Silencing of CG3808, the Drosophila orthologue of TRMT2A, showed promising results in ameliorating the detrimental effects of elongated SCA3 protein. Thus, we addressed the question whether CG3808 knockdown would also prove beneficial during more detailed analysis and in other polyQ models apart from SCA3tr-Q78. Firstly, we assessed the capability of the RNAi to overcome lethality induced by pan-neural expression of SCA3tr-Q78. Indeed, co-expression of shRNA and SCA3tr-Q78 in all neural cells resulted in viable progeny with no overt abnormalities. Additionally, ubiquitous silencing of CG3808 by the means of RNAi did not render the offspring fatal and had no negative influence on overall life time of the respective flies, demonstrating that the protein is not of vital importance in Drosophila (data not shown).
For evaluation of CG3808 RNAi effects we again utilised histological and biochemical methods together with assessment of longevity. Finally, we transferred experiments to a cellular model in an attempt to verify the progress accomplished in Drosophila in a mammalian model.
5.5.1 Impact of TMRT2A silencing on polyglutamine-induced REPs
Expression of SCA3tr-Q78 in the compound eye led to a rough eye phenotype visible with light (Figure 16A) as well as scanning electron microscopy (SEM, Figure 16B). The SEM findings are in line with results obtained from REP pictures, namely the eyes presenting with heavily disarranged surface and even collapsed eye morphology probably due to underlying tissue degeneration. Silencing of CG3808 by RNAi led to amelioration of SCA3tr-Q78-induced REP with restored patterning and morphology of the exterior eye
surface. For another polyQ fly model, inducing a REP with exon 1 of the huntingtin gene under GMR control, similar observations were made. This htt transgene contains 97 glutamine repeats (w[*];P{w[+]=UAS-Q97ex1}K6,9,15R) and downregulation of CG3808 expression was sufficient to rescue the Htt-induced REP (Figure 16C, D), reversing the phenotype to almost wild type (Figure 16H). This was underpinned by SEM analysis, exhibiting a predominantly ordered surface without signs of degeneration (Figure 16I).
Introduction of shRNA against CG3808 into a model for SCA1 with full-length ATXN1 Q82 expression (y[1]w[118] P{[+]=UAS-SCA1.82Q}[F7], Figure 16E) yielded improvement of the degenerative eye phenotype to great extend as well (Figure 16J).
Concluding, knockdown of CG3808 expression is obviously capable of exerting neuroprotective effects in the course of eye degeneration caused by several different polyQ proteins.
Figure 16. Rescue of polyQ-induced REP by shRNA against CG3808.
Elongated polyQ proteins responsible for SCA3 (A, B), HD (C, D) and SCA1 (E) induced an REP in flies visible by light and scanning electron microscopy. Induction of shRNA directed against CG3808 transcripts mitigates this REP to almost wild type situation (F-J).
All scale bars apply to 200 µm.
5.5.2 Evaluation of photoreceptor integrity of polyQ flies with TRMT2A knockdown
Semi-thin sagittal sections of fly eyes expressing variants of Ataxin-1 and Ataxin-3 under control of GMR-GAL4 (GMR_SCA1 Q82 and GMR_SCA3tr-Q78 respectively) display severe degeneration of photoreceptor neurons as a consequence of polyQ neurotoxicity.
Co-expression of shRNA against CG3808 on the contrary was capable of ameliorating the detrimental effects in the eye (Figure 17A). Quantification of photoreceptor neurons per ommatidium showed a severely decreased PR count in GMR_SCA3tr-Q78 (1.16 ± 0.06) flies and a moderately decreased one (4.24 ± 0.27) in the GMR_SCA1Q82 model. Silencing of CG3808 rescued PR degeneration almost to the level of GMR control conditions (6.85 ± 0.03) in GMR_SCA3tr-Q78 (6.31 ± 0.09) and GMR_SCA1 Q82(6.64 ± 0.05) models (Figure 17B). Additionally, stereotypic patterning in the ommatidia was visible again in GMR_SCA3tr-Q78 flies in combination with CG3808 silencing. These results could be recapitulated also in the flies expressing the elongated full-length form of Ataxin-1 (GMR>SCA1 Q82, Figure 17A) and at least qualitatively with a transgene of exon 1 of HTT with 97 glutamine repeats (GMR_HTT Exon1 Q97, not shown). Therefore, silencing of CG3808 seems to have a strong neuroprotective effect opposing polyQ toxicity in the Drosophila eye.
Figure 17. Evaluation of photoreceptor integrity in polyQ flies with CG3808 RNAi.
(A) Depiction of PR neuron degeneration in polyQ models for SCA3 and SCA1 (upper panel) and rescue of number and patterning of PRs by silencing of CG3808 via RNAi (lower panel). (B) Quantification of PR number in SCA3 and SCA1 fly models compared to GMR control. Significant PR loss was rescued to a great extend by expression of shRNA against CG3808.
All scale bars in (A) apply to 50 µm respectively. Kruskal-Wallis test with Dunn’s Multiple Comparison test was used for statistics in (B), significant changes are: *** p < 0.001; n.s., not significant.
