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Human Molecular Genetics Advance Access originally published online on March 9, 2005
Human Molecular Genetics 2005 14(8):1041-1048; doi:10.1093/hmg/ddi096
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

The pathogenic agent in Drosophila models of ‘polyglutamine’ diseases

Catherine J. McLeod1, Louise V. O'Keefe1 and Robert I. Richards1,2,*

1ARC Special Research Centre for the Molecular Genetics of Development and 2ARC/NHMRC Research Network in Genes and Environment in Development, School of Molecular and Biomedical Sciences, The University of Adelaide, Adelaide 5005, South Australia

* To whom correspondence should be addressed. Tel: +618 83037541; Fax: +618 83037534; Email: robert.richards{at}adelaide.edu.au

Received January 19, 2005; Revised February 18, 2005; Accepted February 28, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
A substantial body of evidence supports the identity of polyglutamine as the pathogenic agent in a variety of human neurodegenerative disorders where the mutation is an expanded CAG repeat. However, in apparent contradiction to this, there are several human neurodegenerative diseases (some of which are clinically indistinguishable from the ‘polyglutamine’ diseases) that are due to expanded repeats that cannot encode polyglutamine. As polyglutamine cannot be the pathogenic agent in these diseases, either the different disorders have distinct pathogenic pathways or some other common agent is toxic in all of the expanded repeat diseases. Recently, evidence has been presented in support of RNA as the pathogenic agent in Fragile X-associated tremor/ataxia syndrome (FXTAS), caused by expanded CGG repeats at the FRAXA locus. A Drosophila model of FXTAS, in which 90 copies of the CGG repeat are expressed in an untranslated region of RNA, exhibits both neurodegeneration and similar molecular pathology to the ‘polyglutamine’ diseases. We have, therefore, explored the identity of the pathogenic agent, and specifically the role of RNA, in a Drosophila model of the polyglutamine diseases by the expression of various repeat constructs. These include expanded CAA and CAG repeats and an untranslated CAG repeat. Our data support the identity of polyglutamine as the pathogenic agent in the Drosophila models of expanded CAG repeat neurodegenerative diseases.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mutation of the androgen receptor leading to Kennedy disease was identified 13 years ago as the first neurodegenerative disease caused by the expansion of a CAG repeat within a coding region (1Go). Since then, numerous human neurodegenerative diseases have been identified with an identical mutation mechanism, but with the CAG repeat expansions occurring in quite distinct genes. Presumably, a similar pathogenic pathway is involved, as each of the expanded CAG repeats encodes polyglutamine and the pathogenic threshold for disease is roughly the same, at around 40 copies of the repeat. The role of a toxic gain of function for the polyglutamine-containing protein in these diseases has been supported by a variety of experimental and pathological observations. For example, mutation of a specific amino acid in Ataxin1 that lies outside the repeat region severely diminishes pathology in a mouse model of SCA1 (2Go). In a Drosophila model of Kennedy disease, testosterone (which activates the androgen receptor and translocates it to the nucleus) is necessary to observe pathology (3Go). In addition, analysis of mouse models of SCA7 suggest that polyglutamine–based association between the SCA7 protein containing expanded polyglutamine and the transcription factor CRX results in transcription interference that is a predominant factor in SCA7 cone-rod dystrophy retinal degeneration (4Go).

A prominent feature of polyglutamine diseases is the presence of intranuclear inclusions formed by aggregation of the expanded polyglutamine protein. These inclusions are observed in brain tissue of patients with neurodegenerative diseases and contain cellular components such as ubiquitin, the proteasome, Hsp70 and transcription factors (5Go). Similar inclusions are also observed in Drosophila models of the diseases, where disruption of the inclusions is associated with a dramatic reduction in pathology (6Go). On the other hand, the role of nuclear inclusions in the pathology of neurodegenerative diseases has been controversial almost because their discovery with disagreement as to whether the inclusions are an integral component of the pathology. Rather, some studies have suggested that they may instead play a protective role (7Go,8Go).

