Genetic and Functional Characterisation of ALS-linked Genes TBK1, OPTN and MATR3

Student thesis: Doctoral ThesisDoctor of Philosophy


Amyotrophic Lateral Sclerosis (ALS) is a degenerative disease predominantly affecting motor neurons in the brain and spinal cord. ALS has a clear genetic component, with mutations in four genes accounting for ~50% of familial and ~10% of sporadic cases. Several other rarer ALS genes have been identified but their mechanistic role in causing TDP-43 proteinopathy is unknown. Exome sequencing of 699 index familial ALS cases identified mutations in three recently discovered ALS genes: 16 in Tank Binding Kinase 1 (TBK1), a kinase involved in inflammation and autophagy; five in Matrin 3 (MATR3), a DNA/RNA binding protein; and one in Optineurin (OPTN) in a consanguineous Palestinian family with an aggressive form of ALS. The experiments described in this thesis sought to explore the functional impact of these novel ALS-associated variants with the aim of determining their potential pathogenicity and providing insights into disease mechanisms.
Here I show that missense and nonsense TBK1 mutations can disrupt its kinase activity by interfering with homodimer formation and phosphorylation of its targets IRF3, OPTN and itself, pointing towards a common loss of kinase function mechanism. Furthermore, I show that the novel OPTN p.S174X truncation mutation causes a complete loss of OPTN protein expression in homozygous, and haploinsufficiency in heterozygous patient fibroblasts due to nonsense mediated decay. Heterozygotes show a minor increase in the mitochondrial markers TOM20 and COXIV and increased cellular respiration under a stress test but do not present any striking mitophagy or autophagy defects and homozygous fibroblasts are apparently unaffected. Lastly I explore the effects of MATR3 mutations on protein solubility which was decreased for the p.S85C mutant and that p.R841C forms cytoplasmic inclusions, however no phenotype was elicited for two other mutations.
The findings in this thesis have made a modest contribution to our knowledge of the molecular mechanisms underlying the pathogenesis of ALS, the understanding of which is pivotal in order to develop more effective therapies for this dreadful disease.
Date of Award2018
Original languageEnglish
Awarding Institution
  • King's College London
SupervisorBradley Smith (Supervisor) & Christopher Shaw (Supervisor)

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