Human Molecular Genetics, 2003, Vol. 12, No. 7 749-757
DOI: 10.1093/hmg/ddg074
© 2003 Oxford University Press
Aggresomes protect cells by enhancing the degradation of toxic polyglutamine-containing protein
1Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 10 Center Drive, Building 10, Room 3B-14, Bethesda, MD 20892-1250, USA and 2Neuroimmunology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA
Received November 21, 2002; Accepted January 21, 2003
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ABSTRACT |
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Expression of misfolded protein in cultured cells frequently leads to the formation of juxtanuclear inclusions that have been termed ‘aggresomes’. Aggresome formation is an active cellular response that involves trafficking of the offending protein along microtubules, reorganization of intermediate filaments and recruitment of components of the ubiquitin proteasome system. Whether aggresomes are benevolent or noxious is unknown, but they are of particular interest because of the appearance of similar inclusions in protein deposition diseases. Here we present evidence that aggresomes serve a cytoprotective function and are associated with accelerated turnover of mutant proteins. We show that mutant androgen receptor (AR), the protein responsible for X-linked spinobulbar muscular atrophy, forms insoluble aggregates and is toxic to cultured cells. Mutant AR was also found to form aggresomes in a process distinct from aggregation. Molecular and pharmacological interventions were used to disrupt aggresome formation, revealing their cytoprotective function. Aggresome-forming proteins were found to have an accelerated rate of turnover, and this turnover was slowed by inhibition of aggresome formation. Finally, we show that aggresome-forming proteins become membrane-bound and associate with lysosomal structures. Together, these findings suggest that aggresomes are cytoprotective, serving as cytoplasmic recruitment centers to facilitate degradation of toxic proteins.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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Correct folding requires proteins to assume one particular structure from a constellation of possible but incorrect conformations. The failure of polypeptides to adopt their proper structure is a major threat to cell function and viability. Consequently, elaborate systems have evolved to protect cells from the deleterious effects of misfolded proteins. Molecular chaperones associate with nascent polypeptides as they emerge from the ribosome, promoting correct folding and preventing untoward interactions. A large fraction of newly translated proteins nonetheless fails to fold correctly, generating a substantial burden of defective polypeptide (1). These proteins are degraded primarily by the ubiquitin–proteasome system (UPS), a multicomponent system that identifies and degrades unwanted proteins (2). Such protective mechanisms face a particularly difficult challenge when disease-causing mutations lead to excess production of misfolded protein. This challenge may be met by efficient removal of the offending product by degradation, as is the case for mutant forms of the cystic fibrosis transmembrane conductance regulator (CFTR) (3). Alternatively, misfolded protein may aggregate and accumulate in and around cells. The deposition of misfolded protein in intraneuronal inclusions has emerged as a common feature underlying many neurodegenerative diseases, suggesting that these diseases may share common mechanisms relating to the failure of neurons to cope with excess misfolded protein (4).
Expression of mutant proteins in cultured cells frequently leads to the formation of juxtanuclear inclusion bodies that have been termed ‘aggresomes’ (5,6). These structures develop at a specific location within the cell, the microtubule organizing center (MTOC). In addition to the misfolded protein itself, which is typically ubiquitinated, components of the ubiquitin–proteasome pathway are enriched in aggresomes (7). Aggresome formation is dependent on dynein-mediated, minus end-directed movement along microtubules, and inhibition of this microtubule function blocks aggresome formation (6,8). The development of aggresomes is intimately related to the cell's capacity to handle misfolded protein. Inhibition of proteasome function provokes aggresome formation, suggesting that these structures develop when a threshold of misfolded protein is exceeded (6). Aggresomes are of particular interest because they resemble the intracellular inclusions found in neurodegenerative diseases such as Parkinson's disease, amyotrophic lateral sclerosis and the polyglutamine diseases. It is not known whether aggresomes are benevolent or noxious, and this parallels the debate as to whether neuronal inclusions in neurodegenerative diseases are helpful or harmful. An elegant reporter system designed to monitor proteasome function provided evidence that aggresome formation is accompanied by a defect in proteasome-mediated degradation (9). However, it is unclear whether aggresome formation in this setting is causative or reactive, or whether the proteasomal defect would be lesser or greater in the absence of aggresome formation.
