Somatic mitochondrial DNA mutations do not increase neuronal vulnerability to MPTP in young POLG mutator mice

Abstract

Mitochondrial DNA (mtDNA) mutations are hypothesized to play a pathogenic role in aging and age-related neurodegenerative diseases such as Parkinson's disease (PD). In support of this, high levels of somatic mtDNA mutations in “POLG mutator” mice carrying a proofreading-deficient form of mtDNA polymerase ã (Polg(D257A)) lead to a premature aging phenotype. However, the relevance of this finding to the normal aging process has been questioned as the number of mutations is greater even in young POLG mutator mice, which shows no overt phenotype, than levels achieved during normal aging in mice. Vulnerability of dopaminergic neurons to 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) increases with age, and we hypothesized that this may result in part from the accumulation with age of somatic mtDNA mutations. If correct, then levels of mutations in young (2–3 month old) POLG mutator mice should be sufficient to increase vulnerability to MPTP. In contrast, we find that susceptibility to MPTP in both heterozygous and homozygous POLG mutator mice at this young age is not different from that of wild type littermate controls as measured by levels of tyrosine hydroxylase positive (TH+) striatal terminals, striatal dopamine and its metabolites, a marker of oxidative damage, or stereological counts of TH+ and total substantia nigra neurons. These unexpected results do not support the hypothesis that somatic mtDNA mutations contribute to the age-related vulnerability of dopaminergic neurons to MPTP. It remains possible that somatic mtDNA mutations influence vulnerability to other stressors, or require additional time for the deleterious consequences to manifest. Furthermore, the impact of the higher levels of mutations present at older ages in these mice was not assessed in our study, although a prior study also failed to detect an increase in vulnerability to MPTP in older mice. With these caveats, the current data do not provide evidence for a role of somatic mtDNA mutations in determining the vulnerability to MPTP.

Fig. 1

Young (2~3 month old) POLG mutator mice do not exhibit increased susceptibility to MPTP-induced dopaminergic neurodegeneration. (A) Representative images of TH immunostaining of the striatum from mice treated with either saline or MPTP (n= 3~5 per group). Scale bar= 300 μm. (B) Densitometric quantification indicating that the densities of TH-positive fibers in the striatum are similar across genotypes at baseline and after MPTP treatment. (C) HPLC measurements of striatal levels of DA, DOPAC and HVA in mice treated with saline or MPTP (n= 8 per group). (D) Stereological counts of TH+, TH− and total neurons in SN (n= 3~4 per group). Two-way ANOVA followed by Bonferroni test indicates a significant effect of MPTP treatment in (B), (C) and TH+ and total SN neurons in (D), but the effect of genotype and interaction between treatment and genotype are not significant.

Fig. 2

Oxidative stress caused by MPTP neurotoxicity, mRNA levels of DAT and MAOb in the SN of MPTP-treated mice. (A) Representative images of OxyBlots demonstrate a significant increase in the formation of protein carbonyl derivatives in mice following MPTP treatment (n= 4 per group). (B) Densitometric quantification of OxyBlot. Data were analyzed by two-way ANOVA followed by a Bonferroni test, indicating a significant effect of MPTP treatment, but the effect of genotype and interaction between treatment and genotype are not significant. (C) Levels of malondialdehyde (MDA) in the striatum were assessed for lipid oxidative damage. Data are normalized to the saline-treated WT group. Two-way ANOVA followed by Bonferroni’s test indicates no significant effect of treatment, genotype or interaction between treatment and genotype (n= 4~5 per group). (D)There are no significant differences in mRNA levels of DAT or MAOb (normalized to HPRT) between POLG mutator mice and WT mice after MPTP treatment (n= 3~4 per group). Data were analyzed by one-way ANOVA followed by Bonferroni test.