Human Molecular Genetics, 2001, Vol. 10, No. 16 1679-1692
© 2001 Oxford University Press
SCA7 mouse models show selective stabilization of mutant ataxin-7 and similar cellular responses in different neuronal cell types
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, B.P.163, 67404 Illkirch cedex, CU de Strasbourg, France and 1Department of Neurology, University of Ulm, 89075 Ulm, Germany
Received April 13, 2001; Revised and Accepted June 5, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Accumulation of expanded polyglutamine proteins and selective pattern of neuronal loss are hallmarks of at least eight neurodegenerative disorders, including spinocerebellar ataxia type 7 (SCA7). We previously described SCA7 mice displaying neurodegeneration with progressive ataxin-7 accumulation in two cell types affected in the human pathology. We describe here a new transgenic model with a more widespread expression of mutant ataxin-7, including neuronal cell types unaffected in SCA7. In these mice a similar handling of mutant ataxin-7, including a cytoplasm to nucleus translocation and accumulation of N-terminal fragments, was observed in all neuronal populations studied. An extensive screen for chaperones, proteasomal subunits and transcription factors sequestered in nuclear inclusions (NIs) disclosed no pattern unique to neurons undergoing degeneration in SCA7. In particular, we found that the mouse TAFII30 subunit of the TFIID initiation complex is markedly accumulated in NIs, even though this protein does not contain a polyglutamine stretch. A striking discrepancy between mRNA and ataxin-7 levels in transgenic mice expressing the wild-type protein but not in those expressing the mutant one, indicates a selective stabilization of mutant ataxin-7, both in this model and the P7E/N model described previously. These mice therefore provide in vivo evidence that the polyglutamine expansion mutation can stabilize its target protein.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Spinocerebellar ataxia type 7 (SCA7) is a neurodegenerative disorder characterized by progressive degeneration of the cerebellum, brain stem and spinal cord, belonging to the clinically and genetically heterogeneous group of autosomal-dominant cerebellar ataxias (ADCA I–III). In ADCA type II (SCA7), cerebellar ataxia is associated with blindness secondary to pigmentary macular dystrophy and retinal degeneration. The pathology involves neuronal loss and gliosis mostly affecting the Purkinje cell layer, the nucleus dentatus and the inferior olive (1).
The SCA7 mutation is a CAG trinucleotide expansion within the coding region of the SCA7 gene, which is translated into an expanded polyglutamine stretch. SCA7 shows particularly high sensitivity to the polyglutamine length (2). It belongs to the family of polyglutamine expansion diseases including Huntington’s disease (HD), dentatorubral-pallidoluysian atrophy (DRPLA), spinal and bulbar muscular atrophy (SBMA) and several spinocerebellar ataxias (SCA1, 2, 3 and 6) (3). These diseases are characterized by adult-onset neuronal dysfunction and death in selected but distinct regions of the CNS, despite ubiquitous expression of the target proteins. The pathological threshold ranges from 35 to 40 glutamines in most of these diseases. It corresponds to a conformational shift of the polyglutamine stretch, as suggested by the selective recognition of mutant proteins by a monoclonal antibody (4). This conformational shift correlates with an increased propensity for aggregation in vitro (5), accounting for the formation of nuclear inclusions (NIs) as a common pathological feature of many of these diseases (6). However, the actual toxicity of nuclear aggregates has been disputed, while several experimental models indicate that presence of the mutant protein in the nucleus is a key feature in the pathogenic mechanism (7).
NIs contain ubiquitin, different proteasome subunits as well as some chaperones suggesting that the mutant proteins are misfolded and targeted to degradation. Expanded polyglutamine tracts appear to be more resistant to degradation by the proteasome (8) and aggregate formation could reflect an imbalance of the turnover of the mutant proteins. For several polyglutamine diseases, NIs are stained by antibodies against epitopes close to the polyglutamine tract but not by antibodies against more distant epitopes, suggesting a cleavage of full-length mutated proteins. Such a cleavage has been reported in HD, SBMA, DRPLA, SCA3 and SCA7 and appears crucial for nuclear translocation and aggregate formation (9,10).
The selective pattern of neurodegeneration could be due to many different factors, such as relative levels of the mutant protein, existence of cell-specific interacting proteins, differences in catabolism of the mutant protein or other cell-specific features. So far none of these hypothesis has been confirmed. For instance, the distribution of NIs does not parallel the neuropathology. In HD and SCA7 patients they are observed in both vulnerable and spared neuronal populations (11,12). Clues toward the understanding of this specificity were provided by studies on HD knock-in mice. In these models, although neurological phenotype was very mild or even absent, some molecular abnormalities were observed, restricted to the medium spiny neurons of the striatum (13–15). This specificity may be correlated to a higher somatic instability of the CAG repeat in the striatum (16).
Finally, transcriptional dysregulation is suspected to be involved in the pathogenesis of polyglutamine diseases. Downregulation of specific sets of genes has been observed in cellular and mouse models of HD and SCA1 (17–19), and several studies reported the presence of transcription factors (such as CBP or TAFII130) in nuclear inclusions (20–22), suggesting a transcriptional dysregulation due to their sequestration. Other studies reported a mislocalization of transcription co-factors in autopsied brains from HD patients (23).
We previously described SCA7 mouse models allowing comparison of pathological features in two affected cell types: rod photoreceptors (R7E/N mice) and Purkinje cells (P7E/N mice) (9). We report here novel strains of transgenic mice overexpressing full-length normal and mutant ataxin-7 in many neuronal populations within the brain, including regions spared in human SCA7. We observed in this model and in the P7E/N model a stabilization of ataxin-7 by the expansion of the polyglutamine tract. Control and mutant lines show similar expression levels of transgene mRNA but, in the conditions used, mutant ataxin-7 was easily detectable while normal ataxin-7 was not. Processing of mutant ataxin-7 appears very similar in various neuronal types as all showed relocalization and accumulation in the nucleus of an N-terminal fragment of the protein. We have performed an extensive molecular characterization of ataxin-7 NIs. Their distribution and content in chaperones, proteasome subunits and transcription factors in different neuronal populations did not show any obvious differences that would explain the regional degeneration specificity observed in patients.
This model reproduces key molecular features of polyglutamine expansion diseases and offers the opportunity to unravel early pathological events.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Transgenic mice widely expressing full-length mutant ataxin-7 in the CNS develop a progressive ataxic phenotype
We have generated transgenic mouse lines carrying the full-length human SCA7 cDNA, placed under the transcriptional control of the platelet-derived growth factor chain B promoter (PDGF-B) (24). This promoter was reported to lead to high neuronal expression in all brain regions and was successfully used to generate Alzheimer’s and Parkinson’s disease transgenic mouse models (25–27). The trinucleotide repeat length within the cDNA is (CAG)10 and (CAG)128 in the ‘B7N’ and ‘B7E2’ lines, respectively (Fig. 1A). Injection was done in [C57BL/6xSJL] fertilized embryos, leading to several founders for both constructs (Table 1). Lines were then amplified on the C57BL/6 background. The F1 progenies were genotyped by Southern blot for determination of the number of integration sites and the approximate copy number per site. Crosses were then designed to obtain lines with single integration sites. The resulting lines are listed in Table 1.
