Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on September 2, 2003

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Human Molecular Genetics, 2003, Vol. 12, No. 21 2789-2795
DOI: 10.1093/hmg/ddg300
© 2003 Oxford University Press

Regional differences of somatic CAG repeat instability do not account for selective neuronal vulnerability in a knock-in mouse model of SCA1

Kei Watase^1,2, Koen J. T. Venken³, Yaling Sun⁴, Harry T. Orr⁵ and Huda Y. Zoghbi^1,2,3,4,*

¹Howard Hughes Medical Institute, ²Department of Molecular and Human Genetics, ³Program in Developmental Biology and ⁴Department of Pediatrics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA and ⁵Institute of Human Genetics, University of Minnesota, Minneapolis, MN 55455, USA

Received June 16, 2003; Revised August 15, 2003; Accepted August 25, 2003

	ABSTRACT

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Expression of unstable translated CAG repeats is the mutational mechanism in nine different neurodegenerative disorders. Although the products of genes harboring these repeats are widely expressed, a subset of neurons is vulnerable in each disease accounting for the different phenotypes. Somatic instability of the expanded CAG repeat has been implicated as a factor mediating the selective striatal neurodegeneration in Huntington disease. It remains unknown, however, whether such a mechanism contributes to the selective neurodegeneration in other polyglutamine diseases or not. To address this question, we investigated the pattern of CAG repeat instability in a knock-in mouse model of spinocerebellar ataxia type 1 (SCA1). Small pool PCR analysis on DNA from various neuronal and non-neuronal tissues revealed that somatic repeat instability was most remarkable in the striatum. In the two vulnerable tissues, cerebellum and spinal cord, there were substantial differences in the profiles of mosaicism. These results suggest that in SCA1 there is no clear causal relationship between the degree of somatic instability and selective neuronal vulnerability. The finding that somatic instability is most pronounced in the striatum of various knock-in models of polyglutamine diseases highlights the role of trans-acting tissue- or cell-specific factors in mediating the instability.

	INTRODUCTION

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Spinocerebellar ataxia type 1 (SCA1) is a progressive neurodegenerative disorder, characterized by degeneration of cerebellar Purkinje cells and neurons within the brain stem and the spinal cord. The mutation underlying SCA1 is an expansion of a polymorphic CAG trinucleotide repeat that encodes a polyglutamine tract in the coding region of the SCA1 transcript (1). In the unaffected individuals, the allele sizes range from six to 44 CAG units, and are always interrupted by at least one CAT triplet when the length exceeds 21 repeats. In contrast, SCA1 disease alleles carry uninterrupted CAG tracts ranging in size from 40 to 83 repeat units (2,3).

At least eight other dominantly inherited disorders result from expansions of CAG repeats: spinobulbar muscular atrophy (SBMA), Huntington's disease (HD), dentatorubral pallidoluysian atrophy (DRPLA), and the spinocerebellar ataxias types 2, 3, 6, 7 and 17 (4,5). The repeat size is a major determinant of the severity and pathology in these disorders: the longer the repeat, the more severe the symptoms, the earlier the age of onset, and the more widespread the pathology (6). In most of these diseases, with the exception of SCA6 and 17, the expanded CAG repeats are quite unstable both in germline and in somatic tissues. The expanded repeats often increase in size upon parent-to-offspring transmissions, resulting in earlier manifestation of the clinical symptoms in subsequent generations, a phenomenon called anticipation. The magnitude and direction of the intergenerational repeat instability has been shown to be dependent on the gender of parent-of-origin; paternal transmissions tend to be most unstable and result in further expansion whereas maternal transmissions often lead to contraction of the transmitted alleles. In somatic tissues, the expanded alleles are unstable with variable patterns of expansions and contractions in different tissues (7).

