Truncated forms of the androgen receptor are associated with polyglutamine expansion in X-linked spinal and bulbar muscular atrophy
Truncated forms of the androgen receptor are associated with polyglutamine expansion in X-linked spinal and bulbar muscular atrophyRachel Butler+, P. Nigel Leigh, Michael J. McPhaul1 and Jean-Marc Gallo*
Department of Clinical Neurosciences, Institute of Psychiatry and King's College School of Medicine and Dentistry, De Crespigny Park, London SE5 8AF, UK and 1Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75235-8857, USA
Received September 5, 1997;Revised and Accepted September 29, 1997
X-linked spinal and bulbar muscular atrophy (SBMA) is a rare form of motor neuron degeneration linked to a CAG repeat expansion in the first exon of the androgen receptor gene coding for a polyglutamine tract. In order to investigate the properties of the SBMA androgen receptor in neuronal cells, cDNAs coding for a wild-type (19 CAG repeats) and a SBMA mutant androgen receptor (52 CAG repeats) were transfected into mouse neuroblastoma NB2a/d1 cells. The full length androgen receptor proteins, of 110-112 kDa and 114-116 kDa for the wild-type and mutant protein, respectively, were detected by Western blotting in transfected cells. In addition, the presence of an expanded polyglutamine tract in the SBMA androgen receptor appears to enhance the production of C-terminally truncated fragments of the protein. A 74 kDa fragment was particularly prominent in cells expressing the SBMA androgen receptor. From its size, it can be deduced that the 74 kDa fragment lacks the hormone binding domain but retains the DNA binding domain. The 74 kDa fragment may therefore be toxic to motor neurons by initiating the transcription of specific genes in the absence of hormonal control. Immunofluorescence microscopy on transfected NB2a/d1 cells showed that, after hormone activation, the wild-type androgen receptor translocated to the nucleus whereas the SBMA androgen receptor was mainly localized in the cytoplasm in the form of dense aggregates with very little androgen receptor protein in the nucleus. This could explain the reduction in transcriptional activity of the SBMA mutant as compared with wild-type androgen receptor.
X-linked spinal and bulbar muscular atrophy (SBMA), or Kennedy's disease, is a rare form of adult hereditary motor neuropathy characterized by progressive muscle weakness and atrophy, usually beginning in the third to fifth decade of life (1 ). The main pathological feature of SBMA is a marked loss and atrophy of lower motor neurons, particularly of the anterior horn cells of the entire spinal cord (2 -4 ). SBMA is associated with an expansion of a polymorphic CAG repeat sequence in the first exon of the androgen receptor gene coding for a polyglutamine tract in the N-terminal domain of the protein (5 ). In the normal population, the size of the CAG repeat region ranges from 12 to 30 repeats but, in patients with SBMA, the expanded domain varies in size between 40 and 72 repeats (5 -9 ).
In addition to SBMA, a number of neurogenetic diseases are linked to trinucleotide repeat expansion mutations in the coding region of the gene involved. These include Huntington's disease, spinocerebellar ataxias, dentatorubral-pallidoluysian atrophy and Machado-Joseph disease. These are all slow progressive neurodegenerative disorders affecting selective populations of neurons [for a review see (10 )].
The striking similarity of these mutations suggests a common pathological mechanism leading to the modified protein having acquired a neurotoxic function. Expanded polyglutamine stretches in proteins could modify the affinity for interacting proteins, as shown for huntingtin, the product of the Huntington's disease gene (11 ,12 ). Polyglutamine stretches have been shown to form stable [beta]-pleated sheet or `polar zippers' which enable protein-protein interaction (13 ,14 ). When the polyglutamine repeat tract expands beyond a certain number of glutamines it may become an avid substrate for transglutaminases leading to protein aggregates within the cell (15 -17 ).
The androgen receptor is expressed in diverse neuronal populations within the brain and spinal cord, but is preferentially localized in motor nuclei of cranial nerves and in motor neurons of the spinal cord (18 -20 ). Androgens influence the survival of facial and hypoglossal motor neurons and increase the rate of regeneration of their axons after axotomy (21 -26 ). Androgens also have a trophic effect on the sexually dimorphic neurons of the spinal nucleus of the bulbocavernosus (27 ,28 ).
The capability of the SBMA mutant androgen receptor to transactivationally regulate gene expression in non-neuronal (29 ,30 ) or neuronal cells (31 ) is reduced in comparison with that of the normal androgen receptor and is inversely related to the length of the polyglutamine tract (32 ). This, together with the decrease in maximal androgen binding of the SBMA androgen receptor (33 -35 ), would explain the mild androgen insensitivity symptoms often present in patients with SBMA, such as reduced fertility, gynaecomastia and feminized skin changes.
The reduced activity of the androgen receptor cannot, however, explain the specific cell death of motor neurons in SBMA as there is no sign of motor impairment in complete androgen insensitivity syndromes caused by mutations in the androgen receptor. Therefore, the mutation associated with SBMA is likely to cause a gain, rather than a loss, of function of the androgen receptor, at least in motor neurons.
