Abstract
A hexanucleotide repeat expansion GGGGCC in the non-coding region of C9orf72 is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Toxic dipeptide repeats (DPRs) are synthesized from GGGGCC via repeat-associated non-AUG (RAN) translation. Here, we develop C. elegans models that express, either ubiquitously or exclusively in neurons, 75 GGGGCC repeats flanked by intronic C9orf72 sequence. The worms generate DPRs (poly-glycine-alanine [poly-GA], poly-glycine-proline [poly-GP]) and poly-glycine-arginine [poly-GR]), display neurodegeneration, and exhibit locomotor and lifespan defects. Mutation of a non-canonical translation-initiating codon (CUG) upstream of the repeats selectively reduces poly-GA steady-state levels and ameliorates disease, suggesting poly-GA is pathogenic. Importantly, loss-of-function mutations in the eukaryotic translation initiation factor 2D (eif-2D/eIF2D) reduce poly-GA and poly-GP levels, and increase lifespan in both C. elegans models. Our in vitro studies in mammalian cells yield similar results. Here, we show a conserved role for eif-2D/eIF2D in DPR expression.
Highlights
The GGGGCC (G4C2) hexanucleotide repeat expansion in the non-coding region of C9orf[72] is the most common monogenic cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)[1,2], and causes up to 10% of what appears to be sporadic ALS
A recent study showed that the ribosomal protein eS25 (RPS25), which is required for efficient translation initiation at the internal ribosome entry site (IRES) of a number of viral and cellular RNAs11,12, is required for repeat-associated non-AUG (RAN) translation of poly-GP from G4C2 repeats in yeast, fly, and induced pluripotent human stem cell models[13]
We generated four control C. elegans strains: (a) UAG ubi worms carry an identical sequence to the C9 ubi animals, but the upstream translation initiation codon CUG, which is required for translation of poly-GA in vitro[20,29,30,31], is mutated to UAG (Fig. 1a), (b) ΔC9 ubi worms lack the G4C2 repeats and the intronic sequences flanking the G4C2 repeats (Fig. 1a), (c) ΔG4C2 repeats ubi worms lack the G4C2 repeats, but maintain the intronic sequences (Supplementary Fig. 2a), and (d) the C9 ΔnLuc ubi worms carry an identical sequence to the C9 ubi animals, but lack nLuc (Supplementary Fig. 2a)
Summary
The GGGGCC (G4C2) hexanucleotide repeat expansion in the non-coding region of C9orf[72] is the most common monogenic cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)[1,2], and causes up to 10% of what appears to be sporadic ALS. A second set of transgenic worms expressing the 75 G4C2 repeats and the flanking intronic sequences exclusively in neurons displayed similar phenotypes, suggesting that DPR production in this cell type is sufficient to cause disease phenotypes These C. elegans models provide an opportunity to study in vivo the molecular mechanisms underlying DPR production. Supporting the phylogenetic conservation of our C. elegans findings, in vitro knockdown of eIF2D in mouse and human cells led to a similar decrease in poly-GA steady-state levels Together, these observations suggest eIF2D may play an important role in RAN translation from G4C2 repeats, a finding with biomedical implications for C9orf72-associated ALS and FTD
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