Human Molecular Genetics, 2002, Vol. 11, No. 8 905-914
© 2002 Oxford University Press
Huntingtin inclusions do not deplete polyglutamine-containing transcription factors in HD mice
1Department of Human Genetics, Emory University, School of Medicine, Atlanta, GA 30322, USA
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ABSTRACT |
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A pathological hallmark of polyglutamine diseases is the presence of inclusions or aggregates of the expanded polyglutamine protein. Polyglutamine inclusions are present in the neuronal nucleus in a number of inherited neurodegenerative disorders, including Huntington disease (HD). Recent studies suggest that polyglutamine inclusions may sequester polyglutamine-containing transcription factors and deplete their concentration in the nucleus, leading to altered gene expression. To test this hypothesis, we examined the expression and localization of the polyglutamine-containing or glutamine-rich transcription factors TBP, CBP and Sp1 in HD mouse models. All three transcription factors were diffusely distributed in the nucleus, despite the presence of abundant intranuclear inclusions. There were no differences in the nuclear staining of these transcription factors between HD and wild-type mouse brains. Although some CBP staining appeared as dots in the selective brain regions (e.g. hypothalamus and amygdala), double labeling showed that most CBP was not co-localized with huntingtin nuclear inclusions. Electron microscopy confirmed that CBP was diffusely distributed in the nucleus. Western blots showed that these transcription factors were not trapped in huntingtin inclusions. In the striatum of HD mice, which suffers a significant reduction in the expression of a number of genes, mutant huntingtin was present in both an aggregated and a diffuse form. These findings suggest that altered gene expression may result from the interactions of soluble mutant huntingtin with nuclear transcription factors, rather than from the depletion of transcription factors by nuclear inclusions.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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At least eight inherited neurodegenerative diseases are caused by the expansion of a glutamine repeat in the disease proteins (1). Many polyglutamine diseases, such as Huntington disease (HD) and spinocerebellar ataxia (SCA1, -3 and -7), show features of intranuclear accumulation of polyglutamine proteins and the aggregates or inclusions formed by these mutant proteins. Although the effects of intranuclear inclusions are unknown, it is evident that nuclear accumulation of mutant polyglutamine proteins can lead to pathological changes in neurons (1). The intranuclear localization of mutant huntingtin led to the discovery that mutant huntingtin interacts with transcription factors and alters gene expression (2–7). The cAMP-responsive element-binding protein (CREB)-binding protein (CBP) and its interaction with huntingtin and other polyglutamine proteins have been studied extensively (3,4, 8–10). Huntingtin inclusions were found to recruit CBP and to deplete soluble CBP, resulting in altered gene expression (4). Consistent with these findings, polyglutamine inclusions were found to contain the transcription factors TATA-binding protein (TBP) (11,12) and Sp1 (13). Normal CBP and TBP contain a polyglutamine domain (18–38 glutamines), whereas Sp1 contains glutamine-rich domains. While these studies demonstrated the interactions of soluble polyglutamine proteins with transcription factors, they also led to the hypothesis that polyglutamine inclusions may exert toxic effects by sequestering CBP and other proteins containing non-pathogenic polyglutamine stretches (4,9).
However, this hypothesis is challenged by several important pieces of evidence. First, intranuclear inclusions are not necessarily associated with neurodegeneration (14–18). Thus, it is difficult to explain how these nuclear inclusions contribute to pathology by sequestering transcription factors. Second, although the polyglutamine domain is observed to be responsible for recruiting transcription factors (4), recent studies also show that the interactions between CBP and polyglutamine proteins do not require this repeat (8,10). In addition, CBP is not co-localized with nuclear inclusions formed by the SCA1 proteins (10). In SCA7 transgenic mouse brain that also shows intranuclear inclusions (19,20), the nuclear distribution of CBP is not significantly altered (19). These results raise the question of whether polyglutamine inclusions commonly recruit polyglutamine-containing transcription factors. Furthermore, the lack of double immunostaining and characterization of the ultrastructural distribution of CBP in HD brain also makes it unclear whether CBP-immunoreactive products are indeed present in the nuclear inclusions formed by mutant huntingtin.
These questions prompted us to perform a more detailed examination of the distribution and expression of transcription factors in HD mouse brain. For this study, HD mouse brain provides several advantages over postmortem human brain: it expresses abundant polyglutamine inclusions in the nuclei of certain brain regions, its morphology and protein integrity can be better preserved, and the role of nuclear inclusions in presymptomatic conditions can be investigated. We examined three important transcription factors – CBP, TBP and Sp1 – in various HD mouse models. Our findings provide no evidence for the depletion of polyglutamine-containing transcription factors by nuclear inclusions, suggesting that the interactions of transcription factors with soluble mutant huntingtin rather than their depletion may alter gene expression in HD.
