Skip Navigation


Human Molecular Genetics Advance Access originally published online on December 21, 2005
Human Molecular Genetics 2006 15(3):453-465; doi:10.1093/hmg/ddi460
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Material
Right arrow All Versions of this Article:
15/3/453    most recent
ddi460v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (8)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Berger, Z.
Right arrow Articles by Rubinsztein, D. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Berger, Z.
Right arrow Articles by Rubinsztein, D. C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2005. Published by Oxford University Press. All rights reserved.
For Permissions, please email: journals.permissions@oxfordjournals.org

Deleterious and protective properties of an aggregate-prone protein with a polyalanine expansion

Zdenek Berger1,3,{dagger}, Janet E. Davies1,{dagger}, Shouqing Luo1, Matthieu Y. Pasco3, Irina Majoul2, Cahir J. O'Kane3,{ddagger} and David C. Rubinsztein1,*,{ddagger}

1Department of Medical Genetics and 2Department of Clinical Biochemistry, University of Cambridge, Cambridge Institute for Medical Research, Wellcome Trust/MRC Building, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2XY, UK and 3Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK

* To whom correspondence should be addressed. Tel: +44 1223762608; Fax: +44 1223331206; Email: dcr1000{at}cus.cam.ac.uk

Received November 2, 2005; Revised December 2, 2005; Accepted December 14, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 CONCLUSION
 MATERIALS AND METHODS
 REFERENCES
 
Many aggregate-prone proteins, including proteins with long polyglutamine or polyalanine tracts, cause human diseases. Polyalanine proteins may also be present in the tissue of polyglutamine diseases as a result of frameshifting of the primary polyglutamine-encoding (CAG)n repeat mutation. We have generated a Drosophila model expressing green fluorescent protein tagged to 37 alanines that manifests both toxicity and inclusion formation in various tissues. Surprisingly, we show that this aggregate-prone protein with a polyalanine expansion can also protect against polyglutamine toxicity, which can be explained by induction of heat-shock response. A heat-shock response was also seen in an oculopharyngeal muscular dystrophy mouse model expressing an authentic polyalanine-expanded protein. We also show that long polyalanines can protect against a pro-apoptotic stimulus or the toxicity caused by the long polyalanines themselves. Thus, overexpression of an aggregate-prone protein without any normal functions can result in both pathogenic and protective effects in cell culture and in vivo.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 CONCLUSION
 MATERIALS AND METHODS
 REFERENCES
 
Codon reiteration diseases are a large group of human conditions caused by abnormally long polyglutamine or polyalanine tracts in different proteins. Polyglutamine expansions are seen in Huntington's disease (HD) and eight other conditions including spinocerebellar ataxias (SCA) types 1, 2, 3, 6, 7 and 17 (1Go). HD, the most prevalent polyglutamine disease, is a devastating autosomal dominant neurodegenerative condition that typically strikes in middle age and is characterized by movement disorders, cognitive decline and psychiatric symptoms (2Go). The pathological hallmark of HD and other polyglutamine diseases is the presence of intracellular inclusions (3Go,4Go), although the roles of inclusions are debated. Expansions or duplications of polyalanine tracts leading to stretches of up to 29 repeats cause nine known diseases (5Go). In one of these, oculopharyngeal muscular dystrophy (OPMD), polyalanine expansions in the poly-A-binding protein nuclear 1 (PABPN1) cause it to form intranuclear inclusions that correlate with disease pathology (6Go). In addition, polyalanine expansions in four disease genes have been recently shown to lead to inclusion formation in cell models (7Go), although we are not aware that this possibility has been tested in appropriate tissues of patients with these diseases.

In order to gain insights into polyalanine toxicity in general, we have tested if polyalanine expansions are toxic when expressed alone and also when expressed with polyglutamine expansions. The polyalanine expansions were co-expressed with N-terminal fragments of polyglutamine-expanded mutant huntingtin, to allow us to directly and unequivocally test the overall toxic or protective properties of long polyalanines. In addition, our findings may also have potential relevance to polyglutamine diseases, since frameshifting of the original product encoding CAG/polyglutamines to GCA/polyalanine has been reported in SCA3 (8Go,9Go).

Here, we show that an aggregate-prone protein with a polyalanine expansion [enhanced green fluorescent protein (EGFP) tagged to 37 alanines] can be toxic in vivo in Drosophila and surprisingly, the same mutant protein could also mediate protective effects against polyglutamine expansions, a distinct pro-apoptotic stimulus and also against polyalanines themselves. Our data suggest that this protective effect is mediated by a heat-shock response. Heat-shock responses were seen both in cell culture models expressing expanded polyalanine proteins as well as in an OPMD mouse model expressing an authentic polyalanine expanded protein. However, no heat-shock response was evident in HD mice. In summary, overexpression of an aggregate-prone protein with no normal functions can result in both pathogenic and protective effects in cell culture and in vivo.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 CONCLUSION
 MATERIALS AND METHODS
 REFERENCES
 
Toxic effects of polyalanine expansions
To determine the effects of polyalanine repeats in vivo, we generated transgenic Drosophila expressing 37 or 7 alanines fused to an SV40 nuclear localization signal (NLS) and EGFP, referred to as A37 and A7, respectively. An NLS was used, as the proteins associated with polyalanine diseases are nuclear (5Go,6Go). We used 37 polyalanine repeats, because a number of normal human proteins have at least 20 repeats (thus, toxicity of a given repeat length may be modulated by protein context, as is also suggested with polyglutamine repeats). Our previous studies have shown that A37 is toxic and forms inclusions in cell culture (10Go). Similar A37 inclusions are detergent-insoluble and have fibrillar morphologies by electron microscopy (11Go).

We also selected a slightly longer repeat than the longest polyalanine expansion seen in a human disease, in order to maximize the probability of a reliable phenotype within the short Drosophila lifespan, analogous to what many have used for modelling polyglutamine diseases in vivo (12Go–14Go). It is worth noting that the 146 CAG repeat expansion used by Ordway et al. (13Go) and similar large expansions used in other models are not representative of either adult or even juvenile-onset HD, where expansions of more than 75 repeats are quite rare (2Go). Although such an expansion size may have been reported previously, these are exceptional and are likely to have extreme phenotypes.

We fused the polyalanine expansions to EGFP to investigate the consequences of polyalanine expansions outside the context of the human disease proteins—this allows us to test if long polyalanine expansions could mediate toxicity by gain-of-function mechanisms (as in polyglutamine diseases), as opposed to dominant-negative or loss-of-function effects. We have based our strategy on approaches used to analyse the toxicity of isolated polyglutamine stretches in flies (12Go) and polyglutamine expansions inserted into a non-essential non-disease associated protein in mice (13Go) (HPRT), which have led to major insights into the toxicity of these mutations. Furthermore, a major part of the HD literature involves studies of exon 1 fragments in cells and mice, which may be very similar to isolated polyglutamines, given the small size of this exon (14Go). The A7 and A37 constructs were expressed in Drosophila under the control of a UAS promoter, which is only activated when crossed with flies expressing the GAL4 transcription factor in the tissue of choice (15Go). We selected lines expressing similar levels of A37 and A7 for phenotypic analyses (Fig. S1).

Expression of A37 in the nervous system of adult Drosophila led to a shorter lifespan, compared with Drosophila expressing A7 or the GAL4 driver alone (Fig. 1A). We then looked for behavioural manifestations of nervous system dysfunction. Normal flies display a strong negative geotactic response; when tapped to the bottom of a test tube, they rapidly climb to the top. The climbing response of flies expressing A37 was impaired at 2 days after eclosion compared with flies expressing GAL4 driver alone or A7 (Fig. 1B). Similar results were obtained at 3 and 4 days after eclosion (data not shown).