Recently, considerable progress has been made in understanding some of the pathogenic mechanisms underlying polyglutamine diseases. The use of cellular and animal models revealed that a gain of function mechanism mediates disease and illustrated the importance of protein folding and degradation in this class of diseases. Furthermore, these studies highlighted the importance of the protein context and protein modification for disease expression (8–10). In spite of this exciting progress, however, one of the key features of polyglutamine diseases remains elusive; why is it that certain groups of neurons degenerate in each disease while the mutant expanded proteins are widely expressed in the brain in most of the disorders? One hypothesis posits that expression patterns and levels of proteins that modify and/or interact with the mutant polyglutamine protein contribute to the selective vulnerability in each disease (11). Another hypothesis predicts that the capacity to degrade or process toxic mutant proteins may differ from neuron to neuron (12). Slight variations in the levels of the mutant proteins are also likely to contribute to cellular vulnerability given the large body of data demonstrating a direct correlation between levels of mutant protein and neuronal dysfunction and degeneration. Building on the knowledge that the longer repeats cause more severe disease in humans, another hypothesis proposed somatic instability (and hence further repeat expansion) as a possible mechanism for selective neuronal vulnerability (13). In order to investigate this intriguing hypothesis in vivo, it is necessary to study animal models that carry mutant alleles that are expressed in a spatial and temporal pattern that parallels the human condition. To this end, Kennedy and Shelbourne (13) conducted a study of an accurate genetic model of HD, where 72–80 CAG repeats were inserted into the mouse counterpart of the HD gene. They carefully measured the repeat sizes of individual mutant alleles in various tissues derived from the mutant animals and found that CAG repeats are most unstable, with a tendency to expand in striatal tissue. Wheeler et al. (14) observed a similar dramatic pattern of somatic instability in striatal tissues from two other knock-in HD models, Hdh^Q92 and Hdh^Q111. These results raised the possibility that large expansions may precede HD pathology and that somatic mosaicism of the CAG repeat may be the basis for the selective neurodegeneration seen in HD brains.

To determine if somatic instability accounts for selective neuronal vulnerability in other polyglutamine diseases, it is imperative to evaluate somatic repeat instability in other knock-in models of polyglutamine disorders that reproduce the selective neuronal dysfunction and distinct vulnerabilities seen in the respective human disorders.

Previously, we evaluated the CAG repeat instability in an SCA1 knock-in model carrying 78 CAG repeats (15). Because these mice did not develop progressive neuronal degeneration, the studies were limited to analysis of intergenerational instability. In order to address the issue of selective neuronal degeneration, in the present study, we investigated the somatic and intergenerational repeat instability of the expanded CAG allele in the Sca1^154Q/2Q mice (12). These mice possess 154 CAG repeats in the mouse Sca1 locus, they express mutant ataxin-1 according to the endogenous patterns, and they replicate the progressive selective neuronal vulnerability seen in the human disease.

	RESULTS

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Stability of the Sca1^154Q allele in intergenerational transmissions
We investigated the intergenerational CAG repeat instability in the Sca1^154Q/2Q mice by comparing the length of the repeat tract, amplified from tail DNA, from each mother or father to the length of the alleles observed in their progeny (Fig. 1). We studied the DNA of offsprings from 41 paternal transmissions (mean paternal age; 15.4±0.9 weeks) and 22 maternal transmissions (mean maternal age; 18.3±2.2 weeks). The rate of repeat instability per transmission of the expanded CAG allele was 70.7% (29 transmissions) for the male germline and 86.4% (19 transmissions) for the female germline. These results suggest that the Sca1^154Q allele is more unstable than Sca1^78Q allele, where the mutation rates for the paternal and the maternal transmission were 9.2 and 69.2%, respectively (15). In paternal transmissions, the changes in the size of the repeat ranged from -5 to +3 repeats, with a mean change of -0.6±0.3 repeat and a slight bias toward contraction (18 transmissions for contraction and 12 transmissions for expansion, respectively). On the other hand, all of the changes observed in the maternal transmissions were contractions ranging from -19 to -1 repeat, with a mean change of -5.3±1.1, indicating a strong bias towards contraction during maternal transmission. Notably, three most prominent contractions (-19, -13, -13) were observed in the progeny from the two oldest mothers (29.0 and 35.4 weeks of age, respectively, at the time of mating), while the progeny from young mothers showed moderate or no alteration in the repeat size (data not shown). These results suggest that, similar to the Sca1^78Q allele, the Sca1^154Q allele also features an increase in the magnitude of CAG repeat contraction with increasing age of the mother.

View larger version (18K): [in this window] [in a new window]   Figure 1. The changes of the CAG repeat size during intergenerational transmissions of the expanded Sca1 alleles.

