C9orf72-associated arginine-rich dipeptide repeats induce RNA-dependent accumulation of Staufen in nucleus

Abstract

Accumulation of RNA in the nucleus is one of the pathological features of C9orf72-associated amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD), yet its potential toxic cellular consequences remain largely undefined. RNA accumulated in the nucleus may interact with and increase nuclear localization of RNA-binding proteins (RBPs). Here, we show in C9-ALS/FTD Drosophila melanogaster model that Staufen, a double-stranded RBP normally localized in cytoplasm, accumulates in the nucleus, which is in contrast to many nuclear-localized RBPs, such as TDP-43 and FUS, whose cytoplasmic accumulation is thought to be a pathological hallmark of ALS/FTD. We found that in Drosophila neurons expressing arginine-rich dipeptide repeat proteins (DPRs), Staufen accumulated in the nucleus in an RNA-dependent manner. In the nucleus, Staufen localized closely to, and potentially interacts with, heterochromatin and nucleolus in Drosophila C4 da neurons expressing poly(PR), a proline-arginine (PR) DPR. PR toxicity in C4 da neurons increased Fibrillarin staining in the nucleolus, which was enhanced by stau heterozygous mutation. Furthermore, knockdown of fib exacerbated retinal degeneration mediated by PR toxicity, which suggests that increased amount of Fibrillarin by stau heterozygous mutation is protective. Heterozygotic mutation of stau could also mitigate retinal degeneration and rescue viability of flies exhibiting PR toxicity. Taken together, our data show that nuclear accumulation of cytoplasmic protein, such as Staufen, may also be an important pathological feature of C9-ALS/FTD.\n\nAuthor summaryCytoplasmic accumulation of nuclear RNA-binding proteins (RBPs) is one of the common pathological features of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In C9orf72-associated ALS/FTD fly model, we found that Staufen, a double-stranded (ds) RBP normally localized mostly in cytoplasm, accumulates in the nucleus in an RNA-dependent manner. Next, we checked wherein the nucleus Staufen accumulates and found that Staufen partially co-localizes with heterochromatin and nucleolus. Interestingly, the expression of Fibrillarin, a nucleolar protein, was significantly increased by C9orf72-derived PR toxicity and further augmented by reduction in stau dosage, the gene encoding Staufen. When we knocked down fib, the gene encoding Fibrillarin, PR-induced retinal degeneration was exacerbated. This indicates that increased Fibrillarin expression by stau dosage reduction is protective. Furthermore, when we reduced stau dosage in flies presenting PR toxicity, their retinal degeneration and viability were largely rescued. Based on these data, we suggest that nuclear accumulation of Staufen is an important feature of C9-ALS/FTD and suggest that reducing stau dosage is a promising therapeutic target.