The aim of this work was to investigate the properties of the SBMA mutant androgen receptor in neuronal cells. Here we show that the presence of an expanded polyglutamine tract in the androgen receptor leads to the increased production of C-terminally truncated fragments of the protein, which could trigger the abnormal expression of specific genes. In addition the SBMA androgen receptor appears to have a propensity to aggregate in the cytoplasm of transfected cells rather than translocate to the nucleus after hormone stimulation.
cDNAs coding for a wild-type human androgen receptor containing 19 CAG repeats (hARWT) and a SBMA mutant androgen receptor with 52 CAG repeats (hARSBMA) were subcloned into the mammalian expression vector pCMV under the transcriptional control of the CMV promoter. These plasmids were tested for their ability to drive expression of the androgen receptor in mammalian cells by transiently transfecting COS-7 cells. Western blotting analysis of transfected COS-7 cells with the pAnti-AR antibody revealed high levels of ectopic expression of the androgen receptor (Fig. 1 A). The hARWT protein had the predicted molecular weight of 110-112 kDa (Fig. 1 A, lane 1). In contrast, and as expected from the presence of the expanded polyglutamine tract, the electrophoretic mobility of the hARSBMA protein was slightly reduced, with a molecular weight of ~114-116 kDa (Fig. 1 A, lane 2). The endogenous androgen receptor in untransfected cells was below the level of detection.
In order to determine whether androgen receptor species equivalent to the 74 kDa and 93 kDa forms observed in cells expressing hARSBMA were also generated in cells expressing hARWT, increasing amounts of cell lysates from transfected differentiated NB2a/d1 were analyzed by Western blotting with the antibody pAnti-AR (Fig. 2 ). A single fragment of ~83 kDa was detected at high protein loading in cells expressing hARWT. By contrast, two fragments, of ~93 kDa and 74 kDa, were observed in cells expressing hARSBMA. The band corresponding to the 93 kDa species appeared more intense than the 83 kDa species generated by the wild-type androgen receptor, which could only be visualized at the highest loadings (Fig. 2 , lane 4). Quantification of the ratios of fragment/full-length protein proved to be difficult as the fragments only represent a small proportion of the full-length protein under the experimental conditions used.
The transactivational ability of SBMA mutants of the androgen receptor has been reported to be between 50 and 70% of that of the wild-type androgen receptor (29 ,30 ,41 ). In order to confirm the decrease in transcriptional activity due to expandedpolyglutamine tracts for the constructs used in this study, COS-7 cells were co-transfected with either pCMVneohARWTor pCMVneohARSBMA and with the reporter plasmid pMMTVCAT containing the bacterial chloramphenicol acetyltransferase (CAT) gene downstream to the androgen responsive mouse mammary tumor virus long terminal repeat (MMTV). Androgen-mediated transcription of the CAT gene was stimulated by treating the cells for 48 h with 50 nM testosterone and the levels of CAT enzyme produced were measured. The level of activation due to endogenous androgen receptor was determined in cells transfected with the reporter plasmid alone and subtracted from the values obtained in cells expressing exogenous androgen receptor. The final results were expressed in ng of CAT protein produced/mg of total cellular protein. The activities of hARWT and hARSBMA were 0.308 ± 0.036 (n = 3) and 0.060 ± 0.005 (n = 3), respectively. Therefore, the transactivational ability of the SBMA androgen receptor was ~19% of the ability the wild-type androgen receptor in COS-7 cells, which is consistent with previous reports (29 ,30 ,41 ). Such a reduction is unlikely to be due to differences in the level of expression of hARWT and hARSBMA in transfected cells as shown in Figure 1 A. Lanes corresponding to hARWT and hARSBMA have been loaded with the same amount of total cellular protein and show equivalent expression of the androgen receptor.
The subcellular distribution of the hARWT and hARSBMA proteins in transfected differentiated NB2a/dl cells were compared by immunofluorescence microscopy. Prior to fixation, the cells were treated for 1 h with 10 nM mibolerone, a non-metabolisable synthetic steroid producing permanent activation of the androgen receptor and inducing its translocation to the nucleus. The hARWT protein showed a clear pattern of nuclear staining with hardly any residual staining in the cytoplasm (Fig. 4 A). In comparison, a striking difference in distribution was observed for the hARSBMA protein; after mibolerone treatment there was only weak staining in the nucleus, and the majority of the protein appeared to remain in the cytoplasm in the form of large aggregates (Fig. 4 B). Aggregates were also sometimes observed in the nucleus. These patterns of androgen receptor staining were identical irrespective of the differentiation state of the cells or whether the N-terminal or C-terminal specific antibodies were used. Ectopic expression of the androgen receptor at high levels probably contributes to the formation of cytoplasmic aggregates as some were occasionally observed in cells expressing hARWT; however, this was always accompanied by a clear nuclear staining, which was only faint in cells expressing hARSBMA.
