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Human Molecular Genetics Advance Access originally published online on May 10, 2006
Human Molecular Genetics 2006 15(12):2015-2024; doi:10.1093/hmg/ddl125
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Genetic background modifies nuclear mutant huntingtin accumulation and HD CAG repeat instability in Huntington's disease knock-in mice

Alejandro Lloret1, Ella Dragileva1, Allison Teed1, Janice Espinola1, Elisa Fossale1, Tammy Gillis1, Edith Lopez1, Richard H. Myers2, Marcy E. MacDonald1 and Vanessa C. Wheeler1,,*

1Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114, USA and 2Department of Neurology, Boston University School of Medicine, Boston, MA 02118, USA

* To whom correspondence should be addressed. Tel: +1 6176433103; Fax: +1 6176433203; Email: wheeler{at}helix.mgh.harvard.edu

Received March 28, 2006; Accepted May 6, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Genetically precise models of Huntington's disease (HD), Hdh CAG knock-in mice, are powerful systems in which phenotypes associated with expanded HD CAG repeats are studied. To dissect the genetic pathways that underlie such phenotypes, we have generated HdhQ111 knock-in mouse lines that are congenic for C57BL/6, FVB/N and 129Sv inbred genetic backgrounds and investigated four HdhQ111 phenotypes in these three genetic backgrounds: the intergenerational instability of the HD CAG repeat and the striatal-specific somatic HD CAG repeat expansion, nuclear mutant huntingtin accumulation and intranuclear inclusion formation. Our results reveal increased intergenerational and somatic instability of the HD CAG repeat in C57BL/6 and FVB/N backgrounds compared with the 129Sv background. The accumulation of nuclear mutant huntingtin and the formation of intranuclear inclusions were fastest in the C57BL/6 background, slowest in the 129Sv background and intermediate in the FVB/N background. Inbred strain-specific differences were independent of constitutive HD CAG repeat size and did not correlate with Hdh mRNA levels. These data provide evidence for genetic modifiers of both intergenerational HD CAG repeat instability and striatal-specific phenotypes. Different relative contributions of C57BL/6 and 129Sv genetic backgrounds to the onset of nuclear mutant huntingtin and somatic HD CAG repeat expansion predict that the initiation of each of these two phenotypes is modified by different genes. Our findings set the stage for defining disease-related genetic pathways that will ultimately provide insight into disease mechanism.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG repeat to lengths ≥36 residues, elongating a glutamine tract at the N-terminus of the huntingtin protein (1Go). The disease presents with chorea, dementia and personality disturbances, leading to death 10–15 years after onset (2Go). Post-mortem HD brain shows a characteristic graded loss of medium spiny neurons in the striatum, as well as neuronal cell loss in deep cortical layers (3Go). In common with other ‘polyglutamine diseases’, the expanded glutamine stretch is thought to result in neuronal toxicity via a dominant gain of function.

The length of the HD CAG tract is the major determinant of onset age and disease severity (4Go–8Go). However, for the vast majority of patients (those with <60 repeats), HD CAG repeat length accounts for only ~50% of the variability in onset age, with evidence of strong heritability for that portion of onset age not explained by HD CAG repeat size (9Go). Therefore, as demonstrated by a number of studies, modifier genes play an important role in determining the precise onset age in HD and provide direct insight into disease mechanism (9Go–16Go).

The expanded HD CAG tract itself is highly unstable in both the germline and somatic tissues (4Go,17Go–20Go). Understanding the mechanisms that underlie this instability would be the first step towards reducing the HD CAG tract to non-pathogenic lengths. As for disease onset and severity, HD CAG repeat length is a major determinant of instability (17Go). However, cis- and trans-acting factors can influence instability of the HD CAG repeat in humans and mouse models (21Go–26Go), implicating genetic factors other than HD CAG length in modulating instability.

Hdh CAG knock-in mice, precise genetic models of HD in which expanded HD CAG repeats are inserted into the mouse HD gene homologue (Hdh) (27Go,28Go), provide powerful tools to investigate both HD CAG repeat instability and presymptomatic disease pathways. Extensive characterization of HdhQ111 mice has been carried out on an outbred CD1 genetic background. This has revealed high levels of intergenerational HD CAG repeat instability, with a bias towards repeat expansion upon male transmission, as observed in HD patients (4Go,28Go), and a cascade of early striatal-specific phenotypes that are predictive of later pathological hallmarks (29Go–33Go). Early phenotypes include somatic HD CAG repeat expansion and the nuclear accumulation of full-length mutant huntingtin, preceding intranuclear N-terminal huntingtin inclusions. The striatal-specificity, dominant inheritance and HD CAG length-dependence of these phenotypes in Hdh knock-in mice provide the opportunity to test whether similar pathways underlie these events and the onset of symptoms in HD patients.

