Abstract
A GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Neurodegeneration may occur via transcription of the repeats into inherently toxic repetitive sense and antisense RNA species, or via repeat-associated non-ATG initiated translation (RANT) of sense and antisense RNA into toxic dipeptide repeat proteins. We have previously demonstrated that regular interspersion of repeat RNA with stop codons prevents RANT (RNA-only models), allowing us to study the role of repeat RNA in isolation. Here we have created novel RNA-only Drosophila models, including the first models of antisense repeat toxicity, and flies expressing extremely large repeats, within the range observed in patients. We generated flies expressing ~ 100 repeat sense or antisense RNA either as part of a processed polyadenylated transcript or intronic sequence. We additionally created Drosophila expressing > 1000 RNA-only repeats in the sense direction. When expressed in adult Drosophila neurons polyadenylated repeat RNA is largely cytoplasmic in localisation, whilst intronic repeat RNA forms intranuclear RNA foci, as does > 1000 repeat RNA, thus allowing us to investigate both nuclear and cytoplasmic RNA toxicity. We confirmed that these RNA foci are capable of sequestering endogenous Drosophila RNA-binding proteins, and that the production of dipeptide proteins (poly-glycine–proline, and poly-glycine–arginine) is suppressed in our models. We find that neither cytoplasmic nor nuclear sense or antisense RNA are toxic when expressed in adult Drosophila neurons, suggesting they have a limited role in disease pathogenesis.
Highlights
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two adult onset, neurodegenerative diseases, leading to loss of the upper and lower motor neurons, or frontal and temporal lobe cortical neurons, respectively
Others, have previously demonstrated that overexpression of C9orf72 repeat RNA is extremely toxic to Drosophila neurons, resulting in a dramatically reduced lifespan [19, 37, 56]
We have here demonstrated that the genomic context of repeat RNA affects intranuclear RNA foci formation, presumably because splicing of the intronic repeats prevents the nuclear export of repeat RNA
Summary
Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two adult onset, neurodegenerative diseases, leading to loss of the upper and lower motor neurons, or frontal and temporal lobe cortical neurons, respectively. Neuron-specific, or whole genome knockout of the mouse ortholog of C9orf does not lead to a neurodegenerative phenotype, suggesting that toxicity primarily arises through a gain of function mechanism [3, 8, 26, 29, 31, 51, 52]. Two such mechanisms have been proposed: RNA toxicity, or toxicity arising due to proteins derived from the repeat RNA [39]
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