Human Molecular Genetics, 2000, Vol. 9, No. 19 2811-2820
© 2000 Oxford University Press
Mechanisms of chaperone suppression of polyglutamine disease: selectivity, synergy and modulation of protein solubility in Drosophila
Howard Hughes Medical Institute, Department of Biology, 415 South University Avenue, University of Pennsylvania, Philadelphia, PA 19104-6018, USA and 1Department of Neurology, University of Iowa College of Medicine, Iowa City, IA 52242, USA
Received 16 August 2000; Revised and Accepted 29 September 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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At least eight dominant human neurodegenerative diseases are due to the expansion of a polyglutamine within the disease proteins. This confers toxicity on the proteins and is associated with nuclear inclusion formation. Recent findings indicate that molecular chaperones can modulate polyglutamine pathogenesis, but the basis of polyglutamine toxicity and the mechanism by which chaperones suppress neurodegeneration remains unknown. In a Drosophila disease model, we demonstrate that chaperones show substrate specificity for polyglutamine protein, as well as synergy in suppression of neurotoxicity. Our analysis also reveals that chaperones alter the solubility properties of the protein, indicating that chaperone modulation of neurodegeneration in vivo is associated with altered biochemical properties of the mutant polyglutamine protein. These findings have implications for these and other human neurodegenerative diseases associated with abnormal protein aggregation.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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At least eight human neurodegenerative diseases, including Huntington’s disease, share a common molecular mechanism involving expansion of a polyglutamine tract within the respective disease proteins (1–3). Expanded polyglutamine confers dominant toxicity on the otherwise unrelated disease proteins, leading to neuronal dysfunction and cell loss. To elucidate molecular mechanisms underlying disease, polyglutamine toxicity has been modeled in Drosophila melanogaster through targeted expression of polyglutamine disease proteins (4,5). Expression of pathogenic human polyglutamine protein in Drosophila induces late-onset, progressive neurodegeneration, indicating that mechanisms of polyglutamine-induced neural degeneration are conserved in flies. Drosophila thus provides a genetic system in which to address modulation of neurotoxicity in vivo.
In human disease tissue, transgenic animal models and transfected cells, expanded polyglutamine proteins have been shown to undergo intracellular aggregation, in most cases forming nuclear inclusions (NIs) (1–3). These aggregates are immunoreactive for ubiquitin, various molecular chaperones and components of the proteasome complex (6–9). These data suggest that cells recognize aggregated disease protein as abnormal and that chaperone and proteasome recruitment to inclusions may serve to refold, disaggregate and/or degrade the mutant protein. Consistent with this view, overexpression of select molecular chaperones in mammalian cells in culture has been shown to reduce aggregate formation by several polyglutamine disease proteins (6,8,9).
Using Drosophila as a model, we have begun to address the role of heat shock protein (Hsp) chaperones in polyglutamine disease. We have used the disease protein for one of the most common polyglutamine diseases, spinocerebellar ataxia type 3, also known as Machado–Joseph disease (SCA3/MJD) (10). Normally, the SCA3 protein Ataxin-3 has a polyglutamine domain of 12–40 glutamine residues, which in disease becomes expanded to 60–80 residues. In the fly, expression of a truncated form of Ataxin-3 containing expanded polyglutamine induces progressive neurodegeneration accompanied by the formation of NIs, to which endogenous Hsp70, the major stress-induced molecular chaperone in Drosophila, localizes (4,11).