In addition to the earlier mentioned diseases, a series of expanded repeat diseases have been described, some of which demonstrate clinically indistinguishable phenotypes from the ‘polyglutamine’ disorders, but in each case, the repeat cannot encode polyglutamine. In several cases, such as SCA8 (9Go) and SCA12 (10Go), the repeat is located in the untranslated region of a transcript. In HDL2, which shows marked similarity to Huntington's disease, the repeat occurs in a variably spliced exon and is either in an untranslated region or encodes polyleucine or polyalanine (11Go). Furthermore, SCA10 is due to the expansion of a 5 bp repeat in an intron of another unrelated gene (12Go). In these disorders, polyglutamine cannot be the pathogenic agent. Taken together, these data suggest that a different pathogenic pathway is responsible for the consequences of repeat expansion in these diseases. Alternatively, polyglutamine is not responsible for the ‘polyglutamine’ diseases and some other agent is the toxin in a common pathogenic pathway for all of the neurodegenerative diseases caused by expanded repeats.

If the toxic agent in ‘polyglutamine’ diseases is not polyglutamine, what else could be responsible? One alternative agent that has been proposed to play a role is polyalanine, which expands to cause pathogenesis in the degenerative disorder oculopharyngeal muscular dystrophy (13Go). Investigation into a role for polyalanine in Machado–Joseph disease, a ‘polyglutamine’ disorder, revealed that the nuclear inclusions containing expanded polyglutamine also show immunostaining with anti-polyalanine antibodies (14Go). This study also demonstrated using cultured cells that frameshifting during transcription or translation of expanded CAG repeats occurs in a length-dependent matter, leading to the suggestion that polyalanine contributes to nuclear toxicity in the polyglutamine diseases by virtue of frame-shifted translation of the expanded CAG repeat (14Go). Although toxic polyalanine may also account for the HDL2 phenotype, as this transcript can potentially encode polyalanine, it fails to provide a common mechanism for all of the expanded repeat diseases, as some of the repeat transcripts implicated in the diseases, such as the pentanculeotide repeat responsible for SCA10, cannot encode polyalanine.

Another toxic agent already implicated in expanded repeat-induced pathogenesis is RNA. In the case of myotonic dystrophy, there are two genetically distinct forms, DM1 and DM2, caused by CTG or CCTG repeat expansions in unrelated genes (15Go,16Go). In both forms, RNA containing the repeat expansion is central to pathogenesis (reviewed in 17Go). The ability of the repeat-containing RNA to form a stable hairpin secondary structure appears to be involved in pathology: in DM1, this double stranded structure binds to the splicing factor Muscleblind in vitro (18Go) and co-localizes with it in vivo (19Go), suggesting that Muscleblind is sequestered by the double stranded CUG repeat. In addition, levels of another splicing factor CUG-binding protein (CUG-BP) are altered in DM1 (20Go), and perturbation of normal splicing is observed, suggesting that pathogenesis is caused by the expanded CUG repeat-containing RNA inducing these alterations in splicing via Muscleblind and CUG-BP (21Go,22Go). As in DM1, the repeat-containing transcript present in DM2 affects the levels of CUG-BP and Muscleblind resulting in altered splicing (23Go,24Go). This RNA-based model of pathogenesis accounts for the remarkably similar multisystemic phenotypes seen in DM1 and DM2, despite the causative mutations occurring in unrelated genes.

In addition to myotonic dystrophy, a role for RNA in disease characterized by neurodegeneration has recently been revealed by a study using Drosophila. Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late onset ataxia observed in some male carriers of the FRAXA expanded CGG ‘premutation’ (copy numbers of 55 to 230 repeats) (25Go). RNA has been implicated in a Drosophila model of this repeat expansion, where both neurodegeneration and the presence of nuclear inclusions are observed (26Go). This study demonstrates the ability of expanded repeat-containing RNA to induce neurodegeneration.

In the ‘polyglutamine’ diseases, protein pathogenesis has been assumed, but this assumption has not been tested directly. A possible ‘mechanistic overlap’ between neurodegenerative diseases mediated by RNA and those thought to be mediated by polyglutamine has recently been suggested (27Go). Others have suggested that RNA-mediated mechanisms in Huntington's disease pathogenesis may act in parallel with polyglutamine toxicity to promote cell death (28Go). This suggestion was based on evidence that expanded CAG repeats in the Huntington's disease transcript cause the activation of PKR, a double stranded RNA binding protein that is involved in inducing apoptosis (28Go). More recently, the possibility that CAG repeat-containing RNA could act as a pathogenic agent based on its secondary structure has been investigated. Examination of CAG repeat-containing RNA generated in vitro has revealed that CAG repeats in RNA can form a double stranded hairpin structure (29Go). Furthermore, examination of the secondary structure of RNA transcripts implicated in the ‘polyglutamine’ diseases has demonstrated that the CAG repeats form hairpin structures in the context of these full-length disease transcripts (30Go). These observations have lead us to test the hypothesis that it is RNA that causes pathogenesis in the ‘polyglutamine’ diseases, as has been found to be the case in myotonic dystrophy and FXTAS.

