Ndi1 Protein Research Articles

Effects of Rotenone Exposure on Apoptosis in rAAV-NDI1-infected Neural Stem Cell Line of Minipig

Background: The overall objective of this study is to confirm that normal expression of yeast NADH- dehydrogenase (NDI1) can occur in neural stem cell lines of minipig with resistance to rotenone exposure, an environmental factor responsible for dysfunction of mitochondrial enzyme complex I. In modern society, there are many diseases that cannot be treated. Diseases including LHON, Parkinson disease and dystonia, have been associated with defects in mitochondrial complexes. This experiment was performed to demonstrate that it was not sensitive. The overall objective of this study is to confirm that normal expression of yeast NADH-dehydrogenase (NDI1) can occur in neural stem cell lines of minipig with resistance to rotenone exposure, an environmental factor responsible for dysfunction of mitochondrial enzyme complex I. Methods: A Mini Pig Neural stem cell line (MPV) was used for transfection of the NDI gene, DMEM/F-12 culture medium and MPV was inoculated in a six-well plate (Corning, USA) at a concentration of 1×105 cells/3ml/well, with inoculation of MPV at 37°C for 24 hours, 5% CO2, followed by incubation in an incubator with 95% humidity and attached. The plate containing MPV cells was treated with recombinant adeno-associated virus ndi1 (rAAV-ndi1) for transfection, with periodic replacement with a cell selection culture solution containing 10% FBS, 1% P/S and 0.2 mM rotenone. The experiment was performed for restoration of mitochondrial activity of thawed cells. RNA was extracted from MPV cells and transfection of MPV cells with the NDI1 gene was performed using Trizol (Invitrogen, USA). Reverse transcription PCR (RT-PCR) and Western blot were performed to confirm normal expression of the NDI1 gene in MPV cells transfected with the NDI1 gene. Immunofluorescence was performed to determine the presence of the NDI1 protein in the cell and the cell count was used for LUNA and the rate of cell death for rotenone was determined using the MTS [3-(4,5-dimethylthiazol-2-yl)-5- (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay. Result: rAAV-NDI1 was successfully introduced into MPV cells and the proliferation rates for the cells were compared with those of the transformed cells; after three days, the non-infected cells were killed and the infected cells proliferated. The results after differentiating the cell lines were similar to those of previously reported studies. Toxicity analysis on rotenone was also performed using the MTS assay and the rates of cell death over three days were compared; the results showed significantly lower levels of NDI1-transformed minipig neural stem cells compared with those of minipig. The present work will be a complementary contribution to the comprehensive study of the scorpion sting syndrome. These results were similar to those of previously reported studies. Previous studies have reported that oxidoreductive stress can be a cause of apoptosis. Therefore, conduct of additional studies for measurement of ROS and oxygen utilization to determine oxidative stress in cells will be necessary.

NAD+ Regeneration Rescues Lifespan But Not Ataxia in a Mouse Model of Brain Mitochondrial Complex I Dysfunction

Mitochondrial complex I regenerates NAD+ and proton pumps for TCA cycle function and ATP production, respectively. Mitochondrial complex I dysfunction has been implicated in many brain pathologies including Leigh Syndrome and Parkinson’s disease. We sought to determine whether NAD+ regeneration or proton pumping is the dominant function of mitochondrial complex I in protection from brain pathology. We generated a mouse that conditionally expresses the yeast NDI1 protein, a single enzyme that can replace the NAD+ regeneration capability of the 45-subunit mammalian mitochondrial complex I without proton pumping. NDI1 expression was sufficient to dramatically prolong lifespan without significantly improving motor function in a mouse model of Leigh Syndrome. Therefore, complex I activity in the brain supports organismal survival through its NAD+ regeneration capacity while optimal motor control requires the bioenergetic function of mitochondrial complex I.

Reduction of Infarct Size by the Therapeutic Protein TAT-Ndi1 In Vivo

Lethal myocardial ischemia-reperfusion (I/R) injury has been attributed in part to mitochondrial respiratory dysfunction (including damage to complex I) and the resultant excessive production of reactive oxygen species. Recent evidence has shown that reduced nicotinamide adenine dinucleotide-quinone internal oxidoreductase (Ndi1; the single-subunit protein that in yeast serves the analogous function as complex I), transduced by addition of the TAT-conjugated protein to culture media and perfusion buffer, can preserve mitochondrial function and attenuate I/R injury in neonatal rat cardiomyocytes and Langendorff-perfused rat hearts. However, this novel metabolic strategy to salvage ischemic-reperfused myocardium has not been tested in vivo. In this study, TAT-conjugated Ndi1 and placebo-control protein were synthesized using a cell-free system. Mitochondrial uptake and functionality of TAT-Ndi1 were demonstrated in mitochondrial preparations from rat hearts after intraperitoneal administration of the protein. Rats were randomized to receive either TAT-Ndi1 or placebo protein, and 2 hours later all animals underwent 45-minute coronary artery occlusion followed by 2 hours of reperfusion. Infarct size was delineated by tetrazolium staining and normalized to the volume of at-risk myocardium, with all analysis conducted in a blinded manner. Risk region was comparable in the 2 cohorts. Preischemic administration of TAT-Ndi1 was profoundly cardioprotective. These results demonstrate that it is possible to target therapeutic proteins to the mitochondrial matrix and that yeast Ndi1 can substitute for complex I to ameliorate I/R injury in the heart. Moreover, these data suggest that cell-permeable delivery of mitochondrial proteins may provide a novel molecular strategy to treat mitochondrial dysfunction in patients.

