Characterization of intracellular elevation of glutathione (GSH) with glutathione monoethyl ester and GSH in brain and neuronal cultures: relevance to Parkinson's disease.

Parkinson's disease (PD) is associated with loss of total glutathione (GSH) which may contribute to progressive cell death. Peripheral GSH administration has been used clinically with reported benefits. Despite this, there is little specific information to characterize its cellular uptake or clearance, brain elevation with peripheral delivery or neuroprotective efficacy in PD models. The current study was carried out to provide this information using in vitro and in vivo approaches. In rat mesencephalic culture, the monoethyl ester of GSH (GEE), but not GSH (1-10 mM, 24 h) produced a dose-dependent elevation in GSH. The half-life for clearance was 10.14 h and was not different in cells depleted of GSH prior to loading. Elevation of GSH with GEE protected neurons from oxidative stress with H2O2 or metabolic stress with the complex I and II inhibitors MPP+ and malonate, respectively. To determine if peripheral administration of GEE could elevate brain GSH levels, rats were administered 0.1-50 mg/kg/day GEE via osmotic minipump either subcutaneously (sc) or via a cannula placed into the left cerebral ventricle (icv) for 28 days. Only central delivery of GEE resulted in significant elevations of brain GSH. Elevation of brain GSH by icv infusion of GEE was examined for its neuroprotective effects against chronic central delivery of MPP+. Infusion of 0.142 mg/kg/day MPP+ for 28 days caused a selective ipsilateral loss of striatal dopamine. Co-infusion of MPP+ with 10 mg/kg/day GEE significantly protected against striatal dopamine loss. These findings show that the ethyl ester of GSH but not GSH per se can elevate intracellular GSH, that brain elevation of GSH requires central delivery of the ethyl ester and that this elevation provides neuroprotection against oxidative stress or chronic mitochondrial impairment.

Mitochondrial inhibition and oxidative stress: reciprocating players in neurodegeneration.

Although the etiology for many neurodegenerative diseases is unknown, the common findings of mitochondrial defects and oxidative damage posit these events as contributing factors. The temporal conundrum of whether mitochondrial defects lead to enhanced reactive oxygen species generation, or conversely, if oxidative stress is the underlying cause of the mitochondrial defects remains enigmatic. This review focuses on evidence to show that either event can lead to the evolution of the other with subsequent neuronal cell loss. Glutathione is a major antioxidant system used by cells and mitochondria for protection and is altered in a number of neurodegenerative and neuropathological conditions. This review also addresses the multiple roles for glutathione during mitochondrial inhibition or oxidative stress. Protein aggregation and inclusions are hallmarks of a number of neurodegenerative diseases. Recent evidence that links protein aggregation to oxidative stress and mitochondrial dysfunction will also be examined. Lastly, current therapies that target mitochondrial dysfunction or oxidative stress are discussed.

Differential sensitivity of mesencephalic neurons to inhibition of phosphatase 2A.

Disturbance in phosphorylation/dephosphorylation can trigger apoptosis. Little is known as to its effects on mesencephalic dopamine neurons, the major neurons lost in Parkinson's disease. In this study, okadaic acid (OKA), a phosphatase 1 and 2A inhibitor, with greater potency toward 2A, was toxic to mesencephalic dopamine and gamma-aminobutyric acid (GABA) neurons, however, dopamine neurons were 4-fold more sensitive. The EC(50) for dopamine versus GABA toxicity was 1.5 versus 6.5 nM, respectively, and was consistent with an inhibition of phosphatase 2A. Dopamine neurons were also more sensitive to calyculin-A, a phosphatase inhibitor equipotent toward 1 and 2A. OKA-methyl-ester, which lacks phosphatase inhibitory activity, was without effect. DNA laddering typical of apoptosis was observed in cultures at a concentration that was specifically toxic to dopamine neurons (5 nM). In contrast to the sensitivity of mesencephalic neurons to phosphatase inhibition, inhibition of protein kinase activity with staurosporine or K252a showed little toxicity and protected neurons from OKA. Consistent with in vitro findings, infusion of 32 to 320 pmol of OKA into the left striatum of rats caused a dose-dependent loss of striatal dopamine without any loss of GABA 1 week following infusion. Acutely, OKA increased tyrosine hydroxylase activity, a phosphatase 2A substrate, and increased dopamine turnover. The above-mentioned findings demonstrate that dysregulation of phosphatase activity is detrimental to mesencephalic neurons, with dopamine neurons, in vitro and in vivo, being relatively more sensitive to phosphatase 2A inhibition. Disturbances in the phosphorylation control of proteins unique to dopamine neurons may contribute to their enhanced vulnerability to OKA exposure.

