Abstract
A GGGGCC hexanucleotide repeat expansion within the C9orf72 gene is the most common genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia. Sense and antisense repeat-containing transcripts undergo repeat-associated non-AUG-initiated translation to produce five dipeptide proteins (DPRs). The polyGR and polyPR DPRs are extremely toxic when expressed in Drosophila neurons. To determine the mechanism that mediates this toxicity, we purified DPRs from the Drosophila brain and used mass spectrometry to identify the in vivo neuronal DPR interactome. PolyGR and polyPR interact with ribosomal proteins, and inhibit translation in both human iPSC-derived motor neurons, and adult Drosophila neurons. We next performed a screen of 81 translation-associated proteins in GGGGCC repeat-expressing Drosophila to determine whether this translational repression can be overcome and if this impacts neurodegeneration. Expression of the translation initiation factor eIF1A uniquely rescued DPR-induced toxicity in vivo, indicating that restoring translation is a potential therapeutic strategy. These data directly implicate translational repression in C9orf72 repeat-induced neurodegeneration and identify eIF1A as a novel modifier of C9orf72 repeat toxicity.
Highlights
A GGGGCChexanucleotide repeat expansion within the C9orf72 gene is the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)
To determine which proteins the arginine-containing DPRs form complexes with in an intact neuronal system, DNA sequences encoding recodonised ATG-driven dipeptide proteins [28] were cloned upstream of a tandem affinity purification (TAP) tag containing a protein G module and streptavidin-binding domain separated by an internal tobacco etch virus (TEV) protease cleavage site (GSTAP tag)
After generation of transgenic Drosophila, constructs were expressed in the developing compound eye using the GMR-Gal4 driver and, as expected, strong toxicity was observed for the GRGSTAP and PR-GSTAP constructs but not the GA-GSTAP control construct
Summary
A GGGGCChexanucleotide repeat expansion within the C9orf gene is the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Toxicity has been proposed to arise in patients either due to haploinsufficiency of the C9orf gene, or to two major gains of function mechanisms. Several groups, including our own, have previously demonstrated that expression of ATG-driven arginine-containing dipeptide proteins (polyGR, polyPR), which have been recodonised to lack a repetitive RNA intermediate, is extremely toxic to various model systems including human cell lines, Drosophila and mice [18, 28, 43, 48], whilst polyGA exerts toxicity in some model systems [21, 26, 36, 49]
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