Human Molecular Genetics, 2000, Vol. 9, No. 13 2009-2018
© 2000 Oxford University Press
Polyglutamine length-dependent interaction of Hsp40 and Hsp70 family chaperones with truncated N-terminal huntingtin: their role in suppression of aggregation and cellular toxicity
Laboratory for CAG Repeat Diseases, RIKEN Brain Science Institute, 2–1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
Received 18 April 2000; Revised and Accepted 19 June 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by polyglutamine expansion in the disease protein, huntingtin. In HD patients and transgenic mice, the affected neurons form characteristic ubiquitin-positive nuclear inclusions (NIs). We have established ecdysone-inducible stable mouse Neuro2a cell lines that express truncated N-terminal huntingtin (tNhtt) with different polyglutamine lengths which form both cytoplasmic and nuclear aggregates in a polyglutamine length- and inducer dose-dependent manner. Here we demonstrate that newly synthesized polyglutamine-expanded truncated huntingtin interacts with members of Hsp40 and Hsp70 families of chaperones in a polyglutamine length-dependent manner. Of these interacting chaperones, only Hdj-2 and Hsc70 frequently (Hdj-2 > Hsc70) co-localize with both the aggregates in the cellular model and with the NIs in the brains of HD exon 1 transgenic mice. However, Hdj-2 and Hsc70 do not co-localize with cytoplasmic aggregates in the brains of transgenic mice despite these chaperones being primarily localized in the cytoplasmic compartment. This strongly suggests that the chaperone interaction and their redistribution to the aggregates are two completely different phenomena of the cellular unfolded protein response. This unfolded protein response is also evident from the dramatic induction of Hsp70 on expression of polyglutamine-expanded protein in the cellular model. Transient overexpression of either Hdj-1 or Hsc70 suppresses the aggregate formation; however, suppression efficiency is much higher in Hdj-1 compared with Hsc70. Overexpression of Hdj-1 and Hsc70 is also able to protect cell death caused by polyglutamine-expanded tNhtt and their combination proved to be most effective.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Expansion of CAG triplets within the coding regions of target genes is the cause of several autosomal dominant neurodegenerative diseases including Huntington’s disease (HD), several spinocerebellar ataxias (SCAs), dentatorubral pallidoluysian atrophy and X-linked spinal bulbar muscular atrophy (SBMA) (1–3). One of the common characteristic features of all the above diseases is the formation of insoluble aggregates, in particular intranuclear aggregates or nuclear inclusions (NIs) (4–6). The affected neurons in the brains of HD patients show NIs containing N-terminal huntingtin fragments (7–9), and transgenic mice expressing exon 1 of the HD gene containing >115 CAG repeats also have neuronal NIs even before they develop neurological symptoms (10). These findings led us to postulate that such NIs are toxic and responsible for the pathology of HD. In fact, several cellular models of HD also demonstrate that the nuclear aggregates of polyglutamine protein are associated with cell death (11–15). However, two recent studies have raised the possibility that this aggregation may not be the primary factor causing cell death (16,17).
The mechanism that leads the polyglutamine-expanded proteins to aggregate is unknown. One hypothesis is that the glutamine repeats are able to form a polar zipper, an unusual motif for protein–protein interaction (18,19). This polar zipper was predicted to form either between two different molecules with glutamine repeats or within one molecule, forming a hairpin loop. Aggregate formation would then be enhanced by an expanded polyglutamine tract via transglutaminase-catalyzed cross-linking (20) or by aberrant interaction with other proteins dependent on polyglutamine length (21–23). There is also the possibility that the extended polyglutamine tract may destabilize the native conformation of the protein, thereby causing the protein to misfold and aggregate. The fact that the NIs are ubiquitinated raises the possibility that the polyglutamine-expanded proteins become misfolded, with protein degrading machinery such as proteasomes being affected. Indeed recent reports suggest that NIs in SCA1 (24), SCA3 (25,26) and SBMA (27) co-localize with chaperone Hdj-2 and proteasome components. Furthermore, overexpression of the chaperone suppressed the aggregate formation in those studies.
Correct protein folding is an essential biological process, and for correct folding in the cellular milieu, many proteins interact with molecular chaperones. The heat shock protein 70 (Hsp70) class of molecular chaperones is thought to bind early in the folding process to the extended conformation of a polypeptide chain with a preference for hydrophobic sequences, and to maintain the polypeptide in a soluble conformation (28,29). ATP binding and hydrolysis on Hsp70 are coupled to substrate binding and release by conformational changes in the chaperone that represent cooperation between the substrate binding and ATPase domains of Hsp70 (30,31). To facilitate protein folding, Hsp70 must interact with a co-chaperone protein that regulates its ATPase activity (32–34). A major class of Hsp70 co-chaperone proteins is the Hsp40 family. These chaperones also bind to a misfolded substrate and are capable of initiating refolding and of preventing aggregation without the help of Hsp70 (35). On release from the chaperone, a polypeptide may either fold to its native conformation or enter into the degradation machinery of the cells. Chaperones may also play an active role in directing misfolded or mutant proteins to proteolysis by ubiquitin–proteasome pathways (36,37).
