The broad, long-term objective of this project is to determine the pathophysiological mechanism of Leber's Hereditary Optic Neuropathy (LHON), in the hopes that a mechanistic understanding of this disease will lead to more effective therapy. LHON is the result of the inheritance of primary mutations of the mtDNA in three different subunits of Complex I of the mitochondrial respiratory chain. We have recently demonstrated that the most common LHON pathogenic mutation can be transferred into human Nt2 cells and are differentiable into neuron-like cells. We have also observed in earlier work that LHON cells appear to be activated for apoptosis. Thus there are six Specific Aims: 1) to construct all three LHON mutations in a cell environment differentiable into neurons, and measure the effects of differentiation, of respiratory substrates, and of induction of electron transport chain activity on viability in the neuronal environment; 2) to measure the bioenergetic effects of differentiation into a neuronal environment in cells and mitochondria, by mitochondrial membrane potential, oxygen consumption, and reduction of Alamar blue; 3) To measure the apoptotic activity of Nt2 cells and neurons bearing the LHON mutations, and to determine if a particular step in this pathway is activated in LHON cells; 4) to determine if neuron-specific excitotoxic mechanisms are activated in LHON neurons; 5) to determine if LHON mutations exert a concerted effect on gene expression, by array analysis of mRNA levels in mutant and control neurons, and 6) based on the results of Aims 1-5, to integrate and confirm the understanding of the LHON pathophysiological mechanism by rescue with mechanism-specific pharmacological agents, including bioenergetic supplements, anti- apoptotic agents, glutamate antagonists, antioxidants and chelators.
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