A transgenic mouse model of spinocerebellar ataxia type 3 resembling late disease onset and gender-specific instability of CAG repeats

Neurobiology of Disease 37 (2010) 284–293

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Neurobiology of Disease j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y n b d i

A transgenic mouse model of spinocerebellar ataxia type 3 resembling late disease onset and gender-specific instability of CAG repeats Jana Boy a,1, Thorsten Schmidt a,⁎,1, Ulrike Schumann a, Ute Grasshoff a, Samy Unser a, Carsten Holzmann b, Ina Schmitt c, Tim Karl d,2, Franco Laccone e, Hartwig Wolburg f, Saleh Ibrahim g, Olaf Riess a a

Medical Genetics, University of Tuebingen, Calwerstrasse 7, 72076 Tuebingen, Germany Medical Genetics, University of Rostock, Rostock, Germany Neurology, University of Bonn, Bonn, Germany d Functional and Applied Anatomy, Medical School of Hannover, Germany e Department for Medical Genetics, Medical University Vienna, Vienna, Austria f Institute for Pathology, University of Tuebingen, Tuebingen, Germany g Immunogenetics, University of Rostock, Rostock, Germany b c

a r t i c l e

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Article history: Received 10 March 2009 Revised 31 July 2009 Accepted 10 August 2009 Available online 20 August 2009 Keywords: Spinocerebellar ataxia type 3 Machado–Joseph disease SCA3 MJD Polyglutamine Intranuclear inclusion bodies Transgenic mouse model CAG repeat instability Late onset

a b s t r a c t Spinocerebellar ataxia type 3 (SCA3), or Machado–Joseph disease (MJD), is caused by the expansion of a polyglutamine repeat in the ataxin-3 protein. We generated a mouse model of SCA3 expressing ataxin-3 with 148 CAG repeats under the control of the huntingtin promoter, resulting in ubiquitous expression throughout the whole brain. The model resembles many features of the disease in humans, including a late onset of symptoms and CAG repeat instability in transmission to offspring. We observed a biphasic progression of the disease, with hyperactivity during the first months and decline of motor coordination after about 1 year of age; however, intranuclear aggregates were not visible at this age. Few and small intranuclear aggregates appeared first at the age of 18 months, further supporting the claim that neuronal dysfunction precedes the formation of intranuclear aggregates. © 2009 Elsevier Inc. All rights reserved.

Introduction Spinocerebellar ataxia type 3 (SCA3), or Machado–Joseph disease (MJD), is an autosomal-dominantly inherited neurodegenerative disorder caused by the expansion of a CAG repeat in the MJD1 gene (Kawaguchi et al., 1994). While, in unaffected individuals, the number of CAG repeats is usually less than 45 (Padiath et al., 2005), it increases up to 86 repeats in SCA3 patients (Riess et al., 2008). The expanded CAG repeat results in an expanded polyglutamine stretch in the encoded ataxin-3 protein. SCA3 therefore belongs to the group of polyglutamine diseases, which includes other types of spinocerebellar ataxias as well as SBMA, DRPLA, and Huntington's disease (reviewed in Gatchel and Zoghbi, 2005). The protein harboring the expanded polyglutamine tract has a strong tendency for aggregation (Scherzin-

⁎ Corresponding author. Fax: +49 7071 29 5228. E-mail address: [email protected] (T. Schmidt). 1 These authors contributed equally to this project. 2 Present address: Prince of Wales Medical Research Institute, Randwick, Australia. Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2009.08.002

ger et al., 1999), which usually manifests in the nucleus of neuronal cells in SCA3. Interestingly, while ataxin-3 is ubiquitously expressed throughout the whole brain, the formation of intranuclear inclusion bodies appears in specific brain regions (Paulson et al., 1997b; Schmidt et al., 1998). There is an ongoing discussion whether these intranuclear inclusions are toxic or perhaps even protective for neuronal cells (Michalik and Van Broeckhoven, 2003; Sisodia, 1998). Clinically, SCA3 presents with a highly heterogenous phenotype, leading to differentiation into clinical subtypes (reviewed in Riess et al., 2008). In addition, the disease is characterized by a late onset, usually appearing in the late third decade of life (Dürr et al., 1996; Schöls et al., 1997), and by slow progression. In contrast to the disease course in human patients, most of the previously generated mouse models of SCA3 are characterized by an early onset and rapid progression (Bichelmeier et al., 2007; Cemal et al., 2002; Chou et al., 2008; Goti et al., 2004; Ikeda et al., 1996) with rather distinct regional manifestation. Thus, not all the pathogenic aspects of the disease in humans are reproduced by these models. Here, we present a mouse model for SCA3 with late onset of symptoms that resembles major genetic and pathogenetic characteristics in humans and demonstrates that motor symptoms precede the

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occurrence of intranuclear inclusion bodies. For this new transgenic mouse model, we utilized the well-characterized rat huntingtin promoter (Holzmann et al., 1998, 2001) to express a full-length ataxin-3 construct containing 148 CAG repeats ubiquitously throughout the brain. In transgenic mice, the instability of the CAG repeat expansion was obvious, and clearly correlated with the sex of the transmitting parent. We additionally observed a biphasic course of the disease and detected hyperactivity in HDPromMJD148 mice long before the onset of motor deficits, but the formation of intranuclear inclusion bodies was no prerequisite for the onset of symptoms. The novel mouse model of SCA3 presented in this study is a valuable addition to previous published mouse models, since its slowly progressing phenotype allows us to study aspects of the disease that cannot be analyzed in other models of this disease. Materials and methods Transgenic mice To generate a novel transgenic mouse model for SCA3, a 764 bp XbaI restriction fragment of the rat huntingtin promoter corresponding to nucleotide positions −777 to −14 was cloned to the 5′ end of the full-length ataxin-3 cDNA (isoform ataxin-3c) (Goto et al., 1997; Schmitt et al., 1997) in the pBluescript SK vector. The transcriptional activity of this promoter fragment has been demonstrated before (Holzmann et al., 1998). The SV40 early mRNA polyadenylation signal was amplified from the vector pEGFP-C (Clontech, Mountain View, CA) using the primers pEGFP-C-PolyA Kpn-For (5′-GTT GGT ACC AAC TTG TTT ATT GCA GCT TA-3′) and pEGFP-C-PolyA Kpn-Rev (5′-TTT GGT ACC TAA GAT ACA TTG ATG AGT TT-3′). The primers attach KpnI restriction sites to both sides of the PCR product which were used to insert the polyadenylation signal at the 3′ end of the ataxin-3 cDNA. 148 CAG repeats were cloned into the construct using the BglII and XhoI restriction sites. Extension of the CAG repeat to 148 CAG repeats was performed as described earlier (Laccone, 2002). The transgene construct, comprising the rat huntingtin promoter fragment, the full-length ataxin-3 cDNA with 148 CAG repeats and the polyadenylation signal, were linearized and separated from the vector backbone using the restriction enzymes NotI and PvuI. Seven injections into fertilized murine oozytes of the C57BL/6N mouse strain were performed, and the resulting 25 offspring were genotyped. The transgene was detected in four individuals. After crossing these founders with wildtype mice, one stable mouse line (line 3746, designated HDPromMJD148) was generated that expresses the ataxin-3 transgene. Western blot Mice were killed by CO2 inhalation, the tissue was freshly prepared, immediately snap frozen, and stored at −80 °C. For protein isolation, tissue was homogenized at 30,000 rpm using a tissue homogenizer (Ultra-Turrax; IKA Werke, Staufen, Germany) in TES buffer (50 mM Tris, pH 7.5, 2 mM EDTA, and 100 mM NaCl) supplemented with a mixture of protease inhibitors (complete; Roche Applied Science, Mannheim, Germany). After the addition of Nonidet NP-40 (Sigma-Aldrich, Seelze, Germany) to a final concentration of 1%, and incubation at 4 °C for 15 min, debris was removed by centrifugation (15 min each, 20,000 relative centrifugal force (rcf), 4 °C). The cleared protein extract was supplemented with glycerol (final concentration of 10%; VWR International, Darmstadt, Germany) and stored at − 80 °C. The protein concentration was determined using a protein assay (Protein Assay Dye Reagent Concentrate; BioRad, Munich, Germany) based on the method described by Bradford (1976) according to the manufacturer's instructions. Protein extracts (30 μg of each) were supplemented with loading buffer (80 mM Tris