In the present study, we hypothesized that the polyglutamine-expanded huntingtin protein is probably misfolded and that the cellular chaperone system may be affected. Therefore, we investigated the involvement of various chaperones in the pathogenesis of HD using HD exon 1 transgenic mice and an inducible stable mouse Neuro2a cell line that expresses truncated N-terminal huntingtin (tNhtt) containing different polyglutamine lengths. We demonstrate that several chaperones of the Hsp family interact with newly synthesized tNhtt in a polyglutamine length-dependent manner and that some chaperones are also co-localized with the aggregates. We further show that the transient overexpression of some of these chaperones reduces the aggregate formation as well as cellular toxicity caused by expanded polyglutamine tracts.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Interaction of Hsp40 and Hsp70 chaperone families with polyglutamine-expanded tNhtt
We have established stable and inducible mouse Neuro2a cell lines that express tNhtt with enhanced green fluorescent protein (EGFP) containing 16, 60 and 150 glutamine residues. These cell lines are denoted HD 16Q-23, 60Q-14 and 150Q-28 and the expressed proteins are tNhtt-16Q, -60Q and -150Q, respectively. Using this cellular model, we showed that the formation of aggregates and cell death induced by tNhtt were polyglutamine length- and inducer dose-dependent (38). In the present study, we used these cell lines to examine the interaction of various chaperones with normal and polyglutamine-expanded tNhtt. Each cell line was differentiated with N6,2-O-dibutyryl-cAMP (dbcAMP) and induced with ponasterone A for 2 days and then the total cell lysate prepared for immunoprecipitation by anti-green fluorescent protein (anti-GFP). The immunoprecipitates were analyzed by SDS–PAGE and immunoblotting. The blots were first probed with either anti-huntingtin or anti-GFP to confirm the immunoprecipitation of GFP–huntingtin protein with various glutamine repeats (Fig. 1A). Both anti-GFP and anti-huntingtin detected two major bands in HD 150Q-28 cell lysate because of the instability of the longer CAG repeats which was further confirmed by PCR analysis. The blots were then sequentially probed with different chaperone antibodies. Figure 1 shows the polyglutamine length-dependent co-immunoprecipitation of Hdj-1 and Hdj-2 chaperones. Both Hdj-2 (Fig. 1B) and Hdj-1 (Fig. 1C) precipitated well with tNhtt-150Q, very poorly with tNhtt-60Q, but did not precipitate at all with tNHtt-16Q. The interaction of Hdj-1 was further confirmed by transient transfection of the HD 16Q-23, 60Q-14 and 150Q-28 cells with Hdj-1 expression plasmid, immunoprecipitation by anti-GFP antibody and detection by V5-tag antibody (Fig. 1D). Control experiments containing mouse IgG instead of anti-GFP antibody resulted in no precipitation of the chaperones. In the reverse experiments using both anti-Hdj-1 and anti-Hdj-2 antibodies, we also observed co-immunoprecipitation of tNhtt-150Q with Hdj-1 and Hdj-2 (data not shown). In another experiment, differentiated HD 150Q-28 cells were induced for different time periods (from 5 h to 3 days) and the cells collected at each time-point then processed for immunoprecipitation with anti-GFP antibody. A blot of the precipitated proteins was then probed with anti-Hdj-2 antibody. The result shows that the immunoprecipitated Hdj-2 due to newly synthesized tNhtt-150Q was detected from 10 h onwards and increased steadily up to 2 days (Fig. 1E). The amount of immunoprecipitated Hdj-2 decreased on the third day due to a gradual increase in aggregate formation of the soluble protein.
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Figure 2A and C demonstrate similar polyglutamine length-dependent co-immunoprecipitation of Hsc70 and Hsp70 chaperones with tNhtt. Reverse experiments were also carried out to confirm the results (data not shown). Interactions of Hsp70 (Fig. 2D) and Hsc70 (data not shown) were further confirmed by transfecting cells with their respective expression plasmids, immunoprecipitating with anti-GFP antibody and probing a blot with V5 antibody. Figure 2B shows the time dependency of the immunoprecipitatable Hsc70. Several other chaperones, namely Hsp27, Hsp60, Hsp90
and Hsp104, were tested in similar co-immunoprecipitation experiments, but none was immunoprecipitated by anti-GFP antibody.
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Expanded polyglutamine protein elicits stress response
Surprisingly, during the immunoprecipitation experiment we observed a very high level of induction of Hsp70 in HD 60Q-14 and 150Q-28 cells. Consequently, we examined the expression levels of various other interacting chaperones in cells induced for different time periods as well as in transgenic mice (R6/1 line) with age-matched controls. In the cellular system, although Hsp70 was normally undetectable, its expression was dramatically up-regulated in a polyglutamine length-dependent manner (Fig. 3). However, the expression levels of various other chaperones, namely Hdj-1, Hdj-2 and Hsc70, did not change. No differences in the expression levels of any of the chaperones were observed between control and transgenic mice (Fig. 3). In both control and transgenic mice, Hsp70 was normally expressed at quite high levels and did not show any further increase in transgenic mice until 30–35 weeks when the mice began to display severe HD symptoms.
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Co-localization of Hdj-2 and Hsc70 chaperones with the polyglutamine aggregates in the cellular model
Since the chaperones interacted with the soluble form of polyglutamine-expanded tNhtt, we next examined whether the aggregated form also contained those chaperones. Therefore, we performed immunocytochemical staining of those chaperones. In wild-type Neuro2a cells or HD 16Q-28, 60Q-14 and 150Q-28 cells, endogenous Hdj-2 and Hsc70 were primarily localized in the cytoplasmic compartment with very faint nuclear staining. However, on induction of the mutant protein in HD 60Q-14 or 150Q-28 cells these chaperones redistributed to the aggregates and showed co-localization with GFP aggregates (Fig. 4). The staining pattern of Hsc70 in the aggregates was much weaker and the frequency of the Hsc70 positively stained aggregates were also comparatively lower than Hdj-2. However, we were unable to find any Hdj-1- and Hsp70-positive aggregates in either HD 60Q-14 or 150Q-28 cells, although both interacted with the polyglutamine-expanded protein.
