Correspondence to: Fred H. Gage, The Salk Institute for Biological Studies, 10010 North Torrey Pines Rd., La Jolla, CA, 92037. Tel:(858) 453-4100 ext
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Abstract |
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Proteins with expanded polyglutamine (polyQ) tracts have been linked to neurodegenerative diseases. One common characteristic of expanded-polyQ expression is the formation of intracellular aggregates (IAs). IAs purified from polyQ-expressing cells were dissociated and studied by protein blot assay and mass spectrometry to determine the identity, condition, and relative level of several proteins sequestered within aggregates. Most of the sequestered proteins comigrated with bands from control extracts, indicating that the sequestered proteins were intact and not irreversibly bound to the polyQ polymer. Among the proteins found sequestered at relatively high levels in purified IAs were ubiquitin, the cell cycleregulating proteins p53 and mdm-2, HSP70, the global transcriptional regulator Tata-binding protein/TFIID, cytoskeleton proteins actin and 68-kD neurofilament, and proteins of the nuclear pore complex. These data reveal that IAs are highly complex structures with a multiplicity of contributing proteins.
Key Words: polyglutamine, Huntington's disease, aggregates, inducible expression, ecdysone receptor
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Introduction |
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CAG repeat expansion and concomitant polyglutamine (polyQ)1 expression have been linked to a variety of neurodegenerative conditions including Huntington's disease (HD), dentatorubro-pallidoluysian atrophy, spinocerebellar ataxia (including types 1, 2, 3, 6, 7, and 12), and spinobulbar muscular atrophy (
One common hallmark of these diseases and other neurodegenerative conditions, including Alzheimer's disease and Parkinson's disease, is the formation of insoluble protein aggregates and inclusion bodies in disease-specific regions of the human brain (for reviews see
Some proteins known to be colocalized to polyQ IAs are components of protein-folding and proteolytic pathways. These proteins include ubiquitin, molecular chaperones, and components of the 20S proteasome. Recent studies in transgenic mouse and Drosophila melanogaster models of polyQ-mediated toxicity reveal that inhibition or perturbation of these cellular pathways can significantly suppress cytotoxicity of expressed polyQ proteins in vivo (
Other proposed components of IAs are proteins with nonpathogenic length polyQ tracts and caspases. IA recruitment of synthetic reporter proteins with short polyQ tracts, and sequestration of intracellular factors with tracts as short as 19 glutamines, such as CREB-binding protein, have been demonstrated (
To date, the majority of studies have assayed recruitment or sequestration of proteins to IAs by immunohistochemical colocalization of antibodies to IAs stained with established IA markers, generally antiubiquitin or an antibody against the polyQ protein itself. The benefit of this type of analysis is that differences in the staining pattern or intensity of staining from one IA to another may provide information on heterogeneity within the IA population, which in turn may yield valuable clues as to the pathogenesis of polyQ-mediated cell death. One drawback of this type of analysis is that many antigens may be masked or conformationally altered by association with the IA and could lead to false negative conclusions or misleading staining patterns. Furthermore, it is difficult to assign a quantitative value to positive structures in immunocytochemical studies.
