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Human Molecular Genetics Pages 763-774  

A Huntington's disease CAG expansion at the murine Hdh locus is unstable and associated with behavioural abnormalities in mice
Introduction
Results
   Generation of mice carrying a CAG expansion allele at the mouse Hdh locus
   Genetic analysis of the HD-repeat knock-in mouse lines
   Histological examination of brains from HD-repeat knock-in mice
   Mice expressing mutant huntingtin display abnormal social behaviour
Discussion
Materials And Methods
   Generation of the targeting constructs (Fig. 1A)
   Gene targeting in ES cells and generation of mutant mice
   In vitro excision of loxPneo sequences
   Genotyping and CAG repeat copy number determination
   Recombinant DNA techniques
   Protein analysis
   Histopathology
   Behavioural analyses of mice
   Oligonucleotides
Acknowledgements
References

A Huntington's disease CAG expansion at the murine Hdh locus is unstable and associated with behavioural abnormalities in mice

A Huntington's disease CAG expansion at the murine Hdh locus is unstable and associated with behavioural abnormalities in mice

Peggy F. Shelbourne1,6,*, Nigel Killeen2, Robert F. Hevner3, Heather M. Johnston6, Laurence Tecott4, Mark Lewandoski5, Margaret Ennis6, Lucia Ramirez1, Zhen Li1, Carlo Iannicola1,+, Dan R. Littman7 and Richard M. Myers1

1Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305, USA, 2Department of Microbiology and Immunology, 3Nina Ireland Laboratory of Developmental Neurobiology, 4Department of Psychiatry and 5Department of Anatomy and Program in Developmental Biology, University of California, San Francisco, CA 94143, USA, 6Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G11 6NU, UK and 7Howard Hughes Medical Institute, Skirball Institute of Biomolecular Medicine, New York University Medical Center, New York, NY 10016, USA

Received January 12, 1999; Revised and Accepted February 24, 1999

Huntington's disease (HD) is a dominant disorder characterized by premature and progressive neurodegeneration. In order to generate an accurate model of the disease, we introduced an HD-like mutation (an extended stretch of 72-80 CAG repeats) into the endogenous mouse Hdh gene. Analysis of the mutation in vivo reveals significant levels of germline instability, with expansions, contractions and sex-of-origin effects in evidence. Mice expressing full-length mutant protein display abnormal social behaviour in the absence of acute neurodegeneration. Given that psychiatric changes, including irritability and aggression, are common findings in HD patients, our data are consistent with the hypothesis that some clinical features of HD may be caused by pathological processes that precede gross neuronal cell death. This implies that effective treatment of HD may require an understanding and amelioration of these dysfunctional processes, rather than simply preventing the premature death of neurons in the brain. These mice should facilitate the investigation of the molecular mechanisms that underpin the pathway from genotype to phenotype in HD.

INTRODUCTION

Huntington's disease (HD) is a dominantly inherited neurological disorder that follows a progressive course, leading to death 15-20 years after onset of symptoms (1). Typical clinical features include rapid, uncontrolled movements of the limbs and trunk, cognitive impairment and emotional disturbances such as depression and irritability. These have been attributed to the loss of specific subpopulations of neuronal cells, primarily in the striatum of the basal ganglia and, to a lesser extent, in other extrapyramidal and cortical areas. The onset of symptoms typically occurs in mid-adult life, although this is partially influenced by the sex and age at onset of the transmitting parent.

In 1993, the identification of a gene containing a CAG trinucleotide repeat that is expanded and unstable on HD chromosomes was reported (2). Normal chromosomes have a repeat length of <30 that is inherited in a stable, polymorphic manner. In contrast, almost all (>99%) HD chromosomes have 36 CAG repeats or more (3,4), with the copy number showing an inverse correlation with the age at onset of the disorder (5). The mutation is unstable in >70% of transmissions. The largest length increases occur during paternal transmission, accounting for juvenile-onset cases typically inheriting the disease from their affected father (3,4,6).

The CAG repeat encodes a polyglutamine stretch in a ubiquitously expressed protein of unknown function, called `huntingtin'. Although the presence of an expanded stretch of CAG repeats seems to correlate well with the presence of HD, it is still unclear how it mediates the dominant disease phenotype. Current opinion favours a gain-of-function rather than a loss-of-function effect (7). This notion is supported by the fact that targeted disruption of both alleles of the highly conserved orthologue of the HD gene in mice (Hdh) causes early embryonic death (8-10), whereas patients who are homozygous for the CAG expansion in the human HD gene are viable and apparantly phenotypically indistinguishable from their heterozygous siblings (11).

A number of other neurodegenerative disorders, including spinal and bulbar muscular atrophy (SBMA), dentatorubral-pallidoluysian atrophy (DRPLA) and spinocerebellar ataxia types 1, 3 and 7 (SCA1, SCA3 and SCA7), are also caused by polyglutamine tract expansions (for a review, see ref. 12). Although the mutant protein implicated in each of these diseases is different, a novel gain-of-function property, mediated by the polyglutamine stretch, appears to be responsible for the distinct patterns of neuronal degeneration in each case. Recent observations of mice that are transgenic for the HD mutation (13) have provided intriguing new insights into a possible mechanism. These mice display a neurological phenotype which appears to be associated with the presence of insoluble nuclear aggregates called neuronal intranuclear inclusions (NIIs) (14). Similar NIIs, containing polyglutamine peptides and ubiquitin, subsequently have been demonstrated in the nuclei of affected neurons in HD (15), DRPLA (16), SBMA (17), SCA1 (18), SCA3 (19) and SCA7 (20). Whilst it has been postulated that NIIs may have a role in promoting premature neurodegeneration, it remains a major research challenge to define the precise details of the molecular mechanisms responsible for the pathology in this group of diseases. For example, if nuclear aggregates disrupt the functional integrity of neurons, do aspects of their formation display cell specificity? Can they account for all the clinical features of the disease phenotype or does the mutation have other functional consequences, perhaps mediated by the interactions of the full-length mutant protein?

