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. 2015 Mar 23;2(1):ENEURO.0075-14.2015.
doi: 10.1523/ENEURO.0075-14.2015. eCollection 2015 Jan-Feb.

The Polg Mutator Phenotype Does Not Cause Dopaminergic Neurodegeneration in DJ-1-Deficient Mice

David N Hauser  1 , Christopher T Primiani  1 , Rebekah G Langston  1 , Ravindran Kumaran  1 , Mark R Cookson  1
Affiliations

The Polg Mutator Phenotype Does Not Cause Dopaminergic Neurodegeneration in DJ-1-Deficient Mice

David N Hauser et al. eNeuro. .

Abstract

Mutations in the DJ-1 gene cause autosomal recessive parkinsonism in humans. Several mouse models of DJ-1 deficiency have been developed, but they do not have dopaminergic neuron cell death in the substantia nigra pars compacta (SNpc). Mitochondrial DNA (mtDNA) damage occurs frequently in the aged human SNpc but not in the mouse SNpc. We hypothesized that the reason DJ-1-deficient mice do not have dopaminergic cell death is due to an absence of mtDNA damage. We tested this hypothesis by crossing DJ-1-deficient mice with mice that have similar amounts of mtDNA damage in their SNpc as aged humans (Polg mutator mice). At 1 year of age, we counted the amount of SNpc dopaminergic neurons in the mouse brains using both colorimetric and fluorescent staining followed by unbiased stereology. No evidence of dopaminergic cell death was observed in DJ-1-deficient mice with the Polg mutator mutation. Furthermore, we did not observe any difference in dopaminergic terminal immunostaining in the striatum of these mice. Finally, we did not observe any changes in the amount of GFAP-positive astrocytes in the SNpc of these mice, indicative of a lack of astrogliosis. Altogether, our findings demonstrate the DJ-1-deficient mice, Polg mutator mice, and DJ-1-deficient Polg mutator mice have intact nigrastriatal pathways. Thus, the lack of mtDNA damage in the mouse SNpc does not underlie the absence of dopaminergic cell death in DJ-1-deficient mice.

Keywords: DJ-1; Polg mutator; mtDNA; neurodegeration; parkinsonism; substantia nigra.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
Generation of DJ-1 knockout Polg mutator mice. A, Double heterozygous mice were bred to generate the four genotypes of mice used in this study. B, The number of viable pups born from double heterozygous breeding is shown. A χ2 test was used to determine that the observed proportions did not differ from the expected proportions (n = 208 mice, p = 0.901)a. C, The weights of female mice at one year of age are displayed. The groups were compared with ANOVA (F(3,9) = 29.17, p = 0.00005745)b followed by Tukey’s multiple comparison test (*p < 0.05 vs DJ-1+/+;PolgWT/WT, $p < 0.05 vs DJ-1−/−;PolgWT/WT). D, The weights of male mice at 1 year of age.
Figure 2
Figure 2
Behavioral characterization using the pole test. A, The mice were tested for behavioral deficits using the pole test and the method of descent for each mouse during each of their trials is displayed. Each bar represents an individual animal, and the methods of descent from four to seven trials are reported as a proportion within the bar. B, The mean time to descend the pole for each mouse is displayed (n = 4-7 mice per genotype, n = 4-7 trials per mouse, ANOVA F(3,19) = 1.171, p = 0.347)c. C, Following their first set of trials on the pole test, three mice in the DJ-1+/+;PolgMT/MT group and three mice in the DJ-1−/−;PolgMT/MT were given l-DOPA and retested 30 min later. The results of the test before and after l -DOPA are displayed with each bar representing an individual animal (n = 4-7 trials).
Figure 3
Figure 3
Stereological counts of dopaminergic neurons in the SNpc. Unbiased stereology was performed by a blinded observer to count the number of dopaminergic neurons in the SNpc of the mice after that had reached a year of age. Two separate experiments were performed to analyze the same set of brains. A, TH immunoreactive cells in the midbrain stained brown using DAB (scale bars, 200 μm). B, DAB-stained cell counts for each animal (red circles) along with mean and SEM of each group (n = 6-8 mice per genotype, ANOVA F(3,23) = 2.072, p = 0.1318)d. C, TH immunoreactive cells were detected in the midbrain using fluorescence (TH = green; scale bars, 500 μm). D, The numbers of SNpc dopaminergic neurons counted using stereology for each animal (red circles) are shown with mean and SEM (n = 6-8 mice per group, ANOVA F(3,23) = 0.9124, p = 0.4504)e.
Figure 4
Figure 4
Dopaminergic terminal density in the striatum. A, Representative TH-stained tissue sections through the striatum. The sections were immunostained using an infrared fluorescent dye conjugated secondary antibody and imaged using an infrared imaging system. The sections are pseudo-colored using a heat map, with warmer colors indicating strong TH immunoreactivity. B, Striatal TH staining intensity calculated from infrared imaged tissues. Individual data points represent animals and the mean and SEM are also displayed (three sections per animal were averaged, n = 5-8 animals, ANOVA F(3,22) = 1.189, p = 0.3369)f.
Figure 5
Figure 5
Detection of astrogliosis in the SNpc. A, GFAP-positive astrocytes were immunostained in the SNpc (outlined in white) and surrounding tissue (GFAP = red; scale bars, 500 μm). Unbiased stereology was used to count GFAP-positive cells in the SNpc simultaneously with the TH cells counts shown in Figure 3B. B, GFAP-positive SNpc cell counts per animal (red circles) along with mean and SEM are displayed in the graph (ANOVA F(3,23) = 1.744, p = 0.1860)g.
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