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RESULTS |
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Distribution of CBP in HD mouse brain
The observation of CBP in nuclear inclusions led us to examine the CBP distribution in HD knock-in mice. These mice express mutant huntingtin with a 150-glutamine repeat in the endogenous mouse huntingtin locus (21). The HD knock-in mice at age 7–8 months show nuclear inclusions selectively in the striatum, allowing us to examine whether CBP is specifically recruited into nuclear inclusions in this tissue. We used A-22, an anti-CBP antibody against the N-terminal region of CBP described previously (4). We also immunolabeled the same HD mouse brain with the antibody EM48, which recognizes the N-terminal region of human huntingtin and effectively labels mutant huntingtin and its aggregates (22, 23). Although mutant huntingtin selectively forms intranuclear inclusions in striatal neurons, we did not see any altered distribution of CBP or CBP nuclear inclusions in the striatum of the HD mice (Fig. 1). The diffuse nuclear distribution of CBP in the cortex is also similar in HD and control mice.
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We then examined N171-82Q mice that express the first 171 amino acids of huntingtin with a 82-glutamine repeat, since these mice have been found to have nuclear inclusions containing CBP (4). Mutant huntingtin forms inclusions in various brain regions in these mice, and nuclear inclusions are abundant in the cortex (24). With EM48 staining, we confirmed that most nuclear inclusions were in the cortex (Fig. 2). It has been reported that CBP is not equally distributed in various regions of rat brain (25). For example, some neurons in the hypothalamus, amygdala, cortex and hippocampus are heavily labeled, while most neurons in the striatum and certain parts of the thalamus are weakly labeled by anti-CBP (25). We observed a similar staining pattern of CBP in mouse brain: prominent CBP labeling was seen in the hypothalamic and amygdaloid neurons. Most CBP staining was diffuse in the nuclei of neurons in the brains of both HD and normal mice, and we could not find any significant difference in the nuclear distribution of CBP in the brains of N171-82Q mice. Some CBP-stained puncta or dots were seen in the lateral hypothalamus (LH), but there appeared to be no difference in such punctate staining between normal and HD brains (Fig. 2A). Using various dilutions of A-22 antibody (1:1000–1:8000), we could not observe discrete nuclear inclusions in the cortex either. In contrast, EM48 labeling revealed both diffuse and inclusion-like staining in the nucleus (Fig. 2A).
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We used three different lots of A-22 and observed a similarly diffuse nuclear staining in HD mouse brain. To ensure that most CBP is indeed diffusely distributed in the nucleus in HD mouse brain, we also used two other CBP antibodies: rabbit antibody C-20 and mouse monoclonal antibody C-1, which recognize the C-terminal region of CBP. Use of all these CBP antibodies revealed diffuse CBP staining in the nucleus without discrete nuclear inclusions in N171-82Q mouse brain. In contrast, mouse monoclonal antibody (mEM48), which was generated using the same immunogen as for rabbit antibody EM48, also intensively labeled abundant nuclear inclusions in the same HD mouse brain (Fig. 2B). We used immunocytochemistry to examine the brains of R6/2 mice at ages of 8–12 weeks, which express exon 1 huntingtin with 115–150 glutamines (26). No obviously discrete nuclear inclusions were stained by A-22 (data not shown). Thus, parallel studies using both anti-huntingtin and anti-CBP antibodies to examine three different types of HD models failed to find significant inclusions in the nucleus that could also be labeled by anti-CBP antibodies.
No co-localization of transcription factors withhuntingtin inclusions
Some punctate nuclear staining of CBP in HD brain was observed in a previous study, and was thought to represent huntingtin inclusions that have sequestered CBP (4). If huntingtin inclusions recruit CBP, we should see more CBP-positive inclusions or puncta in HD mouse brain than in wild-type mouse brain, and we should also see the co-localization of CBP with huntingtin inclusions. Thus, we counted the number of CBP-immunoreactive puncta in N171-82Q and normal mouse brains and performed immunofluorescent double labeling using anti-CBP and anti-huntingtin antibodies.
The quantitative assessment of CBP puncta confirmed that there was no difference in the number of these puncta in N171-82Q and wild-type mouse brain (Fig. 3A). Most CBP puncta were found in the lateral hypothalamus and amygdala. Very few puncta or dots were seen in the striatum and cortex. In contrast, huntingtin nuclear inclusions were highly enriched in the cortex and relatively abundant in other regions in N171-82Q mouse brain. Thus, there is apparently no correlation between the increased density of huntingtin nuclear inclusions and the smaller number of CBP puncta.
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To examine whether small CBP puncta were co-localized with any huntingtin inclusions, we performed immunofluorescent double labeling of the lateral hypothalamus of N171-82Q mice. For this assay, we used a mouse monoclonal antibody mEM48 that reacted strongly with nuclear inclusions. However, no co-localization of CBP and huntingtin inclusions was seen in HD mouse brain (Fig. 3B).
