Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disease caused by the expansion of a polyglutamine repeat within the androgen receptor (AR). We have studied the mutant AR in an in vitro system, and find both aggregation and proteolytic processing of the AR protein to occur in a polyglutamine repeat length-dependent manner. In addition, we find the aberrant metabolism of expanded repeat AR to be coupled to cellular toxicity, indicating a likely molecular basis for the toxic gain of AR function that produces neuronal degeneration in SBMA.
Spinal and bulbar muscular atrophy (SBMA) is one of a group of inherited neurodegenerative diseases caused by the expansion of a polyglutamine repeat within the associated disease protein (1-7). In SBMA patients, a normally polymorphic CAG repeat (10-36 CAGs) in exon 1 of the androgen receptor (AR) gene expands to 40-62 CAGs. In other diseases caused by the same mutational mechanism, the CAG trinucleotide repeat that encodes the polyglutamine tract shows a repeat length and expansion range of similar size.
The lack of a neurological phenotype in androgen-insensitive individuals with loss of function mutations in the AR gene (8) suggests that the polyglutamine expansion causes disease through the acquisition of a novel toxic property. In support of this notion, animal models with hemizygous or heterozygous loss-of-function mutations in the AR (9) or Huntington's disease (HD) genes (10-12), respectively, do not display neurological phenotypes. Conversely, transgenic models that express expanded repeat cDNAs for ataxin-1, huntingtin or ataxin-3 reproduce many aspects of the associated neurodegenerative disease (13-15), confirming the hypothesis that neuronal demise is caused by the gain of a toxic function. While substantial evidence indicates that SBMA results from the acquisition of a novel toxic property, most females known to be carriers of the mutation do not demonstrate a neurological phenotype [although a small number of mildly affected females have been described (16)]. However, a majority of carrier females may be protected by a favorable X-inactivation pattern in susceptible neurons, or by low levels of circulating androgens.
While the evidence is strong for a gain-of-function mechanism of toxicity in SBMA and other polyglutamine repeat diseases, additional complicating effects due to a graded loss of function cannot be ruled out. In SBMA, patients show signs of partial androgen insensitivity, indicating that the mutant AR protein is either not completely normal in transducing the effects of its hormonal ligand, androgen, or that it is not present at normal levels, at least in tissues involved in secondary sex characteristics. In fact, a decrease in maximal ligand binding of AR from SBMA patients has been noted by several groups (17,18), and an expanded repeat AR has also been shown to display a decrease in maximal binding in vitro (19,20). While these studies focus on AR function in non-neuronal cells, the growing evidence for a trophic effect of AR in neurons (reviewed by ref. 21) suggests that a partial loss of AR function may contribute to the neurodegenerative phenotype as well.
Our previous studies of full-length normal and expanded repeat AR in neuronal cell culture (19) and in transgenic mice (unpublished observations) revealed no toxic effects of an AR containing 66 glutamine repeats (AR65; an additional CAA extends the glutamine tract by one residue). Not only was the survival of unstressed cells unchanged, but cells challenged with agents that induce oxidative stress (menadione and hydrogen peroxide) also showed no increased sensitivity in the presence of AR65. In addition, it was shown that AR65 demonstrated normal ligand binding (but reduced Bmax), transactivation capability and intracellular location as an AR containing 24 repeats (AR24) (19). Studies of ataxin-3 (14,22) indicate that truncated expanded repeat proteins may be more toxic to cells than the full-length proteins, suggesting that characteristics of the full-length proteins might contribute to repressing a toxic phenotype. Such characteristics probably also contribute to tissue and cell type specificity, perhaps through direct protein-protein interactions.
Biochemical characterization of isolated polyglutamines indicates their propensity to form novel interactions, mediated by hydrogen bonding between amides in a proposed `polar zipper' conformation (23,24). Such predicted interactions recently have been identified for huntingtin and ataxin-3 (22,25-27). We have developed an in vitro system in which both aggregation and proteolytic processing of the AR protein occur in a polyglutamine repeat length-dependent fashion. In addition, the aberrant metabolism of expanded repeat AR is coupled to cellular toxicity, indicating a likely molecular basis for the toxic gain of AR function that produces neuronal degeneration in SBMA.