The structure of the yeast NADH dehydrogenase (Ndi1) reveals overlapping binding sites for water- and lipid-soluble substrates

Bioenergy is efficiently produced in the mitochondria by the respiratory system consisting of complexes I-V. In various organisms, complex I can be replaced by the alternative NADH-quinone oxidoreductase (NDH-2), which catalyzes the transfer of an electron from NADH via FAD to quinone, without proton pumping. The Ndi1 protein from Saccharomyces cerevisiae is a monotopic membrane protein, directed to the matrix. A number of studies have investigated the potential use of Ndi1 as a therapeutic agent against complex I disorders, and the NDH-2 enzymes have emerged as potential therapeutic targets for treatments against the causative agents of malaria and tuberculosis. Here we present the crystal structures of Ndi1 in its substrate-free, NAD(+)- and ubiquinone- (UQ2) complexed states. The structures reveal that Ndi1 is a peripheral membrane protein forming an intimate dimer, in which packing of the monomeric units within the dimer creates an amphiphilic membrane-anchor domain structure. Crucially, the structures of the Ndi1-NAD(+) and Ndi1-UQ2 complexes show overlapping binding sites for the NAD(+) and quinone substrates.

No Immune Responses by the Expression of the Yeast Ndi1 Protein in Rats

BackgroundThe 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 FindingsIn 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/SignificanceWe 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.

Abstract 3795: Mitochondrial complex I modulation regulates autophagy and breast cancer progression

Abstract Tumor cells express altered metabolic activities often linked to mitochondrial dysfunction. Such mitochondrial defects can inhibit oxidative phosphorylation, change the cellular redox state (NAD+/NADH), increase production of reactive oxygen species (ROS), and cause DNA damage that further supports tumorigenesis and a metastatic phenotype. Our study identified mitochondrial complex I (NADH dehydrogenase) as a modulator of tumorigenesis and metastatic progression in breast cancer. We used NDI1, the NADH dehydrogenase from yeast, to augment complex I activity in metastatic human breast cancer cells. The NDI1 protein is known to integrate into the inner mitochondrial membrane of mammalian cells, function as NADH dehydrogenase, pass electrons into the respiratory chain, and thereby fully augment intrinsic complex I activity. Thus, NDI1 identifies critical involvement of complex I NADH dehydrogenase activity in cellular functions that are altered upon its expression. We investigated a panel of aggressive human breast cancer cell lines, including MDA-MB 231, and found that augmentation of NADH dehydrogenase activity through NDI1 resulted in an enhanced NAD+/NADH ratio and inhibition of ROS production. Most importantly, NDI1 expression inhibited metastasis and tumor growth in the mammary fad pad of immune deficient mice, seen by non-invasive imaging and histology. The same effects were achieved by treatment with NAD precursors, demonstrating that normalization of the NAD+/NADH ratio is critical for inhibition of breast cancer progression. We found that the underlying mechanism is based on activation of PTEN, inhibition of the AKT/mTOR survival pathway, and stimulation of autophagy. Autophagy is responsible for NDI1-mediated inhibition of breast cancer progression, as knock-down of ATG5 reversed the anti-metastatic effect of NDI1. Our results demonstrate that tumorigenesis and metastasis in breast cancer are critically influenced by mitochondrial complex I activity. We show that augmentation of tumor cell complex I function can interfere with tumor formation and breast cancer spreading through modulating the NAD+/NADH ratio. Thus, normalization of complex I function appears as a promising new target for prevention and treatment of breast progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3795. doi:10.1158/1538-7445.AM2011-3795

Protective Role of rAAV-NDI1, Serotype 5, in an Acute MPTP Mouse Parkinson's Model

Defects in mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I) have been implicated in a number of acquired and hereditary diseases including Leigh's syndrome and more recently Parkinson's disease. A limited number of strategies have been attempted to repair the damaged complex I with little or no success. We have recently shown that the non-proton-pumping, internal NADH-ubiquinone oxidoreductase (Ndi1) from Saccharomyces cerevisiae (baker's yeast) can be successfully inserted into the mitochondria of mice and rats, and the enzyme was found to be fully active. Using recombinant adenoassociated virus vectors (serotype 5) carrying our NDI1 gene, we were able to express the Ndi1 protein in the substantia nigra (SN) of C57BL/6 mice with an expression period of two months. The results show that the AAV serotype 5 was highly efficient in expressing Ndi1 in the SN, when compared to a previous model using serotype 2, which led to nearly 100% protection when using an acute MPTP model. It is conceivable that the AAV-serotype5 carrying the NDI1 gene is a powerful tool for proof-of-concept study to demonstrate complex I defects as the causable factor in diseases of the brain.