Ultrastructural localization of CBP in HD transgenicmouse brain
We used electron microscopic immunogold labeling to further examine the ultrastructural distribution of CBP and huntingtin in N171-82Q mouse brain. While a large huntingtin inclusion could be found in the nucleus (Fig. 4A), CBP immunogold particles were diffuse in the nucleus (Fig. 4B). We also examined other brain regions, including the cortex, striatum and hypothalamus, but could not find any CBP-positive inclusions or aggregates in the nucleus. A few CBP immunogold particles were occasionally clustered in the lateral hypothalamus (data not shown). However, these clusters were present in the cytoplasm or perinuclear region. They were apparently different from huntingtin inclusions, and may represent the small CBP puncta observed by light microscopy. In addition, control brains from age-matched wild-type mice also showed diffuse CBP immunogold particles in the nucleus, with some clustered immunogold particles in the cytoplasm (data not shown).
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To confirm the above observation, we also performed electron microscopic double labeling of brain cortex of a N171-82Q mouse at 5 months of age. The intense EM48 reaction with huntingtin inclusions allowed us to use diaminobenzidine (DAB) staining to identify nuclear inclusions. The brain section was first labeled with mouse huntingtin antibody mEM48 and then with rabbit CBP antibody A-22. DAB staining for huntingtin and immunogold labeling for CBP were then performed. In this way, we could reveal dark huntingtin inclusions labeled by DAB (Fig. 4C, D). A few CBP immunogold particles were scattered diffusely around the inclusions; however, this distribution appeared to be not different from that in other nuclear regions, where most gold particles were also scattered diffusely. Furthermore, very few or no immunogold particles were seen within the inclusions. Apparently, CBP immunogold particles were not concentrated in the inclusions (Fig. 4D). In contrast, when the same brain section was labeled with EM48 immunogold particles alone, mutant huntingtin was extremely abundant in the nuclear inclusions (Fig. 4E). These results also support the idea that CBP is not sequestered or depleted by these inclusions.
Nuclear distribution of TBP and Sp1 in HD mouse brain
Next, we extended our study to TBP and Sp1, which contain a polyglutamine repeat and glutamine-rich domains, respectively. Immunocytochemistry of N171-82Q mouse cortex showed that these transcription factors were diffusely distributed in the nucleus of the HD brain, in contrast to huntingtin nuclear inclusions, which were highly abundant in the same brain region (Fig. 5). Wild-type mouse brain displayed similar diffuse nuclear staining for these transcription factors (data not shown). These findings thus suggested that TBP and Sp1, like CBP, are not concentrated in huntingtin inclusions.
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Expression level of transcription factors in HD transgenic mouse brain
A previous study, by sequentially probing a western blot with anti-huntingtin and then anti-CBP, revealed the presence of CBP in huntingtin aggregates (4). Since we found that incompletely stripping a blot could lead to the same aggregate-like staining on the blot later probed with anti-CBP, we probed western blots with anti-CBP first. Using Triton X-100-soluble fraction from the cortex of R6/2 mice at 12 weeks of age, which contains a large amount of intranuclear huntingtin, we found no difference in the expression of soluble CBP in HD mouse brains compared with that in normal brain (Fig. 6A). We then examined Triton X-100-insoluble pellets with anti-CBP and did not see CBP signals at the top of the gel (Fig. 6B, upper panel). Probing the same blots with mouse monoclonal antibody to huntingtin, however, gave rise to a considerable labeling of aggregated huntingtin at the top of the gel (Fig. 6B, lower panel). These results suggest that no, or very little, CBP was trapped in huntingtin aggregates.
