Human Molecular Genetics

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Human Molecular Genetics, 2000, Vol. 9, No. 17 2539-2544
© 2000 Oxford University Press

Dramatic mutation instability in HD mouse striatum: does polyglutamine load contribute to cell-specific vulnerability in Huntington’s disease?

Laura Kennedy and Peggy F. Shelbourne⁺

Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G11 6NU, UK

Received 15 June 2000; Revised and Accepted 25 August 2000.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
An unstable CAG triplet repeat expansion encoding a polyglutamine stretch within the ubiquitously expressed protein huntingtin is responsible for causing Huntington’s disease (HD). By quantifying the repeat sizes of individual mutant alleles in tissues derived from an accurate genetic mouse model of HD we show that the mutation becomes very unstable in striatal tissue. The expansion-biased changes increase with age, such that some striatal cells from old HD mice contain mutations that have tripled in size. If this pattern of repeat instability is recapitulated in human striatal tissue, the concomitant increased polyglutamine load may contribute to the patterns of selective neuronal cell death in HD. Our findings also suggest that trinucleotide repeat instability can occur by mechanisms that are not replication-based.

	INTRODUCTION

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Huntington’s disease (HD) is a progressive neurological disorder, characterized by early loss of medium spiny neurons in the striatum (1). The dominant HD phenotype is mediated by an expanded stretch of glutamine residues in a ubiquitously expressed protein of unknown function called huntingtin (2). Disease alleles carry >36 copies of the corresponding CAG trinucleotide repeat mutation (3,4) and display size mosaicism in germline and somatic tissues (5–7). The copy number of the CAG repeat is a primary determinant of the disease-associated pathology, showing a highly significant correlation with age at onset of symptoms (8) and neuronal pathology (9).

Although considerable progress in understanding HD pathogenesis has been made over recent years, one particular feature of the disease remains a puzzle. Given that mutant huntingtin protein is present at similar levels in many central nervous system (CNS) and non-CNS tissues (10), it is unclear as to why medium spiny striatal neurons are selectively vulnerable to the disease process (11). Most hypotheses to date have invoked cell-based differences in processing the mutant polyglutamine peptide (12) or the consequences of abnormal interactions between mutant huntingtin and its brain-specific protein partners (reviewed in ref. 13). We have considered an alternative hypothesis that proposes that a higher polyglutamine load in vulnerable cells may expedite downstream pathological events. As huntingtin expression levels are unaltered in HD, we reasoned that an increased polyglutamine load could be mediated by an increase in the copy number of CAG repeats present in the mutant gene. We therefore investigated the mutation profile in somatic tissues derived from an accurate knock-in mouse model of HD (14). These mice, generated by inserting 72–80 CAG repeats into the mouse counterpart of the human HD gene, express full-length mutant huntingtin appropriately and display behavioural and pathological features reminiscent of early HD (12,14,15). Here we show unexpectedly high levels of CAG repeat length variation in many tissues of the HD mice. The size of the mutations, particularly in some striatal cells, is much greater than previously considered in the corresponding human tissue.

	RESULTS

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Small-pool PCR (SP-PCR) assays (16) revealed tissue-specific differences in the CAG mutation profile of 24-month-old HD mice (Fig. 1A and Table 1). The group of six 24-month-old HD mice examined in this study was made up of males (n = 4) and females (n = 2) from two independently derived lines of HD mice, carrying 72 CAG repeats (n = 5) and 80 CAG repeats (n = 1), respectively. In each case, the mutation profile was very similar; low/medium levels of instability with a clear bias towards expansion in non-CNS tissues and most regions of the brain. Significantly, the highest mutational load and the greatest change in repeat copy number was observed in the striatum, where >80% of the cells displayed an increased allele length (greater than +5 CAG repeats). Some increases were dramatic, as illustrated in Figure 1B. Within the striatum of this HD mouse, 1:20 mutant alleles had >150 CAG repeats whereas 1:250 had >250 CAG repeats (Fig. 1B), three times the size of the progenitor allele. Moreover, accurate sizing of the CAG repeat stretch on individual mutant alleles from various brain regions of the same mouse confirmed that the median mutation length in the striatum was significantly greater than the corresponding measures in both cerebellum (P < 0.0001) and cortex (P < 0.0001) (Fig. 2).

