Human Molecular Genetics, 2002, Vol. 11, No. 9 1075-1094
© 2002 Oxford University Press
YAC transgenic mice carrying pathological alleles of the MJD1 locus exhibit a mild and slowly progressive cerebellar deficit
1Cell and Molecular Biology, 2Leukocyte Biology and 3Biological Structure and Function, Division of Biomedical Sciences, Faculty of Medicine, Sir Alexander Fleming Building, Imperial College, London SW7 2AZ, UK and 4Royal Free and University College Medical School, Neurosciences, Rowland Hill Street, London NW3 2PF, UK
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Machado–Joseph disease (MJD; MIM 109150) is a late-onset neurodegenerative disorder caused by the expansion of a polyglutamine tract within the MJD1 gene. We have previously reported the generation of human yeast artificial chromosome (YAC) constructs encompassing the MJD1 locus into which expanded (CAG)76 and (CAG)84 repeat motifs have been introduced by homologous recombination. Transgenic mice containing pathological alleles with polyglutamine tract lengths of 64, 67, 72, 76 and 84 repeats, as well as the wild type with 15 repeats, have now been generated using these YAC constructs. The mice with expanded alleles demonstrate a mild and slowly progressive cerebellar deficit, manifesting as early as 4 weeks of age. As the disease progresses, pelvic elevation becomes markedly flattened, accompanied by hypotonia, and motor and sensory loss. Neuronal intranuclear inclusion (NII) formation and cell loss is prominent in the pontine and dentate nuclei, with variable cell loss in other regions of the cerebellum from 4 weeks of age. Interestingly, peripheral nerve demyelination and axonal loss is detected in symptomatic mice from 26 weeks of age. In contrast, transgenic mice carrying the wild-type (CAG)15 allele of the MJD1 locus appear completely normal at 20 months. Disease severity increases with the level of expression of the expanded protein and the size of the repeat. These mice are representative of MJD and will be a valuable resource for the detailed analysis of the roles of repeat length, tissue specificity and level of expression in the neurodegenerative processes underlying MJD pathogenesis.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Machado–Joseph disease (MJD; MIM 109150) is a late-onset, dominantly inherited disorder presenting with gait disturbance due to defects in motor neuron activity. MJD, also known as spinocerebellar ataxia 3 (SCA3), is the most common of the dominantly inherited ataxias worldwide (1). Clinically, the neurological presentation is in the third to fifth decade of life, and includes gait ataxia, dysarthria, dysmetria, pyramidal signs, hyperreflexia, dystonia and opthalmoplegia (2).
Neuropathologically, MJD is characterized by neuronal loss and gliosis in the dentate nucleus, vestibular nuclei, spinocerebellar tracts, substansia nigra and other nuclei of the basal ganglia (3,4) and axonal neuropathy of peripheral motor and sensory axons (5).
Molecular analysis has shown that MJD is caused by a pathological expansion of a CAG repeat in exon 10 of the MJD1 gene. The CAG repeats range from 12 to 37 in the normal population and from 55 to 86 in MJD patients. MJD1 encodes ataxin-3, a protein of unknown function (6). MJD1 mRNA is ubiquitously expressed in human tissues in at least four alternatively spliced forms (7). The expansion of a CAG repeat encoding a pathological polyglutamine tract has been identified in a growing group of dominantly inherited neurological disorders, including Huntington's disease (HD), dentatorubropallidoluysian atrophy (DRPLA), spinal and bulbar muscular atrophy (SBMA) and the spinocerebellar ataxias 1, 2, 3 (MJD), 6, 7, 12 and 17. This group of diseases all show a neurological deficit and an inverse correlation between CAG repeat length and age of onset. The mutant polyglutamine proteins have been shown to localise to ubiquitinated neuronal intranuclear inclusions (NIIs) in patients with HD (8–10), MJD (11), SCA1 (12,13), DRPLA (14), SBMA (15), SCA7 (16,17), SCA2 (18) and SCA17 (19) and in animal models of HD (8), MJD (20), SCA1 (12), DRPLA (21), SBMA (22) and SCA7 (23). However, it is not clear whether ubiquitinated NIIs are a primary mechanism or a by-product of the neurodegenerative process.
The first transgenic mouse models of MJD were generated with truncated and full-length human MJD1 cDNAs specifically expressed in mouse Purkinje cells under the control of the L7 promoter (24). Mice expressing an expanded polyglutamine repeat in the context of either a C-terminal fragment (HA-Q79), or the same fragment truncated at the C-terminus (HA-Q79C), showed ataxic posture and gait disturbance as early as the 4 weeks after birth. The cerebellum in these mice was very atrophic occupying, only one-eighth of its normal volume with thinning of the granular, Purkinje and molecular cell layers. However, mice expressing the full-length MJD1a cDNA with a polyglutamine expansion from the L7 promoter appeared normal. In contrast, SCA1 transgenic mice constructed using the same promoter and a full-length ataxin-1 cDNA containing 82 CAG repeats demonstrated an ataxic phenotype 20–40 weeks after birth and progressive Purkinje cell pathology that closely parallels that seen in human SCA1 patients (25,26). Clinically, MJD is similar to SCA1, but it is the dentate and pontine neurons, rather than Purkinje cells, that are the primary sites of cerebellar pathology in MJD (4,27–29). The relative sparing of Purkinje cells in MJD, even though the protein product is expressed in these cells, suggests that the intact protein is important for the neuronal specificity of the degenerative process. It is also apparent from this study that regulatory elements controlling temporal and spatial expression are likely to be critical to the expression of the disease phenotype.
In order to properly mimic the temporal and spatial expression of the human disease gene, we have made transgenic mice with yeast artificial chromosome (YAC) constructs carrying the full-length MJD1 gene with expanded polyglutamine tracts and all the enhancers and long-range regulatory elements needed for cell-specific expression at physiological levels. These animals are the first expressing full-length mutant MJD1 under the control of its own regulatory elements.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Transgenic mice carrying a human MJD1 YAC
A 250 kb YAC containing the intact human MJD1 locus with 15 CAG repeats, and its modification by homologous recombination to include expanded CAG trinucleotide repeat alleles ranging in size from 48–84 repeat units, has been described previously (30). DNA from YAC constructs containing 15, 76 or 84 CAG repeats was purified and used to generate transgenic mice by pronuclear injection. A total of 272 pups were obtained, and these were screened by PCR for the left and right arms of the YAC, and for exons 2 and 10 of the MJD1 gene. Ten transgenic mice were identified, but two of these did not contain the MJD1 gene and were not analysed further. The eight founder mice with the MJD1 gene (Table 1) all contain the left arm of the YAC and exons 2 and 10 of the MJD1 gene. All except MJD76.1 also contain the right arm of the YAC.
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In order to determine precisely the number of CAG repeats in the founder mice, PCR amplification was performed with the primers MJD52 and MJD25 on DNA extracted from tail tissue. The resulting PCR products were cloned and sequenced, and the results for founders MJD76.1 and MJD22.1/84.1 are shown in Figure 1. The data obtained for each of the founders containing MJD1 is summarized in Table 1. The repeat tract in exon 10 of MJD1 usually consists of (CAG)2CAAAAGCAGCAA(CAG)6–80, which encodes Q3KQ2(Q)6–80, the latter part of which is polymorphic.
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The two founder mice (MJD15.1 and MJD15.4) generated after injection of the wild-type YAC clone with the allele (CAG)2CAAAAGCAGCAA(CAG)9 maintained this allele, which encodes Q3KQ11, and for simplicity is referred to as (CAG)15 (Table 1). The length of the repeat tract is indicated in the name of each founder/line.
