Universität zu Köln

Morón Oset, Javier ORCID: 0000-0002-2230-0288 (2022). Investigating the spread and toxicity of glycine-alanine dipeptides in C9orf72 ALS/FTD using Drosophila melanogaster. PhD thesis, Universität zu Köln.

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Abstract

Hexanucleotide repeat expansions of variable size in C9orf72 are the most prevalent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The role of repeat size in disease onset and severity in humans remains controversial. Transcripts of the expansions are translated into five dipeptide repeat (DPR) proteins. Most preclinical studies have used relatively short and tagged poly-DPR constructs to investigate DPR-mediated toxicity, and shown that poly-GR, poly-PR and, to a lesser extent, poly-GA DPRs are neurotoxic. Consequently, a major emphasis has been placed on understanding poly-GR- and poly-PR-mediated toxicity. However, poly-GA is the most abundant DPR in patient tissue. Transmission of protein aggregates may be a major driver of toxicity in neurodegeneration. In this study, I show for the first time that only poly-GA DPRs can spread trans-neuronally in vivo using the adult fly brain. Repeat length and tissue age modulate this phenomenon, and exosomes and synaptic vesicles are relevant in the extracellular release of GA DPRs. I also compared the toxicity, aggregation and cellular responses of GA100 DPRs carrying or not commonly used tags. Expression of tagged GA100 was markedly less toxic. GA100 tagged with GFP and mCherry exhibited aggregation differences and failed to cause DNA damage or proteostasis stress compared to untagged GA100 and GA100FLAG. These findings highlight the need to use untagged DPRs as controls when investigating their pathobiology. Finally, I tested the role of repeat size in modulating GA toxicity, subcellular localization, aggregation and cellular responses by comparing these in flies expressing untagged GA100, GA200 and GA400 DPRs. While aggregation propensity and proteostasis stress hold a positive correlation with repeat length, and GA400 was markedly more toxic than GA100, the latter was in turn more toxic than GA200. This highlights a non-linear correlation between repeat length and toxicity. GA100 and GA200 formed numerous puncta-like aggregates both in the soma and axons of neurons and, especially GA200, exhibited spreading, whereas GA400 resided only in somata and did not spread. Surprisingly, GA200 caused more DNA damage than GA100, but this effect was not observed upon GA400 expression. Collectively, I show that GA DPRs have a unique ability to spread in vivo, and their toxicity may have been previously underestimated by the use of short and tagged constructs. Therefore, my data support the further characterization of GA DPRs of a clinically relevant composition to develop strategies with therapeutic potential for C9orf72 mutation carriers.

Item Type:	Thesis (PhD thesis)
Creators:	Creators Email ORCID ORCID Put Code Morón Oset, Javier javier.moron@age.mpg.de orcid.org/0000-0002-2230-0288 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-640489
Date:	2 December 2022
Publisher:	University of Cologne
Place of Publication:	University of Cologne
Language:	English
Faculty:	External institution
Divisions:	Außeruniversitäre Forschungseinrichtungen > MPI for Biology of Ageing
Subjects:	Life sciences
Uncontrolled Keywords:	Keywords Language Neurodegeneration English Drosophila English C9orf72 English
Date of oral exam:	2 December 2020
Referee:	Name Academic Title Partridge, Linda Professor
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/64048