View larger version (27K): [in this window] [in a new window]   Figure 1. Somatic mosaicism of the CAG repeat mutation in heterozygous HD mice derived from a line in which the wild-type allele carries seven triplet repeats and the progenitor mutant allele carries 72 CAG repeats at the Hdh locus. (A) Representative data from SP–PCR analyses demonstrating the mutation size in various somatic tissues from the same mouse, aged 24 months. Six reactions containing 10 amplifiable mutant molecules from each tissue are shown. Note the clear bias towards expansions; <1% of length changes in all tissues tested displayed contractions in allele size. (B) SP-PCR analyses of striatal tissue derived from a 24-month-old mouse demonstrate that 1 in 250 cells contains a mutant allele of >250 CAG repeats. Each lane contains the products from 50 amplifiable mutant molecules. (C) Age-dependent changes in the somatic instability of the CAG repeat mutation in HD mice. Each lane of the SP-PCR analysis contains the products of 20 amplifiable mutant molecules from the striata of heterozygous HD mice aged between 3 and 19 months. M indicates the size marker; the numbers on the right side of each panel indicate the estimated number of triplet repeats carried by the amplified alleles.

 

View this table: [in this window] [in a new window]   Table 1. CAG mutation instability in somatic tissues of a 24-month-old HD mouse

 

View larger version (30K): [in this window] [in a new window]   Figure 2. A comparison of the CAG repeat copy number on individual mutant alleles within the striatum (A), cortex (B) and cerebellum (C) of the 24-month-old heterozygous HD mouse referred to in Figure 1B. The histograms of CAG repeat length distribution are compiled from SP-PCR data of reactions containing 1–4 amplifiable mutant molecules. The median mutation length in the striatum is significantly larger than the median mutation lengths in the cerebellum and cortex [U = 9430.5, P < 0.0001, two-tailed Mann–Whitney U-test (striatum versus cortex); U = 6881.5, P < 0.0001, two-tailed Mann–Whitney U-test (striatum versus cerebellum)]. There is no significant difference between median mutation lengths in the cortex and cerebellum (U = 4125.5, P = 0.359, two-tailed Mann–Whitney U-test).

 
In order to determine whether the variation in mutation size increased as a function of time, we performed SP-PCR assays on tissue from mice aged between 3 and 19 months. The mutant CAG repeat allele was relatively stable in striatal (Fig. 1C) and other tissues of young mice (data not shown). At later time points, striatal mutation instability increased in an expansion-biased and age-dependent manner (Fig. 1C), although a more detailed study is required to determine whether large numbers of small repeat length changes or small numbers of large repeat length changes are responsible for the highly expanded mutant alleles detected in aged HD mouse striatum. Our findings suggest that selective vulnerability of neurons in HD may be caused by cell-specific determinants that directly or indirectly induce dramatic expansion-biased mutability of the CAG repeat over time. Although other cells in the brain also show CAG repeat instability and are most likely vulnerable to pathogenic polyglutamine effects, the striatal cells may succumb early by virtue of the fact that they exceed a critical concentration threshold first. This argument is further strengthened by the observation that the visible aggregation of mutant polyglutamine peptides in the medium spiny striatal neurons of the HD mice used in this study (12) occurs after significant mutation length variability is detected in striatal tissue. Previous reports have indicated that aggregate formation correlates well with polyglutamine length (17) and proceeds in a concentration-dependent manner (18,19). The proposed temporal gradient of neuronal vulnerability may also help explain the extensive extrastriatal neurodegeneration observed in human HD brain tissue at the end of a prolonged disease course (1).

