NDI1 Expression Research Articles

Yeast NDI1 reconfigures neuronal metabolism and prevents the unfolded protein response in mitochondrial complex I deficiency.

Mutations in subunits of the mitochondrial NADH dehydrogenase cause mitochondrial complex I deficiency, a group of severe neurological diseases that can result in death in infancy. The pathogenesis of complex I deficiency remain poorly understood, and as a result there are currently no available treatments. To better understand the underlying mechanisms, we modelled complex I deficiency in Drosophila using knockdown of the mitochondrial complex I subunit ND-75 (NDUFS1) specifically in neurons. Neuronal complex I deficiency causes locomotor defects, seizures and reduced lifespan. At the cellular level, complex I deficiency does not affect ATP levels but leads to mitochondrial morphology defects, reduced endoplasmic reticulum-mitochondria contacts and activation of the endoplasmic reticulum unfolded protein response (UPR) in neurons. Multi-omic analysis shows that complex I deficiency dramatically perturbs mitochondrial metabolism in the brain. We find that expression of the yeast non-proton translocating NADH dehydrogenase NDI1, which reinstates mitochondrial NADH oxidation but not ATP production, restores levels of several key metabolites in the brain in complex I deficiency. Remarkably, NDI1 expression also reinstates endoplasmic reticulum-mitochondria contacts, prevents UPR activation and rescues the behavioural and lifespan phenotypes caused by complex I deficiency. Together, these data show that metabolic disruption due to loss of neuronal NADH dehydrogenase activity cause UPR activation and drive pathogenesis in complex I deficiency.

Transgenic NADH dehydrogenase restores oxygen regulation of breathing in mitochondrial complex I-deficient mice

The hypoxic ventilatory response (HVR) is a life-saving reflex, triggered by the activation of chemoreceptor glomus cells in the carotid body (CB) connected with the brainstem respiratory center. The molecular mechanisms underlying glomus cell acute oxygen (O2) sensing are unclear. Genetic disruption of mitochondrial complex I (MCI) selectively abolishes the HVR and glomus cell responsiveness to hypoxia. However, it is unknown what functions of MCI (metabolic, proton transport, or signaling) are essential for O2 sensing. Here we show that transgenic mitochondrial expression of NDI1, a single-molecule yeast NADH/quinone oxidoreductase that does not directly contribute to proton pumping, fully recovers the HVR and glomus cell sensitivity to hypoxia in MCI-deficient mice. Therefore, maintenance of mitochondrial NADH dehydrogenase activity and the electron transport chain are absolutely necessary for O2-dependent regulation of breathing. NDI1 expression also rescues other systemic defects caused by MCI deficiency. These data explain the role of MCI in acute O2 sensing by arterial chemoreceptors and demonstrate the optimal recovery of complex organismal functions by gene therapy.

The Origin and Functional Impact of Mitochondrial DNA Mutations in Acute Myeloid Leukemia

