Conditional deletion of the <i>floxed Msh2</i> allele in the striatum. A.

<p>Genotyping for the conditional <i>Msh2</i> allele in genomic DNA extracted from striatum of <i>Msh2</i>+/+, <i>Msh2flox</i>/+, <i>Msh2flox</i>/+ <i>D9-Cre</i> and <i>Msh2flox</i>/flox <i>D9-Cre</i> mice. The deleted (Δ) <i>Msh2</i> allele is present only in mice harboring both the <i>Msh2flox</i> allele and the <i>D9-Cre</i> transgene. <b>B.</b> Genotyping for the conditional <i>Msh2</i> allele in genomic DNA extracted from five different tissues from a <i>Msh2flox</i>/+ <i>D9-Cre</i> mouse shows that the deletion is specific for the striatum. Mice were six weeks of age. <i>flox</i>: <i>Msh2</i> allele flanked by <i>loxP</i> sites; Δ:deleted <i>Msh2</i> allele; wt: wild-type <i>Msh2</i> allele.</p

Deletion of <i>Msh2</i> in medium-spiny striatal neurons eliminates the majority of striatal <i>HTT</i> CAG expansions.

<p>GeneMapper traces of PCR-amplified <i>HTT</i> CAG repeats from striatum, cortex, liver and tail DNA of representative five-month <i>HdhQ111</i>/+ mice (<b>A</b>) or from striatum and tail of representative ten month <i>HdhQ111</i>/+ mice (<b>C</b>) with <i>Msh2</i>+/+, <i>Msh2</i>+/−, <i>Msh2Δ</i>/<i>Δ</i>, <i>Msh2Δ</i>/− and <i>Msh2</i>−/− genotypes. Constitutive CAG repeat lengths, as determined in tail DNA, are indicated. Instability indices were quantified from GeneMapper traces of PCR-amplified <i>HTT</i> CAG repeats from five-month striatum, cortex and liver (<b>B</b>) and ten-month striatum (<b>D</b>) of <i>HdhQ111</i>/+ mice with <i>Msh2</i>+/+, <i>Msh2</i>+/−, <i>Msh2Δ</i>/<i>Δ</i>, <i>Msh2Δ</i>/− and <i>Msh2</i>−/−genotypes. Five-month mice: <i>Msh2</i>+/+ (n = 6, CAG 113, 118, 119, 121, 123, 125), <i>Msh2</i>+/− (n = 4, CAG 114, 114, 120, 123), <i>Msh2Δ</i>/<i>Δ</i>(n = 5, CAG 113, 121, 121, 126, 129), <i>Msh2Δ</i>/−(n = 7, CAG 113, 121, 121, 122, 125, 125, 133) and <i>Msh2</i>−/− (n = 3, CAG 112, 120, 123). Ten-month mice: <i>Msh2</i>+/+ (n = 6, CAG 118, 121, 121, 123, 126, 134), <i>Msh2</i>+/− (n = 4, CAG 116, 118, 123, 131), <i>Msh2Δ</i>/<i>Δ</i> (n = 1, CAG 133), <i>Msh2Δ</i>/− (n = 7, CAG 115, 115, 117, 120, 121, 122, 123) and <i>Msh2</i>−/− (n = 1, CAG 132). Bars represent mean ± S.D. *** p<0.0001, * p<0.05 (Student’s t-test).</p

Deletion of Msh2 in medium-spiny neurons delays nuclear huntingtin phenotypes. A, B.