 

	TISSUE- AND TIME-DEPENDENT SOMATIC MOSAICISM IN Sca1154Q/2Q MICE

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To examine somatic repeat instability in the Sca1^154Q/2Q mice, we harvested tissues from two mutant females (44 and 46 weeks of age) and a mutant male (41 weeks of age) at their advanced stage. Replicate small pool polymerase chain reactions (SP–PCRs) were performed with DNA molecules obtained from various tissues (olfactory bulb, cerebral cortex, striatum, hippocampus, cerebellum, pons, spinal cord, heart, lung, liver, kidney, skeletal muscle, tail, testis and ovary) and the sizes of the amplified products were compared. Somatic repeat instability was found in most of the tissues. Among the brain tissues of the 41-week-old animal, the repeat was dramatically unstable in the striatum with a slight bias toward expansion (Fig. 2A). A subset of cells from the striatum contained alleles in excess of 210 repeats (thus an expansion of more than 56 repeats) which were not observed in the other tissues (based on analysis of 500–2500 amplified mutant alleles per tissue). In the cerebellum and spinal cord, sites where neurodegenerative changes are known to occur in SCA1, there were substantial differences in the pattern and degree of instability. The repeat remained relatively stable in the cerebellum where expansions greater than +20 CAG repeats were never observed and contractions by as many as 50–100 repeats were occasionally seen among a total of more than 2500 amplifiable mutant alleles tested. Compared to the cerebellum, the degree of instability was higher in the spinal cord, where contractions as well as expansions up to 200 CAG repeats were seen. Other brain regions, such as pons, cerebral cortex, olfactory bulb and hippocampus, displayed moderate instability where expansions greater than +20 repeats were often observed. In the tissues outside the brain, the repeat was relatively stable compared with the brain, but large reductions of the CAG repeat size were occasionally observed (Fig. 3A). Among those somatic tissues, expansions greater than +10 repeats were observed almost exclusively in the kidney and rarely in the liver (data not shown). In the 44- and 46-week-old female mice, the repeat instability was slightly more marked than the 41-week-old animal and biased towards expansion, but the patterns of regional changes were generally similar to those seen in the 41-week-old animal (Figs 2B and C and 3B). In the striatum, a considerable number of the mutant alleles, which appeared as intense bands on the blots, showed expansions greater than +20 repeats. The cerebellum displayed relatively stable repeats just like that of the 41-week-old mouse. Expansions up to 210 repeats were occasionally found in the spinal cord of these animals. The frequency of such large expansions, however, was less than those observed in the striatum. In the pons of these animals, both contractions and expansions, sometimes greater than +80 repeats, were found.

View larger version (58K): [in this window] [in a new window]   Figure 2. Somatic mosaicism of the CAG repeat mutation in the central nervous system of Sca1154Q/2Q mice. Shown are representative SP–PCR reactions each containing 10–30 molecules of DNA from mice at 41 (A), 44 (B) and 46 (C) weeks of age. Marker bands indicate 234, 154, 105, 50 and 2 CAG repeats, respectively. Because a probe spanning the polyglutamine tract is used to detect the expanded allele, the signal from the wild-type (2Q) allele is very faint.

 

View larger version (35K): [in this window] [in a new window]   Figure 3. Somatic mosaicism of the CAG repeat mutation in the peripheral organs of Sca1154Q/2Q mice. Shown are representative SP–PCR reactions each containing 10–30 molecules of DNA from mice at 41 (A) and 46 (B) weeks of age. Marker bands indicate 234, 154, 105, 50 and 2 CAG repeats, respectively.

 
In order to determine whether there was a tendency for the mutant alleles to expand more as the animals aged, we also performed SP–PCRs on brain tissues from 7 and 30 weeks of age (Fig. 4). The mutant CAG repeat allele was relatively stable in brain both at 7 and 30 weeks. At 30 weeks of age, small portion of the mutant alleles displayed contraction in these tissues, but expanded alleles were rarely observed in any brain region, suggesting that the CAG repeat instability is age-dependent and most expansions take place at later than 30 weeks of age, well beyond the onset of the neuronal dysfunction and degeneration.

View larger version (123K): [in this window] [in a new window]   Figure 4. SP–PCRs of the CAG repeat mutation in the central nervous system of young Sca1154Q/2Q mice. Shown are representative SP–PCR reactions each containing 10–30 molecules of DNA from mice at 7 and 30 weeks of age. Marker bands indicate 154 and 2 CAG repeats, respectively.