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pH 6.8, 0.1 M DTT, 2% SDS, 10% glycerol, bromphenol blue), denatured, and analyzed in PAGE buffer (192 mM glycine, 25 mM Tris, 1% SDS) using SDS-PAGE (Blue Vertical 100/C; Serva, Heidelberg, Germany), according to the method described by Laemmli (1970). The separated proteins were transferred to a Polyvinylidenedifluoride (PVDF) membrane (Immobilon-P Transfer-Membran, Millipore, Schwalbach, Germany) in transfer buffer (0.2 M glycine, 25 mM Tris, 20% methanol). The detection of the protein was performed essentially as described previously (Schmidt et al., 1998). Briefly, the membrane was blocked in 5% dry milk in TBST buffer (10 mM Tris, pH 7.5, 0.15 M NaCl, 0.1% Tween 20) for 2 h at room temperature. The primary antibody was diluted in TBST. The generation of our anti-ataxin-3 antibody (diluted 1:1000) has been described previously (Schmidt et al., 1998). The 1H9 antibody against ataxin-3, as well as the 1C2 antibody directed against expanded polyglutamines, were purchased from Chemicon (Hofheim, Germany). After incubation for 2 h, the membrane was washed four times with TBST for 15 min. The secondary antibody, coupled to horseradish peroxidase (GE Healthcare, Freiburg, Germany), was incubated with the membrane for 75 min. After four washing steps with TBST (15 min each), bands were visualized using the enhanced chemiluminescence method (ECL; GE Healthcare) and by exposure to Hyperfilm ECL (GE Healthcare). Immunohistochemistry Mice were deeply anaesthetized by CO2 inhalation and transcardially perfused using 4% paraformaldehyde (PFA) in 0.1 M PBS. Brains were removed from the skull and postfixed overnight in fixative, embedded in paraffin and cut into 7 μm sagittal sections. The immunohistochemical staining of paraffin-embedded tissue was performed as described previously (Schmidt et al., 2002). Briefly, after deparaffinization of sections in xylene and rehydration in a graded alcohol series, slides were microwaved for 15 min in 10 mM sodium citrate, pH 6.0. The slides were washed with PBS; endogenous peroxidases were blocked using 1% hydrogen peroxide in 40% methanol for 10 min and blocked using 5% normal goat serum in PBS supplemented with 0.3% Triton. After washing with PBS (three times for 10 min), the primary antibody (diluted in PBS plus 3% goat serum) was added and incubated at 4 °C overnight in a humid chamber. The secondary antibody, coupled with biotin (Vector Laboratories, Burlingame, CA), was diluted the same way in PBS plus 1.5% goat serum and added after washing the slides with PBS. After incubation for 30 min at room temperature and a brief wash with PBS, an ABC enhancer complex coupled with peroxidase (Vector Laboratories) was added and incubated for 30 min at room temperature. After washing with PBS, the substrate (DAB; Sigma-Aldrich) was added, and the reaction was stopped in distilled water after the desired degree of staining was reached. Finally, slides were dehydrated again and mounted using CV mount (Leica, Bensheim, Germany). Staining was visualized using an Axioplan 2 imaging microscope (Carl Zeiss Microimaging, Oberkochen, Germany) equipped with an Axio-Cam MR color digital camera (Carl Zeiss Microimaging) using a 40 × Plan Neofluar and a 63× Plan/Apochromat objective and the AxioVision 4.6 software package (Carl Zeiss Microimaging). Genotyping and fragment analysis For the genotyping of transgenic mice, DNA isolated from ear biopsy tissue and the following primer combination was used: MJDPromA-Int-F (5′-CTT TGG TTC CGC TTC GGT CT -3′), MJDPromAInt-R (5′-CTT CTC CTC CTC ATC CAG CT -3′). In order to determine the CAG repeat number, the primers hMJD1c-CAG-Frag-F (5′-Cy5-GCT AAG TAT GCA AGG TAG TTC C-3′) and hMJD1c-postGAG-R (5′-CAA GTG CTC CTG AAC TGG TG -3′) were used to specifically amplify the CAG repeats. For detection, the forward primer was marked with Cy5, and for size determination, an

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internal standard (DNA-Size Standard Kit-600, Beckman Coulter, Krefeld, Germany) was used. Fragment detection was performed using a CEQ8000 Genetic Analysis System (Beckman Coulter). For additional calibration, we used ataxin-3 constructs for which repeat lengths were confirmed by sequencing (15, 77, or 148 CAG repeats). Home cage activity To analyze the spontaneous home cage activity of the mice, the LabMaster system (TSE Systems, Bad Homburg, Germany) was used. The mouse cages were placed into sensor frames, the number of beam brakes was counted, and the total activity, ambulatory and fine movements, as well as rearings, were quantified. In addition, drinking and feeding behavior was analyzed. The analysis was performed for 23 h, starting with 5 h of light phase, continuing with 12 h of darkness and concluding with an additional 6 h of light. For analysis, beam breaks were counted in 15 min intervals. In each run, four mice were analyzed in parallel using four separate test systems. In order to exclude any bias, to serve as internal control, and for reproduction, both transgenic and control mice were routinely analyzed in parallel and were randomly distributed between the test systems.