Our previous studies of expanded repeat, full-length AR in neuronal MN-1 cells (19) and Cos-7 cells (28) revealed neither toxicity nor aberrant protein metabolism. In addition, a truncated construct encoding 57 amino acids N-terminal and 100 amino acids C-terminal to a 66 repeat polyglutamine tract did not produce toxicity when expressed in Cos-7 cells (28). We further decreased the protein size by creating a truncated human AR cDNA construct that encodes 57 amino acids N-terminal and 52 amino acids C-terminal to the polyglutamine repeat (Fig. 1A). Versions were made containing either 16 or 65 CAG repeats. Initial studies of these encoded proteins expressed in Cos-7 cells also revealed ample expression levels and insignificant toxicity (unpublished observations). Thus, in order to develop a model system in which to study the pathogenesis of polyglutamine expansion in the AR, we created additional constructs containing repeat expansions up to 112 CAGs (see Materials and Methods). While the plasmid into which the longer CAG repeats were introduced (AR65[Delta]HA) was stable during bacterial growth, constructs with repeat lengths of 97 or greater showed marked instability, allowing repeat lengths >97 to be obtained from the AR97[Delta]HA construct.
AR cDNA constructs containing 16, 65, 97, 106 and 112 CAG repeats were transfected into Cos-7 cells in the presence of a plasmid encoding [beta]-galactosidase (pCal-Z-neo), and [beta]-galactosidase activity was assessed at 12, 24 and 48 h. Decreasing [beta]-galactosidase activity was noted with time in cultures transfected with AR97[Delta]HA, AR106[Delta]HA and AR112[Delta]HA, but not in cultures transfected with AR16[Delta]HA, AR65[Delta]HA, or with [beta]-galactosidase in the presence of irrelevant carrier DNA (Fig. 1B). Assessment of cell death by trypan blue exclusion confirmed that the decrease in [beta]-galactosidase activity was due to direct cell toxicity (Fig. 1C). Note that AR65[Delta]HA does not cause a consistent decrease in [beta]-galactosidase activity in Cos-7 cells (Fig. 1B), but does show a toxic effect when assayed by trypan blue exclusion (Fig. 1C), indicating that the latter assay is a more sensitive measure of cell death.
Analysis of protein expression from AR constructs transiently transfected into Cos-7 cells, using an antibody to detect the hemagglutinin (HA) epitope, revealed monomeric AR protein with the expected shifts in migration relative to repeat length (Fig. 2, top). Not only are the overall protein levels of AR97, AR106 and AR112 consistently lower than those of AR16 and AR65, but expression of these proteins at 48 h is nearly undetectable, while AR16 and AR65 proteins show increased expression at 48 h. The loss of AR protein at 48 h, coupled with both the decrease in [beta]-galactosidase activity at this time and the increase in trypan blue-positive cells, is consistent with the death of cells expressing truncated AR proteins containing repeats of 97, 106 and 112.
Immunostaining of transiently transfected Cos-7 cells (Fig. 3A, C and E) and MN-1 cells (Fig. 3B, D and F), with the anti-HA antibody Y-11, revealed the cellular correlate to the aggregates seen by western blot analysis. While AR16[Delta]HA-expressing cells showed a homogeneous cytoplasmic distribution of AR protein (Fig. 3A and B), cells expressing AR65[Delta]HA (Fig. 3C and D) or AR112[Delta]HA (Fig. 3E and F) proteins contained large cytoplasmic or nuclear immunoreactive aggregates. Occasionally many small inclusions were visible, but most cells contained one very large inclusion. In AR112[Delta]HA-expressing cells, 45% of stained cells contained inclusions, while in AR65[Delta]HA-expressing cells, 35% of transfected cells showed aggregates; the remaining cells showed homogeneous cytoplasmic staining similar to that of AR16[Delta]HA. Note the nuclear location of inclusions in MN-1 cells (Fig. 3D and F); such a striking localization was rarely seen in Cos cells, and suggests that MN-1 cells might provide a useful system for identifying determinants of nuclear transport. Consistent with the results seen by western blot analysis, 1C2 did not detect the large inclusions, but rather revealed small, punctate cytoplasmic staining in AR65[Delta]HA and AR112[Delta]HA cells (data not shown). Very rare AR16[Delta]HA-expressing cells stained with 1C2 and showed a diffuse, cytoplasmic protein distribution.
