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Human Molecular Genetics Advance Access originally published online on February 2, 2005
Human Molecular Genetics 2005 14(6):765-774; doi:10.1093/hmg/ddi071
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Human Molecular Genetics, Vol. 14, No. 6 © Oxford University Press 2005; all rights reserved

A structure-based analysis of huntingtin mutant polyglutamine aggregation and toxicity: evidence for a compact beta-sheet structure

Michelle A. Poirier1,2,*, Haibing Jiang1,2 and Christopher A. Ross1,2,3,4

1Division of Neurobiology, 2Department of Psychiatry, 3Department of Neurology and Neuroscience and 4Program in Cellular and Molecular Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA

* To whom correspondence should be addressed at: Division of Neurobiology, Department of Psychiatry, Johns Hopkins University School of Medicine, 618 Ross Research Building, 720 Rutland Avenue, Baltimore, MD 21205, USA. Tel:+1 410614001; Fax: +1 4106140013; Email: mpoirie1{at}jhmi.edu

Received December 15, 2004; Accepted January 24, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Huntington's disease (HD) arises from an expanded polyglutamine (polyQ) in the N-terminus of the huntingtin (htt) protein. Neuronal degeneration and inclusions containing N-terminal fragments of mutant htt are present in the cortex and striatum of HD brain. Recently, a model of polyQ aggregate structure has been proposed on the basis of studies with synthetic polyQ peptides and includes an alternating beta-strand/beta-turn structure with seven glutamine residues per beta-strand. We tested this model in the context of the htt exon-1 N-terminal fragment in both mammalian cell culture and cultured primary cortical neurons. We found our data support this model in the htt protein and provide a better understanding of the structural basis of polyQ aggregation in toxicity in HD.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Huntington's disease (HD) is a member of the family of hereditary neurodegenerative disorders caused by a polyglutamine (polyQ) repeat expansion in the N-terminus of huntingtin (htt) (1Go–4Go). In affected individuals, the htt polyQ region is expanded to 36 or more glutamines and the age of disease onset is inversely correlated to polyQ repeat length (5Go,6Go). Mutant forms of htt have been shown to aggregate both in vitro (7Go,8Go) and in vivo (9Go–11Go). A pathological feature of HD is the presence of intranuclear and cytoplasmic inclusions in postmortem brain tissue. These inclusions consist of fibrillar htt aggregates and are generally seen in affected regions of the brain (10Go,11Go), though are not limited to the neurons most likely to degenerate (12Go). Although some studies have shown a correlation between inclusions and pathology (9Go,11Go), others have found that the two can be dissociated (13Go,14Go). Thus, the role of inclusions in neurotoxicity has been controversial.

Aggregation of mutant htt in vitro proceeds by a nucleation-dependent process and results in the accumulation of beta-sheet-rich fibrillar structures. Many studies leading up to these findings have been carried out with the htt exon-1 protein, an N-terminal fragment of htt comprised of the first 90 amino acids including the polyQ repeat region. This fragment has been shown to cause an HD-like phenotype in transgenic mice (15Go). The formation of htt fibrils in vitro is preceded by the appearance of oligomeric and protofibrillar intermediates that also adopt beta-structure (16Go). Recently, Sanchez et al. (17Go) have found that blocking polyQ-mediated associations has a neuroprotective effect. Thus, even if inclusions are not responsible for toxicity, the aggregation process, as well as the abnormal structure of expanded polyQ, appears to be linked to pathogenesis. It is then critical to understand the aggregation pathway including the abnormal structure of polyQ.

Detailed structural information on polyQ aggregates has remained elusive, as both long and short stretches of synthetic peptide are insoluble under aqueous conditions. More than a decade ago, Perutz et al. (18Go) found a Q15 peptide to adopt beta-structure upon improving solubility of the peptide. An atomic model of polyQ was constructed consisting of anti-parallel beta-strands held together by hydrogen bonds and was referred to as the ‘polar zipper’. Since then, a number of structures have been proposed for aggregated polyQ including parallel beta-sheets, beta-hairpins, compact beta-sheets and beta-helix (reviewed in 19Go).

