Sense and antisense RNA are not toxic in Drosophila models of C9orf72-associated ALS/FTD

Abstract

A GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Neurodegeneration may occur via transcription of the repeats into inherently toxic repetitive sense and antisense RNA species, or via repeat-associated non-ATG initiated translation (RANT) of sense and antisense RNA into toxic dipeptide repeat proteins. We have previously demonstrated that regular interspersion of repeat RNA with stop codons prevents RANT (RNA-only models), allowing us to study the role of repeat RNA in isolation. Here we have created novel RNA-only Drosophila models, including the first models of antisense repeat toxicity, and flies expressing extremely large repeats, within the range observed in patients. We generated flies expressing ~ 100 repeat sense or antisense RNA either as part of a processed polyadenylated transcript or intronic sequence. We additionally created Drosophila expressing > 1000 RNA-only repeats in the sense direction. When expressed in adult Drosophila neurons polyadenylated repeat RNA is largely cytoplasmic in localisation, whilst intronic repeat RNA forms intranuclear RNA foci, as does > 1000 repeat RNA, thus allowing us to investigate both nuclear and cytoplasmic RNA toxicity. We confirmed that these RNA foci are capable of sequestering endogenous Drosophila RNA-binding proteins, and that the production of dipeptide proteins (poly-glycine-proline, and poly-glycine-arginine) is suppressed in our models. We find that neither cytoplasmic nor nuclear sense or antisense RNA are toxic when expressed in adult Drosophila neurons, suggesting they have a limited role in disease pathogenesis.

Keywords: ALS; C9orf72; Drosophila; FTD; Repeat expansion.

Fig. 1

Expression of C9orf72 sense RNA-only repeats is not strongly toxic to Drosophila neurons. a Diagram of the constructs generated. RO repeats were either cloned directly into the vector, forming part of a polyadenylated transcript (PolyA) or cloned into an intron within the eGFP coding sequence (Intronic). 5× Gal4 DNA binding domain (G4BD), heat-shock promoter (Prom), late SV40 termination and polyadenylation sequence (SV40/PolyA). b Fluorescence in situ hybridisation was performed to assess foci formation (GGGGCC, red). Expression was induced in adult Drosophila neurons using the elavGS driver, leading to the formation of largely cytoplasmic RNA puncta in Sense-PolyA-1 flies, and intranuclear RNA foci in Sense-Intronic-1 flies. Scale bar 2.5 μm. c Analysis of lifespan of Sense-PolyA-1 and Sense-Intronic-1 flies fed RU486 to induce expression (+RU) or controls where expression was not induced (−RU). In both cases a significant lifespan extension was observed upon transgene expression (Sense-PolyA-1, median lifespan −RU = 83.5 days +RU = 89 days, P = 6.91E−5, log rank test; Sense-Intronic-1, median lifespan −RU = 79 days +RU = 89 days, P = 9.76E−19, log rank test). d Negative geotaxis assays performed on Sense-PolyA-1 and Sense-Intronic-1 flies expressing the transgene (+RU) and controls (−RU). A slight reduction in climbing ability is observed in Sense-PolyA-1 flies (ordinal logistic regression, interaction of RU status and time P = 0.00025), whilst no significant effect is seen in Sense-Intronic-1 flies P = 0.47). Error bars are ± SEM. Genotypes: w; UAS-Sense-PolyA-1/+; elavGS/+ (Sense-PolyA-1), w; UAS-Sense-Intronic-1/+; elavGS/+ (Sense-Intronic-1)

