Evaluating the Effect of Iron (II) Chloride Induced Oxidative Stress in SH-SY5Y Cells and Its Role in Upregulation of α-Synuclein Expression

Abstract

The α-synuclein (SNCA) gene is a pathogenic gene identified in rare familial Parkinson Disease (PD). Recent studies highlight the role of DNA methylation in the pathogenesis of familial and sporadic PD. Hypomethylation in SNCA gene has been associated with increased SNCA gene expression and was observed in post mortem brains of patients with sporadic PD. This study was aimed at evaluating the effect of iron (II) chloride on SH-SY5Y cell models as pertain to cell death caused by oxidative stress, upregulation of SNCA gene expression and reduced SNCA gene methylation. Result obtained from LDH assay showed significant (p < 0.05) evidence of cell death in treated cells as compared to the control sample. Analysis for SNCA gene quantification using RT-PCR showed significant increases in fold change. Cells treated with 1000µM of FeCl₂ showed the highest fold change of 6.0 while cells treated with 250µM had the lowest fold change of 1.8. In DNA methylation assay using pyrosequencing, cells treated with varying concentrations of FeCl₂ showed significant (p < 0.05) decrease in DNA methylation. At 250µM, 500µM and 750µM concentrations of FeCl₂, an average mean methylation levels of 1.84%, 1.40% and 1.23% was obtained respectively while cells treated with 1000 µM had the lowest average mean methylation level of 1.0%. Thus, the decrease in methylation is linked to the upregulation of the SNCA gene which has been reported to be among the causative factors in the pathogenesis of Parkinson’s disease.