The main function of the androgen receptor is to control expression of target genes by binding to an androgen response element following interaction with the hormone. Expansion of polyglutamine tracts in the androgen receptor has been shown to result in a reduction in transcriptional activity in non-neuronal (29 ,30 ,41 ) and neuronal cellular systems (31 ,32 ). In the present study, we confirmed these findings by showing that an androgen receptor containing 52 CAG repeats has a transcriptional activity reduced by ~80% compared with the activity of an androgen receptor with 19 CAG repeats in transfected COS-7 cells.
As revealed by immunofluorescence microscopy, and as expected, the distribution pattern exhibited by the wild-type androgen receptor after activation was clearly nuclear with little or no residual staining in the cytoplasm. In contrast, after steroid treatment the SBMA androgen receptor protein was mainly localized in the cytoplasm in the form of dense aggregates with very little androgen receptor protein appearing in the nucleus. The clear nuclear staining as well as the high levels of transcriptional activity produced from the reporter plasmid show that the wild-type androgen receptor is functioning normally. The very low level of nuclear translocation achieved by the SBMA mutant androgen receptor and its accumulation as dense cytoplasmic aggregates correlates with its low transactivational capability. A possible explanation for the SBMA androgen receptor aggregation is that the protein is crosslinking with itself or other proteins that bind preferentially to polyglutamine tracts either by the formation of `polar zippers' (13 ,14 ) or by becoming substrates for transglutaminases (15 -17 ). A fragment of MJD1, a protein with an expanded polyglutamine tract in Machado-Joseph disease has also been shown to precipitate in the cytoplasm of transfected cells (42 ). Recently, huntingtin-containing neuronal intranuclear inclusions has been described in transgenic mice expressing exon 1 of the huntingtin gene (43 ,44 ). Similar protein aggregates can also be formed in vitro (44 ). However, we did not detect the presence of high molecular weight complexes of the androgen receptor in western blot analysis of cells transfected with the SBMA mutant androgen receptor.
Western blot analysis of transfected cells revealed that, in addition to the full length androgen receptor proteins, some smaller fragments were also generated. The SBMA androgen receptor gave rise to two fragments, of 93 and 74 kDa, the latter being particularly prominent. In cells transfected with the wild-type androgen receptor, a fragment of ~83 kDa was observed at high protein loadings, and occasionally a faint band of ~71 kDa was also detected, especially with the N-terminal-specific antibody. The 93 and 74 kDa fragments in cells expressing the SBMA androgen receptor are likely to arise from the same mechanisms as the 83 and 71 kDa fragments from cells expressing the wild-type androgen receptor; the presence of the 33 extra glutamine residues would partly account for the molecular weight difference. Analysis with N- and C-terminal- specific antibodies revealed that these fragments retained the N-terminus of the full-length protein but were C-terminally truncated. This confirms that the truncated fragments contain the polyglutamine tract, which starts at position 58.
The 83/93 kDa fragments would correspond in size to a form of the androgen receptor, termed the A-form, found in a number of human tissues, but not in brain (45 ,46 ). However, the A-form is N-terminally truncated, possibly as the result of alternative translation from the first internal methionine residue (Met188). Thus, the 83/93 kDa androgen receptor detected in the present study cannot correspond to the A-form as they retain an intact N-terminal domain.
Truncated fragments have not been reported in earlier studies involving the transfection of the SBMA androgen receptor in cultured cells (30 ,31 ,41 ,47 ). Analysis of the androgen receptor protein in spinal cord (48 ,49 ) or scrotal skin fibroblasts (45 ) from SBMA patients also failed to detect the presence of truncated fragments. This may be because the truncated forms only represent a small proportion of the total androgen receptor protein, and they can only be observed with high protein loadings on gels.
The C-terminally truncated androgen receptor fragments probably result from proteolytic cleavage enhanced by the presence of the extended polyglutamine tract. Indeed, proteins with expanded polyglutamine stretches appear to be good substrates for cysteinyl aspartate proteases (caspases). This has been shown for the product of another gene with CAG repeats, the 350 kDa huntingtin of Huntington's disease. Normal length huntingtin is cleaved by caspase-3, or apopain, but the rate of cleavage is increased with the number of glutamine residues present in the protein (50 ,51 ). Larger polyglutamine tracts are thought to alter the conformation of proteins (50 ,52 ). This could enhance proteolysis by exposing sites which would normally be masked.
A similar mechanism of cysteine protease cleavage could be envisaged for the androgen receptor. It has been suggested that the cleavage of abnormal huntingtin may result in aberrant subcellular trafficking leading to the accumulation of truncated huntingtin in subcellular compartments (53 ). As the SBMA androgen receptor forms aggregates in the cytoplasm, it may be more prone to proteolysis resulting in the production of larger amounts of the truncated forms. Small androgen receptor fragments could be toxic either through general mechanisms of toxicity of proteins with long polyglutamine stretches (42 ,54 ,55 ), alternatively, truncated fragments may have specific properties.