We have, therefore, addressed the possibility that RNA is the toxic agent using a Drosophila model of ‘polyglutamine’ disease. The Drosophila system has been previously established and validated as a useful model of polyglutamine diseases (31Go–33Go) and also exhibits neurodegeneration mediated by RNA containing expanded repeats (26Go). To examine the RNA hypothesis, we have expressed various repeat constructs in the Drosophila eye, comparing the effect of expanded CAG and CAA repeats and an untranslated CAG repeat. By comparing the effects of expressing these various repeats, we have been able to rule out a role for RNA in pathogenesis. Instead, our results suggest that toxicity does occur at the protein level, at least in this model system.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of trinucleotide repeat transgenes in the Drosophila eye
Drosophila has previously been utilized to investigate the mechanisms of polyglutamine toxicity. The expression of an expanded CAG repeat was found to cause neuronal degeneration in a length-dependent manner (33Go). The relevant construct encoded a polyglutamine tract flanked by short amino acid sequences (six on the N-terminal and four on the C-terminal side) and a myc/flag epitope tag of 26 amino acids at the C-terminus. These sequences were placed downstream of an upstream activator sequence (UAS), which allows tissue-specific expression when flies containing the transgene are crossed to a driver strain expressing GAL4 (34Go) (Fig. 1A). We have assembled several versions of this construct containing various repeat tracts (Fig. 1B–D) in a P-element transformation vector and used them to generate multiple lines of transgenic Drosophila. These transgenic lines were then crossed to the driver strain GMR-GAL4, resulting in expression of the repeat transgene in all cells of the developing eye behind the morphogenetic furrow and on into adulthood (35Go).



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  		Figure 1. Schematic representation of repeat constructs. (A) All repeat constructs were inserted downstream of UAS sites to allow expression under the control of GMR-GAL4. The repeats are flanked by six amino acids on the N-terminal side and four on the C-terminal, and a myc/flag epitope tag is downstream. (B) Constructs containing polyglutamines encoded by (CAG)30, (CAG)52 and (CAG)99 were generated. (C) These were compared to constructs containing polyglutamine encoded by (CAA)20 or (CAA)94. (D) A variation of this construct was generated, where a termination codon is inserted immediately upstream of (CAG)93, effectively moving the repeat into the 3' untranslated region. This is referred to as term(CAG)93.

 
Expression of either expanded CAG or CAA causes neurodegeneration
Previously, only polyglutamine repeats encoded by CAG have been expressed using this system (Fig. 1A). To determine whether hairpin-containing RNA and/or some sequence specific attribute of CAG has a role in pathogenesis, we expressed polyglutamine encoded by CAA repeats. When present in RNA, this repeat is not predicted to form a hairpin secondary structure, as the complementary C–G base pairs required for this are lacking. However, the CAA repeat construct encodes an identical protein to that encoded by the CAG repeat. Thus, comparison of the effects of expression of these two constructs will allow us to determine whether RNA sequence composition or structure has a role in pathogenesis. If this is the case, we would expect a milder or even no phenotype to result from the expression of expanded CAA repeats.

Repeat lengths below and above the human pathogenic threshold (of ~40 repeats) were generated for both CAA and CAG (Fig. 1B and C) and expressed in the Drosophila eye. Expression of repeats below the pathogenic threshold had no apparent effect on the external morphology of the eye when compared with a fly carrying GMR-GAL4 alone (Fig. 2E), as expected, for either (CAG)30 (Fig. 2A) or (CAA)20 (Fig. 2B). In contrast, expression of the expanded (CAG)99 transgene caused a dramatic phenotype with disorganization of the ommatidia, loss of pigment cells and a collapsed eye (Fig. 2C). This is in agreement with the previous description of the phenotype resulting from the expression of this construct in the Drosophila eye (33Go). Expression of expanded (CAA)94 driven by GMR-GAL4 resulted in a phenotype which is indistinguishable from the expanded CAG phenotype. Again, a dramatic phenotype was observed with disorganization of the ommatidia, loss of pigment cells and collapse of the eye structure (Fig. 2D). For both the expanded CAG and the expanded CAA constructs, phenotypes ranging in severity from wild-type to lethality resulted when independently generated transgenic lines were crossed to GMR-GAL4 (Table 1). Presumably, this is due to positional effects, where varying levels of expression between independent lines are caused by differences in the site of integration (36Go). However, the same range of phenotypes was seen for expanded CAG and CAA, indicating that the expression of these different constructs has the same effect on the Drosophila eye.



