Use of the NADH-quinone oxidoreductase (NDI1) gene of Saccharomyces cerevisiae as a possible cure for complex I defects in human cells

The Ndi1 enzyme of Saccharomyces cerevisiae is a single subunit rotenone-insensitive NADH-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane. We have shown previously that the NDI1 gene can be functionally expressed in Chinese hamster cells (Seo, B. B., Kitajima-Ihara, T., Chan, E. K., Scheffler, I. E., Matsuno-Yagi, A., and Yagi, T. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9167-9171) and human embryonal kidney 293 (HEK 293) cells (Seo, B. B., Matsuno-Yagi, A., and Yagi, T. (1999) Biochim. Biochem. Acta 1412, 56-65) and that the Ndi1 protein is capable of compensating respiratory deficiencies caused by defects in the host NADH-quinone oxidoreductase (complex I). To extend the potential use of this enzyme to repair complex I deficiencies in vivo, we constructed a recombinant adeno-associated virus vector carrying the NDI1 gene (rAAV-NDI1). With rAAV-NDI1 as the gene delivery method, we were able to achieve high transduction efficiencies (nearly 100%) even in 143B cells that are difficult to transfect by lipofection or calcium phosphate precipitation methods. The NDI1 gene was successfully introduced into non-proliferating human cells using rAAV-NDI1. The expressed Ndi1 protein was shown to be functionally active just as seen for proliferating cells. Furthermore, when cells were cultured under the conditions where energy has to be provided by respiration, the NDI1-transduced cells were able to grow even in the presence of added complex I inhibitor such as rotenone and 1-methyl-4-phenylpyridinium ion. In contrast, control cells that did not receive the NDI1 gene failed to survive as anticipated. The Ndi1 protein has a great potential as a molecular remedy for complex I defects, and it is highly likely that the same strategy can be extended to correction of other mitochondrial disorders.     

In vivo complementation of complex I by the yeast Ndi1 enzyme. Possible application for treatment of Parkinson disease

Recent studies suggest that dysfunction of the NADH-quinone oxidoreductase (complex I) is associated with a number of human diseases, including neurodegenerative disorders such as Parkinson disease. We have shown previously that the single subunit rotenone-insensitive NADH-quinone oxidoreductase (Ndi1) of Saccharomyces cerevisiae mitochondria can restore NADH oxidation in complex I-deficient mammalian cells. The Ndi1 enzyme is insensitive to complex I inhibitors such as rotenone and 1-methyl-4-phenylpyridinium ion, known as a metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). To test the possible use of the NDI1 gene as a therapeutic agent in vivo, we chose a mouse model of Parkinson disease. The NDI1-recombinant adeno-associated virus particles (rAAV-NDI1) were injected unilaterally into the substantia nigra of mice. The animals were then subjected to treatment with MPTP. The degree of neurodegeneration in the nigrostriatal system was assessed immunohistochemically through the analysis of tyrosine hydroxylase and glial fibrillary acidic protein. It was evident that the substantia nigra neurons on the side used for injection of rAAV-NDI1 retained a high level of tyrosine hydroxylase-positive cells, and the ipsilateral striatum exhibited significantly less denervation than the contralateral striatum. Furthermore, striatal concentrations of dopamine and its metabolites in the hemisphere that received rAAV-NDI1 were substantially higher than those of the untreated hemisphere, reaching more than 50% of the normal levels. These results indicate that the expressed Ndi1 protein elicits resistance to MPTP-induced neuronal injury. The present study is the first successful demonstration of complementation of complex I by the Ndi1 enzyme in animals.     

