Human Molecular Genetics, 2001, Vol. 10, No. 26 3063-3074
© 2001 Oxford University Press
Glucocorticoid modulation of androgen receptor nuclear aggregation and cellular toxicity is associated with distinct forms of soluble expanded polyglutamine protein
Departments of Neurology, and Cellular and Molecular Pharmacology and 1Departments of Surgery, Medicine and Physiology, University of California, San Francisco, CA 94143-0450, USA
Received August 29, 2001; Revised and Accepted October 23, 2001.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Spinobulbar muscular atrophy is a progressive motor neuron disease caused by abnormal polyglutamine tract expansion in the androgen receptor (AR) gene, and is part of a family of central nervous system (CNS) neurodegenerative diseases, including Huntington’s disease (HD). Each pathologic protein is widely expressed, but the cause of neuronal degeneration within the CNS remains unknown. Many reports now link abnormal polyglutamine protein aggregation to pathogenesis. A previous study reported that activation of the wild-type glucocorticoid receptor (wtGR) suppressed the aggregation of expanded polyglutamine proteins derived from AR and huntingtin, whereas a mutant receptor containing an internal deletion, GR
108–317, increased polyglutamine protein aggregation, in this case primarily within the nucleus. In this study, we use these two forms of GR to study expanded polyglutamine AR protein in different cell contexts. Using cell biology and biochemical approaches, we find that wtGR promotes soluble forms of the protein and prevents nuclear aggregation in NIH3T3 cells and cultured neurons. In contrast, GR108–317 decreases polyglutamine protein solubility, and causes formation of nuclear aggregates in non-neuronal cells. Nuclear aggregates recruit hsp72 more rapidly than cytoplasmic aggregates, and are associated with decreased cell viability. Limited proteolysis and chemical cross-linking suggest unique soluble forms of the expanded AR protein underlie these distinct biological activities. These observations provide an experimental framework to understand why expanded polyglutamine proteins may be toxic only to certain populations of cells, and suggest that unique protein associations or conformations of expanded polyglutamine proteins may determine subsequent cellular effects such as nuclear localization and cellular toxicity. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Polyglutamine expansion in distinct proteins accounts for nine inherited neurodegenerative diseases including spinobulbar muscular atrophy (SBMA), Huntington’s disease (HD), spinocerebellar ataxia (SCA) 1, 2, 3, 6, 7 and 12, and dentatorubro-pallidoluysian atrophy (1–3). In each disease, CAG repeat expansion in DNA coding sequence results in a polyglutamine tract enlargement within the target protein. With only one exception (SCA6), expansion must exceed 37 repeats to produce disease. SBMA is a slow, progressive neurodegenerative disease that primarily involves motor neurons in the central nervous system (CNS) (4), although sensory neurons of the peripheral nervous system can also be affected (5). It is caused by CAG repeat expansion in the androgen receptor (AR) gene, which enlarges a polyglutamine tract in the N-terminus of the AR protein (6) (Fig. 1A). Polyglutamine expansion may affect AR transcriptional activity (7) or protein expression levels (8). However, neurotoxicity of the expanded AR protein is probably due to a gain of abnormal toxic function, as in other polyglutamine neurodegenerative diseases (1,2). Specifically, the expanded AR protein may acquire a new toxic cellular activity that is independent of its normal function as a transcription factor.
|
A remarkable feature of the polyglutamine expansion diseases is their selective neuronal toxicity: each disorder primarily affects neurons of the CNS while sparing non-neuronal cells, despite ubiquitous expression of the pathologic protein throughout the body. In addition, each disorder selectively targets subgroups of neurons within the CNS. For example, in SBMA an abnormal AR protein is expressed throughout the brain, but exclusively involves motor neurons, whereas HD targets cortical and striatal neurons, but spares motor neurons. Factors that determine regional degeneration within the CNS may be specific for each disease protein. On the other hand, understanding the molecular basis of the selective vulnerability of the CNS (as opposed to other somatic tissue) may be important in the development of effective therapies for multiple polyglutamine expansion diseases.
A unifying pathologic feature of polyglutamine expansion diseases is the formation of nuclear inclusions containing the pathologic protein (9–14). These inclusions are formed primarily, if not exclusively, in affected neurons of the CNS, and thus have been linked to pathogenesis. However, the role of nuclear inclusions in the pathogenesis of disease is controversial: some suggest they may underlie toxicity (15–17) whereas others believe that they are not central to disease (18), and may even be protective (19). Nonetheless, nuclear localization of expanded polyglutamine proteins does appear to be required for their toxicity both in cell culture and transgenic mouse models of disease (18,19).
Regardless of the role of neuronal intranuclear inclusions in pathogenesis, the capacity to form such nuclear aggregates may be related to a unique form of the expanded polyglutamine protein in vulnerable cells. That is, in the appropriate cellular environment, an expanded polyglutamine protein may exist in a form that is both pathogenic and capable of forming aggregates through self-association. This is analogous to fibril formation of prion proteins. In this case a normally folded prion protein may be subverted to a pathogenic form capable of producing detergent-insoluble fibrils, even though the infectious ‘unit’ may actually be a monomer or oligomer (20). Thus, self-association into aggregates might be a characteristic feature of expanded polyglutamine proteins in affected cells. However, despite the correlation of aggregate formation with pathologic changes in vulnerable cells, self-association of abnormal protein does not appear to be required for initial toxicity. For example, inhibition of ataxin-1 aggregation through deletion of a self-association region (18), or elimination of a ubiquitin ligase activity (21) does not prevent its toxicity in animal models. Ultimately, pathogenesis of polyglutamine disease could occur at several levels. Soluble protein might disrupt normal protein–protein interactions, possibly affecting certain transcription factors (22–24). Additionally, aggregates themselves might produce additional toxicity as a consequence of chronic cellular exposure to a misfolded protein (25,26).
In a previous study, one of us (M.I.Diamond) reported that the glucocorticoid receptor (GR) can regulate the aggregation and subcellular localization of both polyglutamine-expanded AR and huntingtin (htt) fragments (27). GR, a ligand-activated transcription factor, exists in the cytoplasm complexed with various molecular chaperones. Upon binding hormone, activated GR moves into the nucleus, where it regulates specific gene expression by binding to DNA sites near regulated genes. When co-expressed with expanded forms of AR or huntingtin (htt), we observed that the wild-type GR (wtGR) reduced aggregate formation upon hormone activation. Those aggregates that did form were restricted to the cytoplasm. In contrast, co-expression of a deletion mutant in the N-terminus of GR, in which a major transcription activation domain has been disrupted (GR108–317), enhanced aggregate formation. We observed these aggregates almost exclusively within the nucleus (27) (Fig. 1B). This data suggested that through modulation of gene expression these different forms of GR dramatically modified the solubility and subcellular localization of the expanded polyglutamine proteins. Thus, GR appears to regulate cellular factors that might modify the toxicity of an expanded polyglutamine protein.
