Akemi Matsuno-Yagi

Mitochondrial complex I activity and NAD+/NADH balance regulate breast cancer progression.

Despite advances in clinical therapy, metastasis remains the leading cause of death in breast cancer patients. Mutations in mitochondrial DNA, including those affecting complex I and oxidative phosphorylation, are found in breast tumors and could facilitate metastasis. This study identifies mitochondrial complex I as critical for defining an aggressive phenotype in breast cancer cells. Specific enhancement of mitochondrial complex I activity inhibited tumor growth and metastasis through regulation of the tumor cell NAD+/NADH redox balance, mTORC1 activity, and autophagy. Conversely, nonlethal reduction of NAD+ levels by interfering with nicotinamide phosphoribosyltransferase expression rendered tumor cells more aggressive and increased metastasis. The results translate into a new therapeutic strategy: enhancement of the NAD+/NADH balance through treatment with NAD+ precursors inhibited metastasis in xenograft models, increased animal survival, and strongly interfered with oncogene-driven breast cancer progression in the MMTV-PyMT mouse model. 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.

Mechanism of Toxicity in Rotenone Models of Parkinson's Disease

Exposure of rats to the pesticide and complex I inhibitor rotenone reproduces features of Parkinsons disease, including selective nigrostriatal dopaminergic degeneration and α-synuclein-positive cytoplasmic inclusions (Betarbet et al., 2000; Sherer et al., 2003). Here, we examined mechanisms of rotenone toxicity using three model systems. In SK-N-MC human neuroblastoma cells, rotenone (10 nm to 1 μm) caused dose-dependent ATP depletion, oxidative damage, and death. To determine the molecular site of action of rotenone, cells were transfected with the rotenone-insensitive single-subunit NADH dehydrogenase of Saccharomyces cerevisiae (NDI1), which incorporates into the mammalian ETC and acts as a “replacement” for endogenous complex I. In response to rotenone, NDI1-transfected cells did not show mitochondrial impairment, oxidative damage, or death, demonstrating that these effects of rotenone were caused by specific interactions at complex I. Although rotenone caused modest ATP depletion, equivalent ATP loss induced by 2-deoxyglucose was without toxicity, arguing that bioenergetic defects were not responsible for cell death. In contrast, reducing oxidative damage with antioxidants, or by NDI1 transfection, blocked cell death. To determine the relevance of rotenone-induced oxidative damage to dopaminergic neuronal death, we used a chronic midbrain slice culture model. In this system, rotenone caused oxidative damage and dopaminergic neuronal loss, effects blocked by α-tocopherol. Finally, brains from rotenone-treated animals demonstrated oxidative damage, most notably in midbrain and olfactory bulb, dopaminergic regions affected by Parkinsons disease. These results, using three models of increasing complexity, demonstrate the involvement of oxidative damage in rotenone toxicity and support the evaluation of antioxidant therapies for Parkinsons disease.

Mechanism of toxicity of pesticides acting at complex I: relevance to environmental etiologies of Parkinson’s disease

Parkinson’s disease (PD) has been linked to mitochondrial dysfunction and pesticide exposure. The pesticide rotenone (ROT) inhibits complex I and reproduces features of PD in animal models, suggesting that environmental agents that inhibit complex I may contribute to PD. We have previously demonstrated that ROT toxicity is dependent upon complex I inhibition and that oxidative stress is the primary mechanism of toxicity. In this study, we examined the in vitro toxicity and mechanism of action of several putative complex I inhibitors that are commonly used as pesticides. The rank order of toxicity of pesticides to neuroblastoma cells was pyridaben > rotenone > fenpyroximate > fenazaquin > tebunfenpyrad. A similar order of potency was observed for reduction of ATP levels and competition for 3H‐dihydrorotenone (DHR) binding to complex I, with the exception of pyridaben (PYR). Neuroblastoma cells stably expressing the ROT‐insensitive NADH dehydrogenase of Saccharomyces cerevisiae (NDI1) were resistant to these pesticides, demonstrating the requirement of complex I inhibition for toxicity. We further found that PYR was a more potent inhibitor of mitochondrial respiration and caused more oxidative damage than ROT. The oxidative damage could be attenuated by NDI1 or by the antioxidants α‐tocopherol and coenzyme Q10. PYR was also highly toxic to midbrain organotypic slices. These data demonstrate that, in addition to ROT, several commercially used pesticides directly inhibit complex I, cause oxidative damage, and suggest that further study is warranted into environmental agents that inhibit complex I for their potential role in PD.