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130 kDa (arrowhead). Mutant ataxin-7 is detected at the expected size (>150 kDa, arrow) when compared to a lymphoblastoid cell line extract (LCL) from an SCA7 patient.
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Expression of ataxin-7[Q128] in each B7E2 line was assessed by western blot using the 1C2 antibody recognizing expanded polyglutamines (4) (Fig. 1B). Highest expression levels were observed in whole brain extracts of lines B and E. We then examined expression throughout brain regions by immunohistochemistry using the 1261, 1262 and 1C1 anti-ataxin-7 antibodies (28) (data not shown). A more intense immunoreactivity (IR) was obtained from lines B, E and G, confirming the western blot analysis. Ataxin-7 IR was detected in many regions, e.g cortex, hippocampus, cerebellum, brainstem and inner retina. Within these regions, expression varied among different cell types. For instance, the outer cortical layer displayed lower ataxin-7 IR compared to the inner cortical layers. Not all neurons within a given region displayed ataxin-7 IR. In the retina, for example, no ataxin-7 IR was observed in photoreceptors. In cerebellum, ataxin-7 IR was undetectable in granule cells, but easily visible in Purkinje cells, in Golgi cells and in neurons of the molecular layer (basket and stellate cells). The three lines B, G and E differed somewhat in the distribution pattern of ataxin-7 IR. Line B, for example, showed an intense immunoreactivity in the cerebellum while animals from lines E and G did not.
At the behavioural level, B7E2 mice displayed no obvious abnormalities at birth, nursed well and developed normally, reaching motor milestones at the expected timepoints. In young adult mice, there were no behavioural or motor abnormalities. At 5 months of age, however, F1 animals from line B started to display some signs suggesting difficulties in motor coordination, appeared to be less active and bradykinetic. Behavioural patterns suggestive of seizures were observed in two F1 animals but not in the later generations. The most striking abnormality, however, was a progressive motor incoordination resulting in frequent slips while beam walking or climbing. The motor incoordination was more marked in the hind limbs compared to forepaws. There was no obvious muscular atrophy or weakness. In addition, there was no evidence for an impairment of vision interfering with daily activities. In general, mice died within 3 months of onset of symptoms. Overall B7E2 mice from line B developed a progressive ataxic phenotype, that suggests a preferential cerebellar involvment contrasting with the widespread expression of the mutant protein. Age at onset was variable among F1 and F2 animals, ranging from 3 to 8 months. We observed a marked delay of this onset in animals from further generations (backcrossed to C57BL/6 mice), with even some F3 animals living >15 months with no obvious neurological signs. This observation is neither due to a meiotic contraction of the pathogenic expansion nor to an overall decrease in transgene expression (data not shown). It might thus be due to a protective effect of the enrichment in C57BL/6 background. We are currently backcrossing the B7E2.B line on SJL background to test this hypothesis. The B7E2.E and G mice did not develop any obvious neurological phenotype. This discrepancy is probably related to differences in cerebellar neuronal populations expressing mutant ataxin-7.
To assess expression of ataxin-7[Q10] in the B7N lines, we performed immunohistochemistry using the same conditions as in the B7E2 lines. In all these control lines, no specific immunoreactivity was observed when compared to wild-type animals (data not shown). By RT–PCR on brain extracts, transgene-specific mRNAs could be detected in three of the seven B7N lines (Table 1). We focused further characterization on line B7N.G which showed the highest mRNA expression level (Table 1).
Mutant and control transgenes have similar ataxin-7 mRNA, but different ataxin-7 protein expression levels
For a direct comparison of protein and RNA levels, we performed western blotting and RT–PCR on the same animals (Fig. 2A and B). A combination of two monoclonal anti-ataxin-7 antibodies recognizing equally well the mutant and normal proteins (28; and data not shown) detected the recombinant protein at the expected size (150 kDa) in B7E2 brain extracts, but not in B7N or wild-type littermates (Fig. 2A). It is to be noted that the endogenous mouse ataxin-7 was not detected in these experiments by the antibodies (monoclonal or polyclonal) raised against human ataxin-7. This may be due to a low affinity to the mouse homolog and/or its low level in tissues. We also did not detect ataxin-7[Q10] from B7N extracts enriched by immunoprecipitation using the 1261 and 1262 antibodies (data not shown). Similarly, in the cerebellum of P7N transgenic animals, normal ataxin-7 was not detected by immunohistochemistry in any of the four lines established. In contrast, the mutant protein was clearly detected in cerebellar sections from P7E lines (9) (Fig. 2F).
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To investigate whether the transgene mRNA was produced in B7N animals, we performed semi-quantitative RT–PCR on brain extracts. Conditions were optimized by using a reverse oligonucleotide striding over the intron of the construct (Fig. 1A), thus avoiding amplification of contaminating genomic DNA. SCA7 transgene mRNA was found in both B7E2 and B7N animals, at a higher level in the B7N brain extract (Fig. 2B). Similarly, RT–PCR on cerebellar RNA extracts from P7N animals showed transgene mRNA in three of the four lines (Fig. 2B).
To compare the RNA levels of mutant and control transgenes in the two models (B-lines and P-lines), we performed comparative semi-quantitative RT–PCR on several animals from lines B7E2.B and B7N.G (Fig. 2C) and from lines P7E.B and P7N.C (Fig. 2D). No significant difference was observed between the B-lines. In contrast, expression was 10 times higher in P7N animals than in P7E animals (significant level P < 0.002; see Materials and Methods).
We also compared RNA expression levels between P7E.B and P7N.C animals by in situ hybridization (Fig. 2E). The SCA7 transgene mRNA was detected with a higher intensity in sections of P7N.C animals. In contrast, immunohistochemistry on P7E.B and P7N.C animals at the same age showed a higher ataxin-7 detection in P7E sections (Fig. 2F).
Although the B7E2 construct, which leads to protein expression in mice, is derived from the B7N plasmid (Materials and Methods), a possible explanation for the presence of RNA but no detectable protein product could be a mutational event occurring during the elaboration of the construct. To verify the integrity of the B7N construct, neural-derived NG108 cells were transfected with the final DNA preparation (the one used to prepare the fragment injected into embryos). These cells displayed a strong immunofluorescent nuclear signal when using anti-ataxin-7 antibodies (Fig. 2G). We conclude that the B7N construct is able to drive protein expression in transfected cells.
