Preferential Extracellular Generation of the Active Parkinsonian Toxin MPP⁺by Transporter-Independent Export of the Intermediate MPDP⁺

1-Methyl-4-phenyl-tetrahydropyridine (MPTP) is among the most widely used neurotoxins for inducing experimental parkinsonism. MPTP causes parkinsonian symptoms in mice, primates, and humans by killing a subpopulation of dopaminergic neurons. Extrapolations of data obtained using MPTP-based parkinsonism models to human disease are common; however, the precise mechanism by which MPTP is converted into its active neurotoxic metabolite, 1-methyl-4-phenyl-pyridinium (MPP(+)), has not been fully elucidated. In this study, we aimed to address two unanswered questions related to MPTP toxicology: (1) Why are MPTP-converting astrocytes largely spared from toxicity? (2) How does MPP(+) reach the extracellular space?In MPTP-treated astrocytes, we discovered that the membrane-impermeable MPP(+), which is generally assumed to be formed inside astrocytes, is almost exclusively detected outside of these cells. Instead of a transporter-mediated export, we found that the intermediate, 1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP(+)), and/or its uncharged conjugate base passively diffused across cell membranes and that MPP(+) was formed predominately by the extracellular oxidation of MPDP(+) into MPP(+). This nonenzymatic extracellular conversion of MPDP(+) was promoted by O2, a more alkaline pH, and dopamine autoxidation products.Our data indicate that MPTP metabolism is compartmentalized between intracellular and extracellular environments, explain the absence of toxicity in MPTP-converting astrocytes, and provide a rationale for the preferential formation of MPP(+) in the extracellular space. The mechanism of transporter-independent extracellular MPP(+) formation described here indicates that extracellular genesis of MPP(+) from MPDP is a necessary prerequisite for the selective uptake of this toxin by catecholaminergic neurons.