Co-expression of Hsp70 with toxic polyglutamine protein suppresses neurodegeneration in flies (11). Because Hsp70 activity is modulated by Hsp40 molecular chaperones, we have now examined the role and specificity of Hsp40 chaperones in modulation of neurodegeneration in Drosophila. Hsp40 chaperones are also called DNAJ proteins, after the bacterial homolog, and contain a J-domain that is important for interaction with Hsp70 (12). The different C-terminal domains of the Hsp40 proteins are suggested to impart substrate specificity to particular Hsp40 chaperones (13). J-domain-containing proteins have been shown to have effects on phenotypes induced by isolated polyglutamine tracts when expressed in the fly eye (14). In yeast and Caenorhabditis elegans, Hsp104, a chaperone not found in flies or humans, affects aggregation of polyglutamine proteins (15,16), although potential effects on neural degeneration are unknown. More recent studies in vitro implicate Hsp70 and Hsp40 as co-chaperones that can inhibit fibril formation by polyglutamine disease protein (17). In contrast, Hsp70 suppression of polyglutamine-induced neural degeneration in vivo is not accompanied by an obvious change in NI formation (11). This raises a discrepancy between chaperone modulation of polyglutamine aggregation in vitro and in yeast, with effects of chaperones on suppression of toxicity in the nervous system in vivo.
Here we address the mechanisms of chaperone-mediated suppression of neurotoxicity. We demonstrate that the fly dHdj1 protein (the Drosophila ortholog of human Hdj1) ameliorates neurotoxicity induced by a human disease protein and that, in contrast, dHdj2 (the fly ortholog of Hdj2) is a poor suppressor of toxicity. Expression of both dHdj1 and Hsp70 was synergistic in suppression, providing in vivo evidence of their linked biological activities. Detailed analysis of rescued flies suggests that, although NIs appear unchanged when viewed by light microscopy, the mechanism of chaperone-mediated suppression of neurotoxicity involves alteration of the biochemical properties of the mutant disease protein.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Suppression of polyglutamine-induced degeneration by the Drosophila Hdj-1 ortholog
We addressed whether Hsp40 chaperones played a role in mitigating polyglutamine-induced degeneration. To do this, we made transgenic flies expressing a cDNA encoding dHdj1, the fly ortholog of the human Hsp40 chaperone family member Hdj1 (see Materials and Methods). Expression was directed with gmr-GAL4, a promoter line that directs expression to all cells of the eye, including pigment cells and photoreceptor neurons. Expression of truncated Ataxin-3 with expanded polyglutamine (MJDtr-Q78) induces massive degeneration with loss of external pigmentation and internal retinal structure (Fig. 1C) (4). On co-expression of dHdj1, external eye structure was restored (Fig. 1D). Internal eye structure, which normally undergoes massive degeneration on expression of the mutant protein, was also restored by dHdj1 co-expression, with the eye showing partial preservation of photoreceptor rhabdomere specializations (data not shown).
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To confirm that suppression was specific, we performed several control experiments. First, we demonstrated that simply co-expressing two proteins by the same GAL4 promoter line has no effect on neurodegeneration (11). Second, dHdj1 did not modulate gene expression by the GAL4/UAS system. For example, co-expression of dHdj1 with other proteins that induce a phenotype, such as the cell death protein Hid, did not modify the reduced eye phenotype caused by Hid expression (data not shown). This result also indicated that dHdj1 does not suppress degeneration by preventing programmed cell death. Immunoblot analysis showed that co-expression of dHdj1 did not decrease levels of polyglutamine protein expression (data not shown). Taken together, these data indicate that dHdj1, like Hsp70, selectively mitigates polyglutamine protein toxicity.
Further analysis revealed that dHdj1 also ameliorated progressive degeneration. To examine progressive loss of neurons, we used flies that express the mutant disease protein less strongly in the eye, such that a more intact eye structure is present on emergence of the adult (Fig. 1E). Over time, however, these flies display dramatic retinal degeneration (Fig. 1F). Co-expression of dHdj1 dramatically restored the structure of the eye at emergence of the adult, as well as mitigating progressive neuronal loss over time (Fig. 1G and H).
To address whether dHdj1 could suppress polyglutamine toxicity throughout the nervous system, we directed expression to all neurons with elav-GAL4. Normally, strong expression of mutant polyglutamine protein by elav-GAL4 is lethal, with no adult flies emerging (4). Co-expression of dHdj1 suppressed lethality such that adult flies emerged and lived as long as 2 weeks (Table 1).
