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

Abstract

Aims: 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? Results: 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. Innovation and Conclusion: 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. Antioxid. Redox Signal. 23, 1001–1016.

Tipping Points and Endogenous Determinants of Nigrostriatal Degeneration by MPTP

“…This contrasts with the lack of astroglial damage typically observed upon MPTP exposure [29,30], although astrocytes are not MPP + resistant. In astrocytes, genetically modified to express DAT, MPP + accumulates intracellularly and elicits a toxic response comparable to that in DA neurons [31,32]. These findings suggest that MPP + toxicity in any cell type is determined mainly by the toxicant concentration reached inside mitochondria.…”

“…Cells have been widely demonstrated to exhibit distinct susceptibilities largely due to variations in MPP + uptake across the plasma membrane, availability of export mechanisms, and intracellular deposition in organelles. Astrocytes might be spared from MPTP-induced cell death because of an efficient export of MPTP metabolites [32] (Figure 4).…”

Reference compounds for alternative test methods to indicate developmental neurotoxicity (DNT) potential of chemicals: example lists and criteria for their selection and use

“…This consideration is pivotal for chemical use in in vitro systems as well as alternative species models in which metabolism can vary from that of humans. For instance, chlorpyrifos may need to be converted to chlorpyrifos-oxon (Yang et al, 2008), heroin may fail to show effects in systems that lack deacetylases that catalyze the formation of the final toxicant morphine, and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) will fail to show any effect, unless it is metabolized by astrocytic monoamine oxidase to 1-methyl-4-phenylpyridinium (MPP + ) (Efremova et al, 2015; Schildknecht et al, 2015). …”

In vitro acute and developmental neurotoxicity screening: an overview of cellular platforms and high-throughput technical possibilities

“…Energy metabolism can be monitored by measurement of ATP content of cells (Volbracht et al 1999;Latta et al 2000), or quantifying the reduction in resazurin or tetrazolium salts (e.g. MTT) by viable cells Pamies et al 2014;Schildknecht et al 2015). The release of lactate dehydrogenase (LDH) from dead cells (Leist et al 1996;Wong et al 2001) is also amenable for highthroughput use and an indicator of irreversible cell damage.…”