Interspecies Behavioral Variability of Medaka Fish Assessed by Comparative Phenomics.

Recently, medaka has been used as a model organism in various research fields. However, even though it possesses several advantages over zebrafish, fewer studies were done in medaka compared to zebrafish, especially with regard to its behavior. Thus, to provide more information regarding its behavior and to demonstrate the behavioral differences between several species of medaka, we compared the behavioral performance and biomarker expression in the brain between four medaka fishes, Oryzias latipes, Oryzias dancena, Oryzias woworae, and Oryzias sinensis. We found that each medaka species explicitly exhibited different behaviors to each other, which might be related to the different basal levels of several biomarkers. Furthermore, by phenomics and genomic-based clustering, the differences between these medaka fishes were further investigated. Here, the phenomic-based clustering was based on the behavior results, while the genomic-based clustering was based on the sequence of the nd2 gene. As we expected, both clusterings showed some resemblances to each other in terms of the interspecies relationship between medaka and zebrafish. However, this similarity was not displayed by both clusterings in the medaka interspecies comparisons. Therefore, these results suggest a re-interpretation of several prior studies in comparative biology. We hope that these results contribute to the growing database of medaka fish phenotypes and provide one of the foundations for future phenomics studies of medaka fish.

Identifying Mitochondrial-Related Genes NDUFA10 and NDUFV2 as Prognostic Markers for Prostate Cancer through Biclustering.

Prostate cancer is currently associated with higher morbidity and mortality in men in the United States and Western Europe, so it is important to identify genes that regulate prostate cancer. The high-dimension gene expression profile impedes the discovery of biclusters which are of great significance to the identification of the basic cellular processes controlled by multiple genes and the identification of large-scale unknown effects hidden in the data. We applied the biclustering method MCbiclust to explore large biclusters in the TCGA cohort through a large number of iterations. Two biclusters were found with the highest silhouette coefficient value. The expression patterns of one bicluster are highly similar to those found by the gene expression profile of the known androgen-regulated genes. Further gene set enrichment revealed that mitochondrial function-related genes were negatively correlated with AR regulation-related genes. Then, we performed differential analysis, AR binding site analysis, and survival analysis on the core genes with high phenotypic contribution. Among the core genes, NDUFA10 showed a low expression value in cancer patients across different expression profiles, while NDUFV2 showed a high expression value in cancer patients. Survival analysis of NDUFA10 and NDUFV2 demonstrated that both genes were unfavorable prognostic markers.

12 days of in vivo caloric reduction can improve important parameters of aging in humans.

Caloric reduction (CR) is considered as the most reasonable intervention to delay aging and age-related diseases. Numerous studies in various model organisms provide the main basis for this hypothesis. Human studies exist, but they differ widely in study design, characteristics of test persons and study outcome. In this study we investigated CR in humans on a molecular level to gain a better understanding in these processes. For that purpose, we analyzed human peripheral blood mononuclear cells of healthy people fasting according to F.X. Mayr. In a previous study our group could show a significantly improved DNA repair capacity after fasting. Here we were able to confirm these findings despite a slightly modified fasting therapy. Furthermore, the function of the mitochondrial respiratory chain and the mRNA levels of the mitochondria-associated genes SIRT3 and NDUFS1 were significantly affected by CR. However, these changes were only detectable in people who exhibited no improvement in DNA repair capacity. In contrast to that we could not observe any changes in ROS levels, mitochondrial DNA copy number and non-mitochondrial respiration. Altogether our results reveal that CR in form of F. X. Mayr therapy is able to positively influence several cellular parameters and especially mitochondrial function.

Improved developmental competence in embryos treated with lycopene during in vitro culture system.

In vitro embryo development remains suboptimal compared to in vivo development due to the challenge from various stressors associated with in vitro culturing of oocytes. When 0.2 µM lycopene was added to oocyte in vitro maturation and embryo culture media, to assess its antioxidant effects on embryo development, we observed a significant (p < 0.05) increase in cleavage and blastocyst development rates compared to the corresponding controls (84.3 ± 0.6% vs. 73.1 ± 1.9% and 41.0 ± 1.4% vs. 33.4 ± 0.7%, respectively). Lycopene also significantly reduced (p < 0.05) intracellular reactive oxygen species concentrations in oocytes and blastocysts, whereas lipid peroxidation and mitochondrial activity increased compared to control conditions. The number of apoptotic nuclei was significantly reduced in the lycopene-treated compared to the control group (1.7 ± 0.1 vs. 4.7 ± 0.3), and the quantity of cells in the trophectoderm (207.1 ± 1.6 vs. 171.3 ± 1.0, respectively) and inner cell mass (41.9 ± 0.4 vs. 36.7 ± 0.4, respectively) was higher following treatment-although the inner cell mass-to-trophectoderm ratio was unchanged (1:3.3 vs. 1:3.4 for lycopene vs. control, respectively). Lycopene supplementation also significantly (p < 0.05) attenuated expression of IKBKB (Inhibitor of nuclear factor kappa B kinase, subunit beta) and reduced Caspase 9 and Caspase 3 protein abundance, while up-regulating GDF9 (Growth and differentiation factor 9), BMP15 (Bone morphogenetic protein 15), SOD2 (Superoxide dismutase 2), NDUFA2 (NADH dehydrogenase), ACADL (Acyl-CoA dehydrogenase, long chain), and ACSL3 (Acyl-CoA synthetase 3, long-chain membrane 3) transcription compared to control. Therefore, co-culturing with lycopene during oocyte maturation improved bovine embryo developmental potential during in vitro culture by improving embryonic resilience to stress.