Four founders were generated following injection of the MJD1/CAG76 YAC construct, with the allele (CAG)2 CAAAAGCAGCAA(CAG)70, referred to as (CAG)76. MJD76.1 retained the original (CAG)76 motif (Fig. 1). MJD72.1 demonstrated differences within the motif, including loss of the lysine residue, resulting in (CAG)4CAA(CAG)67, which codes entirely for polyglutamines (Q72) and is referred to as (CAG)72. MJD64.8 had a contraction of the polymorphic CAG motif by 12 repeat units, and the repeat is referred to as (CAG)64. MJD67.2 had a contraction of the motif by 9 repeat units to (CAG)67.
Two founders were generated with the MJD1/CAG84 YAC construct, which has the allele (CAG)2CAAAAGCAGCAA (CAG)78, referred to as (CAG)84. MJD22.1/84.1 had two discrete alleles comprising (CAG)2CAAAAGCAGCAA (CAG)16, referred to as (CAG)22, and an interrupted tract of 84 repeats corresponding to (CAG)2CAAAAGCAGCAA (CAG)12CAA(CAG)65, referred to as (CAG)84 (Fig. 1). MJD84.2 has an unrearranged (CAG)2CAAAAGCAGCAA (CAG)78 repeat motif, also referred to as (CAG)84.
Transgenic founders were mated with C57BL/6J mice, and the resulting litters were screened by PCR for the presence of the YAC left and right arms and the MJD1 gene. Transmission of the transgene was confirmed in five out of the eight founder mice, namely MJD15.4, MJD72.1, MJD67.2, MJD22.1/84.1 and MJD84.2 (Table 1). In the five transmitting lines, stable transmission of the respective construct has been demonstrated for multiple generations with a predicted frequency of about 50%. Of the other three founders, MJD15.1 and MJD76.1 had many offspring (>50) but never transmitted the transgene, while MJD64.8 did not breed. Southern analysis in all eight founder mice and F1 offspring suggested a single site of transgene integration (data not shown).
YAC copy number was quantified by ‘ratio PCR’ (31), using primers designed to amplify a 166 bp exon 8 product from both the mouse and human genes equally. The PCR product from the transgenic mice was digested with human-specific TaqI and mouse-specific BglII, and the products were quantified to give the approximate transgene copy number in the founder mice as shown in Table 1. The copy number is indicated in the last digit in the name of the transgenic founder/line.
Human MJD1 mRNA is expressed at near endogenous levels in the YAC-transgenic mice
Expression of human MJD1 mRNA was assessed by RT–PCR using primers in exons 4 and 6, which amplify a 175 bp product from both mouse and human sequences equally. The RT–PCR product from human mRNA includes a TaqI site, which is not present in the mouse cDNA sequence. Restriction of the RT–PCR products with TaqI yields 115 and 60 bp human-specific fragments. Examples of the restriction digestion are shown in Figure 2 using cerebellum-derived mRNA from both MJD1 YAC-transgenic and wild-type/non-transgenic mice. Human mRNA expression can be seen in all five MJD1 YAC-transgenic lines. Human transgene-derived products were also clearly observed from heart, skeletal muscle and cerebral cortex mRNA (data not shown), confirming that the human transgene is expressed in all of these tissues.
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The level of expression of each human transgene relative to that of the endogenous mouse gene was quantified by ‘ratio PCR’ (31) in which the 175 bp RT–PCR product was digested with both human-specific ClaI and mouse-specific RsaI restriction enzymes and the products quantified. The levels of expression in each line (Table 1) were found to be largely copy-number-dependent and integration-position-independent, with each copy of the transgene expressing approximately the same amount as each endogenous gene. Thus, MJD72.1 with one copy of the transgene and two mouse genes expresses approximately 50% as much transgenic as endogenous mRNA (60%). MJD67.2, MJD22.1/84.1 and MJD84.2 all carry two copies of the transgene along with two endogenous genes, and express approximately equal amounts of transgenic and endogenous mRNA (134%, 117% and 108%, respectively), while MJD15.4 with four transgene copies expresses more transgenic than endogenous mRNA (169%).
Transgene-derived ataxin-3 is expressed in a tissue-specific pattern similar to endogenous ataxin-3
Western blot analysis with ataxin-3 antiserum (32) was used to investigate the expression of ataxin-3 in tissues from the five transgenic lines. Analysis of cerebellum tissue from all five lines is shown in Figure 3A and summarized in Table 1. The size of the protein was found to correlate with the length of the polyglutamine tract and the amount to correlate with the copy number of the transgene. All lanes show the endogenous mouse ataxin-3 protein at 42 kDa. MJD15.4 mice show abundant YAC-encoded ataxin-3 protein at about 48 kDa (Fig. 3A). The wild-type human protein is predicted to run at 42 kDa, but runs at 48 kDa – presumably because of retardation due to the (CAG)15 repeat motif. MJD67.2 shows a larger approximately 70 kDa transgenic protein [corresponding to (CAG)67], which is less abundant than that in MJD15.4 (two copies versus four). MJD72.1 has a similar approximately 70 kDa protein, but at a lower abundance corresponding to the single copy of the transgene. MJD84.2 shows a protein at about 72 kDa with a minor form at about 75 kDa, and the intensity is equivalent to that in MJD67.2 (both two copies). MJD22.1/84.1 has two copies of the YAC, one with a (CAG)22 and the other a (CAG)84 repeat motif. The two alleles in this line transmit together, and this is reflected in the proteins present at about 48 and 72 kDa. The lower intensity of the larger protein could reflect lower expression or lower stability of the protein.
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Ataxin-3 expression was detected in all tissues analysed from the five lines and a representative western blot analysis of protein lysates from the cerebellum, cerebral cortex, heart, lung, spleen and liver from a single MJD84.2 mouse and a non-transgenic mouse is illustrated in Figure 3B. We did not detect proteolytic fragments of ataxin-3 in any of the lysates using polyclonal antiserum raised against full-length ataxin-3. Similarly, proteolytic cleavage of ataxin-3 was not detected in human MJD lysates using the same antiserum (11). Subsequent experiments with a monoclonal antibody (mAb 1H9) that recognizes amino acids 214–233 of ataxin-3 (32) or 1C2 (33) also did not detect cleavage of ataxin-3.
The monoclonal antibody 1C2 (mAb 1C2) (Chemicon International) was originally raised against TATA-binding protein (TBP), a general transcription initiation factor, but it preferentially recognizes pathological forms of polyglutamine disease proteins, including ataxin-3 (34). We used 1C2 to detect proteins with expanded polyglutamine tracts in the five lines, and a representative western blot analysis of protein lysates from the cerebellum, cerebral cortex, heart, lung, spleen and liver from a single MJD84.2 mouse and a single non-transgenic mouse, together with a MJD15.4 cerebellum control, is illustrated in Figure 3C. 1C2 does not bind to the non-expanded polyglutamine tract, and consequently ataxin-3 was not detected in the MJD15.4 lysate. Detection of a 72 kDa protein in MJD84.2 lysates confirms that mutant ataxin-3 is ubiquitously expressed. The variable band at 50 kDa is probably the TATA-binding protein (Fig. 3C).
