Novel Drosophila Models for Unravelling the Mechanisms of Frontotemporal Dementia and Amyotrophic Lateral Sclerosis

Abstract

A hexanucleotide repeat expansion in a non-coding region of the C9orf72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), neurodegenerative diseases with considerable clinical and pathological overlap. FTD and ALS are invariably fatal and there is no cure. An understanding of the mechanisms through which the C9orf72 mutation leads to FTD/ALS will be crucial for development of effective therapeutics. There is evidence to support three possible pathogenic mechanisms underlying C9orf72 toxicity: (1) haploinsufficiency of C9orf72; (2) repetitive RNA transcripts arising from the repeat are toxic; (3) repeat-associated non-AUG (RAN) translation produces five different toxic dipeptide repeat proteins (DPRs) – AP, GP, GA, PR, and GR – from repeat RNA. The majority of evidence points to a gain of function mechanism and DPRs as the main toxic species, but does not rule out possible contributions from RNA toxicity and haploinsufficiency via synergistic mechanisms. Despite many studies into DPR-mediated toxicity in vitro and in vivo, the precise mechanisms through which DPR expression leads to neurodegeneration are still poorly understood. Drosophila models of DPR toxicity have been instrumental in the discovery that DPRs are the main drivers of toxicity. However, there remains a lack of consistent and physiologically relevant DPR models. Most express up to only 100 repeat units, whereas in patients, the expansion is several hundreds to thousands of repeats in length. Furthermore, despite the average age of onset of C9orf72-related FTD/ALS being 57 years of age, there are few studies investigating the effect of increasing age on DPR toxicity. The aims of this study were to address the lack of Drosophila models expressing DPRs of a physiological repeat length, fully characterise the effect of DPR expression on age-related neurodegenerative phenotypes, and subsequently use these models to unravel the mechanisms underpinning DPR toxicity. Firstly, we showed that 1000 repeat DPRs are stable in the Drosophila genome for over 3 years (~100 generations), and flies pan-neuronally expressing DPRs are viable with a reasonable lifespan. Each DPR was associated with a distinct age-related phenotypic profile. In support of previous studies, GR1000 showed the greatest age-related toxicity. However, AP1000, generally considered the least toxic of the DPRs, showed neurodegeneration, reduced climbing speed, and electrophysiological defects. Given that all DPRs have the capacity to be present in the same cell in patients, we next looked to see if co-expressing DPRs can alter their phenotypic profiles. Co-expression revealed a novel bang-sensitive seizure susceptibility not previously observed in DPR fly models. Subsequently, a dominant modifier screen was designed to identify mutations that could exacerbate seizure susceptibility in DPR flies. The screen implicated genes involved in mitochondrial function, oxidative stress, microtubule dynamics, and synaptic inhibition in DPR-mediated neurotoxicity. In summary, novel Drosophila models expressing physiologically relevant repeat length DPRs have been generated. Characterisation revealed age- and DPR- specific phenotypic profile, and highlights the importance of co-expression. We anticipate that this model will be useful for future studies elucidating the mechanisms underpinning DPR toxicity in C9orf72-related FTD/ALS.

Date of Award	1 Aug 2022
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Ryan West (Supervisor), Andreas Prokop (Supervisor) & Stuart Pickering-Brown (Supervisor)