In the present study we have further characterized the impact of wtGR and GR108–317 on the cell biology and biochemistry of expanded polyglutamine AR proteins. We exploit our ability to manipulate aggregation and nuclear localization of the AR protein in a single cell type to investigate its distinct molecular and biological activities in different cell contexts.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Glucocorticoid receptor mutants differentially control the subcellular localization and aggregation potential of AR protein truncation products
GR and GR
108–317 differentially regulate aggregation and nuclear localization of a truncated form of AR in HEK293 (kidney) and N2a (neuroblastoma) cells (27) (Fig. 1B). We further tested these effects in the NIH3T3 fibroblast cell line. We used a full-length form of AR, and truncation mutants comprised either of its N-terminal 127 or 556 amino acids with a wild-type (25) or expanded (65) tract of glutamines, termed ARN127 and ARN556, respectively (Fig. 1A). All constructs contained an HA epitope tag at the N-terminus to facilitate detection. We focused our attention on truncated forms of AR because investigations using cultured cells (28,29) and SBMA patient tissue (10) have suggested that the AR protein may be cleaved in vivo to generate a short fragment containing the expanded polyglutamine tract. Additionally, we and others have observed that only the most truncated form of the expanded AR protein [ARN127(65)] spontaneously aggregates when expressed in a variety of cell types (27,28). Last, truncated forms of the protein have been reported to produce cell toxicity both in cultured cells (28,29) and in a mouse model of SBMA (30), whereas the full-length molecule has not (31).
We transfected NIH3T3 cells with ARN127, ARN556 and full-length AR, in conjunction with constitutively active wild-type form, GRN525, or the mutant form, GRN525108–317. Full-length and constitutively active forms of GR have identical effects on polyglutamine protein aggregation (data not shown), and constitutively active forms were used purely for convenience. After 48 h cells were fixed in paraformaldehyde or methanol, and then stained for the presence of the HA-labeled AR proteins (Fig. 2). In prior studies we had reported that GR108–317 had no effect on unexpanded forms of AR in HEK293 cells, with most of the protein localized within the cytoplasm, and no apparent formation of protein aggregates. However, in the NIH3T3 cells examined using methanol fixation we detected a small population of cells (
1–5%) that exhibited nuclear accumulation of ARN127(25), but only when co-expressed with GRN525108–317. The vast majority of cells showed soluble ARN127(25) protein in all cases. When cells expressing ARN127(65) were co-transfected with GRN525108–317, 70% of transfected cells showed dense nuclear inclusions. These dense aggregates of ARN127(65) were distinct from the less dense and more granular-appearing accumulations of ARN127(25) that rarely occurred in the nucleus. Co-expression of ARN127(65) with wtGR produced cells that either had cytoplasmic aggregates or only soluble protein. Table 1 summarizes these findings and those described below.
|
|
Expression of a longer truncation derivative of AR containing the N-terminal 556 amino acids, ARN556, revealed similar results (Fig. 2 and Table 1). First, both expanded and unexpanded ARN556 protein exhibited a diffuse cytoplasmic distribution when co-expressed with GRN525 (Fig. 2; data not shown). In contrast, co-expression of GRN525
108–317 led to the formation of nuclear aggregates of ARN556(65) in 10–15% of transfected cells. GRN525108–317 had no apparent effect on the subcellular localization of ARN556(25). In this case, all of the protein was observed within the cytoplasm in a soluble form (data not shown). Finally, full-length AR(25) and AR(65) at equal frequency both spontaneously formed cytoplasmic inclusions that had a distinct appearance from those inclusions formed by the truncated AR proteins. Upon hormone activation with 100 nM dihydrotestosterone, both forms of full-length AR translocated to the nucleus, and did not form inclusions (data not shown). Neither GRN525 nor GRN525108–317 co-expression had any effect on the localization or aggregation potential of full-length AR(25) or AR(65) (Fig. 2 and Table 1; data not shown). Thus, GR and GR108–317 appear to modify the aggregation potential only of the truncated forms of the expanded AR protein. In addition, under these conditions in non-neuronal cells, nuclear inclusions characteristic of SBMA and other polyglutamine expansion diseases only occurred in those cells co-expressing GRN525108–317 (or full-length GR108–317; data not shown).
GR and GR108–317 differentially regulate AR protein detergent solubility
Biochemical fractionation using detergent extraction corroborated our observations with indirect immunofluorescence. We transfected NIH3T3 cells with both wild-type and expanded ARN127 and ARN556, and full-length AR, along with GRN525 or GRN525108–317, or an inactive form of GR harboring a point mutation that abrogates DNA binding, GRN525(R466K). The cells were lysed in buffer containing high levels of non-ionic detergent, and then fractionated by centrifugation. Equivalent amounts of extract (normalized for total SDS-soluble AR protein expression) from both the supernatant and the detergent-insoluble pellet were analyzed by SDS–PAGE and western blotting (Fig. 3). Co-expression of wtGR slightly decreased the amounts of insoluble ARN127 and ARN556 (Fig. 3A). In contrast, GR108–317 co-expression significantly increased the levels of insoluble ARN127 and ARN556 regardless of polyglutamine length, based on gross appearance and densitometry (Fig. 3A). On the other hand, neither GRN525 nor GRN525108–317 affected the solubility of full-length AR with an unexpanded or expanded polyglutamine tract (Fig. 3B). Of note, we found that full-length expanded AR(65) showed much higher levels of spontaneous protein cleavage fragments than did AR(25) (Fig. 3B). In summary, with the exception of ARN556(25), which showed detergent insolubility not detected by immunofluorescence, GR appears to modify the detergent solubility of truncated forms of AR in a manner that parallels our assessment of aggregation by immunofluorescence in cultured cells.
|
In primary neurons, ARN127(65) spontaneously forms nuclear inclusions; wtGR activation prevents their formation
Our experiments in non-neuronal cells indicated that ARN127(65) typically aggregated in the cytoplasm. However, when co-expressed with GR
108–317 the majority of aggregates localized within the nucleus. Because polyglutamine diseases exclusively affect the CNS, we evaluated ARN127(25) and ARN127(65) expression in primary neurons. Cultured primary striatal rat neurons were transfected with ARN127(25) and ARN127(65). Staining for ARN127(25) revealed only a diffuse cytosolic distribution (data not shown). However, expression of ARN127(65) resulted in the spontaneous formation of inclusions that localized almost exclusively within the nucleus (Fig. 4). Co-transfection of wtGR reduced the formation of nuclear inclusions in these cells 60% upon hormone activation (Fig. 4, graph). On the other hand, co-expression of GR108–317 had little or no effect on inclusion formation, which may reflect that the potential for nuclear aggregation in these cells is already at a maximum. Surprisingly, we did not observe polyglutamine-dependent cell death in this assay system, which may be due to a combination of high background rate of cell loss, and low transfection efficiency. To summarize, in neurons (as opposed to non-neuronal cells) the expanded ARN127(65) truncation protein spontaneously formed nuclear inclusions, and co-expression of wtGR inhibited this process in a manner similar to non-neuronal cells.