In this report, we describe the isolation, characterization, and composition of IAs from an inducible in vitro model of polyQ-mediated disease using a human cell line that standardizes several parameters of polyQ-mediated cell death. Through a combination of protein blot analysis, immunohistochemical studies, and proteomics using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI) analysis (
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Materials and Methods |
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Construction of polyQ Reporter Fusion Proteins and Vectors
PolyQ reporter constructs were made by PCR amplification of human Huntingtin (Htt) exon I variants with 13- and 96-CAG tracts, preserving polyproline (polyP) sequences immediately downstream of the polyQ tract (see Fig 1) and fusing these fragments in frame to the NH2 terminus of EGFP (CLONTECH Laboratories, Inc.) in the cloning vector SKSP. Unique AscI and MluI restriction sites were inserted into the 5' and 3' coding regions for insertion of oligonucleotides encoding the SV 40 nuclear localization signal (NLS). For the experiments in this report, only the 3'-localized SV 40 NLS was used. The 96Q tract contains a characterized arginine residue at position 42 of the 96Q tract (
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Cell Transfection, Retroviral Infection, and Cell Culture
Transient transfection by calcium phosphate precipitation and transfection to produce replication-defective retrovirus was as previously described (
TUNEL Staining
DNA fragmentation was detected in situ on cultured cells using the immunocytochemical terminal deoxynucleotidyl transferasemediated dUTPbiotin end labeling (TUNEL) technique (Roche) according to the manufacturer's protocol. Using sheep antifluorescein antibody (Roche), the fluorescein signal was converted into a peroxidase signal for visualization using 0.025% DAB, 0.5% nickel chloride, and 0.018% H2O2 in TBS. Positive and negative controls were included in each experiment.
IA Purification
IAs were purified using a modification of the CsCl gradient procedures described by 1/22/3 from the top of the tube. These bands were collected in a volume of 200300 µl and diluted 50 times (to
15 ml) with 10 mM Tris-Cl, pH 7.5. The diluted IAs were centrifuged at 5 x 103 g three times with repeated dilution (sonicating as needed to break up IA clumps). The final pellet was resuspended in 100300 µl of 10 mM Tris, aliquoted, and frozen at -70°C until use.
Protein Blot Analysis and Quantification
For Western blot analysis, cell lysates and preparations of purified aggregates were heated in loading buffer (50 mM Tris-HCl, pH 6.8, 10% SDS, 0.1% bromophenol blue, 10% glycerol). Equal amounts of protein (7.5 µg) were loaded in each lane of a 10% SDS-PAGE gel and electrophoresed. Gels were subsequently transblotted to nitrocellulose membranes using a liquid transfer apparatus (Bio-Rad Laboratories). Membranes were blocked for 1 h in blocking solution (5% nonfat milk in TBS-Tween) and incubated overnight at 4°C with primary antibodies (Table 1) diluted in blocking solution. After extensive washes, membranes were incubated for 1 h with peroxidase-conjugated secondary antibodies (1:1,0001:5,000), and immunoreactive signals were visualized using a chemiluminescence detection kit (Amersham Pharmacia Biotech). Control experiments were carried out by replacing the primary antibody with normal serum or preadsorbing the primary antibody with the corresponding peptide antigen.
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For quantification of sequestered proteins, each antibody used to detect a sequestered protein species in the Western blot analyses (see Fig 3) was titrated against increasing concentrations of induced 96QN cell whole cell extracts or purified IA samples to determine the linear range of detection (data not shown). Each antibody was reassessed against the same samples as those described in the legend to Fig 3 using an amount of protein and dilution of antibody determined to be within the linear range of detection. From these protein blots, the integrated densities of bands within purified IA lanes and control lanes were determined and used to calculate the percentage of protein for an individual protein species sequestered into IAs relative to the amount of the same protein in whole cell extracts. Since no recruited protein species displayed different band intensities in the 5-d induced 13QN and 96QN uninduced lanes, these two values were averaged to provide the basal protein expression level.
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To calculate degree of sequestration, two parameters were established. The first parameter established the number of whole cell extract equivalents of IAs per unit volume of purified IAs relative to a unit weight of 96QNGFP whole cell extracts. By quantifying the intensity of the polymerized 96QNGFP band for 5 µl of purified IAs sample relative to the polymerized 96QNGFP band for 7.5 µg 96QNGFP whole cell extracts, it was determined that this value averaged 16.3, indicating that the amount of IA protein in 5 µl of purified IAs represents the amount of IA protein in 7.5 µg x 16.3, or 122 µg of 5-d 96QNGFP whole cell extract. For blots in which the amount of purified IAs or whole cell extract was reduced or increased to bring detection within the linear range, the correction factor was proportionally adjusted.
