Investigation into the pathogenic cyclin FE624K sporadic mutation in amyotrophic lateral sclerosis and frontotemporal dementia
thesisposted on 2022-03-28, 15:43 authored by Maria de los Angeles Morales Villalva
Motor neuron disease (MND) is an umbrella term for many neurodegenerative disorders that are characterised with a range of symptoms and signs, from dysfunction to an individual's locomotion to abnormalities in cognitive functions. Two fatal neurodegenerative diseases that share molecular and pathogenic characteristics are amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which have been shown to present dysfunction in mechanisms that regulate protein homeostasis (proteostasis). Dysfunction of proteostasis may arise due to the presence of aggregated protein inclusions, potentially an underlying cause of ALS/FTD pathology. Recently, the CCNF gene has been identified to present various ALS/FTD mutants, that have been discovered in both familial and sporadic cases. The CCNF gene encodes for cyclin F, a protein involved in cell-cycle processes and ubiquitylation, specifically a module of the Skp1-Cul1-F-Box (SCF) E3 ubiquitin ligase complex, which is involved in quality control mechanisms that regulate proteostasis levels. Recent studies have focused on the familial cyclin FS621G mutation, demonstrating hyper-ubiquitylation due to autophagy pathway dysfunction and impairment of regulatory phosphorylation. However, further research is needed to identify whether other CCNF mutants, such as the sporadic cyclin FE624K mutant presents with similar or different familial ALS/FTD pathogenesis. For this thesis, the aim is to generate and characterise the cyclin FE624K mutant in HEK293 cells and investigate whether it causes any perturbations to cellular pathways as well as perturbed protein-protein interactions that may be shared or unique to the familial cyclin FS621G mutant and cyclin F wild-type. Expression of sporadic cyclin FE624K mutant revealed a decrease in phosphorylation and no change in K48-ubiquitylation of proteins. Global proteomic analysis revealed inhibition of pathways involved in actin cytoskeleton regulation. Proteomic analysis of immunoprecipitated cyclin FE624K identified proteins that clustered within a ubiquitin- and proteasome-associated protein network and proteins that clustered within the oxidative stress response. Perturbed cyclin FE624K protein interactors involved in these networks suggests dysregulation may lead to cellular proteostasis impairments and ultimately lead to neurodegeneration. Comparative analysis with the familial cyclin FS621G mutant elucidates differences in cellular mechanisms between sporadic and familial mutations that govern ALS/FTD pathology.