A CAG repeat threshold for therapeutics targeting somatic instability in Huntington's disease

Abstract

The Huntington's disease mutation is a CAG repeat expansion in the huntingtin gene that results in an expanded polyglutamine tract in the huntingtin protein. The CAG repeat is unstable and expansions of hundreds of CAGs have been detected in Huntington's disease post-mortem brains. The age of disease onset can be predicted partially from the length of the CAG repeat as measured in blood. Onset age is also determined by genetic modifiers, which in six cases involve variation in DNA mismatch repair pathways genes. Knocking-out specific mismatch repair genes in mouse models of Huntington's disease prevents somatic CAG repeat expansion. Taken together, these results have led to the hypothesis that somatic CAG repeat expansion in Huntington's disease brains is required for pathogenesis. Therefore, the pathogenic repeat threshold in brain is longer than (CAG)40, as measured in blood, and is currently unknown. The mismatch repair gene MSH3 has become a major focus for therapeutic development, as unlike other mismatch repair genes, nullizygosity for MSH3 does not cause malignancies associated with mismatch repair deficiency. Potential treatments targeting MSH3 currently under development include gene therapy, biologics and small molecules, which will be assessed for efficacy in mouse models of Huntington's disease. The zQ175 knock-in model carries a mutation of approximately (CAG)185 and develops early molecular and pathological phenotypes that have been extensively characterized. Therefore, we crossed the mutant huntingtin allele onto heterozygous and homozygous Msh3 knockout backgrounds to determine the maximum benefit of targeting Msh3 in this model. Ablation of Msh3 prevented somatic expansion throughout the brain and periphery, and reduction of Msh3 by 50% decreased the rate of expansion. This had no effect on the deposition of huntingtin aggregation in the nuclei of striatal neurons, nor on the dysregulated striatal transcriptional profile. This contrasts with ablating Msh3 in knock-in models with shorter CAG repeat expansions. Therefore, further expansion of a (CAG)185 repeat in striatal neurons does not accelerate the onset of molecular and neuropathological phenotypes. It is striking that highly expanded CAG repeats of a similar size in humans cause disease onset before 2 years of age, indicating that somatic CAG repeat expansion in the brain is not required for pathogenesis. Given that the trajectory for somatic CAG expansion in the brains of Huntington's disease mutation carriers is unknown, our study underlines the importance of administering treatments targeting somatic instability as early as possible.

Keywords: Huntington’s disease; MSH3; genetic modifiers; pathogenic CAG repeat length; somatic CAG repeat instability.

Figure 1

Characterization of the Msh3 knockout line. (A) Msh3 knockout allele generation by CRISPR/Cas9 showing the position of guide RNAs (gRNA) targeting Msh3 exons 3 and 8 and the position of the quantitative PCR assays. (B) Expression of the Msh3 mRNA was confirmed, using a quantitative PCR assay with primers in the deleted region (Msh3_756), in the cortex of heterozygous Msh3^+/− and homozygous Msh3^−/− mice at 8 weeks of age (n= 5/genotype). (C) Western blotting with an MSH3 antibody showed that MSH3 levels were decreased in Msh3^+/− heterozygotes and absent in Msh3^−/− homozygotes. MSH3 levels were quantified by normalizing to α-tubulin (n = 5/wild-type, n = 4/Msh3^+/− and Msh3^−/−). (D) Schematic of the genetic cross performed to generate the six experimental genotypes as littermates. One-way ANOVA with Tukey's post hoc test. Error bars = standard error of the mean (SEM). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. The test statistic, degrees of freedom and P-values are summarized in Supplementary Table 3. Full-length blots are shown in Supplementary Fig. 5. WT = wild-type.

Figure 2

CAG repeat instability in zQ175 mice is dependent on Msh3 expression. Representative GeneMapper traces of CAG repeat profiles for brain regions and peripheral tissues from mice at 6 months of age (n = 8/genotype). The modal CAG repeat length in ear samples at 12 days of age (P12) for zQ175 mice was 186 ± 2.3, for the zQ175:Msh3^+/− mice was 185 ± 2.4 and for zQ175:Msh3^−/− mice was 182 ± 2.0. The representative traces are from the same mouse for each genotype with the CAG repeat mode at P12 indicated by the dashed line. Somatic instability in 6-month-old zQ175 mice was most pronounced in the striatum, brainstem, spinal cord and liver, and the repeat had contracted in the pituitary. Somatic CAG expansion was prevented in zQ175 mice in which Msh3 was absent, and contractions had led to a reduction in the CAG repeat mode. Heterozygosity for Msh3 led to a decrease in the extent of repeat expansion.

Figure 3

Tissue-specific changes in the instability index and CAG repeat mode of the of the CAG repeat profiles in zQ175, zQ175:Msh3^+/− and zQ175:Msh3^−/− mice. (A) Instability index quantified for the GeneMapper traces for each brain region and tissue normalized to P12 ear sample for each genotype. (A) Stepwise reduction in the instability index was observed for each brain region/tissue with the reduction and loss in Msh3 expression. (B) Quantification of the change in mode from the P12 ear sample. Modal CAG was reduced with Msh3 genotype, in all regions apart from striatum, heart and liver. n = 8/genotype, 2–3 technical replicates for each mouse. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. Box and whisker plots represent median and 25th and 75th percentiles with bars drawn from minimum to maximum. Statistical analysis was mixed-effects model (REML), with Tukey's correction for multiple comparisons. The test statistic, degrees of freedom and P-values are summarized in Supplementary Table 4.