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It was reported that a large number of genes are significantly downregulated in the striatum of 6-week-old R6/2 mice (7). If nuclear inclusions were involved in the decreased gene expression, we would expect to see the presence of a large amount of nuclear inclusions and perhaps the recruitment of transcription factors by the nuclear inclusions as well. We therefore examined nuclear huntingtin aggregation in R6/2 mice at 6 and 12 weeks of age using both immunocytochemistry and western blots. EM48 immunocytochemistry showed that at 6 weeks, diffuse mutant huntingtin was predominant in the nucleus, with some small nuclear inclusions. However, at 12 weeks, almost all neurons displayed a single large inclusion in the nucleus (Fig. 7A). To quantitatively assess huntingtin aggregation, we performed a western blot analysis of huntingtin aggregates in R6/2 mouse brain. The result showed that soluble mutant huntingtin was prominently present in the nucleus at 6 weeks. At 12 weeks, however, aggregated huntingtin became predominant in the nucleus (Fig. 7B). Densitometric analysis suggested that aggregated huntingtin was at least 4-fold more abundant at 12 weeks than that at 6 weeks. The diffuse nuclear EM48 staining suggests that soluble mutant huntingtin in the nucleus could contribute to the decreased gene expression seen in these HD mice (7). The increased aggregation of mutant huntingtin at 12 weeks could worsen nuclear function if proteasomes, chaperones or other nuclear molecules are recruited into these inclusions. However, CBP immunostaining did not reveal any aggregated CBP at the top of the gel, regardless of increased huntingtin aggregation (Fig. 7C, lower panel). The same brain tissue samples were also analyzed with antibodies to Sp1 and TBP. No aggregated forms of these transcription factors were found on the top of the gel either (Fig. 7D, data are not shown for TBP). Also, TBP and Sp1 did not show any significant change in their expression in the nucleus of the striatum in HD brain as compared with normal brain (Fig. 7D). Thus, Sp1 and TBP did not appear to be trapped in huntingtin inclusions.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The present studies provide several lines of evidence to show that intranuclear huntingtin inclusions do not deplete the transcription factors CBP, TBP and Sp1. First, we did not find any significant alteration in the nuclear localization of these transcription factors in brains of several HD mouse models. Second, there was no significant co-localization of CBP dots or puncta with huntingtin inclusions. Third, the expression levels of the soluble forms of these transcription factors were not altered in HD mouse brains that express abundant intranuclear inclusions. Taken together, the present findings do not support the idea that nuclear huntingtin inclusions can sequester polyglutamine-containing transcription factors, such as CBP, to deplete the expression level of the soluble form of these proteins.
These results are apparently different from the previous finding showing that soluble CBP is recruited into or depleted by huntingtin inclusions (4). Since we also used the same HD transgenic mouse strains, the different results may be due to the different immunoreactivities of various batches of polyclonal antibodies used. We therefore used several anti-CBP antibodies and statistically analyzed the CBP puncta and huntingtin inclusions. Our results suggested that the majority of CBP is not co-localized with huntingtin inclusions. Another possibility would be that HD conditions promote protein degradation such that the loss of the soluble form of CBP occurs in HD brain, especially in postmortem HD patient brain because of a longer period of disease or greater postmortem deterioration. In our study, we compared the expression levels of several proteins, including CBP, TBP, Sp1 and tubulin, in HD mouse brain. We did not observe any significant changes in the expression of these proteins, although nuclear huntingtin inclusions were abundant in all the HD mice we examined.
Most of the previous studies observed CBP inclusions in HD brain at the light microscopic level. These studies, however, examined different neurons with antibodies to CBP and huntingtin (4). Using double immunolabeling and electron microcopy, we did not detect discrete nuclear inclusions stained by anti-CBP in HD mouse brains. It is possible that some CBP may appear as small nuclear dots or bodies under certain circumstances such as stress or cell degeneration. This may explain why, in other studies, CBP nuclear staining showed diffuse nuclear staining with some dots (3) or did not show discrete inclusions in polyglutamine disease mouse brains (19). Thus, it was important to confirm whether nuclear huntingtin inclusions alter the distribution or expression of CBP. We performed double immunolabeling, quantitative assessment of CBP-positive puncta and electron immunogold examination with HD mouse brain. Although we did find some CBP-positive puncta or dots in some regions such as the hypothalamus, we did not see any difference in the density of CBP dots between HD and wild-type mouse brains. Immunofluorescent double labeling did not show that these CBP immunoreactive dots were in nuclear huntingtin inclusions. The light microscopy results were confirmed by immunogold electron microscopy, which also showed a diffuse CBP distribution in the nucleus in HD brain. It is possible that the anti-CBP antibodies we used may not be effective at recognizing CBP in the inclusion because its immunoreactivity has been masked by polyglutamine aggregation. Western blotting would provide another way to detect aggregated proteins under a denaturing condition. Our western blot analysis revealed that CBP, TBP and Sp1 were not trapped in huntingtin inclusions, and that their expression was not altered by huntingtin inclusions. Taken together, these findings support the idea that the majority of these transcription factors are not recruited into nuclear aggregates and that their expression is thus unlikely to be depleted by the inclusions.
There is compelling evidence that CBP is co-localized with polyglutamine inclusions in transfected cells (4,8–10,27). Since polyglutamine proteins and CBP were overexpressed in these studies, the transfected CBP might have been recruited into polyglutamine inclusions that were rapidly formed by overexpressed proteins. Also, in normal cell culture, CBP has been found in the normal promyelocytic leukemia (PML) nuclear body (28–30), which is co-localized with polyglutamine proteins (10,31). Our studies and others (25) did not reveal the PML nuclear body in brain, perhaps because it forms differently in vivo or may be difficult to see with CBP staining in brain. Another possibility is that the immunoreactive properties of the antibodies used and the state of the brain tissue examined are critical to reveal nuclear CBP inclusions and may also account for the difference between our findings and others. Nevertheless, all the antibodies that we used in this study revealed prominently diffuse CBP in the nucleus in HD mouse brain and showed no significant difference of CBP distribution in the HD and control brains, suggesting that at least the majority of CBP is not recruited into nuclear inclusions. This may be because mutant huntingtin is expressed at a level similar to that of endogenous huntingtin, so it forms inclusions slowly in vivo. The slow aggregation of mutant huntingtin or the formation of nuclear inclusions could, instead, reduce the binding affinity of huntingtin for some interacting proteins. One example is HAP1, a huntingtin-associated protein that is co-localized with huntingtin inclusions in transfected cells (32), but is not detectable in huntingtin inclusions in HD brain (33).