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, theNDI1 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.

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 Parkinsons 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.

Expression of the yeast NADH dehydrogenase Ndi1 in Drosophila confers increased lifespan independently of dietary restriction

Mutations in mitochondrial oxidative phosphorylation complex I are associated with multiple pathologies, and complex I has been proposed as a crucial regulator of animal longevity. In yeast, the single-subunit NADH dehydrogenase Ndi1 serves as a non-proton-translocating alternative enzyme that replaces complex I, bringing about the reoxidation of intramitochondrial NADH. We have created transgenic strains of Drosophila that express yeast NDI1 ubiquitously. Mitochondrial extracts from NDI1-expressing flies displayed a rotenone-insensitive NADH dehydrogenase activity, and functionality of the enzyme in vivo was confirmed by the rescue of lethality resulting from RNAi knockdown of complex I. NDI1 expression increased median, mean, and maximum lifespan independently of dietary restriction, and with no change in sirtuin activity. NDI1 expression mitigated the aging associated decline in respiratory capacity and the accompanying increase in mitochondrial reactive oxygen species production, and resulted in decreased accumulation of markers of oxidative damage in aged flies. Our results support a central role of mitochondrial oxidative phosphorylation complex I in influencing longevity via oxidative stress, independently of pathways connected to nutrition and growth signaling.

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

Background Lebers 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.

Characterization of the NuoM (ND4) subunit in Escherichia coli NDH-1: conserved charged residues essential for energy-coupled activities.

The proton-translocating NADH-quinone (Q) oxidoreductase (NDH-1) from Escherichia coli is composed of two segments: a peripheral arm and a membrane arm. The membrane arm contains 7 hydrophobic subunits. Of these subunits, NuoM, a homolog of the mitochondrial ND4 subunit, is proposed to be involved in proton translocation and Q-binding. Therefore, we conducted site-directed mutation of 15 amino acid residues of NuoM and investigated their properties. In all mutants, the assembly of the whole enzyme seemed intact. Mutation of highly conserved Glu144 and Lys234 leads to almost total elimination of energy-transducing NDH-1 activities as well as increased production of superoxide radicals. Their NADH dehydrogenase activities were almost normal. Because these two residues are predicted to be located in the transmembrane segments of NuoM, the results strongly suggest that they participate in proton translocation. Although it is hypothesized that His interacts with a Q head group, mutations at four His moderately inhibited NDH-1 activities and had almost no effect on the Km values for Q or IC50 values of capsaicin-40, a competitive inhibitor for the Q binding site. The data suggest that these His are not involved in the catalytic Q-binding. Functional roles of NuoM and advantages of NDH-1 research as a model for mitochondrial complex I study have been discussed.

Characteristics of the energy-transducing NADH-quinone oxidoreductase of Paracoccus denitrificans as revealed by biochemical, biophysical, and molecular biological approaches.

A comparison of the mitochondrial NADH-ubiquinone oxidoreductase and the energy-transducing NADH-quinone oxidoreductase (NDH-1) ofParacoccus denitrificans revealed that both systems have similar electron-transfer and energy-transduction pathways. In addition, both complexes are sensitive to the same inhibitors and contain similar electron carriers, suggesting that theParacoccus NDH-1 may serve as a useful model system for the study of the human enzyme complex. The gene cluster encoding theParacoccus NDH-1 has been cloned and sequenced. It is composed of 18,106 base pairs and contains 14 structural genes and six unidentified reading frames (URFs). The structural genes, URFs, and their polypeptides have been characterized. We also discuss nucleotide sequences which are believed to play a role in the regulation of the NDH-1 gene cluster andParacoccus NDH-1 subunits which may contain the binding sites of substrates and/or electron carriers.

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.