Impaired Glutamate Clearance as a Consequence of Energy Failure Caused by MPP+ in Astrocytic Cultures

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 α-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.