Figure 4601
View larger version (37K):
[in this window]
[in a new window]
  		Figure 1. Toxic effects of long polyalanine repeats in Drosophila. (A) Survival curves of two A37 lines and one A7 line expressing the transgene in the nervous system with Nrv2-GAL4 or Nrv2-GAL4 alone without any transgene (GAL4). The two different A37 lines were statistically different from the A7 line and from GAL4 alone (Kaplan–Meier survival test, P<0.0001 for both A37 lines). Approximately, 200 individuals were used for each genotype. (B) Climbing abilities of two A37 lines and one A7 line expressing the transgene in the nervous system under the control of Nrv2-GAL4 and Nrv2-GAL4 alone (GAL4) 2 days after eclosion. The climbing ability of flies expressing Nrv2-GAL4 alone was arbitrarily set as 100%. Both A37 lines were different from A7 and GAL4 alone (P<0.001 for both A37, Mann–Whitney U-test), A7 was not different from Nrv2-GAL4. Graph shows summary data and error bars represent standard errors of mean. Approximately 100 flies were used. (C) Survival to adulthood of flies expressing A37 or A7. Flies heterozygous for A7 or A37 and a balancer chromosome were crossed to three GAL4 lines and the proportions of the adult flies with A7 or A37 were determined. The proportion of flies expressing A7 versus the balancer chromosome was arbitrarily set as 1, and the data for A37 lines are expressed as odds ratios relative to 1 (see Materials and Methods). The ratio is decreased in both A37 lines, reflecting the decreased survival of flies with A37 transgene to adulthood (Nrv2-GAL4: P<0.0001, A37a; P=0.0003, A37b; MHC-GAL4: P<0.0001 A37a, P=0.0009 A37b; Ey-GAL4: P<0.0001 A37a and A37b). Error bars represent 95% confidence intervals. Data for each genotype are based on three different bottles, each with at least 300 flies. (D) Photographs of Drosophila eyes. Expression of A37a driven by ey-GAL4 led to rough and small eye phenotypes, which were not seen in flies expressing A7b, or two copies of A7 transgene (A7a+A7b), or GAL4 alone. Eyes of females are shown. (E) A37 forms aggregates in the eye imaginal disc of third instar larvae and A7 shows a diffuse distribution inside the nucleus, when expressed under ey-GAL4 control. (F) Congo red rescues the pre-adult lethality caused by expression of A37 under ey-GAL4 control. Progeny of ey-GAL4 crossed to either UAS-A37/TM6B or A7/TM6B were treated with 250 µM or 36 mM Congo red and the number of adult flies with and without the transgene were counted. 36 mM Congo red increased the proportion of flies with the A37 transgene (P<0.0001, odds ratio: 4.3, 95% confidence interval 2.9–6.3), while it did not have any effect on the proportion of flies with the A7 transgene (proportion of A7 is higher than 50% because of low viability of flies with TM6B). At least three bottles were evaluated per each concentration, with approximately 200 flies in each bottle (also applies to g). (G) Congo red decreases the frequency of rough/small eye phenotype caused by expression of A37 under ey-GAL4 control. Treatment with 250 µM and 36 mM Congo red reduced the frequency of rough/small eye phenotype to 70% or 10% of control (untreated), respectively (P=0.0002 for 250 µM, and P<0.0001 for 36 mM; odds ratio was used). Treatment was done as described in Fig. 1F. (H) Congo red decreases number of cells with inclusions in cell culture. SK-N-SH cells were transfected with 2 µg of EGFP-NLS-A37 and treated with 100 µg/ml Congo red after transfection. Cells were fixed 48 h later and scored for the presence of inclusions (P<0.0001; odds ratio: 0.41; 95% confidence interval 0.33–0.49). Three experiments were done, at least 300 cells were scored in each experiment. Error bars represent standard errors of mean except where otherwise indicated. ***P<0.001. Flies were raised at 29°C in A–F or at 25°C in g.

 
We also evaluated the effects of A37 on survival to adulthood, using Nrv2-GAL4, MHC-GAL4, ey-GAL4, which drive expression in the nervous system, muscles, and the eye-antennal disc and other tissues, respectively (visualized by EGFP, data not shown). Expression of A37 with each of these drivers led to reduced survival to adulthood compared with an A7 line (Fig. 1C). Line A37a has more dramatic effect than A37b, which is likely due to the lower expression when A37b was driven by GAL4 in larvae (data not shown). The effects on survival are unlikely due to leaky expression, as there is ~4-fold difference in survival between different GAL4 drivers; the lethality of A37 flies crossed to ey-GAL4 may be due to expression seen in the abdomen (visualized by GFP, data not shown).

Expression of A37 in the developing Drosophila eye using ey-GAL4 led to the formation of abnormal eyes characterized by smaller size, abnormal shape and rough surface, while expression of A7 or GAL4 alone did not have any visible effects on the external eye morphology (Fig. 1D). We identified at least four A37 lines exhibiting abnormal eye phenotypes when A37 was expressed under the control of ey-GAL4. We analysed 11 independent A7 lines, none of which exhibited any visible eye abnormalities (when driven with ey-GAL4). We did not detect any abnormal eyes even when dosage of the A7 transgene was doubled (Fig. 1D). Also, we observed moderately abnormal eyes with the A37 transgene at 25°C (Fig. 1D), while at 29°C (leading to higher expression of the transgene) we observed in some cases a complete loss of eyes in flies expressing A37 (data not shown). In stark comparison, we never observed any abnormalities with any A7 lines even at this higher temperature. A37 expression resulted in inclusion formation, whereas A7 exhibited diffuse fluorescence in the eye-antennal imaginal disc of third instar larvae (Fig. 1E). When A37 was expressed in the nervous system or muscles, similar inclusions were observed while only diffuse fluorescence was seen with A7 (data not shown).

The toxic effects of long polyalanines were verified with at least two independent insertions and the toxicity of long polyalanines was always compared with the toxicity of short polyalanines expressed at similar levels. Thus, these results clearly demonstrate that expression of long polyalanine repeats causes pathology in vivo and is associated with inclusion formation.

Congo red treatment reduces aggregation and toxicity of both long polyglutamines (in cells and in vivo) (16Go) and long polyalanines (in the context of mutant PABPN1 (in cells) (17Go). We therefore tested if Congo red could rescue the polyalanine toxicity in vivo using the survival to adulthood and rough/small eye phenotype as our readouts. In both cases, Congo red significantly rescued A37 toxicity (Fig. 1F and G). We did not quantify the effects of Congo red on aggregation in the Drosophila eye disc because it appears difficult to quantify changes in aggregation in this system unless they are very large, possibly because of high expression levels of the aggregating protein [see (18Go) for detailed discussion]. For example, while numerous studies observed the effect of heat-shock proteins on polyglutamine aggregation in mouse and cell models (19Go–25Go), such changes were not observed in Drosophila eye disc (26Go). Similarly, Drosophila myeloid leukaemia factor reduced polyglutamine aggregation in cell culture (18Go) but no effects were observed in Drosophila eyes (27Go).

In mammalian cell culture, Congo red decreased the proportion of A37-transfected cells with inclusions using the same EGF-NLS-A37 construct (10Go) as used in flies (Fig. 1H). (In all subsequent experiments using A7 or A37 in cells, we have used such constructs.) This suggests that Congo red-type compounds may be worth considering as therapeutics for diseases caused by long polyalanine repeats, such as OPMD. While this protective effect of Congo red may be due to direct effects against aggregation, we cannot exclude the possibility that Congo red may act by blocking the accessibility of toxic epitopes on monomeric expanded polyalanine species (28Go).

Polyalanines protect against polyglutamine toxicity
We initially tested if polyalanines could modulate polyglutamine toxicity using model systems expressing an N-terminal fragment of huntingtin with expanded polyglutamines, which we and others have extensively previously characterized both in cell culture and in vivo (29Go). Co-transfection of mammalian cells with a 1:1 ratio (1 µg:1 µg) of HD exon 1 construct with 74Q (74 glutamines) and A37 construct resulted in enhanced toxicity—this is not unexpected as both constructs are toxic when transfected alone (Fig. S2 and data not shown).

Could long polyalanines be protective when they are expressed at levels that are not toxic? We decreased the amount of DNA per well so that transfection with A37 construct did not show any obvious toxicity (Fig. S2). Co-transfection of mammalian COS-7 cells with a 1:1/9 ratio (1 µg:111 ng) of HD exon 1 Q74 construct and A37 construct led to decreased toxicity accompanied by decreased percentage of cells with Q74 inclusions (Fig. 2A). A7 did not affect cell death or aggregation (Fig. 2A). Similar results were obtained in a neuronal SK-N-SH cell line using the same ratio (data not shown). Interestingly, co-transfection of mammalian COS-7 cells with a 1:1/3 ratio of HD exon 1 Q74 construct and A37 construct led to an intermediate effect between the 1:1/9 (protective) and 1:1 ratios (enhanced toxicity). The 1:1/3 ratio resulted in no change in toxicity of the Q74 (Odds ratio=1.084; 95% CI 0.89–1.46, P=0.7, data not shown).