Xenotransplantation of mitochondrial electron transfer enzyme, Ndi1, in myocardial reperfusion injury

A significant consequence of ischemia/reperfusion (I/R) is mitochondrial respiratory dysfunction, leading to energetic deficits and cellular toxicity from reactive oxygen species (ROS). Mammalian complex I, a NADH-quinone oxidoreductase enzyme, is a multiple subunit enzyme that oxidizes NADH and pumps protons across the inner membrane. Damage to complex I leads to superoxide production which further damages complex I as well as other proteins, lipids and mtDNA. The yeast, S. cerevisiae, expresses internal rotenone insensitive NADH-quinone oxidoreductase (Ndi1); a single 56 kDa polypeptide which, like the multi-subunit mammalian complex I, serves as the entry site of electrons to the respiratory chain, but without proton pumping. Heterologous expression of Ndi1 in mammalian cells results in protein localization to the inner mitochondrial membrane which can function in parallel with endogenous complex I to oxidize NADH and pass electrons to ubiquinone. Expression of Ndi1 in HL-1 cardiomyocytes and in neonatal rat ventricular myocytes protected the cells from simulated ischemia/reperfusion (sI/R), accompanied by lower ROS production, and preservation of ATP levels and NAD+/NADH ratios. We next generated a fusion protein of Ndi1 and the 11aa protein transduction domain from HIV TAT. TAT-Ndi1 entered cardiomyocytes and localized to mitochondrial membranes. Furthermore, TAT-Ndi1 introduced into Langendorff-perfused rat hearts also localized to mitochondria. Perfusion of TAT-Ndi1 before 30 min no-flow ischemia and up to 2 hr reperfusion suppressed ROS production and preserved ATP stores. Importantly, TAT-Ndi1 infused before ischemia reduced infarct size by 62%; TAT-Ndi1 infused at the onset of reperfusion was equally cardioprotective. These results indicate that restoring NADH oxidation and electron flow at reperfusion can profoundly ameliorate reperfusion injury.     

The single subunit NADH dehydrogenase reduces generation of reactive oxygen species from complex I

Using rat dopaminergic and human neuroblastoma cell lines transduced with the NDI1 gene encoding the internal NADH dehydrogenase (Ndi1) from Saccharomyces cerevisiae, we investigated reactive oxygen species (ROS) generation caused by complex I inhibition. Incubation of non-transduced cells with rotenone elicited oxidative damage to mitochondrial DNA as well as lipid peroxidation. In contrast, oxidative stress was significantly decreased when the cells were transduced with NDI1. Furthermore, mitochondria from the NDI1-transduced cells showed a suppressed rate of ROS formation by the complex I inhibitors. We conclude that the Ndi1 enzyme is able to suppress ROS overproduction from defective complex I.     

Protection by the NDI1 gene against neurodegeneration in a rotenone rat model of Parkinson's disease

It is widely recognized that mitochondrial dysfunction, most notably defects in the NADH-quinone oxidoreductase (complex I), is closely related to the etiology of sporadic Parkinson's disease (PD). In fact, rotenone, a complex I inhibitor, has been used for establishing PD models both in vitro and in vivo. A rat model with chronic rotenone exposure seems to reproduce pathophysiological conditions of PD more closely than acute mouse models as manifested by neuronal cell death in the substantia nigra and Lewy body-like cytosolic aggregations. Using the rotenone rat model, we investigated the protective effects of alternative NADH dehydrogenase (Ndi1) which we previously demonstrated to act as a replacement for complex I both in vitro and in vivo. A single, unilateral injection of recombinant adeno-associated virus carrying the NDI1 gene into the vicinity of the substantia nigra resulted in expression of the Ndi1 protein in the entire substantia nigra of that side. It was clear that the introduction of the Ndi1 protein in the substantia nigra rendered resistance to the deleterious effects caused by rotenone exposure as assessed by the levels of tyrosine hydroxylase and dopamine. The presence of the Ndi1 protein also prevented cell death and oxidative damage to DNA in dopaminergic neurons observed in rotenone-treated rats. Unilateral protection also led to uni-directional rotation of the rotenone-exposed rats in the behavioral test. The present study shows, for the first time, the powerful neuroprotective effect offered by the Ndi1 enzyme in a rotenone rat model of PD.     