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Structural domains of dHdj1 required for suppression of neurotoxicity
Hsp40 proteins have two critical functional domains. The first is the J-domain, which interacts with and stimulates the ATPase activity of Hsp70 (12). The second is the C-terminal domain, which is important for interactions with substrate proteins. To determine whether these domains were important for suppression of neurodegeneration, we made select mutations in dHdj1 (Fig. 2A). First, we deleted the J-domain (dHdj1
J), which is predicted to eliminate interaction with Hsp70 in vivo. In a second construct (dHdj1.G295D), a point mutation was made within the C-terminal region which is predicted to interfere with substrate binding (18). External eye structure remained normal when either mutant dHdj1 protein was expressed on its own (data not shown). However, when co-expressed with expanded polyglutamine protein, both mutant dHdj1 proteins not only failed to suppress, but instead enhanced the disease phenotype. As shown in Figure 2B, weak expression of MJDtr-Q78 normally results in adult flies with only a mildly disrupted eye. However, on co-expression with dHdj1J or dHdj1.G295D, the eye was severely degenerate, seen by the enhanced loss of pigmentation and collapse of the eye (Fig. 2C and D). These studies suggest that both Hsp70 interaction and substrate binding domains are important in dHdj1 modulation of degeneration.
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In order to address the specificity of suppression by the Hsp40 class of chaperones, we also tested the ability of a Drosophila ortholog of Hdj2 to suppress. dHdj1 and dHdj2 share the J-domain, but have divergent C-terminal domains, likely reflecting different substrate specificities. Hdj2 has been shown to decrease aggregate formation of several polyglutamine proteins, including Ataxin-3, in transfected cells (6,8,9). We made several dHdj2 transgenic lines that express dHdj2 at various levels, including levels similar to our best dHdj1 transgenic lines. Co-expression of any dHdj2 transgenic line with polyglutamine protein resulted in only weak suppression of neurodegeneration when compared with suppression by dHdj1 (Fig. 3A and B; compare with Fig. 1D). Therefore, these Hsp40 chaperones showed selectivity in their ability to suppress polyglutamine degeneration in vivo, illustrating the importance of the C-terminal domain.
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Hsp70 suppresses toxicity of the Huntingtin protein
We extended our analysis of chaperone suppression to a second polyglutamine disease protein, the Huntington’s disease protein, Huntingtin. To do this, we co-expressed Hsp70, dHdj1 or dHdj2 with a truncated form of Huntingtin containing an expanded polyglutamine domain of 120 (Htt-Q120) (5). Flies expressing Htt-Q120 show normal eyes at 1 day, but a severely degenerate eye structure by 10 days (Fig. 3C) (5). On co-expression of chaperones, we found that Hsp70 strongly suppressed neuronal degeneration induced by Htt-Q120, restoring a normal photoreceptor rhabdomere structure (Fig. 3D). However, dHdj1 showed partial and dHdj2 showed no ability to suppress neurodegeneration induced by mutant Huntingtin (Fig. 3E and F). These results demonstrate broad effects of Hsp70 on suppression of polyglutamine toxicity and further emphasize substrate specificity among the Hsp40 class of chaperones.
Interactions between dHdj1 and Hsp70 in suppression of neurodegeneration
Hsp40 proteins are co-chaperones that present substrate to and stimulate the ATPase activity of Hsp70 (12,19). Since Hsp70 suppresses neurodegeneration, we were interested in testing for functional interactions between dHdj1 and Hsp70 in suppression of polyglutamine toxicity using our SCA3/MJD disease model. First, we addressed whether dHdj1 mediated suppression through an Hsp70-dependent pathway. To do this, we asked whether overexpression of Hsp70 could suppress the enhanced polyglutamine toxicity observed with mutant forms of dHdj1 and whether dHdj1 could still suppress polyglutamine disease in the presence of mutant Hsp70. The dHdj1J mutant enhances polyglutamine toxicity, presumably because of compromised interaction with Hsp70 as a result of the J-domain deletion. We found that suppression of polyglutamine toxicity by Hsp70 was epistatic to dHdj1J, resulting in a rescued eye phenotype (Fig. 4C). This suggested that a decrease in dHdj1 activity can be overcome by increasing the level of Hsp70, possibly through interactions with other J-domain-containing proteins. Moreover, overexpression of dHdj1 could not suppress the enhanced toxicity observed on co-expression of mutant Hsp70 with polyglutamine protein (Fig. 4B) (11), consistent with a requirement for Hsp70 chaperones in dHdj1 function.
