Human COMT over-expression confers a heightened susceptibility to dyskinesia in mice

Abstract

Catechol-O-methyltransferase (COMT) degrades dopamine and its precursor l-DOPA and plays a critical role in regulating synaptic dopamine actions. We investigated the effects of heightened levels of COMT on dopamine-regulated motor behaviors and molecular alterations in a mouse model of dyskinesia. Transgenic mice overexpressing human COMT (TG) and their wildtype (WT) littermates received unilateral 6-OHDA lesions in the dorsal striatum and were treated chronically with l-DOPA for two weeks. l-DOPA-induced dyskinesia was exacerbated in TG mice without altering l-DOPA motor efficacy as determined by contralateral rotations or motor coordination. Inductions of FosB and phospho-acetylated histone 3 (molecular correlates of dyskinesia) were potentiated in the lesioned striatum of TG mice compared with their WT littermates. The TG mice had lower basal levels of dopamine in the striatum. In mice with lesions, l-DOPA induces a greater increase in the dopamine metabolite 3-methoxytyramine in the lesioned striatum of dyskinetic TG mice than in WT mice. The levels of serotonin and its metabolite were similar in TG and WT mice. Our results demonstrate that human COMT overexpression confers a heightened susceptibility to l-DOPA-induced dyskinesia and alters molecular and neurochemical responses in the lesioned striatum of mice.

Keywords: 22q11.2; ARVCF; Abnormal involuntary movements; COMT; Dopamine; LID; Striatum; TXNRD2; l-DOPA.

Fig. 1

Dyskinetic responses to l-DOPA are potentiated in COMT-overexpressing mice. Scores for axial (A), limb (B), orolingual (C), and the sum total (ALO) (D) dyskinetic behaviors were evaluated for 4 min, 40 min after l-DOPA injections in mice. A repeated two-way ANOVA indicated significant main effects for axial ([A] genotype, F_1,17 = 13.21, P= 0.0021; day, F_4,68 = 34.59, P< 0.0001; interaction,F_4,68 = 0.63, not significant [n.s.]), limb ([B] genotype, F_1,17 = 10.00, P=0.0057; day, F_4,68 = 37.65, P< 0.0001; interaction, F_4,68 = 0.60, n.s.), orolingual ([C] genotype, F_1,17 = 8.73, P = 0.0089; day, F_4,68 = 22.46, P< 0.0001; interaction, F_4,68 = 0.44, n.s.), and total ([D] genotype, F_1,17 = 13.62, P= 0.0018; day, F_4,68 = 78.53, P< 0.0001; interaction, F_4,68 = 0.38, n.s.) dyskinesia. Data are expressed as the means ± SEM. *P< 0.05 and **P< 0.01 vs. WT as determined by Bonferroni post hoc test; n = 9–10/group.

Fig. 2

Overexpression of COMT enhances dyskinesia in mice but not motor coordination or rotational behaviors. (A) Time course of dyskinetic (axial, limb, and orolingual [ALO]) behaviors on day 16, evaluated for 1 min once every 30 min during a 180-min period following l-DOPA treatment. A repeated two-way ANOVA indicated significant main effects (genotype, F_1,17 = 5.88, P = 0.0268; time, F_5,85 = 191.63, P < 0.0001; interaction, F_5,85 = 1.45, P= 0.215), and a genotype effect at each time point, as determined by Bonferroni's tests.*P< 0.05vs. WT. (B) Sum of all scores over the 180-min period (t[17] =2.425)*P = 0.0268vs. WT. (C) Motor coordination and balance assessed on the rotarod before 6-OHDA lesions (prelesion), 3 weeks after lesions (pre-l-DOPA), and 90 min after l-DOPA was injected on day 14 of the chronic treatment (post-l-DOPA). A two-way ANOVA indicates a significant group effect only (genotype, F_1,17 = 0.02, not significant [n.s.]; group, F_2,34= 60.70, P< 0.0001; interaction, F_2,34= 0.06, n.s.). ***P< 0.001 vs. TG pre-l-DOPA, ###P< 0.001 vs. WT pre-l-DOPA as determined by Bonferroni post hoc tests. (D) Contralateral turns were measured for a period of 15 min, 5 min after l-DOPA was injected during chronic treatment. A repeated two-way ANOVA indicates a significant effect of day only (genotype, F_1,17 = 0.46, n.s.; day, F₄,₆8 = 18.12, P< 0.0001; interaction, F₄,₆8 = 1.10, n.s.). Data are expressed as the means ± SEM. n = 9–10/group.

