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. 2012;7(9):e44273.
doi: 10.1371/journal.pone.0044273. Epub 2012 Sep 7.

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

Marina Kovalenko  1 , Ella Dragileva, Jason St Claire, Tammy Gillis, Jolene R Guide, Jaclyn New, Hualing Dong, Raju Kucherlapati, Melanie H Kucherlapati, Michelle E Ehrlich, Jong-Min Lee, Vanessa C Wheeler
Affiliations

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

Marina Kovalenko et al. PLoS One. 2012.

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Conditional deletion of the floxed Msh2 allele in the striatum. A.
Genotyping for the conditional Msh2 allele in genomic DNA extracted from striatum of Msh2+/+, Msh2flox/+, Msh2flox/+ D9-Cre and Msh2flox/flox D9-Cre mice. The deleted (Δ) Msh2 allele is present only in mice harboring both the Msh2flox allele and the D9-Cre transgene. B. Genotyping for the conditional Msh2 allele in genomic DNA extracted from five different tissues from a Msh2flox/+ D9-Cre mouse shows that the deletion is specific for the striatum. Mice were six weeks of age. flox: Msh2 allele flanked by loxP sites; Δ:deleted Msh2 allele; wt: wild-type Msh2 allele.
Figure 2
Figure 2. Msh2 protein levels in the striata of Msh2 conditional knockout mice. A.
Representative immunoblot of striatal lysates from five-month HdhQ111/+ mice with Msh2+/+, Msh2+/−, Msh2Δ/Δ, Msh2Δ/− and Msh2−/− genotypes probed with an anti-Msh2 antibody (upper panel). The membrane was stripped and re-probed with an anti-α-tubulin antibody (lower panel). Arrowhead indicates Msh2 protein. B. Quantification of immunoblots. The density of Msh2 bands from four immunoblots, each containing striatal protein lysates from different HdhQ111/+ mice with Msh2+/+ (n = 6, CAG 114, 116, 120, 123, 123, 124), Msh2+/− (n = 5, CAG 110, 114, 117, 119, 122), Msh2Δ/Δ (n = 3, CAG 118, 124, 126), Msh2Δ/− (n = 7, CAG 111, 113, 119, 120, 123, 124, 124), and Msh2−/− (n = 2, CAG 125 and a Hdh+/+ mouse) genotypes was quantified with QuantityOne software and normalized by the density of the corresponding α-tubulin bands. Four protein blots were run; on each, the Msh2/α-tubulin ratio was normalized to that of Msh2+/+ (100%) on that gel. Normalized Msh2/α-tubulin ratios were averaged across the 4 gels. Bars represent mean ±SD. **p<0.01; ***p<0.001. C. Fluorescent micrographs of striatal sections from five-month HdhQ111/+ mice with Msh2+/+, Msh2+/−, Msh2Δ/Δ, Msh2Δ/− and Msh2−/− genotypes co-stained with anti-MSH2 ab70270 and anti-DARPP-32 D32-6a antibodies. Note the significant overlap in DARPP-32 and Msh2 signals in Msh2+/+ and Msh2+/− striata, the loss of specific Msh2 signal in all cells in Msh2−/− striata and the specific loss of Msh2 signal in DARPP-32-positive cells in Msh2Δ/Δ and Msh2Δ/− striata. D. Mean integrated intensity of Msh2 immunostaining in DARPP-32-positive cells (total integrated intensity of ab70270 staining in D32-6a-positive cells normalized by the number of D32-6a-positive cells) in the striatum of the following five-month mice: Msh2+/+ (n = 6, CAG 113, 118, 119, 121, 123, 125), Msh2+/− (n = 3, CAG 114, 114, 123), Msh2Δ/Δ (n = 4, CAG 121, 121, 126, 129), Msh2Δ/− (n = 7, CAG 113, 121, 121, 122, 125, 125, 133), and Msh2−/− (n = 3, CAG 112, 120, 123). Bars represent mean ±S.D. *** p<0.0001, ** p<0.01, * p<0.05 (Student’s t-test).
