Vesicular Monoamine Transporter 2 (VMAT2) Level Regulates MPTP Vulnerability and Clearance of Excess Dopamine in Mouse Striatal Terminals

Abstract

The vesicular monoamine transporter 2 (VMAT2) packages neurotransmitters for release during neurotransmission and sequesters toxicants into vesicles to prevent neuronal damage. In mice, low VMAT2 levels causes catecholaminergic cell loss and behaviors resembling Parkinson's disease, while high levels of VMAT2 increase dopamine release and protect against dopaminergic toxicants. However, comparisons across these VMAT2 mouse genotypes were impossible due to the differing genetic background strains of the animals. Following back-crossing to a C57BL/6 line, we confirmed that mice with approximately 95% lower VMAT2 levels compared with wild-type (VMAT2-LO) display significantly reduced vesicular uptake, progressive dopaminergic terminal loss with aging, and exacerbated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity. Conversely, VMAT2-overexpressing mice (VMAT2-HI) are protected from the loss of striatal terminals following MPTP treatment. We also provide evidence that enhanced vesicular filling in the VMAT2-HI mice modifies the handling of newly synthesized dopamine, indicated by changes in indirect measures of extracellular dopamine clearance. These results confirm the role of VMAT2 in the protection of vulnerable nigrostriatal dopamine neurons and may also provide new insight into the side effects of L-DOPA treatments in Parkinson's disease.

Keywords: DAT; L-DOPA.; MPTP; VMAT2; dopamine; vesicle.

FIG. 1.

C57BL/6 vesicular monoamine transporter 2 (VMAT2)-LO mice show progressive dopaminergic degeneration with aging and reduced vesicular uptake. A–D, VMAT2-LO mice have significantly reduced VMAT2 levels and, by 22 months of age, also show significant reductions in dopamine transporter (DAT) level in the striatum, as measured by immunoblotting (n = 10). E, VMAT2-LO mice show an 80% reduction in VMAT2-mediated [³H]-dopamine uptake in isolated vesicles compared with wild-type littermates at 12 months of age (P < .01, n = 2–3).

FIG. 2.

VMAT2 level modifies vulnerability to dopamine terminal marker loss following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). A, Representative blots of VMAT2 and dopaminergic markers DAT and tyrosine hydroxylase (TH) following MPTP or saline treatment. S, saline; M, 2 × 10 mg/kg MPTP. B, VMAT2 levels vary with genotypes as expected. C and D, VMAT2-LO mice show a larger decrease in DAT and TH, compared with wild-type and VMAT2-HI mice (n = 4–6). Different letters above bars (A vs B, for example) indicate differences of at least P < .05 between those groups by a 2-way ANOVA and Bonferroni post hoc tests. Bars with the same letter above (A vs A) are not significantly different. Results are % wild-type control. E and F, Results were confirmed with immunohistochemistry for DAT and TH.

FIG. 3.

VMAT2 level modifies stimulated dopamine (1p) release and dopamine clearance following L-DOPA application. A–C, At baseline, VMAT2-LO mice show the smallest dopamine release (n = 3), and VMAT2-HI mice show the largest release (n = 6), compared with wild-type mice (n = 6). Following L-DOPA application (D), all 3 genotypes showed increases in DAmax compared with baseline values, however the fall time of the release traces appeared to be particularly affected between the genotypes. E and F, VMAT2-HI mice show a significant decrease in both the rate constant, tau, and half-width, all of which reflects the steeper fall of the release traces in this genotype compared with wild-type and VMAT2-LO mice. Different letters above the bars indicate differences of at least P < .05 as indicated by a 2-way ANOVA and Bonferroni post hoc tests.

FIG. 4.

There are no changes in DAT expression or binding in synaptosomes prepared from wild-type and VMAT2-HI mice. A, Representative immunoblotting of VMAT2 and dopaminergic markers DAT and TH between wild-type and VMAT2-HI mice from synaptosomal preparations from wild-type and VMAT2-HI mice (n = 4). B, VMAT2 level is significantly increased in the VMAT2-HI mice. C and D, There are no differences in the expression levels of DAT and TH between wild-type and VMAT2-HI mice. E, Using isolated synaptosomal preparations from bilateral dissected striatal homogenates, WIN binding showed no significant differences in DAT level between genotypes (n = 4).

FIG. 5.

VMAT2 level alters dopamine release but has minimal effect on exogenous dopamine handling in lesioned slices. Following a 2 × 10 mg/kg MPTP lesion, all VMAT2 genotypes show significant decreases in peak stimulated dopamine release compared with controls mice (approximately 60% decrease in DAmax compared with baselines from Fig. 3). VMAT2-HI mice show a significantly preserved peak dopamine release following this dosing regimen (P < .05, n = 4).