Nuclear localized C9orf72-associated arginine-containing dipeptides exhibit age-dependent toxicity in C. elegans

Abstract

A hexanucleotide repeat expansion mutation in the C9orf72 gene represents a prevalent genetic cause of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Non-canonical translation of this repeat gives rise to several distinct dipeptide protein species that could play pathological roles in disease. Here, we show in the model system Caenorhabditis elegans that expression of the arginine-containing dipeptides, but not alanine-containing dipeptides, produces toxic phenotypes in multiple cellular contexts, including motor neurons. Expression of either (PR)50 or (GR)50 during development caused a highly penetrant developmental arrest, while post-developmental expression caused age-onset paralysis. Both (PR)50- and (GR)50-green fluorescent protein tagged dipeptides were present in the nucleus and nuclear localization was necessary and sufficient for their toxicity. Using an inducible expression system, we discovered that age-onset phenotypes caused by (PR)50 required both continual (PR)50 expression and an aged cellular environment. The toxicity of (PR)50 was modified by genetic mutations that uncouple physiological aging from chronological aging. However, these same mutations failed to modify the toxicity of (GR)50, suggesting that (PR)50 and (GR)50 exert their toxicity through partially distinct mechanism(s). Changing the rate of physiological aging also mitigates toxicity in other C. elegans models of ALS, suggesting that the (PR)50 dipeptide might engage similar toxicity mechanisms as other ALS disease-causing proteins.

Review: Modelling the pathology and behaviour of frontotemporal dementia

“…To investigate DPR-only toxicity, models have been generated where individual DPRs are expressed. When overexpressed, polypeptides of the arginine-rich DPRs glycine-arginine (GR) and proline-arginine (PR) have the greatest potential to induce neurotoxicity in Drosophila, C. elegans and zebrafish [66,67,80,81,[83][84][85][86][87][88], but polypeptides of glycine-alanine (GA) also cause toxicity in Drosophila, zebrafish, chick embryo and mouse models [67,80,[89][90][91][92][93].The expression of 50 or 69 GA repeats via adenovirus-mediated delivery into neonatal mouse brain or zygote injection for (mostly) neuronal expression of 149 GA repeats induce development of primarily motor phenotypes [92][93][94]. However, memory deficits and increased anxiety were also identified in the (GA) 50 model [93].…”

PolyQ-independent toxicity associated with novel translational products from CAG repeat expansions

“…To evaluate the toxicity of individual CAG-encoded RAN polypeptides, we created C. elegans models for each of the possible CAG RAN products using a previously described codon-variation strategy. This approach maintains the amino acid repeat of the RAN polypeptide but removes the CAG nucleotide repeat [20]. The codon-varied constructs were also designed to minimize computationally predicted hairpin structures in the resulting RNA, as hairpin structures are thought to be required for RAN translation and could lead to the production of non-relevant RAN products [21].…”

“…To begin addressing these questions, we created codon-varied GFP-tagged HD RAN homopolymers at both disease-relevant lengths (38 repeats) and highly expanded lengths (90 repeats) using previously described approaches [20]. We expressed these peptides in multiple cellular settings in C. elegans.…”

Models and mechanisms of repeat expansion disorders: a worm’s eye view

“…However, factors that promote the cytoplasmic localization of the repeat bearing RNA appear to facilitate RAN translation, suggesting that the observed RNA foci may serve a protective role (Mori, Nihei et al 2016, Hautbergue, Castelli et al 2017). The sense-derived DPRs Glycine-Arginine (GR n ) and antisense derived Proline-Arginine (PR n ) are toxic across a wide range of model systems (Mizielinska, Gronke et al 2014, Wen, Tan et al 2014, Freibaum, Lu et al 2015, Jovicic, Mertens et al 2015, Tran, Almeida et al 2015, Boeynaems, Bogaert et al 2016, Lee, Zhang et al 2016, Rudich, Snoznik et al 2017). While RAN DPRs are thought to play a significant role in the toxicity of the C9orf72 repeat expansion, there are some notable inconsistencies that mitigate this hypothesis.…”

“…To address these limitations, a newly reported C. elegans model specifically examined individual RAN translation products in the absence of repeat containing RNA (Rudich, Snoznik et al 2017). In this model, codon variation was utilized to encode a specific amino acid sequence but eliminate (G 4 C 2 ) sequence and any potential RNA secondary structure and therefore RNA based toxicity.…”