The theory that nuclear inclusions sequester transcription factors is also based on the finding that CBP interacts with several polyglutamine proteins. This interaction implies that the polyglutamine stretch is involved in recruiting various polyglutamine-containing transcription factors into nuclear inclusions (4). However, other studies showed that the interactions of CBP with huntingtin and the SCA3 proteins were not dependent on the polyglutamine domain (8,10). It is possible that protein context determines the interactions of polyglutamine proteins. Thus, some proteins can be recruited into inclusions while others interact better with soluble mutant proteins. For example, nuclear polyglutamine inclusions commonly recruit chaperones and ubiquitin (34), whereas the SCA1 protein specifically binds to the nuclear protein LANP (35) and to A1Up, a ubiquitin-like nuclear protein (36). In addition, polyglutamine inclusions in SCA7 mouse brain contain TAFII 30, a transcription factor that does not have a polyglutamine tract (19), whereas the soluble SCA7 protein interacts with a cone–rod homeobox protein, CRX, in the nucleus (20).
Given the findings that GST pull down and co-immunoprecipitation demonstrate the interaction of CBP with huntingtin (3,4), it is possible that soluble mutant huntingtin binds to CBP and affects gene expression. Our recent study suggests that soluble mutant huntingtin binds more tightly to Sp1 than does aggregated huntingtin and affects Sp1-dependent gene expression (37). Since overexpressed chaperones suppress polyglutamine pathology and the solubility polyglutamine proteins but not nuclear inclusions in Drosophila (38), misfolded polyglutamine proteins could mediate cellular pathology before they form obvious nuclear inclusions. This idea is also supported by the evidence that diffuse intranuclear mutant huntingtin was predominantly present in the striatum of 6-week-old R6/2 mouse brain, in which there is a significant decrease in the expression of a number of genes (7). Nuclear inclusions, on the other hand, may worsen cellular function if they recruit chaperones, ubiquitin and other nuclear proteins. Whether and how the nuclear inclusions are involved in HD pathology remains to be further investigated, since several studies have suggested that intranuclear polyglutamine inclusions do not necessarily associate with neuronal degeneration (14–18). For example, in transgenic mice lacking the ubiquitin–protein ligase, E6-AP, the formation of nuclear inclusions is reduced, while neuronal toxicity is enhanced (39). Despite the controversial role of nuclear inclusions, the nuclear toxicity of mutant huntingtin is evident by its interactions with transcription factors and its deleterious effects on gene expression. The results in the current study suggest that the strategy to prevent the early neuropathological changes should focus on the interactions of transcription factors and polyglutamine proteins before the mutant proteins form microscopic inclusions or aggregates.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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HD mice
R6/2 mice [B6CBA-TgN (HDexon1) strain 62], which express exon 1 of the human mutant HD gene containing 115–150 CAGs (40), were obtained from Jackson Laboratory (Bar Harbor, ME). N171-82Q mice [B6C3F1/-TgN (HD82Gln)81Dbo] that express the first 171 amino acids of huntingtin with a 82-glutamine repeat (24) were also obtained from Jackson Laboratory. HD repeat knock-in mice that express a 150-CAG repeat in the endogenous mouse HD gene were generated as described previously (21). Breeding pairs of these HD knock-in mice were provided by Dr Peter Detloff at the University of Alabama at Birmingham. Age-matched control mice (C57B6 for R6/2 and HD knock-in mice and C3H/B16 for N171-82Q mice) were also used. These mice were bred and maintained in the animal facility at Emory University. Genotyping of transgenic mice was performed according to the methods described previously (21,24,40).
Antibodies
EM48, a rabbit polyclonal antibody against amino acids 1–256 of human huntingtin was generated from our previous studies (23). The same antigen was also used for the production of mouse monoclonal antibodies by Auburn University Hybridoma Facility. Of 7 hybridoma cell lines extensively characterized, one produced a mouse monoclonal IgG, mEM48, which had immunoreactivity similar to that of rabbit antibody EM48 and was used in the present study. Antibodies to CBP, which were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), included C-20, C-1 and three different lots of A-22. A-22 is a rabbit polyclonal antibody to the N terminus of CBP, C-20 is a rabbit polyclonal antibody to the C terminus of CBP, and C-1 is a mouse monoclonal antibody to the C terminus of CBP. Other antibodies used in the study are a mouse monoclonal antibody to tubulin (Sigma, St. Louis, MO), a rabbit antibody (SC-59) to Sp1 (Santa Cruz Biotechnology, Santa Cruz, CA) and a rabbit antibody (SL-1) to TBP (Santa Cruz).
