Elsevier

Neuroscience

Volume 96, Issue 2, February 2000, Pages 309-316
Neuroscience

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

https://doi.org/10.1016/S0306-4522(99)00567-9Get rights and content

Abstract

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. 6 h, 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 1 mM glutamate for 1 h 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 μM) 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 24 h pretreatment with 10 μM 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.

Section snippets

Experimental procedures

Every effort was made to minimize animal suffering and to reduce the animals used for this study.

Energy impairment or glutamate exposure produces different alterations in intra- and extracellular glutathione

In order to demonstrate that oxidative stress is an early and potentially causal event in the sequelae of events associated with cell damage due to energy impairment or excitotoxicity rather than a downstream consequence, alterations indicative of oxidative stress should precede or occur concurrently with minimal exposure times necessary for toxicity. Our previous work showed that at least 6 h of exposure to 50 mM malonate was needed to cause dopamine cell loss, whereas longer times were required

Discussion

Defects in mitochondrial energy metabolism have been linked with Parkinson's disease.10., 33., 39., 45., 52. Whether reduced mitochondrial metabolism plays a primary, causative role in the disease is unknown. Regardless, reduced energy metabolism would likely impact on the surviving dopamine population during progression of the disease. Study of the mechanism(s) associated with dopaminergic cell loss due to impaired energy metabolism is, therefore, important in providing insight into their

Conclusions

Our findings indicate that the secondary excitotoxicity caused by impairment of energy metabolism with malonate does not contribute significantly to the oxidative stress that is observed. These findings may have relevance to the sequelae of cellular events that occur during neuronal loss in several neurodegenerative diseases in which mild impairments of mitochondrial metabolism have been described.

Acknowledgements

The authors wish to thank Hoechst Marion Roussel Research Institute (Cincinnati, OH) for their gift of MDL 102,832. This work was supported by U.S. Public Health Service grants NS 36157 and NS 17360.

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