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We then addressed potential synergistic interactions between the two chaperones. When expanded polyglutamine protein is expressed strongly in the eye, co-expression of either Hsp70 or dHdj1 resulted in complete suppression of external eye degeneration, but only partial suppression of the internal eye structural abnormalities (Fig. 5a–c). However, when dHdj1 and Hsp70 were both expressed simultaneously, suppression was more complete with the rescued eye now showing photoreceptor neuron specializations (Fig. 5d and e). This indicates that co-expression of both Hsp40 and Hsp70 chaperones is more effective than expression of either alone. We confirmed this by driving expression of polyglutamine protein more strongly by increasing the gene dosage. In this situation, rescue by either dHdj1 or Hsp70 independently was incomplete, whereas together they showed complete rescue of the external eye (Fig. 5f–h).
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Similar results were found when expression was directed to all neurons with elav-GAL4. Normally, no adult flies emerge when the mutant disease protein is expressed strongly throughout the nervous system (Table 1). Co-expression of either dHdj1 or Hsp70 resulted in partial suppression of the early death phenotype, with rescued female flies surviving up to
2 weeks (Table 1). Co-expressing both dHdj1 and Hsp70 did not significantly extend lifespan in females beyond that seen with either chaperone alone, but there was a striking effect on male survival. Males express twice the level of mutant disease protein because elav-GAL4 is on the X chromosome. Expression of either dHdj1 or Hsp70 alone resulted in some adult male survival relative to females, up to 20% with dHdj1. However, co-expression of both chaperones led to roughly equal numbers of surviving male and female flies (Table 1). In the course of these studies, we also noted that simply further increasing the levels of either dHdj1 or Hsp70 to two doses had deleterious effects on eye morphology, in the absence of disease protein, that were not observed when the two chaperones were co-expressed together (data not shown). These findings are consistent with in vitro studies of the interactions between Hsp40 proteins and Hsp70 proteins (12,19) and also indicated that dHdj1 and Hsp70 have differing, albeit linked, biological activities in vivo.
Chaperones modulate the biochemical properties of mutant polyglutamine protein
To determine whether chaperones altered polyglutamine aggregation, we examined NI formation in developing eye tissue and adult brain sections with the hypothesis that chaperones might alter NI formation. Using immunofluorescence microscopy, we failed to observe a change in the NIs, either during their formation or in the adult retina (Fig. 5i–n). However, we then extended our studies to examine the biochemical properties of the protein, and observed a striking change in the solubility properties of the mutant protein when molecular chaperones were co-expressed.
In the absence of co-expressed chaperones, mutant polyglutamine protein in flies, as in mice and human cells (20,21), ran as an SDS-insoluble complex in the stacking gel, with little protein visible as a monomer (Fig. 6A). However, when dHdj1 and Hsp70 were co-expressed, either separately or together, with expanded polyglutamine protein, significant levels of monomeric protein were seen (Fig. 6A). The normal lack of monomeric protein was not due to loss of eye tissue, because the result was the same whether the polyglutamine protein was expressed weakly (when retinal degeneration has not yet occurred in flies) or strongly (when significant degeneration has occurred) (Fig. 6). Although the amount of monomer seen on co-expression of dHdj1 and Hsp70 was not much greater than that seen with dHdj1 or Hsp70 alone (Fig. 6A), we suggest that this is likely due to the generally strong suppression seen in all of these situations when driving gene expression with gmr-GAL4. We did confirm that dHdj1 and Hsp70 together gave greater amounts of monomer when the dose of mutant polyglutamine protein was increased (data not shown) which was associated with the greater phenotypic suppression as shown in Figure 5f–h.
