Selective expression of mutant huntingtin during development recapitulates characteristic features of Huntington's disease

Abstract

Recent studies have identified impairments in neural induction and in striatal and cortical neurogenesis in Huntington's disease (HD) knock-in mouse models and associated embryonic stem cell lines. However, the potential role of these developmental alterations for HD pathogenesis and progression is currently unknown. To address this issue, we used BACHD:CAG-Cre(ERT2) mice, which carry mutant huntingtin (mHtt) modified to harbor a floxed exon 1 containing the pathogenic polyglutamine expansion (Q97). Upon tamoxifen administration at postnatal day 21, the floxed mHtt-exon1 was removed and mHtt expression was terminated (Q97(CRE)). These conditional mice displayed similar profiles of impairments to those mice expressing mHtt throughout life: (i) striatal neurodegeneration, (ii) early vulnerability to NMDA-mediated excitotoxicity, (iii) impairments in motor coordination, (iv) temporally distinct abnormalities in striatal electrophysiological activity, and (v) altered corticostriatal functional connectivity and plasticity. These findings strongly suggest that developmental aberrations may play important roles in HD pathogenesis and progression.

Keywords: neurodegeneration; plasticity; prodromal.

Fig. 1.

CRE^ER yields highly efficient levels of mHtt excisional recombination in Q97^CRE mice. In contrast to striatal Q97 specimens, RT-PCR amplicons using primers that selectively detect the presence of mutant exon 1 of the Htt cDNA (Top) and Western blot analysis probed with the mAb 2166 anti-Htt antibody (∼350 kDa, Bottom) show no discernible bands in control and Q97^CRE striatal specimens (n_{per strain} = 3).

Fig. S1.

CRE^ER yields highly efficient levels of mHtt excisional recombination in Q97^CRE mice. CRE-mediated excisional recombination after tamoxifen administration at PND21 was examined in 3-mo-old striatal specimens. High Q97^CRE QRT-PCR cycle thresholds of DNA (n_{per strain} = 8, A and B) and RNA (n_{per strain} = 4, C and D) demonstrate very efficient levels of CRE^ER-mediated excisional recombination of mHtt [relative quantification (RQ) = 0.018, CI95% = 0.015–0.021 and RQ = 0.002, CI95% = 0.001–0. 002, fold change of Q97^CRE relative to Q97 for DNA and RNA, respectively]. Rn is the fluorescence of the reporter dye divided by the fluorescence of a passive reference dye.

Fig. S2.

CRE^ERT2 yields highly efficient levels of mHtt excisional recombination in Q97^CRE mice throughout the nervous system and in nonneural tissues. BACHD:CAG-Cre^ERT2 mice received an oral dose (1.125 mg⋅d⁻¹) of tamoxifen given via feeding tube for five consecutive days at PND21 to generate mice expressing mHtt (i) only during development (Q97^CRE) or (ii) during development and adult life (Q97) and (iii) WT controls not expressing mHtt (WT). The efficiency of mHtt tamoxifen-induced brain and body gene ablation was examined at genomic (A) and protein (B) levels, respectively, in a cohort of 3-mo-old mice. In contrast to striatal Q97 specimens, PCR amplicons using primers that selectively detect the presence of mutant exon 1 (A) and Western blot (B) analysis probed with the mAb2166 anti-Htt antibody (∼350 kDa, Bottom) show no discernible bands in control and Q97^CRE specimens. Tissue-specific expression of Cre^ERT2 was also verified in CAG-Cre^ERT2 mice carrying a floxed inducible reporter (zsGreen1). (C) After tamoxifen recombination at PND21, 3-mo-old specimens widely expressed the fluorescence protein throughout the brain and body.

Fig. 2.

Q97^CRE mice display late-life appearance of striatal cellular degeneration and enhanced vulnerability to QA. Semithin sections (1 μm) of 9-mo-old striatum of female mice show that the number of darkly stained degenerating toluidine blue-positive cells was significantly higher in both Q97^CRE (A, arrowheads) and Q97 mice compared with CTL mice (B, percentage ± 95% confidence interval). (C–E) Electron micrographic images showing representative degenerating cells in the striatum of Q97^CRE mouse specimens. D corresponds to the boxed area in C, illustrating the presence of an axosomatic synapse (arrowheads). The asterisk in E identifies the nucleus of a normal striatal cell. (G–I) Representative striatal specimens that received a single intrastriatal injection of QA and were thereafter treated with Fluoro-Jade C to label dying cells (an asterisk depicts the site of the QA lesion). Arrowheads in H and I depict scattered Fluoro-Jade C–positive cells throughout the striatum. (Scale bars: A, 100 μm; D, 0.33 μm; C and E, 1 μm; I, 220 μm.) *P < 0.05 for statistical comparisons between CTL and either Q97^CRE or Q97 specimens.

Fig. 3.

Q97^CRE mice have analogous, although less severe, motor deficits compared with Q97 mice. Behavioral examinations were performed at 3 and 9 mo of age in a single cohort of female mice from each experimental group (n = 10, n = 9, and n = 12 for Q97^CRE, Q97, and CTL mice, respectively). Compared with CTL mice, both Q97^CRE and Q97 mice displayed deficits in limb grip strength (paw grip endurance hanging wire test) at 9 mo of age (A) and a higher number of slips in the balance beam test at 3 and 9 mo of age (B). (C and D) Consistently, repeated rotarod tests at 3 and 9 mo of age also revealed significant deficits in motor coordination in Q97^CRE mice. Individual values represent the mean ± SEM. P values (*) depicted at the top or next to each experimental group in A and B represent the statistical comparison with corresponding age-matched controls.