Fig. 2

Expression of ~ 1000 RNA-only repeats or greater causes the production of large numbers of RNA foci but does not induce strong toxicity when expressed in adult Drosophila neurons. a Fluorescence in situ hybridisation against sense RNA foci reveals abundant RNA foci are present in all lines expressing ~ 1000 RO repeats (Sense-800 PolyA and Sense-1000 PolyA) and Sense > 1000 RO repeats (Sense > 1000 PolyA). Scale bar 2.5 μm. b Quantification of the % of foci containing nuclei (%foci+ nuclei) within each line. No foci+ nuclei are detected in driver alone (elavGS/+) and very few observed in transgene without the driver (Sense-800 PolyA/+) lines. A linear model was fitted to the data (effect of genotype P < 0.0001), and comparisons between groups made using orthogonal contrasts (all contrasts shown in Online Resource Table 1). A significantly higher proportion of nuclei were foci+ in ~ 100 repeat Sense-Intronic flies vs. ~ 100 repeat PolyA flies (**P = 0.0071). A significantly higher proportion of foci+ nuclei were observed in 800 to > 1000 repeat-expressing flies compared to ~ 100 repeat-expressing flies (***P < 0.0001). Fewer foci were observed in > 1000-repeat-expressing flies vs. 800–1000 repeat (**P = 0.0039). 3–4 brains per genotype were examined. Bars are mean ± SEM. c Lifespans of flies expressing long sense constructs (+RU) vs. controls (−RU) using the elavGS driver. Significant lifespan extensions are observed in Sense-800 PolyA (median lifespan −RU = 83.5 days +RU = 90.5 days, P = 4.07E−10 log rank test) and Sense > 1000 PolyA-expressing flies (median lifespan −RU = 86.5 days +RU = 90.5 days, P = 4.21E−06 log rank test). Lifespan of Sense-1000 PolyA-expressing flies was not significantly different (median lifespan −RU = 86.5, +RU = 86.5, P = 0.75, log rank test). d Negative geotaxis assays performed on flies expressing the transgene (+RU) and controls (−RU) using the elavGS driver. In Sense-800 PolyA flies, a slight reduction in climbing ability is observed with age vs. controls (ordinal logistic regression, interaction of RU status and time P = 0.0281); however, no significant differences were observed in Sense-1000 PolyA (P = 0.328) or Sense > 1000 PolyA (P = 0.231)-expressing flies. Error bars are ± SEM. Genotypes: w; +; elavGS/+ (elavGS/+), w; UAS-Sense-800 PolyA/+; + (Sense-800 PolyA/+), w; UAS-Sense-PolyA-1/+; elavGS/+ (Sense-PolyA-1), w; UAS-Sense-Intronic-1/+; elavGS/+ (Sense-Intronic-1), w; UAS-Sense-800 PolyA/+; elavGS/+ (Sense-800 PolyA), w; UAS-Sense-1000 PolyA/+; elavGS/+ (Sense-1000 PolyA), w; UAS-Sense > 1000 PolyA/+; elavGS/+ (Sense > 1000 PolyA)

Fig. 3

Antisense RNA forms foci, but does not induce strong toxicity when expressed in Drosophila neurons. a Fluorescence in situ hybridisation was performed to assess antisense foci formation (GGCCCC, green). Expression was induced in adult Drosophila neurons using the elavGS driver, leading to largely cytoplasmic RNA signal in AS-PolyA-1 flies, and predominantly intranuclear RNA foci in AS-Intronic-1 flies. Scale bar 2.5 μm. b Quantification of the % of foci containing nuclei (%foci+ nuclei) within each line. No foci+ nuclei are detected in driver alone (elavGS/+) and very few observed in transgene alone (Sense-800 PolyA-1/+) lines. A linear model was fitted to the data (effect of genotype P < 0.0001), and comparisons between groups made using orthogonal contrasts (all contrasts shown in Online Resource Table 2). A significantly higher proportion of nuclei were foci+ in AS-Intronic flies vs. AS-PolyA flies (***P < 0.0001). 2–4 brains per genotype were examined. Bars are mean ± SEM. c Lifespans of flies expressing antisense constructs (+RU) vs. controls (−RU) using the elavGS driver. A significant extension of lifespan is observed in AS-PolyA-1 (median lifespan −RU = 89 days +RU = 93.5 days, P = 5.20E−8 log rank test) or AS-Intronic-1 flies (median lifespan −RU = 75.0 days +RU = 82.5 days, P = 4.92E−8 log rank test). d Negative geotaxis assays performed on AS-PolyA-1 and AS-Intronic-1 flies expressing the transgene (+RU) and controls (−RU) using the elavGS driver. In AS-PolyA-1 flies, no significant difference in climbing ability is observed with age vs. controls (ordinal logistic regression, interaction of RU status and time P = 0.988), or AS-Intronic-1-expressing flies (P = 0.439). Error bars are ± SEM. Genotypes: w; +; elavGS/+ (elavGS/+), w; UAS-Sense-800 PolyA/+; + (Sense-800 PolyA/+), UAS-AS-polyA-1/+; elavGS/+ (AS-PolyA-1), UAS-AS-Intronic-1/+; elavGS/+ (AS-Intronic-1)