<p>Nuclear mutant huntingtin immunostaining is decreased in the striata of five-month old <i>HdhQ111</i>/+ mice with deletion of <i>Msh2</i> in MSNs. <b>A.</b> Fluorescent micrographs of striata double-stained with anti-huntingtin mAb5374 and anti-histone H3 antibodies for three CAG repeat length-matched mice (<i>Msh2</i>+/+ CAG 113, <i>Msh2Δ</i>/<i>Δ</i> CAG 112, <i>Msh2−/−</i> CAG 113). <b>B.</b> Box plot showing upper and lower quartiles, median and range for the normalized mAb5374 immunostaining intensity (total mAb5374 staining intensity normalized to the number of H3-positive nuclei). Outlier (circle) is defined by a standard interquartile method and is included in the analysis. Multiple regression analysis was used to determine the effect of <i>Msh2</i> genotype on mAb5374 staining using normalized mAb5374 intensity (continuous variable) as a dependent variable and <i>Msh2</i> genotype (discrete variable), constitutive CAG length (continuous variable) and position (medial versus lateral, discrete variable) as independent variables. Both constitutive CAG length (P<0.05) and medial versus lateral position (P<0.001) were significantly associated with normalized mAb5374 intensity. Asterisks above the bars indicate a significant difference from <i>Msh2</i>+/+ at a p-value cut-off of p<0.05(*), p<0.01 (**), p<0.001 (***) in the regression analysis. <i>Msh2Δ</i>/− was not significantly different from <i>Msh2</i>+/− (p = 0.18). The five-month mice used in the quantitative analysis are as follows: <i>Msh2</i>+/+ (n = 6, CAG 113, 118, 119, 121, 123, 125), <i>Msh2</i>+/− (n = 4, CAG 114, 114, 120, 123), <i>Msh2Δ</i>/<i>Δ</i> (n = 5, CAG 113, 121, 121, 126, 129), <i>Msh2Δ</i>/− (n = 7, CAG 113, 121, 121, 122, 125, 125, 133) and <i>Msh2</i>−/− (n = 3, CAG 112, 120, 123). Note that the relatively “weak” effect of the <i>Msh2</i>−/− genotype likely reflects the small number of mice of this genotype and hence the least accurate estimate of the relationship of mAb5374 intensity to CAG length in the regression analysis. <b>C, D.</b> Intranuclear inclusions are decreased in the striata of ten-month old <i>HdhQ111</i>/+ mice with deletion of <i>Msh2</i> in MSNs. <b>C.</b> Fluorescent micrographs of striata stained with mAb5374 from mice with <i>Msh2</i>+/+ (CAG 121), <i>Msh2</i>+/− (CAG 123), <i>Msh2Δ</i>/<i>Δ</i> (CAG 133), <i>Msh2Δ</i>/− (CAG 123) and <i>Msh2</i>−/− (CAG 132) genotypes. <b>D.</b> Quantification of the percentage of cells containing an inclusion (more than one inclusion per cell was rarely observed). The total number of cells was determined by co-staining with histone H3 (not shown). The ten-month mice used in the quantitative analysis are as follows: <i>Msh2</i>+/+ (n = 6, CAG 118, 121, 121, 123, 126, 134), <i>Msh2</i>+/− (n = 4, CAG 116, 118, 123, 131), <i>Msh2Δ</i>/<i>Δ</i> (n = 1, CAG 133), <i>Msh2Δ</i>/− (n = 7, CAG 115, 115, 117, 120, 121, 122, 123) and <i>Msh2</i>−/− (n = 1, CAG 132). Bars represent mean ±S.D.</p

Mismatch Repair Genes <i>Mlh1</i> and <i>Mlh3</i> Modify CAG Instability in Huntington's Disease Mice: Genome-Wide and Candidate Approaches