Figure 4602
View larger version (31K):
[in this window]
[in a new window]
  		Figure 2. Expanded polyalanine repeats protect against polyglutamine toxicity in mammalian cells and in Drosophila. (A) COS-7 cells were co-transfected with polyalanine constructs (tagged with EGFP) or a EGFPC1 vector, and a construct comprising HD Exon 1 with a Q74 repeat fused to a HA tag (Q74; pHM6(CAG)74) at a ratio of 1:1/9 (Q74 1 µg: polyalanines 111 ng). 48 h post-transfection cells were immunolabelled using an anti-HA antibody and cells expressing both constructs were scored for cell death (based on abnormal apoptotic nuclear morphology) and Q74 aggregation. Unconditional logistical regression analysis was used (see Materials and Methods). ***P<0.0001; NS (not significant) P>0.05. (Comparison of A7 and A37: cell death P=0.009, aggregation P=0.002). Six experiments were done, 200 cells were scored for each experiment. (B) Photographs of visible rhabdomeres in Drosophila eyes at 4 days after eclosion. Expression of N-terminal fragment of huntingtin with 120Q leads to loss of rhabdomeres, which is rescued by A37. A7 does not have any effect. (C) Time-course of rhabdomere loss in flies expressing mutant huntingtin alone, or in presence of A7 or A37. A37 protects against rhabdomere loss over a period of time. The graph is based on approximately 11 500 ommatidia (750 flies) counted at 1, 2 and 4 days after eclosion. Number of rhabdomeres per ommatidium is shown. Genotypes: Q120: elav-GAL4/+; +; gmr-Q120/+; Q120+A7: elav-GAL4/+; UAS-A7b/+; gmr-Q120/+; Q120+A37:elav-GAL4/+; +; gmr-Q120/UAS-A37a; A37: elav-GAL4/+; +; UAS-A37a/+. Photographs from flies 4-days-old (Q120, Q120+A7, Q120+A37) or 8 days old (A37). Note that in the photograph shown, A37 rescues the polyglutamine toxicity almost completely. No loss of rhabdomeres was seen in flies expressing A37 only, even at later time points (data not shown). Expression of elav-GAL4 alone or elav-GAL4 with UAS-A37 did not lead to loss of rhabdomeres (data not shown), further confirming that A37 can protect against polyQ toxicity when expressed at relatively low levels and when it is not toxic on its own. Error bars represent standard errors of mean.

 
Next, we tested if long polyalanines could protect against polyglutamine toxicity in vivo in Drosophila. For these experiments, we had initially considered using polyglutamine models driven by the UAS-GAL4 system (15Go). However, we observed an interaction of A37 with GAL4 that is likely to result in decreased GAL4-dependent transcription (and therefore any modification of polyQ toxicity in such a model may be due to modification of GAL4-dependent transcription of the polyQ transgene) (see Supplementary Data and Fig. S3). The A37-GAL4 interaction necessitated selection of a model where the polyQ transgene was independent of the GAL4 transcription factor. The only published model meeting this criteria expresses the N-terminal part of mutant human huntingtin (120Q) (30Go) placed under the direct control of the gmr promoter. As our cell-based data suggest that A37 could exert protective properties at sub-toxic doses, we chose elav-GAL4 to drive the expression of A37/A7. We have used elav-GAL4C155 (31Go), which drives expression of the UAS transgene in the photoreceptor cells and the nervous system. We have not observed any effects on the survival of flies expressing elav-GAL4 and A37 at the temperature used for the following experiment (25°C, data not shown).

Expression of A37 (or A7) in the eyes using elav-GAL4 did not lead to any rhabdomere loss in Drosophila eyes up to 20 days (which is much later than the time points of any of the following experiments). In addition, elav-GAL4 alone is not toxic and does not lead to any rhabdomere loss (data not shown). The absence of A37 toxicity in the Drosophila eye when using elav-GAL4 is likely to be due to low levels of expression (judged by GFP fluorescence, data not shown). We carefully considered the most appropriate read-out for the effects of A37/A7 on Q120-induced degeneration in this model (see Supplementary data) and selected pseudopupil analysis for subsequent experiments.

Expression of A37 reduced the polyglutamine toxicity by increasing the number of visible rhabdomeres in the eye of the 120Q flies, while A7 did not affect the degeneration at all three time points examined (Fig. 2B and C). A37 increased the visible rhabdomeres by 9% at day 1 after eclosion, 29% at day 2 and by 58% at day 4 (Fig. S4), showing that the protective effect is maintained over a period of time (Fig. 2C). The in vivo data are consistent with cell culture experiments, showing that the A37 aggregate-prone protein rescues the toxicity of polyglutamines, while A7 does not have any effect.

Polyglutamine toxicity may be mediated by aberrant interactions between the polyglutamine protein and other proteins within the cell, such as transcriptional cofactors, heat-shock proteins, or proteasome subunits, leading to their sequestration (2Go). We therefore wanted to test if the effect of A37 on polyglutamine toxicity was due to a direct interaction between polyalanine and polyglutamine repeats, possibly preventing some of the aberrant interactions. We could not detect co-localization of YFP-A37 and CFP-HD89Q in any of the cells examined, and A37 was always excluded from 89Q inclusions and vice versa (Fig. 3A). This finding was consistent with data from confocal 3D reconstruction and FRET between expressed donor (CFP-HD89Q) and acceptor (YFP-A37) constructs—after expression of both constructs, we were not able to register any donor–acceptor proximity using acceptor bleach (data not shown). Failure of co-localization by immunofluorescence was confirmed with an independent pair of constructs: EGFP-A37 and HA-tagged HD-Q74 (data not shown). The FRET data were consistent with experiments where we transfected cells with constructs encoding both expanded polyglutamine and polyalanine tracts but failed to co-immunoprecipitate the polyglutamines with the polyalanines and vice versa (data not shown). Therefore, the protective effect of A37 is not because of the sequestration of the polyglutamine protein.


Figure 4603
View larger version (19K):
[in this window]
[in a new window]
  		Figure 3. Expanded polyalanines do not co-localize with long polyglutamines and mutant PABP2 induces a heat-shock response. (A) COS-7 cells were co-transfected with YFP-A37 and CFP-HD89Q and fixed after 48 h. A37 and 89Q form distinct non-overlapping inclusions, shown by confocal microscopy. (B) The number of Q74 inclusions per cell was scored in COS-7 cells transiently expressing both Q74 and either EGFPC1 (black bar), A7 (white bar) or A37 (grey bar; P<0.0001) at a ration of 1:1/9 (Q74 1 µg: EGFP 111 ng). (Comparison of A7 and A37, P<0.0001, Mann–Whitney U test). (C) Western blots of muscle lysates from a transgenic mouse model of OPMD (A17-1) and non-transgenic littermates (NT) at 9 months of age. hsp70 levels are increased in mice expressing mutant form of PABP2 (under the control of a muscle promoter) compared to non-transgenic littermates. Western blots were probed with an antibody raised against hsp70 and normalized with an actin antibody. (D) Western blots of muscle lysates from a transgenic mouse model of Huntington's diseases R6/1 (Tg) and non-transgenic littermates (NT) at 43 weeks of age. Hsp70 levels are not increased in mice expressing fragment of mutant huntingtin compared to non-transgenic littermates. Western blots were done identically to Fig. 3C.

 
The properties of polyalanine expansions are not common to all proteins with polyamino acid expansions. Polyserines appeared to exhibit very different properties compared with polyalanines in mammalian cell culture. Transfection of COS-7 cells with S5 or S25 led to toxicity (even at 1:1/9 dose) without any visible inclusions (Fig. S6). Co-transfection of either S5 or S25 with HD exon 1 74Q in a 1:1 or 1:1/9 ratio resulted in greater toxicity than with 74Q alone, without affecting inclusion formation, both in COS-7 cells (Fig. S6) and SK-N-SH cells (data not shown). In contrast to polyalanines, polyserines are tightly sequestered to polyglutamine inclusions (Fig. S6). Polyserines do not form inclusions, even in the presence of polyglutamine inclusions; hence the polyserines sequestered in polyglutamine inclusions may only represent a small fraction of the mutant polyserine molecules. The consequences of this are difficult to predict.

Polyalanine expansions induce a heat-shock response
Interestingly, while long polyalanines decreased the proportion of cells with polyglutamine inclusions (Fig. 2A and B), they increased the number of polyglutamine inclusions per inclusion-containing cells (Fig. 3B), a phenomenon reminiscent of the protective effect of heat-shock proteins on ataxin-1 (19Go). In addition, we have generated and characterized a new mouse model of OPMD (32Go) and we observed hsp70 induction in muscles of mutant mice at the age of 9 months (Fig. 3C) and 6 months (data not shown). For comparison, we have used the R6/1 mouse model of HD that bears a transgene comprising exon 1 of the human HD gene with an expanded (CAG)115 repeat under control of the HD promoter (33Go). In addition to the central nervous system, the transgene is ubiquitously expressed (33Go) and inclusions have been demonstrated in peripheral tissues, including skeletal muscle (34Go). While we observed variability in hsp70 levels from a mouse model of HD (R6/1), we did not detect any obvious increase of hsp70 in muscle lysates (Fig. 3D), consistent with previously published data in brain lysates (25Go). This suggests that expanded polyalanines can induce the heat-shock response and that this is not a general property of all aggregate-prone proteins.

These findings prompted us to examine hsp70 induction in our cell model. We observed strong induction of hsp70 by immunocytochemistry in cells transfected with A37 but not in the cells transfected with A7 (Fig. 4A). We further quantified the percentage of transfected cells with high levels of hsp70 (judged by immunocytochemistry) and confirmed that A37 (but not A7) induces hsp70 expression both when transfected alone (Fig. 4B) or when co-transfected with polyglutamine constructs (Fig. 4C). Consistent with these results, A37 (but not A7) increased hsp70 expression on western blots of cells transfected with A37 alone (Fig. 4D) or when co-transfected with a polyglutamine construct (Fig. 4E).


