Proteomic insights into the protective mechanisms of an in vitro oxidative stress model of early stage Parkinson's disease

Abstract

Previous studies in Parkinson's disease (PD) models suggest that early events along the path to neurodegeneration involve activation of the ubiquitin-proteasome system (UPS), endoplasmic reticulum-associated degradation (ERAD), and the unfolded protein response (UPR) pathways, in both the sporadic and familial forms of the disease, and thus ER stress may be a common feature. Furthermore, impairments in protein degradation have been linked to oxidative stress as well as pathways associated with ER stress. We hypothesize that oxidative stress is a primary initiator in a multi-factorial cascade driving dopaminergic (DA) neurons towards death in the early stages of the disease. We now report results from proteomic analysis of a rotenone-induced oxidative stress model of PD in the human neuroblastoma cell line, SH-SY5Y. Cells were exposed to sub-micromolar concentrations of rotenone for 48h prior to whole cell protein extraction and shotgun proteomic analysis. Evidence for activation of the UPR comes from our observation of up-regulated binding immunoglobulin protein (BiP), heat shock proteins, and foldases. We also observed up-regulation of proteins that contribute to the degradation of misfolded or unfolded proteins controlled by the UPS and ERAD pathways. Activation of the UPR may allow neurons to maintain protein homeostasis in the cytosol and ER despite an increase in reactive oxygen species due to oxidative stress, and activation of the UPS and ERAD may further augment clean-up and quality control in the cell.

Fig. 1. MTT viability assay

MTT cell viability assay performed on SH-SY5Y cultures pre-incubated with various concentrations of rotenone for 48 hours. Gray bars indicate cells extracted for proteomic analysis (Table 1). Data (mean ± SEM) are expressed as % of control (no rotenone). Asterisk (*) indicates p<0.0001 by ANOVA: 100nM rotenone-treated compared to 50nM; double asterisk (**) indicates p<0.0006 by ANOVA: 250nM-1µM rotenone-treated compared to 5nM–50nM group.

Fig. 2. ssDNA apoptotic assay (ELISA)

SH-SY5Y cultures pre-incubated with various concentrations of rotenone for 48 hours. Data (mean ± SEM) expressed as % of control (no rotenone).

Fig. 3. Caspase-3 activity assay

Caspase-3 activity measured on SH-SY5Y cells pre-incubated with various concentrations of rotenone for 48 hours. Data expressed as relative fluorescence units (RFU). Asterisk (*) indicates p=0.043 by ANOVA: 50nM rotenone-treated compared to 5nM–20nM; double asterisk (**) indicates p=0.014 by ANOVA: 100nM rotenone-treated compared to 5nM–50nM rotenone group.

Fig. 4. Proteasome activity Assay

Fluorogenic substrate measured chymotrypsin-like activity in 20S proteasomes from SH-SY5Y cells exposed to various concentrations of rotenone for 48 hours. Data expressed as relative fluorescence units (RFU). Asterisk (*) indicates p=0.000012 by ANOVA: 20nM–100nM rotenone-treated group compared to control-10nM group.