Since only a subpopulation of cells at the time of harvest contained large IAs likely to equilibrate within the CsCl gradient band taken during purification, a second parameter was determined that corrected for IA heterogeneity within the 96QNGFP cell population. 18% of cells displayed large IAs at the 5-d time point, so, to normalize to whole cell extracts, the protein adjusted value was divided by 0.18 to produce the final value reported.
Protein Isolation and MALDI Analysis
For isolation of individual protein bands from IA preparations, 40 IA equivalents (relative to the amount used in protein blot analyses above) in a volume of 80 µl (a 2.5x relative concentration) prepared for gel loading as above, were subjected to 10% SDS-PAGE on a 1-mm slab gel and run at low constant voltage (90 V) for 8 h. The gel was then stained overnight with 40% methanol, 10% acetic acid, 0.25% Coomassie brilliant blue and destained for 8 h the next day in 40% methanol, 10% acetic acid solution. Four prominent stained bands that ranged in size from 70 to 30 kD were excised from the gel with a scalpel, transferred to a microfuge tube, treated with TFAacetonitrile for 1 h at 37°C, and vacuum lyophilized. The lyophilized samples were treated with trypsin for 16 h at 37°C, and peptides were extracted according to
-cyano-4-hydroxy-trans-cinnamic acid (
1 µl) directly onto the MALDI plate. MALDI analysis was accomplished using a Voyager RP (PerSeptive Biosystems) with internal calibration using insulin (5,734 Da) and a synthetic peptide LAP 821 (1,740 Da). Between 10 and 23 peaks with closely matching peaks on samples run without internal calibration were used to challenge public protein databases using Protein Prospector (http://prospector.ucsf.edu) and ProFound (http://prowl.rockefeller.edu) (
Immunohistochemistry
Antisera used in these studies are listed in Table 1.
Cell Culture.
Cells were fixed for 20 min in phosphate-buffered 4% paraformaldehyde and rinsed twice in 0.1 M TBS. Primary antibodies were diluted in 0.1 M TBS containing 5% normal donkey serum and 0.3% Triton X-100 (NDST). Cells were preincubated in 5% NDST for 1 h at room temperature and then incubated in the primary antibodies overnight at 4°C. Cells were rinsed three times in 5% NDST and incubated for 2 h at room temperature with donkey secondary antibodies (Jackson ImmunoResearch Laboratories) conjugated to FITC, cyanin-3, or cyanin-5, and diluted 1:250 in 5% NDST. Cells were then washed five times in 0.1 M TBS, with the third wash containing 10 ng/ml 4, 6-diamidino-2-phenylindole (Sigma-Aldrich) for nuclear staining. TRITCphalloidin staining was performed by incubating fixed cultured cells in 30 nM TRITCphalloidin for 30 min followed by extensive rinsing with PBS.
Human Tissue.
Postmortem caudateputamen tissues from control (n = 3) and age matched HD grade 3 (n = 3) patients (mean age: 70.8 yr; range: 6078 yr) were obtained from the Harvard Brain Tissue Resource Center, Cambridge, MA. Tissue blocks were cryoprotected at 4°C in increasing concentrations of phosphate-buffered sucrose (10, 20, and 30%). Coronal serial 40-µm-thick sections were cut on a freezing microtome, collected either in 0.1 M TBS for immediate immunostaining, or in cryoprotectant for further storage at -20°C. Free-floating sections were placed in 0.3% hydrogen peroxide for 20 min and then preincubated in 0.1 M TBS containing 5% normal donkey serum, 5% normal AB human serum, and 0.3% Triton X-100 (5% NHDST) for 24 h at room temperature. Sections were then incubated in the primary antibodies (Table 1) diluted in 5% NDHST for 72 h at 4°C. Sections were rinsed three times in 5% NDHST, incubated for 1 h at room temperature with the adequate biotinylated donkey secondary antibodies (Jackson ImmunoResearch Laboratories) diluted 1:200 in 5% NDHST, rinsed twice in 0.1 M TBS, and incubated with an avidinbiotin peroxidase complex (1:120) for 1 h at room temperature. After intense rinses in 0.1 M TBS, tissue-bound peroxidase was visualized using 0.025% DAB, 0.5% nickel chloride, and 0.018% H2O2 in TBS. Sections were rinsed, mounted on gelatin-coated slides, dried, and coverslipped.