<div><p>The Huntington's disease gene (<i>HTT</i>) CAG repeat mutation undergoes somatic expansion that correlates with pathogenesis. Modifiers of somatic expansion may therefore provide routes for therapies targeting the underlying mutation, an approach that is likely applicable to other trinucleotide repeat diseases. Huntington's disease <i>Hdh^Q111</i> mice exhibit higher levels of somatic <i>HTT</i> CAG expansion on a C57BL/6 genetic background (B6.<i>Hdh^Q111</i>) than on a 129 background (129.<i>Hdh^Q111</i>). Linkage mapping in (B6x129).<i>Hdh^Q111</i> F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR) gene <i>Mlh1</i> as the most likely candidate modifier. Crossing B6.<i>Hdh^Q111</i> mice onto an <i>Mlh1</i> null background demonstrated that <i>Mlh1</i> is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. <i>Hdh^Q111</i> somatic expansion was also abolished in mice deficient in the <i>Mlh3</i> gene, implicating MutLγ (MLH1–MLH3) complex as a key driver of somatic expansion. Strikingly, <i>Mlh1</i> and <i>Mlh3</i> genes encoding MMR effector proteins were as critical to somatic expansion as <i>Msh2</i> and <i>Msh3</i> genes encoding DNA mismatch recognition complex MutSβ (MSH2–MSH3). The <i>Mlh1</i> locus is highly polymorphic between B6 and 129 strains. While we were unable to detect any difference in base-base mismatch or short slipped-repeat repair activity between B6 and 129 MLH1 variants, repair efficiency was MLH1 dose-dependent. MLH1 mRNA and protein levels were significantly decreased in 129 mice compared to B6 mice, consistent with a dose-sensitive MLH1-dependent DNA repair mechanism underlying the somatic expansion difference between these strains. Together, these data identify <i>Mlh1</i> and <i>Mlh3</i> as novel critical genetic modifiers of <i>HTT</i> CAG instability, point to <i>Mlh1</i> genetic variation as the likely source of the instability difference in B6 and 129 strains and suggest that MLH1 protein levels play an important role in driving of the efficiency of somatic expansions.</p></div

The <i>Mlh1</i> locus is highly polymorphic between B6 and 129 strains.

<p>The <i>Mlh1</i> locus is highly polymorphic between B6 and 129 strains.</p

Reduced MLH1 expression in 129 versus B6 mice.

<p>Quantification of MLH1 (A) mRNA and (B, C) protein levels in the striatum of B6.<i>Mlh1^+/+</i>, 129.<i>Mlh1^+/+</i> and B6.<i>Mlh1^+/−</i> 10-week-old mice (<i>n</i> = 3). (A) Striatal <i>Mlh1</i> mRNA levels (TaqMan Mm00503449_m1, exons 11–12) in 129.<i>Mlh1^+/+</i> mice were significantly reduced by approximately 50% when compared to B6.<i>Mlh1^+/+</i> (<i>p</i><0.05), and were comparable to levels in B6.<i>Mlh1^+/−</i> mice. (B, C) Western blot analysis of MLH1 protein revealed significantly reduced levels in 129.<i>Mlh1^+/+</i> striata compared to B6.<i>Mlh1^+/+</i> striata. Bar graphs represent mean ±SD. *, <i>p</i><0.05; **, <i>p</i><0.01.</p

Striatal <i>HTT</i> CAG instability in 10-week-old <i>Hdh^Q111/+</i> mice on different genetic backgrounds.

<p>Graphical representation of striatal CAG instability indices from individual (A) B6, 129, (B6x129).F1 and (B6x129).F2 mice, color-coded based on strain genetic background; and from (B) (B6x129).F2 mice color-coded by genotype at the <i>Mlh1</i>, <i>Msh3</i> and <i>Msh2</i> genes (“undetermined” indicates failed genotype). F2 mice homozygous or heterozygous for B6 <i>Mlh1</i> alleles display significantly higher levels of striatal somatic CAG instability than F2 mice homozygous for 129 <i>Mlh1</i> alleles (<i>p</i><0.0001 for both). No relationship could be established between <i>Msh3</i> or <i>Msh2</i> genotype and striatal CAG instability. B6.<i>Hdh^Q111/+</i>, <i>n</i> = 10, CAG116.9±1.2SD; 129.<i>Hdh^Q111/+</i>, <i>n</i> = 12, CAG110.9±1.2SD; (B6x129).<i>Hdh^Q111/+</i> F1, <i>n</i> = 11, CAG114.7±6.4SD; (B6x129).<i>Hdh^Q111/+</i> F2, <i>n</i> = 69, CAG107.7±3.2SD. dbSNP markers located within MMR genes: <i>Mlh1</i>, rs30131926 and rs30174694 (concordant genotypes detected with both markers); <i>Msh3</i>, rs29551174; <i>Msh2</i>, rs33609112 and rs49012398 (concordant genotypes detected with both markers). Horizontal bars represent the mean CAG instability indices of the respective groups.</p

<i>Mlh1</i> is required for striatal and liver <i>HTT</i> CAG repeat instability in B6.<i>Hdh^Q111/+</i> mice.

