Abstract
Parkinson's disease (PD) is a major human disease associated with degeneration of the central nervous system. Evidence suggests that several endogenously formed 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mimicking chemicals that are metabolic conversion products, especially β-carbolines and isoquinolines, act as neurotoxins that induce PD or enhance progression of the disease. We have demonstrated previously that mitochondrially targeted human cytochrome P450 2D6 (CYP2D6), supported by mitochondrial adrenodoxin and adrenodoxin reductase, can efficiently catalyze the conversion of MPTP to the toxic 1-methyl-4-phenylpyridinium ion. In this study, we show that the mitochondrially targeted CYP2D6 can efficiently catalyze MPTP-mimicking compounds, i.e. 2-methyl-1,2,3,4-tetrahydroisoquinoline, 2-methyl-1,2,3,4-tetrahydro-β-carboline, and 9-methyl-norharmon, suspected to induce PD in humans. Our results reveal that activity and respiration in mouse brain mitochondrial complex I are significantly affected by these toxins in WT mice but remain unchanged in Cyp2d6 locus knockout mice, indicating a possible role of CYP2D6 in the metabolism of these compounds both in vivo and in vitro These metabolic effects were minimized in the presence of two CYP2D6 inhibitors, quinidine and ajmalicine. Neuro-2a cells stably expressing predominantly mitochondrially targeted CYP2D6 were more sensitive to toxin-mediated respiratory dysfunction and complex I inhibition than cells expressing predominantly endoplasmic reticulum-targeted CYP2D6. Exposure to these toxins also induced the autophagic marker Parkin and the mitochondrial fission marker Dynamin-related protein 1 (Drp1) in differentiated neurons expressing mitochondrial CYP2D6. Our results show that monomethylamines are converted to their toxic cationic form by mitochondrially directed CYP2D6 and result in neuronal degradation in mice.
Highlights
Parkinson’s disease (PD) is a major human disease associated with degeneration of the central nervous system
Effects of monomethylamine neurotoxins on mouse brain mitochondrial function We investigated the effect of three neurotoxins, 2-methyl1,2,3,4-tetrahydroisoquinoline HCl (TISQ), 2-methyl-1,2,3,4tetrahydro--carboline (MTHBC), and 9-methyl-9H-pyrido (3,4-)indole (-Carb) (Table 1), all of which are produced endogenously in rodent and primate brains (10 –12) from dietary or environmental components
The in vivo effects of these toxins on mitochondrial function were tested in WT and Cyp2d6KO mice by intraperitoneal injection of TISQ (64 mg/kg/day) for 21 days, and the mitochondrial respiration profiles were measured in a Seahorse XF-24 flux analyzer
Summary
Effects of monomethylamine neurotoxins on mouse brain mitochondrial function We investigated the effect of three neurotoxins, 2-methyl1,2,3,4-tetrahydroisoquinoline HCl (TISQ), 2-methyl-1,2,3,4tetrahydro--carboline (MTHBC), and 9-methyl-9H-pyrido (3,4-)indole (-Carb) (Table 1), all of which are produced endogenously in rodent and primate brains (10 –12) from dietary or environmental components. MTHBC marginally reduced TH-positive neurons in Mc2D6-expressing Neuro-2a cells but had a marked effect on Mt2D6expressing Neuro-2a cells, which showed nearly 70% inhibition Quinidine prevented this loss significantly in Mt2D6-expressing cells (Fig. 6E). Immunoblot analysis of cells treated with MTHBC and -Carb (Fig. 7, B and C) showed similar effects and protection by quinidine These results clearly indicate that cells expressing Mt2D6 are much more susceptible to monomethylamine-induced toxicity and susceptibility to undergo autophagy than Mc2D6-expressing Neuro-2a cells. Drp is a known fission marker and an indicator of the mitochondrial quality control system [41] Both immunocytochemistry and Western blot analysis showed that mitochondrial Drp-1 levels were markedly increased by all three toxins in Mt2D6-expressing cells, but there was only a slight increase in Mc2D6-expressing cells (Fig. 8, A–C). All of these results confirm that mitochondrial CYP2D6 plays a pivotal role in monomethylamine neurotoxin-induced mitochondrial dysfunction
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