Testosterone Upregulates the Expression of Mitochondrial ND1 and ND4 and Alleviates the Oxidative Damage to the Nigrostriatal Dopaminergic System in Orchiectomized Rats.

Testosterone deficiency, as a potential risk factor for aging and aging-related neurodegenerative disorders, might induce mitochondrial dysfunction and facilitate the declines of the nigrostriatal dopaminergic system by exacerbating the mitochondrial defects and increasing the oxidative damage. Thus, how testosterone levels influence the mitochondrial function in the substantia nigra was investigated in the study. The present studies showed that testosterone deficiency impaired the mitochondrial function in the substantia nigra and induced the oxidative damage to the substantia nigra as well as the deficits in the nigrostriatal dopaminergic system. Of four mitochondrial respiratory chain complexes, castration of male rats reduced the activity of mitochondrial complex I and downregulated the expression of ND1 and ND4 of 7 mitochondrial DNA- (mtDNA-) encoded subunits of complex I in the substantia nigra. Supplements of testosterone propionate to castrated male rats ameliorated the activity of mitochondrial complex I and upregulated the expression of mitochondrial ND1 and ND4. These results suggest an important role of testosterone in maintaining the mitochondrial function in the substantia nigra and the vulnerability of mitochondrial complex I to testosterone deficiency. Mitochondrial ND1 and ND4, as potential testosterone targets, were implicated in the oxidative damage to the nigrostriatal dopaminergic system.

Biochemical evidence for a mitochondrial genetic modifier in the phenotypic manifestation of Leber's hereditary optic neuropathy-associated mitochondrial DNA mutation.

Leber's hereditary optic neuropathy (LHON) is the most common mitochondrial disease. Mitochondrial modifiers are proposed to modify the phenotypic expression of primary LHON-associated mitochondrial DNA (mtDNA) mutations. In this study, we demonstrated that the LHON susceptibility allele (m.14502T > C, p. 58I > V) in the ND6 gene modulated the phenotypic expression of primary LHON-associated m.11778G > A mutation. Twenty-two Han Chinese pedigrees carrying m.14502T > C and m.11778G > A mutations exhibited significantly higher penetrance of optic neuropathy than those carrying only m.11778G > A mutation. We performed functional assays using the cybrid cell models, generated by fusing mtDNA-less ρo cells with enucleated cells from LHON patients carrying both m.11778G > A and m.14502T > C mutations, only m.14502T > C or m.11778G > A mutation and a control belonging to the same mtDNA haplogroup. These cybrids cell lines bearing m.14502T > C mutation exhibited mild effects on mitochondrial functions compared with those carrying only m.11778G > A mutation. However, more severe mitochondrial dysfunctions were observed in cell lines bearing both m.14502T > C and m.11778G > A mutations than those carrying only m.11778G > A or m.14502T > C mutation. In particular, the m.14502T > C mutation altered assemble of complex I, thereby aggravating the respiratory phenotypes associated with m.11778G > A mutation, resulted in a more defective complex I. Furthermore, more reductions in the levels of mitochondrial ATP and increasing production of reactive oxygen species were also observed in mutant cells bearing both m.14502T > C and m.11778G > A mutation than those carrying only 11778G > A mutation. Our findings provided new insights into the pathophysiology of LHON that were manifested by interaction between primary and secondary mtDNA mutations.

Breathing air to save energy--new insights into the ecophysiological role of high-affinity [NiFe]-hydrogenase in Streptomyces avermitilis.

The Streptomyces avermitilis genome encodes a putative high-affinity [NiFe]-hydrogenase conferring the ability to oxidize tropospheric H2 in mature spores. Here, we used a combination of transcriptomic and mutagenesis approaches to shed light on the potential ecophysiological role of the enzyme. First, S. avermitilis was either exposed to low or hydrogenase-saturating levels of H2 to investigate the impact of H2 on spore transcriptome. In total, 1293 genes were differentially expressed, with 1127 and 166 showing lower and higher expression under elevated H2 concentration, respectively. High H2 exposure lowered the expression of the Sec protein secretion pathway and ATP-binding cassette-transporters, with increased expression of genes encoding proteins directing carbon metabolism toward sugar anabolism and lower expression of NADH dehydrogenase in the respiratory chain. Overall, the expression of relA responsible for the synthesis of the pleiotropic alarmone ppGpp decreased upon elevated H2 exposure, which likely explained the reduced expression of antibiotic synthesis and stress response genes. Finally, deletion of hhySL genes resulted in a loss of H2 uptake activity and a dramatic loss of viability in spores. We propose that H2 is restricted to support the seed bank of Streptomyces under a unique survival-mixotrophic energy mode and discuss important ecological implications of this finding.