Figure 4604
View larger version (31K):
[in this window]
[in a new window]
  		Figure 4. Expanded polyalanines induce heat-shock response. (A) COS-7 cells were transiently transfected with 111 ng A37 or A7 (green) and 48 h later cells were fixed and immunolabelled with hsp70 antibody (magenta). Cells expressing A7 or untransfected cells have very low levels of hsp70 staining (just above or equal to background, not visible on the photograph). In contrast, a significant proportion of cells expressing A37 have very bright hsp70 signals. These very brightly staining cells were considered immuno-positive in experiments described in Fig. 4B and C and Fig. S5. Bright fluorescence was seen in cells with and without visible inclusions. (B) Transfected cells from (A) were scored for the presence of high levels of hsp70 (shown by immunocytochemistry). Transfection with A37 construct dramatically increases proportion of cells with high levels of hsp70. (Comparison of C1 and A7, P=0.86; C1 and A37, P<0.001; A7 and A37, P<0.001; odds ratio was used). Approximately 150 cells were scored per experiment and this was done at least in duplicate (also applies to C). (C) COS-7 cells were co-transfected with Q74 and EGFP, A7 or A37 constructs (1 µg:111 ng) and immunolabelled as in (A). The percentage of transfected cells with high levels of hsp70 expression (shown by immunocytochemistry), was recorded. The A37 construct increased the proportion of cells with high levels of hsp70 in the presence of Q74. Please note that the experiments in (B) and (C) are separate experiments that were performed on different days and therefore the difference in the percentage of cells with high levels of hsp70 might represent a variation in baseline between experiments. (Comparison of C1 and A7, P=0.894; C1 and A37, P<0.001; A7 and A37, P<0.001; odds ratio was used). (D) COS-7 cells were transfected with 111 ng of EGFP, A7 or A37 construct. After 48 h, transfected (GFP+) cells were sorted using FACS and probed with hsp70 and actin antibody. A37 construct induces expression of hsp70. Approximately 20 000 GFP+ cells were used for the western blot analysis (three independent experiments were done with similar results were). (E) COS-7 cells were co-transfected with Q74 and EGFP, A7 or A37 construct in a ratio 1:1/9 (1 µg:111 ng). After 48 h, transfected cells (GFP+) were sorted using FACS and probed with hsp70 and actin antibody. A37 construct induces expression of hsp70 in the presence of Q74 construct (note that all GFP+ cells will have Q74 due to the transfection ratio). (F) Luciferase reporter assay using a HSE-luciferase construct in the presence of EGFP, A7 or A37 (111 ng). A37 caused activation of the heat-shock element. Mean relative luciferase activity with EGFP was set to 100% with the mean response of A7 and A37 transfected cells expressed as comparative percentages. A representative experiment is shown from six experiments with similar results. Error bars represent the standard error of the mean. (Comparison of C1 and A37, P=0.002; C1 and A7, P=0.7465; A7 and A37, P=0.0001; unpaired T-test). (G) COS-7 cells were transfected with 111 ng of A7 or A37 construct and 48 h later exposed to 2 µM staurosporine for 4 h. Cells were subsequently fixed and number of dead transfected cells (with fragmented nuclei) was counted. Three experiments were done and 200 cells were evaluated for each experiment. Experiments in Fig. 4(A–F) were done in presence of pan-caspase inhibitor zVADfmk. (H) Toxicity of long polyalanines is enhanced by loss of one copy of functional Drosophila Heat-shock factor (Hsf). Flies of genotype ey-GAL4/+; UAS-A37/+ (control) or ey-GAL4/Hsf1; UAS-NLS-A37/+ (Hsf/+) were scored for the presence of abnormal eyes at 25°C. Hsf1 allele alone or in the presence of ey-GAL4 does not lead to any eye abnormalities (data not shown). Flies with Hsf1 allele alone, in the presence ey-GAL4 or in the presence of ey-GAL4 and UAS-A7 never exhibit any visible eye abnormalities. We evaluated 16 bottles (approximately 900 flies). (P<0.001; odds ratio: 4.287, 95% confidence interval 3.194–5.755). (I) Experiment was done as described in (H) and survival to adulthood was evaluated. We evaluated 10 bottles (approximately 1000 flies). (P<0.001; odds ratio: 0.546; 95% confidence interval 0.398–0.749). (J) Loss of one copy of functional Drosophila Hsf does not affect survival to adulthood of flies expressing non-toxic short stretches of alanines. Flies of genotype ey-GAL4/CyO; UAS-A7 were crossed to flies with Hsf1 allele and the survival was evaluated (see Materials and Methods for details). We evaluated 10 bottles (approximately 1500 flies). (P=0.614; odds ratio=1.055; 95% confidence interval 0.856–1.301). *P<0.05; ***P<0.001; NS not significant.

 
To test if hsp70 induction was mediated by HSF-1 driven transcription, we performed luciferase assays using a HSE-luciferase reporter construct. We observed a 3-fold increase in luciferase activity in the presence of A37 compared with EGFP, while A7 had no effect (Fig. 4F). We observed no increase in HSE-luciferase activity in stable inducible lines expressing mutant versus wild-type huntingtin exon 1 or full-length huntingtin (Sugars and Rubinsztein, unpublished data). The above experiments were performed in the presence of the broad-spectrum caspase inhibitor zVADfmk to avoid potential confounds of cell death (caspase activation) but similar results were also obtained without zVADfmk (data not shown). In addition, we observed increase of hsp70 expression when cells were transfected with a low (Fig. 4A–F) or high dose of long polyalanines (Fig. S5). These data show that long polyalanines induce a heat-shock response by activating HSF-1-driven transcription, which is likely to protect against polyglutamine-mediated toxicity.

As hsp-induction is known to be protective against apoptosis (35Go), we tested if transfection with A37 could also protect against staurosporine-induced cell death. Indeed, A37 decreased the percentage of dead cells compared with cells transfected with A7 construct (Fig. 4G). This raises the possibility that enhanced HSF-driven transcription may allow polyalanines to protect against some of their inherent toxicity. Indeed, Drosophila with one loss-of-function Hsf allele had enhanced toxicity of the polyalanine mutation (using both abnormal eyes and survival as our read-outs), compared to flies with two wild-type Hsf alleles (Fig. 4H–J). Therefore, whether polyalanines are toxic or not may be a balance between deleterious effects (increased at high expression) and protective effects (HSF induction).

It would not have been informative to test whether A37 expression still protected against polyglutamine toxicity in mutant flies containing a loss-of-function allele of Hsf (like the flies analysed in Fig. 4H–J). Hsf knockdown and dominant-negative hsp70 (or hsp40) significantly enhance the toxicity of polyglutamines in flies (36Go–38Go). This is consistent with Hsf enhancing the levels of Hsps even under basal conditions (39Go). Thus, polyglutamine flies with knockdown of hsp70/Hsf are likely to have more toxicity irrespective of whether polyalanines are expressed or not, particularly since our data suggest that HSF-1 knockdown enhances polyalanine toxicity (Fig. 4H–J). Thus, enhanced toxicity because of HSF-1 knockdown in polyQ+polyA flies will not be interpretable.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
CONCLUSION
 MATERIALS AND METHODS
 REFERENCES
 
Mechanism of polyalanine toxicity
We have generated a Drosophila model expressing EGFP tagged with 37 alanines, to provide insights into the consequences of such mutations in vivo. Long polyalanines are toxic and form inclusions in Drosophila when expressed in a variety of tissues. The mode by which expanded polyalanines cause toxicity has been widely discussed and the possibilities include gain-of-function, loss-of-function and dominant-negative models (5Go,7Go). The use of a model EGFP protein tagged with polyalanines allows us to provide the first in vivo evidence that polyalanines can cause the disease by a gain-of-function mechanism in vivo. We believe that this conclusion is valid for our EGFP–polyalanine model, as it is a non-native protein that allows us to dispense with the loss-of-function and dominant-negative components of the argument. However, any extension to polyalanine diseases requires the assumption that 37 alanines tagged to EGFP behave in a way that is qualitatively similar to 17 alanines in the protein causing OPMD, or the larger expansions of up to 29 alanines in various transcription factors causing some of the other polyalanine diseases (5Go,7Go). In these diseases, we cannot exclude the possibility that loss-of-function/dominant-negative effects may occur, even if there is gain-of-function toxicity.

Protective properties of expanded polyalanines
Surprisingly, the same polyalanine expansion mutation could also rescue polyglutamine toxicity in two different cell lines and in vivo in Drosophila. In addition, long polyalanines also protect against a pro-apoptotic stimulus (staurosporine) in cells and against long polyalanines themselves. The protective effects induced by long polyalanines could be explained by their ability to induce a heat-shock response even at non-toxic concentrations. Thus, using model proteins (mutant huntingtin fragments) or toxic insults, we have demonstrated that expanded polyalanines can exhibit protective properties.

