MOLECULAR REMEDY OF MITOCHONDRIAL DEFECTS

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It has been reported that many human diseases are associated with defects in the mitochondrial protontranslocating NADH-ubiquinone (UQ) oxidoreductase, also known as complex I. Defects in complex I, which render it dysfunctional, result in the following three problems: (1) impaired ability of the respiratory chain to oxidize NADH back to NAD; (2) impaired ability of this enzyme to pump protons; (3) production of reactive oxygen species (ROS). The overall goal of this grant application is to identify potential areas of treatment and/or remedies for the diseases that result from dysfunctional complex I. Of the three problems described above, impairment of proton pumping at any one of the three proton translocation sites does not appear to present a severe health hazard when compared to the inability of mitochondria to oxidize NADH and/or damage caused by ROS. Yeast (Saccharomyces cerevisiae) mitochondria lack complex I but contain instead a NADH-UQ oxidoreductase composed of a single- subunit (Ndi1). In an initial attempt to tackle the problems associated with dysfunctional complex I, we have attempted to employ the yeast Ndil to transmit electrons from NADH to UQ in mammalian mitochondria lacking a functional complex I. We have demonstrated that the Ndil can be functionally expressed in complex I-deficient Chinese hamster mutant cells (CCL16-B2), complex I deficient human cells (C4T), and human embryonal kidney 293 cells (HEK 293). In all cases the expressed Ndil was correctly localized in the mitochondria. These results indicate that the ND11 gene provides a potentially useful tool for gene therapy of mitochondrial diseases caused by complex I deficiency. The studies planned during this grant period are as follows. (1) Functional expression of the ND11 gene in growth-arrested mammalian cells. (2) Suppression of the ROS in mammalian mitochondria by ND11 transfection. (3) Construction of transgenic mice containing the ND11 gene. (4) Repair of a point mutation in the NDUFA1 gene encoding the MWFE subunit of complex I.