Hexanucleotide repeats in the C9ORF72 gene, which cause both frontotemporal dementia and amyotrophic lateral sclerosis, spew a fusillade of potentially toxic molecules into the cell: two kinds of repetitive RNA from transcription in the sense and antisense directions, plus five different repeat dipeptides encoded by those messages. One of the biggest research questions in the field these days is, which of those biomolecules are dangerous? In the September 23 Neuron, investigators zero in on sense-direction repeat RNAs that assemble foci in the nucleus, but their fly model—carefully mimicking transcription of the repeats in humans—survived without a phenotype.

“We conclude that sense nuclear RNA foci are not a major source of toxicity,” said senior author Fen-Biao Gao of the University of Massachusetts Medical School in Worcester. He added that it remains possible that antisense nuclear RNAs, or non-aggregated repeat RNAs, might be responsible for some element of neurodegeneration.

Repeats in Focus.

C9ORF72 repeats (red) form foci in the nucleus (blue) of neurons. One large spot (arrow) forms at the site of transcription, co-localizing with the large cluster of RNA polymerase (green). [Neuron, Tran et al.]

Researchers have determined the C9ORF72 dipeptides are indeed toxic in research models, though they have not confirmed that the dipeptides are the key killer molecules in people (see Dec 2014 newsAug 2014 news). Others have suggested the RNAs might be the key troublemakers (see May 2015 newsOct 2013 news). 

Like many scientists in the field, Gao and first author Helene Tran started designing their model organism as soon as the C9ORF72 gene was pegged as a risk factor, before the antisense RNAs or atypical translation were known (see Sep 2011 news). First, study co-author Sandra Almeida used neurons differentiated from induced pluripotent cells of C9ORF72 carriers to confirm that the repeats did not affect splicing events around them. The excess hexanucleotides were transcribed, then cut out and abandoned in the nucleus. To mimic that process as closely as possible in flies, Tran engineered a minigene with 160 repeats flanked by the human intronic sequences and exons that occur on either side of the expansion in people. “This is the first model with repeats expressed, in the molecular context similar to that in human patients,” Gao said. Tran expressed the minigene at high levels in the neurons and glia of fruit flies.

She and Gao were initially disappointed. Their flies were fine, despite the 40 or so repeat RNA foci accumulated in the nuclei of their neurons and glia (see image above). No such foci occurred in control flies with zero or five repeats. Tran typically saw one large focus surrounded by smaller ones. The big spot co-localized with RNA polymerase, suggesting it was the site of the minigene’s transcription. Gao advised other scientists studying C9ORF72 that the large focus in animal models like his results from overexpression of the transgene, while the smaller spots correspond to the foci seen in human tissues.

Tran then managed to induce toxic effects in her 160-repeat fly model by keeping the flies at a warmer temperature, 29 degrees Celsius instead of 25. In that balmy environment, several flies died a bit prematurely, with populations reaching the 50-percent survival mark five days earlier than wild-type flies. However, the RNA foci themselves did not seem to cause this increased mortality; their numbers remained the same between the two ambient temperatures. Instead, the warmed-up flies started making dipeptides. The authors measured four times the amount of poly-glycine-proline in the toasty flies as in their cooler counterparts. 

To confirm their suspicion that dipeptides, not RNA foci, were toxic to flies, Tran turned to a Drosophila model engineered by Adrian Isaacs and colleagues at University College London (see Aug 2014 news story). These contain 36 repeats that differ from Tran’s in their context. Instead of putting the hexanucleotides in an intronic milieu, the researchers hooked them directly to the code for a poly-adenosine tail, which stabilizes mRNA and points it toward the cytosol for translation. Thus, these flies contain cytosolic repeats instead of nuclear ones. Expressing Isaacs’ repeat construct all over the fly was lethal, but by expressing it in the eye only, Tran confirmed that it was toxic, and generated repeat dipeptides. “I think we can now firmly say that the current generation of repeat fly models cause toxicity through dipeptide repeats,” commented Isaacs, who was not involved in the new paper (see full comment below).