Our study shows that the same aggregate-prone protein can have pathogenic and protective effects in cell culture and in vivo. This situation is different and complementary to what has been reported before (40Go), where full-length mutant ataxin 3 protein had protective effects. Our protein is non-native and the wild-type counterpart had no obvious protective effect, whereas the protective effect of the mutant ataxin 3 appeared to be a consequence of the wild-type protein function—the wild-type ataxin 3 appeared to protect at least as well as, if not better than, the mutant protein (40Go). Thus, our data suggest that the polyalanine mutation itself (as opposed to the surrounding protein context) has both toxic and protective effects. This is an important principle to consider and the experimental set-up that is required to make this point necessarily has to use a non-native protein.

Expanded polyalanines exhibit protective properties because of induction of a heat-shock response
Long polyalanines induced a heat-shock response that was consistent with the altered aggregation of polyglutamines and the protection against cell death (19Go). Raised levels of hsps were induced both by polyalanines tagged to EGFP and in our OPMD mouse model that expresses an authentic polyalanine expansion mutation with a repeat length seen in patients (41Go). Increased HSF-1 activity leads to increased levels of hsp70 and other inducible hsps like hsp27. While this manuscript was being written, we noted that hsp70 mRNA was one of many transcripts up-regulated in another OPMD cell model (42Go)—however, this study did not confirm upregulation of hsp70 in independent experiments (false positive results occur frequently with microarray studies).

It is important to stress that hsp70 accumulation in inclusions (25Go,43Go,44Go) or in disease cell models exposed to proteasome inhibitors (45Go) does not mean that the relevant proteins are inducing a heat-shock response. Hsps can accumulate in inclusions even when heat-shock responses are compromised [e.g. in the R6/2 HD mice (25Go) and proteasome inhibition by itself leads to a heat-shock response (46Go)]. Furthermore, even though some cell studies may suggest that polyglutamines can induce a heat-shock response, the converse occurs in vivo in mammalian models (25Go,47Go), (and this paper).

HSF-1 inducible hsps like hsp70 and HSF-1 itself have been shown to protect against polyglutamine toxicity in cell models (19Go), in flies (26Go) and in mice (48Go). Hsp70 reduces aggregation in cell models but both hsp70 and hsp27 may protect against polyglutamine toxicity via additional aggregation-independent mechanisms, like inhibition of caspase-9 activation (23Go).

In summary, we demonstrate that long polyalanines induce a heat-shock response and this is a feature of both our model protein and an authentic disease protein containing polyalanine expansion mutation. Our data and those of others (see above) show that this is not a general property of all aggregate-prone proteins.

Effect of polyalanine frameshift products in polyglutamine diseases
Our findings may have disease relevance in SCA3 where polyQ to polyA frameshifts have been reported (8Go). Our data show that low doses of polyalanines can protect against polyglutamine-induced toxicity, while high expression of polyalanines exacerbates polyglutamine toxicity. In order to address the outcome of the polyalanine frameshifts in polyglutamine diseases, it is necessary to know the relative load of polyalanine products in specific neurons in human disease brains. This is technically extremely difficult and is currently unknown. Also, there will be polyalanine products of different lengths, depending on whether the frameshift occurred close to the N-terminus of the CAG tract (when polyalanine tracts will be long), or closer to the C-terminus (when products may be short and inert like A7). So, one would have to somehow quantitate the size distributions of such events. Note that the total amount of these products in the whole brain would not be informative as there may be populations of cells with high and low levels of the polyalanine frameshift products leading to overall toxic and protective effects.

A recent study suggested that polyalanine frameshifts exacerbate polyglutamine toxicity in cell models of SCA3 (9Go). A crucial finding of this study was that anisomycin reduced both frameshifts and toxicity, suggesting a causal effect. However, anisomycin reduces protein synthesis (9Go) and the attenuation of toxicity could have been simply because of a reduction in dose of the toxic transgene product. Also, the analyses of cell death comparing stable inducible cell lines expressing frameshift-competent and -incompetent clonal cell lines was difficult to interpret, as it appears that only one clonal cell line of each was reported. It is not uncommon for different clones of a particular stable cell line to exhibit very different properties. Most importantly, the SCA3 study did not directly test (as we did in cell and Drosophila models) whether polyalanine expansions could modulate polyglutamine toxicity when expressed together—their data were largely inferential.

Thus, our data suggest a new paradigm for the effects of these frameshift products, although this phenomenon is not a primary focus of the paper and we do not address their final outcome in polyglutamine disease pathogenesis.

Expanded polyalanines can exhibit both toxic and protective properties simultaneously
Irrespective of the consequences of these frameshifts or even if they are important in vivo, our experiments enabled us to reach the conclusion that the same disease-causing mutation can have both deleterious and protective properties at the same time (Figs. 3,4 and S5). It is important to emphasize that we observe induction of a heat-shock response both at high and low levels of expression, that is when polyalanines exhibit either overall toxic or protective properties (Figs. 3,4 and S5).

The protective polyalanine-induced heat-shock response was also observed in vivo with an authentic polyalanine expanded protein—mutant PABPN1 in an OPMD mouse model. Furthermore, we have shown that overexpression of HSP70 can protect against mutant PABPN1 toxicity in cell models (17Go). Thus, our data with model proteins are reflected with authentic disease proteins in vivo—we speculate that the induction of a protective heat-shock response by a polyalanine expansion may be one reason for the late onset and mild pathological effects of this mutation in OPMD. These protective consequences may explain the lack of a degenerative phenotype in certain polyalanine diseases associated with mutations in transcription factors, even though these appear to aggregate in cell models (7Go,49Go). However, further experiments will be necessary to confirm that heat-shock protein induction occurs in animal models and people with other polyalanine diseases.

We speculate that the emergence of overall toxicity at higher polyalanine expression levels may be due to dose-dependent increase in the toxic properties overwhelming the protective responses—it is likely that heat-shock responses are saturable as they are physiologically subject to feedback control (50Go). As polyalanine expansions cause dominant diseases, our data suggest that decreased expression levels of such toxic proteins below normal physiological levels may be protective in certain contexts (Fig. 5). In our studies, the same disease-causing mutation exhibits thus both toxic and protective properties. This situation is distinct from other studies where the protective property is a feature of the protein context and not the disease-causing mutation.


Figure 4605
View larger version (16K):
[in this window]
[in a new window]
  		Figure 5. Properties of long polyalanines. Long polyalanines exhibit both protective and toxic properties at the same time. Overall effect depends on expression levels as toxicity is dose-dependent.

 
Protective effect of polyalanines is independent of inclusions
Our data address a distinct issue from a recent report (51Go) finding that cells with inclusions were less likely to die compared with cells expressing the same model constructs without obvious inclusions. We report findings in cells and in vivo and focus on aggregate-prone proteins, as distinct from the inclusions themselves. It is likely that the heat-shock responses we observed are independent of inclusions, as these responses are not obviously attenuated by Congo red treatment (data not shown). Also, the protective responses we report are seen with polyalanine but not with polyglutamine (HD) constructs (either in cells or in vivo).


   CONCLUSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 CONCLUSION
MATERIALS AND METHODS
 REFERENCES
 
In summary, our data suggest that certain disease-associated aggregate-prone proteins simultaneously mediate a diversity of consequences, some deleterious and some protective (Fig. 5). Irrespective of the specific disease context, our findings have important consequences for the way we view the roles of such so-called ‘toxic’ proteins.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 CONCLUSION
 MATERIALS AND METHODS
REFERENCES
 
Mammalian cell culture
African green monkey kidney (COS-7) and human neuroblastoma cell line (SK-N-SH) cells were maintained and transfected as described previously (10Go,52Go). See supplementary methods for details on serine constructs. Other plasmids used were pHM6(CAG)74 and pHM6(CAG)21 (53Go), A7 and A37 (10Go). Forty-eight hours post-transfection, cells were fixed with 4% paraformaldehyde in 0.1M PBS of pH 7.6 and pHM6(CAG)74 expression detected by fluorescent immunocytochemistry using an anti-HA antibody (1:200; Covance), or HSP70 antibody (1:200, Stressgen) and secondary antibody Alexa Fluor red anti-mouse (1:1000; Molecular Probes).

Double-expressing cells with both red (polyglutamines) and green (EGFP-tagged alanines or serines) fluorescence were scored for cell death and aggregation; 200 cells per coverslip were counted with the viewer blinded to slide identity. Pooled estimates for aggregation and cell death (assessed by apoptotic nuclear morphology from multiple (x6) experiments were calculated as odds ratios (OR; the ratios of the proportion of abnormal/normal cells in different experimental conditions) with 95% confidence intervals, as described previously (10Go,52Go). OR and P-values were determined by unconditional logistical regression analysis using the general log-linear analysis option of SPSS Version 6.1 (SPSS, Chicago, IL). The specificity and sensitivity of our methods is reported elsewhere (23Go).