Western blot analysis of protein lysates from Cos-7 cells transiently transfected with truncated AR constructs with various repeat lengths, using an antibody that detects the N-terminal 20 amino acids of AR (N-20), revealed the monomeric forms of AR detected by both Y-11 and 1C2, and the high molecular weight aggregates detected by Y-11 (Fig. 2, bottom). In addition, N-20 identified a new species, an ~14 kDa protein, in protein lysates with pathologically expanded repeat AR constructs. This protein appeared in a repeat length-dependent manner, and contained repeat length-dependent amounts of polyglutamine, seen as a shifted size between AR65[Delta]HA, AR97[Delta]HA, AR106[Delta]HA and AR112[Delta]HA (Fig. 2, bottom). Analysis of this peptide on 15% SDS-PAGE indicates that it ranges in size from ~6.5 (AR65) to 12-13 kDa (AR112). This size is inconsistent with cleavage distal to the polyglutamine tract; thus this finding, coupled with the detection of this peptide with N-20 but not with 1C2, predicts that it represents the product of proteolytic cleavage within the polyglutamine tract. We are in the process of confirming this by sequence analysis.
Immunostaining of transfected Cos-7 cells with N-20 revealed a pattern similar to that seen with the anti-HA antibody Y-11, including large cytoplasmic and nuclear inclusions in cells expressing pathologically expanded repeat AR proteins. Thus there is no evidence to suggest a distinct intracellular localization of the N-terminal proteolytic cleavage product at this time.
In addition to the truncated, expanded AR constructs shown in Figure 1A, we made fusions between truncated, expanded AR cDNAs and glutathione S-transferase (GST). Following induction in Escherichia coli and purification on glutathione-Sepharose beads, the proteins were analyzed by SDS-PAGE and western blotting (Fig. 4). Hybridization with anti-HA antibody Y-11 revealed the expected increase in size of monomeric forms of AR with increased polyglutamine length. In addition, as with the Cos-7 lysates described above, GST-AR fusion proteins exhibited repeat length-dependent aggregation, seen as high molecular weight complexes at the top of the stacking gel.
We labeled GST-AR fusion proteins with [[gamma]-32P]ATP using heart muscle kinase (HMK), in order to assess aggregation in a cell-free system. To this end, we determined soluble and precipitated radioactivity, following centrifugation, immediately after (0 h) and 20 h after labeling GST-HMK, GST-AR16[Delta]HA, GST-AR34[Delta]HA, GST-AR66[Delta]HA and GST-AR119[Delta]HA (see Materials and Methods). While labeled GST-HMK, GST-AR16[Delta]HA and GST-AR34[Delta]HA proteins remained largely soluble following 20 h at 4°C (GST-HMK, 86.9 ± 6.4% original soluble counts; GST-AR16[Delta]HA, 96.4 ± 7.6%; GST-AR34HA, 81.0 ± 8.3%), GST-AR66[Delta]HA and GST-AR119[Delta]HA proteins showed a repeat length-dependent decrease in soluble radioactivity (GST-AR65[Delta]HA, 67.5 ± 19.4%; GST-AR119[Delta]HA, 35.2 ± 0.4%). These data, compiled from three experiments, were significantly different (GST-AR66[Delta]HA versus GST-AR16[Delta]HA, P = 0.04; GST-AR119[Delta]HA versus GST-AR16[Delta]HA, P = 0.003). Despite the fact that AR34 is not within the disease-causing repeat length range, statistical analysis revealed a significantly different soluble fraction for this repeat length as well (GST-AR34[Delta]HA versus GST-AR16[Delta]HA, P = 0.04). We have never seen protein aggregates by SDS-PAGE and western analysis with the 34 repeat sample, while recombinant ARs with 66 and 119 repeats show modest and substantial aggregate formation, respectively. It is likely that the sensitivity of this assay is not high enough to distinguish between the 34 and 66 repeat length samples. Nonetheless, these data indicate that expanded repeat AR constructs form aggregates in vitro, and that aggregation occurs in a repeat length-dependent way.
We have developed a set of expanded repeat AR molecules that display both aggregation and proteolytic processing in a repeat length-dependent manner. In addition, these physical attributes of the expanded repeat proteins are coupled to both cellular toxicity and the appearance of intracellular accumulations of protein in Cos and MN-1 cells. Although protein cleavage, aggregation and toxicity have each been reported previously with polyglutamine expansion, here we demonstrate all three occurring in a repeat length-dependent manner in vitro.