Fig. 3

Induction of FosB and pAcH3 in striata of COMT-overexpressing mice. (A) Immunostaining for TH, FosB, and pAcH3. Photomicrographs of adjacent coronal striatal sections of the lesioned striata at low and high (40x) magnification from WT and TG mice. Scale bar = 100 μm for low-magnification and 50 μm for high-magnification images. The continuous outline represents the dorsal striatum and the dashed outline represents the completely denervated striatum in the low magnification TH pictures. (B) The extent of striatal lesions was assessed by quantifying the percentage of striatal volume that did not stain for TH (t[17] = 0.53, n.s.). The densities of FosB-positive (C) (t[17] = 2.38, P = 0.0292) andpAcH3-positive (D) (t[17] = 2.80, P = 0.0123) cells in the lesioned striata. Data are expressed as the means ± SEM. *P< 0.05 vs. WT mice; n = 9–10/group.

Fig. 4

Levels of DA and its metabolites in lesioned mice after l-DOPA treatment. Levels of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and 3-methoxytyramine (3-MT) in the striata contralateral (C) and ipsilateral (I) to 6-OHDA lesions in sham-operated, lesioned and saline-administered (Park), and lesioned and l-DOPA-administered (dysk) mice. Mice were sacrificed 60 min after the last saline or l-DOPA injection, and levels were measured by HPLC. A three-way ANOVA with significant P levels adjusted by Bonferroni corrections showed genotype× condition interactions for DA (F_2,29= 10.838, P = 0.001) and DOPAC (F_2,28= 6.326, P = 0.005). For 3-MT levels, significant effects were found according to genotype (F_1,29= 21.205, P< 0.001), condition (F_2,29= 11.01, P< 0.001), and hemisphere (F_1,29= 71.61, P< 0.001) without interactions (genotype × condition, F_2,29= 0.659, P = 0.525; genotype × hemisphere, F_1,29= 0.026, P = 0.873; genotype × condition × hemisphere, F_2,29= 0.588, P = 0.562). *P< 0.05 and **P< 0.01 vs. WT ipsilateral striatum. The data are shown as the means± SEM. n = 5–7/group.

Fig. 5

Levels of serotonin and its metabolite in lesioned mice after l-DOPA treatment. Levels of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in the striata contralateral (C) and ipsilateral (I) to 6-OHDA lesions in sham-operated, lesioned and saline-administered (Park), and lesioned and l-DOPA-administered (dysk) mice. Mice were sacrificed 60 min after the last saline or l-DOPA injection, and levels were measured by HPLC. A three-way ANOVA with significant P levels adjusted by Bonferroni corrections showed significant differences for effects of condition (F_2,29= 8.215, P = 0.001) and a condition × hemisphere interaction (F_2,29= 12.61, P< 0.001) for 5-HT; no other effect was significant. For 5-HIAA, no main or interaction effect involving genotype was significant (genotype, F_1,28= 0.018, not significant [n.s.]; genotype × condition, F_2,28= 0.283, n.s.; genotype × hemisphere, F_1,28= 0.415, n.s.; genotype × condition × hemisphere, F_2,28= 0.543, n.s.). *P< 0.05 and **P< 0.01 vs. contralateral striatum. Data are presented as the means ± SEM. n = 5–7/group.