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Human Molecular Genetics, 2003, Vol. 12, No. 3 273-281
© 2003 Oxford University Press

Mismatch repair gene Msh2 modifies the timing of early disease in HdhQ111 striatum

Vanessa C. Wheeler1,*, Lori-Anne Lebel1, Vladimir Vrbanac1, Allison Teed1, Hein te Riele2 and Marcy E. MacDonald1

1Molecular Neurogenetics Unit, Massachusetts General Hospital, Charlestown, MA 02129, USA and 2Division of Molecular Carcinogenesis, The Netherlands Cancer Institute, Amsterdam, The Netherlands

Received September 19, 2002; Accepted November 18, 2002


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Somatic instability of expanded HD CAG repeats that encode the polyglutamine tract in mutant huntingtin has been implicated in the striatal selectivity of Huntington's disease (HD) pathology. Here in HdhQ111 mice, we have tested whether a genetic background deficient in Msh2, expected to eliminate the unstable behavior of the 109 CAG array inserted into the murine HD gene, would alter the timing or striatal specificity of a dominant disease phenotype that predicts late-onset neurodegeneration. Our analyses of HdhQ111/+:Msh2+/+ and HdhQ111/+: Msh2-/- progeny revealed that, while inherited instability involved Msh2-dependent and -independent mechanisms, lack of Msh2 was sufficient to abrogate progressive HD CAG repeat expansion in striatum. The absence of Msh2 also eliminated striatal mutant huntingtin with somatically expanded glutamine tracts and caused an ~5 month delay in nuclear mutant protein accumulation, but did not alter the striatal specificity of this early phenotype. Thus, somatic HD CAG instability appears to be a consequence of a striatal-selective disease process that accelerates the timing of an early disease phenotype, via expansion of the glutamine tract in mutant huntingtin. Therefore Msh2, as a striking modifier of early disease onset in a precise genetic HD mouse model, provides a novel target for the development of pharmacological agents that aim to slow pathogenesis in man.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Huntington's disease (HD), with its dominantly inherited graded loss of striatal neurons and chorea, is initiated by unstable HD CAG repeat expansions that elongate a glutamine tract in huntingtin to more than ~39 residues (1,2). The expanded CAG repeat size is correlated with disease severity such that adult-onset HD typically involves arrays of ~40–50 repeats, whereas large expansions (>65 units) cause the rare juvenile form of the disease (3,4). While the size of the expanded HD CAG repeat is the major determinant of onset age, the ±18 year range found for any given repeat size has implicated modifying factors in the precise timing of symptoms (3,5).

Factors that act at a fundamental level to modify the size of the expanded HD CAG array would be expected to alter the disease trigger, i.e. the size of the glutamine tract in mutant huntingtin. Disease-causing HD CAG repeat arrays frequently undergo small size fluctuations during transmission from parent to child, with occasional large increases through the male germline, explaining the paternal inheritance of most juvenile-onset HD (3,4). Moreover, HD CAG instability in brain has been suggested to explain the neuronal specificity of HD pathology (6,7). This hypothesis is supported by striking somatic mosaicism of HD CAG tracts (6) and somatically expanded isoforms of mutant huntingtin (11) found in tissues of juvenile-onset HD cases, although somatic variation of adult onset HD CAG repeats is more limited (8–10).

Studies in HD mouse models have revealed instability of long HD CAG repeats that differs with the precise context of the array. In HD exon 1 transgenic mice, HD CAG tracts of 150–165 units carried on a random transgene integration exhibit paternally derived size increases and somatic expansions throughout the brain and peripheral tissues (12), along with widespread toxicity due to the encoded mutant amino terminal huntingtin product (13). By contrast, in lines of Hdh knock-in mice, where HD CAG repeats of ~80, 90 or 109 units inserted at the mouse HD locus alter the length of the glutamine tract in murine huntingtin, dominantly inherited instability comprises both contractions and expansions (7,14). Moreover, for each Hdh knock-in line somatic expansion occurs predominantly in striatum (7,14), suggesting that HD CAG repeat size increases may be one outcome of a disease process that has been shown to be dominant and selective for striatal neurons (15–17). Indeed, the striatal specificity and progressive nature of somatic HD CAG repeat expansion in Hdh knock-in mice has provided support for the proposal that mutant huntingtin with somatically expanded glutamine segments might contribute the neuronal specificity of HD pathology (7).