To understand the mechanisms responsible for presymptomatic striatal phenotypes and intergenerational HD CAG repeat instability in Hdh CAG knock-in mice, we are taking the approach of dissecting the genetic pathways that underlie these events. Complementary to a biochemical approach, elucidation of genetic modifiers provides mechanistic insight into different disease pathways and the interactions among them and generates candidate genes that can be tested as modifiers of disease in HD patient populations. We have previously found that a single gene (Msh2) modified intergenerational HD CAG repeat instability, somatic HD CAG repeat instability and the timing of nuclear mutant huntingtin accumulation in HdhQ111 mice (26Go). Now, with the aim of performing unbiased searches for genetic modifiers, we have investigated the influence of genetic background on HdhQ111 phenotypes as a first step towards dissecting the genetic pathways involved. Using three congenic HdhQ111 strains, on C57BL/6 (B6), FVB/N (FVB) and 129Sv (129) inbred genetic backgrounds, we have examined the effect of genetic background on four HdhQ111 phenotypes: intergenerational HD CAG repeat instability, somatic HD CAG repeat instability, the nuclear accumulation of full-length mutant huntingtin and intranuclear N-terminal huntingtin inclusions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Genetic background modifies intergenerational instability of the HD CAG repeat
The HdhQ111 outbred CD1 line was repeatedly backcrossed onto B6, FVB and 129 inbred genetic backgrounds to generate three congenic HdhQ111 lines: B6.HdhQ111, FVB.HdhQ111 and 129.HdhQ111. To investigate the influence of genetic background on intergenerational instability of the HD CAG repeat, we analyzed transmissions from heterozygous B6.HdhQ111/+, FVB.HdhQ111/+ and 129.HdhQ111/+ males to their progeny. The data displayed in Figure 1A show the overall frequencies of expansions, contractions, changed and unchanged alleles in each genetic background. B6.HdhQ111/+ mice showed a small but statistically significant difference in the frequency of changed versus unchanged alleles compared with 129.HdhQ111/+ mice ({chi}2=7.88, P=0.005, df=1). There were no significant differences in the frequencies of changed versus unchanged alleles between 129 and FVB or between FVB and B6 genetic backgrounds. Comparisons of the relative frequencies of expansions versus contractions between the different genetic backgrounds revealed that these genetic backgrounds do not influence the direction of repeat length change.


Figure 1251
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  		Figure 1. Genetic background modifies intergenerational instability. The change in HD CAG repeat size was determined in transmissions from B6.HdhQ111/+ (90 meioses), FVB.HdhQ111/+ (51 meioses) and 129.HdhQ111/+ (39 meioses) males to their progeny by comparing constitutive repeat size in tails at weaning in fathers and their progeny. The HD CAG repeat was amplified using a human-specific PCR assay and repeat size determined using an automated DNA sequencer. In transmissions from B6.HdhQ111/+ males, the repeat length was altered in 74/90 transmissions (82%). Fifty-six alleles were expanded (62%) and 18 were contracted (20%). In transmissions from FVB.HdhQ111/+ males, the repeat length was altered in 39/51 transmissions (76%). Thirty-one alleles were expanded (61%) and 8 were contracted (16%). In transmissions from 129.HdhQ111/+males, the repeat length was altered in 23/39 transmissions (59%). Twenty alleles were expanded (51%) and 3 were contracted (7%). (A) The overall frequencies of repeat expansions (gray bars), contractions (striped bars), changed alleles (black bars) and unchanged alleles (unfilled bars) upon transmission from B6.HdhQ111/+, FVB.HdhQ111/+ and 129.HdhQ111/+ males. The asterisks indicate the significantly greater proportion of changed alleles compared with unchanged alleles in transmissions from B6.HdhQ111/+ males compared with 129.HdhQ111/+ males (P=0.005). (B) Frequency distributions of HD CAG repeat size changes in transmissions from B6.HdhQ111/+ (black bars), FVB.HdhQ111/+ (unfilled bars) and 129.HdhQ111/+ (gray bars) males. A broader distribution of repeat size changes is apparent in B6.HdhQ111 and FVB.HdhQ111 strains than in the 129.HdhQ111 strain.