For detergent resistance experiments, transfected cells were viewed under an inverted confocal microscope and SDS and Triton added to a final concentration of 0.1% (v/v). YFP-A37 was co-transfected with CFP HD exon 1 89Q in COS-7 cells, 1 µg each and fixed after 48 h. FRET analysis (acceptor bleach/increase in donor fluorescence) between donor (CFP HD exon 1 89Q) and acceptor (YFP-A37) was performed with a Zeiss Axiovert 200 microscope, using in-built external laser {lambda}=530 nm for YFP bleach (54Go).

GAL4 construct was co-transfected with A37 or A7 (ratio 3:1, 1.5 µg:0.5 µg), fixed after 48 h and stained using anti-GAL4 antibody against DNA binding domain (Clontech, 1:500) and secondary antibody Alexa Fluor red anti-mouse (1:1000; Molecular Probes).

Congo red was dissolved in DMEM and used at concentration 100 µg/ml. Congo red has been successfully used as an aggregation inhibitor when dissolved in aqueous solution on both cell-based and Drosophila studies (17Go,55Go–57Go).

Western blotting
Mouse muscle protein lysates were prepared and analysed as described in Davies et al. (32Go). Flies that eclosed within 24 h at 29°C were homogenized in a 1.5 ml microtube containing 1x SDS-PAGE sample buffer and treated as for muscle samples. Membranes were probed with primary antibodies raised against hsp70 (StressGen; 1:1000) or GFP (Clontech 1:10000) and, as a loading control, actin (Sigma–Aldrich; 1:5000) or tubulin (Sigma, 1:2000). HRP-conjugated antibodies (Amersham Biosciences; 1:5000) were then added to the blots. Immunoreactive bands were detected with enhanced chemiluminescence reagent (ECL; Amersham Biosciences) and the signal was visualized by exposing the membrane to the ECL Hyperfilm (Amersham Biosciences). Densitometry analysis was performed using Scion Image Beta 4.02 software.

Heat-shock element reporter assay
COS-7 cells were transfected in 24-well plates with 200 ng of a reporter plasmid comprising three tandem copies of the heat-shock element (HSE) consensus sequence fused to a minimal thymidine kinase promoter (PTAL) and firefly luciferase gene (pHSE-Luc; BD Biosciences), 200 ng of a plasmid containing the ß-galactosidase gene driven by a cytomegalovirus promoter (pCMVß BD Biosciences) and either 200 ng (ratio 1) or 25 ng (ratio 1/9) of the EGFP-linked polyalanine constructs (EGFPC1, A7, A37). Cells were harvested 48 h post-transfection and the levels of luciferase and ß-galactosidase activity were determined. Luciferase activity from each well was normalized to its ß-galactosidase activity to control for transfection efficiency. To determine luciferase activity specifically caused by activation of the HSE, the activity obtained from an empty control vector [pTAL-Luc; firefly luciferase gene under the control of a minimal TK promoter (PTAL); BD Biosciences] was subtracted from the test-wells. Experiments were carried out in the presence or absence of a pan-caspase inhibitor (ZVADfmk; 20 µM; Calbiochem).

For each experiment the mean luciferase activities in the presence of A7 and A37 were expressed as a percentage of the luciferase activity in the presence of EGFPC1, which was set to 100.

Drosophila stocks and crosses
Flies were grown on standard corn meal molasses medium supplemented with dry yeast (1.2 l water, 12.5 g agar, 105 g dextrose, 105 g maize, 21 g yeast, 35 g Nipagin) at 25°C or 29°C with 40–70% humidity, with 12/12 h light/dark cycle. Seven or 37 alanines were fused to EGFP and the NLS from SV40 T-antigen as described (10Go). EGFP-NLS-A7 and EGFP-NLS-A37 were subcloned into the pPUAST vector (15Go). Previously described Nrv2-GAL4 (58Go), MHC-GAL4 (59Go), ey-GAL4 (60Go), HSF[1] (61Go) were used.

Lifespan determination
Flies were allowed to lay eggs for one day at 25°C and subsequently reared at 29°C. Flies eclosed within 24 h were used for lifespan determination, 20 individuals of the same sex were placed in each vial; approximately 200 individuals were used for each genotype. Flies were transferred to fresh medium and dead individuals were counted every 2 days.

Climbing assay
Flies were raised as described for lifespan determination. Climbing assays were performed in a set-up originally described for phototaxis experiments with the score reflecting how quickly the fly can climb in a tube in different trials (62Go). Approximately 100 flies were used (same number of males and females). The assay was performed at 25°C with 70% humidity; flies were allowed to acclimatize for approximately 2 h before the assay.

Survival to adulthood
Lines with polyalanine insertions on the third chromosome heterozygous with a TM6B balancer chromosome were used. Flies eclosing as adults with either the transgene (A7 or A37) or with the balancer were counted. The ratio A7:TM6B was arbitrarily taken as 1 and the A37:TM6B ratio was compared to that value. Similarly, in order to quantify the effect of Hsf1 on survival, the abovementioned ratio of A37:TM6B was compared with ratio of: ey-GAL4/Hsf1; A37/+; ey-GAL4/Hsf1; TM6B/+ using the same statistical comparison. Odds ratios were determined as described earlier in mammalian cell culture section.

Pseudopupil analysis
Flies were raised at 25°C and rhabdomeres were counted at indicated times. Values for each genotype were obtained from at least 100 ommatidia counted in 5–10 flies and this was done at least twice (see text), with observer blinded to the identity of the slides. Controls were always done at the same time under exactly the same conditions. For pseudopupil analysis of gmr-GAL4, a gmr-GAL4 homozygous strain was crossed with w[1118] (stock isogenized for the X chromosome and two major autosomes which has been used as the genetics background for the European Drosophila Deletion Kit (63Go). For pseudopupil analysis of gmr-Q120, a stock of elav-GAL4; +; gmr-Q120/TM6B was crossed either with w[1118], A7 or A37 line; w[1118] was used as a parental stock for injection of A7/A37 transgenic lines.

Transgenic mice
Mice were cared for following UK Home Office regulations. Transgenic mice lines used in this study were the HD model R6/1(33Go) (muscle lysates) and a model of OPMD (A17-1) (32Go).

SUPPLEMENTARY MATERIAL
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We thank Drs R. Duden, J. Drummond, J. Rocha, Z. Azstalos and D. Crowther for helpful discussions; M. Gratian, M. Bowen, L. Garcia, E. Perdeaux and O. Chabriol for technical assistance. We thank G. Jackson, P. Salvaterra, C. Schuster and the Bloomington Drosophila stock Center for fly stocks, and A. Acevedo-Arozena for providing muscle of R6/1 mouse. We are grateful to the Wellcome Trust for a Senior Clinical Research Fellowship (DCR), and a Prize Studentship (ZB), BBSRC for a Career Development Award (CJO'K) and an MRC programme grant to DCR and Steve Brown. ZB is a recipient of ORS award.

Conflict of Interest statement. The authors declare that they have no competing financial interests.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back

{ddagger} The last two authors are joint Senior Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 CONCLUSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Gusella, J.F. and MacDonald, M.E. (2000) Molecular genetics: unmasking polyglutamine triggers in neurodegenerative disease. Nat. Rev. Neurosci., 1, 109–115.[Web of Science][Medline]

  2. Rubinsztein, D.C. and Carmichael, J. (2003) Huntington's disease: molecular basis of neurodegeneration. Expert Rev. Mol. Med., 5, 1–21.[Medline]

  3. DiFiglia, M., Sapp, E., Chase, K.O., Davies, S.W., Bates, G.P., Vonsattel, J.P. and Aronin, N. (1997) Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science, 277, 1990–1993.[Abstract/Free Full Text]

  4. Taylor, J.P., Hardy, J. and Fischbeck, K.H. (2002) Toxic proteins in neurodegenerative disease. Science, 296, 1991–1995.[Abstract/Free Full Text]

  5. Brown, L.Y. and Brown, S.A. (2004) Alanine tracts: the expanding story of human illness and trinucleotide repeats. Trends Genet., 20, 51–58.[CrossRef][Web of Science][Medline]

  6. Uyama, E., Tsukahara, T., Goto, K., Kurano, Y., Ogawa, M., Kim, Y.J., Uchino, M. and Arahata, K. (2000) Nuclear accumulation of expanded PABP2 gene product in oculopharyngeal muscular dystrophy. Muscle Nerve, 23, 1549–1554.[CrossRef][Web of Science][Medline]

  7. Albrecht, A.N., Kornak, U., Boddrich, A., Suring, K., Robinson, P.N., Stiege, A.C., Lurz, R., Stricker, S., Wanker, E.E. and Mundlos, S. (2004) A molecular pathogenesis for transcription factor associated poly-alanine tract expansions. Hum. Mol. Genet., 13, 2351–2359.[Abstract/Free Full Text]

  8. Gaspar, C., Jannatipour, M., Dion, P., Laganiere, J., Sequeiros, J., Brais, B. and Rouleau, G.A. (2000) CAG tract of MJD-1 may be prone to frameshifts causing polyalanine accumulation. Hum. Mol. Genet., 9, 1957–1966.[Abstract/Free Full Text]

  9. Toulouse, A., Au-Yeung, F., Gaspar, C., Roussel, J., Dion, P. and Rouleau, G.A. (2005) Ribosomal frameshifting on MJD-1 transcripts with long CAG tracts. Hum. Mol. Genet., 14, 2649–2660.[Abstract/Free Full Text]

  10. Bao, Y.P., Cook, L.J., O'Donovan, D., Uyama, E. and Rubinsztein, D.C. (2002) Mammalian, yeast, bacterial, and chemical chaperones reduce aggregate formation and death in a cell model of oculopharyngeal muscular dystrophy. J. Biol. Chem., 277, 12263–12269.[Abstract/Free Full Text]

  11. Rankin, J., Wyttenbach, A. and Rubinsztein, D.C. (2000) Intracellular green fluorescent protein-polyalanine aggregates are associated with cell death. Biochem. J., 348, 15–19.[Medline]

  12. Kazemi-Esfarjani, P. and Benzer, S. (2000) Genetic suppression of polyglutamine toxicity in Drosophila. Science, 287, 1837–1840.