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Results |
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Expression and Aggregation of polyQGFP Fusion Proteins
Long (96Q) and short (13Q) polyQ tracts derived from human Htt exon I (
To circumvent the toxic consequences of chronic constitutive polyQ expression, the polyQGFP fusion constructs were used in a positively regulated retroviral vector system (1 wk. In the absence of ligand, only widely scattered cells (<0.1%) displayed even low green fluorescence (Fig 1g, Fig i, and Fig k). After ligand stimulation, green fluorescence was rapidly observed and, as predicted from transient transfection studies, displayed diffuse nuclear fluorescence in 13Q/NGFP cells (Fig 1 h) and punctate intense positivity indicative of IAs by 72 h in 96Q- and 96QN-induced cells (Fig 1j and Fig l).
Western blot analysis of the time course of polyQ induction revealed a low level of 13QN expression in the absence of ligand that is increased 50-fold to a steady state level of expression within 48 h of ligand addition (Fig 1 m, left). A similar pattern is observed in 96QN-induced cells (Fig 1 m, right); however, at times >24 h after induction, IA formation was observed with increasing frequency, as evidenced by the increasing accumulation of insoluble material within the stacking gel at the top of the blot. By day 6, there is an apparent loss of monomeric 96QN protein as increasing free protein is trapped within the growing aggregates.
Morphologic Changes and Increased Toxicity Induced by 96QN Expression
Study of induced cells revealed no obvious time-dependent morphological changes in any of the induced lines with the exception of 96QN, which at time points >4 d exhibited irregularities in nuclear morphology, including nuclear hypertrophy, the formation of small satellite structures of nuclear material, and an increase in the formation of multinucleated syncitia. Fig 2, ad, shows examples of the occasional multinucleated syncytia that form at 5 d after induction. One hallmark of these syncytia is the frequent presence of large cytoplasmic IAs at the center of a ring of nuclei (Fig 2, a and c). The number of syncytia within representative cell populations at different times after induction is shown in Fig 2 e. At the 5-d time point, 96QN cells develop over fivefold more multinucleated syncytia than 5-d 13QN cells, 96Q cells, or 96QN cells at the earlier time points.
Changes in cellular/nuclear morphology paralleled a low but detectable increase in cell death within the stimulated polyQGFP populations. Fig 2fi, shows TUNEL staining of a representative 96QN population stimulated for 5 d with tebufenozide. A comparison of 13QN-, 96Q-, and 96QN-stimulated cells reveals that only nuclear-localized long polyQ tract-producing cells displayed increased TUNEL positivity during the 5-d time course of the experiment (Fig 2 j). At 3 d after induction, 97QN cells displayed over twofold more TUNEL-positive profiles than other cell types and over fivefold more TUNEL-positive bodies by day 5, representing 2% of the total cell population.
