Reversible inhibition of mitochondrial complex I activity following chronic dopaminergic glutathione depletion in vitro: Implications for Parkinson's disease

doi:10.1016/j.freeradbiomed.2006.08.002

Free Radical Biology and Medicine

Volume 41, Issue 9, 1 November 2006, Pages 1442-1448

https://doi.org/10.1016/j.freeradbiomed.2006.08.002 Get rights and content

Abstract

The pathogenesis underlying the selective degeneration of nigral dopaminergic neurons in Parkinson's disease is not fully understood but several lines of evidence implicate the role of oxidative stress and mitochondrial dysfunction. Depletion in levels of the thiol reducing agent glutathione (GSH + GSSG) is the earliest reported biochemical event to occur in the Parkinsonian substantia nigra prior to selective loss of complex I (CI) activity associated with the disease believed to contribute to subsequent dopaminergic cell death. Recent studies from our laboratory have demonstrated that acute reduction in both cellular and mitochondrial glutathione levels results in increased oxidative stress and a decrease in mitochondrial function linked to a selective decrease in CI activity through an NO-mediated mechanism ( [8] Jha, N.; Jurma, O.; Lalli, G.; Liu, Y.; Pettus, E. H.; Greenamyre, J. T.; Liu, R. M.; Forman, H. J.; Andersen, J. K. Glutathione depletion in PC12 results in selective inhibition of mitochondrial complex I activity. Implications for Parkinson's disease J. Biol. Chem. 275: 26096-26101; 2000. [9] Hsu, M.; Srinivas, B.; Kumar, J.; Subramanian, R.; Andersen, J. Glutathione depletion resulting in selective mitochondrial complex I inhibition in dopaminergic cells is via an NO-mediated pathway not involving peroxynitrite: implications for Parkinson's disease J. Neurochem. 92: 1091-1103.2005.). However, the effect of prolonged glutathione depletion on dopaminergic cells is not known. In this present study, using low concentrations of buthionine-S-sulfoximine, a chemical inhibitor of the de novo glutathione synthesizing enzyme glutamate cysteine ligase, we developed a chronic model in which glutathione depletion in dopaminergic N27 cells for a 7-day period was found to lead to inhibition of CI activity via a peroxynitrite-mediated event which is reversible by the thiol reducing agent, dithiothreitol, and coincides with increased S-nitrosation of mitochondrial proteins.

Introduction

Reduced glutathione (GSH) is a tripeptide consisting of glutamate, cysteine, and glycine. It is the most abundant nonprotein thiol within cells. GSH is synthesized by a two-step reaction involving the enzymes gamma-glutamylcysteine ligase (γ-GCL) and glutathione synthetase (GS). γ-GCL is the rate-limiting enzyme in this process [1]. GSH is present in high concentrations in both the mitochondrial and the cytosolic compartments of the cell. As mitochondria lack the enzymes for GSH synthesis, the mitochondrial GSH concentration is maintained via uptake from the cytosol [2]. GSH plays an important role in scavenging reactive oxygen and nitrogen species (ROS, RNS) and in recycling other antioxidants [3].

It has been observed that the substantia nigra (SN) of early PD patients displays a significant depletion of total glutathione levels [reduced glutathione (GSH) + oxidized glutathione (GSSG)] [4]. Glutathione depletion is the earliest reported biochemical change in the PD SN [5]. Decreases in glutathione availability in the brain promotes morphological mitochondrial damage most likely via increases in levels of oxidative or nitrosative stress in this organelle [6]. Although glutathione is not the only antioxidant molecule reported to be altered in PD, the magnitude of glutathione depletion appears to parallel the severity of the disease and is the earliest known indicator of nigral degeneration, reportedly preceding detectable losses in both mitochondrial complex I activity and striatal (ST) dopamine content [7]. Glutathione levels are not decreased in other areas of the PD brain nor in other diseases affecting dopaminergic SN neurons such as multiple system atrophy or progressive supranuclear palsy [4].

We previously demonstrated that acute glutathione depletion within both the cytosol and the mitochondria of cultured dopaminergic cells elicits a selective mitochondrial complex I inhibition resulting in mitochondrial dysfunction [8]. Complex I inhibition following acute glutathione depletion appeared to be via an NO-mediated event [9]. These results suggested that the early glutathione depletions observed in the Parkinsonian SN could be responsible for subsequent complex I inhibition, mitochondrial dysfunction, and neuronal cell loss. Earlier in vitro studies also have demonstrated that long-term exposure of mitochondrial membranes to nitric oxide (NO) resulted in selective and persistent inhibition of complex I activity via S-nitrosation of critical thiols in the enzyme complex. Furthermore, inhibition of complex I activity was reversible by addition of reduced glutathione, suggesting that GSH might protect complex I activity by preventing the S-nitrosation of critical thiol groups in the enzyme [10], [11]. Previous studies also demonstrated the importance of cellular antioxidant status, in particular the availability of reduced glutathione (GSH), in determining vulnerability of neurons to NO or peroxynitrite (PN) [12]. In this current study, we assess the effects of more prolonged glutathione depletion on mitochondrial function in dopaminergic cells to better emulate what occurs in PD. We report that following more prolonged glutathione depletion in vitro, CI inhibition is still reversible but appears to be a PN-mediated event which coincides with increased mitochondrial protein S-nitrosation.

Section snippets

Cell culture

N27 cells were grown in RPMI medium 1640 containing 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 μg/ml). The cells were differentiated with 2.0 mM dibutyryl adenosine 3′,5′-cyclic monophosphate (dbcAMP) and 60 μg/ml dehydroepiandrosterone (DHEA) for 2 days. Differentiating agents were added 24 h after plating [13]. Fresh media containing experimental compounds were added every 2–3 days. Cellular glutathione levels were chronically lowered via treatment of N27

Analysis of neuronal cell survival following chronic BSO treatment

To study the effects of chronic GSH depletion on dopaminergic neuronal survival, we first differentiated N27 cells using dbcAMP for 48 h [13]. After differentiation, the susceptibility of dopaminergic N27 cells to glutathione depletion was evaluated by performing MTT assays following 7 days of BSO (L-buthionine sulfoximine, an inhibitor of γ-glutamylcysteine ligase, GCL) exposure at concentrations ranging from 2.5 to 50 μM (Fig. 1). No significant cell death was observed up to a concentration

Discussion

Chronic dopaminergic glutathione depletion in vitro could result in similar effects on CI as those observed following acute glutathione depletion or it may result in alternative oxidative events such as irreversible protein nitration at tyrosine residues (3-nitrotyrosine (3-NT) adduct). To test this, we assessed the effects of more prolonged glutathione levels (7 days) on cell viability, glutathione levels, NO species, and CI activity. Interestingly, 7 day glutathione depletion assessed at a

Acknowledgments

This work was supported by NIH R01s AG12141 and NS045615 to J.K.A.

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Original ContributionReversible inhibition of mitochondrial complex I activity following chronic dopaminergic glutathione depletion in vitro: Implications for Parkinson's disease

Abstract

Introduction

Section snippets

Cell culture

Analysis of neuronal cell survival following chronic BSO treatment

Discussion

Acknowledgments

Methods Enzymol.

J. Biol. Chem.

Biochem. Pharmacol.

Neurosci. Lett.

Neurosci. Lett.

J. Biol. Chem.

Free Radic. Biol. Med.

Anal. Biochem.

Methods Enzymol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Original Contribution
Reversible inhibition of mitochondrial complex I activity following chronic dopaminergic glutathione depletion in vitro: Implications for Parkinson's disease