  13. Ordway, J.M., Tallaksen-Greene, S., Gutekunst, C.A., Bernstein, E.M., Cearley, J.A., Wiener, H.W., Dure, L.S.T., Lindsey, R., Hersch, S.M., Jope, R.S. et al. (1997) Ectopically expressed CAG repeats cause intranuclear inclusions and a progressive late onset neurological phenotype in the mouse. Cell, 91, 753–763.[CrossRef][Web of Science][Medline]

  14. Rubinsztein, D.C. (2002) Lessons from animal models of Huntington's disease. Trends Genet., 18, 202–209.[CrossRef][Web of Science][Medline]

  15. Brand, A.H. and Perrimon, N. (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development, 118, 401–415.[Abstract]

  16. Sanchez, I., Mahlke, C. and Yuan, J. (2003) Pivotal role of oligomerization in expanded polyglutamine neurodegenerative disorders. Nature, 421, 373–379.[CrossRef][Medline]

  17. Bao, Y.P., Sarkar, S., Uyama, E. and Rubinsztein, D.C. (2004) Congo red, doxycycline, and HSP70 overexpression reduce aggregate formation and cell death in cell models of oculopharyngeal muscular dystrophy. J. Med. Genet., 41, 47–51.[Free Full Text]

  18. Kim, W.Y., Fayazi, Z., Bao, X., Higgins, D. and Kazemi-Esfarjani, P. (2005) Evidence for sequestration of polyglutamine inclusions by Drosophila myeloid leukemia factor. Mol. Cell Neurosci., 29, 536–544.[CrossRef][Web of Science][Medline]

  19. Cummings, C.J., Mancini, M.A., Antalffy, B., DeFranco, D.B., Orr, H.T. and Zoghbi, H.Y. (1998) Chaperone suppression of aggregation and altered subcellular proteasome localization imply protein misfolding in SCA1. Nat. Genet., 19, 148–154.[CrossRef][Web of Science][Medline]

  20. Chai, Y., Koppenhafer, S.L., Bonini, N.M. and Paulson, H.L. (1999) Analysis of the role of heat-shock protein (Hsp) molecular chaperones in polyglutamine disease. J. Neurosci., 19, 10338–10347.[Abstract/Free Full Text]

  21. Stenoien, D.L., Cummings, C.J., Adams, H.P., Mancini, M.G., Patel, K., DeMartino, G.N., Marcelli, M., Weigel, N.L. and Mancini, M.A. (1999) Polyglutamine-expanded androgen receptors form aggregates that sequester heat-shock proteins, proteasome components and SRC-1, and are suppressed by the HDJ-2 chaperone. Hum. Mol. Genet., 8, 731–741.[Abstract/Free Full Text]

  22. Wyttenbach, A., Carmichael, J., Swartz, J., Furlong, R.A., Narain, Y., Rankin, J. and Rubinsztein, D.C. (2000) Effects of heat-shock, heat-shock protein 40 (HDJ-2), and proteasome inhibition on protein aggregation in cellular models of Huntington's disease. Proc. Natl Acad. Sci. USA, 97, 2898–2903.[Abstract/Free Full Text]

  23. Wyttenbach, A., Sauvageot, O., Carmichael, J., Diaz-Latoud, C., Arrigo, A.P. and Rubinsztein, D.C. (2002) Heat-shock protein 27 prevents cellular polyglutamine toxicity and suppresses the increase of reactive oxygen species caused by huntingtin. Hum. Mol. Genet., 11, 1137–1151.[Abstract/Free Full Text]

  24. Sittler, A., Lurz, R., Lueder, G., Priller, J., Lehrach, H., Hayer-Hartl, M.K., Hartl, F.U. and Wanker, E.E. (2001) Geldanamycin activates a heat-shock response and inhibits huntingtin aggregation in a cell culture model of Huntington's disease. Hum. Mol. Genet., 10, 1307–1315.[Abstract/Free Full Text]

  25. Hay, D.G., Sathasivam, K., Tobaben, S., Stahl, B., Marber, M., Mestril, R., Mahal, A., Smith, D.L., Woodman, B. and Bates, G.P. (2004) Progressive decrease in chaperone protein levels in a mouse model of Huntington's disease and induction of stress proteins as a therapeutic approach. Hum. Mol. Genet., 13, 1389–1405.[Abstract/Free Full Text]

  26. Warrick, J.M., Chan, H.Y., Gray-Board, G.L., Chai, Y., Paulson, H.L. and Bonini, N.M. (1999) Suppression of polyglutamine-mediated neurodegeneration in Drosophila by the molecular chaperone HSP70. Nat. Genet., 23, 425–428.[CrossRef][Web of Science][Medline]

  27. Kazemi-Esfarjani, P. and Benzer, S. (2002) Suppression of polyglutamine toxicity by a Drosophila homolog of myeloid leukemia factor 1. Hum. Mol. Genet., 11, 2657–2672.[Abstract/Free Full Text]

  28. Schaffar, G., Breuer, P., Boteva, R., Behrends, C., Tzvetkov, N., Strippel, N., Sakahira, H., Siegers, K., Hayer-Hartl, M. and Hartl, F.U. (2004) Cellular toxicity of polyglutamine expansion proteins: mechanism of transcription factor deactivation. Mol. Cell, 15, 95–105.[CrossRef][Web of Science][Medline]

  29. Ravikumar, B., Vacher, C., Berger, Z., Davies, J.E., Luo, S., Oroz, L.G., Scaravilli, F., Easton, D.F., Duden, R., O'Kane, C.J. et al. (2004) Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease. Nat. Genet., 36, 585–595.[CrossRef][Web of Science][Medline]

  30. Jackson, G.R., Salecker, I., Dong, X., Yao, X., Arnheim, N., Faber, P.W., MacDonald, M.E. and Zipursky, S.L. (1998) Polyglutamine-expanded human huntingtin transgenes induce degeneration of Drosophila photoreceptor neurons. Neuron, 21, 633–642.[CrossRef][Web of Science][Medline]

  31. Lin, M.H., Bour, B.A., Abmayr, S.M. and Storti, R.V. (1997) Ectopic expression of MEF2 in the epidermis induces epidermal expression of muscle genes and abnormal muscle development in Drosophila. Dev. Biol., 182, 240–255.

  32. Davies, J.E., Wang, L., Garcia-Oroz, L., Cook, L.J., Vacher, C., O'Donovan D, G. and Rubinsztein, D.C. (2005) Doxycycline attenuates and delays toxicity of the oculopharyngeal muscular dystrophy mutation in transgenic mice. Nat. Med., 11, 672–677.[CrossRef][Web of Science][Medline]

  33. Mangiarini, L., Sathasivam, K., Seller, M., Cozens, B., Harper, A., Hetherington, C., Lawton, M., Trottier, Y., Lehrach, H., Davies, S.W. et al. (1996) Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice. Cell, 87, 493–506.[CrossRef][Web of Science][Medline]

  34. Sathasivam, K., Hobbs, C., Turmaine, M., Mangiarini, L., Mahal, A., Bertaux, F., Wanker, E.E., Doherty, P., Davies, S.W. and Bates, G.P. (1999) Formation of polyglutamine inclusions in non-CNS tissue. Hum. Mol. Genet., 8, 813–822.[Abstract/Free Full Text]

  35. Mosser, D.D., Caron, A.W., Bourget, L., Meriin, A.B., Sherman, M.Y., Morimoto, R.I. and Massie, B. (2000) The chaperone function of hsp70 is required for protection against stress-induced apoptosis. Mol. Cell. Biol., 20, 7146–7159.[Abstract/Free Full Text]

  36. Fernandez-Funez, P., Nino-Rosales, M.L., de Gouyon, B., She, W.C., Luchak, J.M., Martinez, P., Turiegano, E., Benito, J., Capovilla, M., Skinner, P.J. et al. (2000) Identification of genes that modify ataxin-1-induced neurodegeneration. Nature, 408, 101–106.[CrossRef][Medline]

  37. Chan, H.Y., Warrick, J.M., Andriola, I., Merry, D. and Bonini, N.M. (2002) Genetic modulation of polyglutamine toxicity by protein conjugation pathways in Drosophila. Hum. Mol. Genet., 11, 2895–2904.