To test this hypothesis, we have now determined whether elimination of HD CAG repeat instability in HdhQ111 knock-in mice would alter the timing and/or the striatal selectivity of nuclear accumulation of full-length mutant huntingtin. This dominant phenotype, which is detected in medium spiny striatal neurons early in the disease cascade (15), is manifest in a glutamine length-dependent manner (15) that heralds late-onset degeneration (17). To manipulate HD CAG instability, the HdhQ111 allele was placed onto a genetic background deficient in the DNA mismatch repair protein Msh2 by breeding HdhQ111mice with Msh2 knock-out mice. The latter carry a targeted Msh2 inactivating mutation that leads to dinucleotide repeat instability and tumors at ~10–11 months of age (18). This genetic strategy was chosen because lack of Msh2 has been demonstrated to modify CAG repeat instability in bacteria (19) and yeast (20). Moreover, in HD exon 1 transgenic mice, Msh2 deficiency has been shown to prevent both the inherited paternal HD CAG tract size increases and somatic expansions found throughout the brain and periphery (21,23).

Our results reveal that multiple mechanisms must contribute to the complex pattern of inherited instability in HdhQ111 mice and, importantly, disclose Msh2 as a factor that strongly modifies the timing of early disease in HdhQ111 striatum, recommending investigation of this mismatch repair protein as a route to slowing disease in man.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Msh2 deficiency reveals multiple mechanisms for inherited HdhQ111 instability
After generating heterozygous HdhQ111/+ parents that were either wild-type Msh2+/+ or homozygous Msh2-/- knock-out, we first assessed whether Msh2 deficiency would indeed modify instability exhibited by the knock-in HdhQ111 allele, an 109 CAG array that, with penultimate CAACAG codons, extends the glutamine tract in murine huntingtin to 111 residues (15). We first monitored inherited instability, using HD CAG genotyping of tail-clip DNA to determine the size of the expanded CAG repeat tract in parents and their offspring. Figure 1A summarizes the results, depicting the frequency of alleles whose size was unchanged, expanded or contracted, compared with the parental repeat size. The Msh2-deficient background did not alter the frequency of maternally or paternally derived changes (~1–10 units). Instead, the direction of paternal size fluctuations was altered from predominantly increases on the wild-type background to only contractions in the absence of Msh2. By contrast, for female germline HdhQ111 transmissions some expansions and a preponderance of contractions were observed on both genetic backgrounds.



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  		Figure 1. Msh2 deficiency reveals multiple pathways for inherited instability. (A) A bar chart summary of progeny genotypes denoting the frequency of 109 CAG repeat alleles that were unchanged, expanded or contracted when transmitted from HdhQ111/+ males (paternal) or females (maternal) that were either Msh2-/- or Msh2+/+. For paternal Msh2+/+ transmissions (n=52), 36 (69%) alleles were unchanged, 12 (23%) expanded and four (8%) contracted. For paternal Msh2-/- transmissions (n=34), 18 (53%) alleles were unchanged, 0 (<3%) expanded and 16 (47%) contracted. The Msh2-/- background significantly altered the proportion of expansions versus contractions (Fisher's exact test, P<0.0001***), but not the overall frequency of changed alleles (31% Msh2+/+ versus 47% Msh2-/-; {chi}2 test). For maternal Msh2+/+ transmissions (n=71), 28 (39%) alleles were unchanged, eight (11%) expanded and 35 (49%) contracted. For maternal Msh2-/- transmissions (n=14), eight (57%) alleles were unchanged, two (14%) expanded and four (29%) contracted. The Msh2-/- background did not significantly alter the proportion of expanded versus contracted alleles (Fisher's exact test), or the overall frequency of changed alleles (61% Msh2+/+ versus 47% Msh2-/-; {chi}2 test). (B) A schematic describing a possible mechanism for the switch from HD CAG repeat expansions to contractions in the absence of Msh2. Secondary structure hairpin loops may form within a long CAG tract (line) that is tethered at each end by non-repetitive DNA sequence (black rectangles). Msh2 binding and stabilization (gray boxes) (22), recruitment of other mismatch repair proteins, and gap-generation followed by gap-filling DNA synthesis that generates DNA loops results in gain of DNA (dotted line), as described previously (23). In the absence of Msh2, hairpins may not be stabilized, but instead recognized as an abnormal DNA structure by other repair machinery, e.g. nucleotide excision repair (50). Excision of this structure would instead result in contraction of the CAG tract.