Recent whole-genome sequencing studies have identified somatic mutations in the mitochondrial genome of several human malignancies including acute myeloid leukemia (AML). Although mitochondrial DNA (mtDNA) mutations have previously been shown to promote tumorigenicity in some solid cancers, their role in AML development is unknown. To address this issue, we traced the origin of mtDNA mutations in primary human AML samples and determined their potential impact on mitochondrial respiration and cell fate decisions in hematopoietic stem and progenitor cells (HSPCs).We first characterized the spectrum of mtDNA mutations in AML by performing hybrid capture-based next generation sequencing of the 13 protein-coding mtDNA genes in 133 AML patient samples collected at the time of diagnosis. We also sequenced 150 recurrently mutated nuclear genes in parallel. We aligned mtDNA sequences to the revised Cambridge Reference Sequence and identified variants with > 5% variant allele frequency. Since matched normal samples were not available, we filtered out variants with a population frequency of > 0.1% in GenBank to enrich for somatic variants. Of the remaining variants, we filtered out ones that were synonymous or caused an amino acid change at a non-conserved residue to enrich for pathogenic mutations. Based on these criteria, we identified 87 different missense mutations in 39% (n=53) of the patients across 12 of the 13 genes. We did not find any hot spot mutations. Interestingly, mtDNA mutations were mutually exclusive with IDH1 R132 mutations (p < 0.05, Chi-square test). Patients with mtDNA mutations had inferior leukemia-free survival compared to those without mtDNA mutations (134 vs 477 days; p = 0.002, Log-rank test).To trace the origin of these mutations, we developed allele-specific TaqMan assays for 9 of the primary AML samples with known mtDNA mutations. The TaqMan assays were sufficiently sensitive to quantify heteroplasmy at the single cell level. Patient-specific mtDNA mutations were detected in single cells sorted from the hematopoietic stem cell (HSC) compartment (defined as CD45dim/CD33-/CD34+/CD38-/CD90+/CD45RA-) in 8 of the 9 samples. The heteroplasmy levels were generally lower in HSCs than leukemic blasts. Interestingly, mtDNA mutations were also detected at low levels in the T cell compartment of 3 of the 8 samples. Analysis of a matched diagnostic/relapsed pair showed persistence of the mtDNA mutation in HSCs and leukemic blasts. These findings collectively indicate that mtDNA mutations are acquired in HSCs and stable during disease evolution.To investigate the potential impact of mtDNA mutations on mitochondrial respiration, we generated a pair of transmitochondrial cybrids by repopulating mtDNA-depleted 143B osteosarcoma cells with mitochondria from either KG-1α AML cells or peripheral blood cells of a healthy donor. KG-1α cells harbor a nonsense mtDNA mutation (C13396T) in the MT-ND5 gene which encodes a core subunit of Complex I in the electron transport chain (ETC). The KG-1α cybrid had lower levels of basal and maximal respiration compared with the healthy control cybrid as determined by oxygen consumption rates. To confirm that this defect was due to impairment of Complex I activity, we ectopically expressed the yeast NADH dehydrogenase (NDI1) protein which bypasses endogenous Complex I function. NDI1 expression completely rescued the mitochondrial respiration defect in KG-1α cybrids, whereas expression of the AOX protein which bypasses Complex II and III failed to do so. To determine the potential impact of Complex I dysfunction on cell fate decisions in HSPCs, we transduced human CD34+ cord blood cells with shRNA lentiviral vectors targeting the nuclear genes NDUFV1 and NDUFB11 which encode critical subunits of Complex I. Knockdown of the genes resulted in an accumulation of CD34+ cells in ex vivo culture over time.In summary, our results demonstrate that mtDNA mutations are commonly found in AML patients and have potential impact on clinical outcomes. The acquisition these mutations appears to be an early event in AML leukemogenesis. Finally, we showed that mtDNA mutations can negatively impact ETC function which we speculate, may in turn drive clonal hematopoiesis by promoting self-renewal and blocking differentiation of HSCs. DisclosuresNo relevant conflicts of interest to declare.

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, i.e., bioenergetics, is the dominant function of mitochondrial complex I in protection from brain pathology. We generated a mouse that conditionally expresses the yeast NADH dehydrogenase (NDI1), 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 driven by the loss of NDUFS4, a subunit of mitochondrial complex I. Therefore, mitochondrial 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.

Gene Therapy with Single-Subunit Yeast NADH-Ubiquinone Oxidoreductase (NDI1) Improves the Visual Function in Experimental Autoimmune Encephalomyelitis (EAE) Mice Model of Multiple Sclerosis (MS).

Mitochondrial dysfunction mediated loss of respiration, oxidative stress, and loss of cellular homeostasis contributes to the neuronal and axonal degenerations permanent loss of function in experimental autoimmune encephalomyelitis model (EAE) of multiple sclerosis (MS). To address the mitochondrial dysfunction mediated visual loss in EAE mice, self-complementary adeno-associated virus (scAAV) containing the NADH-dehydrogenase type-2 (NDI1) complex I gene was intravitreally injected into the mice after the onset of visual defects. Visual function assessed by pattern electroretinogram (PERGs) showed progressive loss of function in EAE mice were improved significantly in NDI1 gene therapy-treated mice. Serial optical coherence tomography (OCT) revealed that progressive thinning of inner retinal layers in EAE mice was prevented upon NDI1 expression. The 45% optic nerve axonal and 33% retinal ganglion cell (RGC) loss contributed to the permanent loss of visual function in EAE mice were ameliorated by NDI1-mediated prevention of mitochondrial cristae dissolution and improved mitochondrial homeostasis. In conclusion, targeting the dysfunctional complex I using NDI1 gene can be an approach to address axonal and neuronal loss responsible for permanent disability in MS that is unaltered by current disease modifying drugs.

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.

Metformin Inhibits Progression of Head and Neck Squamous Cell Carcinoma by Acting Directly on Carcinoma-Initiating Cells.