Purification of IAs from Day 5 Induced 96QN Cells
IAs were purified from induced polyQ cells at the 5-d time point (corresponding to the maximum change in cellular morphology and cytotoxicity) using a variation of IA isolation procedures described by
Western blot analyses comparing either whole cell extracts or purified aggregates of different cell populations are shown in Fig 3, an. Fig 3 a shows a blot of 5-d induced whole cell extract from 13QN cells (lane 1), uninduced 96QN cells (lane 2), 5-d induced 96QN cells (lane 3), and purified IAs (lane 4) processed for detection of GFP. In Fig 3 a, monomeric 13QN is observed migrating near an apparent molecular mass of 35 kD and monomeric 96QN near 5253 kD as in Fig 1 m. IAs are observed throughout the stacking gel and accumulate at the extreme top of the resolving gel. Anti-GFP staining localizes the purified IAs (lane 4) almost exclusively at the top of the resolving gel with only a weak band corresponding to monomeric 96QN. The low level of monomeric 96QN protein indicates that, although the purified IAs are denatured, they are not resolubilized into free monomers to a significant degree. The possibility that 96QN polymers may be covalently linked together by the action of transglutaminase is further discussed below.
To begin specific identification and characterization of IA components, an identical blot to Fig 3 a was probed with antiubiquitin primary antibody to examine ubiquitin sequestration within the purified IAs. Western blot for ubiquitin (Fig 3 b) revealed a positive signal in all four lanes and substantially increased positivity in induced 96QN whole cell extracts, with most immunoreactive species extending from 5060 kD up and into the stacking gel (Fig 3 b, lane 3). Ubiquitin staining in the purified IA lane (Fig 3 b, lane 4) reveals a significant intensification of the pattern observed in lane 3, indicating a dramatic concentration of ubiquitin in the purified IA sample. The smear of signal from 50 kD up likely represents ligated ubiquitin polymers and other ubiquitinated species, including 96QN polymers and other sequestered proteins.
After published reports of chaperone protein recruitment described in the Introduction, we also examined sequestration of the 70-kD heat shock protein HSP70 (
Another class of protein either predicted or demonstrated to be sequestered within IAs is proteins with nonpathological length polyQ tracts, such as native Htt, TBP, or myocyte-specific enhancer factor (MEF-2a) (38-kD band, suggesting that TBP is not cross-linked to the 96QN polymer at the 5-d time of harvest.
A second class of protein potentially sequestered within IAs are SH2/SH3 proteins capable of binding extended polyP tracts (
Members of the caspase family of cysteine proteases were also examined because they are integral to the progression of apoptosis (
The final group of proteins examined for sequestration were the tumor suppressor protein p53 (4850 kD that was not observed in the control lanes. Mdm-2 was also observed in purified IAs at high levels (Fig 3m and Fig n). Mdm-2 modulates p53 by binding and promoting the ubiquitination and subsequent proteolysis of p53 (
5860-kD form of mdm-2 described in previous reports (
MALDI Analysis of Predominant Protein Species
Fig 4 a shows Coomassie-stained protein species in a concentrated IA sample on a 1-mm 10% SDSpolyacrylamide gel. Four bands at 68, 54, 50, and 41 kD stood out as much more prominent than the
30 other bands of lesser intensity within the sharply resolved region of the gel from 100 kD to the dye front. These four bands were excised from the gel and subjected to MALDI analysis to provide tentative identification of each protein species.
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12 peptide masses were used to query the peptide database for the 6870-kD protein band. 7 of the top 10 matches with 4/12 to 7/12 matched masses covering 1019% of the peptide were from the 68-kD light neurofilament protein (NFL) from a variety of species including human (
Submission of 10 peptide masses for the 4041-kD band at lower mass tolerances returned no strong matches; however, resubmission of the same data set increasing the mass tolerance returned all 10 top matches as various species of the microfilament protein actin (
Identification of the 54-kD band with 10 submitted weights returned no strong matches at low weight tolerance allowances; however, at higher error tolerance, two proteins of the target molecular weight were returned with four to five matches. The first was the DEAD box protein Dbp-5 (5/10 matches) recently described by
The closest return for the remaining band at 50 kD approximating this molecular weight is Tat-binding protein-1 (TatBP-1; 49.1 kD) from human and mouse (8/23 matches, 21% of the peptide). TatBP-1 was characterized early on for its interaction with the HIV Tat protein (
Semiquantitative Analysis of Protein Sequestration into polyQ IAs
Our attempts to accurately quantify the purified IA samples by standard methods were unsuccessful due to the lack of solubility of the sample. However, by titrating different sample volumes, taking multiple exposures of the Western chemiluminescent assay, and performing scanning densitometry on the resulting GFP positivity, we determined that 5 µl of purified IAs contained 16.3-fold more polymerized insoluble 96QNGFP immunoreactivity than 7.5 µg of 5-d 96QN cell whole extract. Using this value and semiquantitative protein blot analysis techniques described in Materials and Methods, we determined the relative levels of individual protein species for each cell type and treatment described in Fig 3 and Fig 4.