Recently, Thakur and Wetzel (20Go) attempted to address the issue of polyQ aggregate structure using a mutational approach. Using Pro–Gly substitutions, they induced beta-turns in synthetic polyQ peptides and, on the basis of their data, proposed a structural model comprised of alternating beta-strand/beta-turn elements, with an optimum of seven or eight glutamine residues per beta-strand. We have tested this model, in the context of htt exon-1 N-terminal fragment, on both aggregation and neurotoxicity in mammalian cell culture and in cultured murine primary cortical neurons. Our findings are consistent with an alternating beta-strand/beta-turn model of aggregated htt polyQ protein and show that this structure is toxic to both mammalian cells and primary neuronal cultures. These findings provide insight into structural rearrangements underlying the conversion of htt polyQ to its toxic conformation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A structural model of polyQ-mediated htt aggregation in mammalian cell culture
To test the alternating beta-strand/beta-turn model of polyQ aggregate structure, four htt exon-1 DNAs were designed for expression in mammalian cells. These constructs, outlined in Figure 1, result in expressed protein with an N-terminal FLAG tag and a C-terminal histidine (His) tag comprised of six His residues (His6). The expressed proteins contain a polyQ stretch that is longer than that used for our previous in vitro studies and is long enough to cause toxicity in cell and animal models of disease (21Go–25Go). On the basis of previous studies by Thakur and Wetzel, a polyQ peptide comprised of four Q9 or Q10 elements interspersed with three Pro–Gly pairs is able to aggregate with similar efficiency and kinetics as an uninterrupted Q45 peptide (20Go). These data support the alternating beta-strand/beta-turn model of polyQ aggregate structure proposed by Thakur and Wetzel and suggest that seven glutamines make up each beta-strand whereas two participate with the Pro–Gly pair to make up the beta-turn.



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  		Figure 1. Schematic diagram of htt exon-1 polyQ and PGQ-polyQ proteins used for cell culture and primary neuron transfection experiments. Domain structure of exon-1 fragment, indicating polyQ region (red). Also shown are two Pro-repeat regions and the Q-rich region. An N-terminal FLAG tag and a C-terminal His6 tag were engineered into each construct. Primary structure for each polyQ repeat region, as well as the predicted secondary structure of the expressed protein, are shown.

 
A Pro/Gly polyQ mutant of htt exon-1 forms aggregates in N2a and HEK293T cells and in murine primary cortical neurons
Transient transfection experiments were carried out to express each protein in neuroblastoma 2a (N2a) cells and in human embryonic kidney (HEK)293T cells. As shown in Figure 2A, a htt exon-1 protein with a polyQ region comprised of four Q9 elements (PGQ9) was able to aggregate as efficiently as a mutant htt with 76 uninterrupted Q residues (76Q), forming both nuclear and cytoplasmic aggregates in N2a cells. Interruption of three out of four internal Q9 elements with a single Pro residue (PGQP) efficiently blocked htt exon-1 aggregation, resulting in a soluble and cytoplasmic pattern of protein expression with infrequent diffuse nuclear labeling. Comparable results were observed in HEK293T cells expressing the htt exon-1 constructs.



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  		Figure 2. Expression of htt exon-1 polyQ and PGQ-polyQ proteins in transiently transfected cells. (A) Confocal images of transfected Na cells showing anti-FLAG (green, left panels) and Hoescht (blue, middle panels) staining 72 h after transfection. Merged images (right panels) indicate that htt polyQ aggregates observed for 76Q and PGQ9-transfected cells were cytoplasmic, perinuclear and nuclear. Diffuse staining observed for PGQP and 16Q was cytoplasmic. Inset shows high magnification images of representative cells expressing PGQ9, 76Q and PGQP, respectively. (B) Quantification of aggregation by cell counting analysis. Data shown are the average of three independent experiments. (C) Western blot analysis of htt exon-1 expression in N2a cells 24H (left panel) and 72H (right panel), 24 and 72th following transfection, respectively. Blots were stained with an anti-htt exon-1 antibody (16Go).