Attenuation of malonate toxicity in primary mesencephalic cultures using the GABA transport blocker, NO-711.

Cultured rat mesencephalic neurons were used to assess the effects of gamma-aminobutyric acid (GABA) transport blockers on toxicity caused by malonate, a reversible, competitive inhibitor of succinate dehydrogenase. Previous studies utilizing an ex vivo chick retinal preparation have shown that GABA release and cell swelling are early consequences of acute energy impairment and that GABA transport blockers attenuate this toxicity. The present results demonstrate that the nonsubstrate GABA transport blocker, NO-711 (1 nM-1 microM), dose-dependently protected cultured mesencephalic dopamine (DA) and GABA neurons from malonate-induced toxicity. Similar protection was demonstrated with nipecotic acid (1 mM) and SKF89976A (100 nM), substrate and nonsubstrate GABA transport blockers, respectively. These compounds by themselves produced no signs of toxicity, although nipecotic acid caused a long-term decrease in GABA uptake not associated with toxicity. Compounds which decrease intracellular reactive oxygen species (ROS) are protective in this model, but NO-711 did not prevent the rise in intracellular ROS induced by malonate, indicating its protective effects were downstream of ROS production. Supplementation of malonate treated cultures with the GABA(A) agonist, muscimol (10 microM), increased the toxicity toward the DA and GABA neuron populations. Antagonists at the GABA(A) and glycine receptors provided partial protection to both the GABA and DA neurons. These findings suggest that the GABA transporter, GABA(A), and/or glycine channels contribute to cell damage associated with energy impairment in this model.

Oxidative stress during energy impairment in mesencephalic cultures is not a downstream consequence of a secondary excitotoxicity.

Past studies have shown that inhibiting energy metabolism with malonate in mesencephalic cultures damages neurons by mechanisms involving N-methyl-D-aspartate receptors and free radicals. Overstimulation of N-methyl-D-aspartate receptors is known to produce free radicals. This study was, therefore, carried out to determine if N-methyl-D-aspartate receptor activation triggered by energy impairment was a significant contributor to the oxidative stress generated during energy inhibition. Exposure of mesencephalic cultures to malonate for the minimal time required to produce toxicity, i.e. 6h, resulted in an increase in the efflux of both oxidized and reduced glutathione, and a decrease in tissue levels of reduced glutathione. In contrast, exposure to 1mM glutamate for 1h caused an increased efflux of reduced glutathione, but no changes in intra- or extracellular oxidized glutathione or intracellular reduced glutathione. Blocking N-methyl-D-aspartate receptors with MK-801 (0.5 microM) during malonate exposure did not modify malonate-induced alterations in glutathione status or free radical generation as monitored by dihydrochlorofluorescein diacetate and dihydrorhodamine 123 fluorescence. In contrast, the increase in dihydrorhodamine fluorescence caused by glutamate was completely blocked by MK-801. Reduction of tissue glutathione with a 24h pretreatment with 10 microM buthionine sulfoxamine, as shown previously, greatly potentiated malonate-induced toxicity to dopamine and GABA neurons, but had no potentiating effect on toxicity due to glutamate. The findings indicate that although oxidative stress mediates damage due either to energy deprivation or excitotoxicity, N-methyl-D-aspartate receptor over-stimulation does not contribute significantly to the oxidative stress that is incurred during malonate exposure.

Excitotoxicity and oxidative stress during inhibition of energy metabolism.