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To correlate the extent of monomer with suppression of polyglutamine toxicity, we compared monomer levels observed on co-expression of dHdj1 to that of dHdj2. In these two conditions, similar levels of chaperone are expressed, but in one case (dHdj1) strong suppression of toxicity was observed, whereas in the other (dHdj2) only weak suppression was seen (Fig. 3). More monomer was observed on dHdj1 co-expression than on dHdj2 co-expression (Fig. 6B), suggesting that the appearance of monomer is linked to the ability of a chaperone to suppress toxicity of the polyglutamine protein.
We further examined chaperone-mediated biochemical changes in polyglutamine protein of the MJDtr-Q78 transgenic lines by fractionating head samples into a high salt extractable supernatant and pellet, which were then analyzed on denaturing SDS gels (Fig. 6C). In the absence of co-expressed chaperones, none of the mutant polyglutamine protein was extractable by high salt. In contrast, in lysates from flies co-expressing chaperones, a portion of the mutant polyglutamine protein was extractable by high salt and migrated as a monomer on gels. Additional mutant protein could be solubilized from the pellet by SDS treatment and also ran as a monomer. These studies indicate that chaperone-mediated suppression of polyglutamine neurotoxicity in vivo is associated with altered solubility properties of the toxic protein.
In human disease, a shorter polyglutamine repeat length is associated with later onset and less severe disease (1–3). We serendipitously isolated a transgenic line that was derived from our strong MJDtr-Q78(S) transgenic insertion, but with a contracted polyglutamine repeat of 61. This transgenic line should therefore express protein at the same levels as our original transgenic line, but of a less toxic form of the protein. On expression of the MJDtr-Q61 protein, these flies, as predicted from humans with disease, showed a milder degenerative phenotype compared with the original MJDtr-Q78(S) line with the longer repeat length (Fig. 7A–C). Analysis of these MJDtr-Q61 flies for protein expression revealed that significant amounts of monomer were detectable in these flies compared with the original line (Fig. 7D). These findings are also consistent with the solubility properties of the mutant protein reflecting its degree of neurotoxicity in vivo.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We have shown that Hsp70 (11) and the Hsp40 chaperone dHdj1 (present study) suppress polyglutamine disease in Drosophila in a synergistic manner that is consistent with known biochemical interactions of Hsp70 and Hsp40 proteins. Our results further suggest selectivity in chaperone recognition of human polyglutamine protein, as two Hsp40 chaperones (dHdj1 and dHdj2) differed in their ability to suppress neurotoxicity. Moreover, we reveal a biochemical basis for the suppression of neurodegeneration: when polyglutamine toxicity was suppressed by chaperones, there was a change in solubility properties of the mutant protein. These findings indicate that the mechanism by which chaperones mediate suppression of polyglutamine-induced neurodegeneration includes altering the structure, and hence the solubility properties, of the mutant disease protein.
Chaperone specificity and interactions in suppression of polyglutamine disease in Drosophila
We previously found that Hsp70 co-localizes to polyglutamine aggregates and that overexpression of Hsp70 suppresses polyglutamine toxicity in vivo (11). Given the well described functional interactions between Hsp40 and Hsp70 proteins, we were prompted to address the role of Hsp40 proteins in modulating disease progression. We found that dHdj1, the fly ortholog of human Hdj1, effectively suppressed toxicity of the MJDtr-Q78 protein, requiring both the J-domain and the substrate binding domain for this activity. Mutant forms of dHdj1, like that of Hsc70 (11), not only failed to suppress, but in fact enhanced polyglutamine disease. They likely function as dominant-negative proteins that compete in a non-productive manner with endogenous proteins for interactions with substrate or Hsp70 (22,23). The fact that toxicity is enhanced suggests that endogenous levels of Hsp40 and Hsp70 chaperones may normally modulate toxicity of polyglutamine protein. Indeed, overexpression of Hsp70 suppressed deleterious effects of dHdj1 mutant protein (Fig. 4), consistent with an involvement of Hsp70 in mediating the effects of dHdj1. Normal cellular levels of Hsp40 and Hsp70, or levels induced as part of a stress response to the mutant protein, may aid in combating deleterious effects of mutant polyglutamine protein and explain, in part, why disease onset is typically delayed in humans. Our studies suggest that augmenting the stress response in vivo may be effective in counteracting deleterious actions of the toxic protein. Indeed, in C.elegans, expression of expanded polyglutamine protein appears to activate the stress response but in a variable manner (16). Normally, the organism may fail to activate heat shock proteins sufficiently to effectively combat the actions of the disease protein.