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Redistribution of Hdj-2 and Hsc70 chaperones to the NIs in HD exon 1 transgenic mice
We next examined the subcellular localization of various chaperones in the brains of HD exon 1 transgenic mice. On immunostaining of the R6/1 transgenic mice brain sections, we found localization of Hdj-2 and Hsc70 chaperones to the NIs (Fig. 5). In control mice, these chaperones were mainly localized to the cytoplasm. A quantitative estimation of the ubiquitin-, Hdj-2- and Hsc70-positive NIs in the cerebral cortex and striatum area is shown in Figure 2B. Immunofluorescence staining was performed to visualize the respective positively stained fluorescein isothiocyanate (FITC)-labeled NIs, and nuclei were counterstained with propidium iodide. The brains of R6/1 transgenic mice at 10–12 weeks contained ~17% of ubiquitin-positive NIs, which increased to 72% at 30–35 weeks. However, Hdj-2- and Hsc70-positive NIs were undetectable at 10–12 weeks and detectable to 51 and 25%, respectively, at 30–35 weeks. In 10- to 12-week-old R6/2 mice, the percentage of ubiquitin-, Hdj-2- and Hsc70-positive NIs were relatively higher than in 30- to 35-week-old R6/1 mice. The frequency of Hdj-2- and Hsc70-positive NIs were much lower compared with that of ubiquitin-positive NIs, and the frequency of Hsc70-positive NIs was ~50% lower than that of Hdj-2-positive NIs in both transgenic mice. Hdj-1 and Hsp70 did not localize to the NIs in both transgenic mice. Figure 6 demonstrates immunofluorescence double labeling of the Hdj-2 chaperone and ubiquitin in transgenic mouse brain sections. Anti-ubiquitin positively stained nuclear and many cytoplasmic aggregates. However, anti-Hdj-2 antibody mostly stained the nuclear aggregates. A similar phenomenon was observed in the case of Hsc70. Several other Hsps, i.e. Hsp27, Hsp60, Hsp90
and Hsp105, did not localize to the NIs.
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Suppression of polyglutamine protein aggregation by Hdj-1 and Hsc70 chaperones
Next we examined whether the interaction of the chaperone with polyglutamine-expanded tNhtt was trying to keep the polyglutamine protein in a soluble form or whether it was enhancing the process of aggregation. We addressed this problem by overexpressing those chaperones into the HD 150Q-28 cells. Overexpression of Hdj-1 efficiently prevented aggregate formation in HD 150Q-28 cells (Fig. 7A). Hsc70 overexpression (Fig. 7B) also had a significant effect on aggregate suppression; however, Hsp70 (Fig. 7C) had no effect. Co-expression of either Hsc70 or Hsp70 with Hdj-1 did not improve the suppressive effect that was observed with overexpression of Hdj-1 alone. The J-domain deleted form of Hdj-1 or ATPase domain deletion mutant of Hsc70 also had a significant effect on suppression of aggregate formation (Fig. 7A and B). However, overexpression of only the J-domain of Hdj-1 or only the ATPase domain of Hsc70 had no effect. The J-domain deletion mutant of Hdj-1 or ATPase domain deletion mutant of Hsc70 both retain their capacity to bind with the polyglutamine-expanded tNhtt as evidenced from immunoprecipitation (data not shown). A similar suppressive effect of Hdj-1 and Hsc70 on polyglutamine protein aggregation was also observed in HD 60Q-14 cell lines. We further confirmed our result by transiently transfecting those chaperones along with pIND-tNhtt-EGFP-150Q at different ratios into Neuro2a cells stably expressing VgRXR (the functional ecdysone receptor). Twenty-four hours after transfection, cells were induced with ponasterone A (1 µM) and, at 48 h post-transfection, cells were fixed and processed for immunofluorescence staining of Hdj-1 and Hsc70 by V5 antibody. As shown in Figure 8, either Hdj-1 or Hsc70 along with polyglutamine-expanded huntingtin efficiently prevent aggregate formation and counting of the aggregates (Fig. 8D) in the transfected cells revealed a dramatic extent of aggregate suppression.
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Hdj-1 and Hsc70 chaperones protect against cell death caused by expanded polyglutamine protein
The ability of the Hdj-1 and Hsc70 chaperones to suppress aggregate formation prompted us to investigate the change in cellular toxicity caused by polyglutamine-expanded tNhtt. We used HD 150Q-28 cells for this investigation. On induction with 1 µM ponasterone A, the differentiated HD 150Q-28 cells showed ~22–36% death on the third and fourth days. Overexpression of Hdj-1 significantly protected against this cell death. Hsc70 also showed a small suppressive effect on cellular toxicity but this was not statistically significant. Co-expression of Hsc70 with Hdj-1 improved the protective effect further in comparison with Hdj-1 alone (Fig. 9A). The expression levels of Hdj-1, Hsc70 and tNhtt-150Q are shown in Figure 9B.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Protein aggregation is the most common characteristic feature among the polyglutamine diseases. The mechanism that causes polyglutamine-expanded proteins to aggregate is not fully understood. Here, we demonstrate that several chaperones of the Hsp family interact with polyglutamine-expanded protein in a glutamine repeat length-dependent manner. This suggests that polyglutamine-expanded proteins are misfolded and become prone to aggregation and that the misfolding propensity is directly proportional to the length of the glutamine repeats. Among several chaperones tested, the members of the Hsp40 (Hdj-1 and Hdj-2) and Hsp70 (both constitutive and inducible form) families bind to the polyglutamine-expanded proteins.