Mechanism of cell death caused by complex I defects in a rat dopaminergic cell line

Defects in the proton-translocating NADH-quinone oxidoreductase (complex I) of mammalian mitochondria are linked to neurodegenerative disorders. The mechanism leading to cell death elicited by complex I deficiency remains elusive. We have shown that expression of a rotenone-insensitive yeast NADH-quinone oxidoreductase (Ndi1) can rescue mammalian cells from complex I dysfunction. By using the Ndi1 enzyme, we have investigated the key events in the process of cell death using a rat dopaminergic cell line, PC12. We found that complex I inhibition provokes the following events: 1) activation of specific kinase pathways; 2) release of mitochondrial proapoptotic factors, apoptosis inducing factor, and endonuclease G. AS601245, a kinase inhibitor, exhibited significant protection against these apoptotic events. The traditional caspase pathway does not seems to be involved because caspase 3 activation was not observed. Our data suggest that overproduction of reactive oxygen species (ROS) caused by complex I inhibition is responsible for triggering the kinase activation, for the release of the proapoptotic factors, and then for cell death. Nearly perfect prevention of apoptotic cell death by Ndi1 agrees with our earlier observation that the presence of Ndi1 diminishes rotenone-induced ROS generation from complex I. In fact, this study demonstrated that Ndi1 keeps the redox potential high even in the presence of rotenone. Under these conditions, ROS formation by complex I is known to be minimal. Possible use of our cellular model is discussed with regard to development of therapeutic strategies for neurodegenerative diseases caused by complex I defects.     

Expression of Ndi1p, an alternative NADH:ubiquinone oxidoreductase, increases mitochondrial membrane potential in a C. elegans model of mitochondrial disease

The NADH:ubiquinone oxidoreductase or complex I of the mitochondrial respiratory chain is an intricate enzyme with a vital role in energy metabolism. Mutations affecting complex I can affect at least three processes; they can impair the oxidation of NADH, reduce the enzyme's ability to pump protons for the generation of a mitochondrial membrane potential and increase the production of damaging reactive oxygen species. We have previously developed a nematode model of complex I-associated mitochondrial dysfunction that features hallmark characteristics of mitochondrial disease, such as lactic acidosis and decreased respiration. We have expressed the Saccharomyces cerevisiae NDI1 gene, which encodes a single subunit NADH dehydrogenase, in a strain of Caenorhabditis elegans with an impaired complex I. Expression of Ndi1p produces marked improvements in animal fitness and reproduction, increases respiration rates and restores mitochondrial membrane potential to wild type levels. Ndi1p functionally integrates into the nematode respiratory chain and mitigates the deleterious effects of a complex I deficit. However, we have also shown that Ndi1p cannot substitute for the absence of complex I. Nevertheless, the yeast Ndi1p should be considered as a candidate for gene therapy in human diseases involving complex I.     

Parkinson’s disease and mitochondrial complex I: a perspective on the Ndi1 therapy

Mitochondrial impairment has been collecting more and more attention as a contributing factor to the etiology of Parkinson’s disease. Above all, the NADH-quinone oxidoreductase, complex I, of the respiratory chain seems to be most culpable. Complex I dysfunction is translated to an increased production of reactive oxygen species and a decreased energy supply. In the brain, the dopaminergic neurons are one of the most susceptible cells. Their death is directly linked to the disease apparition. Developing an effective gene therapy is challenged by harmful actions of reactive oxygen species. To overcome this problem a therapeutic candidate must be able to restore the NADH-quinone oxidoreductase activity regardless of how complex I is impaired. Here we discuss the potency of the yeast alternative NADH dehydrogenase, the Ndi1 protein, to reinstate the mitochondrial respiratory chain compensating for disabled complex I and the benefit Ndi1 brings toward retardation of Parkinson’s disease.     