 
A quantitative comparison of diffuse cytoplasmic or nuclear labeling and cytoplasmic or nuclear aggregation in N2a cells is highlighted in Figure 2B. Although PGQ9 and 76Q htt exon-1-expressing cells showed 75 and 84% aggregation, respectively, only 0.5–1.5% of PGQP and 16Q-expressing cells contained htt aggregates, despite similar levels of protein expression (Fig. 2C). Moreover, SDS–insoluble protein was only observed in those cells expressing the mutant htt proteins that formed aggregates (i.e. PGQ9 and 76Q).

A closer analysis of htt exon-1 expression in mammalian cell culture was carried out using Immunoelectron microscopy (Immuno EM). HEK293T cells have a higher transfection efficiency than N2a cells, so were more suitable for these experiments. HEK293T cell transfected with htt exon-1 PGQ9 is shown in Figure 3. Fibrillar material that was positively stained with anti-FLAG primary and anti-mouse secondary gold-labeled antibodies was observed (Fig. 3, inset), whereas no fibrillar aggregates were detected in cells transfected with PGQP htt or in untransfected cells.



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  		Figure 3. Electron microscopy analysis of HEK293T/htt exon-1 PGQ9 cells. The image depicts a cytoplasmic htt exon-1 PGQ9 aggregate with a fibrillar morphology (boxed region) that is positively labeled with anti-FLAG/anti-mouse 12 nm colloidal gold-conjugate antibodies. Inset shows high magnification image of gold-labeled fibrillar htt aggregate.

 
Although N2a and HEK293T cell lines serve as useful cell models for polyQ-mediated htt aggregation, cultured primary neurons represent a model that more closely resembles in vivo conditions. For this reason, the alternating beta-strand/beta-turn model of polyQ aggregate structure was further examined in cultured murine embryonic primary cortical neurons (E15–E16). Following isolation of cells, neuronal cultures were transfected with the four htt exon-1 DNA constructs and imaged 2 days after transfection. Representative images for PGQ9 and PGQP htt exon-1 are pictured in Figure 4. Htt exon-1 PGQ9 expression resulted mainly in aggregates (Fig. 4, top row) that were frequently observed in either or both the cytoplasm or the nucleus. This expression pattern was identical to that of 76Q and was consistent with data from N2a cells. On occasion, PGQ9 and 76Q showed diffuse labeling. Moreover, htt aggregates could sometimes be seen in the neurites and were usually accompanied by diffuse labeling in the neuronal body and in other areas of the neurites.



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  		Figure 4. Expression of htt exon-1 PGQ constructs in transiently transfected mouse primary cortical neurons. Confocal images of PGQ expression were taken 48 h following transfection. Aggregation of PGQ9 is observed in all four transfected neurons (top panels), whereas PGQP-transfected neurons (bottom panels) contained diffuse labeling.

 
In contrast to results for PGQ9-transfected cells, PGQP expression resulted exclusively in diffuse staining in both the neuronal body and the neurites (Fig. 4, bottom row) and appeared similar to expressed 16Q protein. Taken together, these results demonstrate that PGQ9 and 76Q proteins have a similar ability to aggregate in primary neurons and have a similar cellular localization. Interruption of the putative beta-strand with a Pro residue in the PGQP protein prevented the polyQ region from forming aggregates.

Aggregation of Pro/Gly htt exon-1 mutations parallels cell toxicity in mammalian cells and in cultured primary neurons
We have demonstrated that the htt PGQ9 protein, when expressed in N2a cells, forms visible aggregates as efficiently as the uninterrupted 76Q htt exon-1 fragment. To test for polyQ-induced toxicity in N2a cells, a Tdt-mediated dUDP-X nick end labeling (TUNEL) assay was used to label N2a cells transfected with the htt exon-1 constructs outlined in Figure 1. In addition to TUNEL staining, cells were stained with an anti-FLAG antibody to indicate transfected cells. As demonstrated in Table 1, 72±7.2% of PGQ9 aggregate-containing cells were TUNEL positive, very similar to that for the 76Q cells. Importantly, only 4.5±1.6% of cells expressing PGQ9 with diffuse labeling were TUNEL positive. Similar results were obtained for htt exon-1 76Q-expressing cells. Thus, the formation of an abnormal conformation leading to htt polyQ aggregation seemed to correlate with toxicity.