 
Figure 1B shows frequency distributions for individual HD CAG length changes in each genetic background. The graph clearly demonstrates a broader spread in repeat length changes in B6.HdhQ111 (–5 CAGs to +6 CAGs) and FVB.HdhQ111 strains (–3 CAGs to +6 CAGs) than in the 129.HdhQ111 strain (–2 CAGs to +2 CAGs). Indeed, we found that the magnitude of the repeat length change, irrespective of direction, is significantly different between B6.HdhQ111 and 129.HdhQ111 strains (t-test, P<0.0001) and between FVB.HdhQ111 and 129.HdhQ111 strains (t-test, P<0.005). As the constitutive HD CAG repeat length itself is a factor in determining instability, we performed additional statistical analyses that controls for the paternal HD CAG repeat length (SAS GLM procedure). Using this model, mouse strain was also found to be a significant predictor of the magnitude of repeat length change, independent of paternal HD CAG repeat length (P<0.0001).

Genetic background does not dramatically influence Hdh mRNA levels
Before determining whether genetic background influenced striatal-specific phenotypes, we were interested to test whether genetic background might affect the expression of the Hdh gene itself in the striatum. Therefore, we first investigated Hdh gene expression using a quantitative RT-PCR assay that measures both wild-type and mutant Hdh mRNA. Figure 2 shows Hdh mRNA levels relative to ß-actin in striata from wild-type B6.Hdh+/+, FVB.Hdh+/+ and 129.Hdh+/+ mice and heterozygous mutant B6.HdhQ111/+, FVB.HdhQ111/+ and 129.HdhQ111/+ mice. In striata from wild-type mice, genetic background did not significantly influence Hdh mRNA levels, except for a small (1.3-fold) difference in Hdh mRNA levels between FVB.Hdh+/+ and 129.Hdh+/+ striata (P<0.05). Mutant mice of each genetic background showed a decreased level of striatal Hdh mRNA compared with the respective wild-type mice, consistent with previous observations (34Go). However, striata from mutant mice showed no significant differences in Hdh mRNA levels in the three genetic backgrounds. Therefore, these genetic backgrounds do not appreciably modulate the expression of the Hdh gene.


Figure 1252
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  		Figure 2. Hdh mRNA levels in striata of inbred mouse strains. mRNA was isolated from the striata of B6.Hdh+/+ (n=4), FVB.Hdh+/+ (n=4) and 129.Hdh+/+ (n=4) wild-type mice and B6.HdhQ111/+ (n=4), FVB.HdhQ111/+ (n=4) and 129.HdhQ111/+ (n=4) heterozygous mutant mice at 2.5 months of age. Hdh mRNA levels were determined using a real-time quantitative RT–PCR assay and normalized to the level of ß-actin mRNA. Bar graphs show the mean normalized Hdh mRNA levels (Hdh/ß-actin) for wild-type (unfilled bars) and mutant mice (gray bars) of each congenic strain (B6, FVB and 129) ± standard deviation.

 
Genetic background modifies somatic instability of the HD CAG repeat in the striatum
The HD CAG repeat in HdhQ111 mice undergoes time-dependent striatal-specific somatic expansion (28Go). To determine whether genetic background influences this striatal-specific event, we monitored somatic expansion in DNA isolated from dissected striata of heterozygous B6.HdhQ111/+, FVB.HdhQ111/+ and 129.HdhQ111/+ mice at 5 months of age. Tail DNA, which does not show somatic expansion, was used as a control.

Figure 3 shows representative Genescan traces from mice whose constitutive HD CAG sizes, determined from tail DNA, are 110 (B6), 108 (FVB) and 108 (129) repeats. On average, constitutive HD CAG repeat sizes were found to be 8–9 CAGs higher in FVB.HdhQ111/+ and B6.HdhQ111/+ mice than in 129.HdhQ111/+ mice. However, within each genetic background strain, Genescan traces were identical despite HD CAG repeat length variation of 6–8 CAGs.


Figure 1253
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  		Figure 3. Genetic background modifies somatic instability in the striatum. Representative Genescan traces showing PCR-amplified HD CAG repeat from striatum and tail of heterozygous B6.HdhQ111/+ (n=5), FVB.HdhQ111/+ (n=5) and 129.HdhQ111/+ (n=5) mice at 5 months of age. For each strain, all mice tested showed identical Genescan traces to those shown. The constitutive HD CAG repeat size determined in tail DNA is indicated and its position relative to that in striatal DNA is marked by a red vertical broken line. The HD CAG repeat length of the second peak in B6 and FVB striata is also indicated.