MJD1 YAC-transgenic mice expressing mutant ataxin-3 with expanded CAG repeats develop a progressive neurological phenotype
Behavioural and functional studies were performed at different time points during the life span of mice derived from each line. In addition, mice were generated with higher numbers of copies of the transgene by crossing hemizygous mice. Double-transgenic mice were generated in two crosses. MJD67.2xMJD84.2 gave MJD67.2/84.2 mice, which have four MJD1 YAC-transgenes, two with the (CAG)67 repeat motif and two with the (CAG)84 repeat motif (Table 2). MJD84.2xMJD84.2 gave MJD84.2/84.2 homozygotes with four copies of the MJD84.2 (CAG)84 repeat motifs. The behavioural and functional studies were based on the SHIRPA protocol (35), which mimics the diagnostic process of general, neurological and psychiatric examination in humans.
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There are a number of general behavioural observations that were apparent in the transgenic mice with expanded CAG repeats that differ from non-transgenic control mice. Affected transgenics had a wide gait during grid climbing and lowered pelvis in comparison with non-transgenic mice (Fig. 4A and B). They also had a tremor and were less active, alert and inquisitive than non-transgenics. Transgenics often displayed limb clasping instead of the normal escape reflexes (hindlimbs spread) when suspended by their tails (Fig. 4C), and were either slow or unable to correct body position in the negative geotaxis test while non-transgenic mice were able to correct body position within 30 seconds. Table 2 summarizes the data for the transgenic founders and lines, and the behavioural findings for each line are described below.
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Slow weight gain is a particularly clear phenotype that can be accurately quantified. The weights of at least 10 male and 10 female F3 offspring were measured from the different lines of mice throughout their lifespan, beginning at 4 weeks of age (Fig. 5). The order of severity of the lines is MJD15.4, MJD72.1, MJD67.2, MJD22.1/84.1, MJD84.2, MJD67.2/84.2 and MJD84.2/84.2. It is clear that the degree of reduction of weight gain increases both with length of polyglutamine tract and with level of expression. MJD15.4 control-transgenic mice gain weight at a rate that is indistinguishable from that of wild-type/non-transgenic mice. MJD84.2 mice gain weight more slowly than MJD67.2 mice, showing a repeat length effect on growth. Additionally, MJD67.2 mice gain weight more slowly than male MJD72.1 mice, and MJD84.2/84.2 mice gain weight more slowly than MJD84.2 mice, showing a gene-dosage effect on growth.
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Control transgenic mice MJD15.1 and MJD15.4
Control-transgenic mice were generated with unmodified wild-type YAC with a (CAG)15 repeat motif (Table 1). The MJD15.1 founder had only one copy of the transgene and did not transmit this to offspring. Mice from the MJD15.4 line behaved identically to non-transgenic littermates at all ages tested and did not develop neurological phenotypes. The weight of MJD15.4 mice increases steadily from weaning at the same rate as that of non-transgenic littermates (Fig. 5). They present with a normal gait and no detectable tremor, are active and show normal escape reflexes. Geotaxis is corrected within a few seconds and the mice appear no different to non-transgenic controls. This suggests that overexpression of other genes that may be present on the YAC or the unmodified MJD1 gene does not manifest in a neurological or other deficit. This control line is particularly informative, since it carries four copies of the YAC and gives high levels of ataxin-3 expression with no phenotype, while one copy of the mutant gene with an expanded allele is enough to give a phenotype (see below).
MJD76.1 founder
The MJD76.1 founder animal presented with progressive behavioural symptoms, including a slightly abnormal kyphotic posture and abnormal positioning of hindlimbs from 4 weeks (Fig. 4A). Gait was fluid but abnormal, and progressed in severity. Pelvic elevation was normal at first, flattened and touching base of cage at 46 weeks, and markedly flattened at 60 weeks (Fig. 4B). At 16 weeks, this animal was inactive and showed infrequent rearing behaviour, which progressed to no rearing at all by 46 weeks. A mild intention tremor was noticeable by 46 weeks. Movement was slow, with a reduction in explorative behaviour and extreme passivity by 60 weeks. Locomotor activity was normal at 16 weeks, but significantly reduced by 46 weeks. Startle reflex (90 dB sound) was initially normal, but was reduced at 46 weeks. We also noted a significantly reduced grip in the fore- and hindlimbs, which was progressive. Corneal and toe pinch reflexes were absent at 46 and 60 weeks. Limb clasping of forelimbs was noticeable from 24 weeks (Fig. 4C). Wire manoeuvre was poor with some difficulty to grasp with hindlimbs. Limb tone was markedly reduced in all limbs by 46 weeks and almost absent at 60 weeks. There was a reduced ability to achieve negative geotaxis in this animal.
MJD72.1 line
In the MJD72.1 line (Table 1), there is no male-to-male transmission of the transgene, suggesting that the transgene has integrated into the mouse X chromosome. X inactivation clearly has an effect on transgene expression in this line by reducing expression in half of the cells in females. The growth curves of individual female transgenic mice are very variable, ranging from normal to that seen in males. The growth curve for MJD72.1 transgenic males is shown in Figure 5 and is uniform between mice. Weight gain is essentially the same as for non-transgenic control mice up to 48 weeks of age, after which the mice consistently weigh approximately 5% less than age-matched non-transgenic mice (Fig. 5). MJD72.1 male mice do not manifest with any obvious behavioural phenotype until 34 weeks of age, at which time limb clasping, primarily involving the forelimbs and, in a small percentage (<5%) of mice, all four limbs, has been observed. Limb tone is progressively reduced in all limbs, and most animals have a reduced ability to achieve geotaxis with age. Additionally, MJD72.1 transgenic mice were found to be irritable and aggressive when scruffed, showed provoked biting and were vocal when handled. As this is unique to this line, it is probably due to the position of integration of the transgene.
MJD67.2 line
In MJD67.2 mice, the effect of the transgene on growth is more evident than in the MJD72.1 line, probably owing to the higher transgene copy number. At weaning (4 weeks of age), there is no difference in weight between MJD67.2 mice and non-transgenic littermates. A difference in weight is evident at 48 weeks, where MJD67.2 mice weigh about 8% less than non-transgenic mice. After 48 weeks, the weight difference progressively increases to 20% (Fig. 5). MJD67.2 mice are hypoactive from 4 weeks of age, and present with mild but progressive neurological abnormalities, including abnormal gait, limb clasping and a reduced ability to correct negative geotaxis (Table 2).
MJD64.8 founder
The founder mouse MJD64.8 had an estimated eight copies of the transgene with a (CAG)64 repeat motif (Table 1). MJD64.8 presented with a progressive ataxia and tremor (Table 2), noticeable at 3 weeks of age. The animal failed to thrive, weighed 50% less than age-matched non-transgenic littermates from weaning and failed to breed. At 5 months of age, at which time the animal was showing severe ataxia and weight loss, it was anesthetised and perfused for immunohistological studies. The severe phenotype in this animal is probably associated with the higher copy number, supporting a gene-dosage effect for the modified human transgene. Dissection of the MJD64.8 founder showed atrophy of the ovaries and small brain size (40% less than age-matched control brain), although this was proportional to body weight.
MJD22.1/84.1 line
MJD22.1/84.1 mice have two transgenes, one expressing protein with a (CAG)84 repeat and the other expressing protein with a (CAG)22 repeat motif (Table 1, Fig. 3A). MJD22.1/84.1 transgenic mice have normal growth up to 40 weeks of age, after which they start to gain less weight than non-transgenic mice, consistently weighing approximately 12–20% less than age-matched controls (Fig. 5). This line presents with a relatively mild phenotype much like the MJD72.1 line. Deficits include a mild gait abnormality, tremor and limb clasping, and a reduced ability to correct negative geotaxis with age (Table 2).