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  		Table 1. Quantitative analysisa of toxicity in transiently transfected N2a cells
 
A comparable analysis could not be carried out for cells transfected with either PGQP or 16Q htt exon-1 fragments, as aggregates were not observed with either construct. Therefore, quantitative TUNEL analysis was carried out on all transfected cells, i.e. those cells with cytoplasmic diffuse staining of htt exon-1. As demonstrated in Table 1, expression of either protein had no effect on cell viability. To analyze these data visually, cells were examined by confocal microscopy. Representative images for PGQ9 and PGQP htt exon-1 are presented in Figure 5. PGQ9 aggregates (green, first panel) co-localized with both TUNEL (red, second panel) and DNA/Hoescht (blue, third panel) staining. These cells were rounded, as would be expected for a dead or dying cell, and were occasionally observed on the surface of other healthy cells. Conversely, diffuse staining of PGQP expressed protein was not associated with TUNEL staining. Taken together, this data indicate that htt aggregation parallels toxicity in mammalian cell culture.



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  		Figure 5. Expression of PGQ9 but not PGQP induces toxicity in transfected N2a cells. Htt exon-1-transfected cells were analyzed for toxicity 72 h after transfection by the TUNEL assay. Expression of PGQ9 and PGQP was detected by anti-FLAG immunostaining (left panels) and shows aggregates for PGQ9 (top first panel) transfected cells and diffuse cytoplasmic staining for cells containing PGQP protein (bottom first panel). TUNEL positive cells are shown in red and are observed for the two PGQ9 aggregate-containing cells (top second panel). The merged image of PGQ9 (top third panel) demonstrated that the two TUNEL positive nuclei are co-localized with polyQ aggregates. Phase contrast image of the same cells (top fourth panel) shows that these two cells are rounded and on top of healthy cells. The two cells expressing PGQP were not TUNEL positive (bottom second panel). A phase contrast image (bottom fourth panel) demonstrates that both PGQP-expressing cells were healthy. Images for each panel were merged from multiple confocal Z-series pictures to show the entire cell.

 
To examine the effect of PGQ-htt expression on cell viability in primary cortical neurons, transfected cells were examined for the presence and morphology of primary neurites. These experiments were carried out as co-transfections with a plasmid encoding for DS-Red, a red fluorescent protein that is expressed at high levels in both neuronal cell bodies and neurites. Neurons co-transfected with PGQP and DS-Red had multiple long and smooth (non-fragmented) neurites (Fig. 6A, top panels), indicative of healthy cells. In comparison, neurons expressing PGQ9 and DS-Red had few, if any, visible neurites (Fig. 6A, bottom panels), suggesting that these cells were not healthy. DS-Red was particularly useful as a marker for neurite presence and morphology in PGQ9-expressing neurons, as cells with htt polyQ aggregates were often lacking a visible and healthy neurite. Another indicator of cell viability is nuclear morphology. Nuclei of neurons undergoing cell death appear shrunken and dense. This morphology was observed in PGQ9 aggregate-containing cells (Fig. 6A, bottom right panel, insert), whereas nuclei of viable cells, as seen for PGQP-expressing neurons, were noticeably larger and appeared normal. These data suggest that the expression of PGQ9 protein in primary cortical neurons, leading to polyQ aggregation, had a neurotoxic effect whereas htt PGQP expression did not affect cell viability.