Animal experiments All animal experiments were conducted in accordance with international standards on animal welfare, and comply with the standards defined by the European Communities Council Directive of 24 November 1986 (86/609/EEC). Animal health was routinely monitored by local veterinarians and supervised by the regional board. Results The huntingtin promoter led to transgene expression throughout the whole brain To control the expression of a full-length construct of ataxin-3 containing 148 CAG repeats, a 764-bp fragment of the wellcharacterized rat huntingtin promoter was used (Fig. 1A). This promoter fragment contains numerous conserved putative

Open field analysis For the assessment of the explorative behavior and emotionality of the mice, open field tests were performed. Mice were placed in a 50 × 50 cm arena with 50 cm high walls and their movement activity was recorded for 15 min using the TSE VideoMot2-Video Activity Tracking System (TSE Systems, Bad Homburg, Germany). The light intensity was set to be at least 150 lux in the corners, and not higher than 200 lux in the center of the arena (Brown et al., 2005; Green et al., 2005). To analyze the collected data, the arena was divided into different regions. Region 1 is the border, with a width of 8 cm, region 3 the center, which comprises 16% of the overall area, and region 2 the area between the border and the center. Rotarod To measure the motor coordinative abilities and balance of the transgenic mice, Rotarod analyses were performed. At a maximum illumination of 100 lux, mice were tested on an accelerating Rotarod (TSE Systems, Bad Homburg, Germany) starting at 4 rpm and accelerating to 40 rpm over a period of 300 s (5 min). Three trials per test day, in which the latency to fall off the rotating rod was recorded, were carried out with 15 min rest between trials (Brown et al., 2005; Green et al., 2005). The tests were repeated approximately every 6 weeks. Statistical analysis of the data thus obtained was performed using Excel (Microsoft, Unterschleiβheim, Germany), calculating the mean values (in s) for each group. Both t-tests (Excel) and two-way ANOVA (GraphPad Prism, GraphPad Software, La Jolla, CA) were performed. Data are represented as mean ± SEM. Electron microscopy Electron microscopic analyses were essentially performed as described previously (Lundkvist et al., 2004). Briefly, after being deeply anaesthetized by CO2 inhalation, mice were transcardially perfused using 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4). Brains were removed and postfixed in fixative for 30 min to 1 h. After dehydration in ethanol, brain pieces were incubated overnight in 70% ethanol saturated with uranyl acetate, followed by an additional dehydration in absolute ethanol and propylene oxide. Finally the samples were embedded in Araldite 502 (Sigma-Aldrich) and sectioned on a Leica FCR Ultracut ultramicrotome (Leica). Ultrathin sections were stained with lead citrate and examined with a Zeiss EM 10 electron microscope.

Fig. 1. Generation of HDPromMJD148 transgenic mouse model of SCA3. (A) Construct used for the generation of transgenic mice. A full-length ataxin-3 cDNA with 148 CAG repeats is expressed under the control of a 764-bp fragment of the rat huntingtin promoter. To ensure stability of the encoded mRNA, the construct also contains a polyadenylation (polyA) signal. (B) Expression of transgenic ataxin-3 in different tissues. The expression of the ataxin-3 transgene was analyzed in different tissues. In tissue culture (“construct”, transgene constructs overexpressed in SK-N-AS neuroblastoma cells) and in the brain of our transgenic mouse line, the ataxin-3 transgene is detected using an anti-ataxin-3 antibody (1H9, Chemicon) while no expression can be detected in the negative control (brain of a wildtype mouse), testis, muscle, lung, liver, and kidney. The band detected in the heart is a background band since it is not detected using the 1C2 antibody directed against expanded polyglutamine repeats (polyQ). TBP, which is co-detected by the 1C2 antibody (see below), is not shown on this blot since it was allowed to run out of the gel for optimal separation. (C) Comparison of transgene expression in whole brain lysates of mice transgenic for ataxin-3 with 148 CAG repeats under the control of a prion protein promoter (PrpProm, line 148.19) (Bichelmeier et al., 2007) and of the huntingtin promoter (HDProm, line 3746). The Prp promoter results in about 2.5-fold higher expression than the huntingtin promoter. The transgene band (tg) is not present in the wildtype mouse (wt). (D) The huntingtin promoter results in an even distribution of transgene expression throughout the whole brain. When comparing different brain regions, no difference in transgene expression is observed between the whole brain (brain), the telencephalon (tel), the cerebellum (ceb), and the brainstem (bs). The western blot in B (bottom), C, and D were processed using the 1C2 antibody directed against expanded polyglutamines. TBP marks the TATA binding protein which is usually co-detected when the antibody 1C2 is used, since this antibody was originally generated against TBP (Trottier et al., 1995).

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transcription factor binding sites, which have led to robust expression in a variety of cell lines derived from different rodents (Holzmann et al., 1998). A comparable fragment of this promoter has been successfully used before to generate a transgenic rat model of Huntington's disease (von Hörsten et al., 2003). At the 3′-end of ataxin-3, a SV40 early mRNA polyadenylation signal was introduced. The integrity of the construct was confirmed by sequencing, and the expression of the ataxin-3 transgene was verified in tissue culture (Fig. 1B). Using this construct, one stable mouse line (line 3746, designated HDPromMJD148) was generated. In this mouse line, the transgene is expressed exclusively in the brain, while no expression was observed in peripheral tissue (Fig. 1B). We previously generated a SCA3 transgenic mouse line under the control of a Prion protein promoter, which manifests with early onset and a rapid disease progression (Bichelmeier et al., 2007). Compared to this line, the HDPromMJD148 line has lower expression of ataxin-3 in the brain (Fig. 1C). In order to make sure that this reduced signal is not just due to a weaker expression in specific brain regions, we compared the expression in the whole brain with the telencephalon, the cerebellum, and the brainstem. These analyses revealed that the transgene expression in the HDPromMJD148 line is evenly distributed throughout the brain (Fig. 1D). Immunohistochemical analyses confirmed this observation. In all analyzed brain regions, the transgene can be detected mainly in neuronal cells. In the cerebellum, the transgene is expressed in the

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cerebellar nuclei as well as in all three layers of the cerebellar cortex, including Purkinje cells (Fig. 2). Interestingly, in homozygous mice, the expanded polyglutamine stretch within ataxin-3 gives rise to a predominantly nuclear localization, while in heterozygous mice, the transgene is mainly located in the cytoplasm (Fig. 6). CAG repeats within the HDPromMJD148 transgene behave unstably Analyzing brain lysates from different HDPromMJD148 mice by western blotting revealed mutant transgenic ataxin-3 at slightly different sizes (Fig. 3A, top). To investigate whether these size differences were due to different CAG repeat numbers within the respective transgenes, fragment analyses were performed. These analyses confirmed that the observed size differences of transgenic ataxin-3 protein were due to different CAG repeat numbers: in the analyzed mice, between 135 and 155 CAG repeats were counted (Fig. 3A, bottom). We next wanted to find out whether the inheritance of the transgene was linked to the variation in the CAG repeat number. We therefore analyzed parents and their offspring, and again determined the number of CAG repeats using fragment analysis. In order to follow the repeat number of certain specific transgenes, only heterozygous mice and only breedings between transgenic and wildtype mice were included in this study. In 86% of the analyzed transmissions, the repeat length changed. Interestingly, this analysis