 
Thus, while Msh2 was required for paternal expansions, its absence revealed that the full pattern of inherited HdhQ111 instability must also stem from Msh2-independent mechanisms. A possible explanation for multiple processes in HD CAG instability, which accounts for the ‘switch’ in direction found in male transmissions, is depicted in Figure 1B. Expanded CAG repeat structures may be recognized by Msh2 and ‘repaired', resulting in expansion by a few units (22,23). If Msh2 complexes are not recruited, an excision repair pathway operating on the same repeat DNA structure may instead predominate, producing small contractions.

Progressive somatic expansion of the glutamine tract in mutant huntingtin
Somatic instability in HdhQ111 mice comprises successive expansion of the 109 CAG repeat array in striatum (14). As the repeat encodes the glutamine tract in mutant huntingtin, this finding suggests the progressive generation of isoforms with somatically expanded glutamine segments. To test this prediction, we used immunoblot analyses of striatal extracts from HdhQ111/+ heterozygotes at ages ranging from 1.5 to 28 months of age to assess the size of the glutamine segment in full-length mutant huntingtin. As shown in Figure 2, anti-huntingtin mAb2166 detected the ~340 kDa normal mouse protein (seven-glutamine tract) and a band of mutant protein with decreased mobility conferred by the 111-glutamine segment. By 28 months of age the mutant band signal was obviously decreased (mutant to normal band intensity=0.25), although an upward smear of weak mAb2166-reactivity was also evident. This ‘smear’ was not detected by anti-ubiquitin (data not shown), indicating that mobility shifts did not reflect ubiquitin-conjugation. Instead, probing with mAb1F8, a monoclonal reagent that selectively recognizes long glutamine tracts (24), detected the ‘smear’ bands, revealing altered mobility due to increased glutamine tract length. Thus, the reduction in 111-glutamine mutant protein detected by mAb2166 corresponded to an increasing proportion of mutant huntingtin with somatically expanded glutamine segments, although at younger ages the ‘smear’ bands were not detected, presumably because they are below the sensitivity of the immunoblot format.



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  		Figure 2. The length of the mutant huntingtin glutamine tract increases progressively. Immunoblots of soluble protein extracts from striata of heterozygous HdhQ111/+ mice at 5, 10 and 28 months, probed with mAb2166 or mAb1F8, are shown. The position of the 111-glutamine mutant huntingtin band (Q111), detected with both mAb1F8 and mAb2166, and the position of the 7-glutamine wild-type huntingtin band (Q7) detected with mAb2166, but not by mAb1F8, are indicated. The ‘smear’ of somatically expanded mutant huntingtin (E), evident at 28 months with mAb2166 and mAb1F8, is indicated with a bracket. Analyses at 1.5 months of age indicated bands of wild-type and mutant huntingtin but no ‘smear’ bands (data not shown).