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  		Figure 6. Expression of PGQ9 but not PGQP induces toxicity in mouse primary cortical neurons. (A) Toxicity of htt exon-1 expression in primary cortical neurons was evaluated upon the examination of neurites from only transfected neurons (see Materials and Methods). Immunostaining with an anti-FLAG antibody (green) was used to detect htt exon-1 expression. Neurons were co-transfected with Ds-Red (red). Confocal images taken following PGQ9 expression (bottom panels) show a neuron containing a single large PGQ9 aggregate (green, bottom left panel). DS-Red expression (red, bottom middle panel) indicates that this neuron has no healthy neurites. In contrast, the neuron positive for PGQP expression contains multiple long neurites (green, top left panel) and is positive for DS-Red expression (red, top middle panel). Inset shows magnified image of shrunken and dense nucleus corresponding to unhealthy PGQ9-expressing neuron. Images for each panel were merged from multiple confocal Z-series pictures to show the entire cell. (B) Quantification of toxicity induced by htt exon-1 polyQ and PGQ-polyQ expression in primary cortical neurons. Neurons with aggregates and no Ds-Red staining were likely to be dead when examined for morphology by phase contrast microscopy. Therefore, the toxicity data for PGQ9 and 76Q using the criteria described (see Materials and Methods) may be an underestimate. Data shown are the average of four independent experiments.

 
For the quantification of toxicity, a neuron was considered healthy if at least one smooth primary neurite was observed, with a length greater than two times the length of the cell body. Using these criteria, primary neurites of neurons co-transfected with htt and DS-Red were analyzed for length and morphology. Results from this analysis are shown in Figure 6B. While only 33% of neurons containing the PGQ9 mutant could be considered healthy (compared with 25% for 76Q), >80% of PGQP-expressing neurons were viable (compared with 72% for 16Q). These data are comparable with that observed in N2a cells and suggest that only those proteins that can fold into the beta-strand/beta-turn conformation, resulting in the assembly of htt polyQ aggregates, will have a neurotoxic effect on cells.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study, constructs encoding exon-1 of htt with alterations in the expanded polyQ were used to address the relationship between polyQ structure and aggregation and neurotoxicity in two different cell culture systems expressing the mutant proteins. The current findings indicate that htt exon-1 proteins able to assume a beta-strand/beta-turn structure form aggregates in both cell models, whereas an exon-1 protein unable to form this structure because of Pro interruptions does not form visible aggregates. In addition, exon-1 htt with a normal length polyQ stretch does not form aggregates in either cell model. Furthermore, we find that expression of htt exon-1 mutants capable of forming a beta-strand/beta-turn structure results in dramatic cytotoxicity, while the Pro mutant is only as toxic as exon-1 with a non-pathological polyQ length.

The formation of polyQ-derived htt aggregates was demonstrated both by immunofluorescence, EM and by western blot analysis, as large SDS-insoluble aggregates remain at the top of the gel. Furthermore, western blotting indicated that expression levels were comparable for the different proteins, suggesting that differences in aggregation and toxicity cannot be explained by differences in protein expression levels. In addition, cell toxicity was demonstrated by two independent methods, TUNEL labeling (N2a cells) and neuritic morphology analysis (primary neurons). Both measures identify toxicity in only transfected cells. This approach confirms the effect of the expressed proteins on cell viability. N2a cells have some neuronal-like characteristics and are a good model system to study HD. Although they can be more difficult to handle and transfect, primary cortical neurons differentiate in culture to have neurites and other neuronal features. These neurons are derived from a brain region substantially affected in HD patients, thereby representing a model relevant to study HD.

The current data strongly support the relevance of the beta-strand/beta-turn conformation to aggregation and toxicity for mutant htt. Previous work has demonstrated that synthetic polyQ peptides can cause toxicity when added exogenously to cells in culture (26Go) and that this toxicity can be blocked by a polyQ peptide containing proline interruptions (27Go). However, in the previous study, toxicity was observed with both normal length (Q20) and expanded polyQ, making the relevance to disease pathogenesis uncertain.