|
In NIH3T3 cells nuclear aggregates of polyglutamine protein recruit hsp72 more rapidly than cytoplasmic aggregates
Both nuclear and cytoplasmic aggregates have been described in patient material and transgenic mouse models of HD (9,32,33), but it is still somewhat unclear how these phenomena are linked to cell toxicity. Because accumulation of abnormally folded proteins can activate the cellular heat shock, or stress response, and polyglutamine aggregates are known to recruit heat shock proteins, we examined whether cytoplasmic versus nuclear accumulations of expanded polyglutamine proteins would differentially recruit a stress protein. We relied on hsp72, the most highly inducible member of the hsp protein family, whose expression (in rodent cells) is not detected in the absence of cell stress. NIH3T3 cells were transfected with ARN127(25) or ARN127(65) and GRN525 or GRN525
108–317, and then fixed and double-label stained for AR and hsp72 localization. After 48 or 72 h, cells transfected with ARN127(25) showed no significant aggregation (data not shown). In cells transfected with ARN127(65) and GRN525, soluble protein and cytoplasmic aggregates were observed. After 72 h in culture, these cytoplasmic aggregates clearly co-localized with hsp72 (data not shown). When co-expressed with GRN525108–317, ARN127(65) aggregation occurred almost exclusively within the cell nucleus. In these cells we observed a robust co-localization of hsp72 with the ARN127(65) nuclear aggregates by 36–48 h, while at this earlier time point in cells containing only cytoplasmic aggregates we did not observe strong co-localization with hsp72 (Fig. 5). Quantification of these observations revealed that after 48 h in culture, 75% of cells with nuclear aggregates had strong recruitment of hsp72, whereas <1% of cells with cytoplasmic aggregates showed this phenomenon. Thus, whereas both forms of misfolded polyglutamine protein ultimately recruit hsp72, nuclear aggregates appear to recruit this stress protein more rapidly.
|
ARN127(65) decreases cell viability only in the context of nuclear inclusion formation
Our finding that nuclear aggregates of ARN127(65) were associated with a more rapid co-localization with hsp72 led us to test whether expanded polyglutamine protein expression would decrease cell viability under conditions associated with nuclear inclusion. NIH3T3 cells were transfected with ARN127(25) or ARN127(65), wtGR or GR
108–317, and a GFP expression plasmid to identify transfected cells. We assayed relative survival of the transfected cells by counting the number of cells expressing GFP at 24, 48 and 72 h. No differences in viability were observed at 24 or 48 h. However, by 72 h, ARN127(65) expression produced a 50% decrease in the number of GFP-positive cells, but only in the context of co-expressed hormone-activated GR108–317 (Fig. 6). This was accompanied by clear evidence of cytotoxicity—rounded cell bodies, and cells detaching from the dish. Co-expression of ARN127(25) with either wtGR or GR108–317 had no significant effect on cell viability over the 72 h experiment. Thus, toxicity of the expanded polyglutamine protein is not simply related to its expression within a cell. Rather, this toxicity requires an appropriate cellular context, in this case determined by the hormone-dependent activation of the mutant form of the GR, GR108–317.
|
Distinct forms of expanded AR protein underlie its cellular activities
In an effort to identify molecular characteristics of ARN127(65) that might be associated with its cytotoxicity, we examined some of the biochemical properties of ARN127(65) produced in cells co-expressing either wtGR or GR
108–317. In particular, we attempted to evaluate soluble forms of AR protein before its recruitment into insoluble macro-aggregates. HEK293 cells were transfected with ARN127(25) or ARN127(65) and constitutively active GRN525 or GRN525108–317 (these cells were used for their ease of transfection and high yield of expressed proteins; identical results were obtained with extracts derived from transfected NIH3T3 cells). After 48 h, cells were harvested and lysed in a buffered solution containing 0.05% Triton. Following clarification by centrifugation at 15 000 g to pellet insoluble protein, nuclei and cellular debris, the supernatants were removed and soluble forms of ARN127 examined by limited proteolysis. Extracts were treated with chymotrypsin for 0, 20 or 40 min before stopping the reactions by addition of SDS-sample buffer and heating to 95°C. Samples were then resolved by SDS–PAGE and western blotting.
Figure 7A shows that co-expression of GRN525 or GRN525108–317 had no effect on the gel migration pattern of non-proteolyzed forms of either ARN127(25) or ARN127(65), suggesting that neither covalent modification nor cleavage of the protein had occurred within the cell. However, chymotrypsin digestion revealed a marked difference as a function of co-expressed GR. In particular, ARN127(65) co-expressed with GRN525 generated a 7 kDa digestion product that was reduced by a factor of 10–20 times (based on densitometry) in those samples derived from cells expressing GRN525108–317. Chymotrypsin digestion of unexpanded ARN127(25) protein from cells co-expressing GRN525 also produced a 7 kDa band. However, in this case, co-expression of GRN525108–317 did not affect levels of this 7 kDa product. We also noted that the overall protease sensitivity of the full-length ARN127(65) protein appeared to be increased by co-expression with GRN525108–317, as shown by a decrease in uncleaved protein in the lane with 40 min of digestion. Protein sequence analysis reveals a putative chymotrypsin consensus cleavage site N-terminal to the polyglutamine tract (Fig. 7B). To rule out the possibility that our observations were limited to one protease, we performed a similar experiment using a more non-specific protease (proteinase K). Although the signal intensity was diminished by proteolytic degradation of the HA tag, this also showed preferential formation of a small cleavage product of 7 kDa only in the context of wtGR (Fig. 7C).
|
To exclude the possibility that soluble micro-aggregates of ARN127(65) were responsible for the difference in protease susceptibility, we subjected unexpanded and expanded ARN127 from the two different cell contexts (i.e. GRN525 versus GRN525
108–317) to native (non-denaturing) gel electrophoresis. As shown in Figure 8A, the migration of the two forms of the protein was virtually identical, without evidence for different higher order structures in the gel. This study implies that the clarified extracts contain largely lower order forms of ARN127 (since we cannot exclude that the bands observed on native gels constitute heteromers of protein). Likewise, by this assay there was no observable difference in the aggregation state of the soluble ARN127 protein in the context of either wtGR or GR108–317.
|
We next tested whether the ARN127 protein might have unique protein associations by using chemical cross-linking. Clarified extracts were prepared by non-ionic detergent lysis and centrifugation at 15 000 g and were subjected to chemical cross-linking for 5 or 60 s with glutaraldehyde, followed by SDS–PAGE and western blot. We observed in multiple repetitions a shifted band of
110 kDa in the context of wtGR, whereas no such band was observed in the context of GR108–317 (Fig. 8B). This result implies that under conditions where it fails to form nuclear inclusions, ARN127(65) protein may have a unique association with another protein of 50–60 kDa. However, we cannot exclude the possibility that the 110 kDa shifted band we observe with ARN127(65) represents its self-association. Regardless, our data suggests that soluble ARN127(65) protein may exist in at least two forms, depending on the cellular environment. One form (that which forms a distinct cross-linked species, and produces a 7 kDa digestion product) appears to have less toxic potential, whereas the other form is associated with nuclear inclusion formation and cell toxicity. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
By exploiting wtGR and a mutant that counteracts its function, GR
108–317, we have studied an expanded polyglutamine protein under two conditions. In one condition (in the context of wtGR), we propose the expanded protein exists in a ‘less toxic’ form, wherein it remains largely soluble (or forms cytoplasmic aggregates), and is not particularly toxic to the cell. However, when expressed in the context of GR108–317 it has reduced solubility, forms nuclear aggregates that rapidly recruit a cellular stress protein, and reduces cell viability. These observations indicate that the cellular context in which the expanded polyglutamine protein is expressed likely affects its conformation and/or protein interactions in solution, and subsequently its relative toxicity to the cell. Figure 9 illustrates a model of this hypothesis.
