Depletion of cytosolic GSH decreases the ATP levels and viability of synaptosomes from aged mice but not from young mice

doi:10.1016/0047-6374(95)01644-F

Mechanisms of Ageing and Development

Volume 84, Issue 1, 29 September 1995, Pages 77-81

https://doi.org/10.1016/0047-6374(95)01644-F Get rights and content

Abstract

The effect of glutathione depletion on the viability of freshly isolated synaptosomes from whole brain was investigated in young and aged mice. Aging did not influence the GSH and ATP levels and the viability of these synaptosomes. However depletion of glutathione caused by the cytosolic glutathione inhibitor diethyl maleate (1 mM) resulted in a significant decline, after 60 min of incubation, in ATP levels and viability in the synaptosomes from aged mice but not in those from young mice. When synaptosomes were incubated in the presence of the mitochondrial glutathione inhibitor ethacrynic acid (0.2 mM) there was a similar decline in glutathione, ATP levels and synaptosomal viability, both in young and aged mice. These results emphasize the relative importance of the cytosolic glutathione pool for the maintenance of the plasma membrane integrity in synaptosomes from aged mice.

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As a consequence, compounds that can be metabolized to cysteine could be used as pro-drugs to increase neuronal GSH levels. NAC is the simplest cysteine pro-drug that can be systemically administered to deliver cysteine to the brain [8,34–37], acting as a precursor for glutathione synthesis as well as a stimulator of the cytosolic enzymes involved in glutathione regeneration. Increase in Complex I activities in vivo and in vitro in mitochondria isolated from pre-synaptic terminals of aged mice was proposed 10 years ago as evidence that NAC was able to cross the blood brain barrier having reparative effects on brain mitochondria and against age-associated memory decline [9,34,35].
Parkinson’s disease is an age-related neurodegenerative disorder that is ameliorated with levodopa. However, long-term use of this drug is limited by motor complications, postural instability and dementia resulting in the progression of the disease. Insights into the organization of the basal ganglia and knowledge of the mechanisms responsible for cell death in Parkinson’s disease has permitted the development of putative neuro-protective drugs that might slow the disease progression. Although no drug has yet been established to alter the rate of disease progression, recent publications have confirmed previous results and hypotheses about the probable role of thiolic antioxidants on Parkinson’s disease, demonstrating a significant reduction of dopaminergic neuronal degeneration in α-synuclein over expressing mice treated with oral N-acetyl-cysteine. This thiolic antioxidant is a modified form of the natural amino acid cysteine, which is the precursor of the most potent intracellular antioxidant glutathione. Besides, increasing evidence has been accumulated in the last 10 years about the beneficial effects of this thiolic antioxidant in experimental and pathologic states of the nervous system, including against neurotoxic substances. The present paper put forward the existing rationale evidence for the use of N-acetyl-cysteine alone or in combination with levodopa in the clinical management of this neurodegenerative disorder.
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The relative variations in levels of glutathione in different brain regions are cortex > cerebellum > hippocampus > striatum > substantia nigra [45–47]; however, glutathione profiles change throughout the life span, from high values during growth, dropping to a maturation plateau, and then decreasing 30% during aging [45]. After preparation of synaptosomes from mouse brain, depletion of glutathione in vitro decreased ATP levels and viability of synaptosomes from aged mice but not from young mice [48]. Nevertheless, after in vivo depletion of glutathione and a 24-h postmortem delay in which glutathione levels were decreased by 79%, approximately twofold more than in Parkinson disease, there was no effect on complex I activity or complex II–III and IV activity.