Figure 3
Figure 3. Deletion of Msh2 in medium-spiny striatal neurons eliminates the majority of striatal HTT CAG expansions.
GeneMapper traces of PCR-amplified HTT CAG repeats from striatum, cortex, liver and tail DNA of representative five-month HdhQ111/+ mice (A) or from striatum and tail of representative ten month HdhQ111/+ mice (C) with Msh2+/+, Msh2+/−, Msh2Δ/Δ, Msh2Δ/− and Msh2−/− genotypes. Constitutive CAG repeat lengths, as determined in tail DNA, are indicated. Instability indices were quantified from GeneMapper traces of PCR-amplified HTT CAG repeats from five-month striatum, cortex and liver (B) and ten-month striatum (D) of HdhQ111/+ mice with Msh2+/+, Msh2+/−, Msh2Δ/Δ, Msh2Δ/− and Msh2−/−genotypes. Five-month mice: Msh2+/+ (n = 6, CAG 113, 118, 119, 121, 123, 125), Msh2+/− (n = 4, CAG 114, 114, 120, 123), Msh2Δ/Δ(n = 5, CAG 113, 121, 121, 126, 129), Msh2Δ/−(n = 7, CAG 113, 121, 121, 122, 125, 125, 133) and Msh2−/− (n = 3, CAG 112, 120, 123). Ten-month mice: Msh2+/+ (n = 6, CAG 118, 121, 121, 123, 126, 134), Msh2+/− (n = 4, CAG 116, 118, 123, 131), Msh2Δ/Δ (n = 1, CAG 133), Msh2Δ/− (n = 7, CAG 115, 115, 117, 120, 121, 122, 123) and Msh2−/− (n = 1, CAG 132). Bars represent mean ± S.D. *** p<0.0001, * p<0.05 (Student’s t-test).
Figure 4
Figure 4. Deletion of Msh2 in medium-spiny neurons delays nuclear huntingtin phenotypes. A, B.
Nuclear mutant huntingtin immunostaining is decreased in the striata of five-month old HdhQ111/+ mice with deletion of Msh2 in MSNs. A. Fluorescent micrographs of striata double-stained with anti-huntingtin mAb5374 and anti-histone H3 antibodies for three CAG repeat length-matched mice (Msh2+/+ CAG 113, Msh2Δ/Δ CAG 112, Msh2−/− CAG 113). 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 Msh2 genotype on mAb5374 staining using normalized mAb5374 intensity (continuous variable) as a dependent variable and Msh2 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 Msh2+/+ at a p-value cut-off of p<0.05(*), p<0.01 (**), p<0.001 (***) in the regression analysis. Msh2Δ/− was not significantly different from Msh2+/− (p = 0.18). The five-month mice used in the quantitative analysis are as follows: Msh2+/+ (n = 6, CAG 113, 118, 119, 121, 123, 125), Msh2+/− (n = 4, CAG 114, 114, 120, 123), Msh2Δ/Δ (n = 5, CAG 113, 121, 121, 126, 129), Msh2Δ/− (n = 7, CAG 113, 121, 121, 122, 125, 125, 133) and Msh2−/− (n = 3, CAG 112, 120, 123). Note that the relatively “weak” effect of the Msh2−/− 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. C, D. Intranuclear inclusions are decreased in the striata of ten-month old HdhQ111/+ mice with deletion of Msh2 in MSNs. C. Fluorescent micrographs of striata stained with mAb5374 from mice with Msh2+/+ (CAG 121), Msh2+/− (CAG 123), Msh2Δ/Δ (CAG 133), Msh2Δ/− (CAG 123) and Msh2−/− (CAG 132) genotypes. D. 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: Msh2+/+ (n = 6, CAG 118, 121, 121, 123, 126, 134), Msh2+/− (n = 4, CAG 116, 118, 123, 131), Msh2Δ/Δ (n = 1, CAG 133), Msh2Δ/− (n = 7, CAG 115, 115, 117, 120, 121, 122, 123) and Msh2−/− (n = 1, CAG 132). Bars represent mean ±S.D.
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