Recent histological evidence from patients with spinocerebellar ataxia type 3/Machado-Joseph disease (SCA3/MJD) (22), HD (27), and from a transgenic model for HD (25) indicates the presence of large aggregated structures within the nucleus of diseased neurons, suggesting a histological link between previous findings of polyglutamine-induced aggregation (23), the gain of function hypothesized by genotype-phenotype correlations and the neurodegenerative process. Our data showing aggregation of expanded repeat AR both in cells and in a cell-free system indicate that the AR bearing an expanded polyglutamine tract is also capable of such interactions, and that this aggregation likely represents the same biological phenomenon as that described for other polyglutamine-containing proteins. The demonstration of repeat length-dependent AR aggregation in a cell-free system provides a possible mechanism for both the repeat length dependence and the repeat length threshold of toxicity in polyglutamine repeat diseases.
Unlike the intranuclear inclusions seen in disease tissue, the aggregates occur largely in the cytoplasm in our Cos cell experiments, although MN-1 cells show nuclear aggregation. Nonetheless, the repeat length dependence of this phenomenon indicates that the same basic mechanisms of aggregation and toxicity may be at work. It is possible that the cytoplasmic rather than nuclear localization of inclusions reflects one aspect of cell type specificity; factors required for the nuclear transport of these proteins may be present in MN-1 cells and in susceptible neurons in vivo.
In addition to protein aggregation, we have observed repeat length-dependent proteolytic processing of truncated, expanded repeat AR. This proteolytic cleavage also correlates with cellular toxicity, and appears to occur within the polyglutamine tract itself. The observation of a specific cleavage occurring within the expanded polyglutamine repeat suggests that cleavage specificity is provided by a repeat length-dependent tertiary structure. To our knowledge, this would represent an unprecedented mechanism to define protease specificity. An alternative explanation to proteolytic processing within the polyglutamine tract must also be considered. It is possible that processing occurs C-terminal to the polyglutamine repeats, and the conformation of the expanded repeat is modified in such a way so as to prohibit 1C2 binding, and to increase its migration on SDS-PAGE (the novel peptide migrates faster than an expanded repeat protein would be expected to migrate). Expanded polyglutamines are well known to migrate more slowly on SDS-PAGE than predicted by size alone; this may be related to an abnormal binding of negatively charged SDS molecules (30). It is possible that a change in conformation of an expanded polyglutamine-containing peptide, truncated distal to the polyglutamine tract, might acquire a different structure such that both its migration and epitope availability are altered. We are in the process of purifying and microsequencing this peptide to address these issues.
The identification of intranuclear inclusions in HD brains with antibodies that detect N-terminal huntingtin, but not with antibodies that detect portions of the protein C-terminal to the polyglutamine tract, and the identification of an N-terminal fragment of huntingtin in juvenile cases of HD (27), suggests that a proteolytic processing event eliminates C-terminal epitopes during the molecular pathogenesis of HD. Indeed, previous studies have suggested that truncated expanded repeat proteins are more efficient than full-length proteins in causing cellular toxicity (14,15,22). Our previous experiments with full-length AR containing 65 repeats revealed neither toxicity nor aggregation in Cos or MN-1 cells (19), while the truncated 65 repeat-containing AR protein expressed in the present experiments produced moderate toxicity and cytoplasmic inclusions. These findings are consistent with the idea that in disease tissue, full-length expanded repeat proteins undergo proteolytic processing, allowing smaller polyglutamine repeat-containing peptides to enter the nucleus and form aggregates in a repeat length-dependent way.
In the current study, proteolytic processing of expanded repeat AR results in an N-terminal fragment that contains a portion of the polyglutamine tract. This peptide does not bind the antibody 1C2, and thus probably does not contain pathological lengths of polyglutamine. The other fragment that results from this cleavage might be predicted to contain a pathological polyglutamine tract; it would also contain the HA epitope. This fragment is not detected by western analysis with the antibody Y11, and is presumably located within the high molecular weight aggregate. Thus, it is likely that the aggregates seen in this study contain both the entire truncated AR protein and the C-terminal product of proteolytic cleavage. Recent evidence from Paulson (22) indicates that truncated forms of expanded repeat ataxin-3 are capable of recruiting full-length expanded ataxin-3 into high molecular weight aggregates. It is possible that the cleaved product formed here seeds and then recruits the complete truncated AR protein into aggregates.