Figure 4

Statistical analysis of the effect of heterozygosity and homozygosity for Msh3 knockout on somatic CAG repeat instability in the striatum. (A and B) Scores and loading plots from the principal component analysis of all summary statistics using ear and striatal data. The plots indicate the statistical variables contributing to the differences seen between genotypes: contraction, mode, median, skew, mean, expansion. II = instability index; log(IQR) = log interquartile range; log(var) = log variance, kurtosis; n = sum of peak heights. (C) Least squares means plots of instability index, expansion and variance summary statistics showing means (geometric means for variance) adjusted for ear and 95% confidence intervals for striatum across genotype. These data again confirm the significant effect of Msh3 genotype on instability, expansion and variation. P-value of effect test (genotype) shown.

Figure 5

Transcriptional dysregulation in the striatum of zQ175 mice is not alleviated by loss of Msh3 expression. (A–D) Volcano plots illustrating gene expression changes between (A) zQ175 and wild-type mice (B) zQ175:Msh3^−/− and wild-type mice, (C) zQ175 and zQ175:Msh3^−/− mice and (D) Msh3^−/− and wild-type mice, at 6 months of age. (E) Of the 2486 genes dysregulated in zQ175 mice as compared to wild-type mice at 6 months of age, 2408 showed negligible reversal, with a total of 15 genes showing full reversal and 51 partial reversal, indicating that only 3% of genes were rescued, a level considered to be background. Of the known 266 genes dysregulated in striatal tissues across models, 258 were detected as dysregulated, with none reversed and one exacerbated. (F and G) Expression levels for (F) striatal genes of interest: Cnr1, Drd1, Pde10a, Penk and Ppp1r1b and (G) the DNA mismatch repair genes Msh3, Msh2, Msh6 plotted as variance stabilizing transformed counts derived using DESeq2. Box and whisker plots represent median and 25th and 75th percentiles with bars drawn from minimum to maximum. They show there was no change in expression due to loss of Msh3. n = 8/genotype, four males/four females for each genotype except zQ175:Msh3^−/− at five males/three females. HD = Huntington’s disease; WT = wild-type.

Figure 6

Striatal nuclear HTT aggregation is not reduced or delayed with loss of Msh3. Coronal striatal sections from wild-type (WT), zQ175, zQ175:Msh3^+/− and zQ175:Msh3^−/− at 3 and 6 months of age were immunohistochemically stained with the S830 antibody. A nuclear counterstain was not applied to these images, as this would mask the nuclear signal. (A) HTT aggregation was detectable by 3 months of age in the zQ175 mice and S830 immunostaining levels were comparable irrespective of Msh3 genotype. (B) Thresholding was applied to images to quantify levels of staining of S830 positive objects, average area of object, and the percentage of image covered. The Msh3 genotype had no impact on these three measures of S830 staining. (C) At 6 months of age, in addition to diffuse nuclear aggregation, nuclear inclusions were also prominent in many nuclei. (D and E) Thresholding was applied to quantify the number of (D) S830 positive objects and (E) inclusions. In each case, the number of objects, the average area of the objects and the percentage of an image covered by the S830 signal was determined. One-way ANOVA with Bonferroni correction. Error bars = standard error of the mean (SEM). n = 5/zQ175 genotype, n = 3 wild-type. Scale bar in large image = 10 μm, scale bar in inset = 5 μm. M = months.

Figure 7

Striatal HTTexon1 aggregation is not affected by loss of Msh3 expression. (A) Schematic of epitope location of antibodies used in HTTexon1 homogeneous time-resolved fluorescence (HTRF) assays. (B) Levels of aggregated HTTexon1 as measured by the 4C9-MW8 assay indicated that aggregation increased from 2 to 6 months of age in zQ175 mice. There was no change in the levels of HTTexon1 aggregation detected between zQ175, zQ175:Msh3^+/− and zQ175:Msh3^+/− in the striatum, but small and significant differences were detected in the cortex and brainstem at 6 months of age. (C) High levels of soluble HTTexon1, as detected by the 2B7-MW8 assay, were present by 2 months of age in the zQ175 striatum, cortex and brainstem and had decreased at 6 months in all regions. There was no difference in soluble HTTexon1 at 6 months of age between the zQ175, zQ175:Msh3^+/− or zQ175:Msh3^−/− genotypes. Six month samples: one-way ANOVA with Bonferroni correction; 2-month and 6-month zQ175 and wild-type: two-way ANOVA with Bonferroni correction. Error bars = standard error of the mean (SEM). *P ≤ 0.05, ** P ≤ 0.01, ***P ≤ 0.001. n = 10/genotype. The test statistic, degrees of freedom and P-values are summarized in Supplementary Table 5. AU = arbitrary units; M = months; WT = wild-type.