MJD84.2 line
Of the five hemizygous transmitting lines, the MJD84.2 mice have the most pronounced behavioural and functional abnormalities (Table 2). At weaning, MJD84.2 mice weigh, on average, 12% less than controls, and by 20 weeks they weigh 22% less than non-transgenic mice. After 16 weeks of age, MJD84.2 mice rarely weigh more than 30 g, and from 30 weeks of age, the mice tend to lose weight (Fig. 5).
Gait and pelvic elevation is abnormal from 4 weeks of age. Mice show a displaced or wide gait that is especially noticeable in the hindlimbs (Fig. 4A) and pelvic elevation is reduced (Fig. 4B) with the abdomen flattened during rest, and to some degree locomotion is reduced. The mice are hypoactive from a very early age as compared with age- and sex-matched littermates. This includes spontaneous activity that is somewhat reduced. Trunk curl is abnormal and the MJD84.2 mice tend to clasp their hind- and forelimbs (Fig. 4C). Limb clasping is observed at 24 weeks in the forelimbs, and progresses to all four limbs being involved by 56 weeks in some mice (10%). Wire manouvre is carried out poorly and the mice tend to stay in a fixed position or fall off the wire, suggesting weakening in the limbs and/or lack of coordination. MJD84.2 mice achieve geotaxis very slowly from 16 weeks, which progresses to an inability to turn. After 20 weeks, the mice are unable to turn and often fall off the wire grid, suggesting a defect in co-ordination and general weakness. Additionally, toe pinch response is abnormal, with little or no reaction, and is markedly reduced or absent with increasing age, suggesting a sensory deficit.
MJD67.2/84.2 double-transgenic mice
Crosses between the hemizygous MJD67.2 and MJD84.2 mice yielded non-transgenic, hemizygous and double-transgenic offspring at the expected 1 : 2 : 1 Mendelian ratio. The first sign of phenotypic abnormality in the MJD67.2/84.2 mice is failure to gain weight at the same rate as control mice or mice with one or two copies of a mutated transgene (Fig. 5). The body weight of MJD67.2/84.2 mice is on average approximately 20% less than that of non-transgenic mice at weaning and 25% less at 20 weeks of age. After 32 weeks, the MJD67.2/84.2 mice lose weight. Limb clasping is more frequent in MJD67.2/84.2 mice than in hemizygous mice, and has been observed in hind- and forelimbs by 16 weeks of age, although this phenotype is not completely penetrant (Table 2). MJD67.2/84.2 mice do not correct geotaxis after 12 weeks (Table 2) and fall off the wire grid, suggesting a defect in coordination and/or generalized muscle weakness. Almost all double-transgenic mice show excessive grooming. One MJD67.2/84.2 mouse developed a tight circling behaviour at 48 weeks similar to that described in HD YAC-transgenic mice (36). We cannot be sure at this time if the circling behaviour will be a recurring phenotype or whether it is a singular anomaly.
MJD84.2/84.2 homozygous mice
Crosses between hemizygous MJD84.2 mice yielded non-transgenic, heterozygous and homozygous offspring at the expected 1 : 2 : 1 Mendelian ratio, indicating that this transgene has not disrupted an essential gene. MJD84.2/84.2 mice weigh on average 27% less than non-transgenic control mice at weaning, and by 20 weeks of age they weigh, on average, 39% less than controls (Fig. 5). Limb clasping is more frequent in homozygous mice than in heterozygous mice and has been observed in hind- and forelimbs by 16 weeks of age although this phenotype is not completely penetrant (Table 2). Homozygous mice do not correct geotaxis after 12 weeks (Table 2) and fall off the wire grid, suggesting a defect in coordination and/or generalized muscle weakness. Almost all homozygous mice show excessive grooming.
Neuropathological analysis reveals cell loss in the pons, the deep cerebellar nuclei and the cerebellum
Histological analysis of the MJD1 YAC-transgenic mice was carried out at 3, 6, 12 and 18 months for the transmitting lines and at 5 months for the MJD64.8 founder. The gross organization of the brain in the transgenic lines was no different to controls, and brain size was proportional to body weight. Neuronal degeneration in the mice was assessed by light microscopy in 7 µm thick sections of sagitally cut brain sections, and included analysis of the pontine and dentate neurons and the cerebellar cortex (Table 2).
H&E staining of the pontine neurons in non-transgenic, MJD84.2 and MJD64.8 mice is illustrated in Figure 6A–C. Degenerating neurons were particularly obvious in the pons of the MJD64.8 mouse (5 months of age), where cells were hyperchromatic and shrunken and were observed as scattered dark cells with pyknotic or small, staining nuclei and eosinophillic cytoplasm (Fig. 6C) compared with the non-transgenic pons (Fig. 6A). The extent of pathology in the pons of the MJD64.8 mouse at 5 months of age (40% cell loss) (Fig. 6C) is more severe than in the MJD84.2 mice at 12 months of age (Fig. 6B), where approximately 30% neuronal loss was observed, and is indicative of a gene-dosage effect.
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H&E staining of the dentate neurons in non-transgenic, MJD84.2 and MJD64.8 mice is illustrated in Figures 6D–F. The extent of neuronal loss (approximately 40%) in the dentate nucleus of the MJD64.8 mouse at 5 months of age (Fig. 6F) was similar to that seen in the MJD84.2 mice at 12 months of age (Fig. 6E). The remaining neurons of the dentate nucleus showed grumose alterations (Fig. 6E,F) typical of MJD pathogenesis (37).
GFAP staining of the dentate nuclei in non-transgenic, MJD84.2 and MJD64.8 mice is illustrated in Figure 6G–I. The mild increase in GFAP staining in MJD84.2 (Fig. 6H) could be attributed to neuronal loss and is indicative of gliosis. MJD64.8 mice also had an increase in GFAP staining with some increased numbers of reactive astrocytes (Fig. 6I). There was no evidence of elevated GFAP staining or reactive astrocytosis in non-transgenic mice (Fig. 6G).
In addition, increased numbers of reactive astrocytes were detected, at varying degrees, with GFAP immunostaining, in the cerebellar white matter of all transgenic mice with expanded MJD1 alleles. GFAP staining of the cerebellar white matter in non-transgenic, MJD84.2 and MJD64.8 mice is illustrated in Figure 6J–L. GFAP staining was more intense in the MJD64.8 mouse (Fig. 6L) compared with MJD84.2 mice (Fig. 6K). We did not observe astrocytosis in non-transgenic mice (Fig. 6J). GFAP staining and the number of reactive astrocytes were found to increase with (CAG)n repeat length and age (data not shown). No significant neuronal degeneration or gliosis was observed in MJD15.1 or MJD15.4 control-transgenic mice.