Light microscopy
Mouse brain sections were prepared according to the method described previously (26). Mice were anesthetized and then perfused intracardially with phosphate-buffered saline (PBS, pH 7.2) for 30 s followed by 2% paraformaldehyde/lysine/periodate fixative in 0.1
phosphate buffer (PB) at pH 7.4. Brains were removed, cryoprotected in 30% sucrose at 4°C, and sectioned at 40 µm using a freezing microtome. Free-floating sections were preblocked in 4% normal goat serum (NGS) in PBS, 0.1% Triton X-100 and avidin (10 µg/ml). Brain sections were incubated with EM48 (1:1000 dilution) or anti-CBP (A-22, 1:1000–8000; C-20, 1:100 dilution) at 4°C for 24 h. The immunoreactive product was visualized with the avidin–biotin complex kit (Vector ABC Elite, Burlingame, CA). Controls included brain sections from age-matched wild-type mice.
Electron microscopic immunocytochemistry
Immunogold labeling was performed as described previously (23). Briefly, mice were fixed by perfusion with 0.1 PB containing 4% paraformaldehyde and 0.5% glutaraldehyde. After perfusion, the brain was removed, postfixed with 4% paraformaldehyde in PB for 6–8 h and then sectioned using a vibratome. Brain sections were incubated with primary antibodies (1:1000) in PBS containing 4% NGS for 24 h at 4°C and then with Fab fragments of goat anti-rabbit secon-dary antibodies (1:50) conjugated to 1.4 nm gold particles (Nanoprobes Inc., Stony Brook, NY) in PBS with 4% NGS overnight at 4°C. After rinsing in PBS, sections were fixed again in 2% glutaraldehyde in PB for 1 h, silver-intensified using the IntenSEM kit (Amersham International, Buckinghamshire, UK), osmicated in 1% OsO4 in PB, and stained overnight in 2% aqueous uranyl acetate.
Double electron microscopic labeling was performed using the pre-embedding 3,3'-diaminobenzidine (DAB) reaction for huntingtin combined with the immunogold reaction for CBP. Mouse antibody mEM48 (1:100) was first incubated with tissue section (50 µm) for 24 h at 4°C. The mouse antibody was detected with ABC kit (Vector Laboratories. Burlingame, CA) and DAB. Rabbit CBP antibody (A-22, 1:100) was then added to the section for 24 h at 4°C, followed by incubation with gold–secondary antibody to rabbit antibodies for 2 h at room temperature. The sections were then postfixed with 2.5% glutaraldehyde in PB for 2 h, silver-enhanced for 30 min, and washed in PB for further processing.
All sections used for electron microscopy were dehydrated in ascending concentrations of ethanol and propylene oxide/Eponate 12 (1:1) and embedded in Eponate12 (Ted Pella, Redding, CA). Ultrathin sections (60 nm) were cut using a Leica Ultracut S ultramicrotome. Thin sections were counterstained with 5% aqueous uranyl acetate for 5 min followed by Reynolds lead citrate for 5 min, and were examined using an Hitachi H-7500 electron microscope.
Western blots
Brain tissue of mice was resuspended in lysis buffer (50 m Tris, pH 8.0, 150 m NaCl, 1% Triton X-100) with protease inhibitor cocktail (1000x, Sigma P8340) and PMSF (100 µg/ml). The tissue was homogenized for 30 s and centrifuged at 12 000g for 15 min at 4°C. The supernatant and pellets (50 µg proteins) were used for western blotting with ECL kits (Amersham Inc). For western blot analysis of nuclear fractions, mouse striatum tissue was homogenized in buffer (0.25 sucrose, 15 m Tris–HCl, pH 7.9, 60 m KCl, 15 m NaCl, 5 m EDTA, 1 m EGTA and 100 µg/ml PMSF). The homogenate was spun at 2000g for 10 min at 4°C. The nuclear pellet was resuspended in the homogenizing buffer and spun down again at 2000g for 10 min. The pellets were resuspended in the SDS sample buffer and sonicated for 10 s. About 50 µg of protein were loaded in each lane of a 10% Tris–glycine SDS gel.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr Peter Detloff for providing HD repeat knock-in mice and thank Dr He Li and Ms Hong Yi for technical assistance in using the electron microscope and Ajay Pillarisetti for assistance in genotyping of HD mice. We also thank Dr Gillian Bates for providing us with some A-22 antibody. This work was supported by NIH Grants NS41669 and AG19206.