Fig. 2. Even distribution of transgene expression throughout the brain. Immunohistochemical staining using an antibody against ataxin-3 (1H9, Chemicon) confirmed an even and mainly neuronal distribution of the ataxin-3 transgene throughout the brain. Shown are representative examples from the cortex, the red nucleus (nucleus ruber), the pons, and the cerebellum (cerebellar cortex and cerebellar nuclei). In the cerebellar cortex, the transgene is expressed in all three layers, including Purkinje cells. Interestingly, the long stretch of 148 polyglutamine repeats results in a predominantly nuclear localization of the transgene in homozygous mice. Both the wildtype and the homozygous HDPromMJD148 mice were analyzed at the age of 14 months. No intranuclear inclusion bodies were observed at this timepoint. MolCb, molecular layer of the cerebellar cortex; PuCb, Purkinje cell layer of the cerebellar cortex; GrCb, granular layer of the cerebellar cortex. Bar, 20 μm.

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Fig. 3. Instability of CAG repeats and selective changes after maternal and paternal transmission. Following the number of CAG repeats within the ataxin-3 transgene for several generations, an instability of the CAG repeat number becomes apparent. (A) Western blot analysis showing examples of the unstable transgenic ataxin-3 protein in heterozygous and homozygous animals, detected using the 1C2 antibody directed against expanded polyglutamine repeats. The CAG repeat numbers of the analyzed mice, as determined using fragment analysis, are listed below each lane. TBP marks the TATA binding protein, which is usually co-detected when the 1C2 antibody is used. (B) Chromatograms obtained during fragment analysis. Shown are examples of repeat number changes during paternal transmission (father with 147 CAG and his offspring with 153 CAG) as well as maternal transmission (mother with 148 CAG and her offspring with 143 CAG). (C) Diagram showing the parental CAG repeat number (x axis) and the observed change of repeat number in the offspring (y axis). Interestingly, an increase of CAG repeat number was only observed with paternal inheritance (black squares), while maternal inheritance led to a reduction of the number of CAG repeats (open rhombi). (D) Frequency of observed changes of repeat number. For each change of repeat number (x axis), the number of observed changes during transmission (y axis) is listed. Paternal transmission (black bars) led to no change or an increase of repeat number, while after maternal transmission (white bars) only reductions of repeat numbers were observed.

revealed a clear link (p b 0.001) between the change of the CAG repeat number and the mode of inheritance: while an increase in CAG repeats was only observed with paternal inheritance, maternal transmission was linked to a decreased number of CAG repeats (Fig. 3B, C). No correlation between the original number of CAG repeats and the increase or decrease, and no tendency of long repeats towards a further extension was obvious. On the contrary, a large increase (+ 6) of CAG repeat numbers was observed in a mouse with an average repeat number, and reductions in CAG repeat number were also observed in mice with high numbers of CAG repeats (e.g. from 165 down to 155 CAG repeats). Analyzing the frequency of changes, we found that stable transmission and an increase of two repeat units were most common outcomes of paternal transmission, while maternal transmission always led to a reduction in the number of repeat units, most frequently a reduction of two repeat units (Fig. 3D). Transgenic HDPromMJD148 mice show early hyperactivity, but reduced motor coordination and impaired motor learning in late disease stages In order to investigate whether transgenic HDPromMJD148 mice develop a neurological phenotype, behavioral tests, including Rotarod analysis, beam walking, pole test, and others were performed at the age of 4 and 7 months. These tests revealed no differences between

transgenic HDPromMJD148 mice and controls. Since we and others demonstrated before that mice transgenic for ataxin-3 with a normal repeat length develop no phenotype and are indistinguishable from wild type mice even at older age and irrespective of the applied promoter or ataxin-3 construct (Bichelmeier et al., 2007; Cemal et al., 2002; Chou et al., 2008; Goti et al., 2004; Ikeda et al., 1996), negative (wild type) littermates were used as controls for these analyses. We then recorded the home cage activity of transgenic HDPromMJD148 mice for 23 h at the age of about 4 to 6 months, and observed different activity patterns of HDPromMJD148 mice compared to controls: During the first 2 h in the cage, the HDPromMJD148 mice were more active than the control mice (Fig. 4A). This hyperactivity of HDPromMJD148 mice compared to controls was not only characterized by an increased number of beam breaks, but also by a significantly increased percentage of time spent in the center of the cage, a significantly increased percentage of ambulatory movements, and a significantly increased number of rearings (data not shown). In addition to hyperactivity, these data also indicate a reduced level of anxiety in HDPromMJD148 mice. When analyzing the dark phase, we observed a higher activity of control mice at the beginning of darkness and a higher activity of HDPromMJD148 mice at the end of 12 h of darkness. These differences are due to a stronger decrease in the activity of control mice during the 12 h of darkness compared to HDPromMJD148 mice (Fig. 4B).

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Fig. 4. Activity analyses. (A and B) Home cage activity analyses revealed hyperactivity of transgenic HDPromMJD148 mice at the age of 4 to 6 months. Mice were kept for 23 h in LabMaster cages (TSE Systems) and their activity during the light and the dark phase (hour 5 to hour 17) was recorded by the number of beam breaks. Shown are the total numbers of beam breaks in 15 min intervals (A) During the first 2 h in the LabMaster cages, HDPromMJD148 mice (black squares) were significantly more active than control mice (grey triangles). (⁎⁎p b 0.05; ⁎⁎⁎p = 0.001). Error bars, SEM. (B) At the beginning of the dark phase, control mice (gray line) were more active than HDPromMJD148 mice (black broken line); however, their activity decreased more strongly (− 141 beam breaks/15 min) during the 12 h in the dark phase than the activity of HDPromMJD148 mice (− 88 beam breaks/ 15 min), resulting in a higher activity of HDPromMJD148 mice at the end of the dark phase (⁎⁎p b 0.05). Shown is the mean of 12 transgenic and 10 control mice. For clarity, no error bars are shown. (C–F) Open field analyses revealed hyperactivity and reduced anxiety in HDPromMJD148 mice at the age of 14 months (mean of 18 transgenic and 8 control mice). (C) Plot of the moved track in the arena during 15 min. Shown are two representative examples. While the wildtype mouse (control) spend most of the time in the margin area and avoided the center, the HDPromMJD148 mouse evenly moved throughout the arena without any preferences. (D) HDPromMJD148 mice moved longer distances in the open field arena during the first 8 min. The differences in interval/minute 2 and 7 are significant (p b 0.05). (E) No difference between transgenic and control mice was observed regarding the distance moved in the margin area (region 1) of the arena. (F) Distance mice moved in the transition area between the margin and the center (region 2). HDPromMJD148 mice moved (at four intervals significantly) longer distances in the transition area, indicating frequent changes between the center and the margin.