The development of toxicity in Cos cells in our study indicates that, unlike the disease gene product, the constructs we used are not limited in their toxic effects to neurons. This may be due to any of several factors. First, by truncating the AR, we may have eliminated the need for cell type-specific protein interactions that might be required for conformational changes or proteolytic processing in vivo. The truncated protein encoded here would thus undergo the observed processing and aggregation more easily, as an effect of `unmasking' this part of the protein through truncation. In addition, by creating AR constructs with much longer repeats, and by expressing high levels of protein, it is possible that normal controls to prevent toxicity in non-neuronal cells have been by-passed. Understanding the basis of cell type specificity will be aided by further studies of truncated and full-length protein, in cells and in transgenic mice.
The results described here indicate that expanded repeat AR undergoes both proteolytic processing and intracellular aggregation as it effects cellular demise. Determining whether these processes directly cause cellular toxicity must await experiments that inhibit aggregation and proteolysis. Nonetheless, it is clear that with the identification of aggregate formation of polyglutamine-containing proteins including AR, this family of diseases joins the larger group of neurodegenerative diseases associated with protein aggregation. These include not only the polyglutamine repeat diseases, but Alzheimer's disease, prion diseases and Parkinson's disease. While protein aggregation may represent a common pathogenic mechanism in these neurodegenerative diseases, the molecular effects of intracellular inclusions in each disease may be dissimilar. The system that we have developed in this study should help us to elucidate these molecular effects.
Antibodies used were Y-11 (anti-HA, Santa Cruz Biotechnology, Inc., Santa Cruz, CA), N-20 (anti-AR, Santa Cruz Biotechnology, Inc.) and 1C2 (a kind gift from Y. Trottier, Strasbourg).
PCR-based site-directed mutagenesis was used to introduce an AscI restriction enzyme recognition sequence into the human AR cDNA (containing 65 CAG repeats) at nucleotide 750 of the published sequence (31). Two PCR reactions were carried out using the following primer pairs: hARmet, 5'-GCATAAGCTTGCTCAAGGATGGAAGTGC-3', and hAR130revAscI, 5'-GGCTCCAGGCTCTGGGCGCGCCGGACGCCAACCTCTCTC-3'; hAR130- AscI, 5'-GAGAGAGGTTGCGTCCGGCGCGCCCAGAGCCTGGAGCC-3', and hAR166rev, 5'-GCATAAGCTTGCCCAGCAGGGACAACGTGGA-3'. The PCR products were mixed and used as a template for PCR using primers hARmet and hAR166rev. The resulting PCR product was cleaved with HindIII, filled in with Klenow enzyme to create blunt ends and ligated to pCMV5 (32) that was digested with BglII and BamHI and filled in with Klenow to create blunt ends. Following transformation into DH5[alpha], and characterization of the resulting clones, plasmid DNA from a positive clone was digested with AscI and ligated with double-stranded DNA containing the HA epitope, created by annealing the following oligonucleotides: HA-S 5'-CGCGCCTATCCATACGATGTTCCAGATTACGCATAGGCGAGACTAGG-3' and HA-A 5'-CGCGCCTAGTCTAGACTATGCGTAATCTGGAACATCGTATGGATAGG-3'. The resulting plasmid contains the HA epitope at amino acid 130 (20 glutamines), with stop codons in three reading frames. One of the resulting clones underwent contraction of the CAG repeat to 16 units (AR16[Delta]HA); the 65 repeat construct is designated AR65[Delta]HA.
AR65[Delta]HA was digested with PstI, which cuts AR cDNA immediately 5' to the CAG repeat (5'-CTGCAG-3'), and treated with T4 polymerase in the absence of dNTPs to creat blunt ends. A PCR reaction was performed according to Ordway and Detloff (33), with the following modifications: primers 5'-(AGC)25-3' or 5'-(CAG)25-3', and 5'-(TGC)25-3' were used in a PCR reaction with Pfu DNA polymerase (Stratagene, La Jolla, CA). The resulting PCR products were gel-purified, ligated with the T4 polymerase-treated AR65[Delta]HA, and transformed into DH5[alpha]. Clones were analyzed by restriction digestion and sequencing. While the majority of retrieved clones contained frameshifting mutations, a single clone contained 97 CAG in-frame repeats. This isolated DNA was again transformed into DH5[alpha], and a clone containing 106 CAG repeats retrieved. Additional rounds of transformation resulted in a clone containing 112 repeats.