 

	DISCUSSION

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The hypothesis that somatic mosaicism may contribute to the specificity of neurodegeneration was previously tested by determining the CAG tract size in various brain tissues from SCA1 patients (16,17). Both of these studies noted that the repeat size in the mutant allele was smaller in cerebellar tissue compared with the repeat size in other brain regions, such as cerebral cortex, lending support to the conclusion that there is no causal relationship between the degree of somatic mosaicism and selective neurodegeneration in this disease. However, this conclusion can be challenged because the apparent lack of the expanded allele in the cerebellum might simply be due to the loss of vulnerable neurons in postmortem tissues that had undergone severe neurodegenerative changes. The Sca1^154Q/2Q knock-in mouse is a faithful genetic model of SCA1 and replicates the selective neurodegeneration seen in the human disease (12). Routine histopathological and Nissl studies revealed neurodegenerative changes in the Purkinje cells and gliosis in the spinal cord with minimal neuronal loss in other regions of the brain. It is noteworthy that the mutant Purkinje cells were reduced only by 20% in the advanced stage of the disease (12). Thus this model provided a good opportunity for testing the hypothesis that somatic instability contributes to selective neuronal vulnerability. The knock-in models also have the added advantage in that they preserve the genomic context surrounding the CAG repeat which is known to be a key factor for the instability (18).

We found that the degree of instability was much less remarkable in the cerebellum of the mutant animals compared with that in the striatum. Moreover, there were considerable differences in the pattern and degree of instability between the cerebellum and spinal cord. In the cerebellum, granule neurons represent the most predominant cell population and therefore are the most abundant source of amplifiable DNA. However, given the magnitude of the analysis in the present study (a total of more than 7500 DNA molecules from the three aged mutant cerebella), we believe that somewhere in the vicinity of three dozens mutant alleles derive from the surviving Purkinje neurons based on the estimated ratio of 175 granule neurons for every Purkinje cells in the mouse (19). Therefore, the failure to find even a single ‘hyper’-expanded allele in the cerebellum argues against remarkable expansions in both Purkinje and granule cells. Overall, those results suggest that somatic CAG repeat instability is not directly associated with selective neuronal vulnerability in SCA1. It is interesting that somatic repeat instability was most remarkable in the striatum of aged HD knock-in mice (13,14). Because striatal neurons are the most vulnerable cells in HD, it is conceivable that, in this disease, in contrast to SCA1, somatic repeat instability contributes to the selective loss of striatal neurons.

The molecular mechanisms underlying somatic CAG/CTG repeat instability are still largely unknown. It has been suggested that repeat instability arises via DNA replication slippage during cell division (20,21). However, recent studies of mouse models of Huntington's disease and myotonic dystrophy have found a remarkably higher degree of instability in tissues which contain a large proportion of postmitotic cells, such as brain and kidney (13,22). In the present study, we found remarkable instability in the striatum of the aged mutant animals. Neural stem cells in the subventricular zone (SVZ; which borders the striatum) and in the dentate gyrus within the hippocampus have been shown to be a source of new neural cells in adult mice (23). Although large expanded alleles were found in the striatum, the repeat instability was moderate in the hippocampi of both 41- and 46-week-old animals, and large expansions were also identified in the pons of the 46-week-old animal. Thus the data from our study, consistent with published reports, did not show an association between the degree of instability and the presence or absence of replicating stem cells and/or newly generated neural cells. Moreover, the repeat instability does not appear to be associated with proliferation of glial cells, either. The Sca1^154Q/2Q mice develop astrogliosis in the spinal cord but not in the striatum or the pons by the age of 40 weeks (12). Our results suggest that replication does not play a major role in the regional difference of the CAG repeat instability in the mutant mouse brain.

The pattern of somatic mosaicism in the Sca1^154Q/2Q mice resembles that of other knock-in and transgenic mouse models. For instance, the Dmt-D line, which harbors a transgene construct comprised of 162 CTG repeats and 750 bp of the flanking DNA from the human DM1 locus without any coding DNA, features relatively unstable repeats in the striatum and kidney but stable repeats in the cerebellum (23). The knock-in mouse models of Huntington's disease, which have 72–80 CAG repeats in the mouse counterpart of the human HD gene, also showed relatively high instability in the striatum and kidney compared with other brain areas and peripheral organs, respectively (14). These comparisons highlight the importance of tissue/cell specific factors in determining the tissue specificity of CAG/CTG repeat instability.

