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

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. Electronic supplementary material The online version of this article (10.1007/s00401-017-1798-3) contains supplementary material, which is available to authorized users.


Southern blotting
DNA from approximately 30 female Drosophila was extracted using the Qiagen DNeasy Blood and Tissue Kit following manufacturer's instructions, before being incubated with 20µg/ml RNase A (Qiagen) for 1 hour at 37ºC. 40µg of genomic DNA was digested overnight at 37ºC with 20 units of DdeI and AluI (NEB). ~1152RO samples were run on a 0.8% agarose gel at 60V overnight, whilst ~100RO samples were run on a 1% gel for 6 hours. The gel was agitated for 30 minutes in 1L of denaturation buffer (National Diagnostics) followed by 1L of neutralisation buffer (National Diagnostics). DNA was transferred onto a positively charged nylon membrane (Roche Applied Science) via capillary action overnight using 20X SSC (saline sodium citrate, Fisher) as transfer buffer. Following transfer, DNA was baked onto the nylon membrane at 80ºC for 2 hours. Membrane was briefly wetted in ddH2O before being incubated in DIG easy Hyb buffer (Roche) with 100µg/ml salmon sperm DNA (Thermo) at 48ºC for 4 hours. It was then transferred into fresh Dig easy hyb buffer with salmon sperm and 1µg/ml of 5'-DIG-(GGGGCC) 5 -DIG-3' probe (produced by Eurofins genomics) overnight at 48ºC. The blot was washed in 2xSSC with 0.1% SDS (sodium doecyl sulphate) while ramping in temperature from 48ºC-65ºC, followed by pre-warmed 0.5xSSC with 0.1% SDS at 65ºC for 15 minutes, and then 0.2xSSC w/ 0.1% SDS for 15 minutes at 65ºC. Following this, the blot was processed using the DIG wash and block buffer set (Roche) following manufacturer's instructions. The membrane was incubated in CDPstar ready to use (Roche) before being exposed to film (Roche Lumi-Film chemiluminescent detection film) for >1 hour before being developed using an X4 automatic processor (XOgraph). Due to technical issues with probe synthesis, some blots were incubated with 5'-DIG-(CCCCGG) 5 -DIG-3' (antisense) probe.

Eye GFP fluorescence
Flies were reared at 25°C. Five days after eclosion heads were mounted on glass slides and imaged using a Zeiss Axioskop 2 plus microscope using the same settings for each image.

Eye phenotyping and eclosion experiments
3 GMR-Gal4 virgins were crossed to males of the indicated genotypes on SYA food, 5-6 vials per genotype were set up. Females were allowed to lay for 24 hours and eggs left at 29°C to hatch and eclose. Eye phenotypes were scored 24 hours after eclosion, based on a 5-point grading scale: 1= no rough eye, 2= slight rough eye, 3= moderate rough eye, 4=severe rough eye, 5= very severe / no eye. A minimum of 3-5 eyes were scored per sex per vial (n=21-30 per sex, per genotype). The strong eclosion effect in some vials meant that for GR36 flies less flies were scored (n=10-28 per sex, per experiment). Representative images of eyes were taken 24-48hr after eclosion using a Leica M165 C microscope by taking serial images at different depths and reconstructing the final image using the Leica application suite software. 72 hours after eclosion, the number of pupal cases and the number of adult flies were counted and used to calculate the % of adult flies that had eclosed successfully.
Experimenter was blind to genotype when scoring both assays.
Immunoblotting 24hr after eclosion the retinas of female flies were removed in ice cold PBS, 10 retinas per replicate were transferred to 40µl of 2x laemmli sample buffer (BioRad) with 100mM DTT.
Tissue was homogenised before being boiled at 95°C for 10 minutes. 20µl of sample was loaded per well in a 10 well 4-12% Novex Bis-Tris gel (Invitrogen). Protein was transferred to a nitrocellulose membrane using the Trans-Blot Turbo transfer system (BioRad) and blocked in 2% skimmed milk in TBS for 1 hour at room temperature. Anti-Glorund (5B9, DSHB) was applied at 1/750 concentration in block overnight at 4°C. The blot was washed in TBS-(0.1%) Tween and incubated in anti-mouse-HRP (Abcam, ab6789) at 1/10,000 in block. Glorund was visualised using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo).

Fig. 8
Southern blots confirm insertion size in transgenic ~1000 repeat Drosophila. Southern blots were performed using genomic DNA derived from multiple independent transgenic lines (indicated by tick marks). Insertions were sized by comparison to two DNA ladders (L1 and L2, molecular weight shown in base pairs). a Two lines Sense-800-PolyA and Sense-1000-PolyA carry expansions approximately equivalent to expected size (~800 repeats, and ~1000 repeats respectively, expected band size=7768bp). Some lines were observed to reproducibly carry expansions larger than expected. One of these lines Sense>1000 PolyA was selected for further characterisation (estimated repeat length ~2000-5000). b Southern blot performed on DNA extracted from flies derived from the same cross used in climbing and negative geotaxis assays (genotypes as in Fig. 2). Results indicate that flies carry large expansions equivalent to ~650 repeats, ~800 repeats and >1000 repeats in lines Sense-800-PolyA, Sense-1000-PolyA and Sense>1000 PolyA respectively, demonstrating that repeat lengths had not substantially retracted. Antisense PolyA Antisense Intronic L 2 L 1 L 2 L 1 Fig. 11 Partial Glorund loss of function does not induce toxicity a An RNAi line against Glorund was crossed to the GMR-Gal4 driver and adult retinas subjected to immunoblotting to confirm knockdown of Glorund protein relative to the heterozygous driver alone (GMR/+). Representative blot, and graph depicting relative protein expression are shown. Glorund expression was significantly reduced by RNAi compared to controls (two-tailed Welch's t test, *P=0.0434). Bars are mean ±SEM (n=3 replicates per condition) b Graph depicting % of flies that eclosed from pupae for each line. A significant eclosion defect is observed in GR36 expressing positive control flies compared to controls but no other lines (Kruskall-Wallis effect of genotype P=0.0012, post-hoc Dunn's multiple comparisons between GMR/+ and GR36, **P=0.0073). Bars are mean % eclosion per vial ± SEM. c Representative images of eyes of female flies carrying the indicated insertion expressed under the GMR-Gal4 driver. GMR/+ is the heterozygous driver alone. UAS-GR36 was used as a positive control. Scale bar 100µm. d Eye severity scores for females of the indicated lines. A significant rough eye was observed in GR36 expressing flies but not other lines (Kruskall-Wallis test, effect of genotype P=0.0002, with post-hoc Dunn's multiple comparisons between GMR/+ and other lines, *P=0.0020). Bars are mean eye score per vial ± SEM. e Eye severity scores for males of the indicated lines. A significant rough eye was observed in GR36 expressing flies but not other lines (Kruskall-Wallis test, effect of genotype P<0.0001, with post-hoc Dunn's multiple comparisons between GMR/+ and other lines, *P=0.0028). Bars are mean eye score per vial ±SEM. Genotypes: w; GMR-Gal4/+ (GMR/+), w; UAS-GR36/ GMR-Gal4 (GR36), w; GMR-Gal4/+; UAS-Glo RNAi/+ (Glo RNAi).