 
Impact of Msh2 deficiency on striatal-specific somatic HdhQ111 expansion
We then determined whether Msh2 deficiency would eliminate the progressive CAG repeat expansions that occur in HdhQ111/+ striatum (14) and somatically expanded mutant huntingtin. Notably, lack of Msh2 led to fatal tumors in both HdhQ111/+ and Hdh+/+ mice (data not shown), restricting these analyses to mice up to ~10 months of age. Figure 3A shows typical results of genotyping to determine HD CAG repeat size in striatal DNAs from HdhQ111/+ heterozygotes on a wild-type Msh2+/+ or Msh2-/- genetic background at 1.5, 5 and 10 months of age. The ‘smear’ of somatically expanded HD CAG alleles on the wild-type Msh2 background, evident by 5 months and further increased at 10 months, was not detected on the Msh2-deficient background at any age. Densitometry of the PCR products, shown for 10 months of age in Figure 3A, confirmed the lack of somatic expansions, or contractions, in the absence of Msh2. Thus, striatal HdhQ111 instability comprised successive size increases mediated solely by Msh2-dependent processes.



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  		Figure 3. Msh2 deficiency eliminates striatal HD CAG and glutamine tract expansion. (A) Autoradiograms of HD CAG repeat PCR products amplified from HdhQ111/+:Msh2+/+ and HdhQ111/+:Msh2-/- striatal DNA at 1.5, 5 and 10 months of age. The progressive ‘smear’ of larger products that becomes evident with age was not detected in the absence of Msh2. This effect was quantified by densitometry of autoradiograms. Shown are densitometric traces at 10 months of age (y-axis=arbitrary units; x-axis=distance), with the position of the peak Msh2-/- signal intensity indicted by a vertical dotted line. The range of the mean estimated CAG repeat number (±SD), above (+) and below (-) the peak, was determined (Materials and Methods): 1.5 months Msh2+/+=+15 ±2 to -18±1; Msh2-/- = +11 to -20; 5–10 months Msh2+/+ = +40±7 to -20±6; Msh2-/- = +11±3 to -15±6. For Msh2+/+ striata the mean number of CAG repeats above the peak was significantly increased at 5–10 months, compared with 1.5 months (P<0.05). At 5–10 months, the absence of Msh2 significantly decreased the mean CAG repeat number above the peak (P<0.01). This value did not differ significantly from the mean CAG repeat number above the peak for 1.5 month Msh2+/+ striata. (B) Immunoblots of soluble protein extracts from striata of HdhQ111/+:Msh2+/+ or HdhQ111/+:Msh2-/- mice at 8 months of age, probed with mAb2166 or mAb1F8, and ß-actin for normalization. The positions of the 7-glutamine wild-type (Q7) and 111-glutamine mutant (Q111) huntingtin bands are indicated. In the absence of Msh2 the proportion of Q111-mutant huntingtin was increased relative to wild-type huntingtin or ß-actin. Signals were quantified by densitometry (Materials and Methods). Ratio of mAb2166-reactive mutant compared to wild-type huntingtin: Msh2+/+ 0.7, Msh2-/- 1.0. Ratio of mAb1F8-reactive mutant huntingtin, normalized to ß-actin: Msh2+/+ 0.5, Msh2-/- 1.0. The scatter plot shows the ratios of mAb2166 -reactive mutant/wild-type huntingtin (Htt) signal from striata of five HdhQ111/+:Msh2+/+mice and four HdhQ111/+:Msh2-/- mice at 8 months of age. Black squares represent the ratio for a single mouse. Mean ratios±SD are 0.7±0.1 for Msh2+/+ and 1.4±0.4 for Msh2-/- (P<0.005).

 
Immunoblot analyses were performed at 8 months of age to determine whether Msh2 deficiency affected the generation of somatically expanded mutant protein. As the ‘smear’ of somatically expanded protein isoforms was only evident by 28 months (Figure 2), we tested for the correlated reduction in levels of 111-glutamine mutant huntingtin. The data in Figure 3B compares HdhQ111/+:Msh2+/+ and HdhQ111/+:Msh2-/- striatal extracts. The Msh2-deficient and Msh2 wild-type backgrounds yielded mAb2166 and mAb1F8 patterns that were superficially similar. However densitometry revealed increased intensity of the mutant band in the absence of Msh2. The mAb2166-reactive mutant to wild-type huntingtin ratio was increased (1.4 for Msh2-/- versus 0.7 for Msh2+/+, P<0.005), and when normalized to ß-actin the mAb1F8-reactive mutant band was elevated ~2-fold. Thus, consistent with lack of somatic HD CAG repeat expansion and abrogation of somatically expanded mutant protein, Msh2 deficiency resulted in elevated levels of 111-glutamine mutant huntingtin.