An increased detection of mutant ataxin-7 may correspond to a prolonged half-life of the protein. We compared half-lives of normal and mutant ataxin-7 in transfected COS cells by pulse-chase analysis (Fig. 3). We observed a similar rate of production of the two proteins, and a similar decrease of labelled ataxin-7 with time. Thus, mutant and normal ataxin-7 have similar stabilities in this transfection system.
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Mutant ataxin-7 progressively translocates from cytoplasm to nucleus, and accumulates despite constant expression level
In a time-course experiment, we studied the localization of ataxin-7[Q128] by immunohistochemistry on brain sections from B7E2 animals (Fig. 4A). We found that the subcellular localization of the mutant protein varied with age, and that intensity of the ataxin-7 IR increased over time. At 1 month of age, a faint IR was observed in the cytoplasm of most neurons; the nucleus was stained in only a few scattered neurons. These signals were absent on sections from wild-type littermates (data not shown). At 2 months of age, nuclear staining was encountered more frequently and tended to be more intense, whereas the cytoplasm displayed IR of similar intensity as in younger animals. At 3 months of age, there was almost no cytoplasmic staining detectable, but a strong IR in many nuclei with numerous dense focal accumulations; in neurons containing NIs overall nuclear staining appeared reduced. At 4 months of age, NIs were present in most nuclei; their presence was associated with a global decrease of the overall nuclear signal.
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We previously described the progressive nuclear accumulation of mutant ataxin-7 in Purkinje cells of P7E mice (9). In the latter study, animals were investigated from 1 to 16 months of age. In the present study, we extended these observations by looking at the subcellular distribution of ataxin-7[Q90] in P7E animals at earlier time-points (14 and 17 days) by immunohistochemistry using the 1261 antibody (not shown). We observed a cytoplasmatic distribution of the protein at 14 days, with the appearance of nuclear immunoreactivity in some Purkinje cells at 17 days of age. This indicates that the translocation of mutant ataxin-7 from the cytoplasm to the nucleus also takes place in P7E mice, although at an earlier timepoint than in B7E2 mice.
To determine whether the increasing levels of ataxin-7 protein with age were associated with an increased expression of the transgene, we measured the abundance of transgenic mRNA in B7E2 animals at three different ages by semi-quantitative RT–PCR on whole brain extracts (Figs 2C and 4B). Variabilities up to 2.4-fold were detected between animals but did not correspond to an increased expression over time.
Processing of mutant ataxin-7 and cellular responses elicited by its accumulation are not unique to neurons undergoing degeneration in SCA7 patients
This time-dependent processes of translocation to the nucleus and accumulation were observed in all neuronal populations expressing the transgene and were not restricted to regions affected in the human disease, as could be seen in the cerebellum (affected in SCA7) and in the cerebral cortex (spared in SCA7) (Fig. 4A). We showed previously that ataxin-7 NIs of R7E and P7E mice were immunoreactive for antibodies recognizing the N-terminal part of ataxin-7 but not for those (1597 and 1598) recognizing the C-terminal end (9). Similarly, no NIs from B7E2.B animals could be stained with 1597 and 1598 antibodies, suggesting that this cleavage is not specific of affected neurons (data not shown).
Using a panel of markers for components of the ubiquitin/proteasome machinery, we characterized NIs of B7E2.B animals in all brain regions to assess their immunoreactive signature. The same set of antibodies directed against proteasome subunits and chaperones were used as described previously (9). Most NIs were immunoreactive for ubiquitin, a marker for protein degradation via the proteasomal pathway. Inclusions differed in their IR to antibodies directed against the three major components of the proteasome: a higher proportion of NIs were immunoreactive to antibodies recognizing subunits of the 19S component of the 26S proteasome which is thought to unfold proteins prior to degradation. In comparison, a lower number of NIs displayed IR to subunits of the 20S proteolytic core of the proteasome. Only a few inclusions were labelled with an antibody against the 11S subunit PA28ß. Essentially all inclusions defined by N-terminal ataxin-7 are labelled by an antibody directed against the hsp40 chaperone HDJ-2. In addition, we observed hsc70 chaperone IR in almost all ataxin-7 NIs of B7E2 mice (Fig. 5). These results suggest that most NIs recruit a molecular machinery to assist in unfolding protein (chaperones and 19S) and demonstrate that not all NIs are associated with proteasomal elements critical for proteolytic degradation (20S). We did not observe striking qualitative differences in the cellular responses to NIs in neuronal populations typically affected in human SCA7 like Purkinje cells and neurons typically spared like neurons of the hippocampal formation or the cerebral cortex.
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Several transcription factors are detected in ataxin-7 NIs, but do not appear depleted from the nucleoplasm
We performed co-immunofluorescent labelling of ataxin-7 and several transcription factors to assess whether they are sequestrated or mislocalized in our model. A panel of 24 antibodies generated against basic transcription factors, initiation factors of the TFIID complex or activators were selected for their ability to elicit specific signals by western blot on mouse whole-brain extracts, and were then used for fluorescent-immunohistochemistry on brain sections from old B7E2.B or wild-type animals. Eighteen antibodies resulted in a nuclear staining and were therefore considered to be able to detect the mouse homologous factors under our experimental conditions. We examined whether transcription factors co-localized with ataxin-7 NIs and if any difference could be observed between the immunoreactive patterns of transgenic versus wild-type sections (Table 2). As a positive control, one section was stained for ataxin-7 and the 20S
proteasomal subunits and displayed a double labelling of all NIs (data not shown).
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RNA polymerase II and 9 other factors tested were not enriched in the region of ataxin-7 NIs; their subcellular localization was similar in wild-type and transgenic animals (Fig. 6; Table 2). Notably, we did not detect NIs with two anti-TAFII130 antibodies, whereas other authors reported the sequestration of this factor in polyglutamine aggregates of human SCA3 and DRPLA brains (20).
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A small proportion of NIs were immunoreactive for SNF5, a component of the SWI/SNF chromatin remodelling complex. SNF5-immunoreactivity was detected as a punctate staining throughout the nuclei of cells, with one to three bigger foci of accumulated signal per cell (data not shown). Some of these foci colocalized with ataxin-7 inclusions. Globally, <10% of ataxin-7 NIs were stained with the anti-SNF5 antibody, and <20% of SNF5 foci were stained with the anti-ataxin-7 antibody. Similar observations were made when using an antibody against the p89(XPB) subunit of TFIIH.
We studied the subcellular localization of CREB binding protein (CBP) using two antibodies recognizing different epitopes of the protein. The CBP-IR was restricted to multiple spots of varying size and intensities within the nucleus (Fig. 6). Each ataxin-7 inclusion co-localized with one large CBP-IR spot. The spot-like distribution pattern of CBP-IR did not allow us to distinguish NIs from multiple dots of similar appearance. NIs, however, could be unequivocally identified in double labelling experiments using N-terminal anti-ataxin-7 antibodies. There was no difference in the distribution pattern or the apparent intensity of CBP-IR allowing us to distinguish between sections from wild-type and B7E2.B transgenics.