From its size, it can be deduced that the 74 kDa fragment produced in cells expressing the SBMA androgen receptor lacks the hormone binding domain, which starts around residue 670, but retains the DNA binding domain. C-terminally truncated androgen receptors are constitutively active, but only have 10% of the transactivational ability of the intact protein (56 ,57 ). The 74 kDa fragment may therefore be toxic by initiating the transcription of specific genes in the absence of hormonal control. Identifying such genes will be a crutial importance in understanding the pathogenesis of SBMA.
In summary, this work provides further insight into the altered function of the androgen receptor in spinal and bulbar muscular atrophyleading to motor neuron cell death. The data presented show alterations in androgen receptor function as a result of the CAG repeat expansion in two ways: the mutation appears to interfere with the androgen receptor's ability to translocate to the nucleus and enhances the production of potentially toxic androgen receptor fragments.
cDNAs coding for the wild-type (hARWT, 19 CAG repeats) and mutant (hARSBMA, 52 CAG repeats) human androgen receptors (29 ) (obtained from Dr L. Pinsky, The Lady Davis Research Institute, Montreal) were excised by BamHI digestion from pSG5-based vectors (obtained from Dr S. Guidato, Institute of Psychiatry, London) and subcloned at the BamHI site in the vector pCMVneo (a gift from Dr L.-H. Tsai, Harvard Medical School). The resulting mammalian expression vectors were referred to as pCMVneohARWT and pCMVneohARSBMA, respectively. The vector pMMTVCAT, coding for chloramphenicol acetyltransferase under the transcriptional control of the MMTV long terminal repeat (58 ), was a gift from Dr M. V. Govindan (Laval University, Quebec, Canada).
A polyclonal antibody against a fusion protein corresponding to amino acids 331-572 of the human androgen receptor, pAnti-AR (39 ) was purchased from Biogenesis (Poole, UK). Domain-specific anti-peptide antibodies to the N- and C-terminus of the human androgen receptor have been described previously (40 ).
NB2a/d1 cells (a gift from Dr T.B. Shea, University of Massachusetts at Lowell) and COS 7 cells were maintained in a humidified atmosphere of 95% air, 5% CO2 at 37°C and grown in DMEM containing 10% (v/v) heat inactivated foetal bovine serum (HIFBS, Gibco-BRL/Life Technologies, Paisley, UK), 100 U/ml penicillin/100 µg/ml streptomycin and 2 mM L-glutamine. NB2a/dl cells were differentiated 24 h after plating by substitution of the growth medium for DMEM/10% HIFBS containing 1 mM dibutyryl cyclic AMP(dbcAMP). Cells were incubated in differentiation medium for 3 days prior to analysis.
For transfection, cells were seeded to reach 60-70% confluency after 24 h. Five µg DNA was used per 100 mm dish. COS 7 cells were transfected for 48 h by using the calcium phosphate transfection method. Liposome mediated transfection was used for NB2a/d1 cells (Lipofectamine reagent, Gibco-BRL). In this case, the cells were exposed to the DNA/liposome complex for 5 h before being returned to normal growth medium. Cells were routinely analyzed 48 h after transfection. Transfection of differentiated NB2a/d1 cells was performed on the second day of dbcAMP treatment and the cells were maintained in differentiation medium for a further 24 h.
Transactivational activity of the androgen receptor was determined in COS-7 cells by double transfection of 5 × 108 cells with 2.5 µg of each pMMTVCAT and 2.5 µg of pCMVneohARWT or pCMVneohARSBMA. Transfected cells were cultured with or without 50 nM testosterone for 48 h in phenol-red free DMEM/10% (v/v) dextran-coated charcoal-stripped HIFBS. Cell lysates were then prepared and the levels of CAT enzyme produced were measured using an ELISA technique (Boehringer Mannheim, Lewes, UK) according to the manufacturer's instructions.
Prior to harvesting, cells were treated with 10 nM mibolerone and incubated for 1 h at 37°C in order to activate the androgen receptor and promote translocation to the nucleus. After incubation, the cells were washed once with PBS at 4°C and scraped into an appropriate volume of ice-cold TEDGP buffer [40 mM Tris,pH 7.4, 1 mM EDTA, 10% (w/v) glycerol, 10 mM DTT, 0.6 mM PMSF, 0.5 mM bacitracin]. Androgen receptor samples were stored at -70°C. Proteins were separated in 8% (w/v) SDS-polyacrylamide gels and transferred to nitrocellulose membranes. Membranes were incubated in 10% (w/v) skimmed milk in PBS for 1 h at 37°C. Subsequent antibody incubations were carried out in PBS containing 5% (w/v) skimmed milk and 0.5% (w/v) Tween-20. Membranes were routinely incubated with primary antibodies overnight at 4°C (or for 1 h at 37°C). Three 10 min washes in PBS containing 0.5% (w/v) Tween-20 were carried out between antibody steps, followed by incubation with the appropriate species-specific secondary antibody conjugated to horseradish peroxidase (Amersham, Little Chalfont, UK; 1:2000 dilution) for 1 h at room temperature. Immunoreactivity was detected using the enhanced chemiluminescence development method (Amersham).