Glutamate receptor involvement and oxidative stress have both been implicated in damage to neurons due to impairment of energy metabolism. Using two different neuronal in vitro model systems, an ex vivo chick retinal preparation and dopamine neurons in mesencephalic culture, the involvement and interaction of these events as early occurring contributors to irreversible neuronal damage have been examined. Consistent with previous reports, the early acute changes in the retinal preparation, as well as irreversible loss of dopamine neurons due to inhibition of metabolism, can be prevented by blocking NMDA receptors during the time of energy inhibition. Oxidative stress was suggested to be a downstream consequence and contributor to neuronal cell loss due to either glutamate receptor overstimulation or metabolic inhibition since trapping of free radicals with the cyclic nitrone spin-trapping agent MDL 102,832 (1 mM) attenuated acute excitotoxicity in the retinal preparation or loss of mesencephalic dopamine neurons due to either metabolic inhibition by the succinate dehydrogenase inhibitor, malonate, or exposure to excitotoxins. In mesencephalic culture, malonate caused an enhanced efflux of both oxidized and reduced glutathione into the medium, a significant reduction in total reduced glutathione and a significant increase in total oxidized glutathione at time points that preceded those necessary to cause toxicity. These findings provide direct evidence for early oxidative events occurring following malonate exposure and suggest that the glutathione system is important for protecting neurons during inhibition of energy metabolism. Consistent with this, lowering of glutathione by buthionine sulfoxamine (BSO) pretreatment greatly potentiated malonate toxicity in the mesencephalic dopamine population. In contrast, BSO pretreatment did not potentiate glutamate toxicity. This latter finding indicates dissimilarities in the type of oxidative stress that is generated by the two insults and suggests that the oxidative challenge during energy inhibition is not solely a downstream consequence of glutamate receptor overstimulation.

Role of oxidative stress and the glutathione system in loss of dopamine neurons due to impairment of energy metabolism.

Alterations in the glutathione system and impairment in energy metabolism have both been implicated in the loss of dopamine neurons in Parkinson's disease. This study examined the importance of cellular glutathione and the involvement of oxidative stress in the loss of mesencephalic dopamine and GABA neurons due to inhibition of energy metabolism with malonate, the reversible, competitive inhibitor of succinate dehydrogenase. Consistent with previous findings, exposure to malonate for 24 h followed by 48 h of recovery caused a dose-dependent loss of the dopamine population with little effect on the GABA population. Toxicity was assessed by simultaneous measurement of the high-affinity uptake of [3H]dopamine and [14C]GABA. Total glutathione content in rat mesencephalic cultures was decreased by 65% with a 24-h pretreatment with 10 microM buthionine sulfoxamine. This reduction in glutathione level greatly potentiated damage to both the dopamine and GABA populations and removed the differential susceptibility between the two populations in response to malonate. These findings point to a role for oxidative stress occurring during energy impairment by malonate. Consistent with this, several spin-trapping agents, alpha-phenyl-tert-butyl nitrone and two cyclic nitrones, MDL 101,002 and MDL 102,832, completely prevented malonate-induced damage to the dopamine neurons in the absence of buthionine sulfoxamine. The spin-trapping agents also completely prevented toxicity to both the dopamine and GABA populations when cultures were exposed to malonate after pretreatment with buthionine sulfoxamine to reduce glutathione levels. Counts of tyrosine hydroxylase-positive neurons verified enhancement of cell loss by buthionine sulfoxamine plus malonate and protection against cell loss by the spin-trapping agents. NMDA receptors have also been shown to play a role in malonate-induced dopamine cell loss and are associated with the generation of free radicals. Consistent with this, toxicity to the dopamine neurons due to a 1-h exposure to 50 microM glutamate was attenuated by the nitrone spin traps. These findings provide evidence for an oxidative challenge occurring during inhibition of energy metabolism by malonate and show that glutathione is an important neuroprotectant for midbrain neurons during situations when energy metabolism is impaired.

Energy stress-induced dopamine loss in glutathione peroxidase-overexpressing transgenic mice and in glutathione-depleted mesencephalic cultures.

The role of the glutathione system in protecting dopamine neurons from a mild impairment of energy metabolism imposed by the competitive succinate dehydrogenase inhibitor, malonate, was investigated in vitro and in vivo. Treatment of mesencephalic cultures with 10 microM buthionine sulfoxamine for 24 h reduced total glutathione levels in the cultures by 68%. Reduction of cellular glutathione per se was not toxic to the dopamine population, but potentiated toxicity when the cultures were exposed to malonate. In contrast, transgenic mice overexpressing glutathione peroxidase (hGPE) that received an intrastriatal infusion of malonate (3 mumol) into the left side had significantly less loss of striatal dopamine than their hGPE-negative littermates when assayed 1 week following infusion. These studies demonstrate that manipulation of the glutathione system influences susceptibility of dopamine neurons to damage due to energy impairment. The findings may provide insight into the loss of dopamine neurons in Parkinson's disease in which defects in both energy metabolism and the glutathione system have been identified.