Most inclusions were immunoreactive for TFIIE (Fig. 6) and TFIIFß (not shown). Incubation with antibodies to these factors resulted in a punctated staining pattern throughout the nucleus both in wild-type and transgenic animals. Large dots corresponded to NIs in the latter. A striking co-localization was seen between ataxin-7 inclusions and the TAFII30 subunit of the TFIID initiation complex (Fig. 6). NIs stood out as big immunoreactive spots, from a background of punctated nuclear staining. Overall, there was no apparent decrease or redistribution of anti-TAFII30 IR in B7E2.B compared to wild-type.
With no exception, even intense staining of nuclear inclusions was not associated with obvious change in distribution or intensity of the immunohistochemical signals. In addition, the staining of NIs with transcription factor antibodies did not differ between neuronal populations known to undergo degeneration and those spared in the human pathology.
It has been suggested that transcription factors like CBP that contain a polyglutamine stretch may be selectively trapped in NIs by interacting with the expansion of the mutant proteins (29). Our observations do not support this hypothesis: there was no correlation between the presence of polyglutamine motifs in the sequences of the respective transcription factors (Table 2) and their likelihood to decorate NIs.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We generated SCA7 transgenic mice in which expression of ataxin-7 is targeted into many neuronal populations. In B7E2 and P7E (9) mice overexpressing ataxin-7[Q>90], N-terminal fragments of the mutant ataxin-7 progressively translocated from the cytoplasm to the cell nucleus and accumulated with time. In B7E2 mice, a similar translocation and accumulation was observed in all expressing neurons with the same timing, showing that this process is not specific to cells known to degenerate in SCA7. We screened for the presence of molecular chaperones, proteasomal subunits and transcription factors into ataxin-7 NIs and found similar responses to the accumulation process among the various neuronal types. Surprisingly, in several B7N and P7N (9) control lines, we did not detect the expected ataxin-7[Q10] protein product despite similar or even higher mRNA expression levels when compared to mutant lines.
Stabilizing effect of the polyglutamine expansion mutation
The simplest interpretation of the detection of mRNA but no protein product in a transgenic approach is the possible occurrence of a mutational event affecting the integrity of the construct. Several arguments led us to conclude that this does not account for the present findings. First, all normal transgenes (R7N, P7N and B7N) were derived from the same pIA construct. As R7N leads to high protein expression (9), mutational events would suggest the occurrence of two independent mutations in the final construction steps of both P7N and B7N, which is highly improbable. Secondly, the B7E2 plasmid was derived from the B7N construct (Materials and Methods), and displayed the expected protein expression. Thirdly, recombinant protein expression was observed when cells were transfected with the final B7N plasmid preparation. Fourthly, the P7N and B7N constructs integrated entirely into the mouse genome since their expected size was observed on Southern blot (Materials and Methods). Finally, the entire open reading frame is transcribed in B7N and P7N mice since RT–PCR amplified a fragment from the 3'-UTR of the construct. Because of the number of lines studied, the converging results of the two systems, and the use of several detection techniques, we conclude that recombinant ataxin-7[Q10] is not detected because of a rapid turnover within the cells, and that the increased detection of the mutant protein is due to a stabilization effect of the expansion mutation. Consistent with a rapid turnover of the wild-type protein, SCA7 mRNA is easily detected by northern blot, while low levels of endogenous ataxin-7 protein were observed in human brain (2,28).
One earlier study showed a stabilizing effect of the polyglutamine mutation on ataxin-1 (8). The authors observed in vitro a similar ubiquitin conjugation but a reduced degradation by the proteasome of mutant ataxin-1 when compared to the normal protein. Another study compared, by pulse–chase experiment, the half-lives of mutant and wild-type huntingtin in lymphoblastoid cells derived from HD patients or control individuals (30). No difference was reported between the stabilities of the two proteins. Our observation suggests that mutant ataxin-7 is stabilized as a full-length protein, as it was detected at 1 month of age by western blot (Fig. 2A). However, we did not observe different stabilities between normal and mutant ataxin-7 in transfected COS cells. There are several possible interpretations of this difference with in vivo observations: the stability may depend on neuron-specific factors, the time scale in transient transfection may be too short to observe a difference, or expression in COS cells is too high to reproduce the same degradation steps as in targeted neurons of mice. Indeed, very high expression levels may saturate the mechanisms controlling ataxin-7 turnover. This possibility would also explain why we did not observe different stabilities in our R7E/N models (9). In these mice, expression is controlled by the rhodopsin promoter known to generate very high expression levels (31). So far, there is no in vivo evidence for a stabilization effect of the mutation in other polyglutamine diseases. However, higher levels of the corresponding proteins could hide subtle differences in protein stabilities.
The toxicity of polyglutamine-expanded proteins is known to be concentration dependent since several transgenic models with low expression levels developed no overt or mild neurological phenotypes (32–35). Furthermore, several loss-of-function mutations affecting the ubiquitin/proteasome pathway enhance ataxin-1-mediated neurodegeneration in mice and flies (8,36), possibly by increasing the intracellular concentration of the mutant protein. It is currently not known whether wild-type ataxin-7 is a substrate for the proteasome in vivo, but the sequestration of several proteasomal subunits in ataxin-7 inclusions suggests a dynamic response of the proteasome to mutant ataxin-7 accumulation.
The stabilization of ataxin-7 may contribute to the pathogenesis of SCA7 in several ways. High levels of ataxin-7 may dysregulate the functional pathways, presently unknown, in which this protein is implicated, and may be deleterious to some cells. This would be in agreement with the observation of Fernandez-Funez et al. (36) that ataxin-1[Q30] is toxic to flies and mice when highly overexpressed. In addition, a rise in ataxin-7 levels might trigger a cellular response resulting in proteolytic processing of the protein as an alternative way of degradation, and such a process could then generate a fragment of higher toxicity. In disease, expression of the mutant allele may be ubiquitous but the degradation efficiency, or the threshold of concentration after which a cellular response is triggered may differ between neuronal types. This might explain, at least in part, the selective degeneration observed in each polyglutamine disorder.
The selective degeneration pattern in SCA7 is not explained by a differentiated handling of mutant ataxin-7 in spared and affected cell types
We observed, in all targeted cell types, from 1 to 5 months of age, a progressive increase of anti-ataxin-7 immunoreactivity on B7E2 mouse sections, accompanied by a re-localization of mutant ataxin-7 in the cell nucleus. The increased nuclear immunoreactivity was not seen with antibodies recognizing the C-terminal end of ataxin-7 (data not shown), suggesting accumulation of only N-terminal fragments of the protein. Taken together, our data indicate that the expansion mutation provokes an increased concentration of full-length mutant ataxin-7 in vivo, that this increase is followed by a proteolytic processing of the protein and by a re-localization and progressive accumulation of N-terminal fragments in the cell nucleus. Although the ataxic phenotype of B7E2.B mice suggest a preferential involvement of the cerebellum, a similar pattern of processing was observed in all brain regions and thus does not parallel the selective pattern of degeneration observed in SCA7 patients.