Metformin may reduce the progression of head and neck squamous cell carcinoma (HNSCC); however, whether metformin acts by altering the host metabolism or targets cancer-initiating cells remains poorly understood. This gap in knowledge has prevented the stratification of patient populations who are most likely to benefit from metformin treatment. Here, we explored whether metformin acts directly on HNSCC cells to inhibit aberrant cell growth. To investigate the tumor cell autonomous effects of metformin, we engineered representative HPV- and HPV+ HNSCC cells harboring typical genetic alternations to express the yeast mitochondrial NADH dehydrogenase (NDI1) protein, which is insensitive to metformin. NDI1 expression rescued the inhibitory effects of metformin on mitochondrial complex I, abolished the ability of metformin to activate AMP-activated protein kinase, and inhibited mTOR signaling both in vitro and in vivo, and was sufficient to render metformin ineffective to prevent HNSCC tumor growth. This experimental system provided an opportunity to identify metformin-regulated transcriptional programs linked to cancer cell growth inhibition in the tumor microenvironment. Remarkably, computational analysis of the metformin-induced transcriptome revealed that metformin downregulated gene expression signatures associated with cancer stemness and epithelial-mesenchymal transition, concomitant with increased expression of squamous differentiation genes. These findings support that metformin may act directly on cancer-initiating cells to prevent their progression to HNSCC, which may inform the selection of patients at risk of developing HNSCC in future early-stage clinical trials. SIGNIFICANCE: Metformin's ability to directly target HNSCC-initiating cells instead of exerting cancer preventive activity based solely on its systemic effects may inform the selection of patients in future precision prevention trials.

Differential Effects of Yeast NADH Dehydrogenase (Ndi1) Expression on Mitochondrial Function and Inclusion Formation in a Cell Culture Model of Sporadic Parkinson's Disease.

Parkinson’s disease (PD) is a neurodegenerative disorder that exhibits aberrant protein aggregation and mitochondrial dysfunction. Ndi1, the yeast mitochondrial NADH dehydrogenase (complex I) enzyme, is a single subunit, internal matrix-facing protein. Previous studies have shown that Ndi1 expression leads to improved mitochondrial function in models of complex I-mediated mitochondrial dysfunction. The trans-mitochondrial cybrid cell model of PD was created by fusing mitochondrial DNA-depleted SH-SY5Y cells with platelets from a sporadic PD patient. PD cybrid cells reproduce the mitochondrial dysfunction observed in a patient’s brain and periphery and form intracellular, cybrid Lewy bodies comparable to Lewy bodies in PD brain. To improve mitochondrial function and alter the formation of protein aggregates, Ndi1 was expressed in PD cybrid cells and parent SH-SY5Y cells. We observed a dramatic increase in mitochondrial respiration, increased mitochondrial gene expression, and increased PGC-1α gene expression in PD cybrid cells expressing Ndi1. Total cellular aggregated protein content was decreased but Ndi1 expression was insufficient to prevent cybrid Lewy body formation. Ndi1 expression leads to improved mitochondrial function and biogenesis signaling, both processes that could improve neuron survival during disease. However, other aspects of PD pathology such as cybrid Lewy body formation were not reduced. Consequently, resolution of mitochondrial dysfunction alone may not be sufficient to overcome other aspects of PD-related cellular pathology.

Apoptosis-Inducing-Factor-Dependent Mitochondrial Function Is Required for T Cell but Not B Cell Function

The role of apoptosis inducing factor (AIF) in promoting cell death versus survival remains controversial. We report that the loss of AIF in fibroblasts led to mitochondrial electron transport chain defects and loss of proliferation that could be restored by ectopic expression of the yeast NADH dehydrogenase Ndi1. Aif-deficiency in T cells led to decreased peripheral T cell numbers and defective homeostatic proliferation, but thymic T cell development was unaffected. In contrast, Aif-deficient B cells developed and functioned normally. The difference in the dependency of T cells versus B cells on AIF for function and survival correlated with their metabolic requirements. Ectopic Ndi1 expression rescued homeostatic proliferation of Aif-deficient T cells. Despite its reported roles in cell death, fibroblasts, thymocytes and B cells lacking AIF underwent normal death. These studies suggest that the primary role of AIF relates to complex I function, with differential effects on T and B cells.

Caenorhabditis elegans expressing the Saccharomyces cerevisiae NADH alternative dehydrogenase Ndi1p, as a tool to identify new genes involved in complex I related diseases.