Cells, grown on coverslips, were rinsed in PBS at 37°C and fixed in methanol at -20°C for 5 min. Following fixation, cells were rehydrated in PBS for 10 min and all antibody incubations were carried out in PBS. Cells were incubated at room temperature with anti-androgen receptor antibodies for 30 min, followed by FITC-conjugated anti-rabbit antibody (Vector Laboratories,Peterborough, UK; 1:250 dilution) for 30 min. Between incubations, cells were washed in PBS over 20 min. Coverslips were mounted in VectaShieldTM (Vector Laboratories).
We thank Drs Govindan, L.-H. Tsai and L. Pinsky for providing vectors used in this study and Dr T.B. Shea for the NB2a/d1 cell line. Special thanks to Dr S. Guidato for the pSGAR vectors and her help in subcloning. This work was supported by the Medical Research Council and by NIH grant DK03892 (M.J.M.).
1 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.MEDLINE Abstract
2 Harding, A. E., Thomas, P. K., Baraitser, M., Bradbury, P. G., Morgan-Hughes, J. A. and Pongsford, J. R. (1982) X-linked recessive bulbospinal neuronopathy: a report of ten cases. J. Neurol. Neurosurg. Psychiatry,45,1012-1019.MEDLINE Abstract
3 Nagashima, T., Seko, K., Hirose, K., Mannen, T., Yoshimura, S., Arima, R., Nagashima, K. and Morimatsu, Y. (1988) Familial bulbo-spinal muscular atrophy associated with testicular atrophy and sensory neuropathy (Kennedy-Alter-Sung syndrome). Autopsy case report of two brothers. J. Neurol. Sci.,87, 141-152.MEDLINE Abstract
4 Sobue, G., Hashizume, Y., Mukai, E., Hirayama, M., Mitsuma, T. and Takahashi, A. (1989) X-linked recessive bulbospinal neuropathy. A clinicopathological study. Brain,112, 209-232.MEDLINE Abstract
5 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 Abstract
6 Igarashi, S., Tanno, Y., Onodera, O., Yamazaki, M., Sato, S., Ishikawa, A., Miyatani, N., Nagashima, M., Ishikawa, Y., Sahashi, K., Ibi, T., Miyatake, T. and Tsuji, S. (1992) Strong correlation between the number of CAG repeats in androgen receptor genes and the clinical onset of features of spinal and bulbar muscular atrophy. Neurology,42, 2300-2302.MEDLINE Abstract
7 Doyu, M., Sobue, G., Mukai, E., Kachi, T., Yasuda, T., Mitsuma, T. and Takahashi, A. (1992) Severity of X-linked recessive bulbospinal neuronopathy correlates with size of the tandem CAG repeat in androgen receptor gene. Ann. Neurol.,32, 707-710.MEDLINE Abstract
8 Amato, A. A., Prior, T. W., Barohn, R. J., Snyder, P., Papp, A. and Mendell, J. R. (1993) Kennedy's disease: a clinicopathologic correlation with mutations in the androgen receptor gene. Neurology,43, 791-794.MEDLINE Abstract
9 Garofalo, O., Figlewicz, D. A., Leigh, P. N., Powell, J. F., Meininger, V., Dib, M. and Rouleau, G. A. (1993) Androgen receptor gene polymorphisms in amyotrophic lateral sclerosis. Neuromuscular Disorders,3, 195-199.MEDLINE Abstract
10 Paulson, H. L. and Fischbeck, K. H. (1996) Trinucleotide repeat in neurogenetic disorders. Annu. Rev. Neurosci.,19, 79-107.MEDLINE Abstract
11 Li, X. J., Li, S. H., Sharp, A. H., Nucifora, F. C.,Jr, Schilling, G., Lanahan, A., Worley, P., Snyder, S. H. and Ross, C. A. (1995) A huntingtin-associated protein enriched in brain with implications for pathology. Nature,378,398-402.MEDLINE Abstract
12 Kalchman, M. A., Koide, H. B., McCutcheon, K., Graham, R. K., Nichol, K., Nishiyama, K., Kazemi-Esfarjani, P., Lynn, F. C., Wellington, C., Metzler, M., Golberg, Y. P., Kanasawa, I., Gietz, R. D. and Hayden, M. R. (1997) HIP1, a human homologue of S. cerevisiae Sla2p, interacts with membrane-associated huntingtin in the brain. Nature Genet.,16, 44-53.MEDLINE Abstract
13 Perutz, M. F., Johnson, T., Suzuki, M. and Finch, J. T. (1994) Glutamine repeats as polar zippers: their possible role in inherited neurodegenerative diseases. Proc. Natl Acad. Sci. USA,91, 5355-5358.MEDLINE Abstract
14 Stott, K., Blackburn, J. M., Butler, P. J. and Perutz, M. (1995) Incorporation of glutamine repeats makes protein oligomerize: implications for neurodegenerative diseases. Proc. Natl Acad. Sci. USA,92, 6509-6513.MEDLINE Abstract
15 Green, H. (1993) Human genetic diseases due to codon reiteration: Relationship to and evolutionary mechanism. Cell,74, 955-956.MEDLINE Abstract
16 Kahlem, P., Terre, C., Green, H. and Djian, P. (1996) Peptides containing glutamine repeats as substrates for transglutaminase-catalyzed cross-linking: relevance to diseases of the nervous system. Proc. Natl Acad. Sci. USA,93,14580-14585.MEDLINE Abstract
17 Cooper, A. J. L., Sheu, K.-F. R., Burke, J. R., Onodera, O., Strittmatter, W. D., Roses, A. D. and Blass, J. P. (1997) Polyglutamine domains are substrates of tissue transglutaminase: does transglutaminase play a role in expanded CAG/poly-Q neurodegenerative diseases. J. Neurochem.,69, 431-434.