Knock-in models of HD have successfully produced specific molecular events in medium spiny neurons of the striatum, the most vulnerable neuronal type in HD. In these mice, fragments of mutant huntingtin translocate into the nucleus and accumulate as a diffuse staining preceding formation of NIs (13–15). These events probably represent early steps in HD, as these mice develop no or very mild neurological symptoms. We showed that similar events occurred in our B7E2 model, suggesting that the process also represents early steps of SCA7 pathogenesis. At variance with knock-in models showing these events specifically in vulnerable neurons, no molecular features analysed in B7E2 mice paralleled the SCA7 degeneration pattern. This difference is probably due to different expression levels and patterns resulting from the two different techniques. Indeed, overexpression of mutant ataxin-7 could affect many different neurons because ataxin-7 levels are above the critical threshold inducing neuronal responses. In B7E2 mice, we found a similar set of chaperones, proteasome components and transcription factors in NIs from all neuronal types. This suggests that the cells respond in a similar manner to the accumulation of the mutant fragments.
Several transcription factors are trapped in NIs of SCA7 transgenic mice
Using a large-scale immunohistochemical search for the presence of transcription factors into NIs, we found that components of complexes TFIID, E, F, H and activators could be detected in ataxin-7 neuronal inclusions. This finding confirms the interpretation of previous studies reporting the sequestration of CBP (21,22). If sequestration of transcription factors into NIs contributes to polyglutamine toxicity, this contribution may alter pre-initiation (sequestration of TFIIE, F and H), initiation (sequestration of TAFII30) and activation (sequestration of CBP and SNF5). The combined toxic impact would then be a global alteration of transcription rather than just an impairment of specific pathways of gene expression activation. This could explain why transcription of more than 100 genes was found to be downregulated in transgenic mice overexpressing the Huntington’s disease mutation (19). However, the relevance of such sequestrations to the disease process remains to be clarified. When a transcription factor was found in NIs, we did not observe a marked reduction of the signal corresponding to its normal localization in the nucleus. Thus, the sequestration in NIs does not cause obvious depletion from the nucleoplasm. However, a decreased activity could result from the sequestration of a proportion of the transcription factor molecules, undetectable in our immunofluorescent conditions. The heterogeneity of neuronal populations would render a quantitative assessment of transcription factors by a western blot approach difficult.
In several cell transfection experiments, polyglutamine-expanded proteins have been shown to recruit other polyglutamine containing proteins into NIs (29,37). Here the transcription factors found in NIs were not preferentially those carrying polyglutamine motifs. This difference is probably due to enhanced interactions when proteins are overexpressed in cellular systems.
We did not observe a sequestration of TAFII130 in ataxin-7 NIs. This transcription factor was reported to be sequestered in NIs from human SCA3 and DRPLA brains by others (20). These authors used the anti-TAFII130 antibody 4A6 (Santa Cruz) which did not detect the mouse protein on western blot in our first antibody selection, and which was therefore not used further on our mouse sections. The contradiction between the two studies can be due to the use of different immunoreagents, to a difference between the human and mouse pathologies, or to different properties between ataxin-7 NIs and ataxin-3 or atrophin-1 NIs. Finally, we also observed that the normal distribution of many transcription factors, including TAFII130 and CBP was a dot-like pattern throughout the nucleus. Since Shimohata et al. (20) and Steffan et al. (22) used single labelling immunohistochemistry, it is possible that some of the dots identified as inclusions did not colocalize with the pathogenic proteins. We also did not observe sequestration of TBP which was found in human SCA3 NIs by others (37). Other extensive analyses will be essential in the future to compare the sets of trancription factors trapped in NIs from various polyglutamine diseases, to determine if a subset is commonly sequestered in all of them. It will then be possible to focus investigations on these transcription factors and question their possible contribution to polyglutamine toxicity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation of transgenic animals
The p
aIA construct (9), containing the full-length human SCA7 cDNA followed by an intron and poly(A) signal was used to build the transgenes. The PDGF-B promoter was extracted as a 1.4 kb XbaI–AvrII fragment from the pSIS-1 clone (38) kindly provided by Dr T.Collins and cloned into the SpeI site of paIA to give the B7N construct. To obtain a longer repeat expansion than used previously (9), we searched for expansion events occurring in bacteria. An AatII/NarI digest (cutting on both sides of the CAG repeat) of the P7E construct (9) was run on an agarose gel, and a band above the expected size of 400 bp was blindly cloned into B7N (using the final plasmid preparation of B7N) using the AatII/NarI sites. Resulting clones were selected for their CAG repeat size: the B7E2 clone was the one having received a (CAG)128 repeat. Both B7N and B7E2 constructs were confirmed by sequencing, and the homogeneity of CAG repeats of the final plasmid preparations was checked on Southern blot by probing AatII/NarI digests with a (CAG)10 oligoprobe. The final plasmids were BssHII digested to remove bacterial sequences, purified on sucrose gradient and micro-injected into [C57BL/6xSJL] fertilized eggs as described (39). Mouse tail DNA was screened by PCR. The segregation of different integration sites and the approximate copy numbers were assessed by Southern blot using the SstI enzyme that cuts only once in the transgene, and a transgene-specific probe covering the SCA7 3'-UTR and SV40 maturation cassette (PvuII fragment of paIA construct).
Western blotting
Whole brains were dissected and homogenized in SDS-lysis buffer containing 100 mM Tris–HCl pH 9, 2% SDS, 5% ß-mercaptoethanol and 15% glycerol, and boiled for 15 min. Total protein extracts were analysed on an 8% SDS–polyacrylamide gel. Primary antibodies were used at 1:2000 for 1C2 (4), 1:1000 each for the 1C1 + 2A10 (28) pool and 1:10 000 for 
-tubulin (Sigma) and revealed with peroxidase-conjugated secondary antibody (Jackson Laboratories) and the ECL chemiluminescent reaction (Pierce).
Tissue fixation
Animals were anaesthetized by i.p. injection of a sublethal dose of 100 mg/ml ketamin, 5 mg/ml chlorobutanol, 8 mg/ml xylazin base, 0.4 mg/ml methylparabenzoate (Imalgene1000, Merial and Rompun, Bayer) in 0.9% saline solution, and intracardiac perfusion was carried out with 10 ml of 0.9% saline solution followed by 100 ml of 4% paraformaldehyde and 1x PBS. Brains were then dissected and post-fixed for 1 h by immersion (same fixative).