|
In both established cells and in primary neurons we observed that glucocorticoid-regulated gene expression modified the subcellular distribution and macro-aggregation of AR protein. Activation of GR
108–317 increased ARN127(65) aggregation, with aggregates localized primarily within the cell nucleus. Conversely, wtGR activation reduced the total number of aggregates, and those that did form were restricted to the cytoplasm. These observations at a cellular level had biochemical correlates. wtGR decreased and GR108–317 increased the amount of detergent-insoluble/nuclear forms of ARN127 and ARN556. We also observed that whereas both nuclear and cytoplasmic aggregates ultimately recruited an induced stress protein, hsp72, nuclear aggregates appeared to recruit this protein more readily. Moreover, we observed a clear reduction in cell viability only in the cellular context wherein nuclear inclusions formed. Finally, using independent biochemical assays, we found evidence for distinct forms of the expanded polyglutamine ARN127(65) protein in solution as a function of co-expression of wtGR versus GR108–317. These results may help in our understanding of the pathogenesis of polyglutamine expansion diseases. First, in NIH3T3 cells, formation of nuclear aggregates correlated with greater cell toxicity than did cytoplasmic aggregates, suggesting that such nuclear inclusions mark a uniquely pathological form of the expanded polyglutamine protein. Moreover, we observed that nuclear inclusions more rapidly recruited hsp72, the most stress-inducible member of the heat shock protein family. Whereas numerous prior reports have documented chaperone co-localization with aggregates, this is the first report, to our knowledge, that particularly links nuclear aggregation with stress responses. Thus, our work extends prior research by demonstrating by two independent measures (stress protein recruitment and cell death) that nuclear aggregates are especially associated with cell toxicity. Whereas nuclear inclusions were correlated with decreased cell survival, the precise mechanism underlying the observed toxicity is still unclear. Notably, we observed that in primary neurons, the site of greatest toxicity in vivo, ARN127(65) spontaneously formed almost exclusively nuclear inclusions. Along these lines, Merry et al. (28) found that expression of a truncated AR protein in a motor neuron line produced primarily nuclear inclusions, but that expression in non-neuronal cells produced primarily cytoplasmic inclusions. Finally, in studies of a transgenic mouse model of SBMA, Abel et al. (30) also observed nuclear inclusion formation in brain tissue, but did not observe such inclusions in muscle or testis. These dramatic differences in the subcellular distribution and aggregation of a single protein as a function of cellular milieu underscore the existence of cell-specific factors that control these events.
Whereas glucocorticoid-regulated pathways clearly can modulate the processing of polyglutamine proteins, it remains unclear whether preventing nuclear inclusion formation per se has beneficial effects on neuronal survival in culture. However, our experiments with NIH3T3 cells suggest that survival is improved when aggregation is either prevented or restricted to the cytoplasm. An obvious limitation of our studies in cultured neurons was the lack of detectable polyglutamine-induced cell death. Unfortunately, we were unable to transfect a suitable number of cells to observe quantitative effects in this regard against a background of ongoing polyglutamine-independent cell death. This prevented us from asking directly whether inhibition of nuclear inclusion formation would protect against cell death in culture. The most valid test of any protective effect of glucocorticoid-responsive pathways will come from studies of animal models of expanded polyglutamine disease, which are being pursued.
Perhaps the most important implication of our results is that distinct forms of soluble ARN127(65), detected by independent assays, were linked with its biological activities in the cell. We found that under conditions when it is less likely to form intracellular aggregates or cause toxicity (wtGR activation), expanded polyglutamine AR had a distinct proteolytic susceptibility to chymotrypsin and proteinase K. This was consistent either with an increase in protease accessibility to a site within the N-terminus or with protection of the N-terminus from complete degradation (possibly through a unique protein association). Either scenario could explain the production of the 7 kDa cleavage product we observed primarily in the context of wtGR activation. Cross-linking data also emphasized the different molecular environments for ARN127 in these two contexts, as only with wtGR activation in the cell did a 110 kDa cross-link product form. This shifted band could represent dimers of the ARN127 protein in the context of wtGR that are unable to form in the context of GR108–317, but we feel that this band probably signifies a novel protein association—possibly a factor that prevents the formation of nuclear inclusions.
It is unlikely that differences we detect by proteolysis and cross-linking reflect micro-aggregates of protein that have remained in solution following centrifugation. Using native gel electrophoresis of clarified extract we found no differences in mobility of the proteins, suggesting that the soluble protein is not in large complexes in one of the conditions. We have also found no evidence of higher order structures using gel permeation chromatography, and no significant difference in passage through a 100 kDa size exclusion filter (data not shown), arguing against significant multimerization of the protein. Several prior studies have suggested that ubiquitination (19,21) or proteolytic cleavage (34,35) may underlie expanded polyglutamine aggregation and toxicity, respectively. We found no evidence for covalent modification (e.g. ubiquitination) or proteolytic cleavage of the expanded polyglutamine AR protein in the two cellular environments created by co-expression of wtGR and GR108–317: the proteins migrated identically on denaturing and non-denaturing gels. Although other explanations are possible, the simplest interpretation of our data is that biochemically distinct forms of the expanded polyglutamine AR protein are closely tied to, if not the underlying causes of, the various cellular effects we have observed.
Reports of other investigators have also suggested the existence of distinct conformers of expanded polyglutamine proteins, but have not clearly linked them to cellular toxicity. Recently, Krobitsch and Lindquist (36) showed that polyglutamine protein aggregation in yeast depended on expression of hsp104. Thus, in this system the transition of an expanded polyglutamine protein between aggregation-prone and non-aggregation-prone forms is subject to cellular control, and may involve a specific molecular chaperone. However, polyglutamine proteins expressed in yeast do not exhibit properties found in mammalian cells such as formation of nuclear inclusions and cellular toxicity, and humans lack an hsp104 homolog.
Others have used antibodies that preferentially recognize expanded polyglutamine domains (37,38) or proteolysis (39) to highlight conformational differences between expanded and unexpanded forms of polyglutamine proteins. However, a central point of our work is that an expanded polyglutamine protein itself may have distinct conformers associated with unique cellular consequences. Two prior studies have also raised this issue. Persichetti et al. (38) showed that an antibody, 1F8, failed to recognize expanded polyglutamine in its aggregated form, and concluded that the aggregated protein had a distinct conformation that masked the epitope. Whereas it is perhaps not surprising that an aggregated protein should have distinct conformations in comparison to its soluble counterpart, our study demonstrates distinct forms of expanded polyglutamine AR may exist in solution. In another study, Wheeler et al. (40) have demonstrated in a knock-in mouse model that a distinct epitope on the N-terminus of htt protein becomes accessible to the EM48 antibody when it is expressed in the context of medium spiny neurons, a population of cells involved in human disease. This result may indicate a novel conformation of htt protein (or a novel protein association) occurs in a cell context in which it produces injury. However, in this study, antibody accessibility in fixed tissue specimens was the sole criterion used to identify structural protein differences. Moreover, there was no direct link between htt conformers and cell toxicity, since the animal model used does not have an obvious striatal cellular pathology or behavioral phenotype.