AR[Delta]HA plasmids were co-transfected into triplicate wells of Cos-7 cells in a 4:1 molar ratio with pCal-Z-neo (encoding [beta]-galactosidase) (34), using Lipofectin reagent (Gibco-BRL, Gaithersburg, MD). Cells were harvested at 12, 24 and 48 h for protein analysis, [beta]-galactosidase activity and direct counts of trypan blue-positive cells. [beta]-galactosidase activity was determined using a solution assay, according to Ausubel et al. (35). Protein content was determined using a detergent-compatible assay (Bio-Rad, Richmond, CA), and activity values were adjusted to protein content. Numbers of trypan blue-positive cells were adjusted by relative transfection efficiency, assessed by analysis of expression at 12 h of the co-transfected [beta]-galactosidase construct.
Protein samples were prepared as described (19), and protein concentration determined. Protein samples (50 µg) were electrophoresed on a 12.5% SDS-polyacrylamide gel and the protein transferred to Immobilon-P (Millipore), using a semi-dry transfer apparatus (Fisher). Membranes were blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween-20, and incubated with the appropriate primary antibody. Secondary antibodies were peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Labs) or peroxidase-conjugated sheep anti-mouse IgG (Amersham). Immunoreactive protein was detected using enhanced chemiluminescence reagent (ECL, Amersham).
For immunostaining of cells, cells were plated onto chamber slides before transfection. At 48 h following transfection, the cells were fixed with 4% paraformaldehyde, washed with phosphate-buffered saline (PBS) and incubated for 1 h at room temperature with the primary antibody. After washing with PBS, cells were incubated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody for 30 min, washed with PBS and mounted with Vectashield (Vector Laboratories). Cells were analyzed with a Leitz fluorescence photomicroscope.
Fusion protein plasmids were prepared with a vector consisting of GST (pGEX3X) and a heart muscle kinase (HMK) domain. The vector was prepared by digesting the DNA with EcoRI and filling in with Klenow to create blunt ends. AR inserts were prepared by digesting AR16[Delta]HA, AR65[Delta]HA, AR97[Delta]HA and AR106[Delta]HA with AccI and PvuII. After filling in with Klenow to create blunt ends, inserts were ligated to the prepared vector and transformed into DH5[alpha]. Resulting clones were confirmed by sequencing. Additional rounds of transformation resulted in clones containing 16, 34, 45, 66, 97 and 119 repeats. To induce expression of the resulting fusion proteins in E.coli, 500 ml cultures were inoculated with 20 ml cultures grown overnight. Exponentially growing cultures were induced with 1 mM isopropyl-[beta]-d-thiogalactopyranoside (IPTG), and incubated at 30°C for 2 h. Cells were pelleted and resuspended in ice-cold TEGM (10 mM Tris, pH 7.4, 1 mM EDTA, 10% glycerol, 10 mM sodium molybdate, pH7.2) (36) containing protease inhibitors. Cells were sonicated for 1 min in a sonicator bath, and centrifuged for 30 min at 10 000 g. Supernatants were mixed with glutathione-Sepharose 4B (Pharmacia), and incubated for 1 h at 4°C. The Sepharose beads were then washed with PBS, and stored at -80°C until use. Radioactive labeling of 1 µg of fusion protein was performed according to Blanar (37,38), using [[gamma]-32P]ATP (6000 Ci/mmol) and heart muscle kinase (Sigma, St Louis, MO).
We thank Suping Wang for technical assistance, Yvon Trottier for supplying the 1C2 antibody, Jiping Zha for supplying the GST-HMK vector, and Andy Lieberman for discussions and critical reading of the manuscript. The work was supported by NIH grants NS36248 (D.E.M.), NS32214 (K.H.F.), and by grants from the March of Dimes Birth Defects Foundation and the Muscular Dystrophy Association.