Msh2 deficiency modifies the timing of nuclear mutant huntingtin
To determine the impact of Msh2 deficiency on an early disease event, we assessed the timing of nuclear accumulation of full-length mutant huntingtin, which is fully evident in striatal neurons of HdhQ111/+ heterozygotes at ~4–5 months of age (15). Immunostaining of sections, shown in Figure 4A, revealed the expected nuclear EM48 immunoreactivity in HdhQ111/+: Msh2+/+ striata at 5 months of age, with increased intensity at 10 months. By contrast, nuclei of striatal neurons of HdhQ111/+:Msh2-/- mice exhibited little staining at 5 months, and only very weak EM48 immunoreactivity was detected in some striatal nuclei at 10 months of age. Quantification of the nuclear EM48 signal, shown in Figure 4B, showed that the absence of Msh2 caused a significant 21-fold and 14-fold reduction in the mean nuclear staining index (Materials and Methods) at 5 and 10 months, respectively. To assess the striatal selectivity, we assessed nuclear EM48 immunoreactivity in regions that exhibit this phenotype following onset in striatal neurons (15). At 10 months of age, weak nuclear EM48 staining was apparent in nuclei of a subset of neurons in the olfactory tubercle and pyriform cortex, as expected, but nuclear staining was not found in HdhQ111/+:Msh2-/- sections (data not shown), consistent with delayed onset.



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  		Figure 4. Msh2 deficiency delays striatal nuclear mutant huntingtin accumulation. (A) EM48 immunostaining of striatal sections demonstrates that at 5 months of age diffuse EM48-positive nuclear huntingtin is apparent in HdhQ111/+:Msh2+/+ striatum, but is barely detectable in HdhQ111/+:Msh2-/- striatum. At 10 months of age the EM48 staining in HdhQ111/+:Msh2+/+ striatum shows a progression to an increased intensity, whilst only very weak EM48 staining is seen in HdhQ111/+: Msh2-/- striatum. (B) For quantification of the EM48 immunostaining, the nuclear staining index (SI), the product of the mean nuclear stain intensity (MNI) and mean number of stained nuclei (MN) (Materials and Methods) is plotted as a histogram. HdhQ111/+:Msh2+/+ at 5 months SI=358.4± 4.7 (MNI=5.41±0.26; MN=66.3±17.8); HdhQ111/+:Msh2-/-at 5 months SI=17.16±0.87 (MNI=3.73±0.59; MN=3.29±1.47); HdhQ111/+:Msh2+/+at 10 months SI=1260.9±11.0 (MNI=10.21±0.58; MN=123.3±12.5); HdhQ111/+:Msh2-/- at 10 months SI=88.5±3.8 (MNI=3.59±0.3; MN=24.6±12.5). HdhQ111/+:Msh2+/+ and HdhQ111/+:Msh2-/- mice showed a statistically significant difference between their MN values (P<0.01 at 5 and 10 months of age), and a statistically significant difference between their MNI values (P=0.01 at 5 months of age and P<0.0001 at 10 months of age). The total number of stained nuclei observed and quantified were: HdhQ111/+:Msh2+/+ at 5 month, 263; HdhQ111/+:Msh2-/- at 5 months, 23; HdhQ111/+:Msh2+/+ at 10 months, 490; HdhQ111/+: Msh2-/-at 10 months, 103. Data above are given ±SE.

