetes mellitus (T2DM) in nonobese patients; the aim of the study was to investigate the role of adenosine deaminase (ADA) and see its association with diabetes mellitus. The preliminary case control study comprised of 56 cases and 45 healthy controls which were age and sex matched. 3 mL venous blood samples were obtained from the patients as well as controls after 8-10 hours of fasting. Serum ADA and routine biochemical parameters were analyzed. Serum ADA level was found significantly higher among nonobese T2DM subjects with respect to controls (38.77 ± 14.29 versus 17.02 ± 5.74 U/L; P < 0.0001). Serum ADA level showed a significant positive correlation with fasting plasma glucose (r = 0.657; P < 0.0001) level among nonobese T2DM subjects, but no significant correlation was observed in controls (r = -0.203; P = 0.180). However, no correlation was observed between serum ADA level compared to BMI and HbA1c levels. Our study shows higher serum ADA, triglycerides (TG) and fasting plasma glucose (FPG) levels in nonobese T2DM patients, and a strong correlation between ADA and FPG which suggests an association between ADA and nonobese T2DM subjects.