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FOOTNOTES |
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* To whom correspondence should be addressed at: Department of Human Genetics, Emory University School of Medicine, Whitehead Building 347, 615 Michael Street, Atlanta, GA 30322, USA. Tel: +1 404 727 3290; Fax: +1 404 727 3949; Email: xiaoli{at}genetics.emory.edu
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REFERENCES |
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1 Zoghbi, H.Y. and Orr, H.T. (2000) Glutamine repeats and neurodegeneration. Annu. Rev. Neurosci., 23, 217–247.[Web of Science][Medline]
2
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.
3
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.
4
Nucifora, F.C., Jr, Sasaki, M., Peters, M.F., Huang, H., Cooper, J.K., Yamada, M., Takahashi, H., Tsuji, S., Troncoso, J., Dawson, V.L. et al. (2001) Interference by huntingtin and atrophin-1 with cbp-mediated transcription leading to cellular toxicity. Science, 291, 2423–2428.
5
Cha, J.H., Kosinski, C.M., Kerner, J.A., Alsdorf, S.A., Mangiarini, L., Davies, S.W., Penney, J.B., Bates, G.P. and Young, A.B. (1998) Altered brain neurotransmitter receptors in transgenic mice expressing a portion of an abnormal human huntington disease gene. Proc. Natl Acad. Sci. USA, 95, 6480–6485.
6
Li, S.H., Cheng, A.L., Li, H. and Li, X.J. (1999) Cellular defects and altered gene expression in PC12 cells stably expressing mutant huntingtin. J. Neurosci., 19, 5159–5172.
7
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.
8 Steffan, J.S., Bodai, L., Pallos, J., Poelman, M., McCampbell, A., Apostol, B.L., Kazantsev, A., Schmidt, E., Zhu, Y.Z., Greenwald, M. et al. (2001) Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila. Nature, 413, 739–743.[Medline]
9
McCampbell, A., Taylor, J.P., Taye, A.A., Robitschek, J., Li, M., Walcott, J., Merry, D., Chai, Y., Paulson, H., Sobue, G. and Fischbeck, K.H. (2000) CREB-binding protein sequestration by expanded polyglutamine. Hum. Mol. Genet., 9, 2197–2202.
10
Chai, Y., Wu, L., Griffin, J.D. and Paulson, H.L. (2001) The role of protein composition in specifying nuclear inclusion formation in polyglutamine disease. J. Biol. Chem., 276, 44889–44897.
11 Huang, C.C., Faber, P.W., Persichetti, F., Mittal, V., Vonsattel, J.P., MacDonald, M.E. and Gusella, J.F. (1998) Amyloid formation by mutant huntingtin: threshold, progressivity and recruitment of normal polyglutamine proteins. Somat. Cell Mol. Genet., 24, 217–233.[Web of Science][Medline]
12
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 localization in polyglutamine-mediated aggregation. J. Cell Biol., 143, 1457–1470.
13 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]
14 Klement, I.A., Skinner, P.J., Kaytor, M.D., Yi, H., Hersch, S.M., Clark, H.B., Zoghbi, H.Y. and Orr, H.T. (1998) Ataxin-1 nuclear localization and aggregation: role in polyglutamine-induced disease in SCA1 transgenic mice. Cell, 95, 41–53.[Web of Science][Medline]
15 Saudou, F., Finkbeiner, S., Devys, D. and Greenberg, M.E. (1998) Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions. Cell, 95, 55–66.[Web of Science][Medline]
16 Kuemmerle, S., Gutekunst, C.A., Klein, A.M., Li, X.J., Li, S.H., Beal, M.F., Hersch, S.M. and Ferrante, R.J. (1999) Huntington aggregates may not predict neuronal death in Huntington's disease. Ann. Neurol., 46, 842–849.[Web of Science][Medline]
17 Warrick, J.M., Chan, H.Y., Gray-Board, G.L., Chai, Y., Paulson, H.L. and Bonini, N.M. (1999) Suppression of polyglutamine-mediated neurodegeneration in Drosophila by the molecular chaperone HSP70. Nat. Genet., 23, 425–428.[Web of Science][Medline]
18
Kazemi-Esfarjani, P. and Benzer, S. (2000) Genetic suppression of polyglutamine toxicity in drosophila. Science, 287, 1837–1840.
19
Yvert, G., Lindenberg, K.S., Devys, D., Helmlinger, D., Landwehrmeyer, G.B. and Mandel, J.L. (2001) SCA7 mouse models show selective stabilization of mutant ataxin-7 and similar cellular responses in different neuronal cell types. Hum. Mol. Genet., 10, 1679–1692.
20 La Spada, A.R., Fu, Y., Sopher, B.L., Libby, R.T., Wang, X., Li, L.Y., Einum, D.D., Huang, J., Possin, D.E., Smith, A.C. et al. (2001) Polyglutamine-expanded ataxin-7 antagonizes crx function and induces cone-rod dystrophy in a mouse model of sca7. Neuron, 31, 913–927.[Web of Science][Medline]
21
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.