	DISCUSSION

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So what drives the expansion-biased instability of the CAG mutation in the striatal cells of HD mice? To date, most discussion of the processes responsible for the dynamic behaviour of triplet repeat mutations has focused on replication-associated mechanisms such as slippage (20). Given that >65% of cells in the striatum are neurons (21) and therefore post-mitotic, it is difficult to reconcile our observations with small, successive replication-based expansions such as those postulated to explain germline instability in HD (22). Proliferation of glial cells is absent in the HD mice striata (14) and replication-competent cells in the subventricular zone of the striatum do not preferentially contain the largest mutant alleles (Fig. 3A). Moreover, no instability of the CAG repeat mutation could be detected during the period of greatest proliferation of neuronal precursors in the developing mouse brain (Fig. 3B). Finally, the contrasting mutation profiles of sperm and striatal tissue derived from the same aged HD mouse (Fig. 3C) lead us to conclude that mechanisms of repeat instability in terminally differentiated tissue may differ significantly from those operating in the germline. This notion is consistent with a number of previous reports that hint at non-replication based mechanisms of triplet repeat instability (23–26). The possibility that multiple rounds of DNA damage and repair may mediate mutation instability in post-mitotic cells (25) is made more compelling by the recent observation that the absence of Msh2 (a component of the mismatch–repair pathway) appears to reduce length variability of the CAG mutation in the striata of R6/2 HD mice (27). Normal age-related DNA damage due to increased oxyradical concentrations and diminished antioxidant defences in the striatum (28) could be exacerbated by the functional consequences of the HD mutation, thereby initiating a vicious circle of mutation expansion and associated cellular pathology in vulnerable cells. Significantly, the molecular context of the CAG mutation and/or the encoded polyglutamine stretch appears to be influential as a similar study using tissues derived from transgenic mice carrying a myotonic dystrophy-associated CTG/CAG repeat displayed different patterns of somatic instability (26). However, further studies are required to elucidate the precise nature of the mechanisms responsible for the tissue-specific patterns of mutation instability.

View larger version (24K): [in this window] [in a new window]   Figure 3. Instability of the CAG repeat mutation in the brains of aged HD mice may not be caused by a replication-based mechanism. (A) SP-PCR analyses indicate that the mutation profile obtained with a sample of striatal tissue containing replication-competent cells derived from the subventricular zone (+SVZ) does not differ significantly from that obtained when the SVZ is excluded from the striatal tissue sample (–SVZ). Each lane contains the products from 20 amplifiable mutant molecules; both +SVZ and –SVZ samples were obtained from the same heterozygous animal aged 24 months. (B) SP-PCR analyses demonstrate limited instability of the CAG mutation in the brains of heterozygous embryos at day 14 (E14) and 18 (E18) of gestation, the period of maximal neuronal-precursor cell division. Each lane contains the products from 50 amplifiable mutant molecules. (C) A comparison of somatic (striatum) and germline (sperm) instability of the mutation in a heterozygous HD mouse aged 24 months. Each lane of the SP-PCR analysis contains the products of 10 amplifiable mutant molecules. M indicates the size marker; the numbers on the right side of each panel indicate the estimated number of triplet repeats carried by the amplified alleles.

 
The idea that somatic instability of the CAG mutation may play a role in the cell-specificity of the pathological HD phenotype has been considered previously. Studies using densitometric approaches, including those utilizing Genescan analysis software, have noted that the HD mutation tends to be more unstable in the striatum than other tissues (5,23,25,27,29). However, the conclusions drawn from these data were somewhat limited as the maximum length changes reported were 20% the size of the progenitor mutant HD allele, 10-fold smaller than those detected in the present study. The discrepancy may be due to differences in the methodologies employed or the pathological state of the striatal tissue investigated. It is clear from this (Fig. 4A) and other studies (16,26,30) that amplification of bulk genomic DNA (>1000 template molecules per reaction) masks the level of allele size heterogeneity present in a DNA sample. In addition, the hybridization-based component of SP-PCR analysis allows detection and resolution of signals from single amplified products. Genescan methodology struggles to match this level of sensitivity (Fig. 4B). Advanced neurodegenerative changes in post-mortem brain tissue from end-stage HD patients may preclude detection of the largest mutant alleles since the vulnerable cells are no longer present. Although our study needs to be extended to human HD tissue, the limited availability of suitable autopsy material highlights the potential value of using the HD mice to study molecular changes that pre-date cellular pathology.

View larger version (38K): [in this window] [in a new window]   Figure 4. Comparison of CAG repeat length variation detected in striatal tissue from a 24-month-old HD mouse using SP–PCR analyses and Genescan-based methodology. (A) SP-PCR analyses demonstrate the level of CAG repeat length variation detectable in PCR reactions generated from differing amounts of input genomic DNA. Lanes in the left panel contain products from 3 cells (20 pg genomic DNA), lanes in the middle panel contain products from 50 cells (300 pg genomic DNA) and lanes in the right panel contain products from 17 000 cells (100 ng genomic DNA). M indicates the size marker; the numbers on the far right side of the panels indicate the estimated number of triplet repeats carried by the amplified alleles. (B) Representative Genescan traces generated by PCR products that are equivalent to those shown in a single lane of the three different SP-PCR analyses in (A). The vertical scale on the left side of the trace represents fluorescence intensity (arbitrary units); the numbers below the panel indicate the estimated number of triplet repeats carried by the amplified alleles. Note that the range of allele sizes detected by Genescan is much smaller than that detected by SP-PCR analysis.