Our work has focused on generating a mouse model of HD that permits the study of the behaviour and functional consequences of the mutation in its appropriate genomic and protein contexts. In this report, we demonstrate that the insertion of 70-80 CAG repeats into the murine Hdh gene results in observable behavioural abnormalities-the first time that a phenotype has been associated with the endogenous expression of full-length mutant huntingtin protein in mice. We also provide evidence suggesting that pathological pathways, other than acute neurodegeneration, may contribute to the symptomatology of HD.

RESULTS

Generation of mice carrying a CAG expansion allele at the mouse Hdh locus

   A, B
   C, D

Figure 1. Generation and identification of a mutant CAG expansion allele at the Hdh locus. (A) Schematic representing the stepwise assembly of the constructs used for targeted modification of the Hdh locus, in which the wild-type glutamine-encoding stretch [(CAG)2CAA(CAG)4] is replaced by an expanded tract of 80 perfect CAG repeat units. Details of steps 1-3 are found in Materials and Methods. The final targeting construct was created by insertion of a 1.3 kb pMC1neo cassette flanked by loxP sites (loxPneo) into the downstream intron. The orientations of the neo gene transcription and loxP sequences are indicated by arrows. (B) Schematic representation of the targeting construct, the endogenous Hdh allele and the predicted structure of the mutant CAG expansion allele generated by homologous recombination and a Cre-mediated excision event. The upper line represents a restriction map of the 5[prime] end of the Hdh gene and the stippled boxes depict the probes used to identify correctly targeted ES cell clones. The middle line represents the predicted structure of the modified Hdh locus after homologous recombination. Expected restriction fragment sizes are indicated by horizontal arrows. The lower line represents the predicted genomic structure of the final mutant CAG expansion allele after Cre-mediated excision of the loxPneo sequences. B, BamHI; S, ScaI; H, HindIII. (C) Identification of recombinant ES cell clones. The introduction of the expanded CAG mutation and loxPneo sequences into the Hdh locus increases the size of the ScaI fragment containing this genomic region as well as introducing a novel BamHI site. Genomic DNA from ES cell clones was digested with ScaI and BamHI and hybridized with probes a and b. Fragments derived from correctly (lane 1) and incorrectly (lanes 2 and 3) targeted clones are indicated. These data were confirmed by further screening with probes derived from neo and pBluescript sequences (data not shown). (D) The expanded CAG mutation in the recombinant ES cell clones was detected by PCR analysis. Products, amplified from wild-type (lane 2), recombinant (lanes 3-5) ES cell clone DNAs and a zero DNA control (lane 1), were resolved on an ethidium bromide-stained agarose gel.

A strategy of targeted modification by homologous recombination in murine embryonic stem (ES) cells was used to generate a mutant CAG expansion allele of the Hdh gene. We constructed a series of targeting vectors containing Hdh genomic sequences, modified in the following way. Firstly, a stretch of 72-80 CAG repeats replaced the normal polyglutamine-encoding stretch in exon 1. Secondly, a neomycin resistance expression cassette flanked by 34 bp loxP sites (loxPneo) was inserted ~200 bp downstream of the CAG mutation, 60 bp into intron 1 (Fig. 1A). The construction and configuration of these targeting vectors are detailed in Materials and Methods, Figure 1 and Table 1.

Selection and subsequent screening of transfected ES cells identified three independently targeted clones (Fig. 1B-D and Table 1). However, western blot analyses (Fig. 2) demonstrated that mutant huntingtin protein was only detected in these cells after Cre-mediated deletion of the loxPneo sequences at the modified Hdh locus. Although these data do not elucidate the mechanism by which the loxPneo cassette exerts its influence on the modified Hdh locus, they suggest it is not dependent on the direction of neo transcription since e4A11 and e6C9 cells carry the loxPneo sequences in opposite orientations. Mouse lines (Hdh4/neo and Hdh6/neo) were produced from the recombinant e4A11 and e6C9 cell lines, carrying the neo+ allele with both the CAG expansion and the loxPneo sequences at the Hdh locus. Heterozygous mutant males from these lines were then crossed with homozygous [beta]-actin-cre females (21) to achieve an in vivo deletion of the loxPneo sequences in the developing embryos. PCR and Southern blot analyses of DNA from tail biopsies of the F1 and subsequent progeny indicated that neo sequences were not present in mice carrying the CAG mutation. These data were consistent with previous studies of the cre transgenic lines indicating that recombination occurs in all cells of the embryo, at or before the blastocyst stage (21). The founders were bred to establish the `HD-repeat knock-in' mouse lines, designated Hdh4/Q80 and Hdh6/Q72.


Figure 2. Expression of the Hdh gene in recombinant ES cells before and after in vitro Cre-mediated excision of the loxPneo sequences. Immunodetection of normal and mutant huntingtin protein was performed using the [alpha]HD2 antibody. Western blot analysis of protein lysates from wild-type (RF8 and JM-1, lanes 2 and 5) and recombinant ES cells, before (e4A11 and e6C9, lanes 3 and 6) and after (e4A11[Delta]neo and e6C9[Delta]neo, lanes 4 and 7) excision of the loxPneo sequences, confirmed that deletion of the loxPneo sequences was required for expression of the mutant huntingtin protein, detected as a more slowly migrating band (*). Normal huntingtin protein is detected in the control protein lysate (lane 1) from the mouse fibroblast 3T3 cell line.

Table 1. Homologous recombination of the Hdh gene
Targeting construct pCAG5.1 pCAG5.2 pCAG6.1 pCAG6.2
Length of homology (kb) 5.0 5.0 5.0 5.0
CAG repeat copy number 80 80 72 72
Orientation of neo cassettea - + - +
ES cell lines transfected RF8; JM-1 RF8; JM-1 JM-1 JM-1
G418-resistant ES cell clones analysed 400 400 200 200
Correctly targeted alleles detected:        
(i) Southern blot (for presence of neo cassette) 1 1 1 1
(ii) PCR analysis to detect expanded CAG repeat 0 1 1 1
Identity of recombinant ES cell clone - e4A11 e6C9 e8A2
(parental ES cell line)   (RF8) (JM-1) (JM-1)
Mouse lines generated - Hdh4/neo Hdh6/neo -
    Hdh4/Q80 Hdh6/Q72  
aEither the same (+) or opposite (-) orientation to normal Hdh transcription.