 
Msh2 deficiency, therefore, eliminated somatic HD CAG expansion and delayed the timing of an early disease event by at least 5 months, although the disease process remained selective for striatal neurons. Thus, somatic CAG expansion was not correlated with striatal specificity but rather was associated with more rapid timing, consistent with an increased proportion of mutant huntingtin with somatically expanded polyglutamine tracts.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Genetic modifiers of events that occur early in the disease process would provide additional starting points for unraveling and manipulating the consequences of mutant huntingtin that lead to neurodegeneration. Here we have tested whether somatic HD CAG instability might be involved in the timing or the striatal selectivity of the disease process in HdhQ111 mice. Msh2 was a logical modifier candidate because this mismatch repair protein plays a role in HD CAG instability, and therefore could alter the disease process at a fundamental level by changing the size of the polyglutamine segment in mutant huntingtin. We now report that Msh2 hastens by many months the timing, but not the striatal selectivity, of early disease in a precise genetic HD mouse model, implicating Msh2-dependent pathways in modifying pathogenesis in man.

In altering early disease timing, Msh2 defines a discrete class of modifier that currently also includes the N171-82Q transgene, which expresses mutant amino terminal huntingtin product (17). Indeed, the effect of this transgene implicated mutant polyglutamine product, which eventually accumulates in brains of Hdh CAG knock-in mice (15,16) and HD patients (11), in hastening earlier steps in the disease process (17). The early modifier class is distinguished from neuroprotectants such as wild-type huntingtin and dominant-negative caspase 1, which ameliorated measures of disease in HD YAC and HD exon 1 transgenic mice (25,26), but failed to alter the timing of nuclear mutant huntingtin accumulation in HdhQ111 striatum (17). It will be of interest to determine whether any neuroprotectants that delay phenotypes in HD exon 1 transgenic mice by a few weeks act by altering timing of early disease events. These include environmental enrichment (27) and agents active in neurotoxic models; minocycline, creatine, lipoic acid, dichloroacetate, coenzyme Q10, remacemide, riluzole, dopamine and tauroursodeoxycholic acid (28–36). Thus, tests of modifiers in HdhQ111 mice imply that HD pathogenesis may be attacked at multiple levels, with Msh2 providing a novel target for the development of pharmaco-logical agents aimed at slowing early steps in the disease cascade.

Msh2 seems likely to hasten early disease by generating HD CAG expansions that give rise to mutant huntingtin with somatically expanded glutamine segments. Lack of Msh2 eliminated striatal CAG repeat instability, and non-somatically expanded mutant huntingtin was associated with delayed onset. By contrast, the striatal selectivity of nuclear mutant huntingtin accumulation was unaltered by Msh2 deficiency. This finding suggests that progressive somatic HD CAG expansion may be a consequence of a striatal-specific disease process, rather than a cause of striatal vulnerability. In this scenario, the disease process initiated by mutant huntingtin in striatal neurons would lead to CAG expansions at the HdhQ111 locus that would ‘feedback’ to hasten disease via mutant huntingtin with somatically expanded glutamine tracts. In this case, somatic instability and somatically expanded mutant huntingtin would have to occur before the nuclear accumulation of mutant huntingtin detected by EM48 in striatal neurons. Consistent with this, both striatal-specific HD CAG expansion and nuclear huntingtin accumulation are manifest in HdhQ111/+ heterozygotes at ~4–5 months. However, somatically expanded mutant huntingtin was not detected by immunoblot until very much later. The latter discrepancy may reflect different detection thresholds of the PCR genotyping and immunoblot protein assays. Alternatively, it may be due to an intriguing biological effect, such as differential turnover of mutant protein with somatically expanded glutamine tracts.

An alternative to the ‘feedback’ scenario is the possibility that Msh2 acts to hasten early disease via pathways that are unrelated to its role in HD CAG instability. In this case, somatic expansion at the DNA or protein levels need not precede the nuclear mutant huntingtin phenotype. This possibility is consistent with an emerging role for Msh2 in mediating stress response to a variety of insults, including glutamate mediated excitotoxic neuronal cell death (37,38). Msh2 is also integral to execution of apoptosis, although this function is revealed in conjunction with repair of DNA damage (18,39–43).