Spinobulbar muscular atrophy (SBMA) is an X-linked neurodegenerative disorder characterized by progressive loss of motor neurons in the brainstem and spinal cord. The disease is caused by an expanded polyglutamine tract in the amino terminal domain of the androgen receptor (10). Polyglutamine-containing N-terminal fragments of the androgen receptor accumulate and form inclusions in degenerating motor neurons. SBMA is one of nine known ‘polyglutamine diseases’, and as such is a typical protein deposition disease. We used a model of mutant androgen receptor toxicity to study the fundamental nature of polyglutamine inclusions and to evaluate their role in polyglutamine-mediated toxicity. We demonstrate that (i) inclusions of mutant androgen receptor exhibit key features of aggresomes, (ii) aggresome/inclusion formation is a distinct process from aggregation, (iii) inhibiting aggresome/inclusion formation (but not aggregation) enhances the cytotoxicity of mutant androgen receptor, (iv) aggresome-forming proteins exhibit an enhanced rate of turnover, and (v) inclusions composed of mutant androgen receptor associate with lysosomal structures, implicating autophagy as a possible route of degradation.
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RESULTS AND DISCUSSION |
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Polyglutamine expansion in the androgen receptor leads to inclusion formation, aggregation and cytotoxicity
Truncated androgen receptor containing a 19-glutamine repeat (AR-19Q-GFP) showed a diffuse pan-cellular pattern of expression that remained unchanged up to 1 week following transient transfection (Fig. 1A). In contrast, when the polyglutamine tract was expanded to 112 residues, the protein formed compact, spherical inclusions almost immediately after expression was evident,
15 h post-transfection. The use of truncated AR constructs is supported by the finding of only N-terminal epitopes within inclusions of SBMA motor neurons, suggesting that proteolysis of full-length AR occurs during the course of the disease (11). Thus, the truncated form of AR represents a potential proteolytic fragment of the mutant full-length AR. Importantly, the expression of this truncated form reproduces many aspects of the aggregation and toxicity of SBMA (12–15). The inclusions observed here are similar to those described for other polyglutamine containing proteins (Fig. 1A). Within 48 h 60% of transfected cells contained inclusions and by 96 h post-transfection many cells harboring inclusions assumed apoptotic morphology with fragmented nuclei. Constructs with shorter repeats had a greater lag time before inclusions appeared and a lower percentage of transfected cells that contained inclusions (not shown). The majority of inclusions were cytoplasmic and in a juxtanuclear location, while a small fraction were intranuclear (Fig. 1B). The ratio of cytoplasmic to nuclear inclusions varied to some extent with cell type. We saw no difference in the rate, frequency and location of inclusions formed by GFP-tagged protein when compared with untagged proteins detected by immunofluorescence. Polyglutamine length-dependent inclusion formation also occurred upon expression of full-length androgen receptor, albeit with less frequency, with no apparent toxicity (Fig. 2C). Western blot of extracts prepared from transfected cells showed that a substantial fraction of AR-112Q is retained in the well, the stacking gel, and the interface of the stacking and resolving gel, correlating with the formation of insoluble aggregates as has been described for expanded polyglutamine-containing huntingtin, ataxin-1 and ataxin-3 (16–18).
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To evaluate cell viability, we used fluorescence-activated cell sorting (FACS) analysis to assess the ability of cells to exclude propidium iodide. The use of GFP-tagged constructs allowed us to examine specifically the transfected population of cells. Truncated AR with expanded polyglutamine was found to be cytotoxic, with about 30% of AR-112Q-expressing cells dead by 96 h post-transfection, comparable to the toxicity of the pro-apoptotic protein Bax (Fig. 1D). We observed comparable toxicity in a variety of neuronal and non-neuronal cell lines including the motor neuronal cell line MN-1 (not shown). This general toxicity of expanded polyglutamine has been widely observed; the cell-type specificity of polyglutamine disease is probably determined by protein context. In the case of the androgen receptor, for example, this might relate to the higher level of mutant protein expression in motor neurons or to an increased propensity of motor neurons to generate toxic fragments.