 
Both B6.HdhQ111/+ and FVB.HdhQ111/+ mice displayed significant somatic expansion of the HD CAG repeat in the striatum, as evidenced by a characteristic bimodal distribution of HD CAG repeat sizes, not seen in tail DNA from the same animal. In contrast, 129.HdhQ111/+ mice striata did not display this repeat size distribution, but rather exhibited a slight broadening of the Genescan trace compared with that seen in tail DNA. Therefore, genetic background influences somatic repeat instability in the striatum.

Genetic background modifies the nuclear accumulation of mutant huntingtin and intranuclear inclusions in striatal neurons
We have previously demonstrated the time-dependent appearance of a conformation of mutant huntingtin that is detected with N-terminal anti-huntingtin antibody EM48, in the nuclei of medium spiny striatal neurons of HdhQ111 knock-in mice carrying expanded HD CAG repeats (29Go). The intensity of the diffuse EM48 immunostaining and the number of immunostained nuclei increase with age such that these parameters reflect the timing of this ongoing disease-specific event. To determine whether genetic background modulates this early phenotype, we have compared diffuse EM48 immunostaining in striata from heterozygous B6.HdhQ111/+, FVB.HdhQ111/+ and 129.HdhQ111/+ mice at 6 months of age. As shown in Figure 4A, comparisons of the mean staining index (SI), a measure that represents both the intensity and number of immunostained nuclei, revealed significant differences between all three genetic backgrounds: B6 versus 129, P<0.0001; FVB versus 129, P<0.001; B6 versus FVB, P<0.001. For the two groups that showed the most extreme difference in mean SIs, B6 and 129, there was no overlap in the individual SI values, and the mean SIs differed by greater than two standard deviations. As the timing of diffuse nuclear huntingtin has been shown to be HD CAG repeat length-dependent (29Go), we performed additional statistical analyses using generalized estimating equations (SAS GENMOD procedure) to determine whether the SI is predicted by genetic background independent of HD CAG repeat length. These analyses revealed that, adjusting for HD CAG repeat length, B6 had a significantly higher SI than FVB (P<0.005) and 129 had a significantly lower SI than FVB (P<0.005). Therefore, the accumulation of diffusely immunostaining nuclear mutant huntingtin is modified by genetic background.


Figure 1254
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  		Figure 4. Genetic background modifies nuclear accumulation of mutant huntingtin and intranuclear inclusions in striatal neurons. EM48 immunostaining (left) and quantitation of the immunostaining (right) in striatal sections from HdhQ111 mice on B6, FVB and 129 inbred genetic backgrounds. (A) Representative sections showing diffuse nuclear EM48 immunoreactivity in striata from heterozygous B6.HdhQ111/+ (n=8), FVB.HdhQ111/+ (n=8) and 129.HdhQ111/+ (n=8) mice at 6 months of age. SI was calculated for each mouse as the product of the mean nuclear staining intensity and the number of stained nuclei (see Materials and Methods). The bar graph shows the mean SI value for each congenic strain ± standard deviation. (B) EM48-positive intranuclear inclusions (indicated with arrows) in B6.HdhQ111/+ striata at 6 months of age. (C) Representative sections showing EM48 immunoreactivity in striata from heterozygous B6.HdhQ111/+ (n=7), FVB.HdhQ111/+ (n=4) and 129.HdhQ111/+ (n=5) mice at 12 months of age. Intranuclear inclusions are apparent in B6.HdhQ111/+ and FVB.HdhQ111/+ striata but are rare in 129.HdhQ111/+ striata. The number of intranuclear inclusions was determined for each mouse and normalized to the number of EM48-positive neurons (see Materials and Methods). The bar graph shows the mean normalized inclusion number for each congenic strain ± standard deviation.

 
A later pathogenic hallmark of the disease in Hdh CAG knock-in mice is the appearance of intranuclear inclusions of N-terminal mutant huntingtin, apparent in HdhQ111 striatum on a CD1 background at 10–12 months of age (30Go). As this later phenotype is predicted by early full-length mutant huntingtin EM48-immunoreactivity, we determined whether strain background influences huntingtin intranuclear inclusion formation.