22
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.
23 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]
24
Schilling, G., Becher, M.W., Sharp, A.H., Jinnah, H.A., Duan, K., Kotzuk, J.A., Slunt, H.H., Ratovitski, T., Cooper, J.K., Jenkins, N.A. et al. (1999) Intranuclear inclusions and neuritic aggregates in transgenic mice expressing a mutant N-terminal fragment of huntingtin. Hum. Mol. Genet., 8, 397–407.
25 Stromberg, H., Svensson, S.P. and Hermanson, O. (1999) Distribution of CREB-binding protein immunoreactivity in the adult rat brain. Brain Res., 818, 510–514.[Web of Science][Medline]
26 Davies, S.W., Turmaine, M., Cozens, B.A., DiFiglia, M., Sharp, A.H., Ross, C.A., Scherzinger, E., Wanker, E.E., Mangiarini, L. and Bates, G.P. (1997) Formation of neuronal intranuclear inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell, 90, 537–548.[Web of Science][Medline]
27
Kazantsev, A., Preisinger, E., Dranovsky, A., Goldgaber, D. and Housman, D. (1999) Insoluble detergent-resistant aggregates form between pathological and nonpathological lengths of polyglutamine in mammalian cells. Proc. Natl Acad. Sci. USA, 96, 11404–11409.
28
LaMorte, V.J., Dyck, J.A., Ochs, R.L. and Evans, R.M. (1998) Localization of nascent RNA and CREB binding protein with the PML-containing nuclear body. Proc. Natl Acad. Sci. USA, 95, 4991–4996.
29
Doucas, V., Tini, M., Egan, D.A. and Evans, R.M. (1999) Modulation of CREB binding protein function by the promyelocytic (PML) oncoprotein suggests a role for nuclear bodies in hormone signaling. Proc. Natl Acad. Sci. USA, 96, 2627–2632.
30
Boisvert, F.M., Kruhlak, M.J., Box, A.K., Hendzel, M.J. and Bazett-Jones, D.P. (2001) The transcription coactivator CBP is a dynamic component of the promyelocytic leukemia nuclear body. J. Cell Biol., 152, 1099–1106.
31 Yamada, M., Wood, J.D., Shimohata, T., Hayashi, S., Tsuji, S., Ross, C.A. and Takahashi, H. (2001) Widespread occurrence of intranuclear atrophin-1 accumulation in the central nervous system neurons of patients with dentatorubral–pallidoluysian atrophy. Ann. Neurol., 49, 14–23.[Web of Science][Medline]
32
Li, S.H., Gutekunst, C.A., Hersch, S.M. and Li, X.J. (1998) Interaction of huntingtin-associated protein with dynactin P150Glued. J. Neurosci., 18, 1261–1269.
33
Gutekunst, C.A., Li, S.H., Yi, H., Ferrante, R.J., Li, X.J. and Hersch, S.M. (1998) The cellular and subcellular localization of huntingtin-associated protein 1 (HAP1): comparison with huntingtin in rat and human. J. Neurosci., 18, 7674–7686.
34 Paulson, H.L. (1999) Protein fate in neurodegenerative proteinopathies: polyglutamine diseases join the (mis)fold. Am. J. Hum. Genet., 64, 339–345.[Web of Science][Medline]
35 Matilla, A., Koshy, B.T., Cummings, C.J., Isobe, T., Orr, H.T. and Zoghbi, H.Y. (1997) The cerebellar leucine-rich acidic nuclear protein interacts with ataxin-1. Nature, 389, 974–978.[Medline]
36
Davidson, J.D., Riley, B., Burright, E.N., Duvick, L.A., Zoghbi, H.Y. and Orr, H.T. (2000) Identification and characterization of an ataxin-1-interacting protein: A1Up, a ubiquitin-like nuclear protein. Hum. Mol. Genet., 9, 2305–2312.
37
Li, S.-H., Zhou, H., Rao, M., Lam, S., Li, H. and Li, X.-J. (2002) Interaction of mutant huntingtin and transcriptional activator Sp1. Mol. Cell. Biol. 22, 1277–1287.
38
Chan, H.Y., Warrick, J.M., Gray-Board, G.L., Paulson, H.L. and Bonini, N.M. (2000) Mechanisms of chaperone suppression of polyglutamine disease: selectivity, synergy and modulation of protein solubility in Drosophila. Hum. Mol. Genet., 9, 2811–2820.
39 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]
40 Mangiarini, L., Sathasivam, K., Seller, M., Cozens, B., Harper, A., Hetherington, C., Lawton, M., Trottier, Y., Lehrach, H., Davies, S.W. and Bates, G.P. (1996) Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell, 87, 493–506.[Web of Science][Medline]
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