Cytoplasmic polyglutamine inclusions are aggresomes
The inclusions generated by AR with expanded polyglutamine exhibited characteristics of aggresomes. Nearly all of the AR-112Q inclusions were found in close juxtaposition to the MTOC, as identified by staining for 
-tubulin, a major MTOC component (Fig. 2A). Furthermore, AR-112Q aggresome formation was associated with a striking redistribution of intermediate filaments from the normal lattice network into a cage-like structure around the inclusion. In neuronal cells, AR-112Q aggresomes were encased by a thick cortex of neurofilaments (Fig. 2B), while in HEK-293 cells the aggresomes were encased by vimentin (not shown). As has been observed for prototypical aggresomes, inclusions of AR-112Q were found in association with components of the ubiquitin-proteasome system, including ubiquitin (Fig. 2D–F), Hsp70 (Fig. 2G–I), and the 20 S proteasome subunit (not shown).
Prototypical aggresomes are formed by two misfolded proteins: GFP-250 (a GFP-tagged fragment of the vesicle-associated protein p115) (8) and the 
F508 mutant of the CFTR (6,8). When co-expressed with GFP-250 or F508 CFTR, AR-112Q formed closely apposed, yet distinct inclusions (Fig. 2J–L and M–O, respectively). Thus, the proteins segregated into distinct, homogeneous inclusions, both adjacent to the MTOC, rather than co-mingling in a single aggresome. The formation of distinct, homotypic aggresomes have been previously observed when unrelated misfolded proteins are co-expressed (19).
Key to aggresome formation is dynein-mediated retrograde transport along microtubules (1). Nanomolar concentrations of nocodazole resulted in a pronounced reduction in the formation of inclusions by AR-112Q. In this dose range, nocodazole blocks microtubule function by altering dynamic instability, demonstrating that AR-112Q aggresome formation is dependent on microtubule function (20). In nocodazole-treated cells, the expanded polyglutamine proteins were distributed diffusely throughout the cytoplasm (Fig. 2P) or coalesced into multiple, peripheral spiculated bodies (Fig. 2Q). The inhibitory effect of nocodazole on inclusion formation was dose-dependent, with 2 µg/ml resulting in 80% reduction in inclusion formation (Fig. 2R).
Minus end-directed movement along microtubules is mediated by dynein motors, which bind their cargo via a large macromolecular intermediate called dynactin (21). Overexpression of p50/dynamitin, a subunit of dynactin, disrupts dynactin function and results in a failure of dynein-mediated, minus end-directed movement along microtubules (22). This feature has enabled use of p50/dynamitin in evaluating dynein-mediated movement of cargo within cultured cells. With this approach, we observed a dose-dependent decrease in cytoplasmic inclusion formation by AR-112Q, demonstrating that polyglutamine inclusion formation was not only microtubule-dependent, but also mediated by dynein motor function (Fig. 2S).
Aggresome formation is cytoprotective
The function of aggresomes and their role in toxic protein diseases is unknown. The ability to manipulate aggresome formation with both molecular and pharmacological interventions offered the opportunity to address this central question. We found that increasing doses of nocodazole resulted in a dose-dependent exacerbation of AR-112Q toxicity (Fig. 3A). Similarly, we found that overexpression of p50/dynamitin exacerbated the toxicity of AR-112Q (Fig. 3B). The doses of nocodazole and p50/dynamitin we used strongly suppressed aggresome formation (Fig. 2M and N), but were not toxic to cells expressing AR-19Q (Fig. 3A and B). The inverse relationship between inclusion-formation and AR112 toxicity is consistent with a cytoprotective function for these structures.
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The aggresome-forming proteins GFP-250 and
F508 CFTR were not toxic to cells in culture (Fig. 3C and D, only GFP-250 shown), demonstrating that aggresomes themselves are not inherently toxic. However, inhibiting aggresome formation with either nocodazole or p50/dynamitin resulted in marked toxicity in cells expressing GFP-250 (Fig. 3C and D) or F508 CFTR (not shown). These observations clearly dissociate aggresome/inclusion formation from cytotoxicity and support the notion that aggresomes protect cells from the deleterious effects of misfolded proteins.
