Paediatrics (RCH) - Theses
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ItemDevelopment, characterisation and nicotinamide riboside treatment of Complex I-deficient model systems( 2018)Mitochondrial disorders are the most common inborn error of metabolism, affecting 1 in 5000 live births. Complex I (CI) de ciency is the most common cause of paediatric mitochondrial disease. Deleterious mutations in CI subunits vary in their pathological presentations and current treatments available for these disorders are limited. Investigations here focused on the CI subunits NDUFS4 and NDUFS6, specifi cally the impact of loss on different model systems and response of these models to the NAD+ precursor nicotinamide riboside (NR). NDUFS4 mutations in humans are typically associated with the progressive neurodegenerative disease Leigh Syndrome and early fatality, and Ndufs4fky=fky knockout mice have a similar progressive encephalopathy. Human NDUFS6 mutations are associated with acidosis and neonatal mortality, while the Ndufsgt=gt (gene-trap) mice have a cardiac phenotype resulting from tissue-specific c splicing of NDUFS6. Isolated mitochondria from these mice were investigated alongside a comprehensive characterisation of NDUFS4KO and NDUFS6KO HEK293T cell lines, providing a baseline for treatment investigations. As CI deficiency results in a disruption of the NAD+/NADH ratio, a range of NAD+ precursors have been investigated to attempt to ameliorate this imbalance. Dietary supplementation with NR has shown phenotypic improvement in mouse models of mitochondrial myopathy. Here, dietary supplementation of Ndufs4fky=fky mice with 0.15% NR for 4 weeks did not significantly impact phenotypic progression or biochemical parameters. However, in vitro NR treatment of NDUFS4KO and NDUFS6KO cells resulted in bene cial biochemical changes, including increased respiratory capacity. This difference in response may be due to the models investigated. While HEK293T cells are a good starting point for investigations due to their quick growth and straightforward characterisation, clinically relevant cell types provide a much more powerful tool for investigation of potential treatments and the biochemical basis of mitochondrial disease. To enable these investigations, CRISPR/Cas9 technology was used to disrupt our genes of interest in human embryonic stem cells, thus generating mutants with the potential to form clinically relevant cell types. The NDUFS6 mutant had an apparent defect in ATP synthesis and cardiac differentiation however, supercomplex disruption was not detected. Characterisation of the NDUFS4 mutants is more preliminary and clones appeared to be using an alternative start site to produce protein. More comprehensive characterisation of all mutant lines is required to provide a clear direction for future research. This work has provided additional characterisation across a range of model systems with loss of NDUFS4 or NDUFS6, providing a clear basis for future studies into potential treatments for mitochondrial disorders. While the NR treatment investigations were inconclusive, results suggested that NR can alleviate the biochemical features of CI deficiency, and the ability of NR to meaningfully impact neural cells needs to be further elucidated. The development of human embryonic stem cell mutants will facilitate more relevant models for future treatment investigations.