In contrast, in the current studies, cell toxicity was only observed with a pathological length polyQ. The current data are consistent with previous studies demonstrating that disease phenotype is only caused by htt expanded polyQ in cultured cells and in transgenic mice (reviewed in 28Go,29Go). Finally, these observations are in agreement with data from humans indicating that only those individuals with 36 or more consecutive glutamines in htt are afflicted with HD.

The fragment of htt used for the current study has been used previously and its relevance to the human disease is indicated by several different considerations. First, comparable htt fragments have been found in human postmortem tissue (10Go,30Go,31Go). In addition, fragments of similar length are generated in cell models of HD incorporating full-length expanded htt (30Go–32Go). These fragments aggregate and are highly toxic to the cells in culture (21Go,33Go–37Go). Finally, N-terminal fragments of identical or similar length have been used to generate transgenic mouse models of HD and result in a toxic phenotype (23Go,38Go).

A recent study by Toda and colleagues (39Go) has shown that the interruption of the expanded polyQ stretch by proline insertions reduces its aggregation in vitro similar to the current data. However, this study did not attempt to address the relationship between beta-strand/beta-turn structure and polyQ aggregation and toxicity. In addition, this work involved thioredoxin–polyQ fusion proteins lacking htt protein sequence. Finally, the toxicity data were generated from COS-7 cells, a simian kidney cell line, so cannot be directly correlated with toxicity in neurons.

The current experiments indicate that the expression of proteins capable of assuming a beta-strand/beta-turn structure results in aggregation and toxicity in neuronal cells. Although no direct evidence for beta-strand/beta-turn structure was demonstrated in the current study, previous experiments have shown that similar PGQ proteins are capable of forming fibers with amyloid-like appearance, whereas analogous proteins with Pro interruptions were not (20Go). Thus, this mutational analysis is an alternate approach to study polyQ aggregate secondary structure, as more direct methods including X-ray crystallography and solution NMR spectroscopy have been unsuccessful.

The present data indicate that a relationship exists between mutant polyQ beta-strand/beta-turn structure and aggregation and toxicity. However, it is important to note that our data do not imply that aggregates visible in the light microscope are themselves responsible for toxicity. One possibility is that the aggregation of exon-1 involves several intermediates including oligomeric species and protofibril-like species (16Go), though whether these are sequential or parallel is not yet known. Regardless, these intermediates would not be visible in the light microscope. However, it is quite possible that similar intermediate species can form by the proteins designed in the present work and that these species are responsible for neurotoxicity.

Previous in vitro studies have shown that beta-strand/beta-turn forming polyQ and PGQ variant peptides form fibers with the characteristic appearance of amyloid (20Go). These fibers are very similar to the fibers observed in transgenic mouse models (7Go,9Go) and in human postmortem brain material from HD patients (10Go). The current study demonstrates that a PGQ variant protein (PGQ9) in the context of the htt exon-1 fragment is able to form fibers within mammalian cells, as visualized by Immuno EM. Therefore, we believe our data shed light on the abnormal protein conformation responsible for the disease in vivo and may be relevant not only for HD but also for other polyQ diseases (40Go,41Go). Insight into mutant htt polyQ structure may assist in designing inhibitors of aggregation and toxicity, with a direct relevance to therapeutics (42Go). In fact, recent studies have shown increasing commonalities among the disease-state structures of proteins involved in other neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease (43Go). Thus, the abnormal protein conformations relevant to polyQ disease may have relevance to a better understanding of other neurodegenerative diseases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Plasmid construction
Preparation of htt exon-1 DNA constructs for expression in mammalian cells and primary neurons was a multistep process. To begin, a modified htt exon-1 cDNA with a 42 bp linker flanked by mixed CAG/CAA sequences encoding 15 glutamines and BseRI/BsgI (New England Biolabs) 5'/3' restriction sites was prepared in pBluescript vector (Stratagene) as previously described (16Go). Synthetic DNA fragments comprised of a mixed CAG/CAA sequence and encoding for Q15[PGQ9]4PGQ15 (PGQ9, see Fig. 1), Q15[PGQ4PQ4]2PGQ9[PGQ4PQ4]PGQ15 (PGQP) or Q47 were synthesized and cloned into a modified pUC vector by Genescript, Corp. Next, these fragments were subcloned into the modified htt exon-1 cDNA described earlier using BseRI and BsgI restriction sites. Finally, PGQ9, PGQP and 76Q htt exon-1 constructs were subcloned into a modified pCMV-2B vector (Stratagene) via SalI and NotI restriction sites to allow for protein expression. A 16Q htt exon-1 construct prepared in pBluescript as previously described (16Go) was also subcloned into modified pCMV-2B.