Expression of Ndi1p, an alternative NADH:ubiquinone oxidoreductase, increases mitochondrial membrane potential in a C. elegans model of mitochondrial disease

The NADH:ubiquinone oxidoreductase or complex I of the mitochondrial respiratory chain is an intricate enzyme with a vital role in energy metabolism. Mutations affecting complex I can affect at least three processes; they can impair the oxidation of NADH, reduce the enzyme's ability to pump protons for the generation of a mitochondrial membrane potential and increase the production of damaging reactive oxygen species. We have previously developed a nematode model of complex I-associated mitochondrial dysfunction that features hallmark characteristics of mitochondrial disease, such as lactic acidosis and decreased respiration. We have expressed the Saccharomyces cerevisiae NDI1 gene, which encodes a single subunit NADH dehydrogenase, in a strain of Caenorhabditis elegans with an impaired complex I. Expression of Ndi1p produces marked improvements in animal fitness and reproduction, increases respiration rates and restores mitochondrial membrane potential to wild type levels. Ndi1p functionally integrates into the nematode respiratory chain and mitigates the deleterious effects of a complex I deficit. However, we have also shown that Ndi1p cannot substitute for the absence of complex I. Nevertheless, the yeast Ndi1p should be considered as a candidate for gene therapy in human diseases involving complex I.     

Successful Amelioration of Mitochondrial Optic Neuropathy Using the Yeast NDI1 Gene in a Rat Animal Model

Background

Leber''s hereditary optic neuropathy (LHON) is a maternally inherited disorder with point mutations in mitochondrial DNA which result in loss of vision in young adults. The majority of mutations reported to date are within the genes encoding the subunits of the mitochondrial NADH-quinone oxidoreductase, complex I. Establishment of animal models of LHON should help elucidate mechanism of the disease and could be utilized for possible development of therapeutic strategies.

Methodology/Principal Findings

We established a rat model which involves injection of rotenone-loaded microspheres into the optic layer of the rat superior colliculus. The animals exhibited the most common features of LHON. Visual loss was observed within 2 weeks of rotenone administration with no apparent effect on retinal ganglion cells. Death of retinal ganglion cells occurred at a later stage. Using our rat model, we investigated the effect of the yeast alternative NADH dehydrogenase, Ndi1. We were able to achieve efficient expression of the Ndi1 protein in the mitochondria of all regions of retinal ganglion cells and axons by delivering the NDI1 gene into the optical layer of the superior colliculus. Remarkably, even after the vision of the rats was severely impaired, treatment of the animals with the NDI1 gene led to a complete restoration of the vision to the normal level. Control groups that received either empty vector or the GFP gene had no effects.

Conclusions/Significance

The present study reports successful manifestation of LHON-like symptoms in rats and demonstrates the potential of the NDI1 gene therapy on mitochondrial optic neuropathies. Our results indicate a window of opportunity for the gene therapy to be applied successfully after the onset of the disease symptoms.     

NADH Dehydrogenases: From Basic Science to Biomedicine

This review article is concerned with two on-going research projects in our laboratory, both of which are related to the study of the NADH dehydrogenase enzyme complexes in the respiratory chain. The goal of the first project is to decipher the structure and mechanism of action of the proton-translocating NADH-quinone oxidoreductase (NDH-1) from two bacteria, Paracoccus denitrificans and Thermus thermophilus HB-8. These microorganisms are of particular interest because of the close resemblance of the former (P. denitrificans) to a mammalian mitochondria, and because of the thermostability of the enzymes of the latter (T. thermophilus). The NDH-1 enzyme complex of these and other bacteria is composed of 13 to 14 unlike subunits and has a relatively simple structure relative to the mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I), which is composed of at least 42 different subunits. Therefore, the bacterial NDH-1 is believed to be a useful model for studying the mitochondrial complex I, which is understood to have the most intricate structure of all the membrane-associated enzyme complexes. Recently, the study of the NADH dehydrogenase complex has taken on new urgency as a result of reports that complex I defects are involved in many human mitochondrial diseases. Thus the goal of the second project is to develop possible gene therapies for mitochondrial diseases caused by complex I defects. This project involves attempting to repair complex I defects in the mammalian system using Saccharomyces cerevisiae NDI1 genes, which code for the internal, rotenone-insensitive NADH–quinone oxidoreductase. In this review, we will discuss our progress and the data generated by these two projects to date. In addition, background information and the significance of various approaches employed to pursue these research objectives will be described.     