Of these four interacting chaperones, Hdj-2 and Hsc70 were frequently co-localized with the aggregates in both the cellular and HD exon 1 transgenic mouse model, indicating that their interaction might be involved in enhancing the process of aggregation. The hypothesis was further supported by the fact that the yeast chaperone Hsp104 has been shown to be necessary for the conversion of prions from soluble to insoluble form through the stabilization of certain folding intermediates. This phenomenon was lost when Hsp104 was overexpressed or removed from the system (39,40). However, in both our cellular and transgenic mouse models, the staining pattern of Hsc70 in the aggregates was very weak and the frequency of positively stained aggregates was also quite low compared with that of Hdj-2. Furthermore, immunofluorescence double labeling between Hdj-2 and ubiquitin in the transgenic mouse brain section revealed that the Hdj-2 associated with only nuclear and not cytoplasmic aggregates. This indicates that Hdj-2 and Hsc70 are most likely not involved in the aggregation process, since these chaperones are mostly cytoplasmic, and if their interaction helps polyglutamine proteins to aggregate we would expect at least some cytoplasmic aggregates to be positively stained for these chaperones.
One possible explanation as to why the chaperones bind to the nuclear aggregates follows. The polyglutamine-expanded proteins escape from the cytoplasmic chaperone barrier, gradually accumulate and form aggregates inside the nucleus. When the aggregates are sufficiently large, a stress response is induced. As a result, the chaperone targets the nuclear aggregates but eventually the attempt is unsuccessful. Alternatively, the chaperones might help the expanded polyglutamine protein to enter the nucleus, where the chaperone-bound polyglutamine protein complex associates with the neighboring molecules and forms aggregates. The nuclear environment possibly favors the aggregation process because the nucleus is less efficient than the cytoplasm in refolding or degrading misfolded proteins (16,41,42). From our observations, it is conceivable that the chaperone interaction with the soluble form of polyglutamine-expanded proteins and their recruitment to the polyglutamine aggregates are two completely different phenomena of the cellular unfolded protein response.
The co-localization of Hdj-2 chaperone to the NIs has been demonstrated in SCA1 (24), SCA3 (26) and SBMA (27) and in all the disease models examined overexpression of Hdj-2 chaperone suppresses aggregate formation. Co-localization of Hdj-1 and Hsp70 with the NIs and suppression of aggregate formation by Hdj-1 has also been demonstrated in SCA3 (26). In the cellular model of HD used in the present study, we found suppression of aggregate formation by Hdj-1 and Hsc70. This strongly suggests that the common target of Hsp40 and Hsp70 family members are any polyglutamine-expanded protein and that Hsp40 and Hsp70 are effective in the suppression of polyglutamine-mediated protein aggregation. However, the suppression efficiency differs among the members of these two families of chaperones. Our results suggest that Hsp40 family members are much more effective in aggregation suppression compared with members of the Hsp70 family. Whether these differences also exist among the members of the same family has yet to be determined.
The Hsp40 family chaperones are known to be co-chaperones for the Hsp70 family and modulate the cellular protein folding by binding to misfolded polypeptides via the C-terminal domain and regulating the ATPase activity of the Hsp70 family members through the N-terminal J-domain (33,34). However, there are reports that the members of the Hsp40 family are also capable of refolding the misfolded substrates and preventing aggregation without the help of Hsp70 family members (35,43). The fact that the J-domain deletion mutant of Hdj-1 still suppresses the aggregate formation strongly suggests that the Hsp40 family chaperones can work alone and also possibly in cooperation with the Hsp70 family to suppress aggregation. How those chaperones are involved in the folding process of unfolded polyglutamine-expanded protein is not clear. Possibly, at higher concentrations, the chaperone-bound folding intermediate efficiently prevents the intra- and intermolecular polar zipper formation and keeps the polyglutamine protein in a soluble form while enhancing their degradation by ubiquitin–proteasome pathways at the same time. The Hsp40 and Hsp70 families of chaperones have been shown to play a critical role in the rapid degradation of misfolded proteins by the ubiquitin–proteasome pathway (36,37).
Another interesting observation in the present study was the polyglutamine length-dependent induction of Hsp70 in the cellular model, despite being unable to detect any major changes in Hsp70 levels in the HD exon 1 transgenic mice (R6/1) until 30–35 weeks, when they showed severe HD symptoms. The induction of Hsp70 has also been reported in the cellular model of SCA3 but not in their transgenic model (26). Many stressful conditions induce Hsp70, which increases the cell’s tolerance to stress. Induction of Hsp70 by polyglutamine-expanded protein suggests that the cell is under stress and trying to compensate. However, the factors generating this stress are currently unknown. One possibility is that the utilization and redistribution of chaperones as well as proteasomes (unpublished data) to the aggregates creates a deficit in their normal active cellular pool, which in turn increases the accumulation of misfolded proteins and ultimately creates stress. Our hypothesis is supported by the observation that inhibition of proteasome function leads to the induction of various chaperones (44). Moreover, the cellular deficit of chaperones and proteasomes might ultimately result in disease progression by disturbing vital cellular function.
In the present study, we found that overexpression of the Hsp40 family chaperones significantly reduces the cellular toxicity created by polyglutamine-expanded tNhtt. This protective effect was further improved by overexpressing Hsp40 with Hsc70. Recently, overexpression of the Hsp40 chaperone has also been reported to suppress the cell death due to mutant SCA3 protein (26). Moreover, an in vivo study in a Drosophila SCA3 disease model demonstrated that overexpression of Hsp70 suppresses polyglutamine-induced neurodegeneration and delays the progression of the disease without a visible effect on NI formation (45). In the cellular model of SCA3, Hsp70 overexpression does not have a suppressive effect on aggregate formation (26). In the cellular model for HD, we were unable to detect any significant suppression of aggregate formation or cellular toxicity on Hsp70 overexpression. One possible explanation is that in cellular models, endogenous Hsp70 is highly induced by polyglutamine-expanded protein and therefore further overexpression does not make a further difference. However, the specificity of Hsp70 protection against polyglutamine protein-mediated cell death remains to be explained, since Hsp70 itself is anti-apoptotic and can rescue the cell from stress-induced apoptosis (46,47).