18 Sar, M. and Stumpf, W. E. (1977) Androgen concentration in motor neurons of cranial nerves and spinal cord. Science,197, 77-79.MEDLINE Abstract
19 Simerly, R. B., Chang, C., Muramatsu, M. and Swanson, L. W. (1990) Distribution of androgen and estrogen receptor mRNA-containing cells in the rat brain: An in situ hybridization study. J. Comp. Neurol.,294, 76-95.MEDLINE Abstract
20 Matsuura, T., Ogata, A., Demura, T., Moriwaka, F., Tashiro, K., Koyanagi, T. and Nagashima, K. (1993) Identification of androgen receptor in the rat spinal motoneurons. Immunohistochemical and immunoblotting analyses with monoclonal antibody. Neurosci. Lett.,158, 5-8.MEDLINE Abstract
21 Yu, W. H. A. and Srinivasan, R. (1981) Effect of testosterone and 5[alpha]-dihydro- testosterone on regeneration of the hypoglossal nerve in rats. Exp. Neurol.,71, 431-435.
22 Yu, W. H.-A. (1982) Effects of testosterone on the regeneration of the hypoglossal nerve in rats. Exp. Neurol.,77, 129-141.MEDLINE Abstract
23 Yu, W. H.-A. (1989) Administration of testosterone attenuates neuronal loss following axotomy in the brain-stem motor nuclei of female rats. J. Neurosci.,9, 3908-3914.MEDLINE Abstract
24 Kujawa, K. A., Kinderman, N. B. and Jones, K. J. (1989) Testosterone-induced acceleration of recovery from facial paralysis following crush axotomy of the facial nerve in male hamsters. Exp. Neurol.,105, 80-85.MEDLINE Abstract
25 Kujawa, K. A., Emeric, E. and Jones, K. J. (1991) Testosterone differentially regulates the regenerative properties of injured hamster facial motoneurons. J. Neurosci.,11, 3898-3906.MEDLINE Abstract
26 Kujawa, K. A., Jacobs, J. M. and Jones, K. J. (1993) Testosterone regulation of the regenerative properties of injured rat sciatic motor neurons. J. Neurosci. Res.,35, 268-273.MEDLINE Abstract
27 Nordeen, E. J., Nordeen, K. W., Sengelaub, D. R. and Arnold, A. P. (1985) Androgens prevent normally occurring cell death in a sexually dimorphic spinal nucleus. Science,229, 671-673.MEDLINE Abstract
28 Araki, I., Harada, Y. and Kuno, M. (1991) Target-dependent hormonal control of neuron size in the rat spinal nucleus of the bulbocavernosus. J. Neurosci.,11, 3025-3033.MEDLINE Abstract
29 Mhatre, A. N., Trifiro, M. A., Kaufman, M., Kazemi-Esfarjani, P., Figlewicz, D., Rouleau, G. and Pinsky, L. (1993) Reduced transcriptional regulatory competence of the androgen receptor in X-linked spinal and bulbar muscular atrophy. Nature Genet.,5, 184-188.MEDLINE Abstract
30 Chamberlain, N. L., Driver, E. D. and Miesfeld, R. L. (1994) The length and location of CAG trinucleotide repeats in the androgen receptor N-terminal domain affect transactivation function. Nucleic Acids Res., 22, 3181-3186.MEDLINE Abstract
31 Nakajima, H., Kimura, F., Nakagawa, T., Furutama, D., Shinoda, K., Shimizu, A. and Ohsawa, N. (1996) Transcriptional activation by the androgen receptor in X-linked spinal and bulbar muscular atrophy. J. Neurol. Sci.,142, 12-16.MEDLINE Abstract
32 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.MEDLINE Abstract
33 Warner, C. L., Griffin, J. E., Wilson, J. D., Jacobs, L. D., Murray, K. R., Fischbeck, K. H., Dickoff, D. and Griggs, R. C. (1992) X-linked spinomuscular atrophy: a kindred with associated abnormal androgen receptor binding. Neurology,42, 2181-2184.MEDLINE Abstract
34 Danek, A., Witt, T. N., Mann, K., Schweikert, H. U., Romalo, G., La Spada, A. R. and Fischbeck, K. H. (1994) Decrease in androgen binding and effect of androgen treatment in a case of X-linked bulbospinal neuronopathy. Clin. Investig.,72, 892-897.MEDLINE Abstract
35 MacLean, H. E., Choi, W. T., Rekaris, G., Warne, G. L. and Zajac, J. D. (1995) Abnormal androgen receptor binding affinity in subjects with Kennedy's disease (spinal and bulbar muscular atrophy). J. Clin. Endocrinol. Metab.,80,508-516.MEDLINE Abstract
36 Shea, T. B., Fischer, I. and Sapirstein, V. S. (1985) Effect of retinoic acid on growth and morphological differentiation of mouse NB2a neuroblastoma cells in culture. Brain Res.,353, 307-314.MEDLINE Abstract
37 Shea, T. B., Sihag, R. K. and Nixon, R. A. (1988) Neurofilament triplet proteins of NB2a/d1 neuroblastoma: postranslational modification and incorporation into the cytoskeleton during differentiation. Dev. Brain Res.,43,97-109.