Aggregation is distinct from inclusion formation
The term ‘inclusion’ describes abnormal intracellular structures observed histologically, while the term ‘aggregate’ implies a biochemical phenomenon. Aggregation-prone proteins associated with neurodegenerative diseases are frequently found in inclusions, prompting the frequent (and inappropriate) interchangeable use of the terms ‘aggregate’ and ‘inclusion’. This confusion has complicated the debate as to whether inclusions themselves are cytotoxic. The distinction between aggregation and inclusion formation is underscored by the observation that these processes can be experimentally dissociated. Microtubule inhibition resulted in a marked reduction in inclusion formation by AR-112Q (Fig. 2R and S). Despite this reduction in inclusions, western blot revealed that aggregation of AR-112Q persists (Fig. 4A). Interestingly, reduction of inclusion formation was associated with an increase in the steady-state level of AR-112Q monomer. Thus, it is likely that the toxicity associated with expanded polyglutamine-containing proteins is mediated by monomeric or microaggregated species rather than large histologically detectable inclusions (Fig. 4B).
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Inclusion formation is associated with an accelerated rate of mutant protein turnover
The increased steady-state levels of monomeric AR-112Q with microtubule inhibition, with no apparent change in the amount of aggregated AR-112Q, suggested that the rate of AR-112Q turnover was reduced in the absence of inclusions. To address this directly, we performed pulse-chase experiments to determine the half-life of aggresome-forming proteins in the presence and absence of microtubule inhibition. The same approach was used to look at the impact of microtubule inhibition on proteins not associated with inclusions. AR-112Q was found to have a half-life of
1.8 h, substantially shorter than the half-life of AR-19Q, which was 2.8 h (Fig. 5A). Similarly, full-length AR with an expanded glutamine tract has been shown to have an accelerated rate of turnover relative to wild-type AR (23). Under conditions of microtubule inhibition the half-life of AR-112Q was prolonged to approximately that of AR-19Q, which was unaffected by nocodazole. Similarly, GFP-250 had a short half-life of 1.6 compared with 3 h for 250-GFP, and microtubule inhibition prolonged the half-life of GFP-250 to approximately that of 250-GFP (Fig. 5B). These observations indicate that the turnover rates of inclusion-prone proteins may be particularly sensitive to microtubule inhibition, perhaps because the inclusions serve as cytoplasmic recruitment centers to facilitate degradation.
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A role for aggresomes in accelerating the turnover of misfolded proteins is suggested by their composition. In addition to sequestering misfolded protein, aggresomes extensively recruit from cytosolic pools of proteolytic machinery. As they grow, aggresomes become enriched with ubiquitin and major components of the 26 S proteasome including the 20 S proteosomal subunit and the PA700 and PA28 proteosomal activator complexes (7). Aggresomes are also rich in cellular chaperones that participate in the presentation of misfolded proteins to the proteasome complex including members of the Hsp40 family (Hdj1 and Hdj2), a member of the chaperonin family (TCP1), and Hsp70 (7,8). Based on these observations, aggresomes may represent a site of highly concentrated proteasome activity, protecting the cell in the setting of excess misfolded protein. Alternatively, aggresomes may provide access to an alternative degradative pathway in situations where the capacity of the ubiquitin-proteasome system is exceeded (24). Autophagy is the primary cellular means of disposing of large structures such as organelles. Large structures are first engulfed by autophagosomes, which then fuse with lysosomes allowing degradation by lysosomal hydrolases. Evidence supporting a role for autophagy in the degradation of aggregation-prone polyglutamine-containing and polyalanine-containing proteins has recently been reported (25). Consistent with these findings, electron microscopy revealed relatively large accumulations of electron-dense material in double-membraned structures adjacent to nuclei in transfected HEK-293 cells (Fig. 6A). The large double-membraned structures were frequently found fused with lysosomes (Fig. 6B). Only infrequently did we observe non-membrane bound spherical clusters of electron-dense material, as previously described for aggresomes formed by CFTR and GFP-250 (6,8). The size, frequency and location of these double-membraned structures correlated with the size, frequency and location of AR-112Q inclusions observed by immunofluorescence, and the presence of AR-112Q protein within these structures was verified by immunolabeling (Fig. 6C). In addition, labeled material was found in association with lysosomal structures in the periphery of the cytoplasm. Frequently, positively labeled small densities consistent with microaggregates were found adjacent to lysosomes (Fig. 6D) and in some cases within lysosomes (Fig. 6E). These observations suggest that lysosomal-mediated degradation may contribute to the turnover of expanded polyglutamine.