Careful examination of the EM48-immunostained B6.HdhQ111/+, FVB.HdhQ111/+ and 129.HdhQ111/+striata from 6-month mice revealed a few intranuclear inclusions of mutant huntingtin only in the B6.HdhQ111 strain (Fig. 4B). To quantify better the strain differences, we immunostained striata from 12-month heterozygous B6.HdhQ111/+, FVB.HdhQ111/+ and 129.HdhQ111/+ mice with EM48 (Fig. 4C). A comparison of the number of inclusions, normalized to the number of EM48-positive neurons, revealed significant differences between all three genetic backgrounds: B6 versus 129, P<0.0001; FVB versus 129, P<0.005; B6 versus FVB, P<0.001 strains. For all of the three genetic background groups, there was no overlap in the individual normalized inclusion numbers between groups, and the mean normalized inclusion numbers differed from one group to the other by greater than two standard deviations. Therefore, genetic background also modifies the nuclear inclusion phenotype.

Separate genetic effects of inbred background strain on the onset of nuclear mutant huntingtin and somatic expansion
Our data implicate genetic factors that modulate two early striatal phenotypes, the nuclear accumulation of mutant huntingtin and somatic HD CAG repeat expansion. We are most interested in understanding the very earliest mutant HD gene-related events in Hdh CAG knock-in mice. Therefore, we further investigated nuclear mutant huntingtin and somatic expansion at a young age (2.5 months), predicted to reveal phenotypic onset (29Go). For this purpose, we investigated only the B6.HdhQ111 and 129.HdhQ111 strains that showed the most extreme difference in nuclear mutant huntingtin accumulation. To determine whether differences in B6 and 129 might reflect inherited genetic variation, we crossed B6 and 129 strains and examined nuclear mutant huntingtin and somatic expansion in the (B6x129).HdhQ111/+ F1 progeny.

The histogram in Figure 5A shows the SI values quantified from EM48 immunostaining of striatal sections of 2.5-month B6.HdhQ111/+, 129.HdhQ111/+ and (B6x129).HdhQ111/+ F1 heterozygous mice. As predicted from the results obtained in 6-month mice, B6.HdhQ111/+ striata showed extensive accumulation of nuclear mutant huntingtin. In contrast, 129.HdhQ111/+ striata exhibited little evidence of nuclear EM48 immunostaining. A comparison of the mean SIs revealed a significant difference between these two strains (P<0.005), demonstrating that the onset of nuclear mutant huntingtin is delayed in 129.HdhQ111/+ striata in comparison with B6.HdhQ111/+ striata. The mean SI in (B6x129).HdhQ111/+ F1 striata was found to be significantly different from that in B6.HdhQ111/+ striata (P<0.01), but not significantly different from that in 129.HdhQ111/+ striata (P=0.49). There was no significant difference in constitutive HD CAG repeat lengths in B6 and F1 mice (P=0.63), strengthening the finding that strain differences are independent of constitutive HD CAG repeat length. Therefore, the similar SI in 129 and F1 striata imply that at 2.5 months of age, 129 gene(s) are dominant over B6 gene(s) in determining the onset of nuclear mutant huntingtin.


Figure 1255
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  		Figure 5. The onset of nuclear huntingtin accumulation and somatic expansion are driven by different genetic effects. (A) Quantification of the EM48 immunostaining of striatal sections from B6.HdhQ111 (n=7), 129.HdhQ111 (n=7) and (B6x129).HdhQ111 F1 (n=6) mice at 2.5 months of age. SI was calculated for each mouse as the product of the mean nuclear staining intensity and the number of stained nuclei (see Materials and Methods). The bar graph shows the mean SI value for each congenic strain ± standard deviation. (B) Representative Genescan traces showing PCR-amplified HD CAG repeat from striatum and tail of heterozygous B6.HdhQ111/+ (n=5), 129.HdhQ111/+ (n=3) and (B6x129).HdhQ111 F1 (n=4) mice at 2.5 months of age. For each strain, all mice tested showed identical Genescan traces to those shown. The constitutive HD CAG repeat size determined in tail DNA is indicated and its position relative to that in striatal DNA is marked by a red vertical broken line.

