| Popis: |
Mitochondrial diseases affect the core energy generating pathways and can result in significant cognitive impairment and neurodegeneration. The mechanisms underlying the neurological and pathological consequences of mitochondrial disease are not understood and current treatment of mitochondrial diseases is limited in scope and efficacy. pyruvate dehydrogenase (PDH) and Complex I are key mitochondrial enzymes pivotal for cellular energy metabolism, and mutations in genes coding for PDH and Complex I proteins are common causes of mitochondrial disease. This thesis describes the use oftwo techniques, gene targeting, and RNA interference, to generate in vitro models of Complex I and PDH deficiency in pluripotent cell lines. Gene targeting of the ndufal gene was unsuccessful, however, a number of cell lines were isolated with significant reductions in PDH activity mediated by RNAi targeted against the Pdhal transcript. These cells lines were neurodifferentiated in vitro and used to characterise the developmental and degenerative consequences of PDH deficiency on neural cultures. The cultures were found to successfully differentiate into neural cells, but were developmentally abnormal with defective neuronal migration and neurite extension, and neuritic varicosities, an indication of neuritic degeneration in many neurological disease models. These features were activity-dependent with the most severe phenotype found in the cell line with the least residual PDH activity. These cultures were used to explore the nature of energy metabolism in PDH deficient neurons, and therapeutic strategies were successfully tested. This research has successfully established a reproducible and practical model to assess neurodegeneration in PDH deficiency, to test t reatments and to model theories of disease mechanisms. As such it provides promise for the improvement of the understanding of and treatment of disorders of brain energy metabolism in the future. |
