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Characterisation of the Cyclin F and Sequestosome-1/p62 protein interaction and its contribution to ALS/FTD pathogenesis

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posted on 2023-11-26, 23:09 authored by Jennilee Mary-Ann Davidson

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases, part of the motor neuron disease (MND) spectrum, that share genetic and pathologic features which lead to the rapid degeneration of motor neurons in the brain and spinal cord. Thus, at both a clinical and molecular level, patients may present with pure ALS, pure FTD, or a combination thereof (ALS/FTD). Only about 10% of cases are genetically linked, whereas the remaining 90% are classified as sporadic. Despite the unknown aetiology, a characteristic hallmark of ALS/FTD pathology is the presence of insoluble protein aggregates. This implicates the protein clearance pathways, the ubiquitin proteasome system (UPS) and autophagy in the pathogenesis of ALS/FTD. 

An ALS/FTD-causing mutation in the gene encoding Cyclin F, CCNF S621G, causes impairments in the UPS and autophagy pathways, and interacts with the key autophagy receptor, Sequestosome-1/p62 (p62). However, there was no experimental evidence on the functional relationship between Cyclin F and p62, neither on the co-causality between the S621G mutation in Cyclin F affecting p62 and ALS/FTD pathogenesis. This study sought to determine whether this was a direct protein interaction and uncover its functional role, to reveal whether Cyclin F S621G affects p62, and ultimately how they may contribute to ALS/FTD pathogenesis.

Cyclin F is a component of the Cyclin F-E3 ligase complex (SCFCyclin F) which facilitates the ubiquitylation of substrates for proteasomal turnover. At the commencement of this PhD project, it was not known whether Cyclin F and p62 were direct protein interactors. Accordingly, the overarching aims of this thesis concern whether p62 is a direct ubiquitylation substrate of Cyclin F, the effect of the mutant Cyclin F S621G on p62 homeostasis, and how Cyclin F S621G is involved in ALS/FTD pathogenesis. Experimentally, this involved a series of mechanistic and biochemical studies, proteomic workflows and bioinformatic analyses. Various ALS/FTD relevant models were used including neuronal-like cells and primary neurons, as well as induced pluripotent stem cells derived from S621G patient fibroblasts and a mouse model, which both employed CRISPR/Cas9 technology to study the effect of the missense S621G mutation. Taken together, results demonstrate a novel role that Cyclin F is involved in the crosstalk between the UPS and autophagy pathways both under basal conditions and in response to proteasome inhibition, through regulating p62, and the effect of the mutant Cyclin F S621G. Notably, this work provides a direct mechanistic link between dysregulated Cyclin F-mediated ubiquitylation of p62 and p62 dyshomeostasis. This involves defective p62 proteasomal turnover and altered aggregation propensity in neurons. This thesis is the first experimental work to provide evidence of how the ALS/FTD-mutant Cyclin F S621G directly affects the proteome in the brain, including p62, by using a mouse model. Finally, this study provides strong evidence of an ALS/FTD-like molecular phenotype in the CRISPR/Cas Cyclin F S621G mouse brain and propose this may be reflective of a pre-symptomatic model. By shining light on the dysregulated Cyclin F and p62 protein interaction, this work expands our understanding of the molecular underpinnings of ALS/FTD pathogenesis.


Table of Contents

Chapter 1: General Introduction -- Chapter 2: General Methods -- Chapter 3: P62/Sequestosome-1 Is a Novel and Direct Ubiquitylation Substrate of Cyclin F for Proteasome-Dependent Turnover -- Chapter 4: Discerning the Effect of the Cyclin F S621G ALS/FTD Disease-Variant on P62 Homeostasis -- Chapter 5: ALS/FTD-Linked Cyclin F S621G Causes an ALS/FTD -Like Molecular Phenotype In Vivo -- Chapter 6: General Discussion -- Chapter 7: Supplementary -- Chapter 8: Appendix

Awarding Institution

Macquarie University

Degree Type

Thesis PhD


Doctor of Philosophy

Department, Centre or School

Department of Biomedical Sciences

Year of Award


Principal Supervisor

Roger Chung

Additional Supervisor 1

Albert Lee


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321 pages

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