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In order to confirm that these differences in activity persist at older ages, we analyzed mice at 14 months of age. Since in younger mice, significant differences were already observed at the beginning of the home cage activity test, we confined this activity analysis to a shorter time period in a novel environment and performed open field analyses. In this test, we again observed hyperactivity and reduced anxiety in HDPromMJD148 mice. During 15 min, control mice spend most of the time in the margin area and only rarely visited the center of the arena. HDPromMJD148 mice, however, moved through the whole arena without any regional preference (Fig. 4C). Statistical analysis revealed an increased level of activity in HDPromMJD148 mice, indicated by an increased distance moved in the arena (Fig. 4D). While no difference was observed regarding the margin area (Fig. 4E), HDPromMJD148 mice showed a significantly increased level of activity in the transition area between the center and the margin, indicating a reduced anxiety in these mice (Fig. 4F). We then wanted to know whether older HDPromMJD148 mice develop a motor phenotype and therefore performed Rotarod analyses. These analyses revealed a significantly reduced Rotarod performance in HDPromMJD148 mice at the age of about 1 year (Fig. 5A). The Rotarod performance of HDPromMJD148 mice was generally impaired. Comparing mice with shorter or longer CAG repeat lengths within the transgene, and comparing heterozygous and homozygous mice, did not reveal any significant correlation between the number of CAG repeats or the zygosity of the transgene and the severity of symptoms (data not shown). The ability of mice to learn novel motor skills can be studied by analyzing the first two trials of naïve mice on a Rotarod (Buitrago et al., 2004). While control mice significantly improved during these first trials, no improvement was detectable in HDPromMJD148 mice, indicating a reduced motor learning capability of these mice (Fig. 5B). Correlation of the neurological phenotype with neuropathological signs We expected that the occurrence of neurological symptoms would be reflected by the formation of intranuclear inclusion bodies in neurons of certain brain regions. However, our analysis at the age of 14 months revealed no intranuclear aggregates in any of the brain regions examined (Fig. 2). In homozygous mice, intranuclear inclusion bodies were observed from the age of 18 months on but in heterozygous mice, inclusion bodies were not detected until the age of

25 months. Intranuclear inclusions formed in certain brain regions like the red nucleus, the pons, and the cerebellum including Purkinje cells (Fig. 6A). We therefore wanted to know whether other signs of neurodegeneration or dysfunction are present in phenotypical HDPromMJD148 mice, and observed using electron microscopy darkly stained Purkinje cells considered as a sign for degeneration (“dark cell degeneration”, Garthwaite and Garthwaite, 1991a,b; PetraschParwez et al., 2007). No darkly stained cells were observed in control mice (Fig. 6B). Taken together, while HDPromMJD148 mice show hyperactivity and reduced anxiety already at 4–6 months of age, motor symptoms can only be detected at the age of 12–14 months. At this age, no intranuclear aggregates could be detected, indicating the occurrence of motor symptoms in this mouse model before the formation of intranuclear aggregates (Fig. 7). Discussion Spinocerebellar ataxia type 3 is a neurodegenerative disease with late onset, slow progression and a heterogeneous clinical phenotype (Riess et al., 2008). As previously generated mouse models of SCA3 do not reproduce all aspects of the disease in humans (Bichelmeier et al., 2007; Cemal et al., 2002; Chou et al., 2008; Goti et al., 2004; Ikeda et al., 1996) we generated a novel mouse model for SCA3 to reflect more closely the late manifesting and slowly progressing phenotype. To control the expression of a full-length ataxin-3 protein with 148 CAG repeats, we used the well-characterized rat huntingtin promoter (Holzmann et al., 1998) with high similarity between the mouse and the rat sequence (Lin et al., 1995). As in human SCA3 patients, the expression of the transgene is evenly distributed throughout the whole brain without differences between affected and non-affected brain regions (Schmidt et al., 1998; Schmitt et al., 1997). Normal ataxin-3 is mainly localized in the cytoplasm (Paulson et al., 1997a; Schmidt et al., 1998), with nuclear localization in some cells (Tait et al., 1998; Trottier et al., 1998). Interestingly, in heterozygous HDPromMJD148 mice, the mutant transgene product is mainly localized in the cytoplasm, but in homozygous mice mainly in the nucleus. Nuclear localizations of the SCA3 transgene with expanded polyglutamines were also described for other mouse models of SCA3

Fig. 5. Motor and motor learning deficits in HDPromMJD148 mice. (A) Sensorimotor coordination in HDPromMJD148 mice was analyzed using the Rotarod test. Starting at the age of about 1 year (57 weeks), a motor phenotype in transgenic mice was apparent in these mice. HDPromMJD148 mice (Tg) performed significantly weaker, as shown in a reduced retention time on the rotating rod. (t-test: ⁎⁎p b 0.05; ⁎⁎⁎p b 0.001; two-way ANOVA: p b 0.005). Shown is the mean of 8 control and 20 transgenic mice, respectively. (B) To assess restrictions of the ability of HDPromMJD148 mice to learn novel motor skills, the first two trials of the first Rotarod analysis (see rectangle in figure A) were analyzed separately. While control mice significantly improved from the first to the second trial, no improvement was observed in HDPromMJD148 mice.

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Fig. 6. Histopathological findings in HDPromMJD148 mice. (A) Formation of intranuclear aggregates in HDPromMJD148 mice. Brain sections of both heterozygous and homozygous mice at the ages of 7 months, 18 months, and 25 months were stained with antibodies against ataxin-3 and analyzed for the formation of intranuclear aggregates (see arrow heads). In heterozygous mice, the ataxin-3 transgene is mainly localized in the cytoplasm. No aggregates were detected at the age of 7 and 18 months. Only at the age of 25 months, a few small aggregates were detected in restricted brain regions, like the brain stem (shown), the red nucleus, and pontine nuclei (not shown). About 10% of Purkinje cells contained intranuclear aggregates. In homozygous mice, the ataxin-3 transgene is mainly located in the nucleus. However, intranuclear aggregates were not detected at 7 months or 14 months (see Fig. 2) of age. Intranuclear inclusion bodies were apparent from 18 months of age. No aggregates were observed in control mice. Bar, 20 μm. (B) Electron microscopical analysis of HDPromMJD148 mice. At the age of 20 months, dark cell degeneration of Purkinje cells was observed in HDPromMJD148 mice. No dark cells were noticed in controls. Bar, 2 μm or 5 μm.