Cell culture and transfection
Mouse N2a cells (American-type culture collection, ATCC) and HEK293T cells (Invitrogen) were maintained in minimum essential medium Eagle (ATCC) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin/streptomycin (BioWhitaker). Cells were grown on coverslips in six-well plates (Corning Incorporated Life Sciences) to 50% confluency at the time of transfection. Transfections were performed using lipofectamine plus (Invitrogen) according to the manufacturer's protocol. Cells were then fixed and processed for indirect immunofluorescence (IIF) 72 hr after transfection.

Cell lysis and immunoblotting
Protein extracts from transfected N2a cells were prepared in buffer containing 50 mM Tris–Cl (pH 7.5), 2 mM EDTA, 100 mM NaCl, 1% Triton-X 100 and a protease inhibitor cocktail containing 4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatinA, E-64, bestatin, leupeptin and aprotinin (Sigma). Following a 5 min centrifugation of the cell lysates at 4°C and 14 000g, detergent-soluble supernatant and detergent-insoluble pellet fractions were collected. Protein determination was completed on each lysate using a Bradford protein assay (BioRad). Detergent-soluble supernatants were prepared for SDS–PAGE by the addition of SDS–sample buffer (BioRad) to a final concentration of 1x(1x=62.5 mM Tris–Cl (pH 6.8), 2% SDS, 25% glycerol, 0.01% Bromophenol Blue). The insoluble fractions were solubilized in 1xSDS–sample buffer supplemented with Benzonase nuclease (0.5 µl per 100 µg protein) (Novagen). For both soluble and insoluble fractions, identical amounts of total protein were analyzed by SDS–PAGE and western blotting using an anti-htt exon-1 antibody as previously described (16Go).

Indirect immunofluorescence
IIF was carried out in N2a cells and HEK293T cells as previously described (44Go). Cells grown on glass coverslips were fixed and permeabilized using 2% paraformaldehyde (Sigma) and 0.1% Triton X-100 (ICN Biomedicals) in PBS (pH 7.4). Fixed cells were incubated with mouse anti-FLAG antibody at 1 : 500 for 1 h at 37°C. Cells were then washed and incubated with FITC anti-mouse IgG (Chemicon International) at 1 : 400 for 1 h at 37°C. Both antibodies were diluted in PBS containing 10% goat serum (Sigma). Cells were washed and stained for 5 min with Hoescht (Sigma) at 1 µg/ml in PBS. Finally, coverslips were mounted with Vectashield (Vector Laboratories). IIF staining was viewed on a Noran confocal microscope. For quantification, images were captured from 10 random selected fields from each coverslip (~100 cells total). Duplicate coverslips for the transfection of each construct were examined in each individual experiment. Data are presented as mean±SEM of three separate experiments with statistical analysis performed using Standard Student's t-test.

