Differential Toxicity of Nuclear RNA Foci versus Dipeptide Repeat Proteins in a Drosophila Model of C9ORF72 FTD/ALS

Abstract

SUMMARY Dipeptide repeat (DPR) proteins are toxic in various models of FTD/ALS with GGGGCC (G4C2) repeat expansion. However, it is unclear whether nuclear G4C2 RNA foci also induce neurotoxicity. Here, we describe a novel Drosophila model expressing 160 G4C2 repeats (160R) flanked by human intronic and exonic sequences. Spliced intronic 160R formed nuclear G4C2 sense RNA foci in glia and neurons about 10 times more abundantly than in human neurons; however, they had little effect on global RNA processing and neuronal survival. In contrast, highly toxic 36R in the context of poly(A)+ mRNA were exported to the cytoplasm where DPR proteins were produced at >100-fold higher level than that in 160R flies. Moreover, the modest toxicity of intronic 160R expressed at higher temperature correlated with increased DPR production but not RNA foci. Thus, nuclear RNA foci are neutral intermediates or possibly neuroprotective through preventing G4C2 RNA export and subsequent DPR production.

SRSF1-dependent nuclear export of C9ORF72 repeat transcripts: targeting toxic gain-of-functions induced by protein sequestration as a selective therapeutic strategy for neuroprotection

“…Our work clearly reinforces this concept using an unbiased approach of investigation which did not rely on modifying expression levels or repeat-length of repeat transcripts but rather on selectively manipulating transport across the nuclear pore to either promote nuclear retention of transcripts or production of DPRs. The increased number of nuclear RNA foci upon SRSF1 depletion did not appear to induce cytotoxicity in neuronal cell models (not shown) or astrocytes in agreement with another study in Drosophila [51] . While no detrimental effects of SRSF1 depletion were observed in neuronal cells or patient-derived neurons, the reduced RAN translation of DPRs specifically led to decreased cytotoxicity and to neuroprotection of C9ORF72-ALS Drosophila and .…”

“…A growing body of evidence implicate DPRs as one of the primary drivers of pathogenesis in cellular and animal models of C9ORF72-ALS/FTD [46,[49][50][51] . Our work clearly reinforces this concept using an unbiased approach of investigation which did not rely on modifying expression levels or repeat-length of repeat transcripts but rather on selectively manipulating transport across the nuclear pore to either promote nuclear retention of transcripts or production of DPRs.…”

RAN translation—What makes it run?

“…From yeast (Jovičić et al , 2015), to Drosophila (Mizielinska et al , 2014; Wen et al , 2014; Freibaum et al , 2015; Tran et al , 2015; Yang et al , 2015), cultured cells (Zu et al , 2013; Zhang et al , 2014; Tao et al , 2015; Yamakawa et al , 2015), and primary mammalian neurons (May et al , 2014; Wen et al , 2014; Zhang et al , 2014), DRP expression leads to cell death and/or reduced survival. Significantly, in many of these systems, DRP expression is sufficient to trigger toxicity, as demonstrated by the use of alternative codons in place of GGGGCC that allow for DRP production in the absence of the potentially toxic repeat-containing RNA species (May et al , 2014; Mizielinska et al , 2014; Wen et al , 2014; Zhang et al , 2014; Jovičić et al , 2015; Tao et al , 2015; Yamakawa et al , 2015; Yang et al , 2015).…”

RNA versus protein toxicity in C9orf72 ALS/FTLD

“…Thus, RNA foci of even large sense and antisense repeat transcripts cause no overt phenotype in Drosophila in the absence of DPR expression, although they bind at least one RNA-binding protein like in humans. Therefore, in fly models, (G4C2) n toxicity is clearly predominantly driven by poly-GR toxicity [18,30]. A possible explanation for the lack of RNA toxicity in this fly model is the pronounced formation of RNA foci which is in contrast to the more diffusely distributed cytoplasmic repeat RNA in zebrafish.…”