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Expanded G4C2 repeats linked to C9ORF72ALS and FTD form length-dependent RNA foci, sequester RNA binding proteins and are neurotoxic


The GGGGCC (G4C2) intronic repeat expansion within C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) [1, 2]. The mechanism by which the G4C2 intronic repeats cause neurodegeneration is unknown. Decreased tissue levels of the C9ORF72 transcript implicate a loss of protein function due to haploinsufficiency, intranuclear neuronal RNA foci have been observed in ALS and FTD tissues, suggesting that G4C2 RNA may be toxic [1].

Materials and methods

In order to determine whether expanded G4C2 transcripts might be toxic and sequester RNA binding proteins we generated G4C2 repeats with 8x, 38x and 72x and expressed them in a range of neuronal and non-neuronal cell lines, primary neurons and zebrafish embryos. We used fluorescent in situ hybridisation probes for G4C2 to detect RNA foci in transfected cells and human brain tissue form C9ORF72 mutation carriers. We assessed the toxicity by cell counting, activated caspase, PARP cleavage and Annexin V expression transfected cells and by TUNEL assay in zebrafish embryos. We probed transfected cells with antibodies to 30 RNA binding proteins and then tested the best candidates by immunohistochemistry in human brain tissues. We used biotinylated G4C2 x72 probes to pull down repeat binding proteins and confirmed binding by western blot.


Cellular toxicity was associated with the nuclear retention of transcripts containing 38x and 72x repeats and the appearance of RNA foci. By placing the repeats 3’ to EGFP we were able to monitor nuclear export and show that transfected neuroblastoma cells, but not HEK cells, declined in number over time due to apoptotic cell death. This was most marked in those showing the greatest nuclear retention and foci, indicating cell type and neuron-specific toxicity of longer G4C2 repeats.

In order to explore the sequestration hypothesis we screened antibodies to 30 RNA binding proteins on 72x transfected neuroblastoma cells and demonstrated colocalization of G4C2 RNA foci with three proteins. Using biotinylated 72x RNA we were only able to pull down one of these indicating that it is able to bind G4C2 RNA directly. On probing human cerebellar ALS and FTD tissues we detected a striking colocalization of this protein with 70% of RNA foci.


Our observation that longer stretches of G4C2 RNA form neurotoxic foci and bind specific RNA binding proteins is similar to other intronic microsatellite expansion disorders where CUG (DM1) and CCUG (DM2) intronic repeats generate RNA foci in cells, animal models and in patients with myotonic dystrophy.


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Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( ) applies to the data made available in this article, unless otherwise stated.

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Lee, YB., Chen, HJ., Peres, J.N. et al. Expanded G4C2 repeats linked to C9ORF72ALS and FTD form length-dependent RNA foci, sequester RNA binding proteins and are neurotoxic. Mol Neurodegeneration 8 (Suppl 1), O14 (2013).

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