The challenge is to determine whether the Hsp40 and Hsp70 family chaperones really suppress the aggregation and protect the neurodegeneration in the in vivo HD transgenic mouse model. If so, and if there are no adverse effects of overexpression, then these chaperones may prove to be effective therapeutic molecules by which to slow the progression of HD and also other polyglutamine diseases.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mice
Heterozygous HD exon 1 transgenic male mice of R6/1 (115 CAG repeats) and R6/2 (145 CAG repeats) lines were obtained from the Jackson Laboratory (Bar Harbor, ME) (Jackson codes: B6CBA-TgN [Hdexon1]61 and B6CBA-TgN[Hdexon1]62) and maintained by crossing carrier males with CBA females. The genotyping and CAG repeat sizing was carried out using a PCR assay and Genescan, respectively, as described previously (48). Mice (transgenic and their age-matched controls) were sacrificed using ether anesthesia, and their brains carefully removed and collected in Tissue-Tek (Sakura Finetek, Tokyo, Japan), frozen with powdered solid CO2 and stored at –80°C.
Antibodies
Antibodies utilized in this study were purchased from the following sources. The rabbit polyclonal anti-Hdj-1 (SPA-400) and mouse monoclonal anti-Hsp70 (SPA-810) were purchased from StressGen Biotechnologies (Victoria, British Columbia). Mouse monoclonal anti-Hdj-2 (MS-225) was from Neomarkers (Union City, CA). Goat polyclonal anti-Hsc70 (sc-1059), goat polyclonal anti-Hsp60 (sc-1052), rabbit polyclonal anti-Hsp105 (sc-1805), goat polyclonal anti-Hsp90 and goat polyclonal anti-Hsp27 (sc1798) were from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse monoclonal anti-GAPDH (MAB374) was from Chemicon International (Tenecula, CA). Rabbit polyclonal anti-huntingtin (corresponding to N-terminal amino acid sequences) was raised in our laboratory. Goat anti-rabbit IgG–Cy3, goat anti-mouse IgG–Cy3 and donkey anti-goat IgG–Cy3 (Molecular Probes, Eugene, OR) were utilized as secondary antibodies in indirect immunofluorescence. Horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG (Amersham Life Science, Little Chalfont, UK) and anti-goat IgG (Santa Cruz Biotechnology) were utilized as secondary antibodies in immunoblotting.
Expression plasmids
The tNhtt expression constructs pIND-tNhtt-EGFP-16Q, pIND-tNhtt-EGFP-60Q and pIND-tNhtt-EGFP-150Q have been described previously (39). Each construct contains 1–90 amino acids of tNhtt with a different polyglutamine length fused to the N-terminus of EGFP. The Hsp70 and Hsp40 expression vectors in pcDNA3.1/GS were obtained from Invitrogen (Carlsbad, CA). The full-length Hsc70 cDNA was isolated from human brain total RNA by using RT–PCR. Primer sequences were 5'-GTGGCTTCCTTCGTTATTGG-3' (sense) and 5'-TTAATCAACCTCTTCAATGGTGGG-3' (antisense). Proof reading polymerase (TaKaRa, Kyoto, Japan) was used in the PCR reaction. PCR fragments were A-tailed and ligated into pGEM-T Easy Vector (Promega, Madison, WI) and confirmed by sequencing. The full-length, ATPase domain (amino acids 1–384) and the ATPase domain deletion (amino acids 385–646) mutant of Hsc70 expression vector in pcDNA6/V5-His were prepared using PCR and subcloning the product in frame into the KpnI–XhoI site of the vector. The primer sequences were: 5'-ACGGGGTACCATGGCCAAGGGACCTGCAC-3' (sense, for full-length and ATPase domain mutant); 5'-ACGGGGTACCATGGTGTCTGGAGACAAGTC-3' (sense, for ATPase domain deletion mutant); 5'-ACCGCTCGAGCGGATCAACCTCTTCAATGG-3' (antisense for full-length and ATPase domain deletion mutant); and 5'-ACCGCTCGAGGGCTGCCTGGACAGCTGCACCATAAGC-3' (antisense for ATPase domain mutant). The J-domain mutant (amino acids 1–70) or J-domains deletion (amino acids 70–341) mutant of Hdj-1 was also constructed from full-length Hdj-1 by subcloning the PCR product into the BamHI–XhoI site of the pcDNA6/V5-His plasmid. Primer sequences were: 5'-ACGCGGATCCACCATGGGTAAAGACTACTACCA-3' (sense for J-domain mutant); 5'-ACCGCTCGAGTTCCTCCCCGTAGCGGTCGA-3' (antisense for J-domain mutant); 5'-ACGCGGATCCACCATGGGCCTAAAGGGGAGTGGCC-3' (sense for J-domain deletion mutant); and 5'-ACCGCTCGAGATATATTGGAAGAACCTGCT-3' (antisense for J-domain deletion mutant). Underlined sequences represent the sites for restriction enzymes.