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  		Figure 2. Effect of expressing various repeat constructs on the external appearance of the Drosophila eye. All repeats are downstream of UAS sites, and expression is driven by GMR-GAL4. (A) No alteration from the wild-type eye phenotype is observed in flies expressing polyglutamine encoded by (CAG)30. (B) Expression of polyglutamine encoded by (CAA)20 also has no effect on the external appearance of the eye. (C) Expanded polyglutamine encoded by (CAG)99 causes degeneration of the eye, seen here as a lack of pigmentation and collapsed eye structure. (D) Expression of expanded polyglutamine encoded by (CAA)94 repeats also causes degeneration, with a phenotype indistinguishable from that caused by (CAG)99. (E) Control fly, expressing only GAL4 in the eye. (F) Expression of an untranslated CAG repeat encoded by term(CAG)93 has no effect on the appearance of the eye.

 

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  		Table 1. Effect of expressing expanded repeats in the Drosophila eye driven by GMR-GAL4 varies between lines
 
Expanded CAG and CAA-induced pathology involves protein aggregation
The observation that the expression of expanded CAA and CAG repeats in the Drosophila eye has the same effect suggests that pathogenesis is independent of RNA sequence and secondary structure and that it is indeed polyglutamine-containing protein which is the pathogenic agent. We determined the distribution of the polyglutamine protein in the eye of these flies by examining horizontal sections of the eye stained with anti-myc antibody, which recognizes the myc epitope tag on the C-terminus of the polyglutamine protein. In flies expressing polyglutamine encoded by either CAG or CAA repeats that are below the pathogenic threshold, the polyglutamine protein appeared to be diffusely distributed throughout the eye (Fig. 3A and B). However, when the repeats are expanded to ~100 copies, the protein forms aggregates in the eye, a result that has been reported previously for polyglutamine encoded by CAG repeats (37Go). We observed such aggregation for expanded polyglutamine proteins encoded by both CAG repeats (Fig. 3C) and CAA repeats (Fig. 3D). This suggests that the behaviour of the encoded proteins is a major determinant of the phenotypes observed in this Drosophila model of the CAG repeat expansion diseases.



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  		Figure 3. Distribution of polyglutamine proteins in the Drosophila eye. Expression of the UAS repeat constructs is driven by GMR-GAL4. Horizontal cryosections are stained with Hoechst (blue) to show DNA and Myc antibody (green), which recognizes the Myc epitope tag on the C-terminus of the polyglutamine protein. Short polyglutamine proteins encoded by (CAG)30 (A) or (CAA)20 (B) show a diffuse distribution. Polyglutamine proteins above the pathogenic threshold form tight aggregates, whether the polyglutamine is encoded by (CAG)99 (C) or by (CAA)94 (D).

 
Untranslated CAG repeats do not induce pathology in Drosophila
The results described earlier suggest that pathogenesis occurs at the protein level. However, it is possible that the pathogenesis observed in this Drosophila model involves multiple pathways and that both RNA and the encoded polyglutamine both contribute to pathogenesis via independent pathways. This possibility can be addressed by expressing a transcript containing expanded repeats in a non-coding region: if RNA has a role in pathogenesis, we would expect to observe a phenotype in the Drosophila eye. Therefore, a construct was generated in which a premature termination codon is inserted before the expanded CAG repeat, referred to as ‘term(CAG)93’. This has the effect of moving the CAG repeats into the 3'-untranslated region of the transcript (Fig. 1D). When the expression of this transgene was driven in the eye using GMR-GAL4, no phenotype was observed (Fig. 2F), with the resulting eye having an appearance identical to the eyes of control flies expressing GAL4 alone (Fig. 2E). This confirms that it is indeed the encoded polyglutamine that is the pathogenic agent and rules out a role for RNA containing expanded repeats in this Drosophila model of expanded CAG repeat diseases.