The expression profile of the mutant huntingtin protein was examined in the HD-repeat knock-in mice. Immunoprobing of tissue lysates from brain (Fig. 3), heart, liver and testes (data not shown) with an anti-huntingtin antibody ([alpha]HD2) (22) revealed an expression pattern that was consistent with the genotype at the Hdh locus. The [alpha]HD2 antibody was raised against a peptide corresponding to the region predicted to lie between amino acid positions 2531 and 2539 of human huntingtin. When the blots were stripped and reprobed with IC2 antibody (23) which specifically detects long polyglutamine tracts, only the more slowly migrating mutant protein bands displayed immunoreactivity. These data confirmed that targeted modification of the Hdh locus followed by in vivo deletion of loxPneo sequences resulted in a mutant allele that expressed full-length huntingtin containing an expanded polyglutamine stretch.


Figure 3. Expression of the Hdh gene in the Hdh4/Q80 and Hdh6/Q72 knock-in mouse lines. Brain protein lysates from wild-type (+/+), heterozygous (+/-) and homozygous (-/-) mice were immunoprobed with [alpha]HD2 antibody (left panel), and reprobed with IC2 antibody (right panel). Although both isoforms of the huntingtin protein were present in all the heterozygous tissues tested, levels of the mutant form were consistently lower than the normal form (for an example, see Fig. 3). Optical densitometry of the immunoblots indicated that this reduction was ~45% in the Hdh6/Q72 line and ~55% in the Hdh4/Q80 line. Although the single 34 bp loxP site, left at the site of the Cre-mediated excision event in intron 1 (Fig. 1B), could exert a residual influence on the expression of the mutant Hdh gene, it is significant that studies of human HD tissue show a similar reduction in levels of mutant huntingtin relative to the normal protein (24,25). This phenomenon appears to be exacerbated as the size of the mutation increases, and has been attributed to a relative decrease in expression levels of the mutant allele, rather than alterations in stability of the resulting mutant protein isoform (25). Although no quantitative or qualitative differences in degradation products were detected in protein lysates derived from the brains of either wild-type or mutant littermates when probed with the [alpha]HD2 and IC2 antibodies, further analysis with an extended panel of anti-huntingtin antibodies is merited to clarify further the molecular basis of this observation.


Since HD is a dominant disease, we have concentrated on characterizing HD-repeat knock-in mice that are heterozygous for the CAG expansion allele. As such, all data from mutant mice in this report were derived from heterozygotes, unless otherwise stated. Studies of homozygous mice currently are under way and will be reported elsewhere.

   A
   B

Figure 4. Intergenerational instability of the CAG repeat mutation in the Hdh4/Q80 and Hdh6/Q72 knock-in mouse lines. (A) Genescan traces representing the size of the CAG mutation in tail biopsy DNA. Analyses of a mutant male mouse (no. 1) and three of his heterozygous progeny (nos 2-4) are shown. The parental CAG repeat length of the mutant allele was estimated to be 77 CAGs; one offspring shows an increase of +8 CAG repeats (no. 2), one shows an increase of +1 CAG repeat unit (no. 3) and one shows no change in repeat length (no. 4). (B) Histograms of the distribution of CAG repeat copy number changes at the mutant Hdh locus, relative to the size of the mutant parental allele. The left panel shows the results for paternal inheritance of the mutant allele (n = 160) and the right panel shows results for maternal inheritance (n = 59).

Genetic analysis of the HD-repeat knock-in mouse lines

Genotypic analysis of the progeny (n = 261) from heterozygous intercrosses of the mutant mice showed no obvious deviation from the expected Mendelian ratio ([chi]2 = 2.30, df = 2, P = 0.3), indicating that the mutation is not associated with neonatal or perinatal lethality. The intergenerational stability of the CAG mutation was investigated by backcrossing heterozygous mice and determining the repeat copy number of the transmitted mutant allele in a PCR-based assay (Fig. 4A). Results obtained from 219 progeny indicated that the mutation rate (per transmission) of the CAG expansion allele was 22.5 ± 3.3% for the male germline and 27.1 ± 5.8% for the female germline (±1 SD, binomial distribution). Although there was no significant difference in the observed mutation rates for the male and female germlines ([chi]2 = 0.5, df = 2, P > 0.1), there was a strong sex-of-parent effect on whether the mutation increased or decreased in size on transmission (P < 0.001; 3×2 contingency [chi]2 for direction of change against sex of parent, [chi]2 = 51.8, df = 2). The majority of differences in the size of the mutation transmitted through the female germline were small contractions of up to four repeats in length. In contrast, only increases in the CAG repeat copy number were observed when the mutation was transmitted through the male germline (Fig. 4B). Whilst most of these were between +1 and +4 repeats, the largest expansion detected was +8 CAG repeats (Fig. 4A). The mutation rate of the CAG expansion in these mice was not as high as the 70-90% rates reported for human HD (3,6,26). Nonetheless, a number of other features that characterize the transmission of the human HD mutation were mirrored in the mice: the rates of maternally and paternally transmitted instability were similar; expansions were associated more frequently with paternal transmission whereas contractions tended to be inherited through the maternal germline, and the most dramatic increases in repeat size were paternally inherited.