Cell culture and treatments
Mouse Neuro2a cells stably expressing tNhtt-EGFP-16Q, tNhtt-EGFP-60Q and tNhtt-EGFP-150Q were regularly maintained in Dulbecco’s modified Eagle’s medium (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum, 0.4 mg/ml zeocin and 0.4 mg/ml G418. Establishment of stable cell lines has been described earlier (39). Cells were differentiated by treating 5 mM dbcAMP (N6,2'-O-dibutyryladenosine-3':5'-cyclic monophosphate sodium salt (Nacalai Tesque, Kyoto, Japan) and induced with different concentrations (0.1–2 µM) of ponasterone A (Invitrogen).
Co-immunoprecipitation experiments
Cells were washed with cold phosphate-buffered saline (PBS), scraped, pelleted by centrifugation and lysed on ice for 30 min with RIPA buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 10 mM EDTA, 2.5 mM EGTA, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 10 mM NaF, 5 mM Na4P2O7, 0.1 mM Na3VO4, 1 mM PMSF, 0.1 mg/ml Aprotinin). Cell lysate was briefly sonicated, centrifuged for 10 min at 15 000 g at 4°C and the supernatants (total soluble extract) were used for immunoprecipitation. Protein concentration was measured according to the method of Bradford using Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA) and bovine serum albumin as a standard. For each immunoprecipitation experiment, 200 µg of protein in 0.2 ml of RIPA buffer was incubated either with 5 µl (2 µg) of anti-GFP antibody or 4 µl (2 µg) of normal mouse IgG. After 5–6 h of incubation at 4°C with rotation, 10 µl of magnetic protein G beads (Perspective Biosystems, Framingham, MA) were added and incubation was continued at 4°C overnight. The beads were pulled down with a magnet (Dynal, Oslo, Norway) and washed six times with RIPA buffer. Bound proteins were eluted from the beads with SDS (1x) sample buffer, vortexed, boiled for 5 min and analyzed by immunoblotting.
Immunoblotting
The total cell lysate, mouse brain total soluble extracts (homogenate after centrifugation at 15 000 g for 10 min) or the immunoprecipitated proteins were separated through SDS–polyacrylamide gel (7.5–20%) electrophoresis and transferred onto PVDF membrane (Immobilon-P; Millipore, Bedford, MA). The membranes were successively incubated in blocking buffer [5% skim milk in TBST (50 mM Tris pH 7.5, 0.15 M NaCl, 0.05% Tween)], with primary antibody in TBST, and then with secondary antibody conjugated with HRP in TBST. Detection was carried out with enhanced chemiluminescence (ECL) reagent (Amersham Life Science).
Immunofluorescence techniques
Cells grown in chamber slides were differentiated and induced together for 2 days. Cells were washed twice with PBS, fixed with 4% paraformaldehyde in PBS for 20 min, permeabilized with 0.5% Triton X-100 in PBS for 5 min, washed extensively and then blocked with 5% non-fat dried milk in TBST for 1 h. Primary antibody incubation was carried out overnight at 4°C. After several washings with TBST, cells were incubated with appropriate secondary antibody for 1 h, washed several times and mounted in antifade solution (Vectashield Mounting Media; Vector, Burlingame, CA). The primary antibodies, anti-Hdj-2 and anti-Hdj-1, were used at 1:1000 dilution and anti-Hsc70, anti-Hsp70, anti-Hsp60, anti-Hsp105 and anti-Hsp90 were used at 1:250 dilution. Secondary antibodies conjugated with Cy3 were used at 1:1000 dilution. Samples were observed using a confocal microscope (Fluoview; Olympus, Tokyo, Japan) and digital images were assembled using Adobe Photoshop. For immunofluorescence staining of various chaperones and ubiquitin in the transgenic mouse brain sections, the sections were fixed and incubated with different primary antibody in a similar manner to that described for the immunohistochemistry. The appropriate FITC- or Cy3-conjugated secondary antibody was used to visualize the expression and localization. In some experiments, nuclei were counterstained with propidium iodide.
Immunohistochemistry
The frozen brains mounted on Tissue-Tek were sectioned in freezing microtome to 20 µm thickness. Sections were fixed with 4% paraformaldehyde in PBS for 20 min, washed several times, blocked with 5% non-fat dried milk for 1 h and then incubated overnight with primary antibody. Staining was carried out using ABC Elite kit (Vector). Briefly, after primary antibody incubation, sections were washed and incubated for 1 h at room temperature with the appropriate biotinylated secondary antibody, washed, incubated with ABC reagent, washed, exposed for several minutes to DAB substrate, washed, dehydrated, cleared and mounted. Dilutions of the different primary antibodies used were the same as described for immunofluorescence.
Transfections, quantitation of aggregate formation and cell viability
Transfection of various chaperones (2 µg/well each) was carried out in 60 mm tissue cultured plates using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. Twenty-four hours after transfection, cells were collected, counted by Trypan blue and re-plated into the chamber slides (for counting aggregation) or 96-well tissue cultured plates (for cell viability assay). Forty-eight hours post-transfection, cells were treated with dbcAMP and ponasterone A. For counting aggregation, 1 x 103 cells were seeded into each well of the chamber slides and, for cell viability assay, 5 x 103 cells were seeded into each well of 96-well plates. Both aggregate formation and cell viability were monitored at different time-points. Aggregate formation was manually counted under a fluorescence microscope and the cells containing more than one aggregate were considered to have a single aggregate. Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay described previously (39). Statistical analysis was performed using paired t-test, with P < 0.05 considered statistically significant.
In other experiments, we transiently transfected 0.5 µg of pIND-tNhtt-EGFP-150Q along with different concentrations of various chaperones (0.5–2 µg) into mouse Neuro2a cells stably transfected with pVgRXR which express a functional ecdysone receptor. Twenty-four hours after transfection, cells were differentiated with 5 mM dbcAMP and induced with 1 µM of ponasterone A. At 24 h post-transfection, cells were processed for immunofluorescence staining of Hdj-1 and Hsc70 using V5 antibody. The appropriate Cy3-conjugated secondary antibody was used to visualize the transfected cells. The number of aggregates was counted using ~100 transfected cells for quantitative estimation.