The untranslated CAG repeat transcript is expressed
To determine whether the untranslated CAG repeat construct was expressed at a sufficiently high level to induce toxicity, quantitative (real time) PCR was performed. This was necessary as the lack of phenotype observed upon expression of this transgene could possibly be due to a difference in the stability of the mRNA. In particular, the introduced (premature) termination codon could cause the transcript to be recognized as nonsense and degraded via the nonsense mediated decay mechanism (38Go). Two independently generated lines carrying the term(CAG)93 transgene were crossed to GMR-GAL4, and the level of expression of the repeat transcript relative to the GAL4 transcript was compared to two lines expressing translated (CAG)99. One of these (CAG)99 lines caused a moderate phenotype when expressed in the eye driven by GMR-GAL4, and one caused no phenotype, presumably owing to differences in expression of the transgene caused by variation in the site of integration. Real-time PCR demonstrated that the expression of the (CAG)99 transcript was higher in the line showing a phenotype, whereas the line showing no phenotype demonstrated a lower level of expression (Fig. 4A and B). Furthermore, the two lines expressing the untranslated CAG repeat did so at a higher level than the translated (CAG)99 transcripts (Fig. 4). Thus, the steady state levels of these transgene mRNAs are comparable to and actually exceed those sufficient to induce toxicity. The lack of phenotype observed can, therefore, be interpreted to mean that untranslated CAG repeats are not pathogenic in this Drosophila model.



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  		Figure 4. Expression levels of translated and untranslated CAG repeat transcripts. GMR-GAL4 is used to drive expression of UAS repeat constructs. Quantitative (real time) PCR was performed to determine levels of repeat transcripts in the following flies: (CAG)99 showing a phenotype (A) in light grey, (CAG)99 showing no phenotype (B) in white and two independent lines of term(CAG)93 shown in dark grey. The expression level of the repeat transcripts are normalized to the level of the GAL4 transcript, and the expression level of each line is expressed as the fold difference relative to the (CAG)99 line that shows a phenotype (A), which is set at one.

 
Expanded polyalanine does not make a major contribution to the observed neurodegeneration
In addition to examining the pathogenic RNA hypothesis, these experiments address another question relating to polyglutamine pathogenesis. An alternative model has been proposed whereby the expanded CAG repeat undergoes a frameshift during transcription and/or translation, resulting in the expression of protein encoded by the alternative reading frame GCA (14Go). These alternative proteins contain a polyalanine repeat, which is suggested to be more toxic than the polyglutamine repeat, and therefore to contribute towards pathogenesis in the ‘polyglutamine’ diseases. This frameshifting has been shown to occur in vivo in a patient with Machado–Joseph disease and also in transfected COS-7 cells in a length-dependent manner (14Go). This mechanism could account for the observation that either polyglutamine (encoded by CAG) or polyalanine are the expanded amino acid repeats found in a growing number of human genetic diseases.

Expression of a polyglutamine protein encoded by CAA repeats in Drosophila also indirectly addressed this hypothesis, as CAA is unable to code for polyalanine in any alternative frame. If polyalanine proteins are contributing to the phenotype observed when expanded CAG repeats are expressed, a milder (or no) phenotype would be expected when CAA repeats are expressed, as the contribution made by polyalanine would be lacking. However, both CAG and CAA repeats cause a phenotype when expressed in the Drosophila eye (Fig. 2C and D). Furthermore, no obvious difference in severity was observed between the CAG and CAA flies, as we saw the same range of phenotypes for each type of repeat. This suggests that polyalanine is not making a major contribution to the pathogenesis observed. Thus, although we cannot rule out a role for polyalanine in the human polyglutamine disorders or the suggestion that it may contribute in a minor way to pathogenesis in Drosophila, it certainly does not appear to play a principle role in pathogenesis in the Drosophila system.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The expanded repeat disorders are a group of diseases caused by the expansion of a repeat above a roughly common pathogenic threshold and characterized by late-onset neurodegeneration. It seems parsimonious that, considering the common features shared by the disorders, they would share a common pathogenic agent or at least a common pathogenic pathway. A good candidate for a common toxic agent in these diseases is RNA, as it has previously been implicated in expanded repeat disorders including a recent Drosophila model of FXTAS that is characterized by neurodegeneration. Furthermore, the disease-causing repeats are always located within the transcriptional unit of the gene, and therefore each repeat is present in an RNA sequence. However, in this study, we have demonstrated that the pathogenic agent in ‘polyglutamine’ disease is indeed polyglutamine-containing protein, ruling out a role for RNA in the Drosophila model. Expanded polyalanine is another candidate previously suggested to act as a pathogenic agent in the polyglutamine diseases and may also be implicated in HDL-2. However, our results also suggest that polyalanine does not play a major role in polyglutamine disease in the Drosophila system. Thus, it appears that there may be at least two major pathways of pathogenesis in the expanded repeat disorders, one caused by protein containing expanded polyglutamine and a separate pathway, possibly still involving RNA, in those disorders where polyglutamine is not present.