Histological examination of brains from HD-repeat knock-in mice


Figure 5. Absence of neuropathological changes in control and Hdh4/Q80 and Hdh6/Q72 knock-in mice at 4-6 months of age (A-D) and 17-18 months of age (E-G). Each panel shows wild-type (+/+) brain tissue on the left, and mutant (+/-) on the right. (A) Dorsal view of whole brains; the olfactory bulbs (OB), cerebral cortex (Ctx) and cerebellum (Cb) appear normal in size. Scale bar: 2.5 mm. (B) Cresyl violet (Nissl) stain. There was no obvious atrophy of cortex (Ctx) or caudate-putamen (CPu), nor enlargement of the lateral ventricle (v). Scale bar: 1 mm. (C) Leucine-enkephalin immunohistochemistry. Projections to the globus pallidus (GP) and ventral pallidum (VP) showed normal immunoreactivity. Scale as in (B). (D) Substance P immunohistochemistry. The entopeduncular nucleus (EP) contained normal immunoreactivity. Scale bar: 0.25 mm. (E) Dorsal view of whole brains at 17 months, showing no obvious cerebral atrophy. Scale as in (A). (F)Glial fibrillary acidic protein (GFAP) immunohistochemistry. Moderate numbers of fibrillary astrocytes (arrows) were detected in both wild-type and mutant white matter fibre bundles. There was no reactive gliosis to suggest neuron loss. Scale bar: 50 µm. (G) Ubiquitin immunohistochemistry showed no ubiquitinated intranuclear or cytoplasmic inclusions in the striatum or other brain regions. Faint cytoplasmic accumulations of ubiquitin were detected in some brainstem centres of both wild-type and mutant mice (data not shown). Scale as in (F). (H) Positive control for ubiquitin immunohistochemistry. Neuritic plaques (arrowhead) and neurofibrillary tangles (arrow) were labelled in human Alzheimer disease tissue. Scale as in (F). To investigate possible neuropathological changes associated with the expression of mutant huntingtin protein, brains from HD-repeat knock-in mice were examined and compared with brains from wild-type littermates. Two age groups were studied: 4-6 months (early adulthood) and 16-17 months (mid-to-late adulthood).


Although no external cerebral abnormalities were apparent at either time point (Fig. 5A and E), mutant brains were, on average, 10-15% smaller than normal in both age groups, although this difference reached statistical significance only in the younger group (P < 0.01; independent two-tailed t-test, t = 4.111, df = 10).

Histological studies were performed to screen for general neuropathological deficits and for specific changes commonly associated with human HD. Haematoxylin and eosin (H&E) and cresyl violet (Nissl) stains revealed no gliosis, ventricular enlargement or neuron loss in any brain area of the mutant mice (Fig. 5B). Immunohistochemistry for glial fibrillary acidic protein (GFAP) confirmed the absence of any pathological gliosis, demonstrating only normal populations of fibrous astrocytes in the white matter of wild-type and mutant mice (Fig. 5F).

Further histochemical and immunohistochemical studies failed to reveal any changes in the brains of the mutant mice. Leucine-enkephalin, a marker of striatopallidal projection neurons that are preferentially lost early in the course of human HD (27), and substance P, a marker of striato-entopeduncular projection neurons, showed no decrement of immunoreactivity (Fig. 5C and D). Nor was there any loss of calbindin immunoreactivity, as reported in human HD (28). Neurons containing NADPH-diaphorase activity, which are spared in human HD (29), were present in normal numbers, and cytochrome oxidase activity, which reflects changes in neuronal activity (30), was unaffected.

In order to detect the presence of ubiquitinated NIIs associated with HD and other triplet repeat diseases (15-20), ubiquitin immunohistochemistry was performed on both paraffin and frozen sections. No abnormalities were detected with this antibody (Fig. 5G and H) or with the anti-pCAG53b antibody (31) (data not shown), which recognizes NIIs in mice transgenic for the HD mutation (14).

Mice expressing mutant huntingtin display abnormal social behaviour

General observation of the HD-repeat knock-in lines (Hdh4/Q80 and Hdh6/Q72), including eating and drinking habits, body weight, fertility and life span, revealed no significant differences between the mutant mice and their normal littermates over a period of ~18 months.

Whilst establishing the colony, we observed that mutant males, and to a lesser extent mutant females, engaged in chronic aggressive behaviour from ~3 months of age. Although they were housed with littermates from the time of weaning, the persistent delivery of bite wounds to the rumps and genitals of their cage-mates often necessitated their removal and maintenance in separate cages.

To investigate this behaviour further, we subjected mutant and wild-type male littermates from both lines to the resident-intruder test (32). Following a 4 week period of isolation, these males (residents) were exposed to a communally housed normal mouse (intruder) and aggressive behaviours such as biting, aggressive grooming, digging and tail rattling were noted. Comparison of the results revealed that mutant males displayed aggressive behaviour significantly earlier (P < 0.025) and more frequently (P < 0.05) than their wild-type littermates (Fig. 6A and B). Moreover, the elevated intensity of the attacks demonstrated by the mutant group led to the premature termination of three tests.


Figure 6. Isolation-induced aggressive behaviour of Hdh4/Q80 and Hdh6/Q72 knock-in mice in the resident-intruder assay. (A) Scatterplots for the latency to the first aggressive act and aggression score indicate that the data are not normally distributed. (B)The latency to the first aggressive act (left panel) and aggression score (right panel), depicted as means ± SEM. Mutant mice (n = 24; hatched bars) displayed significantly shorter latency and more aggressive behaviour than their wild-type littermates (n = 14; solid bars). Statistical analysis was performed using the combined Wilcoxon-Mann-Whitney test[z = -2.29, **P < 0.025, two-tailed test (latency); z = 2.015, *P < 0.05, two-tailed test (aggression score)].

The test cohort of male mice used in this assay had a mixed (75% C57BL/6, 25% 129) strain background and so care was taken to select males from the same intercross generation in order to minimize any bias contributed by independently segregating 129 and C57BL/6 alleles. However, it remains a formal possibility that the 129-derived Hdh gene or closely linked locus may be responsible for the aberrant behaviour, although there were no significant differences in behaviour between the genotypic groupings of the Hdh4/Q80 and Hdh6/Q72 mice which differ in the sub-strain of 129 from which they were derived (129/Sv/J and 129/Sv/ter), suggesting that sub-strain differences are not contributing to this abnormal behaviour.

This caveat notwithstanding, the qualitative observations of heightened aggression amongst group-housed males and females and the increased incidence of isolation-induced aggression in the resident-intruder test led us to conclude that the mutant mice exhibit abnormal social behaviour.

