C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins

Abstract

An expanded GGGGCC repeat in C9orf72 is the most common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis. A fundamental question is whether toxicity is driven by the repeat RNA itself and/or by dipeptide repeat proteins generated by repeat-associated, non-ATG translation. To address this question we developed in vitro and in vivo models to dissect repeat RNA and dipeptide repeat protein toxicity. Expression of pure repeats in Drosophila caused adult-onset neurodegeneration attributable to poly-(glycine-arginine) proteins. Thus expanded repeats promoted neurodegeneration through neurotoxic proteins. Expression of individual dipeptide repeat proteins with a non-GGGGCC RNA sequence showed both poly-(glycinearginine) and poly-(proline-arginine) proteins caused neurodegeneration. These findings are consistent with a dual toxicity mechanism, whereby both arginine-rich proteins and repeat RNA contribute to C9orf72-mediated neurodegeneration.Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are adult-onset, neurodegenerative diseases associated with personality change, language dysfunction and † Corresponding authors. a.isaacs@prion.ucl.ac.uk; l.partridge@ucl.ac.uk. Europe PMC Funders Group Europe PMC Funders Author ManuscriptsEurope PMC Funders Author Manuscripts progressive muscle weakness. These syndromes overlap genetically and pathologically, and can also co-occur in individuals, and within families (1). An intronic GGGGCC hexanucleotide repeat expansion in C9orf72 is the most common genetic cause of both FTD and ALS (C9FTD/ALS) (2-4), and can be found in patients diagnosed with all common neurodegenerative diseases (5). Healthy individuals carry fewer than 33 hexanucleotide repeats, with 2 repeats being the most common, but C9FTD/ALS cases carry between 400 and 4400 repeats (2, 5, 6).The repeat expansion could cause disease by three possible mechanisms: i) toxic sense and/or antisense repeat RNA species that sequester key RNA-binding proteins, ii) toxic dipeptide repeat (DPR) proteins, generated by repeat-associated, non-ATG (RAN) translation, or iii) reduced expression of C9orf72. The absence of a severe phenotype in a homozygous C9orf72 mutation case (7), and the lack of C9orf72 coding mutations (8) argue against loss-of-function as a primary mechanism. Neuronal aggregates of RNA, termed RNA foci, generated from both sense and antisense repeat transcripts are frequent in C9FTD/ALS patient brain (9-13). The GGGGCC repeat can be translated in all sense and antisense frames, two of which encode the same DPR, resulting in five DPR proteins, all of which form inclusions in widespread brain regions (10,12,(14)(15)(16)(17)(18). It is therefore of fundamental importance to understand the contributions of repeat RNA and DPR proteins to C9orf72-mediated neurodegeneration.A major obstacle in the investigation of large expanded repeats is that they are inherently unstable. We used recombination-deficient E. coli and a cloning strategy termed recursive directional ligati...

A zebrafish model for C9orf72 ALS reveals RNA toxicity as a pathogenic mechanism

“…Furthermore, elimination of the ATG initiation codon abolished the toxic effect of the PR construct. The finding that mostly arginine-containing DPRs display neurotoxicity in this in vivo model is in line with the findings in Drosophila [4,33] and provides evidence for a potential role of these DPRs in C9 ALS/FTD pathology. Despite the observation of GA forming abundant aggregates, it failed to induce motor axon toxicity in our model.…”

“…It has previously been shown that these constructs are not RAN translated and do not generate DPRs, while they still adopt the same stable conformation as the uninterrupted GGG GCC repeat RNA [33]. Interestingly, in spite of not being able to generate DPRs, sense RO repeat RNA (70RO and 108RO) still significantly affected axonal length and axonal branching, although to a somewhat lesser degree than the uninterrupted repeat RNA (Fig.…”

“…However, as explained above, this is not supported by our data. Of note, while these 'RNA only' constructs display toxicity in zebrafish, they do not in Drosophila [33], suggesting a different sensitivity to RNA toxicity in zebrafish versus Drosophila models.…”

“…The p35-S and p35-AS plasmids, containing ~ 35 GGG GCC or ~ 35 CCC CGG repeats, were generated by EcoRI digestion of the pATG75 construct [25] and ligation into the pCMV6-Entry vector. Plasmids pUAST91, containing 91 GGG GCC hexanucleotide repeats, and pUAST108RO [33], containing 108 GGG GCC stop codon-interrupted repeats, were digested with EcoRI/NotI and KpnI, respectively, and the repeat was subcloned into the pCMV6-Entry vector to generate the p90-S and the p108RO-S plasmids. In both plasmids, a reverse complementary sequence of the T3 promoter was inserted 30 bp after the repeats to generate antisense transcripts.…”

Linking RNA Dysfunction and Neurodegeneration in Amyotrophic Lateral Sclerosis

“…The G 4 C 2 repeat is translated in all reading frames in both the sense and antisense strands, resulting in several different dipeptides that accumulate specifically within the cells of patients carrying pathogenic (>22) repeat lengths. These dipeptides are toxic to cells in vitro and in vivo [69,[136][137][138], causing cell death in some cases by disrupting RNA processing within nucleoli [69], or by altering the function of essential proteins such as Unc119 [137], a trafficking factor required for axon development [139]. Moreover, the sequestration of RNA binding proteins by expanded G 4 C 2 RNA may be synergistic with the toxicity induced by RAN translation products [138]: many of the proteins bound by G 4 C 2 RNA catalyze the nuclear export of RNA, potentially facilitating cytoplasmic RAN translation of expanded C9orf72 repeats and accentuating toxicity [121].…”