Human Molecular Genetics
Pages
Introduction
Results
Construction of AR plasmids with very long CAG repeats
Toxicity of truncated AR containing very long CAG repeats
Protein expression
Cellular distribution of AR aggregates
Proteolytic processing of expanded, truncated AR protein
GST fusion proteins
Repeat length-dependent AR aggregation occurs in a cell-free system
Discussion
Materials And Methods
Cell lines and reagents
Creation of truncated AR
Expansion of the CAG repeat
Transfection of Cos-7 cells and analysis of toxicity
Immunoblotting and immunofluorescence
Creation of GST fusion constructs, expression, purification and labeling
Acknowledgements
References
Figure
REFERENCES
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K.J. Cowan, M.I. Diamond, and W.J. Welch Polyglutamine protein aggregation and toxicity are linked to the cellular stress response Hum. Mol. Genet., June 15, 2003; 12(12): 1377 - 1391. [Abstract] [Full Text] [PDF]
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T. Okuda, H. Hattori, S. Takeuchi, J. Shimizu, H. Ueda, J. J. Palvimo, I. Kanazawa, H. Kawano, M. Nakagawa, and H. Okazawa PQBP-1 transgenic mice show a late-onset motor neuron disease-like phenotype Hum. Mol. Genet., April 1, 2003; 12(7): 711 - 725. [Abstract] [Full Text] [PDF]
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J. P. Taylor, F. Tanaka, J. Robitschek, C. M. Sandoval, A. Taye, S. Markovic-Plese, and K. H. Fischbeck Aggresomes protect cells by enhancing the degradation of toxic polyglutamine-containing protein Hum. Mol. Genet., April 1, 2003; 12(7): 749 - 757. [Abstract] [Full Text] [PDF]
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J. L. Walcott and D. E. Merry Ligand Promotes Intranuclear Inclusions in a Novel Cell Model of Spinal and Bulbar Muscular Atrophy J. Biol. Chem., December 20, 2002; 277(52): 50855 - 50859. [Abstract] [Full Text] [PDF]
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H.Y. E. Chan, J. M. Warrick, I. Andriola, D. Merry, and N. M. Bonini Genetic modulation of polyglutamine toxicity by protein conjugation pathways in Drosophila Hum. Mol. Genet., November 1, 2002; 11(23): 2895 - 2904. [Abstract] [Full Text] [PDF]
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 Y. L. Giwercman, A. Nordenskjold, E. M. Ritzen, K. O. Nilsson, S.-A. Ivarsson, U. Grandell, and A. Wedell An Androgen Receptor Gene Mutation (E653K) in a Family with Congenital Adrenal Hyperplasia due to Steroid 21-Hydroxylase Deficiency as well as in Partial Androgen Insensitivity J. Clin. Endocrinol. Metab., June 1, 2002; 87(6): 2623 - 2628. [Abstract] [Full Text] [PDF]
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C. K. Bailey, I. F. M. Andriola, H. H. Kampinga, and D. E. Merry Molecular chaperones enhance the degradation of expanded polyglutamine repeat androgen receptor in a cellular model of spinal and bulbar muscular atrophy Hum. Mol. Genet., March 1, 2002; 11(5): 515 - 523. [Abstract] [Full Text] [PDF]
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N. J. Caplen, J. P. Taylor, V. S. Statham, F. Tanaka, A. Fire, and R. A. Morgan Rescue of polyglutamine-mediated cytotoxicity by double-stranded RNA-mediated RNA interference Hum. Mol. Genet., January 1, 2002; 11(2): 175 - 184. [Abstract] [Full Text] [PDF]
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W. J. Welch and M. I. Diamond Glucocorticoid modulation of androgen receptor nuclear aggregation and cellular toxicity is associated with distinct forms of soluble expanded polyglutamine protein Hum. Mol. Genet., December 1, 2001; 10(26): 3063 - 3074. [Abstract] [Full Text] [PDF]
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 R. W. Orrell and D. A. Figlewicz Clinical implications of the genetics of ALS and other motor neuron diseases Neurology, July 10, 2001; 57(1): 9 - 17. [Abstract] [Full Text] [PDF]
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A. Abel, J. Walcott, J. Woods, J. Duda, and D. E. Merry Expression of expanded repeat androgen receptor produces neurologic disease in transgenic mice Hum. Mol. Genet., January 1, 2001; 10(2): 107 - 116. [Abstract] [Full Text] [PDF]