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ACKNOWLEDGEMENT |
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This work was supported partly by a grant from the Ministry of Health and Welfare.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +81 48 467 9702; Fax: +81 48 462 4796; Email: nukina@brain.riken.go.jp
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Y. Zhang, N. Champagne, L. K. Beitel, C. G. Goodyer, M. Trifiro, and A. LeBlanc Estrogen and Androgen Protection of Human Neurons against Intracellular Amyloid {beta}1-42 Toxicity through Heat Shock Protein 70 J. Neurosci., June 9, 2004; 24(23): 5315 - 5321. [Abstract] [Full Text] [PDF]
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Y. Liu and D. Vollrath Reversal of mutant myocilin non-secretion and cell killing: implications for glaucoma Hum. Mol. Genet., June 1, 2004; 13(11): 1193 - 1204. [Abstract] [Full Text] [PDF]
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M. T. Berciano, N. T. Villagra, J. L. Ojeda, J. Navascues, A. Gomes, M. Lafarga, and M. Carmo-Fonseca Oculopharyngeal muscular dystrophy-like nuclear inclusions are present in normal magnocellular neurosecretory neurons of the hypothalamus Hum. Mol. Genet., April 15, 2004; 13(8): 829 - 838. [Abstract] [Full Text] [PDF]
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J. Magrane, R. C. Smith, K. Walsh, and H. W. Querfurth Heat Shock Protein 70 Participates in the Neuroprotective Response to Intracellularly Expressed {beta}-Amyloid in Neurons J. Neurosci., February 18, 2004; 24(7): 1700 - 1706. [Abstract] [Full Text] [PDF]
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K. L. Sugars, R. Brown, L. J. Cook, J. Swartz, and D. C. Rubinsztein Decreased cAMP Response Element-mediated Transcription: AN EARLY EVENT IN EXON 1 AND FULL-LENGTH CELL MODELS OF HUNTINGTON'S DISEASE THAT CONTRIBUTES TO POLYGLUTAMINE PATHOGENESIS J. Biol. Chem., February 6, 2004; 279(6): 4988 - 4999. [Abstract] [Full Text] [PDF]
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K. F. Winklhofer, I. H. Henn, P. C. Kay-Jackson, U. Heller, and J. Tatzelt Inactivation of Parkin by Oxidative Stress and C-terminal Truncations: A PROTECTIVE ROLE OF MOLECULAR CHAPERONES J. Biol. Chem., November 21, 2003; 278(47): 47199 - 47208. [Abstract] [Full Text] [PDF]
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A. Abu-Baker, C. Messaed, J. Laganiere, C. Gaspar, B. Brais, and G. A. Rouleau Involvement of the ubiquitin-proteasome pathway and molecular chaperones in oculopharyngeal muscular dystrophy Hum. Mol. Genet., October 16, 2003; 12(20): 2609 - 2623. [Abstract] [Full Text] [PDF]
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A. Michalik and C. Van Broeckhoven Pathogenesis of polyglutamine disorders: aggregation revisited Hum. Mol. Genet., October 15, 2003; 12(90002): R173 - 186. [Abstract] [Full Text] [PDF]
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W. S. Jackson, S. J. Tallaksen-Greene, R. L. Albin, and P. J. Detloff Nucleocytoplasmic transport signals affect the age at onset of abnormalities in knock-in mice expressing polyglutamine within an ectopic protein context Hum. Mol. Genet., July 1, 2003; 12(13): 1621 - 1629. [Abstract] [Full Text] [PDF]
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K. Ishihara, N. Yamagishi, Y. Saito, H. Adachi, Y. Kobayashi, G. Sobue, K. Ohtsuka, and T. Hatayama Hsp105{alpha} Suppresses the Aggregation of Truncated Androgen Receptor with Expanded CAG Repeats and Cell Toxicity J. Biol. Chem., June 27, 2003; 278(27): 25143 - 25150. [Abstract] [Full Text] [PDF]
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Y. C. Tsai, P. S. Fishman, N. V. Thakor, and G. A. Oyler Parkin Facilitates the Elimination of Expanded Polyglutamine Proteins and Leads to Preservation of Proteasome Function J. Biol. Chem., June 6, 2003; 278(24): 22044 - 22055. [Abstract] [Full Text] [PDF]
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H. Adachi, M. Katsuno, M. Minamiyama, C. Sang, G. Pagoulatos, C. Angelidis, M. Kusakabe, A. Yoshiki, Y. Kobayashi, M. Doyu, et al. Heat Shock Protein 70 Chaperone Overexpression Ameliorates Phenotypes of the Spinal and Bulbar Muscular Atrophy Transgenic Mouse Model by Reducing Nuclear-Localized Mutant Androgen Receptor Protein J. Neurosci., March 15, 2003; 23(6): 2203 - 2211. [Abstract] [Full Text] [PDF]
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W. Duan, Z. Guo, H. Jiang, M. Ware, X.-J. Li, and M. P. Mattson Dietary restriction normalizes glucose metabolism and BDNF levels, slows disease progression, and increases survival in huntingtin mutant mice PNAS, March 4, 2003; 100(5): 2911 - 2916. [Abstract] [Full Text] [PDF]
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K. Mitsui, H. Nakayama, T. Akagi, M. Nekooki, K. Ohtawa, K. Takio, T. Hashikawa, and N. Nukina Purification of Polyglutamine Aggregates and Identification of Elongation Factor-1alpha and Heat Shock Protein 84 as Aggregate-Interacting Proteins J. Neurosci., November 1, 2002; 22(21): 9267 - 9277. [Abstract] [Full Text] [PDF]