(Cemal et al., 2002; Goti et al., 2004). It could be that the expanded polyglutamine itself mainly contributes to the translocation of transgenic ataxin-3 to the nucleus (Fujigasaki et al., 2000; Goti et al., 2004). In accordance with this nuclear localization, intranuclear inclusion bodies were observed much earlier (at 18 months of age) in homozygous mice than in heterozygous mice (25 months). In neurons with aggregates, the nuclear staining was concentrated in the

inclusion body, while the remaining nucleus was mainly unstained, as we have previously observed in human SCA3 patients (Schmidt et al., 2002). While mice at the age of about 1 year still lack intranuclear inclusion bodies, they develop a neurological phenotype, indicating that the formation of aggregates is no prerequisite for the development of symptoms also in transgenic mouse models for SCA3. While

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Fig. 7. Time line of phenotype development in HDPromMJD148 mice. In HDPromMJD148 mice, hyperactivity was already observed with 4 months of age. However, no motor symptoms were present at this age. At 12 months of age, motor learning deficits become apparent. Neuronal nuclear inclusions (NII), however, were only detected at 18 months of age in homozygous and 25 months of age in heterozygous mice, respectively, and dark cell degeneration of Purkinje cells was observed at 20 months of age.

this is the first observation in mouse models of SCA3, it has previously been reported that neurological symptoms, cellular dysfunction, or cell loss are independent of the formation of inclusion bodies in transgenic mouse and tissue culture models for other polyglutamine diseases as well (Cummings et al., 1999; Kim et al., 1999; Klement et al., 1998; Saudou et al., 1998; Takahashi et al., 2005). Instability of CAG repeats For our HDPromMJD148 mouse model, we observed an instability of the CAG repeats which is linked to the mode of inheritance. While paternally transmitted CAG repeats tend to increase, maternal transmission led to a reduction of the CAG repeat number. In human cases of other polyglutamine diseases, a tendency towards greater instability of the expanded allele during paternal and a tendency towards only slight changes or decrease during maternal transmission has been reported in SCA1 (Chung et al., 1993), SCA7 (Gouw et al., 1998), DRPLA (Koide et al., 1994; Komure et al., 1995), and HD (Trottier et al., 1994). In SCA3, however, the situation seems to be not as clear as in other polyglutamine diseases (Cancel et al., 1995; Dürr et al., 1996; Maciel et al., 1995; Maruyama et al., 1995; Matilla et al., 1995; Takiyama et al., 1995). Inconsistent data between different publications are possibly explainable by the theory that the sex of the transmitting parent has less influence on the repeat instability in SCA3 than in other polyglutamine diseases (Dürr et al., 1996). It was shown that a certain haplotype of the C/G polymorphism adjacent to the CAG repeat favours the repeat instability (Igarashi et al., 1996). A comparable haplotype is also present in our HDPromMJD148 mice (C in the transgenic and G in the endogenous ataxin-3). No data concerning the respective polymorphism are available regarding the constructs used for the generation of other transgenic SCA3 mouse models (Cemal et al., 2002; Chou et al., 2008; Goti et al., 2004; Ikeda et al., 1996). However, one cannot exclude the possibility that this haplotype gave rise to the instability we observed in our mouse model. No data regarding the in-/stability in other mouse models for SCA3 are available yet (Bichelmeier et al., 2007; Cemal et al., 2002; Chou et al., 2008; Goti et al., 2004; Ikeda et al., 1996; Torashima et al., 2008). Therefore, this is the first report on inter-generational repeat instability in mouse models for SCA3 and our mouse model will therefore be an important tool to study the mechanisms of repeat instability in SCA3. Behavioral phenotype in HDPromMJD148 mice In the HDPromMJD148 mouse model, we observed a mild and late manifesting phenotype with slow progression. Since no developmental defects or a phenotype was present in young mice, we can largely exclude that other factors (e.g. integration site) except for the transgene itself induce the phenotype of HDPromMJD148 mice. Hyperactivity was observed at 4 to 6 months of age without any additional motor phenotype. At the age of about 14 months, however, motor deficits as well as impaired motor learning were detected using

Rotarod analyses. For good Rotarod performance, an intact cerebellar function is required (Picciotto and Wickman, 1998). Histopathologically, we indeed observed dark cell degeneration of Purkinje cells in older mice. Degenerated Purkinje cells were also observed in human SCA3 patients (Munoz et al., 2002; Rüb et al., 2002a,b) as well as in mouse models for SCA3 (Bichelmeier et al., 2007; Cemal et al., 2002; Chou et al., 2008). Intranuclear inclusion bodies, however, were not detected in homozygous mice until the age of 18 months, and in heterozygous mice, not before 25 months. As motor coordination is known to be impaired in aged rodents (Gage et al., 1984) no further Rotarod analyses were performed at this age. However, our data confirm that the formation of intranuclear inclusion bodies is not required for the manifestation of behavioral symptoms. The mild progression of the phenotype in the HDPromMJD148 mouse model might be due to the lower expression of the mutant transgene protein compared to the previous SCA3 mouse model generated by us using the Prp promoter (Bichelmeier et al., 2007). In contrast to the rapid progression of symptoms and premature death in other mouse models of SCA3 (Bichelmeier et al., 2007; Goti et al., 2004), the slow progression in the HDPromMJD148 mice therefore better reproduces late manifesting forms of the disease in humans (Riess et al., 2008) and allows us to study pathological symptoms before the onset of measurable motor symptoms. In addition, in mouse models of SCA3 with stronger transgene expression, inclusion bodies were observed in multiple brain regions (Bichelmeier et al., 2007; Goti et al., 2004) including regions that are typically spared from the formation of inclusion bodies in humans. However, in the HDPromMJD148 mouse model, although the transgene is ubiquitously expressed, inclusion bodies only occur in restricted regions of the brain stem (red nucleus, pontine nuclei) and in Purkinje cells, regions also affected in SCA3 patients (Munoz et al., 2002; Rüb et al., 2002a,b) implying a more “native” situation in HDPromMJD148 mice. In both home cage analyses and open field tests, we observed hyperactivity of HDPromMJD148 mice compared to controls. Hyperactivity was also observed in a mouse model for DRPLA (Schilling et al., 2001) and can be an indication of reduced emotionality (van der Staay et al., 1990) as observed in human SCA3 patients (Zawacki et al., 2002). In other mouse models of SCA3, rather a hypoactivity than a hyperactivity was reported (Bichelmeier et al., 2007; Cemal et al., 2002; Goti et al., 2004). However, in late disease stages, HDPromMJD148 mice also develop hypoactivity, indicating that the early onset of symptoms in the other mouse models mentioned above might have covered the hyperactivity in earlier disease stages. A comparable biphasic course of the disease was also reported for rat and mouse models of Huntington's disease (Lüesse et al., 2001; Menalled et al., 2003; Nguyen et al., 2006; Reddy et al., 1999; Slow et al., 2003), reflecting comparable observations in human HD patients (Kirkwood et al., 2001). It remains to be studied whether this biphasic disease course is also relevant for human SCA3 patients. Taken together, the new HDPromMJD148 mouse model of SCA3 reflects many features of the disease in humans, in particular, some aspects that were not reproduced by previous mouse models of SCA3. We observe a biphasic course of the disease with hyper- and hypoactivity, reduced anxiety, the formation of intranuclear inclusion bodies in restricted brain regions and a measurable motor phenotype, which seems to be at least partly due to Purkinje cell dysfunction or degeneration. Due to the late onset of symptoms in this model, the HDPromMJD148 mice allow the study of pathological processes occurring before the actual onset of symptoms. In addition, HDPromMJD148 mice allow the study of factors affecting the repeat instability in SCA3. We therefore recommend that the use of the Huntingtin promoter instead of the commonly used Prion protein promoter be considered for the generation of mouse models for diseases with late onset, since the Huntingtin promoter results in an evenly distributed but weaker expression of the transgene, and therefore in a transgenic mouse model that more closely resembles the phenotype in man.