We have corroborated and extended this prior work using independent biochemical assays of expanded polyglutamine protein in solution. We have found that distinct forms of ARN127(65) are closely associated with its behavior within the cell, and may determine its toxic potential, as well as its propensity for nuclear inclusion formation. What might be the determinants of these distinct conformers? Our cross-linking data suggest that ARN127(65) may have a unique binding partner in its ‘non-toxic’ form. Such a factor might be expected to have reduced levels or activities within tissue of the CNS. Several molecular chaperones, notably hsp70 and hsp40, have been implicated in preventing polyglutamine protein aggregation (41–49), but they are widely expressed in all tissues, including the brain. It is possible, alternatively, that other proteins may facilitate a conformation of the protein that leads to nuclear inclusion formation and cellular toxicity. Such a factor could constitute a binding partner that promotes nuclear localization of the expanded polyglutamine protein or leads to its sequestration within the nucleus. Along these lines, preliminary work using extract complementation in vitro has suggested that another factor (possibly a molecular chaperone) may actively convert ARN127(65) from a non-toxic to a toxic form that allows the expanded polyglutamine tract to manifest its pathologic activities.
Through which pathways might wtGR mediate its effects? Based on prior published (27) and unpublished work, we feel it is unlikely that GR or GR108–317 directly interact with polyglutamine proteins. Instead, it may be helpful to consider the role of glucocorticoid-dependent gene expression in the regulation of cell catabolic functions. In certain cells, GR has been described to upregulate proteasome function (50,51), and may also control the levels of certain chaperones, such as 
-B crystallin (52). In ongoing work we are investigating specific gene regulation by wtGR and GR108–317, which may provide clues as to the mediators of the activities we have observed. Whereas prevention of aggregation could result from increased polyglutamine protein degradation, we have not observed evidence for increased polyglutamine protein turnover in response to glucocorticoid activation, as reflected in changes in steady-state levels of protein (although we have not measured protein half-life in these different conditions). In addition, factors involved in protein turnover might have additional roles in determining aggregation potential of the polyglutamine protein, as formation of aggregates has been linked to cellular ubiquitin ligase activity by several investigators (19,21), and multiple reports describe co-localization of proteasome components with polyglutamine aggregates (41,46,53,54). Uncovering the GR-regulated factors that control expanded polyglutamine protein behavior within the cell may prove informative both from the standpoint of understanding glucocorticoid effects on cell physiology and pathogenesis of polyglutamine expansion diseases.
In summary, our studies show that when expressed in a non-neuronal cell, especially in association with wtGR activation, an expanded AR protein has a distinct pattern of cytoplasmic distribution and aggregation. Following alteration of cellular homeostasis (by expression of GR108–317), this same protein may have markedly different activities that parallel the phenotype observed in neurons. Each cellular environment produces distinct biochemical features of the expanded AR protein. Specifically, wtGR promotes a form that may have a unique protein partner, and is limited to soluble forms or cytoplasmic aggregates, whereas GR108–317 promotes a form that does not have the same protein associations, and is capable of nuclear aggregation and cellular toxicity. Thus, this work provides an experimental framework to begin to consider the problem of selective cellular toxicity of expanded polyglutamine proteins in molecular terms. In the future it may be possible to identify the factors that regulate the prevalence of these different forms of ARN127 using biochemical or genetic approaches, which could be of great utility in defining the cellular determinants of polyglutamine protein toxicity.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Plasmids
Plasmids encoding HA-tagged ARN127 (pHA.ARN127.ires.NEO) were created by cloning HA-tagged fragments of 127 N-terminal amino acids from human AR with either 25 or 65 glutamines into pIRES.NEO (Clontech). Truncation derivatives containing the N-terminal 556 amino acids of AR with either 25 or 65 glutamines (pHA.ARN556.ires.NEO) were similarly cloned. Glucocorticoid receptor expression plasmids were as described previously (27,55), and consisted of either full-length GR (p6RGR, p6RGR
108–317) or a truncated, constitutively active form which lacks the ligand-binding domain (p6RGRN525, p6RGRN525108–317) driven by the RSV promoter. GFP expression plasmid (pGL-1) consists of GFP cDNA driven by the CMV promoter. Constructs were checked by sequencing and functional testing using transcription assays and immunofluorescence.
Cell culture and transfection
NIH3T3 and HEK293 cells were grown in DME-H21 medium (4.5 g/l glucose) supplemented with 10% fetal bovine serum. For immunofluorescence and cell viability experiments, NIH3T3 cells were grown on glass coverslips in 24-well dishes. For immunofluorescence, cells were transfected with a total of 0.15 µg DNA (0.12 µg of HA.ARN127.IRES.NEO and 0.3 µg of p6RGRN525 or p6RGRN525108–317). For cell death experiments, using a total of 0.17 µg DNA per well (0.1 µg of HA.ARN127.IRES.NEO, 0.025 µg of p6RGR or p6RGR108–317, and 0.045 µg of pGL-1). Cells were transfected using Effectene reagent (Qiagen), using 8 µl/µg DNA of Enhancer and 10 µl/µg DNA of Effectene reagent. For transfection of HEK293 cells, cells were grown in 3.5 cm dishes, and they were transfected with Lipofectamine Plus reagent (Gibco BRL) using 1 µg total DNA and 4 µl Plus reagent, with 2.67 µl Lipofectamine reagent.
Primary striatal neurons were cultured from E18 fetal rats as previously described by Saudou et al. (19). They were grown on glass coverslips for 5 days prior to transfection in 24-well plates. Cells were transfected with 1.5 µg each of pHA.ARN127.ires.NEO expression plasmid and full-length p6RGR or p6RGR108–317 expression plasmid, using calcium phosphate precipitation according to methods previously described by Saudou et al. (19). Cells were treated either with 100 nM dexamethasone or EtOH vehicle control at the time of transfection. After 6 days in culture, cells were fixed and stained. Aggregate counts were performed by assessing the number of transfected neurons exhibiting macro-aggregates of protein as compared to the total number of transfected neurons. Approximately 50–100 transfected neurons were counted on each coverslip over multiple fields. Each data point represents the average counts from four coverslips.