Isolated complex I deficiencies are one of the most commonly observed biochemical features in patients suffering from mitochondrial disorders. In the majority of these clinical cases the molecular bases of the diseases remain unknown suggesting the involvement of unidentified factors that are critical for complex I function. The Saccharomyces cerevisiae NDI1 gene, encoding the mitochondrial internal NADH dehydrogenase was previously shown to complement a complex I deficient strain in Caenorhabditis elegans with notable improvements in reproduction and whole organism respiration. These features indicate that Ndi1p can functionally integrate the respiratory chain, allowing complex I deficiency complementation. Taking into account the Ndi1p ability to bypass complex I, we evaluate the possibility to extend the range of defects/mutations causing complex I deficiencies that can be alleviated by NDI1 expression. We report here that NDI1 expressing animals unexpectedly exhibit a slightly shortened lifespan, a reduction in the progeny, and a depletion of the mitochondrial genome. However, Ndi1p is expressed and targeted to the mitochondria as a functional protein that confers rotenone resistance to those animals without affecting their respiration rate and ATP content. We show that the severe embryonic lethality level caused by the RNAi knockdowns of complex I structural subunit encoding genes (e.g., NDUFV1, NDUFS1, NDUFS6, NDUFS8, or GRIM-19 human orthologs) in wild type animals is significantly reduced in the Ndi1p expressing worm. All together these results open up the perspective to identify new genes involved in complex I function, assembly, or regulation by screening an RNAi library of genes leading to embryonic lethality that should be rescued by NDI1 expression.

Increased longevity mediated by yeast NDI1 expression in Drosophila intestinal stem and progenitor cells.

A functional decline in tissue stem cells and mitochondrial dysfunction have each been linked to aging and multiple aging-associated pathologies. However, the interplay between energy homeostasis, stem cells, and organismal aging remains poorly understood. Here, we report that expression of the single-subunit yeast alternative NADH dehydrogenase, ndi1, in Drosophila intestinal stem and progenitor cells delays the onset of multiple markers of intestinal aging and extends lifespan. In addition, expression of ndi1 in the intestine increases feeding behavior and results in organismal weight gain. Consistent with increased nutrient uptake, flies expressing ndi1 in the digestive tract display a systemic reduction in the activity of AMP-activated protein kinase (AMPK), a key cellular energy sensor. Together, these results demonstrate that ndi1 expression in the intestinal epithelium is an effective strategy to delay tissue and organismal aging.

Abstract IA3: Normalizing tumor cell metabolism in breast cancer metastasis: A novel therapeutic approach

Abstract Despite advances in clinical therapy, metastasis is still the leading cause of death in breast cancer patients. A better understanding of mechanisms that drive metastasis is a prerequisite for new approaches to effectively prevent and inhibit this most dangerous advancement of the disease. While alterations in the nuclear genome are pivotal in oncogenesis, a role of mitochondria in cancer progression has remained largely unexplored. Mutations in mitochondrial DNA are found in breast tumors and other cancers, however their involvement in driving the disease is unclear. Our study identifies mitochondrial complex I as critical for defining an aggressive phenotype in breast cancer cells. Complex I is the gate-keeper of the respiratory chain and catalyzes the first step of NADH oxidation. It elevates the cellular NAD+/NADH ratio and translocates protons across the inner mitochondrial membrane, which ultimately leads to energy production. We used a unique approach to define contributions of mitochondrial complex I activity to breast cancer progression, based on expression of yeast NADH dehydrogenase Ndi1. Ndi1 encodes a single protein that translocates to mitochondrial, faces the inner mitochondrial matrix and oxidizes NADH from the Krebs cycle. Specific enhancement of mitochondrial complex I activity by Ndi1 expression inhibited tumor growth and metastasis through regulation of the tumor cell NAD+/NADH redox balance, mTORC1 activity, and autophagy. Conversely, non-lethal reduction of NAD+ levels by interfering with nicotinamide phosphoribosyltransferase expression to disturb the NAD+ synthesis and recycling pathway, rendered tumor cells more aggressive and increased metastasis. Thus, the results indicate a cause-and-effect relationship between reduced NAD+/NADH ratios and metastatic activity. Having established that enhancement of NAD+/NADH levels by augmenting breast cancer cell complex I activity inhibits tumorigenicity and metastasis, we used this new concept therapeutically and hypothesized that supplementing tumor cell nutrients with NAD+ precursors, such as nicotinic acid (NIC) or nicotinamide (NAM), could interfere with breast cancer progression. We demonstrate that enhancing NAD+ levels through NAD+ precursor treatment effectively inhibits experimental metastasis of human breast cancer cells in xenograft models. Importantly, this treatment also inhibited spontaneous metastasis, and increased animal survival when the therapy was started after surgical removal of primary tumors. Furthermore, NAD+ precursor treatment strongly interferes with oncogene driven breast cancer development and progression in transgenic MMTV-PyMT mice. Thus, aberration in mitochondrial complex I NADH dehydrogenase activity can profoundly enhance the aggressiveness of human breast cancer cells while therapeutic normalization of the NAD+/NADH balance can inhibit metastasis and prevent disease progression. Our study demonstrates that mitochondrial complex I regulation of tumor cell NAD+/NADH levels impacts breast cancer growth and metastasis, and translates into a new therapeutic approach for preventing breast cancer progression. This is highly relevant as current standard of care for cancer patients relies primarily on chemo- and radiation therapies aimed at killing the tumor cells. Evolutionary models predict that selective pressure imposed by these approaches causes survival of resistant clones that eventually re-activate the disease. Based on the central involvement of metabolic tumor cell alterations in cancer, therapeutic normalization of tumor cell metabolism might interfere with the expansion of residual and break-through clones. Thus, a combination of standard therapy with NAD+ precursor treatment may halt breast cancer progression and prevent relapse. Citation Format: Antonio F. Santidrian, Akemi Matsuno-Yagi, Melissa Ritland, Byoung B. Seo1,2, Sarah E. LeBoeuf, Laurie J. Gay, Takao Yagi, Brunhilde Felding-Habermann. Normalizing tumor cell metabolism in breast cancer metastasis: A novel therapeutic approach. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr IA3.