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Acknowledgments The technical help of Gabi Frommer-Kästle in electron microscopy is greatly appreciated. This study was supported by the German Research Foundation (DFG) to OR, and by a grant from the European Union (6th Framework Programme, EUROSCA). References Bichelmeier, U., et al., 2007. Nuclear localization of ataxin-3 is required for the manifestation of symptoms in SCA3: in vivo evidence. J. Neurosci 27, 7418–7428. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Brown, S.D., et al., 2005. EMPReSS: standardized phenotype screens for functional annotation of the mouse genome. Nat. Genet. 37, 1155. Buitrago, M.M., et al., 2004. Short and long-term motor skill learning in an accelerated rotarod training paradigm. Neurobiol. Learn. Mem. 81, 211–216. Cancel, G., et al., 1995. Marked phenotypic heterogeneity associated with expansion of a CAG repeat sequence at the spinocerebellar ataxia 3/Machado–Joseph disease locus. Am. J. Hum. Genet. 57, 809–816. Cemal, C.K., et al., 2002. YAC transgenic mice carrying pathological alleles of the MJD1 locus exhibit a mild and slowly progressive cerebellar deficit. Hum. Mol. Genet. 11, 1075–1094. Chou, A.H., et al., 2008. Polyglutamine-expanded ataxin-3 causes cerebellar dysfunction of SCA3 transgenic mice by inducing transcriptional dysregulation. Neurobiol. Dis. 31, 89–101. Chung, M.Y., et al., 1993. Evidence for a mechanism predisposing to intergenerational CAG repeat instability in spinocerebellar ataxia type I. Nat. Genet. 5, 254–258. Cummings, C.J., et al., 1999. Mutation of the E6-AP ubiquitin ligase reduces nuclear inclusion frequency while accelerating polyglutamine-induced pathology in SCA1 mice. Neuron 24, 879–892. Dürr, A., et al., 1996. Spinocerebellar ataxia 3 and Machado–Joseph disease: clinical, molecular, and neuropathological features. Ann. Neurol. 39, 490–499. Fujigasaki, H., et al., 2000. Ataxin-3 is translocated into the nucleus for the formation of intranuclear inclusions in normal and Machado–Joseph disease brains. Exp. Neurol 165, 248–256. Gage, F.H., et al., 1984. Spatial learning and motor deficits in aged rats. Neurobiol. Aging 5, 43–48. Garthwaite, G., Garthwaite, J., 1991a. AMPA neurotoxicity in rat cerebellar and hippocampal slices: histological evidence for three mechanisms. Eur. J. Neurosci. 3, 715–728. Garthwaite, G., Garthwaite, J., 1991b. Mechanisms of AMPA neurotoxicity in rat brain slices. Eur. J. Neurosci 3, 729–736. Gatchel, J.R., Zoghbi, H.Y., 2005. Diseases of unstable repeat expansion: mechanisms and common principles. Nat. Rev. Genet. 6, 743–755. Goti, D., et al., 2004. A mutant ataxin-3 putative-cleavage fragment in brains of Machado–Joseph disease patients and transgenic mice is cytotoxic above a critical concentration. J. Neurosci. 24, 10266–10279. Goto, J., et al., 1997. Machado-Joseph disease gene products carrying different carboxyl termini. Neurosci. Res. 28, 373–377. Gouw, L.G., et al., 1998. Analysis of the dynamic mutation in the SCA7 gene shows marked parental effects on CAG repeat transmission. Hum. Mol. Genet. 7, 525–532. Green, E.C., et al., 2005. EMPReSS: European mouse phenotyping resource for standardized screens. Bioinformatics 21, 2930–2931. Holzmann, C., et al., 1998. Isolation and characterization of the rat huntingtin promoter. Biochem. J. 336 (Pt. 1), 227–234. Holzmann, C., et al., 2001. Functional characterization of the human Huntington's disease gene promoter. Brain Res. Mol. Brain Res. 92, 85–97. Igarashi, S., et al., 1996. Intergenerational instability of the CAG repeat of the gene for Machado-Joseph disease (MJD1) is affected by the genotype of the normal chromosome: implications for the molecular mechanisms of the instability of the CAG repeat. Hum. Mol. Genet. 5, 923–932. Ikeda, H., et al., 1996. Expanded polyglutamine in the Machado–Joseph disease protein induces cell death in vitro and in vivo. Nat. Genet. 13, 196–202. Kawaguchi, Y., et al., 1994. CAG expansions in a novel gene for Machado–Joseph disease at chromosome 14q32.1. Nat. Genet. 8, 221–228. Kim, M., et al., 1999. Mutant huntingtin expression in clonal striatal cells: dissociation of inclusion formation and neuronal survival by caspase inhibition. J. Neurosci. 19, 964–973. Kirkwood, S.C., et al., 2001. Progression of symptoms in the early and middle stages of Huntington disease. Arch. Neurol. 58, 273–278. Klement, I.A., et al., 1998. Ataxin-1 nuclear localization and aggregation: role in polyglutamine-induced disease in SCA1 transgenic mice. Cell 95, 41–53. Koide, R., et al., 1994. Unstable expansion of CAG repeat in hereditary dentatorubralpallidoluysian atrophy (DRPLA). Nat. Genet. 6, 9–13. Komure, O., et al., 1995. DNA analysis in hereditary dentatorubral-pallidoluysian atrophy: correlation between CAG repeat length and phenotypic variation and the molecular basis of anticipation. Neurology 45, 143–149. Laccone, F., 2002. A fast polymerase chain reaction-mediated strategy for introducing repeat expansions into CAG-repeat containing genes. Methods Mol. Biol. 192, 217–223. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.