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

BackgroundLeber'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 FindingsWe 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/SignificanceThe 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.

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.

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.

Can a Single Subunit Yeast NADH Dehydrogenase (Ndi1) Remedy Diseases Caused by Respiratory Complex I Defects?

The proton-translocating NADH-quinone oxidoreductase (complex I) is one of five enzyme complexes in the oxidative phosphorylation system in mammalian mitochondria. Complex I is composed of 46 different subunits, 7 of which are encoded by mitochondrial DNA. Defects of complex I are involved in many human mitochondrial diseases; therefore, the authors 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 10 mammalian cell lines (two of which were complex I-deficient mutants). 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. It was further shown that the Ndi1 protein can be functionally expressed in tissues such as skeletal muscles and brain of rodents. The Ndi1 expression scarcely induced an inflammatory response as assessed by hematoxylin and eosin (H&E) staining. The Ndi1 protein expressed in the substantia nigra (SN) elicited protective effects against neurodegeneration caused by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment. The Ndi1 protein has a great potential as a molecular remedy for complex I deficiencies.

In Vivo Complementation of Complex I by the Yeast Ndi1 Enzyme

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.

Functional expression of the single subunit NADH dehydrogenase in mitochondria in vivo: a potential therapy for complex I deficiencies.

It has been reported that defects of mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I) are involved in many human diseases (such as encephalomyopathies and sporadic Parkinson's disease). However, no effective remedies have been established for complex I deficiencies. We have adopted a gene therapy approach utilizing the NDI1 gene that codes for the single subunit NADH dehydrogenase of Saccharomyces cerevisiae (Ndi1). Our earlier experiments show that the Ndi1 protein can replace or supplement the functionality of complex I in various cultured cells. For this approach to be useful, it is important to demonstrate in vivo that the mature protein is correctly placed in mitochondria. In this study, we have attempted in vivo expression of the NDI1 gene in skeletal muscles and brains (substantia nigra and striatum) of rodents. In all tissues tested, the Ndi1 protein was identified in the injected area by immunohistochemical staining at 1-2 weeks after the injection. Sustained expression was observed for at least 7 months. Double-staining of the sections using antibodies against Ndi1 and F(1)-ATPase revealed that the expressed Ndi1 protein was predominantly 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 impairments.

A single-subunit NADH-quinone oxidoreductase renders resistance to mammalian nerve cells against complex I inhibition.

Numerous studies suggest that dysfunction of mitochondrial proton-translocating NADH-ubiquinone oxidoreductase (complex I) is associated with neurodegenerative disorders, such as Parkinson's disease and Huntington's disease. Development of methods to correct complex I defects seems important. We have previously shown that the single-subunit NADH dehydrogenase of Saccharomyces cerevisiae (Ndi1P) can work as a replacement for complex I in mammalian cells. Using a recombinant adeno-associated virus vector carrying the NDI1 gene, we now demonstrated that the Ndi1 enzyme was successfully expressed in the dopaminergic cell lines rat PC12 and mouse MN9D. The cells expressing the Ndi1 protein were resistant to known inhibitors of complex I, such as rotenone and pyridaben. In addition, the NDI1-transduced cells were still capable of morphological maturation as examined by induction of neurite outgrowth. Also, it was possible to infect the cells after the maturation. The expressed Ndi1 protein was located both in cell bodies and in neurites and was functionally active. It is conceivable that the NDI1 gene will be a promising tool in the treatment of neurodegenerative conditions caused by complex I inhibition.

Use of the NADH-Quinone Oxidoreductase (NDI1) Gene ofSaccharomyces 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.

Modulation of oxidative phosphorylation of human kidney 293 cells by transfection with the internal rotenone-insensitive NADH–quinone oxidoreductase ( NDI1) gene of Saccharomyces cerevisiae

In contrast to the mitochondrial proton-translocating NADH–quinone oxidoreductase (complex I), which consists of at least 43 different subunits, the internal rotenone-insensitive NADH–quinone oxidoreductase (Ndi1) of Saccharomyces cerevisiae is a single polypeptide enzyme. The NDI1 gene was stably transfected into the human embryonal kidney 293 (HEK 293) cells. The transfected NDI1 gene was then 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 NADH oxidase activity of the NDI1-transfected HEK 293 cells was not affected by rotenone but was inhibited by flavone. The ADP/O ratios coupled to NADH oxidation were lowered from 2.4 to 1.8 by NDI1-transfection while the ADP/O ratios coupled to succinate oxidation (1.6) were not changed. The NDI1-transfected HEK 293 cells were able to grow in media containing a complex I inhibitor such as rotenone and 1-methyl-4-phenylpyridinium ion. The potential usefulness of incorporating the Ndi1 protein into mitochondria of human cells is discussed.