Immunohistochemistry
Fixed tissues were embedded in paraffin and cut into 5 µm sections which were stained as follows: after deparaffination, slides were rehydrated in a graded series of ethanol (100% twice, 95% twice, 80%, 70%; 5 min each), boiled in 10 mM citrate buffer (pH 6.0) in a microwave, washed in 1x PBS, blocked of the endogenous peroxidase with methanol/H2O2 (40%/1% in H2O) for 10 min, washed in 1x PBS, blocked for 30 min in 1x PBS + 0.3% Triton X-100, 5% normal goat serum (primary polyclonal rabbit antibody) or 5% normal horse serum (primary monoclonal mouse antibody). Sections were then rinsed in 1x PBS, incubated in a humid chamber at 4°C with the primary antibody for 36 h. Secondary antibodies [Vector Laboratories biotinylated anti-mouse IgG (H+L), biotinylated anti-rabbit IgG (H+L), respectively] (diluted 1:200) were applied for 30 min at room temperature and visualized using the avidin–biotin–peroxidase complex method (Vectastain elite kit; Vector Laboratories). The colour reaction was carried out using FAST DAB tablets (Sigma). Some sections were lightly counterstained with hematoxylin, sections were dehydrated through graded ethanols and xylene, coverslipped and viewed using a light microscope.
Immunofluorescence
Fixed brains were incubated overnight (30% sucrose, 1x PBS), snap-frozen on dry ice. Free-floating sections (50 µm) were blocked (5% goat serum, 0.3% Triton X-100, 1x PBS), washed in PBS, incubated with primary and secondary antibodies (Cy3, FITC or Oregon Green conjugated from Jackson ImmunoResearch Laboratories, Inc.) and diluted in 5% goat serum and 1x PBS. Sources and working dilutions of primary antibodies are indicated in Table 3.
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Semi-quantitative RT–PCR
Total RNA was extracted with guanidinium thiocyanate and isolated by CsCl centrifugation according to Delidow et al. (40). RNA concentration was determined by OD260 nm absorbance. Reverse transcription was performed on 1 µg total RNA using SuperScriptII reverse transcriptase (Gibco BRL) and random hexamers (Pharmacia Biotech) in a 20 µl volume reaction following the manufacturer’s procedure. A sample of 1 µl of the product was used for PCR amplification in 10 mM Tris–HCl pH 8.5, 50 mM KCl, 1.5 mM MgCl2, 100 µM each dNTP and 400 nM each primer. To avoid amplification of contaminating genomic DNA, SCA7 cDNA was amplified using a reverse primer (AAQ213, 5'-TTCCATAGGTTGGAATCTTAGAGCT-3') overlapping the intron of the transgene construct. The forward primer (AAO290, 5'-AGACAGCAAGTATCGACAGCAACGGAA-3') hybridized to the 3'-UTR of the SCA7 human cDNA. The HPRT cDNA was amplified using primers: QG197, 5'-GTAATGATCAGTCAACGGGGGAC-3' and QG198, 5'-CCAGCAAGCTTGCAACCTTAACCA-3' located in different exons of the HPRT mouse gene. HPRT and SCA7 products were amplified through 18 and 22 cycles, respectively (10 s at 94°C, 15 s at 58°C, 20 s at 72°C) using Taq DNA polymerase (Sigma). PCR products were run on a 2% agarose gel and transfered to Hybond N+ (Amersham) membranes. The blots were hybridized with 32P-labelled internal primer probes and washed according to standard procedures. Quantification of radioactivity was made on a Bio Imaging Analyser BAS2000 (Fujifilm).
The difference between P7N and P7E lines, and between B7E2 and B7N lines, was tested on the log(XSCA7/XHPRT) values, where XSCA7 and XHPRT are intensities obtained in the same experiment after amplification of the SCA7 and HPRT mRNA, respectively. Values were acquired in four independent experiments with duplicates in each experiment. The test applied was a repeated-measure ANOVA considering two factors, genotype (fixed) and animals (random and nested in genotype), with eight repetitions.
Pulse–chase experiment
The full-length human SCA7 cDNA with 10 or 60 CAG repeats was cloned into a derivative of the pSG5 eukaryotic expression vector and tagged in its N-terminus with the flag coding sequence 5'-ATGGACTACAAAGACGATGACGATAAA-3' to give the p7FN and p7FE constructs, respectively. COS-7 cells were transiently transfected with these constructs, and washed 16 h later. Forty hours post-transfection, cells were starved in methionine–cysteine-free DMEM. Cells from each 60 mm dish were then labelled for 1 h with 8 µl of Pro-mixL-35S-labelling mix (Amersham) and then washed and incubated in DMEM with 5% FCS and 15 mg/ml cold methionine. After indicated times, cells were lysed in immunoprecipitation buffer (10 mM Tris–HCl pH 8.0, 10% glycerol, 1 mM EDTA, 150 mM KCl, 0.5% NP-40 and protease inhibitors). Equal amounts of labelled lysate (5 x 106 c.p.m.) were immunoprecipitated with anti-flag M2 beads (Sigma). Immunoprecipitated proteins were separated on an 8% SDS–polyacrylamide gel, subjected to fluorography by incubation in Amplify solution (Amersham) and analysed by autoradiography.
In-situ hybridization
The P1 cRNA probe was constructed as described previously (28). Frozen sections (20 µm) were fixed in 4% paraformaldehyde in 0.1 M PBS pH 7.4 for 10 min, washed in PBS, acetylated (0.25% acetic anhydride in 0.1 M triethanolamine buffer pH 8.0), dehydrated in a graded series of ethanol, delipidated in chloroform, partially rehydrated in 99 and 95% ethanol and air dried. Sections were hybridized in a humid chamber for 4 h at 50°C in a buffer containing 50% formamide, 0.3 M NaCl, 20 mM Tris–HCl pH 7.4, 5 mM EDTA, 10% dextransulfate, 1x Denhardt’s solution, 100 mM dithiothreitol, 0.1% SDS, 0.1% sodium thiosulfate, 100 µg/ml salmon sperm DNA, 250 µg/ml yeast tRNA, 200 µg/ml yeast total RNA and 150 000 c.p.m. of 35S-labelled probe per microlitre of hybridization buffer. After hybridization, sections were washed in two changes of 2x SSC (0.3 M NaCl, 300 mM Na-citrate) at room temperature and then in 0.1x SSC at 60°C for 30 min, followed by an RNase A treatment [100 µg/ml in RNase-buffer (0.5 M NaCl, 10 mM Tris–HCl, 1 mM EDTA, pH 8.0)] for 30 min at 37°C, rinsed in RNase buffer for 15 min at room temperature, washed in two changes of 0.1x SSC at 60°C for 30 min each, and partially dehydrated in 70 and 95% ethanol. Slides were apposed to HyperfilmTMß-max (Amersham) for 2–5 weeks.