Placing the HdhQ111 allele on an Msh2-deficient background has also revealed new information about HD CAG tract instability. Msh2 had previously been shown to be required for expansions through the male germline and for widespread somatic HD CAG tract expansions exhibited in the context of an HD exon1 transgene (21), expressing mutant amino terminal huntingtin product (12,13). Our data demonstrate that, while Msh2 is required for paternal size increases and somatic expansions, the full spectrum of HD CAG repeat instability in Hdh CAG knock-in mice must also comprise Msh2-independent processes that generate maternally inherited size fluctuations, increases and decreases, as well as contractions through the male germline, revealed in the absence of Msh2. Thus, our findings strongly indicate that Msh2-independent DNA repair processes, such as non-homologous end-joining, nucleotide excision repair and base excision repair, warrant investigation for their role in intergenerational HD CAG repeat instability.

Multiple mechanisms are consistent with the variety of cell types implicated in CAG repeat instability. For example, maternal changes may arise in oocytes arrested in prophase of the first meiotic cell division (44), while paternal expansions appear to occur during the post-meiotic maturation of haploid spermatids to spermatozoa (23). Somatic instability arises in post-mitotic cells (7,14). In yeast, Msh2 deficiency can result in an increased frequency of CAG tract deletion during DNA replication (20). Therefore, the deletions that occur at an increased frequency in the HdhQ111 male germline in the absence of Msh2 may also arise during DNA replication, in contrast to replication-independent expansion (23). In addition, Msh3 and Msh6, which function in a dimeric complex with Msh2 (45), differentially affect trinucleotide repeat expansion in a myotonic dystophy mouse model (46), suggesting that these proteins be investigated as mediators of HD CAG instability.

In summary, our analyses of HD CAG tract instability have demonstrated that Msh2 is a dramatic modifier of the timing of the striatal-selective disease process initiated by mutant huntingtin in a precise genetic HD mouse model. Thus Msh2 and other proteins that alter HD CAG repeat instability merit investigation in the human disease.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Mice
Generation, maintenance on a CD1 background and genotyping of HdhQ111 knock-in mice were carried out as described previously (14,47) Msh2 knockout mice (129Ola/FVB) (18) were genotyped as described (42). To place the HdhQ111 allele on the Msh2-deficient background HdhQ111/+:Msh2+/- mice, generated by crossing HdhQ111/Q111 and Msh2+/- mice, were intercrossed to give HdhQ111/+:Msh-/-, Hdh+/+:Msh2-/-, HdhQ111/+:Msh2+/+ and Hdh+/+:Msh2+/+ littermates. For assessment of intergenerational instability, breeding pairs were established between HdhQ111/+:Msh2-/- (four females, five males) and Hdh+/+:Msh2-/- parents or between HdhQ111/+: Msh2+/+ (four females, two males) and Hdh+/+:Msh2+/+ control littermates, and the size of the HD CAG repeat determined in HdhQ111/+ progeny. Somatic instability, EM48 immunostaining and immunoblot analyses were assessed in HdhQ111/+:Msh2-/- and control HdhQ111/+:Msh2+/+ mice.

PCR amplification of the Hdh CAG repeat and analyses of instability
Genomic DNA was isolated from tail biopsies taken at birth or weaning and from striatum dissected from adult mice brains using the PureGene DNA isolation kit (Gentra, Minneapolis, MN, USA). Amplification of the CAG repeat was carried out using a specific PCR assay (12) that amplifies the human HD CAG repeat, and radiolabeled PCR products were resolved in denaturing 6% polyacrylamide gels. For analysis of intergenerational instability, CAG repeat size in HdhQ111/+ progeny was scored as unchanged, expanded or contracted relative to the migration of PCR products amplified from parental HdhQ111/+ DNAs and run on the same gels. Statistical analyses of expanded and contracted alleles were performed by a {chi}2 test or the Fisher exact test using the SigmaStat package (Jandel Scientific). For analysis of somatic instability autoradiograms were scanned using an LKB UltroScan XL Laser Densitometer to obtain peak traces with subtracted background. The range over which the densitometer scans extended was determined using an arbitrary intensity cut-off value of 10% of the peak signal intensity. The range of the PCR product signal was converted into CAG number by reference to a sequencing ladder, in order to determine the number of CAG repeats above and below the peak signal intensity (~109 CAGs). These analyses were performed with striatal genomic DNA isolated from the following number of mice: 1.5 months (Msh2+/+, n=2; Msh2-/-, n=1); 5 months (Msh2+/+, n=2; Msh2-/-, n=2); 10 months (Msh2+/+, n=1; Msh2-/-, n=1), with 5 and 10 month results considered together for statistical analysis. Comparison of the mean number of CAG repeats above and below peak signal intensity in HdhQ111/+:Msh2+/+ and HdhQ111/+:Msh2-/- striata was carried out using a Student's t-test (Microsoft Excel software).