A second Hsp40 chaperone, dHdj2, was less effective at suppressing MJDtr-Q78 toxicity. Moreover, whereas Hsp70 was effective at suppressing neurodegeneration induced by the Huntington’s disease protein, dHdj1 was partially effective and dHdj2 was a poor suppressor. The presence of divergent C-terminal domains in different Hsp40 proteins is thought to underlie differential substrate specificity of various Hsp40 chaperones (13). In vitro studies of chaperone modulation of fibril formation by polyglutamine protein also show specificity to select chaperones of the Hsp40 class (17). Our studies provide in vivo evidence supporting this specificity in combating neurotoxicity. A genetic screen in Drosophila recently identified two J-domain-containing proteins, dHdj1 and tetratricopeptide repeat protein 2 (dTPR2), as suppressors of a polyglutamine-mediated phenotype, although their effectiveness and mechanism of action were not addressed (14). Our findings suggest that, for polyglutamine disease proteins, the J-domain may impart suppression through interactions with Hsp70.
Our data support a synergistic interaction between dHdj1 and Hsp70 in suppressing neurodegeneration. Although we anticipated that we might observe strong stimulation of Hsp70-mediated suppression by low levels of added dHdj1, we failed to detect a synergy with such characteristics. Instead, we found that co-expressing high levels of both dHdj1 and Hsp70 restored a more complete eye structure, with greater preservation of cellular structure over time. When we expressed either dHdj1 or Hsp70 alone at two doses, there were deleterious effects on eye structure that were minimized on co-expression of the two proteins together. This indicates an interaction between Hsp40 and Hsp70 in vivo, as predicted by biochemical studies of the linked functions of the two chaperones in protein folding (12,19). These observations underscore the potential importance of targeting chaperone activity in a tissue-specific manner and with an appropriate balance of chaperone proteins (3). Given this, perhaps a more effective approach may be to upregulate the stress response at the transcriptional level through heat shock factors (24), as this might achieve the correct physiological levels of appropriate chaperones to combat the protein toxicity.
Chaperones modulate neurodegeneration by altering the solubility properties of mutant polyglutamine protein in vivo
Expanded polyglutamine proteins typically form insoluble aggregates within cells, although the relationship between NI formation and toxicity is still not clear. Our experiments suggest that modulation of aggregation may be essential to chaperone suppression of neural toxicity, but occurs in a more subtle manner than complete dissolution of NIs. Indeed, we found that chaperone-mediated suppression of toxicity did not result in an obvious change in NI formation at the light microscopic level: NIs were as prominent in the suppressed condition as in the disease situation, supporting other observations that the presence of visible protein aggregates does not correlate with disease progression (11,14,25,26). However, in further studies, we found that chaperone suppression of neurotoxicity is associated with the appearance of protein with distinct biochemical properties. In the absence of co-expressed chaperones, little or none of the mutant protein is detectable in gels because the protein is fully complexed into highly insoluble aggregates. When neurodegeneration was suppressed by chaperones, a significant amount of polyglutamine protein could be solubilized by SDS and was detected as a monomer on denaturing gels. Importantly, little monomer was observed on co-expression of dHdj2, which only weakly suppresses MJDtr-Q78 degeneration. This strongly supports our hypothesis that the extent of monomer in the gel reflects biological suppression of neurotoxicity. Similarly, a contracted repeat protein, MJDtr-Q61, expressed at the same levels as MJDtr-Q78(S) protein, displayed a mild degenerative phenotype and more monomer compared with MJDtr-Q78(S). Although we do not anticipate that the detailed mechanisms of disease mitigation by repeat contraction and by suppression with added chaperone are the same, nevertheless both are associated with greater SDS-solubility of mutant protein. Taken together, these findings indicate that the amount of SDS-soluble monomer is correlated with degree of protein toxicity, such that less monomer is associated with greater toxicity (reflected by greater aggregation), whereas more monomer indicates less toxicity (reflected by less aggregation).