Yeast NDI1 improves oxidative phosphorylation capacity and increases protection against oxidative stress and cell death in cells carrying a Leber's hereditary optic neuropathy mutation

G11778A in the subunit ND4 gene of NADH dehydrogenase complex is the most common primary mutation found in Leber's hereditary optic neuropathy (LHON) patients. The NDI1 gene, which encodes the internal NADH-quinone oxidoreductase in Saccharomyces cerevisiae, was introduced into the nuclear genome of a mitochondrial defective human cell line, Le1.3.1, carrying the G11778A mutation. In transformant cell lines, LeNDI1-1 and -2, total and complex I-dependent respiration were fully restored and largely resistant to complex I inhibitor, rotenone, indicating a dominant role of NDI1 in the transfer of electrons in the host cells. Whereas the original mutant Le1.3.1 cell grows poorly in medium containing galactose, the transformants have a fully restored growth capacity in galactose medium, although the ATP production was not totally recovered. Furthermore, the increased oxidative stress in the cells carrying the G11778A mutation was alleviated in transformants, demonstrated by a decreased reactive oxygen species (ROS) level. Finally, transformants were also shown to be desensitized to induction to apoptosis and also exhibit greater resistance to paraquat-induced cell death. It is concluded that the yeast NDI1 enzyme can improve the oxidative phosphorylation capacity in cells carrying the G11778A mutation and protect the cells from oxidative stress and cell death.     

Metformin suppresses glucose-6-phosphatase expression by a complex I inhibition and AMPK activation-independent mechanism

Metformin is widely used as a hypoglycemic agent for the treatment of type 2 diabetes. Both metformin and rotenone, an inhibitor of respiratory chain complex I, suppressed glucose-6-phosphatase (G6pc), a rate limiting enzyme of liver glucose production, mRNA expression in a rat hepatoma cell line accompanied by a reduction of intracellular ATP concentration and an activation of AMP-activated protein kinase (AMPK). When yeast NADH-quinone oxidoreductase 1 (NDI1) gene was introduced into the cells, neither inhibition of ATP synthesis nor activation of AMPK was induced by these agents. Interestingly, in contrast to rotenone treatment, G6pc mRNA down-regulation was observed in the NDI1 expressing cells after metformin treatment. Since NDI1 can functionally complement the complex I under the presence of metformin or rotenone, our results indicate that metformin induces down-regulation of G6pc expression through an inhibition of complex I and an activation of AMPK-independent mechanism.     

Assay of mitochondrial respiratory chain complex I in human lymphocytes and cultured skin fibroblasts

Respiratory chain complex I (NADH:ubiquinone oxidoreductase) deficiency is one of the most frequent causes of mitochondrial disease in humans. The activity of this complex can be confidently measured in most tissue samples, but not in cultured skin fibroblasts or circulating lymphocytes. Highly contaminating non-mitochondrial NADH-quinone oxidoreductase activity in fibroblasts and the limited access of substrates to complex I in lymphocytes hinder its measurement in permeabilized cells. Complex I assay in these cells requires the isolation of mitochondria, which in turn necessitates large quantities of cells and is not feasible when studying circulating lymphocytes. Here we report a simple method to measure complex I activity in a minute amount of either cell type. The procedure strongly reduces contaminating NADH:quinone oxidoreductase activity and permits measuring high rates of rotenone-sensitive complex I activity thanks to effective cell permeabilization.     

Lack of complex I activity in human cells carrying a mutation in MtDNA-encoded ND4 subunit is corrected by the Saccharomyces cerevisiae NADH-quinone oxidoreductase (NDI1) gene