  38. Chan, H.Y., Warrick, J.M., Gray-Board, G.L., Paulson, H.L. and Bonini, N.M. (2000) Mechanisms of chaperone suppression of polyglutamine disease: selectivity, synergy and modulation of protein solubility in Drosophila. Hum. Mol. Genet., 9, 2811–2820.

  39. Yan, L.J., Christians, E.S., Liu, L., Xiao, X., Sohal, R.S. and Benjamin, I.J. (2002) Mouse heat-shock transcription factor 1 deficiency alters cardiac redox homeostasis and increases mitochondrial oxidative damage. EMBO J., 21, 5164–5172.[CrossRef][Web of Science][Medline]

  40. Warrick, J.M., Morabito, L.M., Bilen, J., Gordesky-Gold, B., Faust, L.Z., Paulson, H.L. and Bonini, N.M. (2005) Ataxin-3 suppresses polyglutamine neurodegeneration in Drosophila by a ubiquitin-associated mechanism. Mol. Cell., 18, 37–48.[CrossRef][Web of Science][Medline]

  41. Brais, B., Bouchard, J.P., Xie, Y.G., Rochefort, D.L., Chretien, N., Tome, F.M., Lafreniere, R.G., Rommens, J.M., Uyama, E., Nohira, O. et al. (1998) Short GCG expansions in the PABP2 gene cause oculopharyngeal muscular dystrophy. Nat. Genet., 18, 164–167.[CrossRef][Web of Science][Medline]

  42. Corbeil-Girard, L.P., Klein, A.F., Sasseville, A.M., Lavoie, H., Dicaire, M.J., Saint-Denis, A., Page, M., Duranceau, A., Codere, F., Bouchard, J.P. et al. (2005) PABPN1 overexpression leads to upregulation of genes encoding nuclear proteins that are sequestered in oculopharyngeal muscular dystrophy nuclear inclusions. Neurobiol. Dis., 18, 551–567.[CrossRef][Web of Science][Medline]

  43. Tsai, H.F., Lin, S.J., Li, C. and Hsieh, M. (2005) Decreased expression of Hsp27 and Hsp70 in transformed lymphoblastoid cells from patients with spinocerebellar ataxia type 7. Biochem. Biophys. Res. Commun., 334, 1279–1286.[CrossRef][Web of Science][Medline]

  44. Merienne, K., Helmlinger, D., Perkin, G.R., Devys, D. and Trottier, Y. (2003) Polyglutamine expansion induces a protein-damaging stress connecting heat-shock protein 70 to the JNK pathway. J. Biol. Chem., 278, 16957–16967.[Abstract/Free Full Text]

  45. Abu-Baker, A., Messaed, C., Laganiere, J., Gaspar, C., Brais, B. and Rouleau, G.A. (2003) Involvement of the ubiquitin-proteasome pathway and molecular chaperones in oculopharyngeal muscular dystrophy. Hum. Mol. Genet., 12, 2609–2623.[Abstract/Free Full Text]

  46. Kawazoe, Y., Nakai, A., Tanabe, M. and Nagata, K. (1998) Proteasome inhibition leads to the activation of all members of the heat-shock-factor family. Eur. J. Biochem., 255, 356–362.[Web of Science][Medline]

  47. Vacher, C., Garcia-Oroz, L. and Rubinsztein, D.C. (2005) Overexpression of yeast hsp104 reduces polyglutamine aggregation and prolongs survival of a transgenic mouse model of Huntington's disease. Hum. Mol. Genet., 14, 3425–3433.[Abstract/Free Full Text]

  48. Fujimoto, M., Takaki, E., Hayashi, T., Kitaura, Y., Tanaka, Y., Inouye, S. and Nakai, A. (2005) Active HSF1 significantly suppresses polyglutamine aggregate formation in cellular and mouse models. J. Biol. Chem., 280, 34908–34916.[Abstract/Free Full Text]

  49. Nasrallah, I.M., Minarcik, J.C. and Golden, J.A. (2004) A polyalanine tract expansion in Arx forms intranuclear inclusions and results in increased cell death. J. Cell Biol., 167, 411–416.[Abstract/Free Full Text]

  50. Morimoto, R.I. (2002) Dynamic remodeling of transcription complexes by molecular chaperones. Cell, 110, 281–284.[CrossRef][Web of Science][Medline]

  51. Arrasate, M., Mitra, S., Schweitzer, E.S., Segal, M.R. and Finkbeiner, S. (2004) Inclusion body formation reduces levels of mutant huntingtin and the risk of neuronal death. Nature, 431, 805–810.[CrossRef][Medline]

  52. Wyttenbach, A., Swartz, J., Kita, H., Thykjaer, T., Carmichael, J., Bradley, J., Brown, R., Maxwell, M., Schapira, A., Orntoft, T.F. et al. (2001) 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., 10, 1829–1845.[Abstract/Free Full Text]

  53. Narain, Y., Wyttenbach, A., Rankin, J., Furlong, R.A. and Rubinsztein, D.C. (1999) A molecular investigation of true dominance in Huntington's disease. J. Med. Genet., 36, 739–746.[Abstract/Free Full Text]

  54. Majoul, I., Straub, M., Duden, R., Hell, S.W. and Soling, H.D. (2002) Fluorescence resonance energy transfer analysis of protein-protein interactions in single living cells by multifocal multiphoton microscopy. J. Biotechnol., 82, 267–277.[CrossRef][Medline]

  55. Smith, D.L., Portier, R., Woodman, B., Hockly, E., Mahal, A., Klunk, W.E., Li, X.J., Wanker, E., Murray, K.D. and Bates, G.P. (2001) Inhibition of polyglutamine aggregation in R6/2 HD brain slices-complex dose-response profiles. Neurobiol. Dis., 8, 1017–1026.[CrossRef][Web of Science][Medline]

  56. Poirier, M.A., Li, H., Macosko, J., Cai, S., Amzel, M. and Ross, C.A. (2002) Huntingtin spheroids and protofibrils as precursors in polyglutamine fibrilization. J. Biol. Chem., 277, 41032–41037.[Abstract/Free Full Text]

  57. Crowther, D.C., Kinghorn, K.J., Miranda, E., Page, R., Curry, J.A., Duthie, F.A., Gubb, D.C. and Lomas, D.A. (2005) Intraneuronal Abeta, non-amyloid aggregates and neurodegeneration in a Drosophila model of Alzheimer's disease. Neuroscience, 132, 123–135.[CrossRef][Web of Science][Medline]

  58. Sun, B., Xu, P. and Salvaterra, P.M. (1999) Dynamic visualization of nervous system in live Drosophila. Proc. Natl Acad. Sci. USA, 96, 10438–10443.

  59. Schuster, C.M., Davis, G.W., Fetter, R.D. and Goodman, C.S. (1996) Genetic dissection of structural and functional components of synaptic plasticity. II. Fasciclin II controls presynaptic structural plasticity. Neuron, 17, 655–667.[CrossRef][Web of Science][Medline]

  60. Bonini, N.M., Bui, Q.T., Gray-Board, G.L. and Warrick, J.M. (1997) The Drosophila eyes absent gene directs ectopic eye formation in a pathway conserved between flies and vertebrates. Development, 124, 4819–4826.[Abstract]

  61. Jedlicka, P., Mortin, M.A. and Wu, C. (1997) Multiple functions of Drosophila heat-shock transcription factor in vivo. EMBO J., 16, 2452–2462.

  62. Benzer, S. (1967) Behavioural mutants of Drosophila isolated by countercurrent distribution. Proc. Natl Acad. Sci. USA, 58, 1112–1119.[Free Full Text]

  63. Ryder, E., Blows, F., Ashburner, M., Bautista-Llacer, R., Coulson, D., Drummond, J., Webster, J., Gubb, D., Gunton, N., Johnson, G. et al. (2004) The DrosDel collection: a set of P-element insertions for generating custom chromosomal aberrations in Drosophila melanogaster. Genetics, 167, 797–813.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Am. J. Pathol.Home page
D. W. Dickson
Linking Selective Vulnerability to Cell Death Mechanisms in Parkinson's Disease
Am. J. Pathol., January 1, 2007; 170(1): 16 - 19.
[Full Text] [PDF]

Home page
 J. Med. Genet.Home page
J E Davies and D C Rubinsztein
Polyalanine and polyserine frameshift products in Huntington's disease
J. Med. Genet., November 1, 2006; 43(11): 893 - 896.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Material
Right arrow All Versions of this Article:
15/3/453    most recent
ddi460v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (8)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Berger, Z.
Right arrow Articles by Rubinsztein, D. C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Berger, Z.
Right arrow Articles by Rubinsztein, D. C.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?