Comparison of individual protein species in the three control lanes revealed that only ubiquitin displayed a difference greater than twofold between lanes. Ubiquitin was increased 20-fold in 5-d 96QN whole cell extracts (Fig 3 b, lane 3), compared with 13QN and uninduced 96QN cells (Fig 3 b, lanes 1 and 2). This increase likely represents an accumulation of ubiquitin protein on IAs as opposed to a dramatic increase in ubiquitin expression.
Relative sequestration of proteins to IAs is summarized in Table 2. Of the proteins tested (and excepting 96QNGFP itself and ubiquitin), p53 and Nup-p62 are the most sequestered as a function of total cellular protein, with a level equivalent to or exceeding the entire intracellular pool sequestered in IAs. Other protein species are recruited at levels of 1358% of cellular stores with caspase-3 sequestered at the lowest level, <1% of cellular stores. For all of these proteins, but in particular for those that appear sequestered at lower levels, it must be kept in mind that, if there is significant heterogeneity in IA sequestration, then the relative percentage of recruitment of an individual protein species may be dramatically increased in a subpopulation of IA-containing cells.
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Immunohistochemical Analysis of Sequestration into IAs In Vitro and In Vivo
Ubiquitin, HSP70, TBP, actin, and NPCPs could be localized to IAs in 5-d 96QN cells or human postmortem tissue from HD individuals. Ubiquitin (Fig 5 a) and HSP70 (Fig 5 b) were detected in vitro colocalizing with IAs of 96QN-expressing cells, whereas HSP70 visualization required the use of an alternate antibody from the antibody used for protein blot analysis. Although not directly detected within 96QN cell IAs in situ, an intensification of TBP positivity was observed in induced 96QN cells relative to uninduced cells (Fig 5c and Fig d). This intensification was determined to localize with cells in later stages of apoptotic cell death (Suhr, S., manuscript in preparation). Parallel immunohistochemical localization experiments were performed with postmortem striatal tissue samples from HD individuals (n = 3) and non-HD age-matched controls (n = 3). Striatal tissue samples were examined for altered intracellular distribution of each of the putative sequestered proteins. Punctate ubiquitin-positive profiles were seldom observed throughout the striatum of all three control cases (Fig 5 e) but were frequently observed in HD striatal samples (Fig 5 f). Although HSP70 and caspase-3 displayed a weak intensity of immunopositive staining in all tested tissues, no obvious differences in the distribution pattern of staining were observed between HD or control samples (data not shown). Immunohistochemistry for TBP, on the other hand, revealed the presence of scattered darkly positive profiles exclusively within the striatal samples of the three HD patients that were not observed in the age-matched controls or after preabsorption with antigen (Fig 5, gi). Higher magnification of these inclusions revealed two distinct types of structure: diffusely stained spheres of various size and labeling intensity (Fig 5j and Fig k) and intensely stained inclusion-like objects with a halo of lighter immunostaining (Fig 5l and Fig m). These positive profiles might correspond to a combination of the intensification of antigen observed in the in vitro studies and localization of TBP to polyQ IAs in a subset of these cells.