293

Lin, B., et al., 1995. Structural analysis of the 5′ region of mouse and human Huntington disease genes reveals conservation of putative promoter region and di- and trinucleotide polymorphisms. Genomics 25, 707–715. Lüesse, H.G., et al., 2001. Evaluation of R6/2 HD transgenic mice for therapeutic studies in Huntington's disease: behavioral testing and impact of diabetes mellitus. Behav. Brain Res. 126, 185–195. Lundkvist, A., et al., 2004. Under stress, the absence of intermediate filaments from Muller cells in the retina has structural and functional consequences. J. Cell Sci. 117, 3481–3488. Maciel, P., et al., 1995. Correlation between CAG repeat length and clinical features in Machado–Joseph disease. Am. J. Hum. Genet. 57, 54–61. Maruyama, H., et al., 1995. Molecular features of the CAG repeats and clinical manifestation of Machado–Joseph disease. Hum. Mol. Genet. 4, 807–812. Matilla, T., et al., 1995. Molecular and clinical correlations in spinocerebellar ataxia type 3 and Machado-Joseph disease. Ann. Neurol. 38, 68–72. Menalled, L.B., et al., 2003. Time course of early motor and neuropathological anomalies in a knock-in mouse model of Huntington's disease with 140 CAG repeats. J. Comp. Neurol. 465, 11–26. Michalik, A., Van Broeckhoven, C., 2003. Pathogenesis of polyglutamine disorders: aggregation revisited. Hum. Mol. Genet. 12 (Spec No 2), R173–R186. Munoz, E., et al., 2002. Intranuclear inclusions, neuronal loss and CAG mosaicism in two patients with Machado–Joseph disease. J. Neurol. Sci. 200, 19–25. Nguyen, H.P., et al., 2006. Behavioral abnormalities precede neuropathological markers in rats transgenic for Huntington's disease. Hum. Mol. Genet. 15, 3177–3194. Padiath, Q.S., et al., 2005. Identification of a novel 45 repeat unstable allele associated with a disease phenotype at the MJD1/SCA3 locus. Am. J. Med. Genet. B Neuropsychiatr. Genet. 133B, 124–126. Paulson, H.L., et al., 1997a. Machado–Joseph disease gene product is a cytoplasmic protein widely expressed in brain. Ann. Neurol. 41, 453–462. Paulson, H.L., et al., 1997b. Intranuclear inclusions of expanded polyglutamine protein in spinocerebellar ataxia type 3. Neuron 19, 333–344. Petrasch-Parwez, E., et al., 2007. Cellular and subcellular localization of Huntingtin [corrected] aggregates in the brain of a rat transgenic for Huntington disease. J. Comp. Neurol. 501, 716–730. Picciotto, M.R., Wickman, K., 1998. Using knockout and transgenic mice to study neurophysiology and behavior. Physiol. Rev. 78, 1131–1163. Reddy, P.H., et al., 1999. Transgenic mice expressing mutated full-length HD cDNA: a paradigm for locomotor changes and selective neuronal loss in Huntington's disease. Philos. Trans. R Soc. Lond. B Biol. Sci. 354, 1035–1045. Riess, O., et al., 2008. SCA3: Neurological features, pathogenesis and animal models. Cerebellum 125–137. Rüb, U., et al., 2002a. Degeneration of the external cuneate nucleus in spinocerebellar ataxia type 3 (Machado–Joseph disease). Brain Res. 953, 126–134. Rüb, U., et al., 2002b. Spinocerebellar ataxia type 3 (Machado–Joseph disease): severe destruction of the lateral reticular nucleus. Brain 125, 2115–2124. Saudou, F., et al., 1998. Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions. Cell 95, 55–66. Scherzinger, E., et al., 1999. Self-assembly of polyglutamine-containing huntingtin fragments into amyloid-like fibrils: implications for Huntington's disease pathology. Proc. Natl. Acad. Sci. U. S. A. 96, 4604–4609. Schilling, G., et al., 2001. Distinct behavioral and neuropathological abnormalities in transgenic mouse models of HD and DRPLA. Neurobiol. Dis. 8, 405–418. Schmidt, T., et al., 1998. An isoform of ataxin-3 accumulates in the nucleus of neuronal cells in affected brain regions of SCA3 patients. Brain Pathol. 8, 669–679. Schmidt, T., et al., 2002. Protein surveillance machinery in brains with spinocerebellar ataxia type 3: redistribution and differential recruitment of 26S proteasome subunits and chaperones to neuronal intranuclear inclusions. Ann. Neurol. 51, 302–310. Schmitt, I., et al., 1997. Characterization of the rat spinocerebellar ataxia type 3 gene. Neurogenetics 1, 103–112. Schöls, L., et al., 1997. Autosomal dominant cerebellar ataxia: phenotypic differences in genetically defined subtypes? Ann. Neurol. 42, 924–932. Sisodia, S.S., 1998. Nuclear inclusions in glutamine repeat disorders: are they pernicious, coincidental, or beneficial? Cell 95, 1–4. Slow, E.J., et al., 2003. Selective striatal neuronal loss in a YAC128 mouse model of Huntington disease. Hum. Mol. Genet. 12, 1555–1567. Tait, D., et al., 1998. Ataxin-3 is transported into the nucleus and associates with the nuclear matrix. Hum. Mol. Genet. 7, 991–997. Takahashi, T., et al., 2005. Polyglutamine represses cAMP-responsive-elementmediated transcription without aggregate formation. Neuroreport 16, 295–299. Takiyama, Y., et al., 1995. Evidence for inter-generational instability in the CAG repeat in the MJD1 gene and for conserved haplotypes at flanking markers amongst Japanese and Caucasian subjects with Machado–Joseph disease. Hum. Mol. Genet. 4, 1137–1146. Torashima, T., et al., 2008. Lentivector-mediated rescue from cerebellar ataxia in a mouse model of spinocerebellar ataxia. EMBO Rep. 9, 393–399. Trottier, Y., et al., 1994. Instability of CAG repeats in Huntington's disease: relation to parental transmission and age of onset. J. Med. Genet. 31, 377–382. Trottier, Y., et al., 1995. Polyglutamine expansion as a pathological epitope in Huntington's disease and four dominant cerebellar ataxias. Nature 378, 403–406. Trottier, Y., et al., 1998. Heterogeneous intracellular localization and expression of ataxin-3. Neurobiol. Dis. 5, 335–347. van der Staay, F.J., et al., 1990. Genetic correlations in validating emotionality. Behav. Genet. 20, 51–62. von Hörsten, S., et al., 2003. Transgenic rat model of Huntington's disease. Hum. Mol. Genet. 12, 617–624. Zawacki, T.M., et al., 2002. Executive and emotional dysfunction in Machado–Joseph disease. Mov. Disord. 17, 1004–1010.