Calbindin D-28k immunostaining was used to investigate neuronal degeneration in the cerebellar cortex in non-transgenic and MJD1 YAC-transgenic mice. Calbindin D-28k staining was ubiquitous except in areas of cell shrinkage. Normal cerebellar morphology is illustrated in Figure 7A in a non-transgenic mouse brain. The brains of MJD 15.1 and MJD 15.4 mice containing the wild-type YAC construct with a (CAG)15 repeat motif also exhibited normal cellular morphology with virtually no Purkinje cell loss and normal granular and molecular cell layers (Fig. 7B). However, small areas of Purkinje cell loss were noted at the top of the gyri, together with elevated hyperchromasia in some MJD1 YAC-transgenic mice with expanded alleles. There were areas of Purkinje cell shrinkage (Fig. 6C–H, arrows), cell body displacement and dendritic atrophy. Where Purkinje cell loss was marked, it was confirmed by the observation of empty baskets. Purkinje cell loss was mild and variable in MJD72.1 mice (Fig. 7C). MJD22.1/84.1 mice also had mild but variable cell loss in the cerebellum, including Purkinje cell loss (<10%) and some thinning of the molecular cell layer (Fig. 7D). Purkinje cell loss in MJD67.2 (Fig. 7E) and MJD84.2 (Fig. 7F) mice was more obvious than in the MJD72.1 mice (Fig. 7C). The MJD67.2/84.2 mice showed even more extensive Purkinje cell loss, with dendritic atrophy affecting approximately 20% of cells (Fig. 7G). The phenotypically most severely affected founder animal, MJD64.8, had gross cell loss in the cerebellum affecting both the molecular and Purkinje cell layers. Most of the Purkinje cells were shrunken; however, the small granule cells were normal in the MJD64.8 founder (Fig. 7H). Our data suggest that Purkinje cells are mildly involved in pathogenesis when the mutated transgene is expressed at single copy. Pathology in the cerebellar cortex increases markedly with increasing transgene copy number, suggesting that Purkinje cells are particularly sensitive to the effects of gene dose.
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Peripheral neuropathy characterized by de- and remyelination of the sciatic nerves and degeneration in the dorsal root ganglia (DRG) is a feature in MJD1 YAC-transgenic mice
Peripheral nerve fibre loss has previously been implicated in MJD (38–41). The sciatic nerves and dorsal root ganglia (DRG) were examined in MJD15.4, MJD67.2, MJD84.2 and MJD67.2/84.2 mice by light and electron microscopy as summarized in Table 3. Minor myelination changes were common in all mice, including non-transgenics, aged 17 months or more, so these changes were discounted. At 9 months of age, MJD15.4 mice showed normal sciatic nerve morphology. MJD67.2 mice had normal sciatic nerves at 9 months of age, but there were occasional tomacula and eccentric nuclei in the dorsal root ganglia. At the same age, MJD84.2 mice had a minor degree of de- and remyelination in the sciatic nerves and occasional thin myelin in the DRG, suggesting a small but detectable effect of CAG repeat length on myelination.
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MJD67.2/84.2 double-transgenic mice showed the most extensive myelin abnormalities of the lines investigated. A minor degree of de- and remyelination was detected at 6, 9 and 12 months of age, and this progressed to extensive myelin abnormalities by 14 months of age. There were demyelinated and also some very thinly myelinated axons, probably resulting from remyelination (Fig. 8B and D). Some of these fibres showed typical onion bulbs, indicating that there had been several episodes of de- and remyelination (Fig. 8E). The MJD67.2/84.2 mice had occasional axonal abnormalities and even rarer axonal degeneration. There were no axonal or neuronal inclusions other than occasional ones indicating early axonal degeneration. The increased severity of the myelin abnormalities in the MJD67.2/84.2 mice is evidence of greater effect by higher levels of transgene expression.
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Mutant ataxin-3 is predominantly nuclear and accumulates in neuronal intranuclear inclusions (NIIs)
Polyclonal ataxin-3 antiserum (11) was used to analyse the subcellular localization of ataxin-3 in a number of cell types in the transgenic and non-transgenic mice. Ataxin-3 expression was highest in the substantia nigra, hippocampus, cerebellum, and pontine and dentate nuclei in the MJD15.4, MJD67.2/84.2, MJD84.2/84.2 and MJD64.8 mice (data not shown), which have an additional 4, 4, 4 and 8 copies of transgene-encoded ataxin-3, respectively. In non-transgenic and MJD15.1 and MJD15.4 mice, the subcellular 1ocalization of ataxin-3 was predominantly cytoplasmic in the pontine and dentate neurons and in Purkinje cells (Fig. 9). In diseased brain, including tissue from MJD72.1, MJD67.2, MJD22.1/84.1, MJD84.2, MJD67.2/84.2 and MJD64.8 mice, the subcellular distribution of ataxin-3 was predominantly within the nucleus of pontine and dentate neurons and Purkinje cells (Fig. 9). In individual neurons, this staining in addition took the form of one or more spherical inclusion bodies, within the nucleus, varying in size from 0.5 to about 6 µm in diameter and identical to those described in MJD patients (42). These structures were commonly observed as a single or doublet structure, near to, yet clearly distinct from, the haematoxylin-stained nucleolus. Intranuclear inclusions were seen only in neurons and never in glial cells, and they were found predominantly in dentate and pontine neurons, the primary target neurons in MJD (4,10,11). NIIs were not found in regions typically spared in the disease (and also not affected in the mice), including cerebral cortex, hippocampus and striatum, and were rarely seen in cerebellar Purkinje cells (Fig. 9). Inclusions could be detected at every age analysed in the MJD72.1, MJD67.2, MJD22.1/84.1, MJD84.2, MJD67.2/84.2 and MJD64.8 mice. Inclusions were more frequent (>50% of cells analysed) in MJD84.2 (Fig. 9K), MJD67.2/84.2 and MJD64.8 mice, and there was a clear correlation of frequency of inclusion formation with both gene dosage and repeat size. For example, inclusions were more frequent in MJD67.2 mice in comparison with age-matched MJD72. 1 males, probably due to greater gene dosage, and there were more inclusions in MJD84.2 mice in comparison with MJD67.2 mice – probably because of greater repeat length (Table 2). NIIs were also detected in the cranial nerve nuclei in MJD84.2 and MJD67.2/84.2 mice (data not shown). NIIs were ubiqutin-positive (Fig. 9K), suggesting that they contain abnormally folded protein and/or proteins targeted for degradation. We did not observe inclusions in skeletal muscle (quadriceps, soleus or gastrocnemius) in any of the transgenic animals. NIIs were absent from non-transgenic and control-transgenic tissues (Fig. 9).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have made transgenic mouse models of MJD by inserting the intact, mutant, human MJD1 gene, which expresses ataxin-3. These mice have allowed us to study the effects of expression of full-length ataxin-3 with expanded polyglutamine tracts under the control of its endogenous cis-acting regulatory elements. It was possible that overexpression of other genes within the YAC, or the wild-type gene itself, would give rise to some form of pathological consequence. However, no pathology was observed in the control-transgenic MJD15.4 mice carrying four copies of the unmodified YAC.
Homologous recombination in the yeast host was used to generate three YAC constructs with 15, 76 and 84 repeats, corresponding to the human wild-type, intermediate and early-disease-onset MJD alleles respectively. The wild-type (CAG)15 allele, with the normal variant CAA and AAG triplet repeats, was integrated unrearranged into the MJD15.1 and MJD15.4 founders. The expanded (CAG)76 allele tended to have contracted in the transgenic mice, giving rise to alleles with 64, 67 and 72 repeats as well as the original 76 repeats. The (CAG)84 allele was unrearranged in the MJD84.2 mice, but the MJD22.1/84.1 mice carried two copies of the gene – one with only 22 repeats and the other with 84. The degree of instability of the expanded CAG repeats through successive generations of mice is presently being investigated. The mice with different expansions has allowed us to analyse the effects of repeat length on disease severity in the size range found in human patients.
