Levodopa Deactivates Enzymes That Regulate Thiol−Disulfide Homeostasis and Promotes Neuronal Cell Death: Implications for Therapy of Parkinson’s Disease

Abstract

Parkinson’s disease (PD), characterized by dopaminergic neuronal loss, is attributed to oxidative stress, diminished glutathione (GSH), mitochondrial dysfunction, and protein aggregation. Treatment of PD involves chronic administration of Levodopa (L-DOPA) which is a pro-oxidant and may disrupt sulfhydryl homeostasis. The goal of current studies is to elucidate the effects of L-DOPA on thiol homeostasis in a model akin to PD, i.e., immortalized dopaminergic neurons (SHSY5Y cells) with diminished GSH content. These neurons exhibit hypersensitivity to L-DOPA-induced cell death, which is attributable to concomitant inhibition of the intracellular thiol disulfide oxidoreductase enzymes. Glutaredoxin (Grx) was deactivated in a dose-dependent fashion; but its content was unaffected. Glutathione disulfide (GSSG) reductase (GR) activity was not altered. Selective knockdown of Grx resulted in increased apoptosis, documenting the role of the Grx system in neuronal survival. L-DOPA treatments also led to decreased activities of thioredoxin (Trx) and thioredoxin reductase (TR), concomitant with diminution of their cellular contents. Selective chemical inhibition of TR activity led to increased apoptosis, documenting the Trx system’s contribution to neuronal viability. To investigate the mechanism of inhibition at the molecular level the isolated enzymes were each treated with oxidized L-DOPA. GR, Trx, and TR activities were little affected. However, Grx was inactivated in a time- and concentration-dependent fashion indicative of irreversible adduction of dopaquinone to its nucleophilic active site Cys-22, consistent with intracellular loss of Grx activity but not Grx protein content after L-DOPA treatment. Overall L-DOPA is shown to impair the collaborative contributions of the Grx and Trx systems to neuron survival.

Mechanisms of Altered Redox Regulation in Neurodegenerative Diseases—Focus on S-Glutathionylation

“…Treatment of PD to replenish dopamine and alleviate symptoms involves chronic administration of Levodopa (L-DOPA) which itself is a pro-oxidant. Therefore, we examined the effects of L-DOPA in a model akin to PD, [i.e., immortalized dopaminergic neurons (SHSY5Y cells) with diminished GSH content (154)]. These neurons exhibited hypersensitivity to L-DOPA-induced cell death, attributable to concomitant loss of activity of the intracellular thiol disulfide oxidoreductases.…”

“…Concomitantly, ASK 1 is activated wherein autophosphorylated (activated) ASK1 initiates a phosphorylation cascade of downstream mediators, which induces apoptosis (shown at the right). L-DOPA treatment has been shown to cause loss of Grx1 and Trx1 activities (154), which would impede reduction of other oxidized negative regulators of ASK1, resulting in prolonged ASK1 activation. Details of this model, including the potential involvement of Daxx and DJ-1, are presented in the text.…”

The Roles of Redox Enzymes in Parkinson’s Disease: Focus on Glutaredoxin

“…To test this concept, Grx1 was knocked down in SH-SY5Y cells. The cells treated with the Grx1-siRNA showed an increased level of apoptosis compared to control cells with non-targeting siRNA [42] . This finding was soon confirmed as knockdown of Grx1 in Neuro-2a cells via shRNA also resulted in cell death [43] .…”

“…Treatment of SH-SY5Y cells in culture with the pro-oxidant drug L-DOPA was shown to increase apoptosis. Investigation into the molecular mechanism of this drug-induced cell death revealed that Grx1 was selectively inactivated relative to other redox enzymes by oxidized L-DOPA, which covalently adducted the Grx1 active site (Cys-22) [42] . These findings led to the hypothesis that Grx1 plays a critical role in maintaining neuronal cell viability.…”

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

“…Downregulation of GRX1 itself has been shown to induce a decrease in complex I activity, mitochondrial membrane potential, DJ-1 loss (a putative gene recessively linked to early onset of PD), translocation of DAXX (a deathassociated protein), and cell death (46,68,69). Mieyal's research group recently reported that levodopa (L-DOPA), a dopamine precursor used in the clinical treatment for PD, directly inactivates GRX1 and induces dopaminergic cell death by activation of the apoptosis signaling kinase 1 (ASK-1) (66,67). On the other hand, downregulation of mitochondrial GRX2 has been shown to disrupt iron-sulfur center biogenesis and complex I activity in dopaminergic cells, while its overexpression protects against MPTP-induced toxicity (45,48).…”