Recently, several studies on the animal models of HD and myotonic dystrophy have pointed to the possible involvement of components of the DNA repair machinery in the repeat expansion (24–26). Manley et al. (24) showed that age-dependent somatic repeat instability is abrogated in various somatic tissues of transgenic mice carrying exon 1 of HD but lacking the mismatch repair enzyme Msh2. Similarly, the absence of Msh2 eliminated the somatic repeat expansion in the striatum of Hdh^Q92 and Hdh^Q111 knock-in mice (25). Other key proteins in the DNA repair machinery have also been implicated in the somatic repeat instability (26). Msh3 deficiency led to complete block of the somatic repeat instability in DM1 (CTG)₈₄ knock-in mice. In contrast, Msh6 deficiency resulted in an enhancement of the somatic repeat instability in the same model. So far, there is only limited knowledge on the distribution and/or role of these mismatch repair enzymes in the mammalian brain. It is of note that Msh2 immunoreactivity is distributed heterogeneously in the rat brain (27). The difference in the DNA repair activity and/or the incidence of DNA damage during the aging process among various tissues or cell types may be the basis for the tissue specificity of somatic CAG/CTG repeat instability.

It is noteworthy that the SCA1 CAG repeats undergo contractions in the female germline in three different mouse models: an SCA1 transgenic line carrying 82 repeats in a full-length cDNA (D02) (28), a knock-in allele with 78 repeats (Sca1^78Q/2Q) (15), and a knock-in allele with 154 repeats (Sca1^154Q/2Q) (12). Although the frequency and extent of intergenerational instability in the Sca1^154Q/2Q mice is more remarkable than either of the two other alleles, the similar trends in all three models argue that maternal contractions might be the default state and/or that sequences within the cDNA are sufficient to promote contractions in maternal germline.

In summary, in Sca1^154Q/2Q mice, we did not detect a causal relationship between the degree of somatic repeat instability and selective neuronal vulnerability. Future studies on other mouse models will be helpful to clarify the relationships between the somatic mosaicism and neurodegeneration in polyglutamine diseases. Moreover, Sca1^154Q/2Q mice along with other knock-in models will be a good tool for identifying the trans-acting factors which govern the somatic repeat mosaicism in vivo.

	MATERIALS AND METHODS

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Mice
All mice used in this study were heterozygous for the mutation on a mixed (C57BL6/Jx129Sv/Ev) backgound.

Size determination of the Sca1^154Q allele for intergenerational instability
Intergenerational repeat instability was examined as previously described elsewhere (12). Briefly, genomic DNA was isolated from tails taken at 4 weeks of age. DNA fragments containing the CAG repeats were amplified with primers 5'repU and 3'repL with the reported conditions, using incorporation of [-³²P]dCTP and displayed in a denaturing polyacrylamide gel.

SP–PCR analysis
Genomic DNA was obtained using standard Proteinase K digestion followed by phenol/chloroform extraction. After amplifiable genome equivalence (a.g.e.) of input DNA was determined by SP–PCR using 3–9 pg of DNA, 10–30 a.g.e. of DNA was analyzed per each reaction. Primers sca1-oU (5'-GTCACCAGTGCAGTAGCCTCAG-3') and sca1-oL (5'-ATGTACTGGTTCTGCTGGGTG-3') were used to determine the repeat length. The Sca1 CAG alleles were amplified from geneomic DNA in a reaction mixture containing 11% DMSO, 200 µM dNTPs, 0.2 µM sca1-oU and sca1-oL primers, buffer (10 mM Tris–HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl₂) and 1 U of HotStarTaq^TM DNA polymerase (Qiagen, Germany). PCR cycling conditions are as follows: 94°C for 12 min followed by 35 cycles of denaturing at 94°C for 30 s, annealing at 61°C for 30 s, extension at 72°C for 1 min and final extension at 72°C for 7 min. The PCR products were loaded on 2% agarose gels and electrophoresed at 70 V for 18 h. Southern blot analysis was performed using a radiolabeled probe containing 20 ng of a fragment containing the 154 CAG repeat and 196 bp flanking Sca1 DNA. All PCR reactions were set up in a laminar flow hood.

	ACKNOWLEDGEMENTS

 
We are grateful to D. Shinde and N. Arnheim for help determining the appropriate primers for PCR reactions by performing single-sperm analysis of the Sca1^154Q/2Q mice. This work is supported by grants from the National Institute of Neurological Disorders and Stroke of NIH to H.Y.Z. (NS27699) and H.T.O. (NS22920). H.Y.Z. is an Investigator and K.W. is a Postdoctoral Research Associate with the Howard Hughes Medical Institute.

	FOOTNOTES

 
^* To whom correspondence should be addressed. Tel: +1 7137986558; Fax: +1 7137988728; Email: hzoghbi{at}bcm.tmc.edu

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