The 250 kb YAC construct contains the intact MJD1 gene, which spans approximately 50 kb, and also 30 and 170 kb of flanking genomic DNA (30). The transgenic mice carry one, two, four or eight copies of the transgene, and the levels of MJD1 mRNA and protein expression are approximately proportional to the copy number of the transgenes, with each copy of the transgene expressing approximately the same amount as each endogenous mouse copy of the gene (Table 1). This suggests that the large YAC insert contains all the long-range controlling elements needed for full levels of expression from each copy of the transgene, independently of the position of integration. Human MJD autopsy tissues have clearly demonstrated expression of both the normal and mutated protein at approximately comparable levels, consistent with the heterozygous state (11,34). The mice with one copy of the transgene may model the human disease state in that they express mutant protein from a single gene as would be found in heterozygous patients. Mice with two copies of the transgene express roughly equal amounts of transgenic as endogenous MJD1 mRNA, like heterozygous patients, but they express more protein overall. The mice with four or eight copies may resemble the situation in homozygous humans in that they have higher levels of mutant protein; however, they also express two copies of the wild-type gene.
Human MJD1 expression was identified in cerebellum, cerebral cortex, heart, lung, spleen and liver. This is comparable to the ubiquitous expression reported in human tissues (6), and suggests that the YAC carries all the long-range controlling elements needed for tissue-specific expression. Expression of mutant ataxin-3 in lung, spleen and liver that do not show pathology matches the widespread expression of ataxin-3 in human tissues spared in the disease (11), and suggests that in tissues where cells are replaced, the mutant protein does not cause tissue pathology. Neuronal cells do not divide and therefore the cell loss caused as a consequence of a toxic protein would be marked and irreversible. It has been suggested that a cleavage product of the mutant androgen receptor, huntingtin and atrophin- 1 proteins is a potent inducer of cell death in SBMA (43), HD (44) and DRPLA (45) respectively. We did not detect a cleavage product of ataxin-3 in any of the lysates analysed from the MJD1 YAC-transgenic mice with the antibodies used in our study. We shall use a range of antisera in future studies to determine whether a cleavage product of ataxin-3 is involved in MJD pathogenesis.
Interestingly, human transgene-specific mRNA expression was not detected in testis from any of the transgenic mice, although the murine-specific product had clearly amplified (data not shown). Studies of rat MJD1 expression have indicated alternative splicing involving exons 1–6 in rat testis (46). Failure to amplify the human product from testis-derived cDNA from the transgenic mice could therefore be consistent with a similar pattern of alternative splicing for the transgene, suggesting that sequences present in the YAC construct are able to drive tissue-specific expression and splicing. Different spliced forms have also been observed in other human tissues, and the presence of these will be analysed in the mice.
Behavioural and functional studies revealed a number of findings in the mice expressing expanded (CAG)n repeat alleles that differed from non-transgenic and control-transgenic mice. In over 90% of transgenic mice, we found that the mutated MJD1 gene was associated with animals of lower weight, suggesting that this is a high-penetrance trait. Lower weight is associated with both increased transgene copy number and increased polyglutamine repeat length (Fig. 5). Progressive weight loss is a common finding in MJD patients despite a normal appetite (46). This finding has also been observed in HD patients (47), and may suggest a common underlying metabolic defect in these polyglutamine disorders.
Other differences in the transgenic mice include abnormal gait, tremor, hypoactivity, limb clasping, an inability to correct geotaxis, reduced grip strength and abnormal toe pinch responses. A wide gait, sometimes associated with tremor, is one of the first clinical manifestations seen in MJD patients, and is due to unsteadiness or impaired balance. Defects in gait and limb position (wide gait and limb clasping) in the symptomatic mice could suggest abnormalities in muscle and lower motor neuron function, spinocerebellar and sensory function (35).
The limb clasping behaviour, where the mice hold their paws pressed against their body as shown in Figure 4C (MJD84.2 mouse), was observed in all the transgenic lines/founders with expanded repeats. The clasping phenotype is reminiscent of the abnormal clasping reflex observed in cyclinD1-/-(48), mf3-/-(49) and Hox8-/-(50) mutant mice, although in the mf3-/- mice the clasping is only observed in the hindlimbs. The phenotype in all three mutant strains is coupled with a neurological deficit. More relevant to this study and with regard to triplet repeat models, limb clasping was observed in a number of HD mouse models, including R6-HD (51), full-length HD cDNA (52), YAC-transgenic HD (36) and HD knock-in (53) and SBMA (22) mutant mice.
For all the behavioural findings, the mice with higher gene dosage and or longer repeats are more severely affected and have an earlier age of onset. In MJD, a gene-dosage effect is apparent in individuals homozygous for the mutation, who exhibit juvenile onset and more rapid progression of the disease (54–58). MJD patients also show anticipation in which longer repeat lengths in successive generations are associated with more severe and earlier onset of symptoms (59–61).
Pathological examination of the brains of YAC-transgenic mice with expanded (CAG)n alleles showed degeneration and mild gliosis of the dentate and pontine nerve nuclei. These findings are consistent with the pathological findings reported in MJD patients, where the degeneration involves neuronal loss and gliosis and the commonly affected regions include the dentate and pontine nuclei, cranial motor nerve nuclei, globus pallidus, subthalmic nucleus, substantia nigra, anterior horn cells and Clarke's column (54,61–63). Immunostaining against the astrocytic marker GFAP confirmed the presence of increased numbers of reactive astrocytes in the cerebellar white matter of symptomatic mice, and the immunostaining was found to increase with gene dose (Fig. 6), CAG repeat length and age. This supports the involvement of an inflammatory process in MJD pathogenesis (64) and will be investigated further with other inflammatory markers such as interleukin-1ß (IL-1ß) and CD68. Additionally, there was atrophy of the cerebellar Purkinje and molecular cell layers, which was influenced by CAG repeat length and gene dose (Fig. 7). Changes in cerebellar cortex have been noted in some European MJD kindreds (46), but the degree of degeneration is less than in SCA1. It is generally accepted that Purkinje cells appear to be moderately involved in MJD pathogenesis (65).
It was clear that CAG repeat length and level of expression were both strongly influential on the degree of gliosis and cell loss in the brains of the transgenic mice. This is consistent with the close correlation of degree of atrophy of the brainstem and cerebellar vermis in MJD patients with the size of the expanded CAG repeat (61) and the additional pathological findings in patients homozygous for mutations in the MJD1 gene (54). The availability of a representative mouse model for MJD will allow a more detailed investigation of the pathological processes in specific subpopulations of neurons involved in MJD pathogenesis.
Immunohistochemical analysis showed that ataxin-3 was predominantly cytoplasmic in dentate and pontine neurons of non-transgenic and in control-transgenic mice (Fig. 9). In the diseased MJD1 mouse brains, ataxin-3 was found to be predominantly nuclear and formed neuronal intranuclear inclusions (NIIs) that are ubiquitinated (Fig. 9). This is concordant with the situation seen in human MJD patients (11) and is a common feature of polyglutamine repeat disorders (8,9,12–17,19,36,66). It is not clear whether NIIs play a critical role in the neurodegenerative process in MJD or the other polyglutamine diseases. Until recently, the role of NIIs in neurodegeneration was contentious, since they had not been identified in SCA2 brain and speculation surrounded their role in the degenerative process (67). The identification of NIIs in SCA2 brain (18) strengthens their role in the pathogenesis of the polyglutamine-repeat diseases. Consistent with this, the distribution of the inclusions is enriched in the vulnerable brain cells of patients with the various disorders (9,11,12,68). In other transgenic mice, the data are also consistent with a central role of inclusions in the degenerative process. For example, in ataxin-1 and HD transgenic mice, the inclusions are present before the onset of phenotype and neuronal degeneration (8,12).