 
Figure 5B shows Genescan traces of the HD CAG repeat PCR products amplified from striatal DNA of 2.5-month B6.HdhQ111/+, 129.HdhQ111/+ and (B6x129).HdhQ111/+ F1 heterozygous mice. B6.HdhQ111/+ striatal DNA shows a significant broadening of the Genescan traces compared with those from tail DNA, indicating the presence of somatic expansion in the striatum. In contrast, 129.HdhQ111/+ striatal DNA exhibits no somatic expansion when compared with tail DNA, confirming the more rapid somatic expansion in B6.HdhQ111/+ than in 129.HdhQ111/+ striatum. Notably, Genescan traces from (B6x129).HdhQ111/+ F1 striata are clearly distinguishable from those of 129.HdhQ111/+ striata, but closely resemble those from B6.HdhQ111/+ striata. Therefore, in contrast to the onset of nuclear huntingtin, the similar somatic expansion profiles from B6 and F1 striata imply that at 2.5 months of age, B6 gene(s) are dominant over 129 gene(s) in determining the onset of somatic expansion.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
We have previously characterized intergenerational HD CAG repeat instability and early striatal-specific presymptomatic phenotypes associated with the presence of expanded HD CAG repeats in HdhQ111 CAG knock-in mice. Here, as a first step towards understanding the underlying genetic pathways, we have dissected these events by investigating the influence of three inbred genetic backgrounds on the instability of the HD CAG repeat inherited through the male germline; somatic HD CAG repeat expansion in the striatum; the nuclear accumulation of full-length mutant huntingtin in striatal neurons and N-terminal intranuclear huntingtin inclusions in striatal neurons. We find that genetic background influences all of these phenotypes; intergenerational and somatic instability of the HD CAG repeat are elevated in FVB and B6 backgrounds compared with the 129 background. Both nuclear mutant huntingtin accumulation and intranuclear inclusion formation are fastest in the B6 background, slowest in the 129 background and show intermediate phenotypes in the FVB background. The inbred strain-specific differences in phenotype cannot be explained by the size of the constitutive HD CAG repeat, implying that genetic factors underlie the phenotypic differences. Strain-specific differences in phenotype do not correlate with the level of expression of Hdh mRNA, suggesting that the genetic factors do not influence the transcriptional regulation of the Hdh gene.

The three inbred genetic backgrounds had similar effects on intergenerational instability and somatic instability, suggesting that similar pathways may be involved in determining both types of instability. Inbred strain-specific differences in instability could be the result of differences in the genomic context of the HD CAG repeat (21Go–23Go,35Go–38Go) or in protein factors that mediate or protect against repeat instability, such as members of the mismatch repair pathway (24Go–26Go,39Go–41Go). Interestingly, although mismatch repair protein Msh2 altered HD CAG repeat instability in HdhQ111 mice (26Go), we did not observe significant differences in Msh2 protein levels in striata of the three congenic lines (data not shown), suggesting that Msh2 is unlikely to be a major modifier in these strains. Future studies will determine the genetic loci involved in modifying HD CAG repeat instability, which may lead to novel insight into the underlying mechanisms.

With the aim of dissecting the genetic pathways that cause different HdhQ111 phenotypes, it would be of interest to quantify in the three congenic lines additional phenotypes that have been identified in HdhQ111 mice (30Go–33Go) and other knock-in mouse models (42Go–47Go). In the present study, an early (diffuse nuclear huntingtin) and a late (intranuclear inclusions) phenotype showed the same B6>FVB>129 pattern of relative severity in the three genetic backgrounds. These findings implicate an early-acting modifier(s) that influence an ongoing degenerative process. Similarly, additional phenotypes that show the same B6>FVB>129 outcome would support an early-acting modifier(s), whereas the finding that other phenotypes do not fall into the same pattern of B6>FVB>129 would suggest that they are caused by independent mechanisms that can be modified by different genes. Thus, an appreciation of the genetic pathways underlying multiple HdhQ111 phenotypes can lead to an understanding of the relationship between them.

The relationship between somatic expansion and disease is unclear. In the present study, we have examined the onset of somatic HD CAG repeat expansion and nuclear mutant huntingtin and show that B6 and 129 genetic backgrounds contribute differently to the onset of each of these two phenotypes. This finding predicts that different gene(s) modify these two events and, therefore, that each event may be initiated by an independent underlying mechanism(s). These data are consistent either with the initiation of somatic expansion and nuclear mutant huntingtin accumulation as two independent consequence of the disease process, as implied by the striatal-specificity and age-dependent accumulation of somatic HD CAG repeat expansions (20Go,28Go,48Go), or with the initiation of somatic expansion as an event unrelated to the disease. The latter is supported by observations that the striatum appears to be particularly susceptible to CAG repeat expansion in other triplet repeat disorders in which brain regions other than the striatum succumb to disease (49Go,50Go). The data would argue against the idea that somatic expansion initiates the disease process. However, once initiated, the accumulated HD CAG repeat expansions may act further downstream to accelerate disease phenotypes, as demonstrated by the delay in nuclear mutant huntingtin accumulation in an Msh2–/– background in which somatic HD CAG repeat expansion is eliminated (26Go). Significantly, these data demonstrate that genetic variants can distinguish early presymptomatic phenotypes in HdhQ111 mice, providing valuable insight into the ways in which the underlying pathways interconnect.

