Co-expression of C9orf72 related dipeptide-repeats over 1000 repeat units reveals age- and combination-specific phenotypic profiles in Drosophila

Abstract

A large intronic hexanucleotide repeat expansion (GGGGCC) within the C9orf72 (C9orf72-SMCR8 Complex Subunit) locus is the most prevalent genetic cause of both Frontotemporal Dementia (FTD) and Motor Neuron Disease (MND). In patients this expansion is typically hundreds to thousands of repeat units in length. Repeat associated non-AUG translation of the expansion leads to the formation of toxic, pathological Dipeptide-Repeat Proteins (DPRs). To date there remains a lack of in vivo models expressing C9orf72 related DPRs with a repeat length of more than a few hundred repeats. As such our understanding of how physiologically relevant repeat length DPRs effect the nervous system in an ageing in vivo system remains limited. In this study we generated Drosophila models expressing DPRs over 1000 repeat units in length, a known pathological length in humans. Using these models, we demonstrate each DPR exhibits a unique, age-dependent, phenotypic and pathological profile. Furthermore, we show co-expression of specific DPR combinations leads to distinct, age-dependent, phenotypes not observed through expression of single DPRs. We propose these models represent a unique, in vivo, tool for dissecting the molecular mechanisms implicated in disease pathology, opening up new avenues in the study of both MND and FTD.

Modeling C9orf72-Related Frontotemporal Dementia and Amyotrophic Lateral Sclerosis in Drosophila

“…In this model, GA(64) was also shown to co-aggregate with TBPH (Solomon et al, 2018). This is supported by findings from West et al (2020a) which showed that GA(1000) similarly colocalizes with TBPH in the cytoplasm (West et al, 2020a). However, in contrast to arginine rich DPRs [GR(1000) and PR(1000)] GA(1000) did not cause a significant mislocalisation of TBPH (West et al, 2020a).…”

“…The authors suggest that this points to a selective susceptibility of glutamatergic neurons to PR and GR toxicity mediated through excitotoxicity. In contrast, however, larvae pan-neuronally expressing 1,000-repeat GR and PR, showed no significant changes in bouton number, although PR(1000) flies did show an increase in the number of bruchpilot positive active zones at the larval neuromuscular junction (West et al, 2020a). However, a capacity for excitotoxicity to potentiate neurodegeneration in a cell-autonomous capacity is an area of interest that remains relatively unexplored.…”

“…The discrepancies in GA toxicity across these different models highlight the need for a consistent and robust model system. Commonly, expression of GA in the eye causes no toxic effects, and this is consistent up to 1,000 repeats Freibaum et al, 2015;West et al, 2020a). This lack of a clear phenotype, when expressed in the eye, limits the ability to employ this model for genetic modifier screens, in particular those looking to identify suppressors.…”

“…Drosophila have been extensively used to study a range of neurodegenerative and neurodevelopmental disorders including autism ( Vilidaite et al, 2018 ), Alzheimer’s ( Cao et al, 2008 ; Chakraborty et al, 2011 ), Parkinson’s ( West et al, 2015 ), Huntington’s, FTD ( West et al, 2020a , b ), and ALS ( West et al, 2018 ). In addition to their short lifespan, rapid generation times, low cost and ability to display complex behaviors including learning and memory, the power of Drosophila as a model organism lies in their genetic tractability.…”

Multiple pathways of toxicity induced by C9orf72 dipeptide repeat aggregates and G4C2 RNA in a cellular model

“…The G 4 C 2 hexanucleotide repeat is thought to exert toxic effects in part by forming higher-order RNA assemblies in the nucleus that sequester RNA-binding proteins ( Almeida et al, 2013 ; Cooper-Knock et al, 2015 ; Cooper-Knock et al, 2014 ; Donnelly et al, 2013 ; Haeusler et al, 2014 ; Mori et al, 2013b ; Reddy et al, 2013 ; Rossi et al, 2015 ; Sareen et al, 2013 ; Xu et al, 2013 ). Even if longer G 4 C 2 sequences may mediate additional effects ( West et al, 2020 ), the G 4 C 2 constructs tested here resulted in a pronounced inhibition of protein synthesis, even when coding for cytoplasmic poly-GA protein (NES-GA 65 ), which did not impair protein biogenesis when produced from a synthetic (non-G 4 C 2) construct. However, because expression constructs based on G 4 C 2 repeats also generate the different RAN translation DPR products, a clear distinction between RNA and DPR toxicity in previous studies had been difficult.…”