Immunofluorescence studies
NIH3T3 cells growing on glass coverslips were transfected as described above. Samples expressing full-length GR or AR protein were treated either with 100 nM dexamethasone or dihydrotestosterone, respectively, or EtOH vehicle control. Thirty-six to 48 h later the cells were washed in PBS and fixed either by immersion in –20°C absolute methanol for 2 min or by incubation in 4% paraformaldehyde dissolved in PBS for 10 min followed by immersion in PBS containing 0.1% Triton X-100. Cells were stained for their distribution of the HA-tagged proteins using a mouse monoclonal antibody against HA diluted 1:4000 in PBS containing 5 mg/ml bovine serum albumin (BSA). Primary antibody was detected via incubation with a rhodamine-conjugated rabbit anti-mouse antibody (ICN/Cappel) diluted 1:50 in 5 mg/ml BSA PBS. For double-label staining, the fixed and permeabilized cells were incubated with a mouse monoclonal antibody specific for hsp72 (StressGen C92) diluted 1:800 in BSA/PBS plus rat anti-HA (Roche rat anti-HA antibody 3F10) diluted 1:1000. The mouse hsp72 antibody was detected using a Cy-conjugated goat anti-mouse antibody (rat preabsorbed; Jackson ImmunoResearch) diluted 1:2000. The rat HA antibody was detected via incubation with a fluorescein-conjugated rabbit anti-rat (mouse preabsorbed; ICN/Cappel) diluted 1:50. Primary neurons were fixed in paraformaldehyde and permeabilized as above, and stained with mouse anti-HA monoclonal antibody (mouse HA.11 anti-HA antibody; Covance) at 1:1000 dilution, and secondary antibody (Alexa 546 goat anti-mouse) from Molecular Probes at 1:500 dilution. DAPI stain was used at 0.5 µg/ml, included in the secondary antibody mix.
To measure relative recruitment of hsp72 to cytoplasmic versus nuclear aggregates, NIH3T3 cells were transfected as described above with ARN127(65), GRN525 or GRN525108–317 expression plasmids and cultured for 36–72 h. Cells were then rinsed with PBS/0.1% Triton and fixed in 100% methanol. Immunofluorescence was performed as described above using rat anti-HA antibody and mouse anti-Hsp72 antibody, with a DAPI counterstain. Cells were then visualized by standard fluorescence microscopy. The percentage of nuclear aggregates co-localized with Hsp72 was compared to the percentage of cytoplasmic aggregates co-localized with Hsp72 by counting 100–150 aggregate-containing cells on three different coverslips.
Imaging was accomplished using a Deltavision deconvoluting microscope and a 60x objective for Figures 2 and 5. Images for Figure 4 were acquired using standard fluorescence microscopy and a 100x objective.
Detergent fractionation
NIH3T3 cells growing in 6 cm dishes were transfected with 0.8 µg of plasmid DNA encoding either ARN127, ARN556 or full-length AR with 25 or 65 glutamines. In each case the cells were co-transfected with either p6RGRN525, p6RGR108–317 or p6RGRN525(R466K), which harbors a point mutation in the DNA-binding domain that abrogates DNA binding and transcriptional regulation. Forty-eight hours post-transfection the medium was removed, the cells washed in PBS and then lysed by the addition of 0.4 ml of PBS supplemented with 1% Triton X-100, 1% sodium deoxycholate and 5 mM MgCl (to stabilize chromatin). After vortexing, an aliquot of the cell lysate was removed (total) and the remainder centrifuged at 15 000 g for 15 min at 4°C. The supernatant was removed and adjusted with 5x concentrated Laemmli sample buffer. The pellet was resuspended in 2x Laemmli sample buffer. A sample of the total cell lysate was analyzed by SDS–PAGE and western blotting with the anti-HA antibody to determine the relative expression of the different HA-tagged AR species based on quantification of bands by densitometry. Subsequently, SDS–PAGE analysis of the different fractions (loading of the gel based on relative HA expression) was performed and the relative amounts of the HA-tagged species again determined by western blotting using mouse anti-HA antibody. Densitometry was performed by scanning gels and analyzing bands using NIH Image 1.62 software.
Cell viability assay
Cell viability assays were performed on transfected cells in triplicate minus and plus 100 nM dexamethasone. NIH3T3 cells were transfected as above with ARN127 and full-length GR and GR108–317 expression plamids and cultured for 72 h. At this time cells were fixed in 4% paraformaldehyde as described above. Cells were then examined using a 40x objective with a FITC filter set in place, which allowed visualization of GFP-positive cells. An average of 15 separate fields were counted for each coverslip and the total number of cells tabulated per field. Cell viability was assayed by comparing average GFP-positive cell number minus and plus hormone for three pairs in each condition. The number of viable GFP-positive cells after hormone treatment was reflected as a percentage of the total number of transfected cells untreated with hormone. This controlled for differences in transfection efficiency between different coverslip pairs, and thus normalized hormone effects over the experiment.
Proteolysis assays
Limited digestion with chymotrypsin and proteinase K was performed as follows. HEK293 cells were transfected as above in 3.5 cm dishes. After washing cells two times in chilled PBS in the dish, cells were scraped into PBS, concentrated by centrifugation and the pellets frozen for later use. To create lysates, the cell pellet was resuspended in ice-cold lysis buffer containing: protease inhibitors (1 µg/ml each of leupeptin, pepstatin and aprotinin), 25 mM HEPES pH 7.6, 100 mM KCl, 0.5 mM EDTA, 20% glycerol (v/v) and 0.05% Triton X-100. The cell lysate was clarified by centrifugation at 15 000 g for 10 min at 4°C, and the resultant supernatant used for the assays. Total protein concentration was then determined by the method of Bradford. Digests of ARN127 protein were performed at 25°C using 0.5 ng chymotrypsin/µg total protein or 0.03 ng proteinase K/µg total protein, using 10–20 µg of total cell extract in a 20 µl reaction volume. Undigested samples (i.e. those with no added protease) were incubated at room temperature for 40 min to control for endogenous protease activity. Reactions were stopped by the addition of SDS-sample buffer containing 5 mM PMSF and immediate heating at 95°C for 5 min. Approximately one-third or each reaction was run out on a 15% denaturing polyacrylamide gel, and protein was detected by western blot. Western blots were performed using primary antibody (mouse HA.11 anti-HA antibody; Covance) at 1:7500 dilution and secondary antibody (goat anti-mouse HRP conjugated antibody; Amersham) at 1:10 000 dilution. Identical results were obtained from repetition of this experiment at least five times from separate transfections.
Native gel electrophoresis
Cell lysates were prepared in an identical fashion as for limited proteolysis. Approximately 10 µg of total lysate from each experimental condition was loaded on a 3–10% gradient acrylamide gel, with a buffer consisting of 50 mM sodium acetate/50 mM boric acid pH 7.4, that was recirculated throughout the run. Gels were run at 4°C. Following the run, the gel was soaked in denaturing transfer buffer, transferred and subjected to western blot in the usual fashion as described above. These experiments were repeated more than five times, with identical results from separate transfections.