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ACKNOWLEDGEMENTS |
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We thank Y.Trottier, A.Lunkes, S.Picaud and J.Sahel for fruitful discussions, T.Collins for the gift of the PDGF-B promoter region, M.Vigneron, J.-M.Egly, L.Tora, I.Davidson, J.Dillworth and R.Losson for antibodies against transcription factors and advice, M.Le Meur, E.Metzger and staff at the IGBMC animal facility for micro-injections and mouse care, C.Weber and M.Gendron for priceless technical assistance, and J.-L.Vonesh, D.Hentsch and R.Knoth for confocal imaging. This work was supported by funds from the Institut National de la Recherche Medicale, the Centre National de Recherche Scientifique, the Hôpital Universitaire de Strasbourg, Fondation Louis Jeantet, and by a grant of the DFG (SFB 505, TP C2) to K.S.L and G.B.L. K.S.L. was, in addition, supported by the Graduiertenkolleg Moleculare Medicine, University of Ulm. G.Y. was supported by a fellowship from the Fondation pour la Recherche Médicale.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +33 3 88 65 32 44; Fax: +33 3 88 65 32 46; Email: mandeljl@igbmc.u-strasbg.frPresent address: Gaël Yvert, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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1 Gouw, L.G., Digre, K.B., Harris, C.P., Haines, J.H. and Ptacek, L.J. (1994) Autosomal dominant cerebellar ataxia with retinal degeneration: clinical, neuropathologic, and genetic analysis of a large kindred. Neurology, 44, 1441–1447.
2 David, G., Abbas, N., Stevanin, G., Durr, A., Yvert, G., Cancel, G., Weber, C., Imbert, G., Saudou, F., Antoniou, E. et al. (1997) Cloning of the SCA7 gene reveals a highly unstable CAG repeat expansion. Nat. Genet., 17, 65–70.[Web of Science][Medline]
3 Wells, R.D. and Warren, S.T. (1998) Genetic Instabilities and Hereditary Neurological Diseases. Academic Press, San Diego, CA.
4 Trottier, Y., Lutz, Y., Stevanin, G., Imbert, G., Devys, D., Cancel, G., Saudou, F., Weber, C., David, G., Tora, L. et al. (1995) Polyglutamine expansion as a pathological epitope in Huntington’s disease and four dominant cerebellar ataxias. Nature, 378, 403–406.[Medline]
5 Scherzinger, E., Lurz, R., Turmaine, M., Mangiarini, L., Hollenbach, B., Hasenbank, R., Bates, G.P., Davies, S.W., Lehrach, H. and Wanker, E.E. (1997) Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo. Cell, 90, 549–558.[Web of Science][Medline]
6 Zoghbi, H.Y. and Orr, H.T. (2000) Glutamine repeats and neurodegeneration. Annu. Rev. Neurosci., 23, 217–247.[Web of Science][Medline]
7 Sisodia, S.S. (1998) Nuclear inclusions in glutamine repeat disorders: are they pernicious, coincidental, or beneficial? Cell, 95, 1–4.[Web of Science][Medline]
8 Cummings, C.J., Reinstein, E., Sun, Y., Antalffy, B., Jiang, Y., Ciechanover, A., Orr, H.T., Beaudet, A.L. and Zoghbi, H.Y. (1999) Mutation of the E6-AP ubiquitin ligase reduces nuclear inclusion frequency while accelerating polyglutamine-induced pathology in SCA1 mice. Neuron, 24, 879–892.[Web of Science][Medline]
9 Yvert, G., Lindenberg, K.S., Picaud, S., Landwehrmeyer, G.B., Sahel, J.A. and Mandel, J.L. (2000) Expanded polyglutamines induce neurodegeneration and trans-neuronal alterations in cerebellum and retina of SCA7 transgenic mice. Hum. Mol. Genet., 9, 2491–2506.
10 Zoghbi, H.Y. and Orr, H.T. (1999) Polyglutamine diseases: protein cleavage and aggregation. Curr. Opin. Neurobiol., 9, 566–570.[Web of Science][Medline]
11 Gutekunst, C.A., Li, S.H., Yi, H., Mulroy, J.S., Kuemmerle, S., Jones, R., Rye, D., Ferrante, R.J., Hersch, S.M. and Li, X.J. (1999) Nuclear and neuropil aggregates in Huntington’s disease: relationship to neuropathology. J. Neurosci., 19, 2522–2534.
12 Holmberg, M., Duyckaerts, C., Dürr, A., Cancel, G., Gourfinkel-An, I., Damier, P., Faucheux, B., Trottier, Y., Hirsch, E.C., Agid, Y. et al. (1998) Spinocerebellar ataxia type 7 (SCA7): a neurodegenerative disorder with neuronal intranuclear inclusions. Hum. Mol. Genet., 7, 913–918.
13 Li, H., Li, S.H., Johnston, H., Shelbourne, P.F. and Li, X.J. (2000) Amino-terminal fragments of mutant huntingtin show selective accumulation in striatal neurons and synaptic toxicity. Nat. Genet., 25, 385–389.[Web of Science][Medline]
14 Wheeler, V.C., White, J.K., Gutekunst, C.A., Vrbanac, V., Weaver, M., Li, X.J., Li, S.H., Yi, H., Vonsattel, J.P., Gusella, J.F. et al. (2000) Long glutamine tracts cause nuclear localization of a novel form of huntingtin in medium spiny striatal neurons in HdhQ92 and HdhQ111 knock-in mice. Hum. Mol. Genet., 9, 503–513.
15 Lin, C.H., Tallaksen-Greene, S., Chien, W.M., Cearley, J.A., Jackson, W.S., Crouse, A.B., Ren, S., Li, X.J., Albin, R.L. and Detloff, P.J. (2001) Neurological abnormalities in a knock-in mouse model of Huntington’s disease. Hum. Mol. Genet., 10, 137–144.
16 Kennedy, L. and Shelbourne, P.F. (2000) Dramatic mutation instability in HD mouse striatum: does polyglutamine load contribute to cell-specific vulnerability in Huntington’s disease? Hum. Mol. Genet., 9, 2539–2544.
17 Li, S.-H., Cheng, A.L., Li, H. and Li, X.-J. (1999) Cellular defects and altered gene expression in PC12 stably expressing mutant huntingtin. J. Neurosci., 19, 5159–5172.
18 Lin, X., Antalffy, B., Kang, D., Orr, H.T. and Zoghbi, H.Y. (2000) Polyglutamine expansion down-regulates specific neuronal genes before pathologic changes in SCA1. Nat. Neurosci., 3, 157–163.[Web of Science][Medline]
19 Luthi-Carter, R., Strand, A., Peters, N.L., Solano, S.M., Hollingsworth, Z.R., Menon, A.S., Frey, A.S., Spektor, B.S., Penney, E.B., Schilling, G. et al. (2000) Decreased expression of striatal signaling genes in a mouse model of Huntington’s disease. Hum. Mol. Genet., 9, 1259–1271.