Glutathione is an important antioxidant in the brain that appears to be decreased, in conjunction with mitochondrial complex I activity, in Parkinson disease patients. In postmortem analysis, measurement of glutathione levels and complex I activity can be delayed up to 20 h. We investigated whether depletion of glutathione in the preweanling rat induces a reduction in complex I activity in brain mitochondria and the effects that postmortem delay has on glutathione levels and electron transport chain activity. After injection with the glutamate-cysteine ligase inhibitor, buthionine sulfoximine (L-BSO), glutathione levels were decreased by 53% compared to the control values in whole-brain homogenates. During postmortem delay of 24 h, in which animals were kept at 4 °C, the levels of glutathione decreased in the control group by 58% and in the L-BSO-treated group by 79%. However, during this period, there were no changes in mitochondrial electron transport chain complex I, II–III, or IV activity in either group. These results suggest that a preexisting deficiency of glutathione or a loss of glutathione during postmortem delay does not influence mitochondrial respiratory chain activity in the brain.
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Decreased GSH availability in the brain is believed to promote mitochondrial damage most likely via increases in levels of oxidative stress to this organelle. Depletion of brain GSH has been shown to result in decreases in mitochondrial enzyme activities in preweaning rats as well as losses in ATP production in the aging murine brain [65,66]. Previous studies have shown that complex I in bovine heart submitochondrial particles is particularly affected by oxidative stress [67,68].
Parkinson’s disease (PD) is a progressive neurodegenerative disease involving neurodegeneration of dopaminergic neurons of the substantia nigra (SN), a part of the midbrain. Oxidative stress has been implicated to play a major role in the neuronal cell death associated with PD. Importantly, there is a drastic depletion in cytoplasmic levels of the thiol tripeptide glutathione within the SN of PD patients. Glutathione (GSH) exhibits several functions in the brain chiefly acting as an antioxidant and a redox regulator. GSH depletion has been shown to affect mitochondrial function probably via selective inhibition of mitochondrial complex I activity. An important biochemical feature of neurodegeneration during PD is the presence of abnormal protein aggregates present as intracytoplasmic inclusions called Lewy bodies. Oxidative damage via GSH depletion might also accelerate the build-up of defective proteins leading to cell death of SN dopaminergic neurons by impairing the ubiquitin–proteasome pathway of protein degradation. Replenishment of normal glutathione levels within the brain may hold an important key to therapeutics for PD. Several reports have suggested that iron accumulation in the SN patients might also contribute to oxidative stress during PD.
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In the present study the effect of ascorbate (0.8 mM)/iron (2.5 μM) on lipid and protein oxidation, in Synaptosomes isolated from rat brain cortex, was evaluated. Vitamin E, idebenone and reduced glutathione were used as free radicals scavengers, in order to analyze the mechanism involved in ascorbate/iron-induced oxidative stress. An increased formation of reactive oxygen species (ROS) in the cytosol and in the mitochondria was observed, in ascorbate/iron treated synaptosomes. Idebenone (50 μM) prevented the increased formation of ROS in both synaptosomal compartments, vitamin E (150 μM) protected partially this formation in mitochondria, whereas reduced glutathione (250 μM) (GSH) was ineffective. After ascorbate/iron treatment an increase in lipid peroxidation occurred as compared to control, which was completely inhibited by idebenone. A decrease in protein-SH content was also observed, and it was prevented by Vitamin E, idebenone and GSH. When synaptosomes were treated with ascorbate/iron the levels of GSH decreased, and the levels of oxidized glutathione (GSSG) increased as compared to controls under these conditions. Glutathione peroxidase activity was unchanged, whereas an inhibition of glutathione reductase activity was observed. These data suggest that the increased formation of free radicals in synaptosomes leads to lipid and protein oxidation, the role of the endogenous GSH being essential to protect protein thiol- groups against oxidative damage in order to maintain enzyme activity.
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Depletion of cytosolic GSH decreases the ATP levels and viability of synaptosomes from aged mice but not from young mice

Abstract

Brain Res.

Exp. Gerontol.

Eur. J. Pharmacol.

Arch. Biochem. Biophys.

J. Chromatogr.

J. Biol. Chem.

Arch. Biochem. Biophys.

Blebbing, free Ca2+ and mitochondrial membrane potential preceding cell death in hepatocytes

Nature

Blebbing, free Ca²⁺ and mitochondrial membrane potential preceding cell death in hepatocytes