DISCUSSION

We have generated mice that express full-length mutant huntingtin protein and display chronic aggressive behaviour towards their cage-mates. Although similar behaviour has been reported in a number of different transgenic mouse lines (for an example, see ref. 33), it is unlikely to be a non-specific effect of mouse genome modification as many mutations are not associated with behavioural changes and some result in decreased levels of aggression (34). Mice have a limited repertoire of measurable behavioural interactions/reactions, and it is likely that `aggression' can result from perturbations in many different molecular and physiological mechanisms. However, in the context of modelling HD in mice, it is significant that HD patients often exhibit behavioural and psychiatric symptoms such as irritability and aggression (1,35). Moreover, it has been estimated that ~50% of HD patients present with psychiatric symptoms (36,37). This leads us to speculate that expression of mutant huntingtin protein in our mice may recapitulate aspects of the psychiatric disorder in human HD. The mice provide a convenient model system in which to investigate further the neurobiological basis and possible pharmacological treatments of these behavioural disturbances.

Interestingly, a recent series of electrophysiological studies demonstrated reduced long-term potentiation in hippocampal neurons derived from these HD-repeat knock-in mice (38). This measure of synaptic plasticity is thought to be linked mechanistically to the processes of learning and memory. Although the mice have not been subjected to formal cognitive testing at this time, these observations, along with the behavioural abnormalities, are consistent with our hypothesis that the mutant mice may be modelling the early manifestation of some of the physiological and molecular pathways disrupted in human HD.

Vonsattel, in his seminal study of striatal neuropathology in clinically diagnosed human HD cases, demonstrated that clinical changes can occur prior to the detection of abnormalities on conventional neuropathological examination (39). A more detailed analysis of brain tissue from these HD cases revealed a subtle neuronal loss, the nature of which most likely reflected a deficiency in some aspect of early central nervous system (CNS) development (40). This led the authors to postulate that the onset of the HD may be influenced by two separate variables: the degree of compromise in the development of the CNS and the rate at which additional neurons are lost as a consequence of mid-life effects of the HD mutation.

Do the HD-repeat knock-in mouse data support the hypothesis that the pathogenesis may be `multi-layered', and that contributions from a number of continuous or discontinuous dysfunctional processes may be superimposed in a sequential fashion over a period of time? Certainly, the abnormal phenotypic features displayed by the mice have strong parallels in the human clinical HD picture, and they occur in the absence of the progressive striatal loss, commonly observed in post-mortem tissue from end-stage human HD patients. The nature of the putative pathological processes, responsible for the abnormalities observed in the HD-repeat knock-in mice, is less clear. The preliminary observation of a non-progressive reduction of brain weight in the mutant mice is consistent with the notion that some deficiency in the early development of the CNS may be involved. However, neuronal dysfunction, which would not be revealed by conventional histological analysis, might also contribute to the behavioural and electrophysiological changes observed. It is also prudent to note that a decreased vulnerability of mouse neurons to the effects of the mutation or differences in the development and physiology between mouse and man could also modify the symptomatology of the resulting phenotype.

A comparison with other transgenic mouse studies is instructive when considering putative models of pathogenesis in HD. R6 transgenic mice express an N-terminal fragment of human huntingtin with an expanded stretch of polyglutamine residues (13). NIIs are detected in neurons just prior to the onset of a progressive neurological phenotype that includes resting tremor, stereotypic involuntary movements and severe handling-induced seizures (14). This has led to speculation that formation of NIIs is a primary pathogenic event in polyglutamine repeat disorders. However, recent evidence has cast some doubts on this notion with the demonstration that mutant protein-induced pathological processes can be separated from the formation of NIIs in both an in vivo and an in vitro model system (41,42). The observation of phenotypic changes despite the absence of NIIs in the brains of our mutant mice corroborates these findings. However, the precise nature of the pathogenic agent remains unclear at this time. Our immunoblot data reveal no increased levels of N-terminal fragments of mutant huntingtin in whole brain lysates from the mice. Sensitive immunolocalization studies may help to determine whether more subtle changes in levels of proteolytic degradation products and/or cellular location of the mutant protein may be responsible for the phenotypic changes.

Why are there no NIIs in the brains of the HD-repeat knock-in mice? The ectopic expression of the truncated mutant huntingtin peptide in the R6 mice may expedite accumulation of the inclusions, bypassing the requirement for proteolytic cleavage of full-length mutant huntingtin prior to nuclear translocation and aggregation. Assuming the latter events are time-dependent, it is likely that the life span of a mouse does not provide sufficient time for the manifestation of these `later' effects of the CAG mutation, when present in the context of the full-length protein expressed at normal endogenous levels. Indeed, a recent study has demonstrated that transgenic mice overexpressing (up to five times endogenous levels) a mutated full-length HD cDNA do exhibit neuronal cell loss, NIIs and motor disturbances (43). Moreover, the neuropathological and phenotypic differences between the mutant mice described in this study and other transgenic models strongly support the idea that the CAG mutation can have disparate functional consequences, depending on the expression profile of the cognate gene and protein context in which it occurs. The HD-repeat knock-in mice should provide a useful resource to determine how full-length mutant huntingtin initiates pathogenesis and the role of the endogenous spatial and temporal expression profile in the resulting pathological processes.

A further mouse line (HdhQ50) recently has been generated using an approach similar to that adopted in this study (44). Targeted insertion of a chimeric human-mouse exon 1 into the Hdh gene resulted in full-length mutant huntingtin protein containing a stretch of 50 glutamine residues. In contrast to our study, the HdhQ50 mice were indistinguishable from their wild-type littermates at 6 months of age. These findings hint that genotype-phenotype correlations in human HD may be mirrored in the mutant mice, i.e. larger CAG repeat mutations are associated with an earlier age at onset of `symptoms'. Alternatively, mice may generally exhibit a greater threshold to the functional consequences of the CAG mutation.