 
We have established that the CAG mutation is particularly prone to expansion in the striata of aging HD mice. We predict that the cellular consequences of such large expansions (>250 CAG repeats) will be grave since a similar sized human HD mutation, the largest reported to date, is associated with a particularly severe, early-onset clinical picture (31). Understanding the molecular mechanisms that underlie mutation instability may help explain the distinctive neuropathological profiles of HD and other CAG repeat-associated diseases.

	MATERIALS AND METHODS

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Mouse tissue samples
The following heterozygous mice of different ages from line Hdh6/Q72 (14) with a hybrid 129/Sv/ter x C57BL/6 background were used in this study: 24 months (n = 5), 19 months (n = 1), 15 months (n = 2), 9 months (n = 2), 3 months (n = 3), embryonic days 14 (E14, n = 1) and 18 (E18, n = 1). A further 24-month-old heterozygous mouse from line Hdh4/Q80 (14) with a hybrid 129/Sv/J x C57BL/6 background was also included in the study. Tissue samples were harvested and frozen at –80°C prior to DNA extraction. Striatal tissue excluding the subventricular zone was microdissected from coronal sections (2 mm thick) using a tissue punch.

Preparation of genomic DNA and CAG repeat copy number determination
DNA from all samples except sperm was obtained using standard Proteinase K digestion followed by phenol/chloroform extraction. Sperm DNA was prepared using a differential lysis technique (16) followed by phenol/chloroform extraction. The Hdh CAG alleles were amplified from genomic DNA in a reaction mixture containing 10% DMSO, 200 µM dNTPs, 1 µM MHD16 and MHD18 primers (14), buffer (10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1 mM MgCl₂) and 2 U of Taq polymerase (Sigma, Poole, UK). The reactions were subjected to a touchdown PCR protocol: 94°C for 4 min followed by 20 cycles of 94°C for 30 s, 71–61°C (ramping down –0.5°C per cycle) for 30 s, 72°C for 30 s, 10 cycles of 94 °C for 30 s, 60°C for 30 s, 72°C for 30 s and, finally, an extension step of 72°C for 7 min.

For SP-PCR analyses, DNA was digested with HindIII and diluted in 10 mM Tris–HCl, pH 7.5, 1 mM EDTA and 0.1 µM carrier primer MHD16 to a final concentration of between 6 pg and 100 ng per reaction prior to amplification. The number of molecules amplified in a reaction was predicted using Poisson analysis, as previously described (16,30). The PCR products were loaded on 1.5% agarose gels (40 cm long) and electrophoresed in 0.5x TBE buffer at 180 V for 18 h at 4°C. The marker (200 ng) was loaded in both outside lanes and one middle lane of the gel (1 kb Plus or 1 kb size ladders; Gibco BRL, Paisley, UK). Southern blot analysis was performed using a radiolabeled probe comprising 20 ng of a small fragment containing the mutant CAG repeat and 70 bp flanking Hdh DNA [i.e. the amplicon defined by MHD4 and MHD5 primers (14)] plus 800 pg of the size marker. The amplified Hdh CAG alleles were revealed by autoradiography and sized using Kodak (Newhaven, CT) 1D software. All PCR reactions were set up in a laminar flow hood. No contaminating alleles were detected in the 20% of PCR reactions per run that were zero DNA controls.

Genescan analysis of PCR products was performed as previously described by Shelbourne et al. (14). Due to the limited volume of the polyacrylamide gel wells, the amplified products were concentrated by ethanol precipitation using glycogen as a carrier, prior to loading.

Statistical analysis
The statistical significance of CAG mutation length differences between striatal, cortical and cerebellar brain regions was calculated using the Mann–Whitney U-test.

	ACKNOWLEDGEMENTS

 
We thank Colin Hughes, Dennis Duggan, Giorgia Riboldi Tunnicliffe, Peggy Ennis and Heather Johnston for expert technical assistance and Darren Monckton and the rest of the University of Glasgow Dynamic Mutation Group for helpful discussions. This study was funded in part by grants from the Cunningham Trust and Tenovus-Scotland. L.K. is supported by a studentship from the Huntington’s Disease Association of Great Britain.

	FOOTNOTES

 
⁺ To whom correspondence should be addressed. Tel: +44 141 330 6200; Fax: +44 141 330 6871; Email: ps17z@udcf.gla.ac.uk

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