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Although NFL colocalization to polyQ IAs has been described in an earlier report (
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NPCP immunohistochemistry revealed several distinctive characteristics of IA colocalization not observed with other antigens. First, it is apparent from the examples shown in Fig 6fh, that NPCP only colocalizes strongly with a subset of IAs. Using a multiplicity of fluorescent labels, two distinct populations of IAs are observed in Fig 6 f: green fluorescent IAs, with little or no detectable contribution of NPCP to the IA, and yellow IAs, indicating a combination of the GFP fluorescence and CY3 red-labeled NPCP. Secondly, in Fig 6g and Fig h, a magnification of this field indicates that some IAs are labeled around the edges by NPCP antibody resulting in a halo pattern sometimes appearing associated with nuclei and, in other cases, appearing separate from DAPI-stained nuclei. Third, in addition to these halo structures, there are also examples of smaller IAs in which NPCP positivity colocalizes with the extent of the IA (Fig 6g and Fig h, orange arrows). These fainter IAs may not yet be true IAs but, instead, areas of polyQGFP association with high density NPCP islands on the nuclear lamina. These areas of NPCP density at the nuclear periphery are not dependent on the presence of polyQGFP accumulation, since numerous NPCP-dense regions are observed with no apparent polyQGFP colocalization (Fig 6 g, blue arrows).
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Discussion |
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The observation that cell death increases shortly after the widespread formation of IAs does not necessarily imply a causal link between IAs and cell death; however, it also does not rule out the possibility (discussed in
It is clear from these experiments that many different proteins, in addition to the 96Q and 96QNGFP fusion peptide, are sequestered within IAs. In addition, after denaturation of IAs, and with the exception of ubiquitin and the 96Q peptides themselves, the proteins that are sequestered present at least one band comigrating with proteins from whole cell extracts, suggesting that at least some of the sequestered proteins are likely to be full length and intact. It can be further inferred that these proteins have not been targeted by ubiquitin, have not been cross-linked to 96QN molecules (i.e., via transglutaminase;
MALDI Identification of NFL and Actin
Identification of NFL as a component of the IAs was unexpected, given the nonneuronal nature of the HEK293 cell; however, it is tempting to speculate on the existence of a pathological polyQNFL interaction that might explain limitation of polyQ-mediated disease manifestation to neural tissues. The connection between polyQ, NFL, and toxicity in HD remains to be established, but it is unlikely that polyQ interaction with NFL is the sole mechanism of cytotoxicity, since polyQ expression is toxic to essentially every cultured cell type tested, and it is unlikely that all cultured cells express NFL.
Actin, on the other hand is much more attractive as a universal pathogenic component of polyQ-mediated toxicity since it is expressed in every cell type and across phyla. In addition, we have observed aberrant staining of 96QNGFP-expressing cells with an antimyosin monoclonal antibody, suggesting possible effects on the actin/myosin cytoskeleton (Suhr, S., manuscript in preparation). Actin and myosin are also involved in mitosis and cytokinesis, raising the possibility that the low-level syncitia formation we observed arises from a cytokinesis defect due to changes in actin dynamics. We have not observed a clear difference in nonsequestered actin expression, distribution between control and 96QN-expressing cells, or brain sections from human HD tissue, however, precluding us from definitively identifying actin as an important component of polyQ-mediated cytotoxicity.