Immunohistochemisty
Immunohistochemistry was carried out on 7 µm paraffin-embedded coronal sections of brains perfused with periodate–lysine–paraformaldehyde as described (14,47). Immunostaining with polyclonal anti-huntingtin antibody EM48 (amino acids 1–256) (48) was as described (15). All EM48 immunostaining experiments were performed under identical conditions. To quantify the EM48 immunostaining, the mean nuclear stain intensity (MNI) in two 750 x 500 µm regions of striatum from two to three mice of each age and genotype was determined using the ‘histogram’ function in Adobe Photoshop to convert the signal intensity in all stained nuclei observed to arbitrary units. The background signal, the mean value for 10 fields within each 750 x 500 µm region analyzed, was subtracted from the nuclear signal. Data from each 750 x 500 µm region were pooled and data from multiple mice of each age and genotype were pooled. The MNI was calculated from the pooled dataset with standard error. To quantify the total amount of nuclear stain in a way that reflects both the intensity of the immunostaining and the number of stained nuclei, we calculated the staining index (SI) as the product of the MNI and the mean number of stained nuclei (MN) over all the 750 x 500 µm regions analyzed for the mice of each age and genotype. The MNI and MN of HdhQ111/+: Msh2+/+ mice were compared with those of HdhQ111/+: Msh2+/+ mice at each age using Student's t-test (Microsoft Excel software).

Immunoblot analyses
Soluble protein was extracted from dissected striata in 50 mM Tris–HCl pH 7.5, 10% glycerol, 5 mM magnesium acetate, 0.2 mM EDTA with protease inhibitors (Complete protease inhibitor cocktail, Boehringer Mannheim GmbH, Germany) supplemented with 170 µM PMSF. Sixty micrograms of protein extract were resolved in 6% SDS–polyacrylamide gels and detected by immunoblot as previously described (15) using ECL (KPL, Gaithersburg, MD, USA). Antibodies used were anti-huntingtin mAb2166 (amino acids 181–810) (Chemicon International, Temecula, CA, USA), mAb1F8 (glutamine segments>39) (24,49), anti-ubiquitin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and ß-actin (Sigma, St Louis, MO, USA). Protein was quantified using the Bradford protein assay from BioRad (Hercules, CA). Quantification of immunoblot signals was carried out by determining the peak intensity values, after background subtraction, of traces obtained from densitometer scans using an LKB UltroScan XL Laser Densitometer. The peak intensity values were used to determine the ratio of the mAb2166-positive mutant:wild-type huntingtin signal (three exposures) and the ratio of the mAb1F8:ß-actin signal. Statistical analysis was carried out using Student's t-test (Microsoft Excel software).


   ACKNOWLEDGEMENTS
 
We thank Dr X.-J. Li for the EM48 antibody and Ms C. Chakrabarti for technical assistance. This work was supported by NINDS grants NS16367 (HD Center Without Walls) and NS32765, an Anonymous Donor, and The Huntington's Disease Society of America (Coalition for the Cure). V.C.W. received a postdoctoral fellowship from the Hereditary Disease Foundation.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Molecular Neurogenetics Unit, Building 149, 13th St, Charlestown, MA 02129, USA. Tel.: +1 6177265726; Fax: +1 6177265736; Email: wheeler{at}helix.mgh.harvard.edu Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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