Investigating primary OXPHOS Complex I deficiencies and the succeeding secondary FAO pathway defects contributing to disease pathology.

It has become increasingly evident that within mitochondrial disease, relationships between defects in oxidative phosphorylation Complex I (OXPHOS CI) pathways and fatty acid -oxidation (FAO) pathways are complex and interconnected based on emerging research. Disease pathology pertaining to these metabolic pathway defects presents itself in many forms and degrees of severity. In this study, the use of blue native polyacrylamide gel electrophoresis (BN-PAGE) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is used to observe the isolated protein complexes housed in the inner-membrane of the mitochondria and to determine the effects the knock-out mutations have on the stability of these complexes and complex subunits. The mutants used were gene knockouts of NDUFS2 (core subunit), ACAD9 (assembly factor) and NDUFV1 (core subunit), all associated with the assembly of OXPHOS CI, and chosen for the purpose of identifying what effects defects in CI have on FAO function. It has so far been established that a correlation between defects in the FAO pathway and CI stability breakdown is occurring in some of the metabolic diseases encapsulated by mitochondrial disease overall. In this case, it is thought that the primary defects in OXPHOS CI stability are the direct cause of secondary defects in the FAO pathway. Using these methods, it was determined that the expression of critical proteins essential for the stability and assembly of OXPHOS CI subunits were compromised by the gene knock-out. Steady-state levels in the CI subunit and FAO protein expression was disrupted and resulted in either total loss or crippled formation of the OXPHOS CI. Significant differences were observed between mutant cell lines and controls in both aspects of this study. Subsequent failure to oxidise fatty acids was also observed in multiple samples across both basal and maximal oxygen (O2) consumption due to the defects identified in CI as a result of the gene knockouts. This was the case using both energy sources; glucose, for OXPHOS CI respiration and palmitoyl-L-Carnitine, for FAO respiration. Findings concluded that significant reductions in FAO function occurred when CI defects were present, indicative of the enhanced disease pathology in these cases.