20 Shimohata, T., Nakajima, T., Yamada, M., Uchida, C., Onodera, O., Naruse, S., Kimura, T., Koide, R., Nozaki, K., Sano, Y. et al. (2000) Expanded polyglutamine stretches interact with TAFII130, interfering with CREB-dependent transcription. Nat. Genet., 26, 29–36.[Web of Science][Medline]
21 McCampbell, A., Taylor, J.P., Taye, A.A., Robitschek, J., Li, M., Walcott, J., Merry, D., Chai, Y., Paulson, H., Sobue, G. et al. (2000) CREB-binding protein sequestration by expanded polyglutamine. Hum. Mol. Genet., 9, 2197–2202.
22 Steffan, J.S., Kazantsev, A., Spasic-Boskovic, O., Greenwald, M., Zhu, Y.Z., Gohler, H., Wanker, E.E., Bates, G.P., Housman, D.E. and Thompson, L.M. (2000) The Huntington’s disease protein interacts with p53 and CREB-binding protein and represses transcription. Proc. Natl Acad. Sci. USA, 97, 6763–6768.
23 Boutell, J.M., Thomas, P., Neal, J.W., Weston, V.J., Duce, J., Harper, P.S. and Jones, A.L. (1999) Aberrant interactions of transcriptional repressor proteins with the Huntington’s disease gene product, huntingtin. Hum. Mol. Genet., 8, 1647–1655.
24 Sasahara, M., Fries, J.W., Raines, E.W., Gown, A.M., Westrum, L.E., Frosch, M.P., Bonthron, D.T., Ross, R. and Collins, T. (1991) PDGF B-chain in neurons of the central nervous system, posterior pituitary, and in a transgenic model. Cell, 64, 217–227.[Web of Science][Medline]
25 Games, D., Adams, D., Alessandrini, R., Barbour, R., Berthelette, P., Blackwell, C., Carr, T., Clemens, J., Donaldson, T., Gillespie, F. et al. (1995) Alzheimer-type neuropathology in transgenic mice overexpressing V717F ß-amyloid precursor protein. Nature, 373, 523–527.[Medline]
26 Duff, K., Eckman, C., Zehr, C., Yu, X., Prada, C.M., Perez-tur, J., Hutton, M., Buee, L., Harigaya, Y., Yager, D. et al. (1996) Increased amyloid-ß42(43) in brains of mice expressing mutant presenilin 1. Nature, 383, 710–713.[Medline]
27 Masliah, E., Rockenstein, E., Veinbergs, I., Mallory, M., Hashimoto, M., Takeda, A., Sagara, Y., Sisk, A. and Mucke, L. (2000) Dopaminergic loss and inclusion body formation in -synuclein mice: implications for neurodegenerative disorders. Science, 287, 1265–1269.
28 Lindenberg, K.S., Yvert, G., Muller, K. and Landwehrmeyer, G.B. (2000) Expression analysis of ataxin-7 mRNA and protein in human brain: evidence for a widespread distribution and focal protein accumulation. Brain Pathol., 10, 385–394.[Web of Science][Medline]
29 Kazantsev, A., Preisinger, E., Dranovsky, A., Goldgaber, D. and Housman, D. (1999) Insoluble detergent-resistent aggregates form between pathological and non-pathological lengths of polyglutamine in mammalian cells. Proc. Natl Acad. Sci. USA, 96, 11404–11409.
30 Persichetti, F., Carlee, L., Faber, P.W., McNeil, S.M., Ambrose, C.M., Srinidhi, J., Anderson, M., Barnes, G.T., Gusella, J.F. and MacDonald, M.E. (1996) Differential expression of normal and mutant Huntington’s disease gene alleles. Neurobiol. Dis., 3, 183–190.[Web of Science][Medline]
31 Olsson, J.E., Gordon, J.W., Pawlyk, B.S., Roof, D., Hayes, A., Molday, R.S., Mukai, S., Cowley, G.S., Berson, E.L. and Dryja, T.P. (1992) Transgenic mice with a rhodopsin mutation (Pro23His): a mouse model of autosomal dominant retinitis pigmentosa. Neuron, 9, 815–830.[Web of Science][Medline]
32 Wheeler, V.C., Auerbach, W., White, J.K., Srinidhi, J., Auerbach, A., Ryan, A., Duyao, M.P., Vrbanac, V., Weaver, M., Gusella, J.F. et al. (1999) Length-dependent gametic CAG repeat instability in the Huntington’s disease knock-in mouse. Hum. Mol. Genet., 8, 115–122.
33 Shelbourne, P.F., Killeen, N., Hevner, R.F., Johnston, H.M., Tecott, L., Lewandoski, M., Ennis, M., Ramirez, L., Li, Z., Iannicola, C. et al. (1999) A Huntington’s disease CAG expansion at the murine Hdh locus is unstable and associated with behavioural abnormalities in mice. Hum. Mol. Genet., 8, 763–774.
34 Bingham, P.M., Scott, M.O., Wang, S.P., Mcphaul, M.J., Wilson, E.M., Garbern, J.Y., Merry, D.E. and Fischbeck, K.H. (1995) Stability of an expanded trinucleotide repeat in the androgen receptor gene in transgenic mice. Nat. Genet., 9, 191–196.[Web of Science][Medline]
35 Merry, D.E., McCampbell, A., Taye, A.A., Winston, R.L. and Fischbeck, K.H. (1996) Toward a mouse model for spinal and bulbar muscular atrophy: effect of neuronal expression of androgen receptor in transgenic mice. Am. J. Hum. Genet., 59, A271.
36 Fernandez-Funez, P., Nino-Rosales, M.L., de Gouyon, B., She, W.C., Luchak, J.M., Martinez, P., Turiegano, E., Benito, J., Capovilla, M., Skinner, P.J. et al. (2000) Identification of genes that modify ataxin-1-induced neurodegeneration. Nature, 408, 101–106.[Medline]
37 Perez, M.K., Paulson, H.L., Pendse, S.J., Saionz, S.J., Bonini, N.M. and Pittman, R.N. (1998) Recruitment and the role of nuclear localisation in polyglutamine-mediated aggregation. J. Cell Biol., 14, 1457–1470.
38 Ratner, L., Thielan, B. and Collins, T. (1987) Sequences of the 5' portion of the human c-sis gene: characterization of the transcriptional promoter and regulation of expression of the protein product by 5' untranslated mRNA sequences. Nucleic Acids Res., 15, 6017–6036.
39 Hogan, B., Costantini, F. and Lacy, E. (1986) Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
40 Delidow, B.C., Lynch, J.P., Peluso, J.J. and White, B.A. (1993) Polymerase Chain Reaction: Basic Protocols. Humana Press, Totowa, NJ.
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