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 G. Cancel, C. Duyckaerts, M. Holmberg, C. Zander, G. Yvert, A.-S. Lebre, M. Ruberg, B. Faucheux, Y. Agid, E. Hirsch, et al. Distribution of ataxin-7 in normal human brain and retina Brain, December 1, 2000; 123(12): 2519 - 2530. [Abstract] [Full Text] [PDF]
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A. McCampbell, J. P. Taylor, A. A. Taye, J. Robitschek, M. Li, J. Walcott, D. Merry, Y. Chai, H. Paulson, G. Sobue, et al. CREB-binding protein sequestration by expanded polyglutamine Hum. Mol. Genet., September 1, 2000; 9(14): 2197 - 2202. [Abstract] [Full Text] [PDF]
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H.N. Lim, H. Chen, S. McBride, A.M. Dunning, R.M. Nixon, I.A. Hughes, and J.R. Hawkins Longer polyglutamine tracts in the androgen receptor are associated with moderate to severe undermasculinized genitalia in XY males Hum. Mol. Genet., March 22, 2000; 9(5): 829 - 834. [Abstract] [Full Text] [PDF]
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Y. Kobayashi, A. Kume, M. Li, M. Doyu, M. Hata, K. Ohtsuka, and G. Sobue Chaperones Hsp70 and Hsp40 Suppress Aggregate Formation and Apoptosis in Cultured Neuronal Cells Expressing Truncated Androgen Receptor Protein with Expanded Polyglutamine Tract J. Biol. Chem., March 17, 2000; 275(12): 8772 - 8778. [Abstract] [Full Text] [PDF]
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 M. I. Diamond, M. R. Robinson, and K. R. Yamamoto Regulation of expanded polyglutamine protein aggregation and nuclear localization by the glucocorticoid receptor PNAS, January 18, 2000; 97(2): 657 - 661. [Abstract] [Full Text] [PDF]
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S. Simeoni, M. A. Mancini, D. L. Stenoien, M. Marcelli, N. L. Weigel, M. Zanisi, L. Martini, and A. Poletti Motoneuronal cell death is not correlated with aggregate formation of androgen receptors containing an elongated polyglutamine tract Hum. Mol. Genet., January 1, 2000; 9(1): 133 - 144. [Abstract] [Full Text] [PDF]
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M. D. Kaytor and S. T. Warren Aberrant Protein Deposition and Neurological Disease J. Biol. Chem., December 31, 1999; 274(53): 37507 - 37510. [Full Text] [PDF]
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 L. V. Nazareth, D. L. Stenoien, W. E. Bingman III, A. J. James, C. Wu, Y. Zhang, D. P. Edwards, M. Mancini, M. Marcelli, D. J. Lamb, et al. A C619Y Mutation in the Human Androgen Receptor Causes Inactivation and Mislocalization of the Receptor with Concomitant Sequestration of SRC-1 (Steroid Receptor Coactivator 1) Mol. Endocrinol., December 1, 1999; 13(12): 2065 - 2075. [Abstract] [Full Text]
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P.-W. Hsiao, D.-L. Lin, R. Nakao, and C. Chang The Linkage of Kennedy's Neuron Disease to ARA24, the First Identified Androgen Receptor Polyglutamine Region-associated Coactivator J. Biol. Chem., July 16, 1999; 274(29): 20229 - 20234. [Abstract] [Full Text] [PDF]
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K. L. Moulder, O. Onodera, J. R. Burke, W. J. Strittmatter, and E. M. Johnson Jr Generation of Neuronal Intranuclear Inclusions by Polyglutamine-GFP: Analysis of Inclusion Clearance and Toxicity as a Function of Polyglutamine Length J. Neurosci., January 15, 1999; 19(2): 705 - 715. [Abstract] [Full Text] [PDF]
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M. K. Perez, H. L. Paulson, S. J. Pendse, S. J. Saionz, N. M. Bonini, and R. N. Pittman Recruitment and the Role of Nuclear Localization in Polyglutamine-mediated Aggregation J. Cell Biol., December 14, 1998; 143(6): 1457 - 1470. [Abstract] [Full Text] [PDF]
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V. Heiser, E. Scherzinger, A. Boeddrich, E. Nordhoff, R. Lurz, N. Schugardt, H. Lehrach, and E. E. Wanker Inhibition of huntingtin fibrillogenesis by specific antibodies and small molecules: Implications for Huntington's disease therapy PNAS, June 6, 2000; 97(12): 6739 - 6744. [Abstract] [Full Text] [PDF]
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A. McCampbell, A. A. Taye, L. Whitty, E. Penney, J. S. Steffan, and K. H. Fischbeck Histone deacetylase inhibitors reduce polyglutamine toxicity PNAS, December 18, 2001; 98(26): 15179 - 15184. [Abstract] [Full Text] [PDF]
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