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Concentration of protein aggregates within inclusions may facilitate elimination of harmful proteins by multiple degradative pathways, such as the ubiquitin–proteasome system and the lysosomal system, and this mechanism may be relevant to a wide array of neurodegenerative diseases. Recognition of the protective role of inclusions is of particular relevance in ongoing efforts at identifying therapeutic interventions for protein deposition diseases—and caution that elimination of inclusions should not be used as a surrogate marker of therapeutic efficacy. Indeed, compounds that increase the process of inclusion formation may even be beneficial.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSREFERENCES |
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DNA constructs
pCMV-ARNGFP plasmids (N=19 or 112 CAG repeats) were generated by subcloning an Xba1/EcoR1 fragment of pAR65
HA (13) into pEGFP-N1 (Clontech), generating a fusion cDNA of truncated androgen receptor and enhanced green fluorescent protein (EGFP). A variety of polyglutamine repeat lengths was generated by spontaneous expansions and contractions of the CAG repeat within the bacterial strain DH5
. pBAX and pBAX-GFP were a gift from Richard Youle (NINDS/NIH, Bethesda, MD, USA). p50/dynamitin was a gift from Richard Vallee (University of Massachussetts, Worcester, MA, USA). p250-GFP and pGFP-250 were gifts from Elizabeth Sztul (University of Alabama at Birmingham, AL, USA).
Cell culture and transfections
HEK-293 cells and MN-1 cells (mouse motor neuron-neuroblastoma cells) (26) were maintained in DME medium (Gibco BRL) with 10% fetal bovine serum (Gibco BRL). Cells were plated at a density of 60% in six-well culture dishes the day prior to transfection. Transfection with 1.0 µg of pCMV-ARNGFP or pBax-GFP DNA was carried out using Lipofectamine Plus following the manufacturers protocol (Gibco BRL). For the experiments indicated, growth media was supplemented with 5–100 ng/ml nocodazole (Sigma).
Immunofluorescence microscopy
HEK-293 and MN-1 cells were grown on coverslips or two-well chamber-slides (Nunc). At the times indicated the cells were fixed in 4% paraformaldehyde for 15 min at room temperature then washed three times in phosphate-buffered saline (PBS). Cells were permeabilized with 0.1% Triton X-100 in PBS for 15 min, rinsed with PBS, and incubated for 30 min in blocking buffer (2% goat serum, 0.1% Triton X-100, 0.2% dry milk, and 1% bovine serum albumin). The cells were then incubated with primary antibody in blocking buffer for 1 h, washed three times with PBS, followed by incubation with secondary antibody in blocking buffer for 30 min. All cells were stained with 1 µg/ml Hoechst 33342 (Sigma) in the final PBS wash for 10 min. Rabbit polyclonal antibody which recognizes human androgen receptor (N20, Santa Cruz Biotechnologies) was used at a 1:100 dilution. -Tubulin monoclonal antibody (clone GTU-88 from Sigma) was used at 1:10 000 dilution. Neurofilament 200 monoclonal antibody (clone NE14 from Sigma) was used at 1:40 dilution. Ubiquitin monoclonal antibody NCL-Ubiqm (Novocastra) was used at 1:50 dilution. Texas Red-conjugated goat anti-rabbit and rabbit anti-mouse secondary antibodies (Fischer Scientific) were used at 1:100 dilution. Deconvolved images were produced on an Olympus microscope using a 60 x water-immersion objective and Deltavision software (Applied Precision) on a Silicon Graphics workstation. Multiple 0.2 µm optical sections were analyzed.
Electron microscopy
HEK-293 cells were plated at 60% density on permanox chamber slides (Nunc) and transfected with pAR-112Q as described above. Ninety-six hours post-transfection, the cells were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer for 45 min, washed three times with 0.1 M phosphate buffer, then blocked and permeablized with PBS+5% goat serum and 0.1% saponin for 1 h. The slides were then incubated with rabbit polyclonal antibody N20 (1:1000, Santa Cruz Biotechnologies) which recognizes the N-terminus of human androgen receptor in PBS+5% normal goat serum + 0.1% saponin for 1 h at room temperature. The cells were then washed three times with PBS+1% goat serum, followed by three washes with PBS+5% dry milk. The cells were then incubated with 4 µl anti-rabbit Nanogold (Nanoprobes) in 1 ml of PBS+5% dry milk for 1 h at room temperature, followed by one wash in PBS+5% dry milk, then three washes in PBS. The samples were then fixed a second time in 2% glutaraldehyde in PBS for 30 min, washed three times in PBS, rinsed with deionized water, and subjected to staining and silver enhancement as described (27). After dehydration, embedding and sectioning, the samples were examined on a JEOL 1200EX electron microscope.
