Tran H, Almeida S, Moore J, Gendron TF, Chalasani U, Lu Y, Du X, Nickerson JA, Petrucelli L, Weng Z, Gao FB. Differential Toxicity of Nuclear RNA Foci versus Dipeptide Repeat Proteins in a Drosophila Model of C9ORF72 FTD/ALS. Neuron. 2015 Sep 23;87(6):1207-14. PubMed.
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Institut Jožef Stefan
This interesting paper takes us through all three major potential disease mechanisms arising from the C9ORF72 mutation. The initial analysis of the expression levels of the three transcript variants of C9ORF72 shows a mutation-associated reduction of variant 2, which is the major transcript of this gene. This gives the haploinsufficiency mechanism a boost in disease relevance.
However, the major focus of the paper is on differentiation between the other two mechanisms—RNA toxicity and DPR toxicity. Through use of the C9 minigene, the authors show that RNA toxicity is not detectable in the fruitfly model system, although there is an abundance of RNA foci. Raising the environmental temperature leads to production of DPRs and toxicity.
The use of the C9ORF72 minigene gets us a step closer to modeling the disease; however, numerous pathology papers show that both RNA and DPRs accumulate mainly in regions that are not that relevant to ALS and FTD. So DPRs and RNA may be toxic in certain models, but the question remains which of the three disease mechanisms occurs in C9 patients.
View all comments by Boris RogeljUniversity College London
This paper provides compelling evidence that DPRs drive toxicity in C9ORF72 repeat fly models. This is consistent with the conclusions from our studies investigating C9ORF72 repeat toxicity in flies.
The authors generated a very nice model by expressing 160 GGGGCC repeats within an intron to avoid polyA addition and export to the cytoplasm. This results in abundant RNA foci, but still no toxicity, clearly implicating DPRs as the toxic species. Therefore I think we can now firmly say that the current generation of repeat fly models cause toxicity through DPRs.
This has important implications for the recent modifier screens performed in flies, as it implies that the modifiers (nucleocytoplasmic transport genes) specifically modify DPR toxicity.
However, we can’t conclude from studies performed in Drosophila that RNA foci play no role in human disease. It is certainly still possible that RNA toxicity (including repeat RNA species other than large foci) contributes to neuronal dysfunction, but clearly more work is required in complementary systems.
View all comments by Adrian IsaacsStanford University
This is a provocative new paper and presents highly important experimental findings of immediate interest to the ALS and FTD field.
The authors use Drosophila to model expression of C9ORF72 mutant transgenes. This is not new (there already have been several papers on fly C9ORF72 models), and the authors' results are negative. However, these negative results are tremendously important and essential for the ALS/FTD community to consider. This is why: The authors generated flies with a transgene harboring 160 GGGGCC repeats embedded within an intron. This transgene was expressed, and spliced, and the GGGGCC repeat formed many sense RNA foci in the nucleus. But there was no neurodegeneration, in contrast to flies produced by other labs (e.g., Isaacs et al., 2014).
A key difference between the Tran et al. flies and the Isaacs ones is the presence of the repeat within the intron. The flies from Isaacss’ paper are made to express the GGGGCC expansion in the context of an mRNA with a 3'UTR, which allows it to be efficiently exported to the cytoplasm. This leads to the production of high levels of RAN-translated dipeptide repeat proteins (DPRs) and causes neurodegeneration. The flies made by Tran and colleagues express the repeat from within an intron, have high levels of sense RNA foci in the nucleus, low levels of RAN translation, and no neurodegeneration. This means that accumulation of sense RNA foci in the nucleus is not sufficient to drive neurodegeneration in this fly model.
This result is highly important and comes at a critical time in the field, where several groups are discussing the relative contributions of RNA vs. proteotoxicity caused by C9ORF72 mutations (see recent papers from our group [Jovičić et al., 2015], Jeff Rothstein’s group [Zhang et al., 2015], and Paul Taylor and Fen-Biao Gao’s groups [Freibaum et al., 2015]) and a comprehensive review of the work by Fox and Tibbetts (Fox and Tibbetts, 2015).
All three papers demonstrate nucleocytoplasmic transport impairments caused by C9ORF72 mutations but disagree over the cause of the defect (we say it is the DPRs, Zhang et al. say it’s the sense RNA, and Freibaum et al. say that their phenotypes can be caused by toxic RNAs, DPRs, or some combination of both).
The authors' C9ORF72 intron fly model does not seem to produce antisense RNA foci, which appears to be an important feature of C9ORF72 FTD/ALS (Cooper-Knock et al., 2015). Before we can conclude that RNA foci in the nucleus do not contribute to neurodegeneration, it will be important in the future to test the effect of a similar level of antisense RNA transcripts in the fly model.
A parsimonious explanation for the authors' findings, together with other published work (especially the stop codon interrupted flies produced by Isaacs and colleagues) is that that pathologies seen in the fly C9ORF72 models are due in large part (if not mostly) to translation products from the repeat. Whether this is the situation in human cells and mouse (e.g., Chew et al., 2015) remains to be determined.
References:
Mizielinska S, Grönke S, Niccoli T, Ridler CE, Clayton EL, Devoy A, Moens T, Norona FE, Woollacott IO, Pietrzyk J, Cleverley K, Nicoll AJ, Pickering-Brown S, Dols J, Cabecinha M, Hendrich O, Fratta P, Fisher EM, Partridge L, Isaacs AM. C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins. Science. 2014 Sep 5;345(6201):1192-1194. Epub 2014 Aug 7 PubMed.
Jovičić A, Mertens J, Boeynaems S, Bogaert E, Chai N, Yamada SB, Paul JW 3rd, Sun S, Herdy JR, Bieri G, Kramer NJ, Gage FH, Van Den Bosch L, Robberecht W, Gitler AD. Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS. Nat Neurosci. 2015 Sep;18(9):1226-9. PubMed.
Zhang K, Donnelly CJ, Haeusler AR, Grima JC, Machamer JB, Steinwald P, Daley EL, Miller SJ, Cunningham KM, Vidensky S, Gupta S, Thomas MA, Hong I, Chiu SL, Huganir RL, Ostrow LW, Matunis MJ, Wang J, Sattler R, Lloyd TE, Rothstein JD. The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature. 2015 Sep 3;525(7567):56-61. Epub 2015 Aug 26 PubMed.
Freibaum BD, Lu Y, Lopez-Gonzalez R, Kim NC, Almeida S, Lee KH, Badders N, Valentine M, Miller BL, Wong PC, Petrucelli L, Kim HJ, Gao FB, Taylor JP. GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport. Nature. 2015 Sep 3;525(7567):129-33. Epub 2015 Aug 26 PubMed.
Fox BW, Tibbetts RS. Neurodegeneration: Problems at the nuclear pore. Nature. 2015 Sep 3;525(7567):36-7. Epub 2015 Aug 26 PubMed.
Cooper-Knock J, Higginbottom A, Stopford MJ, Highley JR, Ince PG, Wharton SB, Pickering-Brown S, Kirby J, Hautbergue GM, Shaw PJ. Antisense RNA foci in the motor neurons of C9ORF72-ALS patients are associated with TDP-43 proteinopathy. Acta Neuropathol. 2015 Jul;130(1):63-75. Epub 2015 May 6 PubMed.
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