Prior to this study, the question of a role for RNA in the polyglutamine disorders had not been directly addressed. Evidence from transgenic mouse models suggested that protein is required to mediate pathogenesis, but this evidence was not entirely conclusive. An early mouse model expressing the human Huntington's disease transcript containing 44 CAG repeats, which failed to produce any protein, also failed to demonstrate a disease phenotype (39Go). However, this may simply be due to the fact that 44 copies of the repeat is insufficient to induce a phenotype in the mouse, an explanation which has been suggested previously (28Go) and is supported by the failure of repeats of similar length to cause a phenotype in other mouse models, despite translation of the mRNA (40Go). Further evidence of a role for protein in pathogenesis comes from a SCA1 mouse model in which mutation of the nuclear localization signal in Ataxin1 resulted in mice that did not display the disease phenotype, an observation difficult to explain if pathology is RNA-based (41Go). However, these results are contradicted by an earlier report from the same group describing five lines of SCA1 mutant transgenic mice in which Ataxin1 mRNA but not protein could be detected, and which displayed the disease phenotype (42Go). The possibility that the mutation of the nuclear localization signal (41Go) also had some effect on the structure or localization of the mRNA cannot be ruled out. Therefore, it is difficult to conclusively rule out a role for expanded repeat-containing RNA in polyglutamine pathogenesis from these studies.

We have utilized a Drosophila model of polyglutamine disease, which has previously been well characterized. The neurodegeneration caused by the expression of expanded CAG repeats in this model system is late onset and progressive (33Go), as are the human disorders. However, in the Drosophila model, expanded polyglutamine proteins are expressed at a high level driven by GMR-GAL4 which does not necessarily reflect the human disease state. Thus, it is possible that this level of expression causes degeneration in Drosophila via a mechanism which is not involved in the human diseases or which plays a minor role. In addition, the fly has a much shorter lifespan, and the expanded repeat disorders generally do not occur in humans until later in life. Therefore, care must be taken when extrapolating results from Drosophila to human.

In contrast with our results, two groups have recently reported RNA-mediated neurodegeneration in Drosophila. As discussed earlier, a Drosophila model of FXTAS has been generated, in which rCGG repeats mediate neurodegeneration. A surprising aspect of this model is the presence of aggregates containing Hsp70 and the ability of Hsp70 overexpression to decrease severity (26Go). A similar decrease in severity is also seen when Hsp70 is overexpressed in a Drosophila model of polyglutamine disease (43Go). As Hsp70 is a molecular chaperone, with a central function in protein folding (44Go), this decrease in severity was suggested to imply that misfolding of the polyglutamine-containing protein is central to pathogenesis. However, in FXTAS, no altered protein sequence or expanded polyglutamine is present, making the role of Hsp70 unclear. Nonetheless, it does provide a possible link between polyglutamine disease and RNA-mediated neurodegeneration, as Hsp70 can clearly modulate pathogenesis in both cases, but whether this modulation implies a specific role in pathogenesis, and therefore a common pathway shared by both types of disorders, remains to be determined.