Fig. 4

Glorund colocalises with sense RNA foci in adult Drosophila neurons. Fluorescence in situ hybridisation against sense foci (GGGGCC, red) was coupled with immunofluorescence against glorund (green). Bright puncta of glorund staining were found to colocalise with sense RNA foci (examples of colocalising puncta shown by white arrowheads). An average of 14.8% of sense RNA foci was found to colocalise with glorund puncta (± 4.26% SEM, based on 167 foci scored across 3 separate brains). Scale bar 5 μm. Genotype: w; UAS-Sense-800 PolyA/+; elavGS/+

Fig. 5

RANT is suppressed in RNA-only flies. a Assessment of poly-GP expression. Heads of flies induced on RU486 for 7 days (+RU) and controls (−RU) were lysed and dipeptide protein concentration measured by immunoassay. Poly-GP is detectable in significantly higher abundance in 36R-expressing flies compared to non-induced controls (***P = 0.0002, two-tailed t test, +RU n = 2, −RU n = 3). Additionally a significantly higher level of poly-GP was observed in lines Sense-PolyA-1 (****P < 0.0001, two-tailed t test, +RU n = 4, −RU n = 4), AS-PolyA-1 (**P = 0.0017, Welch’s two-tailed t test, +RU n = 4, −RU n = 3), Sense-Intronic-1 (****P < 0.0001, two-tailed t test, both conditions n = 3), and Sense-800 PolyA (**P = 0.0036, two-tailed t test, both conditions n = 3). No differences in poly-GP levels were observed in lines expressing AS-Intronic-1, Sense-1000 PolyA and Sense > 1000 PolyA vs. controls (Sense > 1000 PolyA and AS-Intronic-1 +RU n = 4, n = 3 for other conditions). b Assessment of poly-GR expression. Poly-GR expression was assessed by immunoassay. Poly-GR was significantly higher in 36R-expressing flies vs. −RU control (**P = 0.0019, Welch’s two-tailed t test, +RU n = 4, −RU n = 3). No significant difference in poly-GR was observed in Sense-PolyA-1 (P = 0.4006, Welch’s two-tailed t test, +RU n = 4, −RU n = 3), Sense-Intronic-1 (P = 0.4226, Welch’s two-tailed t test, both conditions n = 3) or Sense-800 PolyA (P = 0.4226, Welch’s two-tailed t test, +RU n = 4, −RU n = 3). Bars are Mean ± SEM, individual replicates are shown as circles. Genotypes: w; UAS-Sense-PolyA-1/+; elavGS/+ (Sense-PolyA-1), w; UAS-Sense-Intronic-1/+; elavGS/+ (Sense-Intronic-1), w; UAS-Sense-800 PolyA/+; elavGS/+ (Sense-800 PolyA), w; UAS-Sense-1000 PolyA/+; elavGS/+ (Sense-1000 PolyA), w; UAS-Sense > 1000 PolyA/+; elavGS/+ (Sense > 1000 PolyA), w; UAS-AS-PolyA-1/+; elavGS/+ (AS-PolyA-1), w; UAS-AS-Intronic-1/+; elavGS/+ (AS-Intronic-1), w; UAS-36R/+; elavGS/+ (36R)