Transgenic mice with mutated alleles also presented with features suggesting peripheral neuropathy. Wire manouvre was poorly executed and response to toe pinch was abnormal, with lines expressing mutant MJD1 showing little or no reaction, and this response was markedly reduced or absent with increasing age. In addition, grip strength in the fore- and hindlimbs was reduced in mice expressing mutated MJD1. This finding was noticeable at weaning and progressed as the mice aged, suggesting distal muscle weakness. These findings may be due to the peripheral nerve loss due to demyelination or degeneration of the dorsal root ganglia. Clinically, neuropathy causes weakness and atrophy of muscle, and this may explain the differences in weight/growth in the transgenic mice. Additionally, we found evidence of de- and remyelination and premature neuropathy in symptomatic mice (Fig. 8). The neuropathy observed in the mouse models described here is a significant finding, since demyelination is increasingly becoming an important finding in MJD (5,38–41). Moreover, there is a gene-dosage and repeat-length effect on the extent of peripheral neuropathy in MJD1 YAC-transgenic mice. This is consistent with the situation reported in MJD patients, where the number of CAG repeats was found to have an inverse relationship to the extent of pathological changes of the peripheral nerves (40).
The phenotype of the transgenic animals described here is quite different to that which was reported in the models where truncated or full-length MJD1 were expressed under the control of the L7 promoter (24,25). Mice expressing full-length ataxin-3, with 79 glutamine repeats, under the control of the L7 promoter, developed no ataxia or pathological changes (24). In contrast, overexpressing truncated ataxin-3 with 79 glutamines (24) caused a very early-onset and aggressive disease progression. The mice become ataxic by 4 weeks of age, and by 8 weeks they showed massive degeneration of all three layers of the cerebellum, in particular the Purkinje cells, a phenomenon not generally observed in MJD, and not observed in our mice expressing near endogenous levels of transgene encoded ataxin-3. Furthermore, it was not reported whether NIIs were detected. Our YAC transgenic MJD mice have a mild and progressive phenotype involving widespread but specific neuronal subpopulations such as the dentate and pontine neurons as occurs in MJD, with NIIs present in the areas involved in MJD pathogenesis.
The model described here will therefore be an invaluable resource for the detailed analysis of the neurodegenerative processes underlying MJD pathogenesis and a useful reagent to test the efficacy of future therapeutic strategies for MJD and other polyglutamine disorders.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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YAC DNA purification and injection and identification of transgenic mice
Characterization of the 250 kb human MJD1-YAC clone, 9AF12, and generation of the modified clones that contain 76 and 84 CAG repeats, have previously been described (30). YAC DNA was isolated and purified using previously described methods (69). YAC DNA was then microinjected at low concentrations (0.5–1.0 ng/µl) into mouse pronuclei in (C57BL/6JxCBA/Ca)F2 oocytes that were then implanted into foster mothers using standard techniques (70). Transgene-positive mice were backcrossed to C57BL/6J mice.
Offspring resulting from the injections were weaned and tail-clipped at 3 weeks of age and DNA prepared by proteinase K digestion, phenol/chloroform extraction and ethanol precipitation (71). Initially, mouse genomic DNA samples were PCR screened with (i) primers specific for the YAC left and right vector arms (72), (ii) primers specific for human MJD1 exon 2; EXON2NF 5'-GAATATTTTAGCCCTGTGGAATT-3'/EXON2NR 5'-GTGCGATAATCTTCACT AGTAAC-3' and (iii) two sets of primer pairs that amplify across the CAG repeat motif in exon 10 of the human MJD1 gene (MJD52 5'-CCAGTGACTACTTTGATTCG-3'/MJD25 5'-TGGCCTTTCACATGGATGTGAA-3' and/or MJD52/CKC1 5'-GATGTGAGCCACCACATCT-3'). PCR was carried out in 50 µl reactions containing 100 ng genomic DNA, 200 µM dNTPs, 1xQiagen PCR buffer, 50 pM oligonucleotide primers and 0.05 U/ml Taq DNA polymerase (Qiagen). As the primers are all vector- or human-specific, only those mice carrying the human transgene were detected.
Repeat analysis and ratio PCR
To determine the size of the triplet repeats, MJD52/MJD25 PCR products (see above) were subcloned into the PCR II vector (Invitrogen). The inserts (20–100 ng) were then sequenced using a T7 sequencing kit (Pharmacia Biotech) incorporating 0.5 µl [
-33P]dATP/reaction using standard techniques. The products of the reaction were resolved by electrophoresis through a 6% Sequagel polyacrylamide gel (National Diagnostics).
Transgene copy number and expression were estimated by ‘ratio PCR’ (31) using primers designed to amplify both the mouse and human genes equally in the reaction mix so neither product would be preferentially amplified during PCR. Therefore, the ratio between the two products present after PCR should be the same as the ratio between the starting human and murine DNA/cDNAs, even outside the exponential phase of amplification.
Transgene copy number was determined with primers EXONVNF 5'-CAGACCTGGAACGAATGTTAGAA-3' and EXON8R 5'-TTGCATACTTAGCTGAATAGCCCTGCGG-3' derived from exon 8, which amplify a 166 bp product that includes a human-specific TaqI site and a mouse-specific BglII site. The primers have 100% identity to the human and mouse MJD1 homologues. PCR was performed as described above with 100 ng mouse genomic DNA (see above) with an annealing temperature of 55°C. Double digestion of the l66 bp product yields 111 and 55 bp human and 133 and 33 bp mouse fragments.
Expression of the human MJD1 gene was quantified with the primers CK4 5'-GTTATAAGCAATGCCTTGAAA-3' and CK11 5'-GAGAGAATTCAAGTTAAACC-3' located in exons 4 and 6, which amplify a 175 bp product spanning nucleotides 270–445, which includes human-specific ClaI and TaqI sites and a mouse-specific RsaI site. The sequence matching primer CK4 has a single nucleotide difference between mouse and human cDNA, while the CK11 sequence is identical in the two species. PCR was performed as described above but with 50 ng of cDNA (see below) with an annealing temperature of 55°C.
For quantification, PCR was performed as above except that we included 0.5 µl [-33P]dATP/reaction and electrophoresed the entire digested product on a 4% 1xTBE MetaPhor agarose (BMA) midigel. Agarose gels were dried and exposed overnight to a phosphoimaging screen, which was scanned using a CYCLONE phosphorimager (Packard) and ImageQuant software.
Isolation of mRNA from mouse tissues
Messenger RNA (mRNA) was isolated with the Micro-FastTrack Kit (Invitrogen) according to the manufacturer's protocol. Mouse tissue was rinsed in phosphate-buffered saline (PBS), weighed and immediately frozen in liquid nitrogen. The tissues were thawed and immediately homogenized in lysis buffer using a polytron PT1200 homogenizer (KINEMATICA PT). The homogenized cells were transferred to a sterile 30 ml tube and incubated at 55°C for 15 minutes. The extracted mRNA was stored as an ethanol precipitate at -70°C until ready for use. It was then centrifuged at 16 000 g for 15 minutes at 4°C. The mRNA pellet was resuspended in 10–20 µl of elution buffer (10 mM Tris, pH 7.5) and was used immediately. RT-PCR was performed on 50 ng of Poly(A)+ mRNA according to the Micro-FastTrack Kit (Invitrogen).