Fig. 4.

Both Q97^CRE and Q97 mice exhibit abnormal striatal electrophysiological activity. (A and B) Drivable microarrays were implanted within the dorsolateral striatum to record the single-unit activity of awake, behaving 3- to 4-mo-old and 9- to 12-mo-old mice. (C) In 3- to 4-mo-old mice, MSN firing rates from Q97 (n = 22) and Q97^CRE (n = 20) mice were similar to CTL (n = 27) mice. (D) ISI CV was increased in Q97 and Q97^CRE MSNs compared with CTL MSNs. (E) GIN firing rates from Q97 (n = 21) and Q97^CRE (n = 15) mice were significantly lower than in CTL (n = 27) mice. (F) In comparison to CTL units, the SD of the ISI of GINs was higher for both Q97 and Q97^CRE units. In 9- to 12-mo-old mice, MSN firing rates from Q97 (n = 23) and Q97^CRE (n = 27) mice were similar to CTL (n = 26) mice (G); however, the CV of the ISI was decreased in both Q97 (n = 23) and Q97^CRE (n = 27) mice in comparison to CTL (n = 26) units (H). No differences were detected in the firing rate (I) or the CV of the ISI of GINs (J). Data are shown as whisker box plots representing the median (at the center), interquartile range (outer box), and minimum and maximum values (whiskers). *P < 0.05; **P < 0.001.

Fig. 5.

Q97^CRE mice display impairments in corticostriatal connectivity and plasticity. (A) Proportion of cells exhibiting an excitatory response to cortical stimulation was reduced in both Q97 and Q97^CRE mice in comparison to CTL mice (CTL = 47, Q97 = 49, and Q97^CRE = 47 cells). (B) Correspondence to the summary data for the number of extra spikes elicited in striatal units displaying excitation by cortical stimulation (CTL = 32, Q97 = 22, and Q97^CRE = 17 cells). The number of extra spikes was similar among all experimental groups. (C) Correspondence to the summary data for latency to elicited excitation in striatal units by cortical stimulation. The latency was similar among all experimental groups. (D) Proportion of striatal cells undergoing LTP is increased in both Q97 and Q97^CRE mice in comparison to CTL mice (CTL = 27, Q97 = 22, and Q97^CRE = 15 cells). (E and F) Summary data for striatal units undergoing LTD or LTP, respectively, for all experimental groups. (E) Ratio of spikes before and after HFS for cells undergoing LTD was not statistically different among genotypes. (F) Ratio of spikes before and after HFS for cells undergoing LTP among all experimental groups was not statistically different among genotypes. We used six to eight mice per genotype in all of the experimental paradigms. The mean and SEM are displayed in B, C, E, and F, and each individual point represents a striatal unit.

Fig. S3.

Types of striatal response to cortical stimulation and effects of HFS on striatal units. A depiction of types of responses of striatal cells to cortical stimulation [excitation (A), inhibition (B), and no response (C)] is shown. (D) Peristimulus histogram (PSTH) for a striatal cell displaying LTD. (Upper) Striatal unit before HFS. (Lower) Same striatal unit displaying a reduction in the number of spikes elicited after HFS. (E) PSTH for a striatal unit displaying LTP. (Upper) Striatal unit before HFS. (Lower) Same striatal unit displaying an increase in the number of spikes elicited. FR, firing rate.

Fig. S4.

C57BL Q97^CRE mice exhibit motoric and striatal neurodegenerative changes. The FVB-NJ genetic background was outbred for 10 successive matings using double-heterozygous BACHD⁺/CAG⁻CRE^ER+ offspring with WT C57BL mice. (A) At 9 mo of age, the motoric performance of tamoxifen-treated CTL, Q97, and Q97^CRE mice was examined with the balance beam test. Compared with controls, Q97^CRE and Q97 mice have significantly higher numbers of slips (mean ± SEM; mean comparisons were performed with the Kruskal–Wallis test, whereas post hoc analyses used Dunn’s multiple comparisons test). (B) At 12 mo of age, the striata of Q97 and Q97^CRE mice were examined with electron microscopy and exhibited enhanced neuronal degeneration (bars depict percentage ± 95% confidence interval; each comparison was made using the χ² test). *P < 0.05. (C and D) Representative normal and degenerating neurons in CTL and Q97^CRE specimens, respectively. (Scale bars: C and D represent 2 μm.)

Fig. S5.

WT and CAG-CRE^ER mice display similar performance in motor coordination tests and exhibit comparable body weight. Female mice (n = 8 per genotype: WT and CAG-CRE^ER) were examined at 3 and 9 mo of age for motor coordination. Mouse performance in the grip strength (A), balance beam (B), and rotarod (C) tests is depicted. (D) Mouse body weight of both genotypes. P values in A, B, and D were calculated using the Student t test. F and P values in C represent the genotype effects calculated using a general linear ANOVA model. All values depicted correspond to mean ± SEM.