Abnormalities of motor function, transcription and cerebellar structure in mouse models of THAP1 dystonia

Abstract

DYT6 dystonia is caused by mutations in THAP1 [Thanatos-associated (THAP) domain-containing apoptosis-associated protein] and is autosomal dominant and partially penetrant. Like other genetic primary dystonias, DYT6 patients have no characteristic neuropathology, and mechanisms by which mutations in THAP1 cause dystonia are unknown. Thap1 is a zinc-finger transcription factor, and most pathogenic THAP1 mutations are missense and are located in the DNA-binding domain. There are also nonsense mutations, which act as the equivalent of a null allele because they result in the generation of small mRNA species that are likely rapidly degraded via nonsense-mediated decay. The function of Thap1 in neurons is unknown, but there is a unique, neuronal 50-kDa Thap1 species, and Thap1 levels are auto-regulated on the mRNA level. Herein, we present the first characterization of two mouse models of DYT6, including a pathogenic knockin mutation, C54Y and a null mutation. Alterations in motor behaviors, transcription and brain structure are demonstrated. The projection neurons of the deep cerebellar nuclei are especially altered. Abnormalities vary according to genotype, sex, age and/or brain region, but importantly, overlap with those of other dystonia mouse models. These data highlight the similarities and differences in age- and cell-specific effects of a Thap1 mutation, indicating that the pathophysiology of THAP1 mutations should be assayed at multiple ages and neuronal types and support the notion of final common pathways in the pathophysiology of dystonia arising from disparate mutations.

Figure 1.

Generation of Thap1^C54Y and Thap1⁻ alleles and mice. (A) Schematic representation of THAP1 gene and Thap1 protein with the THAP domain (purple), low-complexity proline-rich region (black), coiled-coil domain (red) and nuclear localization signal (green). The exon positions are indicated corresponding to the numbers of the amino acid sequence. (B) B6;129Thap1^{C54YtmLoxPFrt} mutant mouse construct with the inserted LoxP-FRT-PGK-NeoR-FRT-loxP cassette. The C54Y mutation is located in Exon 2 flanked by two LoxP sites. The Thap1^C54Y allele was generated by crossing with the Flp(o) mouse, and subsequently, the Thap1⁻ allele was generated by crossing with the CMV-Cre mouse. Green arrows represent the position of the forward and reverse primers used for genotyping. Orange line represents the probe used for Southern blot analysis. E, EcoRV; K, KpnI; F, Frt site; L, LoxP site. (C) Southern blot analysis of 10 and 20 µg of KpnI/EcoRV digested genomic DNA from Thap1^+/+ and the B6;129Thap1^{C54YtmLoxPFrt} ES clone ultimately used to produce the mice. The Thap1^+/+ sample has bands at 12- and 3-kb bands whereas the positive clone has an additional band at 7.2 kb. (D) Representative Thap1^+/+, Thap1^C54Y/−, Thap1^+/− and Thap1^−/− embryos at E14.5 or E13.5. Rare, homozygote mutant survivors at this age are small with multiple malformations. (E) Transillumination of a Thap1^+/− skull compared with a Thap1^C54Y/− skull at E12 shows that the brain is grossly smaller in size, with relatively increased ventricular size (arrows). (F) Immunofluorescence with anti-Tuj1 of the cerebral cortex in sections derived from embryos pictured in (E) shows a paucity of positively stained neurons and projections in the Thap1^C54Y+/− brain. The immunopositive ‘neurons’ are of abnormal shape and size. Regions identified by arrows in upper panels are shown at higher power in lower panels. Scale bars: 50 µm. (G) Brains of Thap1^C54Y/+ and Thap1^+/− mice are of normal size relative to Thap1^+/+, and the exterior is grossly normal.

Figure 2.

The projection neurons of the DNC are abnormal in Thap1^C54Y/+ and Thap1^+/− mice. (A) Nissl stain of the adult cerebellum (a, a′) shows grossly normal architecture in the adult Thap1^C54Y/+ mouse. Notably, whereas cellularity in the folia appears normal, the deep cerebellar nuclei appear abnormal (arrows, a, a′). The dentate nucleus (arrows) also appears hypocellular at P1 in the Thap1^C54Y/+, as detected with Nissl stain (b, b′) and an anti-Tbr1 antibody (c, c′), a marker of the projection neurons, which appears less intense. Immunostaining with anti-Tbr1 in the cortex of the P1 Thap1^C54Y/+ is similar to the WT (arrows, d, d′). (B) Under higher power, Nissl stain of cerebellum shows hypocellularity in the deep nuclei of the cerebellum of the Thap1^C54Y/+ and Thap^+/− mouse, most prominent in the dentate (lateral) nucleus, indicated by arrows (a–d) and more obvious at higher power (e–h). (C) Hypocellularity is confirmed by stereologic counts of large projection neurons in the dentate nucleus of the Thap^+/− mouse, which reveals a 40% decrease relative to WT (n = 5 of each genotype; **P < 0.01) and not in the Thap1^C54Y/+ mouse (n = 5 of each genotype, P > 0.05). (D) The large projection neurons in the dentate nucleus of the Thap1^C54Y/+ mouse are of increased volume (Bonferroni's multiple comparison test; ***P < 0.001), but the large projection neurons in the Thap^+/− mouse are of normal volume. Note: each genotype had its own set of litter-matched controls for each assay. **P < 0.01;***P < 0.001. Scale bars: (A) a = 500 µm; b–d = 100 µm. (B) a = 250 µm, e = 100 µm.