In conclusion, we have carried out the first study that directly investigates the influence of different inbred genetic backgrounds on phenotypes associated with an expanded HD CAG repeat allele in the mouse. The results obtained in this study will pave the way towards dissecting the genetic pathways that contribute to the disease. Quantitative trait locus (QTL) mapping of the genetic modifier loci is being pursued and will allow us to determine the complexity of the genetic loci that underlie the inbred strain-specific differences. Elucidation of the modifier genes involved will provide direct insight into the disease mechanism and the relationship between different mutant HD gene phenotypes. Importantly, modifier genes identified in a precise genetic knock-in model of HD will generate highly relevant candidates to test as modifiers of disease or repeat instability in HD patients.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Mouse breeding and generation of congenic strains
Congenic HdhQ111 strains were generated by at least eight generations of repeated backcrossing of heterozygous HdhQ111/+ mice, originally on an outbred CD1 background (27Go,28Go) to C57BL/6N, FVB/N or 129Sv inbred strains (all purchased from Charles River Laboratories). Resulting congenic strains are named B6.HdhQ111, FVB.HdhQ111 and 129.HdhQ111, respectively. (B6x129).HdhQ111 F1 mice were generated by crossing B6.HdhQ111/+ heterozygous females with wild-type 129Sv males. All phenotypic and instability analyses were performed using heterozygous HdhQ111/+ mice.

Mouse genotyping and HD CAG repeat length determination
Genomic DNA was isolated from tail biopsies at weaning, from adult tail, or from striatum dissected from adult mice brains, using the PureGene DNA isolation kit (Gentra, Minneapolis, MN, USA). Routine genotyping was carried out as described previously (27Go). The size of the HD CAG repeat was determined using a human-specific PCR assay that amplifies the HD CAG repeat from the knock-in allele but does not amplify the mouse sequence (51Go). The forward primer was fluorescently labeled with 6-FAM (Perkin Elmer), and products were resolved using either the ABI 377 or the AB1 3730xl automated DNA analyzer (Applied Biosystems). Genescan and Genotyper software packages with GeneScan 500-TAMRA as internal size standard (ABI 377) or GeneMapper v3.7 with GeneScan 500-LIZ as internal size standard (ABI 3730) were used to assign repeat size. The HD CAG size was assigned as the highest peak in the Genescan trace.

Analysis of intergenerational instability
Crosses were established between B6.HdhQ111/+, FVB.HdhQ111/+ or 129.HdhQ111/+ heterozygous males and wild-type B6, FVB or 129 females, respectively. Intergenerational instability was determined by comparing HD CAG repeat size, in tail biopsies at weaning, from the transmitting HdhQ111/+ male with those in his HdhQ111/+ progeny. Transmitting parent and progeny were compared in the same ABI automated DNA sequencer run (as described above) and three control DNAs of known HD CAG size were included in every run. At least five different transmitting males of each strain were used to minimize bias from individual mice. The ages of the males upon transmission were 129.HdhQ111/+, 2.5–6.5 months, mean 4.2 months; FVB.HdhQ111/+, 1.6–7.9 months, mean 3.9 months; B6.HdhQ111/+, 1.5–8.5 months, mean 4.0 months. There was no significant difference in the ages of the males between strains. For statistical analyses, t-tests were performed using Microsoft Excel software. {chi}2 analyses, used to compare the frequency of allele changes for different strains, were performed using the SAS statistical software package (52Go). Models evaluating the relationship of instability to strain controlling for paternal constitutive CAG repeat size were performed by linear regression using the general linear models (GLM procedure in SAS).

Immunohistochemistry
Immunohistochemistry was carried out on 7 µm paraffin-embedded coronal sections of brains perfused with periodate-lysine-paraformaldehyde as described (29Go). Immunostaining with polyclonal anti-huntingtin antibody EM48 (amino acids 1–256) (53Go) was as described (29Go). All EM48 immunostaining experiments were performed under identical conditions.