The gene for the single subunit, rotenone-insensitive, and flavone-sensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1) can completely restore the NADH dehydrogenase activity in mutant human cells that lack the essential mitochondrial DNA (mtDNA)-encoded subunit ND4. In particular, the NDI1 gene was introduced into the nuclear genome of the human 143B.TK(-) cell line derivative C4T, which carries a homoplasmic frameshift mutation in the ND4 gene. Two transformants with a low or high level of expression of the exogenous gene were chosen for a detailed analysis. In these cells the corresponding protein is localized in mitochondria, its NADH-binding site faces the matrix compartment as in yeast mitochondria, and in perfect correlation with its abundance restores partially or fully NADH-dependent respiration that is rotenone-insensitive, flavone-sensitive, and antimycin A-sensitive. Thus the yeast enzyme has become coupled to the downstream portion of the human respiratory chain. Furthermore, the P:O ratio with malate/glutamate-dependent respiration in the transformants is approximately two-thirds of that of the wild-type 143B.TK(-) cells, as expected from the lack of proton pumping activity in the yeast enzyme. Finally, whereas the original mutant cell line C4T fails to grow in medium containing galactose instead of glucose, the high NDI1-expressing transformant has a fully restored capacity to grow in galactose medium. The present observations substantially expand the potential of the yeast NDI1 gene for the therapy of mitochondrial diseases involving complex I deficiency.     

Mitochondrial release of the NADH dehydrogenase Ndi1 induces apoptosis in yeast

Saccharomyces cerevisiae NDI1 codes for the internal mitochondrial ubiquinone oxidoreductase, which transfers electrons from NADH to ubiquinone in the respiratory chain. Previously we found that Ndi1 is a yeast homologue of the protein apoptosis-inducing factor–homologous mitochondrion-associated inducer of death and displays potent proapoptotic activity. Here we show that S. cerevisiae NDI1 is involved in apoptosis induced by various stimuli tested, including H₂O₂, Mn, and acetate acid, independent of Z-VAD-fmk (a caspase inhibitor) inhibition. Although Ndi1 also participates in respiration, its proapoptotic property is separable from the ubiquinone oxidoreductase activity. During apoptosis, the N-terminal of Ndi1 is cleaved off in the mitochondria, and this activated form then escapes out to execute its apoptotic function. The N-terminal cleavage appears to be essential for the manifestation of the full apoptotic activity, as the uncleaved form of Ndi1 exhibits much less growth-inhibitory activity. Our results thus indicate an important role of Ndi1 in the switch of life and death fates in yeast: during normal growth, Ndi1 assimilates electrons to the electron transport chain and initiates the respiration process to make ATP, whereas under stresses, it cleaves the toxicity-sequestering N-terminal cap, is released from the mitochondria, and becomes a cell killer.     

Isolation and characterization of complex I, rotenone-sensitive NADH: ubiquinone oxidoreductase, from the procyclic forms of Trypanosoma brucei.

Additional characterization of complex I, rotenone-sensitive NADH:ubiquinone oxidoreductase, in the mitochondria of Trypanosoma brucei brucei has been obtained. Both proline:cytochrome c reductase and NADH:ubiquinone oxidoreductase of procyclic T. brucei were inhibited by the specific inhibitors of complex I rotenone, piericidin A, and capsaicin. These inhibitors had no effect on succinate: cytochrome c reductase activity. Antimycin A, a specific inhibitor of the cytochrome bc1 complex (ubiquinol:cytochrome c oxidoreductase), blocked almost completely cytochrome c reductase activity with either proline or succinate as electron donor, but had no inhibitory effect on NADH:ubiquinone oxidoreductase activity. The rotenone-sensitive NADH:ubiquinone oxidoreductase of procyclic T. brucei was partially purified by sucrose density centrifugation of mitochondria solubilized with dodecyl-beta-D-maltoside, with an approximately eightfold increase in specific activity compared to that of the mitochondrial membranes. Four polypeptides of the partially purified enzyme were identified as the homologous subunits of complex I (51 kDa, PSST, TYKY, and ND4) by immunoblotting with antibodies raised against subunits of Paracoccus denitrificans and against synthetic peptides predicted from putative complex I subunit genes encoded by mitochondrial and nuclear T. brucei DNA. Blue Native polyacrylamide gel electrophoresis of T. brucei mitochondrial membrane proteins followed by immunoblotting revealed the presence of a putative complex I with a molecular mass of 600 kDa, which contains a minimum of 11 polypeptides determined by second-dimensional Tricine-SDS/PAGE including the 51 kDa, PSST and TYKY subunits.     