Chemical cross-linking
Cell lysates were prepared as for limited proteolysis and native gel electrophoresis. Instead, 15 µg of sample was diluted into a 10 µl reaction mix containing 5 mM ATP. A 1.1 µl aliquot of 3% glutaraldehyde solution (Sigma) was added (for final concentration of 0.3%), and the reactions were allowed to proceed for either 5 or 60 s. At this time the glutaraldehyde was quenched by addition of 3 µl of 180 mM Tris–HCl pH 7.4. A 4 µl aliquot of 5x SDS-sample buffer was added and SDS–PAGE was performed using a 10% gel, followed by western blot. Cross-linking experiments were performed more than five times with samples from different transfections with the same results.
|
ACKNOWLEDGEMENTS |
|---|
The authors wish to thank Steven Finkbeiner, for technical assistance and resources in transfections of primary neurons, and Sonia Pollitt for technical assistance with deconvoluting microscopy. For constructive comments on the manuscript we also thank Ivan Diamond, Brian Freeman, Sangram Sisodia, Jonathan Weissman, Alfred Goldberg and Keith Yamamoto. This work was supported by grants from The American Philosophical Society (M.I.D.), Muscular Dystrophy Association (M.I.D.) and the National Institutes of Health (M.I.D., W.J.W.).
|
FOOTNOTES |
|---|
+ To whom correspondence should be addressed. Tel: +1 415 476 2251; Fax: +1 415 502 8644; Email: marcd@itsa.ucsf.edu
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Zoghbi, H.Y. and Orr, H.T. (1999) Polyglutamine diseases: protein cleavage and aggregation. Curr. Opin. Neurobiol., 9, 566–570.[Web of Science][Medline]
2 MacDonald, M. and Gusella, J.F. (2000) Molecular genetics: unmasking polyglutamine triggers in neurodegenerative disease. Nat. Genet., 1, 109–115.
3 Evert, B.O., Wüllner, U. and Klockgether, T. (2000) Cell death in polyglutamine diseases. Cell Tissue Res., 301, 189–204.[Web of Science][Medline]
4 Kennedy, W.R., Alter, M. and Sung, J.H. (1968) Progressive proximal spinal and bulbar muscular atrophy of late onset. A sex-linked recessive trait. Neurology, 18, 671–680.
5 Harding, A.E., Thomas, P.K., Baraitser, M., Bradbury, P.G., Morgan-Hughes, J.A. and Ponsford, J.R. (1982) X-linked recessive bulbospinal neuronopathy: a report of ten cases. J. Neurol. Neurosurg. Psychiatry, 45, 1012–1019.
6 La Spada, A.R., Wilson, E.M., Lubahn, D.B., Harding, A.E. and Fischbeck, K.H. (1991) Androgen receptor gene mutations in X-linked spinal and bulbar muscular atrophy. Nature, 352, 77–79.[Medline]
7 Kazemi-Esfarjani, P., Trifiro, M.A. and Pinsky, L. (1995) Evidence for a repressive function of the long polyglutamine tract in the human androgen receptor: possible pathogenetic relevance for the (CAG)n-expanded neuronopathies. Hum. Mol. Genet., 4, 523–527.
8 Brooks, B.P., Paulson, H.L., Merry, D.E., Salazar-Grueso, E.F., Brinkmann, A.O., Wilson, E.M. and Fischbeck, K.H. (1997) Characterization of an expanded glutamine repeat androgen receptor in a neuronal cell culture system. Neurobiol. Dis., 3, 313–323.[Web of Science][Medline]
9 DiFiglia, M., Sapp, E., Chase, K.O., Davies, S.W., Bates, G.P., Vonsattel, J.P. and Aronin, N. (1997) Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science, 277, 1990–1993.
10 Li, M., Miwa, S., Kobayashi, Y., Merry, D.E., Yamamoto, M., Tanaka, F., Doyu, M., Hashizume, Y., Fischbeck, K.H. and Sobue, G. (1998) Nuclear inclusions of the androgen receptor protein in spinal and bulbar muscular atrophy. Ann. Neurol., 44, 249–254.[Web of Science][Medline]
11 Duyckaerts, C., Durr, A., Cancel, G. and Brice, A. (1999) Nuclear inclusions in spinocerebellar ataxia type 1. Acta Neuropathol. (Berl.), 97, 201–207.[Medline]
12 Gourfinkel-An, I., Cancel, G., Duyckaerts, C., Faucheux, B., Hauw, J.J., Trottier, Y., Brice, A., Agid, Y. and Hirsch, E.C. (1998) Neuronal distribution of intranuclear inclusions in Huntington’s disease with adult onset. Neuroreport, 9, 1823–1826.[Web of Science][Medline]
13 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.
14 Becher, M.W., Kotzuk, J.A., Sharp, A.H., Davies, S.W., Bates, G.P., Price, D.L. and Ross, C.A. (1998) Intranuclear neuronal inclusions in Huntington’s disease and dentatorubral and pallidoluysian atrophy: correlation between the density of inclusions and IT15 CAG triplet repeat length. Neurobiol. Dis., 4, 387–397.[Web of Science][Medline]
15 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]
16 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]
17 Perutz, M.F. (1999) Glutamine repeats and neurodegenerative diseases: molecular aspects. Trends Biochem. Sci., 24, 58–63.[Web of Science][Medline]
18 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]
19 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]
20 Horwich, A.L. and Weissman, J.S. (1997) Deadly conformations—protein misfolding in prion disease. Cell, 89, 499–510.[Web of Science][Medline]
21 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]
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 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.
24 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.
25 Welch, W.J. and Gambetti, P. (1998) Chaperoning brain diseases. Nature, 392, 23–24.[Medline]
26 Sherman, M.Y. and Goldberg, A.L. (2001) Cellular defenses against unfolded proteins: a cell biologist thinks about neurodegenerative diseases. Neuron, 29, 15–32.[Web of Science][Medline]
27 Diamond, M.I., Robinson, M.R. and Yamamoto, K.R. (2000) Regulation of expanded polyglutamine protein aggregation and nuclear localization by the glucocorticoid receptor. Proc. Natl Acad. Sci. USA, 97, 657–661.
28 Merry, D.E., Kobayashi, Y., Bailey, C.K., Taye, A.A. and Fischbeck, K.H. (1998) Cleavage, aggregation and toxicity of the expanded androgen receptor in spinal and bulbar muscular atrophy. Hum. Mol. Genet., 7, 693–701.
29 Ellerby, L.M., Hackam, A.S., Propp, S.S., Ellerby, H.M., Rabizadeh, S., Cashman, N.R., Trifiro, M.A., Pinsky, L., Wellington, C.L., Salvesen, G.S. et al. (1999) Kennedy’s disease: caspase cleavage of the androgen receptor is a crucial event in cytotoxicity. J. Neurochem., 72, 185–195.[Web of Science][Medline]
30 Abel, A., Walcott, J., Woods, J., Duda, J. and Merry, D. (2001) Expression of expanded repeat androgen receptor produces neurologic disease in transgenic mice. Hum. Mol. Genet., 10, 107–116.
31 Bingham, P.M., Scott, M.O., Wang, S., 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]
32 Li, H., Li, S.H., Johnston, H., Shelbourne, P.F. and Li, X.J. (2000) N-terminal fragments of mutant huntingtin show selective accumulation in striatal neurons and synaptic toxicity. Nat. Genet., 25, 385–389.[Web of Science][Medline]
33 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.
34 Ellerby, L.M., Andrusiak, R.L., Wellington, C.L., Hackam, A.S., Propp, S.S., Wood, J.D., Sharp, A.H., Margolis, R.L., Ross, C.A., Salvesen, G.S. et al. (1999) Cleavage of atrophin-1 at caspase site aspartic acid 109 modulates cytotoxicity. J. Biol. Chem., 274, 8730–8736.