On expression of chaperones, three distinct forms of polyglutamine protein were observed: high salt-extractable, SDS-extractable and SDS-insoluble protein. The amount of mutant disease protein detected as a monomer on gels was roughly comparable to the amount of non-mutant disease protein in control samples, although insoluble aggregates were still detected. This suggests that a significant fraction of the mutant protein is being solubilized on extraction. Most of the monomeric protein, however, remained tightly complexed, requiring SDS treatment for dissociation. Our data thus suggest that chaperone-mediated suppression of polyglutamine toxicity does not require complete refolding of mutant polyglutamine protein to its native, soluble state. In vitro studies of polyglutamine protein structure indicate that chaperones are able to inhibit fibril formation, promoting instead the formation of amorphous globules (17). In yeast, endogenous Hsp70 and Hsp40 chaperones have effects on protein solubility properties, where they have been demonstrated to directly interact with polyglutamine protein, again without having much effect on aggregation as observed by immunocytochemistry (17). Our data strongly argue that chaperone-mediated changes in the solubility properties of polyglutamine protein indeed reflect attenuation of the neurotoxic properties of polyglutamine disease protein in vivo.
Recent studies in an inducible mouse model of disease show reversal of behavioral deficits concomitant with clearance of polyglutamine aggregates following transgene silencing (27). Though the cellular mechanisms of clearance in this inducible model have not been identified, it seems likely that endogenous chaperone activity plays a role and that modulation of protein solubility may be critical for achieving disease suppression. As many neurodegenerative diseases are associated with abnormal protein aggregation, there may be common pathogenic mechanisms underlying these different late-onset, progressive conditions that can be counteracted by molecular chaperone activity.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Drosophila strains
Flies were grown on standard corn meal molasses medium supplemented with dry yeast, at 25°C. Fly lines bearing UAS-MJDtr-Q27, UAS-MJDtr-Q78 and Hsp70 (UAS-HspA1L) have been described (4,11). For lines expressing dHdj1 and dHdj2, cDNA clones encoding these proteins were obtained from Research Genetics (Huntsville, AL), a FLAG tag was added to the C-terminus of the protein by PCR, the constructs were sequenced and subcloned into the pUAST transformation vector (28). Any existing mutations in the Research Genetics clones were also corrected by site-directed mutagenesis. For dHdj1, primers used were: N-terminal, 5'-CGGAATTCGTCGACATGGGCAAAGACTTCTACAAGATTCTGGGCC-3', and C-terminal, 5'-GGTCTAGACTACTTGTCGTCGTCGTCATCCTTGTAATCGTTGGGCAGCAGCTCCGACAGCTGATTCTGC-3'. For dHdj2, primers used were: N-terminal, 5'-CGCTCGAGGTCGACATGGACAACCTAAATTTATACGACGTTCTTAAAGTG-GC-3', and C-terminal, 5'-GGTCTAGACTACTTGTCGTCGTCGTCATCTTGTAATCAGCCGTCTGGCACTGTACGCCCTCAAAGTGAGACG-3'. The J-domain deletion mutation in dHdj1 was made using a PCR primer to delete the N-terminus of the protein (5'-CGGAATTCGTCGACATGGGACCAGATGGCGGCGGTAGCCG-3'). The G295D mutation was made using primers to the sequence 5'-GGATCAACGGACTAGATCTGCCGGTGCCC-3' and the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The constructs bearing mutant proteins were sequenced and subcloned into the pUAST vector. Transgenic lines were generated in the w1118 background (29). Fly lines bearing gmr-GAL4 and elav-GAL4 (line C155) were obtained from the Drosophila Bloomington Stock Center. GenBank accession numbers are U34904 for dHdj1 and AAF53247 for dHdj2. Expression of Hsp40 by gmr-GAL4 had a deleterious effect on pigment cell nuclear position similar to, but more severe than, that of Hsp70 (11).