As well as attempting to model disease pathogenesis, transgenic mouse studies have focused on investigating the mechanisms that underlie trinucleotide repeat instability. It is becoming increasingly clear that a number of factors have major influences on the instability of these mutations in mice. For example, transgenes with triplet repeat copy numbers of >110 tend to show much higher levels of instability (45,46) than those containing <46 (47,48). The CAG repeat copy number in the Hdh4/Q80 and Hdh6/Q72 mouse lines falls between these values and shows intermediate levels of instability. Furthermore, a number of studies have demonstrated that the sex of the transmitting parent and parental age can modulate the magnitude and frequency of mutation (45,46,49-51), although the mechanistic basis of these sex-specific differences remains unclear. In common with many of the studies mentioned above, the rate of intergenerational instability of the repeat length in the HD-repeat knock-in mice appears to be less than that expected for a similar sized mutation in humans. There is increasing evidence that either the presence of cis-acting sequences or a particular chromatin conformation may have a role in influencing the instability of triplet repeat mutations (52,53). Indeed, evidence for haplotype effects at the human HD locus is accumulating with the report of an unusually stable HD mutation in a geographically isolated population in Crete (54) and the demonstration that similar sized intermediate alleles display very different rates of expansion into the disease-associated range (55). Our observations suggest that if specific cis-acting sequences do influence CAG repeat instability at the HD locus in humans, they may not be conserved at the 129 strain-derived Hdh locus in our mice.

In summary, we describe mice that have been genetically modified to carry a large HD-like mutation in the appropriate endogenous protein and genomic contexts. As the mutation shows germline instability, selective breeding of mice with different numbers of CAG repeats should afford us the opportunity to examine further the correlation between genotype and phenotype and to investigate potential genetic modifiers of the disease process and triplet repeat instability. HD traditionally has been designated a neurodegenerative disease on the basis of the dramatic neuronal cell loss observed in post-mortem brain tissue from affected patients. Our findings suggest that some of the clinical consequences of the mutation may precede the onset of progressive neurodegenerative changes. This implies that effective treatment of HD may require an understanding and amelioration of these dysfunctional processes, rather than simply preventing the premature death of neurons in the brain. These mice provide an excellent resource in which to address this important issue.

MATERIALS AND METHODS

Generation of the targeting constructs (Fig. 1A)

Step 1. Low-stringency PCR was performed using a subclone of genomic DNA, containing the first exon of the mutant HD gene from a human HD patient (C. Iannicola, unpublished data), as template. The primers used (MHD4 and MHD5; sequences given below) corresponded to the sequence immediately flanking and abutting the imperfect CAG repeat present in the first exon of the mouse Hdh gene. A fragment (CV1.6) containing ~70 CAG repeats, flanked by the sequence of the MHD4 and MHD5 oligonucleotides, was identified.

Step 2. The 3[prime] end of CV1.6 was extended by 174 bp to incorporate the unique BalI site in intron 1, using a modified version of a primer extension protocol termed cross-over PCR (56). Asymmetric PCR reaction conditions were optimized empirically for: (i) CV1.6: 1 ng of DNA was mixed with 25 pmol of primer A, 0.25 pmol of primer B, 1 U of Pfu DNA polymerase (Stratagene, Cambridge, UK), 200 µM dNTPs [50% 7-deaza dGTP (Gibco BRL, Paisley, UK)] and the manufacturer's recommended buffer; and (ii) the 3[prime] template: 5 ng of DNA was mixed with 2.5 pmol of primer C, 25 pmol of primer D, 1 U of Pfu DNA polymerase (Stratagene), 200 µM dNTPs, 10% dimethyl sulfoxide (DMSO) and the manufacturer's recommended buffer. Amplification reactions were subjected to 94°C for 3 min followed by 40 cycles at 94°C for 45 s, 68°C for 45 s, 72°C for 30 s and, finally, 72°C for 10 min. The single-stranded DNA templates produced were mixed in a titration series and added to 25 pmol of primer A, 25 pmol of primer D, 1 U of Pfu DNA polymerase (Stratagene), 200 µM dNTPs, 10% DMSO and the manufacturer's recommended buffer. The primer extension step consisted of 95°C for 3 min, followed by a 5 min ramping down to 68°C, 68°C for 1 min, ramping up to 72°C over 1 min and a final hold at 72°C for 10 min. The resulting reaction mixtures were subjected immediately to 25 cycles at 94°C for 45 s, 68°C for 45 s, 72°C for 30 s and, finally, 72°C for 10 min. Two products that had the correct sequence flanking a 72 and 80 CAG repeat stretch, respectively, were cloned.

Step 3. The 5[prime] end of these clones was extended by digestion with XmnI followed by ligation to the adjacent SfiI-XmnI wild-type mouse genomic fragment (containing 297 bp of 5[prime] upstream sequences). The resulting subclones (called pGU6 and pGU5, respectively) contained a stretch of between 72 and 80 CAG repeats precisely inserted into the SfiI-BalI genomic fragment that contained exon 1 of the Hdh gene.

Primers MHD4 and MHD5, corresponding to the sequences present in the first exon of the mouse Hdh gene, were used to screen a 129/Sv mouse genomic library (kindly provided by P. Soriano, Seattle, WA). A 5 kb HindIII fragment containing exon 1 was subcloned to generate plasmid pH1.1. After digestion with SfiI and BalI, the released 536 bp genomic fragment was replaced by the corresponding SfiI-BalI fragments derived from pGU5 and pGU6 to form pHICAG5 and pHICAG6, respectively.

Digestion of pL2-neo-2, a modified version of pL2-neo (kindly provided by H. Gu, Institut fur Genetik, Koln, Germany), with SacI and AatII released the neo gene flanked by directly repeated 34 bp loxP sites (loxPneo). This fragment was blunt ended and ligated to BalI-digested pHICAG5 and pHICAG6. Correctly configured clones were designated pCAG5.1 and pCAG5.2 (from pHICAG 5), and pCAG6.1 and pCAG6.2 (from pHICAG6).

Gene targeting in ES cells and generation of mutant mice

NotI-linearized targeting vector (20 µg) was electroporated into 2 × 107 RF8 (57) and JM-1 (58) ES cells using a Bio-Rad (Hemel Hempsted, UK) Gene Pulser at 500 µF and 250 V. Standard methods were used for ES cell maintenance, manipulation and selection (59). DNA from G418-resistant ES cell clones was extracted and screened for correctly targeted events by diagnostic PCR assays and Southern blot analyses (Fig. 1B-D). Probes a (1.7 kb) and b (2.0 kb) were unique genomic ScaI-HindIII fragments, external to the targeting construct homology. Blastocyst injection of recombinant e4A11 and e6C9 ES cells and subsequent generation of mutant mice were achieved using standard methods (59).

