Glutaredoxin 1 Protects Dopaminergic Cells by Increased Protein Glutathionylation in Experimental Parkinson's Disease

Abstract

Aims: Chronic exposure to environmental toxicants, such as paraquat, has been suggested as a risk factor for Parkinson's disease (PD). Although dopaminergic cell death in PD is associated with oxidative damage, the molecular mechanisms involved remain elusive. Glutaredoxins (GRXs) utilize the reducing power of glutathione to modulate redox-dependent signaling pathways by protein glutathionylation. We aimed to determine the role of GRX1 and protein glutathionylation in dopaminergic cell death. Results: In dopaminergic cells, toxicity induced by paraquat or 6-hydroxydopamine (6-OHDA) was inhibited by GRX1 overexpression, while its knockdown sensitized cells to paraquat-induced cell death. Dopaminergic cell death was paralleled by protein deglutathionylation, and this was reversed by GRX1. Mass spectrometry analysis of immunoprecipitated glutathionylated proteins identified the actin binding flightless-1 homolog protein (FLI-I) and the RalBP1-associated Eps domain-containing protein 2 (REPS2/POB1) as targets of glutathionylation in dopaminergic cells. Paraquat induced the degradation of FLI-I and REPS2 proteins, which corresponded with the activation of caspase 3 and cell death progression. GRX1 overexpression reduced both the degradation and deglutathionylation of FLI-I and REPS2, while stable overexpression of REPS2 reduced paraquat toxicity. A decrease in glutathionylated proteins and REPS2 levels was also observed in the substantia nigra of mice treated with paraquat. Innovation: We have identified novel protein targets of glutathionylation in dopaminergic cells and demonstrated the protective role of GRX1-mediated protein glutathionylation against paraquat-induced toxicity. Conclusions: These results demonstrate a protective role for GRX1 and increased protein glutathionylation in dopaminergic cell death induced by paraquat, and identify a novel protective role for REPS2.

Alterations in Energy/Redox Metabolism Induced by Mitochondrial and Environmental Toxins: A Specific Role for Glucose-6-Phosphate-Dehydrogenase and the Pentose Phosphate Pathway in Paraquat Toxicity

“…Proteomic analysis was done as explained in ref (7). Heat maps of proteins with a significant up- or downregulation of at least 25% were created using GENE-E software ().…”

Overexpression of alpha-synuclein at non-toxic levels increases dopaminergic cell death induced by copper exposure via modulation of protein degradation pathways

“…Adenovirus containing only the CMV promoter (Ad-Empty) was used as negative control. Adenoviruses were amplified and titered in HEK293T cells as described previously (Barde et al, 2010; Rodriguez-Rocha et al, 2012). Cells were infected with adenoviral particles at indicated multiplicity of infection (MOI), washed and treated under the specified experimental conditions at 24 h post-infection.…”

“…Isolation of whole cell lysates, western-immunoblotting (WB) and SDS-PAGE of SK-N-SH were performed as explained previously (Rodriguez-Rocha et al, 2012). Blots were incubated with the corresponding primary antibodies overnight (1:1000): p-AMPK1α (Thr172, 2535), cleaved-caspase 3 (Asp 175, 9661), α-synuclein (2642, carboxy-terminal sequence), ubiquitin (3936), and p-Ulk1 (Ser555, 5869) (Cell Signaling); AMPK1α (Ab32047), ATP7A (Ab42486), Nrf2 (Ab62352), and Ulk1 (Ab65050) (Abcam); LC3B (L7543, SIGMA); and Ctr1 (sc66847, Santa Cruz Biotechnology).…”

Inhibition of Protein Ubiquitination by Paraquat and 1-Methyl-4-Phenylpyridinium Impairs Ubiquitin-Dependent Protein Degradation Pathways

“…Protein extraction and quantification were done as explained before [39]. For the detection of sulfenic acid modified proteins (PSOH) cells were incubated with the cell-permeable nucleophilic reagent dimedone prior to harvesting.…”

“…Adenovirus containing only the CMV promoter (Ad-Empty) was used as a negative control. Viruses were amplified and tittered in HEK293T cells as previously described [52,39]. Cells were infected with viral particles at the indicated multiplicity of infection (MOI), and 24 h post-infection, they were washed and treated under the specified experimental conditions.…”