38 Carter, J. E., Gallo, J.-M., Anderson, V. E. R., Anderton, B. H. and Robertson, J. (1996) Aggregation of neurofilaments in NF-L transfected neuronal cells. Regeneration of the filamentous network by a protein kinase C inhibitor. J. Neurochem.,67, 1997-2004.MEDLINE Abstract
39 Chang, C., Whelan, C. T., Popovich, T. C., Kokontis, J. and Liao, S. (1989) Fusion proteins containing androgen receptor sequences and their use in the production of poly- and monoclonal anti-androgen receptor antibodies. Endocrinology,123, 1097-1099.
40 Husmann, D. A., Wilson, C. M., McPhaul, M. J., Tilley, W. D. and Wilson, J. D. (1990) Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein to the nuclei of target cells in the rat and human prostate. Endocrinology,126, 2359-2368.MEDLINE Abstract
41 Gao, T. S., Marcelli, M. and McPhaul, M. J. (1996) Transcriptional activation and transient expression of the human androgen receptor. J. Steroid Biochem. Mol. Biol.,59, 9-20.
42 Ikeda, H., Yamaguchi, M., Sugai, S., Aze, Y., Narumiya, S. and Kakizuka, A. (1996) Expanded polyglutamine in the Machado-Joseph disease protein induces cell death in vitro and in vivo. Nature Genet.,13, 196-202.MEDLINE Abstract
43 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 inclusions underlies the neurological dysfunction in mice transgenic for the HD mutation. Cell,90, 537-548.MEDLINE Abstract
44 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.MEDLINE Abstract
45 Wilson, C. M. and McPhaul, M. J. (1994) A and B forms of the androgen receptor are present in human genital skin fibroblasts. Proc. Natl Acad. Sci., USA,91, 1234-1238.MEDLINE Abstract
46 Wilson, C. M. and McPhaul, M. J. (1996) A and B forms of the androgen receptor are expressed in a variety of human tissues. Mol. Cell. Endocrinol.,120, 51-57.MEDLINE Abstract
47 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.MEDLINE Abstract
48 Matsuura, T., Demura, T., Aimoto, Y., Mizuno, T., Moriwaka, F. and Tashiro, K. (1992) Androgen receptor abnormality in X-linked spinal and bulbar muscular atrophy. Neurology,42, 1724-1726. MEDLINE Abstract
49 Nakamura, M., Mita, S., Murakami, T., Uchino, M., Watanabe, S., Tokunaga, M., Kumamoto, T. and Ando, M. (1994) Exonic trinuleotide repeats and expression of androgen receptor gene in spinal cord from X-linked spinal and bulbar muscular atrophy. J. Neurol. Sci.,122, 74-79.MEDLINE Abstract
50 Goldberg, Y. P., Nicholson, D. W., Rasper, D. M., Kalchman, M. A., Koide, H. B., Graham, R. K., Bromm, M., Kazemi-Esfarjani, P., Thornberry, N. A., Vaillancourt, J. P. and Hayden, M. R. (1996) Cleavage of huntingtin by apopain, a proapoptotic cysteine protease, is modulated by the polyglutamine tract. Nature Genet.,13, 442-449.MEDLINE Abstract
51 Nasir, J., Goldberg, Y. P. and Hayden, M. R. (1996) Huntington disease: new insights into the relationship between CAG expansion and disease. Hum. Mol. Genet.,5, 1431-1435.MEDLINE Abstract
52 Burright, E. N., Clark, H. B., Servadio, A., Matilla, T., Feddersen, R. M., Yunis, W. S., Duvick, L. A., Zoghbi, H. Y. and Orr, H. T. (1995) SCA1 transgenic mice: a model for neurodegeneration caused by an expanded CAG trinucleotide repeat. Cell,82, 937-948.MEDLINE Abstract
53 Nasir, J., Floresco, S. B., O'Kusky, J. R., Diewert, V. M., Richman, J. M., Zeisler, J., Borowski, A., Marth, J. D., Phillips, A. G. and Hayden, M. R. (1995) Targeted disruption of the Huntington's disease gene results in embryonic lethality and behavioral and morphological changes in heterozygotes. Cell,81, 811-823.MEDLINE Abstract
54 Mangiarini, L., Sathasivam, K., Seller, M., Cozens, B., Harper, A., Hetherington, C., Lawton, M., Trottier, Y., Lehrach, H., Davies, S. W. and Bates, G. P. (1996) Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell,87, 493-506.MEDLINE Abstract
