This proposal represents two essential approaches to achieve our long-term goals of elucidating the mechanism of action of MPTP, and the relevance of its biologic effects to the neurodegenerative process which underlies Parkinson's disease. Key questions will be addressed on the sequence of biological events which are thought to play essential roles in the occurrence of MPTP neurotoxicity. These include its metabolic activation, the disposition of its metabolites within the intracellular and extracellular compartments, the harmful consequences of the generation and accumulation of its putative toxic metabolite, MPP+, and the biochemical mechanisms of MPTP/MPP+-induced cell death. In vitro studies will be carried out in primary cultures of astrocyte. This model was chosen because astrocyte are the leading candidates as the site of the MAO-B mediated conversion of MPTP and therefore they are directly implicated in the processes making MPP+ available to neurons. These events, although generally accepted, have yet to be demonstrated. In this proposal, metabolic reactions involved in the biotramformation of MPTP, as well as the further conversion of metabolic intermediates(s) (i.e., MPDP+) by astrocyte in culture, will be further characterized by quantitative analysis of the metabolites formed under different experimental conditions (e.g., selective inhibition of metabolic pathways). Accumulation of metabolites will be monitored in the intracellular and extracellular compartments in order to determine,-for example, the process(es) responsible for the presence of MPP+ in the extracellular compartment and the relationship between the intracellular level of MPP+ and cytotoxicity. The latter two events could be linked, since the release of MPP+ into the medium could result from astrocyte death. In addition, the mechanisms involved in MPTP/MPP+ toxicity to astrocyte cultures will be evaluated by monitoring biochemical signs of oxidative stress and mitochondrial injury, respectively. In vivo studies of relative roles of oxygen radical generation and failure of mitochondrial respiration in MPTP neurotoxicity will also be determined using C57B1/6 mice. A relatively recent technique for rapid fixation of brain tissue will be used to determine neurochemical effects induced by MPTP administration. Microwave irradiation of mouse brain will allow rapid inactivation of enzymes, thus preventing post-mortem changes in the levels of neurotransmitters and endogenous-substrates (e. g., dopamine, Vitamin E, ATP and NADH). The proposed studies should greatly advance our knowledge of the relationship between metabolic and toxic events following exposure to MPTP, and could provide basic information on molecular events related to Parkinson's disease.