35 Wellington, C.L., Singaraja, R., Ellerby, L., Savill, J., Roy, S., Leavitt, B., Cattaneo, E., Hackam, A., Sharp, A., Thornberry, N. et al. (2000) Inhibiting caspase cleavage of huntingtin reduces toxicity and aggregate formation in neuronal and nonneuronal cells. J. Biol. Chem., 275, 19831–19838.
36 Krobitsch, S. and Lindquist, S. (2000) Aggregation of huntingtin in yeast varies with the length of the polyglutamine expansion and the expression of chaperone proteins. Proc. Natl Acad. Sci. USA, 97, 1589–1594.
37 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]
38 Persichetti, F., Trettel, F., Huang, C.C., Fraefel, C., Timmers, H.T., Gusella, J.F. and MacDonald, M.E. (1999) Mutant huntingtin forms in vivo complexes with distinct context-dependent conformations of the polyglutamine segment. Neurobiol. Dis., 6, 364–375.[Web of Science][Medline]
39 Abdullah, A., Trifiro, M.A., Panet-Raymond, V., Alvarado, C., de Tourreil, S., Frankel, D., Schipper, H.M. and Pinsky, L. (1998) Spinobulbar muscular atrophy: polyglutamine-expanded androgen receptor is proteolytically resistant in vitro and processed abnormally in transfected cells. Hum. Mol. Genet., 7, 379–384.
40 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.
41 Cummings, C.J., Mancini, M.A., Antalffy, B., DeFranco, D.B., Orr, H.T. and Zoghbi, H.Y. (1998) Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1. Nat. Genet., 19, 148–154.[Web of Science][Medline]
42 Kobayashi, Y., Kume, A., Li, M., Doyu, M., Hata, M., Ohtsuka, K. and Sobue, G. (2000) 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., 275, 8772–8778.
43 Chai, Y., Koppenhafer, S.L., Bonini, N.M. and Paulson, H.L. (1999) Analysis of the role of heat shock protein (Hsp) molecular chaperones in polyglutamine disease. J. Neurosci., 19, 10338–10347.
44 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]
45 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.
46 Stenoien, D.L., Cummings, C.J., Adams, H.P., Mancini, M.G., Patel, K., DeMartino, G.N., Marcelli, M., Weigel, N.L. and Mancini, M.A. (1999) Polyglutamine-expanded androgen receptors form aggregates that sequester heat shock proteins, proteasome components and SRC-1, and are suppressed by the HDJ-2 chaperone. Hum. Mol. Genet., 8, 731–741.
47 Jana, N.R., Tanaka, M., Wang, G. and Nukina, N. (2000) Polyglutamine length-dependent interaction of Hsp40 and Hsp70 family chaperones with truncated N-terminal huntingtin: their role in suppression of aggregation and cellular toxicity. Hum. Mol. Genet., 9, 2009–2018.
48 Muchowski, P.J., Schaffar, G., Sittler, A., Wanker, E.E., Hayer-Hartl, M.K. and Hartl, F.U. (2000) Hsp70 and hsp40 chaperones can inhibit self-assembly of polyglutamine proteins into amyloid-like fibrils. Proc. Natl Acad. Sci. USA, 97, 7841–7846.
49 Kazemi-Esfarjani, P. and Benzer, S. (2000) Genetic suppression of polyglutamine toxicity in Drosophila. Science, 287, 1837–1840.
50 Auclair, D., Garrel, D.R., Chaouki Zerouala, A. and Ferland, L.H. (1997) Activation of the ubiquitin pathway in rat skeletal muscle by catabolic doses of glucocorticoids. Am. J. Physiol., 272, C1007–C1016.
51 Thompson, M.G., Thom, A., Partridge, K., Garden, K., Campbell, G.P., Calder, G. and Palmer, R.M. (1999) Stimulation of myofibrillar protein degradation and expression of mRNA encoding the ubiquitin-proteasome system in C(2)C(12) myotubes by dexamethasone: effect of the proteasome inhibitor MG-132. J. Cell Physiol., 181, 455–461.[Web of Science][Medline]
52 Scheier, B., Foletti, A., Stark, G., Aoyama, A., Döbbeling, U., Rusconi, S. and Klemenz, R. (1996) Glucocorticoids regulate the expression of the stressprotein B-crystallin. Mol. Cell. Endocrinol., 123, 187–198.[Web of Science][Medline]
53 Chai, Y., Koppenhafer, S.L., Shoesmith, S.J., Perez, M.K. and Paulson, H.L. (1999) Evidence for proteasome involvement in polyglutamine disease: localization to nuclear inclusions in SCA3/MJD and suppression of polyglutamine aggregation in vitro. Hum. Mol. Genet., 8, 673–682.
54 Wyttenbach, A., Carmichael, J., Swartz, J., Furlong, R.A., Narain, Y., Rankin, J. and Rubinsztein, D.C. (2000) Effects of heat shock, heat shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington’s disease. Proc. Natl Acad. Sci. USA, 97, 2898–2903.
55 Pearce, D. and Yamamoto, K.R. (1993) Mineralocorticoid and glucocorticoid receptor activities distinguished by nonreceptor factors at a composite response element. Science, 259, 1161–1165.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  Y. M. Ayala, P. Zago, A. D'Ambrogio, Y.-F. Xu, L. Petrucelli, E. Buratti, and F. E. Baralle Structural determinants of the cellular localization and shuttling of TDP-43 J. Cell Sci., November 15, 2008; 121(22): 3778 - 3785. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Shao, W. J. Welch, N. A. DiProspero, and M. I. Diamond Phosphorylation of Profilin by ROCK1 Regulates Polyglutamine Aggregation Mol. Cell. Biol., September 1, 2008; 28(17): 5196 - 5208. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Shao and M. I. Diamond Polyglutamine diseases: emerging concepts in pathogenesis and therapy Hum. Mol. Genet., October 15, 2007; 16(R2): R115 - R123. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
U. A. Desai, J. Pallos, A. A. K. Ma, B. R. Stockwell, L. M. Thompson, J. L. Marsh, and M. I. Diamond Biologically active molecules that reduce polyglutamine aggregation and toxicity Hum. Mol. Genet., July 1, 2006; 15(13): 2114 - 2124. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. LaFevre-Bernt and L. M. Ellerby Kennedy's Disease: PHOSPHORYLATION OF THE POLYGLUTAMINE-EXPANDED FORM OF ANDROGEN RECEPTOR REGULATES ITS CLEVAGE BY CASPASE-3 AND ENHANCES CELL DEATH J. Biol. Chem., September 12, 2003; 278(37): 34918 - 34924. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
|
|
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]
|
|
|
|
 |
|
 S. Dejager, H. Bry-Gauillard, E. Bruckert, B. Eymard, F. Salachas, E. LeGuern, S. Tardieu, R. Chadarevian, P. Giral, and G. Turpin A Comprehensive Endocrine Description of Kennedy's Disease Revealing Androgen Insensitivity Linked to CAG Repeat Length J. Clin. Endocrinol. Metab., August 1, 2002; 87(8): 3893 - 3901. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||