In addition to these results, a Drosophila model of SCA8 has also been recently generated. SCA8 is caused by the expansion of a CTG repeat, and it is unique in that the repeat occurs in a predicted non-coding RNA. When the SCA8 RNA containing 112 CUG repeats was expressed in the Drosophila eye, progressive neurodegeneration was observed (45Go). Similar neurodegeneration was observed when the SCA8 transcript contained nine CUG repeats, in the normal range, making it difficult to determine whether the degeneration is due to the repeats or rather some property of the SCA8 transcript itself. However, when the SCA8(CTG)112 phenotype was used to conduct a screen for genetic interactions, genes were identified which exhibited length-dependent interactions with SCA8(CTG)112 and SCA8(CTG)9 (45Go). One of these interactors was Muscleblind, which is implicated in myotonic dystrophy pathogenesis and has been shown to bind to CUG or CCUG repeats in the DM1 and DM2 transcripts. This finding demonstrates that there are apparent parallels between myotonic dystrophy, which is RNA-mediated, and SCA8, further strengthening the case for RNA-mediated SCA8 pathogenesis. Furthermore, this SCA8 model and the FXTAS Drosophila model demonstrate that the pathways involved in RNA-mediated neurodegeneration are present in Drosophila. Therefore, the lack of phenotype we observed upon the expression of rCAG repeats is not due to the Drosophila system missing some components of the pathways involved in RNA-mediated degeneration, but instead suggests that rCAG repeats are not toxic in the manner that rCGG and possibly rCUG repeats appear to be. This may be due to an intrinsic property of the repeat itself, although it is able to form a hairpin secondary structure (29Go) like the CUG repeat (18Go), or alternatively it could be due to the context in which the repeat occurs, with surrounding RNA sequences necessary for toxicity in some way.

In conclusion, it seems likely that the true pathogenic agent in polyglutamine disorders is indeed polyglutamine, an assumption which had largely been accepted, but until now remained unproved. However, it also seems likely that RNA will be uncovered as the pathogenic agent in a growing number of diseases including those caused by the expansion of untranslated repeats. It will be interesting to see whether there is a point of convergence between the otherwise distinct mechanisms of pathogenesis in these two closely related groups of disorders.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of repeat constructs
Repeats were generated by ligating together shorter repeat oligonucleotides and expanded using PCR-based techniques (46Go). These were subcloned into the Drosophila transformation vector pUAST (34Go), which had been modified to contain the amino acid sequence surrounding the repeat, including the myc and flag epitope tags, which has been described previously (33Go).

Fly stocks and crosses
The length and integrity of repeat constructs were confirmed by DNA sequencing and then microinjected into a w1118 strain by standard methods to obtain germ line transformants. Multiple transgenic lines were obtained for each construct, and the repeat length from each line was confirmed by PCR and sequencing. The GMR-GAL4 line used in this study was obtained from the Bloomington Drosophila stock centre (Indiana University, Indiana, PA, USA). For all figures, the expression of UAS repeat constructs is driven by GMR-GAL4, represented by GMR>repeat. Flies were maintained on cornmeal medium and all crosses were performed at 25°. For light microscopy and cryosectioning, all flies were 24–48 h old.

Cryosectioning and antibody staining
Cryosections 10 µm thick of whole Drosophila heads were cut using a Leipzig 1500 cryostat. They were fixed in 4% formaldehyde and stained using a 9E10 monoclonal anti-myc antibody (47Go) obtained from Dr David Lawrence, IMVS, Adelaide, Australia at 1 : 250 dilution. The secondary antibody used was goat anti-mouse IgG conjugated with Alexa488 (Rockland), at 1 : 300 dilution. Hoechst 33258 (Sigma) was used at 10 µg/ml to stain DNA.

Quantitative PCR
To test the expression levels of transgenes, heads of flies 0–24 h old were collected. Trizol (Invitrogen) and the RN easy mini kit (Qiagen) was used to isolate total RNA, which was then treated with DNAse I (Invitrogen) and reverse-transcribed with oligo(dT)12–18 and SuperScript II (Invitrogen). Real-time PCR was carried out in a LightCycler (Roche Molecular Biochemicals) using primers specific for the repeat-containing transcript and GAL4-specific primers (forward: 5'-CACTGACCCCGTCTGCTTTG-3', and reverse: 5'-GGTTCGGACCGTTGCTACTG-3'). The transgene expression level was quantified using the standard curve method for relative quantitation and expressed relative to the level of GAL4 transcript. The expression level of the GMR-GAL4; UAS(CAG)99 line which shows an eye phenotype was arbitrarily set at 1, and levels of other lines examined are expressed as the fold difference relative to this line.


   ACKNOWLEDGEMENTS
 
L.V.O. is the recipient of a Peter Doherty Post-doctoral Research Fellowship (207830) from the National Health and Medical Research Council, Australia. C.J.M. is the recipient of an Australian Postgraduate Award. The authors would like to thank V. Evci for technical assistance and members of the Richards lab for their constructive comments on drafts of this manuscript. This work was supported by a B3 grant from the Faculty of Health Sciences, Adelaide University, Australia to R.I.R. and by infrastructure support from the ARC Special Research Centre for the Molecular Genetics of Development.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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