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

The Huntington's disease gene (HTT) 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 HdhQ111 mice exhibit higher levels of somatic HTT CAG expansion on a C57BL/6 genetic background (B6.HdhQ111) than on a 129 background (129.HdhQ111). Linkage mapping in (B6x129).HdhQ111 F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR) gene Mlh1 as the most likely candidate modifier. Crossing B6.HdhQ111 mice onto an Mlh1 null background demonstrated that Mlh1 is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. HdhQ111 somatic expansion was also abolished in mice deficient in the Mlh3 gene, implicating MutLγ (MLH1–MLH3) complex as a key driver of somatic expansion. Strikingly, Mlh1 and Mlh3 genes encoding MMR effector proteins were as critical to somatic expansion as Msh2 and Msh3 genes encoding DNA mismatch recognition complex MutSβ (MSH2–MSH3). The Mlh1 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 Mlh1 and Mlh3 as novel critical genetic modifiers of HTT CAG instability, point to Mlh1 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

Mismatch Repair Genes Mlh1 and Mlh3 Modify CAG Instability in Huntington\u27s Disease Mice: Genome-Wide and Candidate Approaches

The Huntington\u27s disease gene (HTT) 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\u27s disease Hdh(Q111) mice exhibit higher levels of somatic HTT CAG expansion on a C57BL/6 genetic background (B6.Hdh(Q111) ) than on a 129 background (129.Hdh(Q111) ). Linkage mapping in (B6x129).Hdh(Q111) F2 intercross animals identified a single quantitative trait locus underlying the strain-specific difference in expansion in the striatum, implicating mismatch repair (MMR) gene Mlh1 as the most likely candidate modifier. Crossing B6.Hdh(Q111) mice onto an Mlh1 null background demonstrated that Mlh1 is essential for somatic CAG expansions and that it is an enhancer of nuclear huntingtin accumulation in striatal neurons. Hdh(Q111) somatic expansion was also abolished in mice deficient in the Mlh3 gene, implicating MutLγ (MLH1-MLH3) complex as a key driver of somatic expansion. Strikingly, Mlh1 and Mlh3 genes encoding MMR effector proteins were as critical to somatic expansion as Msh2 and Msh3 genes encoding DNA mismatch recognition complex MutSβ (MSH2-MSH3). The Mlh1 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 Mlh1 and Mlh3 as novel critical genetic modifiers of HTT CAG instability, point to Mlh1 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

A novel approach to investigate tissue-specific trinucleotide repeat instability

Abstract Background In Huntington's disease (HD), an expanded CAG repeat produces characteristic striatal neurodegeneration. Interestingly, the HD CAG repeat, whose length determines age at onset, undergoes tissue-specific somatic instability, predominant in the striatum, suggesting that tissue-specific CAG length changes could modify the disease process. Therefore, understanding the mechanisms underlying the tissue specificity of somatic instability may provide novel routes to therapies. However progress in this area has been hampered by the lack of sensitive high-throughput instability quantification methods and global approaches to identify the underlying factors. Results Here we describe a novel approach to gain insight into the factors responsible for the tissue specificity of somatic instability. Using accurate genetic knock-in mouse models of HD, we developed a reliable, high-throughput method to quantify tissue HD CAG repeat instability and integrated this with genome-wide bioinformatic approaches. Using tissue instability quantified in 16 tissues as a phenotype and tissue microarray gene expression as a predictor, we built a mathematical model and identified a gene expression signature that accurately predicted tissue instability. Using the predictive ability of this signature we found that somatic instability was not a consequence of pathogenesis. In support of this, genetic crosses with models of accelerated neuropathology failed to induce somatic instability. In addition, we searched for genes and pathways that correlated with tissue instability. We found that expression levels of DNA repair genes did not explain the tissue specificity of somatic instability. Instead, our data implicate other pathways, particularly cell cycle, metabolism and neurotransmitter pathways, acting in combination to generate tissue-specific patterns of instability. Conclusion Our study clearly demonstrates that multiple tissue factors reflect the level of somatic instability in different tissues. In addition, our quantitative, genome-wide approach is readily applicable to high-throughput assays and opens the door to widespread applications with the potential to accelerate the discovery of drugs that alter tissue instability

Large-Scale Phenotyping of an Accurate Genetic Mouse Model of JNCL Identifies Novel Early Pathology Outside the Central Nervous System

Cln3Δex7/8 mice harbor the most common genetic defect causing juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive disease involving seizures, visual, motor and cognitive decline, and premature death. Here, to more thoroughly investigate the manifestations of the common JNCL mutation, we performed a broad phenotyping study of Cln3Δex7/8 mice. Homozygous Cln3Δex7/8 mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10–14 weeks of age. Homozygous Cln3Δex7/8 mice also displayed electroretinographic changes reflecting cone function deficits past 5 months of age and a progressive decline of retinal post-receptoral function. Metabolic analysis revealed increases in rectal body temperature and minimum oxygen consumption in 12–13 week old homozygous Cln3Δex7/8mice, which were also seen to a lesser extent in heterozygous Cln3Δex7/8 mice. Heart weight was slightly increased at 20 weeks of age, but no significant differences were observed in cardiac function in young adults. In a comprehensive blood analysis at 15–16 weeks of age, serum ferritin concentrations, mean corpuscular volume of red blood cells (MCV), and reticulocyte counts were reproducibly increased in homozygous Cln3Δex7/8 mice, and male homozygotes had a relative T-cell deficiency, suggesting alterations in hematopoiesis. Finally, consistent with findings in JNCL patients, vacuolated peripheral blood lymphocytes were observed in homozygous Cln3Δex7/8 neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3Δex7/8 mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3Δex7/8 mice that merit further study for JNCL biomarker development

Msh2 acts in medium-spiny striatal neurons as an enhancer of CAG instability and mutant huntingtin phenotypes in Huntington's disease knock-in mice.

The CAG trinucleotide repeat mutation in the Huntington's disease gene (HTT) exhibits age-dependent tissue-specific expansion that correlates with disease onset in patients, implicating somatic expansion as a disease modifier and potential therapeutic target. Somatic HTT CAG expansion is critically dependent on proteins in the mismatch repair (MMR) pathway. To gain further insight into mechanisms of somatic expansion and the relationship of somatic expansion to the disease process in selectively vulnerable MSNs we have crossed HTT CAG knock-in mice (HdhQ111) with mice carrying a conditional (floxed) Msh2 allele and D9-Cre transgenic mice, in which Cre recombinase is expressed specifically in MSNs within the striatum. Deletion of Msh2 in MSNs eliminated Msh2 protein in those neurons. We demonstrate that MSN-specific deletion of Msh2 was sufficient to eliminate the vast majority of striatal HTT CAG expansions in HdhQ111 mice. Furthermore, MSN-specific deletion of Msh2 modified two mutant huntingtin phenotypes: the early nuclear localization of diffusely immunostaining mutant huntingtin was slowed; and the later development of intranuclear huntingtin inclusions was dramatically inhibited. Therefore, Msh2 acts within MSNs as a genetic enhancer both of somatic HTT CAG expansions and of HTT CAG-dependent phenotypes in mice. These data suggest that the selective vulnerability of MSNs may be at least in part contributed by the propensity for somatic expansion in these neurons, and imply that intervening in the expansion process is likely to have therapeutic benefit

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