55 Onodera, O., Roses, A. D., Tsuji, S., Vance, J. M., Strittmatter, W. J. and Burke, J. R. (1996) Toxicity of expanded polyglutamine-domain proteins in Escherichia coli. FEBS Lett.,399, 135-139. MEDLINE Abstract
56 Simental, J. A., Sar, M., Lane, M. V., French, F. S. and Wilson, E. M. (1991) Transcriptional activation and nuclear targeting signals of the human androgen receptor. J. Biol. Chem.,266, 510-518.MEDLINE Abstract
57 Marcelli, M., Tilley, W. D., Wilson, C. M., Griffin, J. E., Wilson, J. D. and McPhaul, M. J. (1990) Definition of the human androgen receptor gene structure permits the identification of mutations that cause androgen resistance: premature termination of the receptor protein at amino-acid residue 588 causes complete androgen resistance. Mol. Endocrinol.,4,1105-1116.MEDLINE Abstract
58 Govindan, M. V. (1990) Specific region in hormone binding domain is essential for hormone binding and trans-activation by human androgen receptor. Mol. Endocrinol.,4, 417-427.MEDLINE Abstract
*To whom correspondence should be addressed. Tel: +44 171 919 3404; Fax: +44 171 703 9989; Email: j.gallo@iop.bpmf.ac.uk
+Present address: Department of Urology Research, Mayo Clinic, Rochester, MN 55905, USA
J. P. Chapple, K. Anthony, T. R. Martin, A. Dev, T. A. Cooper, and J.-M. Gallo Expression, localization and tau exon 10 splicing activity of the brain RNA-binding protein TNRC4
Hum. Mol. Genet.,
November 15, 2007;
16(22):
2760 - 2769.
[Abstract][Full Text][PDF]
R. P. Pelley, K. Chinnakannu, S. Murthy, F. M. Strickland, M. Menon, Q. P. Dou, E. R. Barrack, and G. P.-V. Reddy Calmodulin-Androgen Receptor (AR) Interaction: Calcium-Dependent, Calpain-Mediated Breakdown of AR in LNCaP Prostate Cancer Cells
Cancer Res.,
December 15, 2006;
66(24):
11754 - 11762.
[Abstract][Full Text][PDF]
D. Goti, S. M. Katzen, J. Mez, N. Kurtis, J. Kiluk, L. Ben-Haiem, N. A. Jenkins, N. G. Copeland, A. Kakizuka, A. H. Sharp, et al. A Mutant Ataxin-3 Putative-Cleavage Fragment in Brains of Machado-Joseph Disease Patients and Transgenic Mice Is Cytotoxic above a Critical Concentration
J. Neurosci.,
November 10, 2004;
24(45):
10266 - 10279.
[Abstract][Full Text][PDF]
C. M. Everett and N. W. Wood Trinucleotide repeats and neurodegenerative disease
Brain,
November 1, 2004;
127(11):
2385 - 2405.
[Abstract][Full Text][PDF]
D. K. Lee and C. Chang Expression and Degradation of Androgen Receptor: Mechanism and Clinical Implication
J. Clin. Endocrinol. Metab.,
September 1, 2003;
88(9):
4043 - 4054.
[Abstract][Full Text][PDF]
D.M. Avila, D.R. Allman, J.-M. Gallo, and M.J. McPhaul Androgen Receptors Containing Expanded Polyglutamine Tracts Exhibit Progressive Toxicity when Stably Expressed in the Neuroblastoma Cell Line, SH-SY 5Y
Experimental Biology and Medicine,
September 1, 2003;
228(8):
982 - 990.
[Abstract][Full Text][PDF]
V. Gorbunova, A. Seluanov, V. Dion, Z. Sandor, J. L. Meservy, and J. H. Wilson Selectable System for Monitoring the Instability of CTG/CAG Triplet Repeats in Mammalian Cells
Mol. Cell. Biol.,
July 1, 2003;
23(13):
4485 - 4493.
[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]
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]
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]
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]
M. Becker, E. Martin, J. Schneikert, H. F. Krug, and A. C.B. Cato Cytoplasmic Localization and the Choice of Ligand Determine Aggregate Formation by Androgen Receptor with Amplified Polyglutamine Stretch
J. Cell Biol.,
April 17, 2000;
149(2):
255 - 262.
[Abstract][Full Text][PDF]
R. A. Irvine, H. Ma, M. C. Yu, R. K. Ross, M. R. Stallcup, and G. A. Coetzee Inhibition of p160-mediated coactivation with increasing androgen receptor polyglutamine length
Hum. Mol. Genet.,
January 22, 2000;
9(2):
267 - 274.
[Abstract][Full Text][PDF]
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]
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]
CiteULike 
Connotea 
Del.icio.us What's this?