In vitro excision of loxPneo sequences

In vitro excision of loxPneo sequences from the targeted allele of recombinant ES cells was accomplished by transient transfection with a Cre recombinase-expressing plasmid clone. A 20 µg aliquot of pMC-Cre (kindly provided by H. Gu) was electroporated into e4A11 and e6C9 ES cells, using a Bio-Rad Gene Pulser at 250 V and 960 µF. The resulting cells were plated on feeder layers at 10-1000 cells per 10 cm plate. Seven days later, portions of individual colonies were placed in two gelatin-treated wells, one of which was exposed to G418 selection. Loss of loxPneo sequences in ES cell clones that were no longer resistant to G418 selection (dubbed e4A11[Delta]neo and e6C9[Delta]neo) was confirmed by Southern blot analysis.

Genotyping and CAG repeat copy number determination

Genotyping of mouse progeny was performed on DNA obtained from tail biopsies using standard procedures. The length of the Hdh CAG alleles was assessed by PCR: 100-300 ng of genomic DNA, 10% DMSO, 200 µM dNTPs [50% 7-deaza dGTP (Gibco BRL)], 1 µM MHD16 and MHD18 primers, buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1 mM MgCl2) and 2 U of Taq polymerase (Promega, Southampton, UK) were subjected to 95°C for 4 min followed by 30 cycles at 94°C for 30 s, 60°C for 45 s, 72°C for 30 s and, finally, 72°C for 10 min. A 1 µl aliquot of the resulting mixture was mixed with the Rox-1000 size standard (Perkin Elmer, Warrington, UK) and separated on a 4.75% polyacrylamide gel. An Applied Biosystems 373 DNA Sequencer system and Genescan software (Perkin Elmer) permitted accurate sizing of the PCR products. The predicted size of the CAG repeat length was 278 bp less than the overall size of the PCR product. Each parent-progeny transmission was analysed initially in duplicate on two separate gels. About 8% of duplicates generated discordant results, typically due to `edge' effects of the gel. If a consensus result was not obtained by running the samples on a third gel, the parent-offspring transmission was disregarded (<1% transmissions tested). A 500 bp fragment was amplified from loxPneo sequences by PCR: 100-300 ng of genomic DNA, 200 µM dNTPs, 1 µM primers (Neo1 and Neo2), buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1 mM MgCl2) and 2 U of Taq polymerase (Promega) were subjected to 95°C for 4 min followed by 30 cycles at 94°C for 45 s, 58°C for 45 s, 72°C for 30 s and, finally, 72°C for 10 min. A 400 bp fragment was amplified from the Cre transgene by PCR: 100-300 ng of genomic DNA, 200 µM dNTPs, 1 µM primers (Cre1 and Cre2), buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2) and 2 U of Taq polymerase (Promega) were subjected to 95°C for 4 min followed by 30 cycles at 94°C for 60 s, 61°C for 60 s, 72°C for 60 s and, finally, 72°C for 10 min. Where stated, Southern blot analysis was performed with the following combinations of restriction digests and probes: EcoRI and a 1.6 kb XhoI fragment released from pMC-Cre; EcoRI and EcoRI-linearized pBluescript II SK+ (Stratagene); and HindIII and a 700 bp PstI fragment released from pL2-neo-2.

Recombinant DNA techniques

Unless stated, DNA from tissues and cells was extracted and manipulated using standard protocols. Manual sequencing was performed using 35S-labelled dATP and the T7 Sequenase 7-deaza dGTP DNA sequencing kit (Amersham International, Little Chalfont, UK).

Protein analysis

Frozen tissue or cultured cells were homogenized in ice-cold EBC buffer (50 mM Tris-HCl, pH 8, 120 mM NaCl, 1% NP-40) containing protease inhibitors [1 µg/ml pepstatin A, 10 µg/ml leupeptin, 60 µM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 1 mM benzamidine]. Insoluble material was removed by centrifuging the homogenates at 16 500 g for 15 min at 4°C. The protein concentration of the supernatant was quantified using Coomassie Protein Assay reagent (Pierce, Chester, UK). Approximately 50 µg of protein was fractionated on 4.5% SDS-PAGE gels alongside pre-stained high molecular weight markers (Gibco BRL), electroblotted onto Immobilon-P membranes (Millipore, Bedford, MA) and blocked for 6 h in 0.2% casein/Tris-buffered saline (Pierce) containing 1% donkey or rabbit serum at room temperature. Primary antibodies were used as follows: [alpha]HD2 diluted to 1:100 for 12 h at 4°C and the IC2 antibody diluted to 1:5000 for 3 h at room temperature. After washing, immunoblots were probed with horseradish peroxidase-conjugated secondary antibodies (Jackson Laboratories, West Grove, PA) and visualized using SuperSignal ECL substrate (Pierce). Stripping and reprobing were performed according to the manufacturer's recommendations.

Levels of normal and mutant huntingtin in brain extracts from heterozygous mice were estimated from densitometric scans (Omnimedia XRS scanner and BioImage software) of immunoblots. The integrated intensity of the mutant band was calculated relative to that of the normal band in the same lane.

Histopathology

Tissue preparation. A total of 18 mice was used for the comparison of brain weight and subsequent histopathological analyses; of the 12 male mice aged 4-6 months, eight were derived from the Hdh6/Q72 line [four wild-type (+/+) and four heterozygotes (+/-)] and four were derived from the Hdh4/Q80 line (two +/+ and two +/-). Of the six female mice aged 16-18 months, two were derived from the Hdh6/Q72 line (one +/+ and one +/-) and four were derived from the Hdh4/Q80 line (two +/+ and two +/-). The animals were anesthetized with 4% chloral hydrate (10 ml/kg, i.p.), and perfused transcardially with cold phosphate-buffered saline (PBS) containing 4% paraformaldehyde and 4% sucrose. Brains were post-fixed overnight at 4°C, weighed and cryoprotected in PBS with ascending concentrations of sucrose (10, 20 and 30%). Slabs 2-5 mm thick were cut and embedded in paraffin or frozen in Tissue-Tek OCT embedding matrix (Sakura Finetek USA, Torrance, CA). Slabs from 11 brains (five at 4-6 months, two +/+, three +/-; six at 16-18 months, three +/+, three +/-) were sectioned coronally on a freezing sliding microtome at 30-40 µm thickness. Free-floating sections were placed in ice-cold PBS, then either stained with cresyl violet or used for further histochemistry or immunohistochemistry. Slabs from seven brains (three at 4-6 months, one +/+, two +/-; four at 16-18 months, two +/+, two +/-) were embedded in paraffin, sectioned at 8 µm and stained with H&E, cresyl violet, or by immunohistochemistry as described below.