Sequestration of p53 and mdm-2
The recruitment of three of the identified proteins (p53, mdm-2, and NPCP/nucleoporins) may have particular relevance to potential mechanisms of polyQ-mediated toxicity. With regard to p53 and mdm-2, it is unknown at this time whether their sequestration or interaction with polyQ proteins is directly mediated through binding with the polyQ protein, or if they represent proteins that are brought into IAs through association with other highly recruited proteins. Four highly recruited proteins found or confirmed in this study to be sequestered within IAsubiquitin, HSP70, TBP, and mdm-2are also known to bind p53 (
A second potential mediator of net increased p53 activity could arise from the sequestered mdm-2 protein species. Sequestered 5860-kD mdm-2 and a lower molecular mass 5556-kD form are observed within IAs. One 5860-kD isoform of mdm-2 has been found to be formed by caspase cleavage in nonapoptotic cells (
Interaction of Polyglutamine with NPCPs
The possibility of polyQ interaction with proteins of the nuclear pore and matrix could answer many questions with regard to localization and toxicity of long-tract polyQ both in vivo and in cultured cell models. Three lines of evidence link the nucleus to polyQ-mediated toxicity: (a) the overwhelming majority of reports examining the mechanism of polyQ-mediated toxicity in HD, either through study of human brain tissue, cultured cells, and animal models, agree that the onset of symptoms and cytotoxicity is concurrent with cleavage of the polyQ tract from the large cytoplasmic Htt molecule and subsequent translocation of this NH2-terminal fragment into the nucleus; (b) addition of an NLS accelerates polyQ-mediated toxicity in cultured cell models; and (c) even though lacking evident NLSs within the Htt NH2-terminal fragment (or any other region of Htt), the NH2-terminal polyQ fragment of Htt (or synthetic polyQ reporters) tends to accumulate within or surrounding the nucleus, often producing an inpocket when located adjacent to the nuclear envelope. Indentation of the nuclear envelope has also been found at high levels in ultrastructural studies of HD brain (
It is our hope that deciphering the contents of IAs in a cellular model of HD will provide new insight into the population of proteins that interact with polyQ-bearing proteins, irrespective of whether sequestration of these proteins within the IA directly contributes to cell death. Much of the speculation about potential mechanisms of polyQ-mediated cell death in this report has focused on p53 and NPCP because they copurify with IAs at higher relative levels than most of the other proteins tested; however, it is equally possible that proteins found at lower levels interact preferentially with soluble or monomeric polyQ protein and are consequently underrepresented in IAs. For this reason, even the most weakly recruited factors should not be dismissed as insignificant to polyQ-mediated pathology until thoroughly studied.
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Footnotes |
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Steven T. Suhr and Marie-Claude Senut contributed equally to this work.
1 Abbreviations used in this paper: CMV, cytomegalovirus; GFP, green fluorescent protein; HD, Huntington's disease; Htt, Huntingtin; IA, intracellular aggregate; MALDI, matrix-assisted laser desorption ionization time of flight mass spectrometry; MEF-2a, myocyte-specific enhancer factor; NDST, normal donkey serum and Triton X-100; NFL, light neurofilament protein; NLS, nuclear localization signal; NPC, nuclear pore complex; NPCP, NPC protein; polyP, polyproline; polyQ, polyglutamine; TatBP-1, Tat-binding protein-1; TBP, TATA-binding protein; TUNEL, terminal deoxynucleotidyl transferasemediated dUTPbiotin end labeling.
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Acknowledgements |
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The authors wish to thank Scott Zeitlin (Columbia University, New York, NY) for polyQ cDNAs and useful advice. We also wish to thank Ethan Signer, Brian Kaspar and Andrew Willhoite in our laboratory, Harry Higgs and Tom Pollard, and Leslie Thompson for helpful discussions. Thanks also to James Lessard (Children's Hospital Medical Center, Cincinnati, OH) for the actin monoclonal antibody. Thanks also to Mary Lynn Gage for help with the manuscript.
Human tissues were provided by the Harvard Brain Tissue Resource Center (Boston, MA), which is supported, in part, by Public Health Services grant number MH/NS 31862. This work was supported by grants from the Hereditary Disease Foundation and the National Institute of Aging. Purchase of the MALDI instrument at University of California at Los Angeles, Los Angeles, CA, was possible through partial support by National Cancer Institute (National Institutes of Health) Cancer Center Support grant CA 16042-20 to the Jonsson Comprehensive Cancer Center.
Submitted: 9 May 2000
Revised: 5 February 2001
Accepted: 5 February 2001
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References |
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