Figure 3.

Thap1^C54Y/+ and Thap1^+/− mice display abnormalities of motor function. (A) On the accelerating rotarod testing days, the female Thap1^C54Y/+ performance was significantly worse compared with their Thap1^+/+ littermates (error bars indicate SEM; two-way ANOVA followed by Bonferroni post hoc test) (n = 9 males and n = 12 females for all assays). (B) On the beam walking test days, male and female Thap1^C54Y/+ mice exhibited an increased number of foot-slips on the first test day on the small 7 mm beam compared with their Thap1^+/+ littermates (unpaired t-test, P < 0.05). Males were not able to learn, showing a worse performance also on the second day of testing (P < 0.05). (C) In the gait analysis, Thap1^C54Y/+ males showed a decrease in stride length and the females had a wider base width of the hind paws compared with Thap1^+/+ (t-test: stride length males: P < 0.001; females: P > 0.05 and base width males: P > 0.05; females: P < 0.01; overlap: n.s.). (D) On the pole test, Thap1^C54Y/+ male and female mice displayed a lower total time to descend the pole, i.e. descended faster, compared with Thap1^+/+ in both sexes (unpaired t-test: P < 0.05). (E) Thap1^+/− males also displayed a lower total time to descend the pole compared with Thap1^+/+ (n = 6; P < 0.01) and also decreased t-turn time (P < 0.01). In contrast, Thap1^+/− females (n = 8) were slower to orient downwards (P < 0.05) and to descend (P < 0.01), i.e. greater t-turn and t-total. *P < 0.05; **P < 0.01.

Figure 4.

Protein levels of torsinA, Gαolf, DARPP-32 and TH are unchanged in Thap1^C54Y/+ and Thap1 ^+/− mice. (A) Western blot analysis of Thap1 in 30 μg of striatal total cellular homogenates showed a significant decrease of the 50-kDa isoform at 2 months of age in Thap1^+/− mice but no difference at 3 months of age, at which time it was also unchanged in Thap1^C54Y/+ mice relative to Thap1^+/+. GAPDH is displayed as a loading control. Blots are representative of n = 3 per genotype for Thap1^C54Y/+ and n = 5 per genotype for Thap1^+/− mice. Densitometry values were normalized to GAPDH, and Thap1^+/+ levels were set arbitrarily at 100%. (B) Western blot analysis of total cellular homogenates from adult Thap1^C54Y/+ and Thap1^+/− striatum (20 μg protein per lane). Baseline levels of TH, DARPP-32, Gαolf and TorsinA are similar in the two recombinant mice compared with their Thap1^+/+ littermates. GAPDH is displayed as loading control. n = 4 per genotype. (C) Densitometry values were normalized to GAPDH and Thap1^+/+ levels were set arbitrarily at 100%. P > 0.05 (unpaired two-tailed t-test).

Figure 5.

Thap1^C54Y/+ mice display altered response to injection of the β-adrenergic antagonist propranolol followed by d-amphetamine. (A) Locomotor, ambulatory and stereotypy activities of Thap1^C54Y/+ male mice following administration of d-amphetamine (i.p., 2 mg/kg) after the nonselective β-adrenergic antagonist (S)-(−)-propranolol injection (PRO) (i.p., 20 mg/kg) or saline (2-month-old males: n = 8 of each genotype per treatment). Locomotion was quantified for 30 min after saline or propranolol injections and 45 min after amphetamine injection. Mice were habituated to the cages for 15 min prior to the first injection. There was a significant interaction between saline and propranolol groups in the Thap1^+/+ (two-way ANOVA), but individual time points were not significant with Bonferroni post hoc test. Data points represent mean activity ± SEM. (B) Thap1^C54Y/+ female mice and littermate controls (n = 8) were treated similarly to males in (A). Genotype-dependent differences were noted in locomotor or ambulatory activity following amphetamine in the absence of propranolol, indicating an increased response of the recombinant mice to amphetamine but a relatively lower contribution of NA activity to cAMP production following the psychostimulant. In the locomotor activity panel, asterisk represents Thap1^+/+ saline/amphetamine versus Thap1^C54Y/+ saline/amphetamine and hash represents Thap1^+/+ saline/amphetamine versus Thap1^+/+ propranolol/amphetamine. In the ambulatory panel, asterisk represents Thap1^+/+ saline/amphetamine versus Thap1^C54Y/+ saline/amphetamine and hash represents Thap1^+/+ saline/amphetamine versus Thap1^+/+ propranolol/amphetamine (Bonferroni post hoc test * or ^#P < 0.05; **P < 0.01).