Histochemistry. Previously described methods for cytochrome oxidase (29) and NADPH-diaphorase (28) histochemistry were used.

Immunohistochemistry. Frozen sections were blocked in PBS containing 3% normal goat serum, 0.4% Triton X-100 for 30 min at room temperature and then incubated with primary antibodies [anti-leucine enkephalin (Incstar, Stillwater, MN), 1:10 000; anti-substance P (Incstar), 1:10 000; anti-calbindin (SWant, Bellinzona, Switzerland), 1:10 000; anti-ubiquitin (Dako, Carpinteria, CA), 1:2000; anti-GFAP (Incstar), 1:20; anti-pCAG53b, 1:2000] overnight at 4°C. After rinsing in PBS, sections were reacted with biotinylated goat anti-rabbit IgG (1:200; Vector, Burlingame, CA) for 4 h at room temperature prior to using the ABC kit (Vector) and diaminobenzidine, according to the manufacturer's instructions. Finally, sections were rinsed in distilled water, mounted on glass slides coated with gelatin-chrome alum, air dried, cleared through ethanol and xylene and coverslipped in Permount.

Behavioural analyses of mice

When establishing the colony of Hdh4/Q80 and Hdh6/Q72 mice from the progeny of the F1 founders, only mice that did not carry the Cre transgene were used as breeding stock in backcrosses. Pups were weaned and housed with littermates of the same sex, 18-21 days after birth. At this time, they were marked by ear tagging, and a tail biopsy was obtained for DNA genotyping. Animals were housed in a temperature-controlled environment with a 12 h light-dark cycle and free access to food and water. The mice were tested between 11.00 and 17.00 h, during the light phase.

The cohort tested in the resident-intruder assay comprised 24 heterozygous (Hdh4/Q80, n = 12; Hdh6/Q72, n = 12) and 14 wild-type (Hdh4/Q80, n = 6; Hdh6/Q72, n = 8) littermates. These 9-12-month-old male mice were isolated for 4 weeks prior to testing in opaque cages (12 × 25 × 42 cm). Litter was changed once a week, but not during the week prior to the test. Wild-type DBA/2 male mice, 12 weeks old and housed at >6 per cage, were used as intruders. The mice received a single training session and then a single test session 1 week later, both of which were videotaped. The intruder was introduced in the resident cage and aggressive behaviour assessed during the 6 min test session by noting incidents of tail rattling, aggressive grooming, digging and biting. Two different measures were scored: latency to the first aggressive action and the intensity/duration of aggressive behaviour expressed as an aggression score. If no aggressive behaviour was displayed during the 6 min test period, it was extended until the latency to the first aggressive action was recorded, after which the session was terminated. The aggression score was obtained by dividing each test session into 10 s blocks; the number of blocks during which aggressive behaviour occurred was expressed as a percentage of the total number of blocks that comprised the session. This percentage value was then converted directly to an aggression score that ranged from 0 to 100. Expression of the aggression score in this manner allowed the inclusion of data derived from sessions that required premature termination due to the excessive aggression (e.g. when blood was drawn). The behaviour was scored independently by two observers who were unaware of the genotype of the mice.

Oligonucleotides

A, 5[prime]-GGGATCCGCGGGAAAAGCTGATG-3[prime]; B, 5[prime]-GGCGCCTGCGGCGGTGGCTGCTG-3[prime]; C, 5[prime]-CAGCAGCCACCGCCGCAGGCGCC-3[prime]; D, 5[prime]-CTGCATGGCCATGCCCAGCACG-3[prime]; MHD4, 5[prime]-GCGGTGGCGGCGCCTGCGGCGGTGGCTG-3[prime]; MHD5, 5[prime]-GAAAAGCTGATGAAGGCTTTCGAGTCGCTCAAGTCGTTTCAG-3[prime]; MHD16, 5[prime]-CCCATTCATTGCCTTGCTGCTAAG-3[prime]; MHD18, 5[prime]-GACTCACGGTCGGTGCAGCGGTTCC-3[prime]; Neo1, 5[prime]-CTTTTTGTCAAGACCGACCTG-3[prime]; Neo2, 5[prime]-AATATCACGGGTAGCCAACGC-3[prime]; Cre1, 5[prime]-TGATGAGGT-TCGCAAGAACC-3[prime]; Cre2, 5[prime]-CCATGAGTGAACGAACCTGG-3[prime].

ACKNOWLEDGEMENTS

We thank John Scarborough, Sue MacAuley, John McAbney, Derek Milroy and Colin Hughes for excellent technical advice and assistance; Jean-Louis Mandel and Erich Wanker for kindly providing the IC2 and anti-pCAG53b antibodies; Robert Farese and Roger Pederson for the RF8 and JM-1 ES cell lines; and past and present colleagues, in particular Ros John, Yuh-Shan Jou, Nila Patil, Richard Wilson, Joe Gray, Darren Monckton and Keith Johnson, for helpful discussions and support. This work was supported by an award from the Wills Foundation and NIH grant NS262237 to R.M.M. and awards from the Royal Society, Cunningham Trust and Tenovus-Scotland to P.F.S.

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*To whom correspondence should be addressed at Division of Molecular Genetics, Institute of Biomedical and Life Sciences, University of Glasgow, 56 Dumbarton Road, Glasgow G11 6NU, UK. Tel: +44 141 330 6200; Fax: +44 141 330 6871; Email: ps17z@udcf.gla.ac.uk
+Present address: Dipartimento di Biologia, Università degli Studi di Roma `Tor Vergata', Via della Ricerca Scientifica, 00133 Roma, Italy

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