Gao suggested that the repeat RNAs cause problems if they leak out into the cytosol and get translated. This, he speculated, might happen in older individuals. However, Gao and other researchers who spoke with Alzforum were not ready to fully retire the idea of toxic RNAs in favor of toxic dipeptides. “We cannot conclude from studies performed in Drosophila that RNA foci play no role in human disease,” Isaacs noted. As a point against killer dipeptides, Christopher Donnelly of the University of Pittsburgh, who did not participate in the Neuron paper, pointed out that two recent studies found little evidence that dipeptide aggregates correlate with pathology in the brain or in motor neurons (Mackenzie et al., 2015; Gomez-Deza et al., 2015).

Donnelly suspects improper transport of intronic repeat RNA from the nucleus to the cytosol might be an issue. “The export seems to be a major problem,” he said. Donnelly theorized that as people age, defects in splicing might allow the repeat-containing intron to escape. It could cause problems in the cytosol or at the nucleocytoplasmic pore, which papers by Donnelly, Gao, and others recently implicated as a major site of C9ORF72 toxicity (see Aug 2015 news story). 

Donnelly added that the new work provides a reminder to scientists designing or selecting a model animal. Tran’s fly full of repeats was fine, while Isaacs’ died, all due to the particularities of each construct. A third group, who examined intronic repeats in primary mouse neurons, saw no translation into dipeptide repeats but nonetheless observed increased cell death (Wen et al., 2014). However, they used an artificial intron and exons from green fluorescent protein, rather than directly from the C9ORF72 gene. “We have to be very careful in what models we choose, understanding the details and caveats of each system,” Donnelly said.—Amber Dance

Comments

  1. 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.

  2. 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.

     

  3. 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:

    . C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins. Science. 2014 Sep 5;345(6201):1192-4. Epub 2014 Aug 7 PubMed.

    . Modifiers of C9orf72 dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS. Nat Neurosci. 2015 Sep;18(9):1226-9. PubMed.

    . The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature. 2015 Sep 3;525(7567):56-61. Epub 2015 Aug 26 PubMed.

    . GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport. Nature. 2015 Sep 3;525(7567):129-33. Epub 2015 Aug 26 PubMed.

    . Neurodegeneration: Problems at the nuclear pore. Nature. 2015 Sep 3;525(7567):36-7. Epub 2015 Aug 26 PubMed.

    . 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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References

News Citations

  1. Live-Cell Studies Blame Arginine Peptides for C9ORF72’s Crimes
  2. C9ORF72 Killer Dipeptides Clog the Nucleolus
  3. Antisense RNA from C9ORF72 Repeats Is Likely Culprit in Patient Neurons
  4. RNA Deposits Confer Toxicity in C9ORF72 ALS
  5. Corrupt Code: DNA Repeats Are Common Cause for ALS and FTD
  6. C9ORF72’s Dirty Work Done by Problem Proteins
  7. ALS Gene Repeats Obstruct Traffic Between Nucleus and Cytoplasm

Paper Citations

  1. . Quantitative analysis and clinico-pathological correlations of different dipeptide repeat protein pathologies in C9ORF72 mutation carriers. Acta Neuropathol. 2015 Dec;130(6):845-61. Epub 2015 Sep 15 PubMed.
  2. . Dipeptide repeat protein inclusions are rare in the spinal cord and almost absent from motor neurons in C9ORF72 mutant amyotrophic lateral sclerosis and are unlikely to cause their degeneration. Acta Neuropathol Commun. 2015 Jun 25;3:38. PubMed.
  3. . Antisense proline-arginine RAN dipeptides linked to C9ORF72-ALS/FTD form toxic nuclear aggregates that initiate in vitro and in vivo neuronal death. Neuron. 2014 Dec 17;84(6):1213-25. PubMed.

Further Reading

Papers

  1. . Cerebellar c9RAN proteins associate with clinical and neuropathological characteristics of C9ORF72 repeat expansion carriers. Acta Neuropathol. 2015 Oct;130(4):559-73. Epub 2015 Sep 8 PubMed.
  2. . Expanded GGGGCC repeat RNA associated with amyotrophic lateral sclerosis and frontotemporal dementia causes neurodegeneration. Proc Natl Acad Sci U S A. 2013 May 7;110(19):7778-83. PubMed.
  3. . FTD/ALS-associated poly(GR) protein impairs the Notch pathway and is recruited by poly(GA) into cytoplasmic inclusions. Acta Neuropathol. 2015 Oct;130(4):525-35. Epub 2015 Jun 2 PubMed.

Primary Papers

  1. . 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.