Microscopy and immunohistochemistry
Eye discs were dissected and stained with antibodies as described (4). Hemagglutinin (HA)-tagged polyglutamine protein was detected with a rabbit polyclonal antibody to HA (Y-11, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA). Drosophila Hsp70 protein was detected with a monoclonal antibody to the fly protein (MAB-007, 1:200; Affinity BioReagents, Golden, CO). Human Hsp70 was detected with an antibody specific to the human protein (SPA812, 1:200; StressGen, Victoria, Canada). FLAG-labeled proteins were detected using anti-FLAG M2 (1:100; Sigma, St Louis, MO). Secondary antibodies were conjugated to fluorescein (1:500) or Texas red (1:50; Jackson ImmunoResearch, West Grove, PA). For staining of frozen sections, adult heads were embedded in OCT, 12 µm serial sections were cut and collected on subbed slides. Tissue was fixed in 4% paraformaldehyde, then stained with appropriate antibodies and DAPI (0.5 µg/ml for 5 min). For plastic sections, adult heads were fixed in paraformaldehyde and embedded in epon for 1 µm horizontal sections, then stained with toluidine blue and methylene blue as described (4). Confocal microscopy was performed on a Leica model TCS SP ultraviolet and visible confocal imaging spectrophotometer microscope. For light microscopy, tissue was viewed on a Leica DMRBE compound fluorescence microscope or a Leica MZ12 stereomicroscope, equipped with a Leica DC200 digital camera.
Immunoblot analysis and sample fractionation
Adult fly heads of the appropriate genotypes were prepared and analyzed by SDS–PAGE and immunoblot as described (11). Gels were 12.5% polyacrylamide, with a 4% stacking gel. Antibodies used were anti-HA (12CA5, 1:3000; Boehringer Mannheim, Indianapolis, IN), anti-FLAG M2 (1:440), anti-H-Hsp70 (1:10 000) and anti-ß-tubulin (E7, 1:1000; Developmental Studies Hybridoma Bank, Iowa City, IA, with funding from the NICHD). Secondary antibodies were goat anti-mouse and goat anti-rabbit (1:4000; Boehringer Mannheim). For fractionation of adult head samples, 32 heads for each condition were homogenized with a plastic pestle in 100 µl high salt (HS) buffer (50 mM Tris pH 7.4, 250 mM NaCl, 19 mM NaF, 5 mM EDTA, pH 8.0 with protease inhibitors) and centrifuged at 100 000 g for 30 min at 4°C. The pellet was then re-extracted with 100 µl of HS buffer, re-spun and supernatants were pooled for the HS fraction. The HS-insoluble pellet was then solubilized in SDS buffer (100 mM Tris pH 6.8, 2% SDS, 20% glycerol, 5% ß-mercaptoethanol). Twenty microliters of each fraction was then analyzed by SDS–PAGE and immunoblot. For DNA sequence analysis of MJDtr-Q61 flies, single fly genomic DNA templates from UAS-MJDtr-Q78 and UAS-MJDtr-Q61 transgenic flies were prepared. UAS-MJD transgenes were amplified using primers that anneal to the ends of the multiple cloning sites of the pUAST vector (5'-CTACTGAAATCTGCCAAGAAGT-3' and 5'-CCAATTATGTCACACCACAGA-3'). Amplification products were purified by standard procedures and then sequenced using the same primers above.
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ACKNOWLEDGEMENTS |
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We thank A. Cashmore for critical reading of the manuscript. This research was funded, in part, by grants from the R.J. Carver Charitable Trust, the University of Iowa Howard Hughes Medical Institute Resources Program, the Ellison Medical Foundation and the National Institutes of Health (to H.L.P.), Wellcome Trust (to H.Y.E.C.), Wills Foundation (to J.M.W.), HDSA Coalition for the Cure, Hereditary Disease Foundation, Alzheimer’s Foundation and the David and Lucile Packard Foundation (to N.M.B.). N.M.B. is an assistant investigator of the Howard Hughes Medical Institute.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 215 573 9267; Fax: +1 215 898 8780; Email: nbonini@sas.upenn.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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