Possibility of transkingdom gene therapy for complex I diseases

Defects of complex I are involved in many human mitochondrial diseases, and therefore we have proposed to use the NDI1 gene encoding a single subunit NADH dehydrogenase of Saccharomyces cerevisiae for repair of respiratory activity. The yeast NDI1 gene was successfully introduced into mammalian cell lines. The expressed NDI1 protein was correctly targeted to the matrix side of the inner mitochondrial membranes, was fully functional and restored the NADH oxidase activity to the complex I-deficient cells. The NDI1-transduced cells were more resistant to complex I inhibitors and diminished production of reactive oxygen species induced by rotenone. It was further shown that the NDI1 protein can be functionally expressed in tissues such as skeletal muscles and the brain of rodents, which scarcely induced an inflammatory response. The use of NDI1 as a potential molecular therapy for complex I-deficient diseases is briefly discussed, including the proposed animal model.     

No immune responses by the expression of the yeast Ndi1 protein in rats

Background

The rotenone-insensitive internal NADH-quinone oxidoreductase from yeast, Ndi1, has been shown to work as a replacement molecule for complex I in the respiratory chain of mammalian mitochondria. In the so-called transkingdom gene therapy, one major concern is the fact that the yeast protein is foreign in mammals. Long term expression of Ndi1 observed in rodents with no apparent damage to the target tissue was indicative of no action by the host''s immune system.

Methodology/Principal Findings

In the present study, we examined rat skeletal muscles expressing Ndi1 for possible signs of inflammatory or immune response. In parallel, we carried out delivery of the GFP gene using the same viral vector that was used for the NDI1 gene. The tissues were subjected to H&E staining and immunohistochemical analyses using antibodies specific for markers, CD11b, CD3, CD4, and CD8. The data showed no detectable signs of an immune response with the tissues expressing Ndi1. In contrast, mild but distinctive positive reactions were observed in the tissues expressing GFP. This clear difference most likely comes from the difference in the location of the expressed protein. Ndi1 was localized to the mitochondria whereas GFP was in the cytosol.

Conclusions/Significance

We demonstrated that Ndi1 expression did not trigger any inflammatory or immune response in rats. These results push forward the Ndi1-based molecular therapy and also expand the possibility of using foreign proteins that are directed to subcellular organelle such as mitochondria.     

Roles of bound quinone in the single subunit NADH-quinone oxidoreductase (Ndi1) from Saccharomyces cerevisiae

To understand the biochemical basis for the function of the rotenone-insensitive internal NADH-quinone (Q) oxidoreductase (Ndi1), we have overexpressed mature Ndi1 in Escherichia coli membranes. The Ndi1 purified from the membranes contained one FAD and showed enzymatic activities comparable with the original Ndi1 isolated from Saccharomyces cerevisiae. When extracted with Triton X-100, the isolated Ndi1 did not contain Q. The Q-bound form was easily reconstituted by incubation of the Q-free Ndi1 enzyme with ubiquinone-6. We compared the properties of Q-bound Ndi1 enzyme with those of Q-free Ndi1 enzyme, with higher activity found in the Q-bound enzyme. Although both are inhibited by low concentrations of AC0-11 (IC(50) = 0.2 microm), the inhibitory mode of AC0-11 on Q-bound Ndi1 was distinct from that of Q-free Ndi1. The bound Q was slowly released from Ndi1 by treatment with NADH or dithionite under anaerobic conditions. This release of Q was prevented when Ndi1 was kept in the reduced state by NADH. When Ndi1 was incorporated into bovine heart submitochondrial particles, the Q-bound form, but not the Q-free form, established the NADH-linked respiratory activity, which was insensitive to piericidin A but inhibited by KCN. Furthermore, Ndi1 produces H(2)O(2) as isolated regardless of the presence of bound Q, and this H(2)O(2) was eliminated when the Q-bound Ndi1, but not the Q-free Ndi1, was incorporated into submitochondrial particles. The data suggest that Ndi1 bears at least two distinct Q sites: one for bound Q and the other for catalytic Q.