<p>(A) Representative GeneMapper profiles of <i>HTT</i> CAG repeat size distributions in the tail, striatum and liver of 22-week-old B6.<i>Hdh^Q111/+</i> mice on different <i>Mlh1</i> genetic backgrounds. <i>Mlh1^+/+</i>, CAG113; <i>Mlh1^+/−</i>, CAG113; <i>Mlh1^−/−</i>, CAG110. (B) Quantification of striatal and liver <i>HTT</i> CAG instability indices in these mice reveals a statistically significant decrease in <i>HTT</i> CAG instability in the absence of <i>Mlh1</i>. <i>Mlh1^+/+</i>, CAG115.3±4.9SD, <i>n</i> = 6; <i>Mlh1^+/−</i>, CAG112.0±2.1SD, <i>n</i> = 6; <i>Mlh1^−/−</i>, CAG109.3±2.6SD, <i>n</i> = 6. Bar graphs represent mean ±SD. ****, <i>p</i><0.0001.</p

Repair of a single CTG slip-out in a cell-free MMR assay is MLH1 dose-dependent.

<p>(A) Short slipped-DNA repair using HeLa or HEK293T (MutLα-deficient) whole cell extracts complemented with equal amounts (100 ng) of purified MutLα protein complexes: hMLH1-hPMS2, mMLH1.B6-hPMS2 or mMLH1.129-hPMS2. Both B6 and 129 MLH1 proteins show ability to repair the mismatch when in a complex with hPMS2. The individual lanes represented are from the same blot. (B) Repair using MutLα-deficient HEK293T cell extracts complemented with increasing concentrations (5, 25 and 100 ng) of either mMLH1.B6-hPMS2 or mMLH1.129-hPMS2 protein complexes. Quantification of repair suggests that both B6 and 129 MLH1 proteins are comparably efficient at repairing CTG slip-outs. In addition, it suggests a MutLα dose-dependency, with higher concentrations of mMLH1-hPMS2 resulting in higher levels of MMR activity (<i>p</i> = 0.0013). The individual lanes represented are from the same blot and the experiment was reproduced three times. Bars graphs represent mean ±SD.</p

The 129 and B6 3′-flanking regions of <i>Mlh1</i> confer differential mRNA regulation.

<p>Investigation of the regulatory potential of B6 and 129 immediate (A) 5′- and (B) 3′-flanking regions of <i>Mlh1</i> using dual luciferase reporter assays. (A) The immediate 5′-flanking region of <i>Mlh1</i> containing 17 B6-129 polymorphisms (2,441 bp) was used to drive firefly luciferase expression. (B) The immediate 3′-flanking region of <i>Mlh1</i> (<i>i–iv</i>) containing either 19, 15, 4 or 1 B6-129 polymorphism(s) (1,676 bp, 1,280 bp, 591 bp and 205 bp, respectively) was cloned downstream of a firefly luciferase gene. “Swap” constructs (<i>v</i>) of the immediate 3′-flanking region of <i>Mlh1</i> containing either 4, 5 or 10 129 polymorphisms (530 bp, 438 bp and 708 bp, respectively; total 1676 bp) were cloned downstream of a firefly luciferase gene. Relative luciferase activity was determined by normalization to internal <i>Renilla</i> luminescence and determined relative to the analogous B6 construct. B6-129 polymorphisms are represented by open triangles. Bar graphs represent mean ±SD. *, <i>p</i><0.05; **, <i>p</i><0.01; ***, <i>p</i><0.001.</p