Identification of the molecular pathway(s) from mutation to clinical symptoms in the dominantly inherited neurodegenerative diseases has proved extremely difficult. A contributing factor to this is that the sensitive cells of affected individuals are lost in the course of the disease. Animal models therefore afford the opportunity to access cells in which the pathogenic pathways are active and also to explore alternative hypotheses as to the nature of the pathogenic agent(s) responsible for these diseases. RNA is such a potential pathogen in the dominantly inherited expanded repeat neurodegenerative diseases. The use of animal models such as Drosophila enables the identification of pathways through which such potential pathogens act and the identification of biomarkers of the responsible pathways. These biomarkers can subsequently be tested in the respective human diseases to validate the role of the pathway and its contribution to the disease. Using this approach, we have modelled repeat RNA pathology in Drosophila and have identified common pathways perturbed by the expression of expanded repeat RNAs.
Analyses of transcriptional changes in a number of models of expanded repeat disease have previously been reported (34
). These studies have largely modelled toxicity of polyglutamine, which induces severe, early phenotypes in both mouse and Drosophila
models, and therefore transcriptional changes are likely to partially reflect downstream effects of cell death. More recently, evidence for a role of RNA-mediated pathogenesis in the polyglutamine diseases has been reported (18
). Since expression of each of the repeat sequences as untranslated RNA either in the Drosophila
eye or throughout the nervous system does not result in gross developmental or degenerative phenotypes, this model can be used to investigate markers of cellular dysfunction attributable to these repeat sequence RNAs which precede cell death and are therefore more likely to represent causative changes in disease progression.
Given the ability of all of the disease-associated repeat sequences to form hairpin secondary structures at the RNA level and the phenotypic overlap seen in the expanded repeat diseases, despite the presence of the repeat tracts within unrelated genes, we predicted that there are likely to be common, intrinsic, sequence-independent cellular effects of expression of expanded repeat sequences. In support of this prediction, pan-neuronal expression of rCAG, rCUG and rAUUCU expanded repeat RNAs was found to elicit a number of common transcriptional changes. Strikingly, a comparison of transcripts showing altered expression in flies expressing rAUUCU repeat RNA revealed a minimum of 40.7% and maximum of 71.4% overlap with genes altered in flies expressing either rCAG or rCUG repeats (Fig. E). This result is strongly suggestive of common mechanisms of toxicity of expanded repeat RNAs.
In the untranslated expanded repeat diseases where there is no toxic peptide expressed, RNA-mediated pathogenesis is presumably sufficient to induce all of the cellular changes leading to neurodegeneration and disease progression. This Drosophila
model of RNA repeat pathogenesis investigates some components of pathogenesis in these diseases, but there are also likely to be specific effects of expression of the repeat-containing transcript in each disease which are dependent on the context of the repeat tract. Nevertheless, at least one candidate which showed a strong interaction with context-independent repeat RNAs used in this study, mod(mdg4)
, has been previously identified as transcriptionally altered in another Drosophila
model which used repeats within the context of the SCA8 transcript (24
In addition, changes identified in these microarray analyses support a role for more generalized transcriptional dysregulation in toxicity of expanded repeat RNAs. In particular, altered transcript levels of histones (H3 and H1), histone acetylating enzymes (msl-2
), chromatin modifiers (mod(mdg4)
), a number of transcription factors (including mef2
, a member of the SP1/SP3-like transcription factor family and a Drosophila
orthologue of PAX5) and transcriptional co-regulators (tna
) were detected in flies expressing more than one of the repeat sequences (Supplementary Material, Tables S1 and S2
). This is consistent with observations in several models of polyglutamine pathogenesis in which wide-spread transcriptional dysregulation has been reported (35
) and suggests that this sort of effect may be an intrinsic property of expanded repeat sequences.
Examination of common transcriptional changes detected in this model also revealed changes to a number of other components of the cell that have been previously implicated in polyglutamine pathogenesis, including several cellular transport and cytoskeletal components. For example, the actin-binding proteins hu li tai shao
, which is an orthologue of mammalian Adducin 1, and cut up
, a component of the dynein complex, both showed altered expression in flies expressing more than one of the expanded repeat sequences (Fig. F). Hu li tai shao
has been previously demonstrated to suppress a phenotype associated with expression of an expanded N-terminal fragment of Huntingtin in the Drosophila
), while cut up
and its human orthologue, DYNLL1
, showed altered expression in a comparison of Drosophila
and human cell culture models of polyglutamine pathogenesis (37
). We therefore predict that some of the pathogenic pathways previously identified in models of expanded repeat disease may be common to both polyglutamine and untranslated repeat diseases and therefore some of these effects may be at least partially mediated through RNA pathogenesis.
Recently published data (42
) demonstrate that expanded CAG repeat alleles are able to be translated internally in all three reading frames, irrespective of whether or not they are located in coding regions and without requiring an initiation AUG, through a mechanism known as repeat-associated non-ATG translation (RAN translation). It is thought that the hairpin structure formed by the expanded repeat RNAs is acting as an Internal Ribosome Entry Site (IRES). In our model, expression of up to four transgene insertions of untranslated CAG and CUG repeat sequences does not result in a phenotype, while expression of a single polyglutamine or polyleucine-encoding transgene is sufficient to induce a visible phenotype in the Drosophila
eye (Fig. ; 8
). Therefore, RAN translation does not appear to play a major role in RNA toxicity in this model. However, as a consequence of these recent findings, homopolymeric amino acid sequences have emerged as a potential mediator of repeat RNA toxicity in the ‘untranslated’ repeat diseases.
Altered transcription of components of the Akt/GSK3-β regulatory pathway was consistently observed in flies expressing rCAG, rCUG and rAUUCU repeat RNAs by microarray analysis, suggesting that this is a key component of cellular dysfunction in this Drosophila
model of untranslated repeat disease pathogenesis. While the ability of CUG repeat RNA to disrupt Akt/GSK3-β signalling has been described, this is the first evidence that expression of other hairpin-forming RNA species can also influence activity of this pathway. The initial stimulus resulting in the disruption of Akt/GSK3-β signalling in this model is unclear; however, there is precedent for similar effects in fragile X syndrome where increased levels of stimulation of the mGluR5 receptor have been demonstrated to increase GSK3-β activity (43
). A disruption to mGluR5 signalling has also been described in a pre-symptomatic model of HD (44
), and in other HD models alterations to N
-aspartate receptor (45
), brain-derived neurotrophic factor (46
) and nerve growth factor (48
) signalling, all of which are associated with activation of the Akt/GSK3-β pathway, have also been observed. Our observations indicate that expression of expanded repeat RNA is sufficient to cause transcriptional changes to the Akt/GSK3-β pathway, and therefore that the hairpin RNAs expressed in the disease situation might also interact with components of this pathway to disrupt normal signalling.