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5.5.3 Assessment of adult-onset polyQ fly longevity
In order to more closely mimic the disease situation in humans with late onset and progressive degeneration, further experiments in a pan-neural adult-onset model for polyQ diseases were performed. Therefore an elav-GAL4 fly strain with additional ubiquitous expression of the temperature-sensitive yeast transcriptional repressor GAL80ts (P{w[+mW.hs]=GawB}elav[C155]; P{w[+mC]=tubP-GAL80[ts]}20) [285] (referred to as elav-GAL80 in the text) was used. elav-GAL80 is a competitor of GAL4 in binding to the UAS without activating properties, thereby preventing subsequent induction of gene expression at permissive temperature (≤ 20 °C). Upon shifting to restrictive temperature (≥ 25 °C), GAL80 is unfolded, which prevents blockage of GAL4, consequently allowing GAL4-UAS binding and gene expression. Protein aggregation analysis and longevity experiments were performed making use of this system, facilitating pan-neural expression of SCA3tr-Q78 only following temperature shift from 18 °C to 29 °C.
Induction of polyQ expression could be shown in Western blot experiments climaxing four days post temperature shift and declining afterwards probably due to cell demise (Figure 18A). Aggregation of truncated Ataxin-3 was shown to be absent before induction of SCA3tr-Q78 expression on permissive temperature and to increase rapidly within a timeframe of 7 days after temperature shift (Figure 18B, C).
Overall lifetime of polyQ-expressing flies is a feasible tool for evaluation of toxicity and neurodegeneration. SCA3tr-Q78 flies showed no abnormalities at restrictive temperature due to absent toxic protein expression. However, locomotive abilities of the flies deteriorated fast after induction of expression concomitantly with a rapid decline in survival time resulting in a median survival time (timepoint when 50 % of flies of overall flies are still alive) of only 10 days. Flies with adult-onset expression of a non-toxic control transgene (P{w[+mC]=UAS-eGFP}) had an almost three times longer mean survival (27 days). Eventually, co-expression of SCA3tr-Q78 and shRNA against CG3808 increased median survival significantly to about 18 days. Therefore, silencing of this methyl transferase proved to be beneficial in alleviating detrimental polyQ effects on longevity, despite limitations compared to non-polyQ transgene expression.
5.5.4 Influence of CG3808 downregulation on aggregate formation in Drosophila
As already demonstrated, targeting of SCA3tr-Q78 expression to the eye leads to aggregate formation and gives rise to inclusion bodies of elongated polyQ protein and eye degeneration. By co-expression of shRNA against CG3808 the anti-aggregation properties of this gene knockdown could be shown in situ and in filter retardation analysis.
Paraffin frontal fly head sections were probed with an antibody directed against the HA-tag of the polyQ protein. It could be observed that induction of CG3808 RNAi is capable Figure 18. Adult-onset model of SCA3tr-Q78 in Drosophila and extension of polyQ fly life time by CG3808 RNAi.
(A) Protein levels of truncated Ataxin-3 in adult-onset fly model are detectable one day post induction (dpi) by temperature shift and increase until 4 dpi. At 7 dpi, levels have already declined. (B, C) Aggregate load of SCA3tr-Q78 in adult-onset fly heads increases steadily after induction over a course of 7 days. (D) Expression of shRNA against CG3808 is sufficient to significantly prolong median survival and overall lifetime of pan-neural adult-onset SCA3tr-Q78 flies, although not to control levels (eGFP).
Log-rank test was used for statistics in (D), significant changes are: **** p < 0.0001.
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B C
D
of preventing assembly of inclusion bodies in the compound eye retina, concomitantly preserving the structure and architecture of the tissue to great extend. Additionally, aggregation of elongated full-length Ataxin-1 in the retina and impact of CG3808 induction thereon was estimated. Ataxin-1 did not show pronounced formation of inclusion, yet rather was localised to the nucleus. CG3808 RNAi did not feature an obvious change of Ataxin-1 distribution or amount, however, retinal structure appeared improved (Figure 19A).
Figure 19. Overview of anti-aggregation effects of CG3808 RNAi in different polyQ models and settings.
(A) Induction of CG3808 RNAi leads to a prominent decrease of inclusion number in the retina of SCA3 model flies (upper row). Ataxin-1 protein does not seem to form inclusion in SCA1 flies and CG3808 RNAi does not influence distribution or protein amount in the retina. (B) Adult-onset co-expression of CG3808 shRNA with SCA3tr-Q78 ameliorates aggregate load in fly head lysates also compared to an RNAi control (white shRNA). (C) Quantification of aggregate load in adult-onset SCA3 flies after introduction of control and CG3808 shRNA.
Scale bar in (A) applies to 50 µm. Red in (A), F-actin stained with Alexa Fluor® 568-linked phalloidin; green in (A) upper row, SCA3tr-Q78 stained with mouse anti-HA antibody; lower row, SCA1 Q82 stained with mouse anti-polyQ antibody. t-test was used for statistics in (C), significant changes are: * p < 0.05; ** p < 0.01; n.s., not significant.
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The potent aggregate-reducing capacity of CG3808 downregulation has already been shown for eye-expressed polyQ protein (see chapter 5.3.2, Figure 14 B). Nevertheless, expression of SCA3tr-Q78 and of CG3808 shRNA under GMR control does not reflect the pathogenic situation in humans with respect to late disease onset. Therefore, elav-GAL80 fly strains for pan-neural adult-onset expression were utilised. Induction of polyQ expression alone by temperature shift produced a significant increase in SDS-insoluble aggregates five days post induction as detected by filter retardation assay (Figure 19B, C).
Introduction of a control shRNA against white gene expression also showed a significant rise in aggregate load, whereas the moderate increase in flies expressing both polyQ and CG3808 RNAi was not statistically significant (Figure 19C). From these findings one can conclude that silencing of CG3808 expression is capable of decelerating the formation and/or accumulation of potentially toxic polyQ aggregates.