Western blotting and pulse-chase
Forty-eight hours post-transfection, HEK-293 cells were harvested by scraping in lysis buffer [2% SDS, 10 mM Tris pH 6.8, 10% glycerol, Complete protease inhibitor cocktail (Roche)] and sonicated with two 5 s pulses. Fifty micrograms of extract was boiled for 5 min and resolved by 12% SDS–PAGE. Proteins were transferred to an Immobilon-P membrane (Millipore) for 90 min at 150 mA. The membranes were blocked in 5% milk, and probed with anti-AR N-20 polyclonal antibody (1:1000, Santa Cruz Biotechnologies). After three 10 min washes in Tris-buffered saline (TBS) with 0.5% Tween 20, HRP-labeled anti-rabbit antibody secondary (1:3000, Santa Cruz Biotechnologies) was applied for 45 min. After three 10 min washes in TBS+0.5% Tween 20 and three 5 min washes in TBS, ECL (Amersham/Pharmacia) was used for detection. For pulse-chase experiments, 48 h post-transfection, HEK-293 cells were washed twice with methionine-free DMEM (Gibco) and incubated in methionine-free DMEM for 10 min. The cells were then incubated with pulse media (methionine-free DMEM supplemented with 2 mCi of [35S] labeled methionine) for 10 min, followed by incubation in chase media (methionine-free DMEM supplemented with 15 mg/l unlabeled methionine). At time zero and multiple time points post-chase the cells were harvested by scraping, pelleted and lysed by sonication in 500 µl of 1x PBS containing 0.1% Triton X-100 and Complete protease inhibitor cocktail (Roche). One milligram of lysates was used for immunoprecipitation with 30 µl of agarose-conjugated rabbit polyclonal anti-GFP antibody (catalog no. sc-8334 AC, Santa Cruz Biotechnologies) in NET-gel buffer in a total volume of 1 ml as described (28). The resuspended immunoprecipitate was subjected to SDS–PAGE and transferred to an Immobilon-P membrane (Millipore) for 90 min at 150 mA. A Storm 560 phosphorimager (Molecular Dynamics) was used to detect radioactivity, and the signal was quantified using ImageQuant 1.2 software. The average and standard deviation of three experiments is plotted. The half-life of AR protein in each experiment was estimated using the equation derived from a logarithmic plot of percentage of protein remaining versus time using Microsoft Excel software.
FACS survival assay
To measure directly the toxicity of expanded polyglutamine in the context of truncated androgen receptor we used a fluorescence-activated cell sorting (FACS)-based survival assay. The use of GFP-fused constructs allowed specific analysis of the transfected population. At 48, 72 or 96 h after transient transfection cells were harvested with trypsin, gently pelleted by centrifugation and resuspended in PBS with 1% serum on ice at a concentration 106/ml. The cells were stained with propidium iodide 1 µg/ml (PI, Sigma), gently vortexed and incubated for 15 min at room temperature in the dark. For each sample 50 000 non-gated events were acquired (Beckman Coulter XL instrument and software package used for analysis). The results were expressed as a percentage of PI-negative (viable) cells (FL-2 channel) relative to total GFP positive (transfected) cells (FL-1 channel).
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ACKNOWLEDGEMENTS |
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Electron microscopy was performed with the help and advice of the NINDS EM Facility. We thank Richard Vallee, Elizabeth Sztul and Diane Merry for contributing the p50/dynamitin, p250-GFP and pAR65
HA expression constructs, respectively. J.P.T. is supported by NINDS Career Transition Award K22-NS44125-01. J.R. and C.M.S. were participants in the Howard Hughes Medical Institute medical student scholars program.
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FOOTNOTES |
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* To whom correspondence should be addressed. Tel: +1 3014359288; Fax: +1 3014803365; Email: taylorjp{at}ninds.nih.gov
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