Immuno EM of transfected cells
HEK293T cells transfected for postembedding immuno EM were fixed 72 h after transfection in 4% paraformaldehyde+0.05% glutaraldehyde +0.1 M phosphate buffer (pH 7.2–7.4) overnight at 4°C. Cells were gently scraped and pelleted following a 30 min rinse in wash buffer (0.1 M phosphate+3% sucrose), then incubated for 30 min with 0.25% tannic acid (Malinkrodt) in wash buffer. The cells were rinsed, reduced with 50 mM NH4Cl in wash buffer and rinsed with 0.1 M maleate buffer. Cells were stained en bloc with 2% uranyl acetate in 0.1 M maleate buffer. Following dehydration in graded ethanol, cells were embedded in LR Gold. Polymerized blocks were trimmed and cut into 70–80 nm thin sections collected on formvar-coated 200 mesh nickel grids. For immunolabeling, grids were treated with 50 mM NH4Cl in TBS buffer (50 mM Trizma base+150 mM NaCl, pH 7.4), blocked with 1% BSA/0.5% fish gelatin/TBST (10 mM Tris+500 mM NaCl+0.05% Tween 20, pH 7.2) and incubated with a mouse anti-FLAG monoclonal antibody overnight at 4°C at a dilution of 1 : 50. Following a wash in TBS buffer, samples were stained for 2 h at room temperature with a 12 nm gold conjugated goat anti-mouse antibody (Jackson Laboratories) at a dilution of 1 : 40, rinsed once in TBS buffer, followed by a final rinse in distilled water (dH2O). Finally, grids were incubated in 2% glutaraldehyde, rinsed in dH2O and stained with lead citrate. Samples were visualized on a Phillips CM 120 transmission electron microscope operating at 80 kV.

Cell viability in N2a cells
TUNEL staining was performed as previously described (44Go) using an in situ Cell Death Detection Kit, Texas Red (Roche Molecular Biochemicals). Seventy-two hours after transfection, cells grown on coverslips were fixed using 4% paraformaldehyde in PBS at 37°C for 1 h. Cells were then washed and permeabilized with 0.1% Triton X-100 in PBS on ice for 2 min. Cells were first labeled with a mouse anti-FLAG antibody for 1 h at 37°C followed by TUNEL staining for 1 h at 37°C according to the manufacturer's instructions. Finally, cells were incubated with FITC anti-mouse secondary antibody and Hoescht before mounting. For counting, images were taken from 10 randomly selected fields from each coverslip by conventional fluorescence microscopy (Zeiss). Analysis was carried out on duplicate coverslips in each individual experiment. Data are presented as mean±SEM of three separate experiments with statistical analysis performed using Standard Student's t-test.

Isolation and transfection of murine primary cortical neurons
Dissection of cortex from E15–E16 mouse embryos was performed according to standard procedures (45Go). Transfection of PGQ constructs into mouse primary cortical neurons was carried out using Nucleofector (Amaxa Biosystem), an electroporation-based method. Isolation and preparation of primary neurons from cortex was done according to the recommended manufacturer's procedures. Neurons were then maintained in Dulbecco's modified Eagle medium (Invitrogen) supplemented with 10% FBS and grown on laminin and polyD-lysine pre-coated coverslips (Biocoat). Under these conditions, >99% of cells are neurons. Forty-eight hours after transfection, neurons on coverslips were fixed and labeled to view PGQ construct expression using the same procedures as for N2a cells.

Assessment of toxicity in murine primary cortical neurons
Primary neurons were co-transfected with each htt exon-1 construct and a plasmid encoding Ds-Red (Clontech). Neurons were fixed and stained using an anti-FLAG antibody. For assessment of cell viability, the following criteria were used: only neurons that were co-transfected with both htt exon-1 DNA and Ds-Red were included in the toxicity analysis, a healthy neuron had at least one healthy neurite that was at least two times the length of the same neuronal body, neurites were smooth and not fragmented. For cells that contained only aggregates, neurites were examined only from the Ds-Red field. For cell counting, images were taken from five randomly selected fields from each coverslip. Data are presented as mean±SEM of four separate experiments with statistical analysis performed using Standard Student's t-test.


   ACKNOWLEDGEMENTS
 
We thank C. Elowsky for assistance with Confocal imaging, Michael Delanoy for EM sample preparation and analysis, A. Davidson and Z. Hou for technical assistance, W. Smith, G. Schilling and D. Borchelt for helpful advice and R. Hirschhorn for critical reading of the manuscript. This work was supported by NINDS NS16375, the Hereditary Disease Foundation, the High-Q Foundation and the HD Society of America.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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