To quantify diffuse EM48 immunostaining in mice at 2.5 months and at 6 months of age, the mean nuclear stain intensity was determined in one to six 750 µmx500 µm regions of striatum from 7–8 mice of each age and genetic background using the ‘histogram’ function in Adobe Photoshop to convert the signal intensity in all stained nuclei observed to arbitrary units. Background signal, the mean value for 10 fields within each 750 µmx500 µm region analyzed, was subtracted from the nuclear signal. Striatal regions were matched in terms of their dorsal/ventral and anterior/posterior locations. To quantify the total amount of nuclear stain in a way that reflects both the immunostaining intensity and the number of immunostained nuclei, we calculated a staining index (SI) as the product of the mean nuclear stain intensity and the number of stained nuclei for each 750 µmx500 µm region analyzed. SI values for each 750 µmx 500µm region were averaged to give an SI value for each mouse.

To quantify EM48-positive inclusions, the number of intranuclear inclusions in four 750 µmx500 µm regions of striatum from 4–7 mice of each genetic background at 12 months of age was counted and normalized to the total number of EM48-positive neurons. Striatal regions were matched in terms of their dorsal/ventral and anterior/posterior locations. Normalized inclusion numbers from each 750 µmx500 µm region were averaged to give a value for the normalized number of inclusions for each mouse.

SI values and normalized inclusion numbers in mice of different genetic backgrounds were compared statistically using t-tests (Microsoft Excel software). Models evaluating the relationship of SI to strain controlling for HD CAG repeat size were performed using the generalized estimating equations (GENMOD procedure in SAS) (52Go).

Determination of Hdh mRNA levels
Striata from B6.HdhQ111/+, B6.Hdh+/+, FVB.HdhQ111/+, FVB.Hdh+/+, 129.HdhQ111/+ and 129.Hdh+/+ (n=4 per group) mice at 2.5 months of age were dissected, snap-frozen on dry ice and stored at –80°C until use. Total RNA was extracted with TRIZOL reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's recommendations. RNA was quantified spectrophotometrically using a Nanospec instrument and analyzed for quality by agarose gel electrophoresis.

Levels of Hdh mRNA were quantified by real-time quantitative RT–PCR performed on the MyIQ iCycler instrument (Bio-Rad Laboratories, Hercules, CA, USA) using the TaqMan® assay as previously described (31Go). Briefly, cDNA synthesis and amplification were done in a one-step reaction using the Superscript One-Step RT-PCR with Platinum Taq kit (Invitrogen). Each reaction was performed in 50 µl volume containing 200 ng of total RNA, 200 nM of each primer and probe, the reaction mix and 1 µl of the Superscript II/Pl Taq enzyme. Cycling parameters were 15 min at 50°C, 5 min at 95°C and 45 cycles as follows: 30 s at 95°C, 1 min at the Tm specific for each primer set. Each reaction was performed in triplicate. A standard curve with known dilutions of total mouse brain RNA (Clontech, Palo Alto, CA, USA) was generated to quantify the mRNA levels. The amount of Hdh mRNA in each sample was normalized to the levels of ß-actin, used as an endogenous control. The normalized Hdh/ß-actin ratios were then used to compare the relative levels of Hdh mRNA in all the samples. Primers and probes were designed using the Primer Express V 1.5 software (ABI, Foster City, CA, USA); each probe was dual-labeled with a 5'-FAM fluorophore and a BHQ-1-3' quencher. The sequences of the specific primers (Integrated DNA Technologies) and probes (Biosearch Technologies, Novato, CA, USA) were as follows: murine ß-actin (GenBank accession no. NM_007393), forward 5'-AGAGGGAAATCGTGCGTGAC-3', reverse 5'-CAATAGTGATGACCTGGCCGT-3', probe 5'-FAM-CACTGCCGCATCCTCTTCCTCCC-BHQ-1-3'; mouse Hdh (GenBank accession no. NM_010414) forward 5'-ACGGAAAGGGAAGGAGAA-3', reverse 5'-CCAGTGAGGATGATTTACTTG-3', probe 5'-FAM-AAGAAAGTTGGTGAGGCCAGTGC-BHQ-1-3'.

Statistical analysis was performed using a t-test (Microsoft Excel software).


   ACKNOWLEDGEMENTS
 
We would like to thank Dr X.-J. Li for providing the polyclonal EM48 antibody and Mr M. Hakky and Mr S. Chaudhury for technical assistance. This work was supported by the Cure HD Initiative, the HighQ Foundation, NINDS grants NS049206, NS032765 and P50NS016367 (Huntington Disease Center Without Walls), the Huntington's Disease Society of America and the Jerry McDonald Huntington's Disease Research Fund. A.L. was supported by CONTACyT postdoctoral fellowship 020076.

Conflict of Interest statement. The authors declare that they have no competing financial or other interests.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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