Supplementary Data Supplementary Figures and Figure Legends

Ogg1-KO

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Supplemental Figure 1. Immunohistochemical detection of MTH1 and OGG1 in the mouse striatum. (A) MTH1 expression was examined using the NB-109 antibody, which reacts with human MTH1; the human protein has 91% sequence identity with mouse and rat proteins. MTH1 was preferentially localized cytoplasm, but no signal was detected in Mth1-KO mice. Arrows, MTH1 immunoreactive signals. (B) OGG1 expression was examined using anti-OGG1 antibody. In wild-type striatum, OGG1 was detected in nucleus and cytoplasm, but no signal was detected in Ogg1-KO mice. Arrows, OGG1 immunoreactive signals. Bar, 20 µm. Dark blue signals represent immunoreactivivity. (A) (B) The images are visualized by differential interference contrast microscopy.

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Supplemental Figure 2. Detection of MTH1 and OGG1 in the mouse striatum by immunoelectron microscopy. (A) MTH1 expression was examined using the NB-109 antibody, MTH1 was preferentially localized in cytoplasm and some in nucleus, but no signal was detected in Mth1-KO striatum. Arrows, MTH1 immunoreactive signals. Bar, 500 nm. C, cytoplasm; N, nucleus. (B) OGG1 expression was examined using anti-Ogg1 antibody. In wild-type striatum, OGG1 was detected in nucleus, cytoplasm, but no signal was detected in Ogg1-KO striatum. Arrows, OGG1 immunoreactive signals. C, cytoplasm; N, nucleus. Bar, 500 nm.

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Supplemental Figure 3. Behavioral assessments of mice by open field after exposure to 3-nitropropionic acid in various mouse strains Motor impairments in various mouse strains assessed by open field test after exposure to 3-nitropropionic acid (3-NP, 120 mg/kg) for one week. Wild-type (N=5 for PBS, N=5 for 3-NP), Mth1-KO (N=4 for PBS, N=4 for 3-NP), Ogg1-KO (N=5 for PBS, N=7 for 3-NP), and Mth1/Ogg1-DKO (N=5 for PBS, N=5 for 3-NP) were tested. Compared to wild-type mice exposed to 3-NP, Ogg1-KO 3-NP (p<0.02), Mth1/Ogg1-DKO 3-NP(p<0.005) (Student’s t-test), ns, not significant. Relative traveling distance (%) indicates the traveling distance obtained on the 7th day after implantation of an osmotic mini-pump as a percentage of that obtained on the day before the implantation. Data are least squares means (LSMeans) ± SE.

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Supplemental Figure 4. Motor impairment observed on the 7th day of chronic exposure to 3-NP Wild-type mice (N=10 for PBS, N=12 for 3-NP) and Mth1/Ogg1-DKO mice (N=11 for PBS, N=12 for 3-NP) were monitored for 10 min on the 7th day of exposure to 3-NP (120 mg/kg). Neurological score was defined as follows: (score=0, normal behavior; 1, general slowness of displacement resulting from mild hindlimb impairment; 2, incoordination and marked gait abnormality; 3, hindlimb paralysis; 4, incapacity to move resulting from forelimb and hindlimb impairment shown in A; 5, recumbency shown in B (Guyot MC et al., 1997). C, Mth1/Ogg1-DKO mice exhibited significantly higher neurological scores compared to wild type (p<0.05, Mann-Whitney U-test). Data are shown with means ± SE.

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Supplemental Figure 5. Impairment in spontaneous locomotor activity after exposure to 3-NP. (A) Night-phase locomotor activity observed in the home cage. (B) Day-phase locomotor activity observed in the home cage. Wild-type (N=6 for PBS, N=9 for 3-NP) and Mth1/Ogg1-DKO mice (N=6 for PBS, N=8 for 3-NP) were examined. In comparison to wild-type mice, the DKO mice exhibited greater decrease in night-phase locomotor activity on the day 5 (D5) and day 6 (D6) after exposed to 3-NP, however, there was no a significant difference on day-phase locomotor activity. Data are shown with mean ± SE. *p, Student’s t test (DKO 3-NP vs Wild type 3-NP).

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Supplemental Figure 6. Motor impairment in wild-type and Mutyh-KO mice on the 7th day of exposure to higher dose of 3-NP . Wild-type mice (N=6 for PBS, N=10 for 3-NP) and Mutyh-KO mice (N=6 for PBS, N=10 for 3-NP) were monitored for 10 min on the 7th day of exposure to 3-NP (150 mg/kg). Wild-type mice exhibited more severe score compared to Mutyh-KO (p<0.05, Mann-Whitney U-test). Data are shown with mean ± SE.

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Genotype=0.1784;Treatment, p<.0001 Genotype*treatment =0.3413

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Supplemental Figure 7. Behavioral assessments of mice by open field test after exposure to 3-nitropropionic acid between Mutyh-KO and Ogg1/Mutyh-DKO. Mutyh-KO (N=7 for PBS, N=5 for 3-NP) and Ogg1/Mutyh-DKO (N=6 for PBS, N=7 for 3-NP) were subjected to open field test on the 7th day of exposure to 3-NP (120 mg/kg). There was no a significant effect of genotype, ns, not significant. Relative performnce (%) indicates the traveling distance or number of rearing obtained on the 7th day after implantation of an osmotic mini-pump as a percentage of those obtained on the day before the implantation. Data are least squares means (LSMeans) ± SE.

Genotype=0.7669; Treatment, p<.0001 Genotype*treatment=0.2967

Mutyh-KO Mutyh/Ogg1-DKO Mutyh-KO Mutyh/Ogg1-DKO

Traveling distance Rearing

Supplementary materials:

Supplemental Movie 1. A control Mth1/Ogg1-DKO mouse that received PBS infusions for a week exhibited normal spontaneous motor activity in open field.  

Supplemental Movie 2. An Mth1/Ogg1-DKO mouse that received 3-NP infusions for 1 week (120 mg/kg/day) exhibited a dramatic decrease in the amount of spontaneous motor activity in the open field.