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Impaired glutamate clearance as a consequence of energy failure caused by MPP(+) in astrocytic cultures
D A Di Monte1, I Tokar, J W Langston
1The Parkinson's Institute, Sunnyvale, California, 94089, USA.
Abstract:
Astrocytes are the site of bioactivation of the parkinsonism-inducing agent 1-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine (MPTP) into its toxic 1-methyl-4-phenylpyridinium (MPP(+)) metabolite. The mechanism by which MPP(+) is capable of decreasing astrocytic glutamate uptake was evaluated in this study using primary cultures of astrocytes. Addition of glutamate to these cultures was followed by its efficient clearance from the extracellular space. However, when astrocytes were preincubated with MPP(+), glutamate clearance was significantly impaired. This effect was concentration-dependent, became more pronounced by prolonging the incubation in the presence of MPP(+) and occurred at a time when cell membrane integrity was still preserved. No evidence was found that reactive oxygen species contributed to MPP(+)-induced decrease in glutamate clearance. Indeed, neither the spin trapping agent alpha-phenyl-tert-butyl nitrone, the lazaroid antioxidant U-74389G, nor the disulfide-reducing agent dithiothreitol was capable of restoring glutamate net uptake. The effect of MPP(+) on glutamate clearance: (i) was accompanied by a decrease in cellular ATP; (ii) could be enhanced by withdrawing glucose from the incubation medium or by inhibiting glycolysis with 2-deoxyglucose, and (iii) could be reproduced using the mitochondrial complex I inhibitor rotenone. Taken together, these results indicate that, by acting as a mitochondrial poison, MPP(+) impairs energy metabolism of astrocytes and significantly reduces their ability to maintain low levels of extracellular glutamate.
Insights
The parkinsonism-inducing agent MPP(+) impairs astrocyte glutamate uptake by disrupting energy metabolism. This mitochondrial toxicity reduces the cells' ability to clear extracellular glutamate, a key process in neurological health.
Area of Science:
- Neuroscience
- Cell Biology
- Toxicology
Background:
- Astrocytes play a crucial role in neurotransmitter homeostasis, including glutamate uptake.
- 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a neurotoxin that induces parkinsonism.
- MPTP is bioactivated to its toxic metabolite, MPP(+), within astrocytes.
Purpose of the Study:
- To investigate the mechanism by which MPP(+) impairs astrocytic glutamate uptake.
- To determine if MPP(+) affects astrocyte cell membrane integrity.
- To elucidate the role of cellular energy metabolism in MPP(+)-induced glutamate uptake dysfunction.
Main Methods:
- Primary astrocyte cultures were used to assess glutamate clearance.
- Cells were preincubated with varying concentrations of MPP(+) for different durations.
- Glutamate uptake, cellular ATP levels, and cell membrane integrity were measured.
- Experiments involved manipulating glucose availability and using metabolic inhibitors (rotenone, 2-deoxyglucose).
- The role of reactive oxygen species was assessed using antioxidants and spin traps.
Main Results:
- MPP(+) significantly impaired glutamate clearance by astrocytes in a concentration-dependent and time-dependent manner.
- This impairment occurred without compromising cell membrane integrity.
- MPP(+)-induced glutamate uptake reduction was linked to decreased cellular ATP levels.
- Withdrawing glucose or inhibiting glycolysis exacerbated the effect.
- The mitochondrial complex I inhibitor rotenone mimicked the effect of MPP(+).
- Reactive oxygen species did not appear to mediate the MPP(+)-induced decrease in glutamate uptake.
Conclusions:
- MPP(+) acts as a mitochondrial poison, disrupting astrocyte energy metabolism.
- Impaired energy metabolism leads to a significant reduction in astrocytic glutamate uptake capacity.
- This mechanism contributes to elevated extracellular glutamate levels, potentially exacerbating neurotoxicity in Parkinson's disease.