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V. Fonte, V. Kapulkin, A. Taft, A. Fluet, D. Friedman, and C. D. Link Interaction of intracellular beta amyloid peptide with chaperone proteins PNAS, July 9, 2002; 99(14): 9439 - 9444. [Abstract] [Full Text] [PDF]
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Y. Kouroku, E. Fujita, A. Jimbo, T. Kikuchi, T. Yamagata, M. Y. Momoi, E. Kominami, K. Kuida, K. Sakamaki, S. Yonehara, et al. Polyglutamine aggregates stimulate ER stress signals and caspase-12 activation Hum. Mol. Genet., June 15, 2002; 11(13): 1505 - 1515. [Abstract] [Full Text] [PDF]
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J.-Z. Chuang, H. Zhou, M. Zhu, S.-H. Li, X.-J. Li, and C.-H. Sung Characterization of a Brain-enriched Chaperone, MRJ, That Inhibits Huntingtin Aggregation and Toxicity Independently J. Biol. Chem., May 24, 2002; 277(22): 19831 - 19838. [Abstract] [Full Text] [PDF]
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A. Wyttenbach, O. Sauvageot, J. Carmichael, C. Diaz-Latoud, A.-P. Arrigo, and D. C. Rubinsztein Heat shock protein 27 prevents cellular polyglutamine toxicity and suppresses the increase of reactive oxygen species caused by huntingtin Hum. Mol. Genet., May 1, 2002; 11(9): 1137 - 1151. [Abstract] [Full Text] [PDF]
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C. Zabel, D. C. Chamrad, J. Priller, B. Woodman, H. E. Meyer, G. P. Bates, and J. Klose Alterations in the Mouse and Human Proteome Caused by Huntington's Disease Mol. Cell. Proteomics, May 1, 2002; 1(5): 366 - 375. [Abstract] [Full Text] [PDF]
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Y. P. Bao, L. J. Cook, D. O'Donovan, E. Uyama, and D. C. Rubinsztein Mammalian, Yeast, Bacterial, and Chemical Chaperones Reduce Aggregate Formation and Death in a Cell Model of Oculopharyngeal Muscular Dystrophy J. Biol. Chem., March 29, 2002; 277(14): 12263 - 12269. [Abstract] [Full Text] [PDF]
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C. K. Bailey, I. F. M. Andriola, H. H. Kampinga, and D. E. Merry Molecular chaperones enhance the degradation of expanded polyglutamine repeat androgen receptor in a cellular model of spinal and bulbar muscular atrophy Hum. Mol. Genet., March 1, 2002; 11(5): 515 - 523. [Abstract] [Full Text] [PDF]
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V. C. Wheeler, C.-A. Gutekunst, V. Vrbanac, L.-A. Lebel, G. Schilling, S. Hersch, R. M. Friedlander, J. F. Gusella, J.-P. Vonsattel, D. R. Borchelt, et al. Early phenotypes that presage late-onset neurodegenerative disease allow testing of modifiers in Hdh CAG knock-in mice Hum. Mol. Genet., March 1, 2002; 11(6): 633 - 640. [Abstract] [Full Text] [PDF]
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H. Zhou, S.-H. Li, and X.-J. Li Chaperone Suppression of Cellular Toxicity of Huntingtin Is Independent of Polyglutamine Aggregation J. Biol. Chem., December 14, 2001; 276(51): 48417 - 48424. [Abstract] [Full Text] [PDF]
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W. J. Welch and M. I. Diamond Glucocorticoid modulation of androgen receptor nuclear aggregation and cellular toxicity is associated with distinct forms of soluble expanded polyglutamine protein Hum. Mol. Genet., December 1, 2001; 10(26): 3063 - 3074. [Abstract] [Full Text] [PDF]
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K. Sathasivam, B. Woodman, A. Mahal, F. Bertaux, E. E. Wanker, D. T. Shima, and G. P. Bates Centrosome disorganization in fibroblast cultures derived from R6/2 Huntington's disease (HD) transgenic mice and HD patients Hum. Mol. Genet., October 1, 2001; 10(21): 2425 - 2435. [Abstract] [Full Text] [PDF]
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A. Wyttenbach, J. Swartz, H. Kita, T. Thykjaer, J. Carmichael, J. Bradley, R. Brown, M. Maxwell, A. Schapira, T. F. Orntoft, et al. Polyglutamine expansions cause decreased CRE-mediated transcription and early gene expression changes prior to cell death in an inducible cell model of Huntington's disease Hum. Mol. Genet., August 1, 2001; 10(17): 1829 - 1845. [Abstract] [Full Text] [PDF]
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A. Sittler, R. Lurz, G. Lueder, J. Priller, M. K. Hayer-Hartl, F. U. Hartl, H. Lehrach, and E. E. Wanker Geldanamycin activates a heat shock response and inhibits huntingtin aggregation in a cell culture model of Huntington's disease Hum. Mol. Genet., June 1, 2001; 10(12): 1307 - 1315. [Abstract] [Full Text] [PDF]
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J.-M. Lecerf, T. L. Shirley, Q. Zhu, A. Kazantsev, P. Amersdorfer, D. E. Housman, A. Messer, and J. S. Huston Human single-chain Fv intrabodies counteract in situ huntingtin aggregation in cellular models of Huntington's disease PNAS, April 10, 2001; 98(8): 4764 - 4769. [Abstract] [Full Text] [PDF]
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