Research in recent years has shown that structural and functional defects of complex I are involved in many human diseases. These diseases include Parkinson's disease, dystonia, severe lactic acidosis, various encepharomyopathies, Leber's hereditary optic neuropathy, and probably Huntington's disease. Dysfunction of complex I causes three problems: (1) impairment of the ability of the respiratory chain to oxidize NADH back to NAD; (2) impairment of the ability of complex I to pump protons, resulting in a decrease in ATP synthesis; (3) production of superoxide radicals. Our overall goal is to find a remedy for the diseases caused by dysfunction of complex I.

Of the three problems, impairment of proton pumping by one of the three proton translocation sites does not appear to be as severe a health hazard as the inability of mitochondria to oxidize NADH and damage by superoxide production. Yeast (Saccharomyces cerevisiae) mitochondria lack complex I and contain instead an NADH dehydrogenase composed of a single subunit (Ndi1). We intend to use yeast Ndi1 enzyme to transmit electrons from NADH to ubiquinone-10 in mammalian mitochondria that lack a functional complex I.

Using the complex I deficient Chinese hamster CCL16-B2 mutant cells, we were able to express NDI1 gene by transfection. The expressed Ndi1 enzyme was localized into mitochondria and functionally active. The transfected cells, but not non-transfected control cells, exhibited the electron transfer activities with glutamate/malate as the respiratory substrates. Therefore, it is concluded that the NDI1 gene provides a potentially useful tool for gene therapy of mitochondrial disease caused by complex I deficiency.

It is important to verify functional expression of Ndi1 in human cells. For this purpose, the NDI1 gene was stably transfected into the HEK 293 cells. The transfected NDI1 gene was transcribed and translated in the HEK 293 cells to produce the functional enzyme. The immunochemical and immunofluorescence analyses indicated that the expressed Ndi1 polypeptide was located to the inner mitochondrial membranes. The expression of Ndi1 did not alter the content of existing complex I in the HEK 293 mitochondria, suggesting that the expressed Ndi1 enzyme does not displace the endogenous complex I. The NDI1-transfected HEK 293 cells were able to grow in media containing a complex I inhibitor such as rotenone and MPP⁺ (1-methy-4-phenylpyridium ion). These results strongly support the potential usefulness of NDI1 gene to repair complex I deficiency in human cells.

Since our goal is to use the NDI1 gene as a therapeutic agent to rescue complex I defects, a next step is to attempt in vivo expression of the Ndi1 enzyme in tissues. We carried out in vivo experiments using rodents to investigate whether the Ndi1 can be introduced in tissues in functional form. We injected a recombinant adeno-associated virus carrying the NDI1 gene into skeletal muscles and brain (substantia nigra and striatum). In all tissues tested, the Ndi1 protein was identified in the injected area by immunostaining at 1-2 weeks after the injection.

The expressed Ndi1 was localized to mitochondria. In addition, the tissue cells expressing the Ndi1 protein stimulated the NADH dehydrogenase activity, suggesting that the expressed Ndi1 is functionally active. It was also confirmed that the Ndi1 expression induced no inflammatory response in the tissues examined. The data indicate that the NDI1 gene will be a promising therapeutic tool in the treatment of encephalomyopathies and neurodegenerative diseases caused by complex I defects.