Expression of yeast NDI1 rescues a Drosophila complex I assembly defect.

Defects in mitochondrial electron transport chain (ETC) function have been implicated in a number of neurodegenerative disorders, cancer, and aging. Mitochondrial complex I (NADH dehydrogenase) is the largest and most complicated enzyme of the ETC with 45 subunits originating from two separate genomes. The biogenesis of complex I is an intricate process that requires multiple steps, subassemblies, and assembly factors. Here, we report the generation and characterization of a Drosophila model of complex I assembly factor deficiency. We show that CG7598 (dCIA30), the Drosophila homolog of human complex I assembly factor Ndufaf1, is necessary for proper complex I assembly. Reduced expression of dCIA30 results in the loss of the complex I holoenzyme band in blue-native polyacrylamide gel electrophoresis and loss of NADH:ubiquinone oxidoreductase activity in isolated mitochondria. The complex I assembly defect, caused by mutation or RNAi of dCIA30, has repercussions both during development and adulthood in Drosophila, including developmental arrest at the pupal stage and reduced stress resistance during adulthood. Expression of the single-subunit yeast alternative NADH dehydrogenase, Ndi1, can partially or wholly rescue phenotypes associated with the complex I assembly defect. Our work shows that CG7598/dCIA30 is a functional homolog of Ndufaf1 and adds to the accumulating evidence that transgenic NDI1 expression is a viable therapy for disorders arising from complex I deficiency.

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

Neuronal expression of a single‐subunit yeast NADH–ubiquinone oxidoreductase (Ndi1) extends Drosophila lifespan

The 'rate of living' theory predicts that longevity should be inversely correlated with the rate of mitochondrial respiration. However, recent studies in a number of model organisms, including mice, have reported that interventions that retard the aging process are, in fact, associated with an increase in mitochondrial activity. To better understand the relationship between energy metabolism and longevity, we supplemented the endogenous respiratory chain machinery of the fruit fly Drosophila melanogaster with the alternative single-subunit NADH-ubiquinone oxidoreductase (Ndi1) of the baker's yeast Saccharomyces cerevisiae. Here, we report that expression of Ndi1 in fly mitochondria leads to an increase in NADH-ubiquinone oxidoreductase activity, oxygen consumption, and ATP levels. In addition, exogenous Ndi1 expression results in increased CO2 production in living flies. Using an inducible gene-expression system, we expressed Ndi1 in different cells and tissues and examined the impact on longevity. In doing so, we discovered that targeted expression of Ndi1 in fly neurons significantly increases lifespan without compromising fertility or physical activity. These findings are consistent with the idea that enhanced respiratory chain activity in neuronal tissue can prolong fly lifespan.

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.

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.

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.