Protein extraction and western blot analysis
To prepare protein extract, frozen samples of tissue were homogenized in 50 mM Tris–HC1, pH 8.0, 1% Triton X-100, 0.5% NP-40, 1 mM dithiothreitol (DTT), 4% (v/v) glycerol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 2 µg/ml leupeptin and 4 µg/ml aprotonin. Samples were sonicated for 2 minutes on ice and then centrifuged at 18 000 g for 30 minutes at 4°C. Aliquots of supernatant were stored at -80°C until further analysis.
Protein concentration was determined by the Pierce bicinchoninic acid (BCA) assay. Total protein extracts (25 µg) were analysed on 10% SDS–PAGE gels. Proteins were transferred to PVDF membrane (Millipore). Blots were then incubated with affinity purified ataxin-3 antiserum (38) at 1 : 1000 dilution or mAb 1C2 (Chemicon International) at 1 : 2000 dilution in 6% non-fat milk, followed by horseradish peroxidase-conjugated goat anti-mouse (1 : 10 000) or goat anti-rabbit (1 : 1000) antiserum (DAKO). Immunoreactive bands were visualized by chemiluminescence (PIERCE).
Phenotypic evaluation
Behavioural and functional evaluation of the mouse phenotype was a modification of the SHIRPA protocol (35), which mimics the neurological examination in humans and includes an investigation of the integrity of muscle and lower motor neuron, spinocerebellar, sensory and autonomic functions. Examination was undertaken every four weeks throughout the natural life span of mice weighing less than 30 g.
Locomotor activity testing was carried out in a clear Perspex (550 mmx300 mmx180 mm) box in which a Perspex sheet on the floor is marked with 15 squares. The number of 11 cm2 squares entered by all four feet in 30 seconds was recorded.
Positional passivity was assessed by the struggle response of the mice to sequential handling including when held by tailor neck (finger grip, not scruffed), when laid supine (on back) and when held by hind legs.
A Startle response observation was to a 90 dB sound from a clickbox 30 cm above a Perspex arena. This analysis involves assessment of the Preyer reflex (backward flick of pinnae).
Toe pinch reaction was monitored by placing the mouse on a wire grid and lifting the hindlimbs clear of the grid by gripping the tail between the thumb and forefinger. A gentle lateral compression of the mid digit of the hindfoot was applied with fine forceps and the withdrawal reaction monitored.
The ability to correct body position was analysed by a negative geotaxis test, this is the orientation response of an animal to a gravitational stimulus (73). A mouse was placed on a horizontal grid, which was raised to an inclined plane of 45° with the animal facing head down. An observation of the animal's ‘angle of orientation’ upon leaving its starting position was recorded for 30 seconds – normal mice should orient in a head-up direction. Before the next trial, the wire mesh was cleaned with ethanol to ensure that odour trails left from previous mice did not influence the behaviour of subsequent animals.
Limb clasping was assessed by gripping the tail between the thumb and forefinger and suspending the mouse for 30 seconds and observing escape reflexes.
Wire manouvre was observed by holding the mice by tail suspension and lowering them to allow the forelimbs to grip a horizontal wire. The mouse was held in extension and rotated around to the horizontal and released. The ability to grip with the hindlimbs was observed.
Limb tone, skin colour, heart rate, lacrimation and salivation were monitored during supine restraint. Resistance to gentle fingertip pressure on the plantar surface of the left right hind paw was used to assess limb tone.
Tissue fixation
Mice were anesthetised by intraperitoneal injection with pentobarbitone (Pentoject) (Animalcare Ltd.) and intracardiac perfusion was carried out with 10 ml heparinized PBS [0.2 ml heparin stock (5000 units/ml, Leo Laboratories Ltd) in 500 ml PBS] for 2 minutes. Perfusion was continued for a further 15 minutes with 100 ml 4% paraformaldehyde through the left cardiac ventricle. Brains were then dissected and post-fixed for 1 hour (same fixative).
Mice destined for peripheral nerve studies were treated as described by Robertson et al. (74). The perfusate contained 1% paraformaldehyde, 1% glutaraldehyde and 1% dextran in PIPES buffer. After dissection, tissues were fixed overnight in the same solution before being post-fixed in buffered 1% osmium tetroxide also containing 1.5% potassium ferricyanide and 3% sodium iodate. They were dehydrated and processed into epoxy resin by standard techniques. Semithin sections for light microscopy were stained with thionin and acridine orange. Ultrathin sections stained with uranyl acetate and lead citrate were examined in a Zeiss EM902C electron microscope.
Immunohistochemistry
Fixed tissue (not including brain) was processed for paraffin wax embedding using a Shandon duplex processor. The final wax change was in a vacuum chamber (Hearson) at a pressure of 400 mmHg to ensure thorough infiltration. Paraffin wax was from BDH (melting point 57–58°C). Brain tissue was removed, post-fixed in 4% paraformaldehyde overnight, and then transferred in PBS. The brains were cut sagitally and processed for paraffin embedding.
Brain and spinal cord were processed and dehydrated by hand with 50% industrial methylated spirit (IMS) for 30 minutes, 70% IMS for 30 minutes, 90% IMS for 30 minutes and three times in 100% IMS for a total of 90 minutes, and left overnight. Processing was continued with two 90-minute washes in chloroform. Brain was wax-embedded under vacuum as described above. Spinal cord (cervical, lumbar and sacral regions) was cut with a double-edged blade (TAAB Laboratories). Processing of DRG and spinal cord was as described above.
Tissue sections were cut on a rotary microtome (Anglia Scientific AS325) and floated on distilled water in a waterbath. The temperature was maintained at 37°C for brain and spinal cord, and 40–45°C for all other tissues. Sections were picked up on subbed slides to aid adhesion, and dried at 37°C on a Hearson hotplate before transferring to a slide-drying oven overnight at 37°C. Peripheral nerves were cut at 5 µm, brain and spinal cord at 7 µm, DRG at 5 µm and all other tissues at 3 µm.
Brain sections were stained with haematoxylin–eosin (H&E) and Holmes' (silver) staining according to Drury and Wallington (75). Calbindin D-28k immunostaining was used specifically to stain cerebellum Purkinje cells, molecular layer dendrites and axonal fibres. The antibodies and dilutions used in this study were as follows: rabbit polyclonal antibody to glial fibrillary acidic protein (GFAP) (1 : 200; DAKO, Hamburg, Germany); anti-calbindin D-28k monoclonal antibody (1 : 200; Chemicon, Temecula, CA); anti-ataxin-3 polyclonal antiserum [1 : 100; Henry Paulson, described previously (11)]; and polyubiquitin antiserum (1 : 300; DAKO, Hamburg, Germany). Sections were typically counterstained with haematoxylin. All antibodies were screened against the appropriate control brain sections to verify that the specific antigen was detected at the concentration used.
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ACKNOWLEDGEMENTS |
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We should like to thank Amanda McGuigan, Mark Hay and Zoe Webster for carrying out pronuclear microinjection of YAC DNA to generate the founder mice. We should also like to thank Henry Paulson for generously providing ataxin-3 antiserum, Michelle Nourallah for assistance with tissue processing, and Gillian Bates and Patricia Maciel for their advice. The help from P.K. Thomas and Franchesco Scaravilli for histological evaluation is also gratefully acknowledged. We should like to thank Mathew Hodges for helpful discussion. This work is supported by The Ataxia Group of Great Britain and Northern Ireland.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +44 20 7594 3235; Fax: +44 20 7594 3015; Email: c.cemal{at}ic.ac.uk
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REFERENCES |
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