The mitochondrial mutation G11778A encodes a missense mutation in complex I of the electron transport chain. This mutation is associated with a maternally inherited form of blindness known as Leber's Hereditary Optic Neuropathy (LHON). Vision loss in LHON results from a selective degeneration of the retinal ganglion cells (RGC) which convey visual information from the retina to vision centers in the brain. In the century and a half since it's description, a tremendous amount of information has been generated regarding the genetics of LHON and mitochondrial diseases in general. However, our understanding of the pathophysiological mechanisms underlying the loss of RGC in LHON, and developments of effective treatments has lagged considerably behind. This is largely a consequence of the difficulty of modeling mitochondrial diseases due to the unique nature of mitochondrial DNA. Experiments were designed to thoroughly characterize the phenotype of patient-derived fibroblasts harbouring the G11778A mutation of LHON as a means of assessing the utility of this experimental model. Cytoplasmic ATP and production of reactive oxygen species (ROS) were assayed by genetically encoded biosensors. Mitochondrial morphology was assessed by quantitative network morphology analysis while ultrastructure was examined by transmission electron microscopy. Additionally, colocalization of mitochondrially targeted fluorescent markers with fluorescent markers for autophagosomes and lysosomes was used to assay mitochondrial turnover. Patient-derived fibroblasts harbouring the G11778A mutation faithfully replicate a phenotype consistent with LHON. These changes are revealed by transition from glucose containing media to galactose containing media. The G11778A mutation produces a substantial decrease in fibroblast proliferation rate in culture. This effect is masked under normal culture conditions in the presence of glucose but is unmasked by substitution with galactose in the culture medium. Galactose-replacement also produces a bioenergetic deficit after 24 hours in culture and an increased sensitivity to exogenous ROS. Decline in ATP corresponds to a failure in compensatory mitophagy, which is increased initially but declines by 48 hours. Furthermore, mitochondria from LHON fibroblasts exhibit an impairment in morphological plasticity being unable to recapitulate the changes observed in control fibroblasts upon transition to galactose.
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Thesis advisor: Rintoul, Gordon
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