Functional Expression of Electron Transport Chain and FoF1-ATP Synthase in Optic Nerve Myelin Sheath.

Our previous studies reported evidence for aerobic ATP synthesis by myelin from both bovine brainstem and rat sciatic nerve. Considering that the optic nerve displays a high oxygen demand, here we evaluated the expression and activity of the five Respiratory Complexes in myelin purified from either bovine or murine optic nerves. Western blot analyses on isolated myelin confirmed the expression of ND4L (subunit of Complex I), COX IV (subunit of Complex IV) and ß subunit of F1Fo-ATP synthase. Moreover, spectrophotometric and in-gel activity assays on isolated myelin, as well as histochemical activity assays on both bovine and murine transversal optic nerve sections showed that the respiratory Complexes are functional in myelin and are organized in a supercomplex. Expression of oxidative phosphorylation proteins was also evaluated on bovine optic nerve sections by confocal and transmission electron microscopy. Having excluded a mitochondrial contamination of isolated myelin and considering the results form in situ analyses, it is proposed that the oxidative phosphorylation machinery is truly resident in optic myelin sheath. Data may shed a new light on the unknown trophic role of myelin sheath. It may be energy supplier for the axon, explaining why in demyelinating diseases and neuropathies, myelin sheath loss is associated with axonal degeneration.

Mitochondrial complex I and III mRNA levels in bipolar disorder.

BACKGROUND: Studies that have focused on the mitochondrial electron transport chain indicate that bipolar disorder (BD) is associated with pathology in mitochondrial function. These pathological processes occur in the brain circuits that regulate affective functions, emotions, and motor behaviors. The present study aimed to determine the relationship between mitochondrial complex dysfunction and BD. METHODS: The BD group included 32 male patients diagnosed with first-episode manic BD. The control group included 35 sociodemographically matched healthy males. Messenger ribonucleic acid (mRNA) was isolated from peripheral blood samples obtained from the patients and control group, and the mRNA levels of the NDUFV1, NDUFV2, and NDUFS1 genes of mitochondrial complex I and the UQCR10 gene of mitochondrial complex III were investigated. RESULTS: Significant differences were identified in complex I gene mRNA levels between the BD group (n = 32) and the control group (n = 35) for the following genes: NDUFV1 (P = 0.01), NDUFV2 (P < 0.01), and NDUFS1 (P = 0.02). The UQCR10 gene (complex III) mRNA level did not differ between the groups (P = 0.1). The mRNA levels of the four genes studied were lower at the 3-month follow-up; however, these differences were not significant (P > 0.05). LIMITATIONS: All of the BD patients were in manic episodes; thus, we were unable to separately compare these levels with those during depressive and euthymic episodes. CONCLUSIONS: The mRNA levels of all of the genes representing the subunits of mitochondrial complex I (NDUFV1, NDUFV2, and NDUFS1) were significantly higher in the present study's BD patients during manic episodes than in the controls. With the data obtained from further research, biomarkers that could be used for the diagnosis and follow-up of neuropsychiatric disorders may be discovered.

Low intensity training of mdx mice reduces carbonylation and increases expression levels of proteins involved in energy metabolism and muscle contraction.

High intensity training induces muscle damage in dystrophin-deficient mdx mice, an animal model for Duchenne muscular dystrophy. However, low intensity training (LIT) rescues the mdx phenotype and even reduces the level of protein carbonylation, a marker of oxidative damage. Until now, beneficial effects of LIT were mainly assessed at the physiological level. We investigated the effects of LIT at the molecular level on 8-week-old wild-type and mdx muscle using 2D Western blot and protein-protein interaction analysis. We found that the fast isoforms of troponin T and myosin binding protein C as well as glycogen phosphorylase were overcarbonylated and downregulated in mdx muscle. Some of the mitochondrial enzymes of the citric acid cycle were overcarbonylated, whereas some proteins of the respiratory chain were downregulated. Of functional importance, ATP synthase was only partially assembled, as revealed by Blue Native PAGE analysis. LIT decreased the carbonylation level and increased the expression of fast isoforms of troponin T and of myosin binding protein C, and glycogen phosphorylase. In addition, it increased the expression of aconitate hydratase and NADH dehydrogenase, and fully restored the ATP synthase complex. Our study demonstrates that the benefits of LIT are associated with lowered oxidative damage as revealed by carbonylation and higher expression of proteins involved in energy metabolism and muscle contraction. Potentially, these results will help to design therapies for DMD based on exercise mimicking drugs.

Stable enhanced green fluorescent protein expression after differentiation and transplantation of reporter human induced pluripotent stem cells generated by AAVS1 transcription activator-like effector nucleases.

Human induced pluripotent stem (hiPS) cell lines with tissue-specific or ubiquitous reporter genes are extremely useful for optimizing in vitro differentiation conditions as well as for monitoring transplanted cells in vivo. The adeno-associated virus integration site 1 (AAVS1) locus has been used as a "safe harbor" locus for inserting transgenes because of its open chromatin structure, which permits transgene expression without insertional mutagenesis. However, it is not clear whether targeted transgene expression at the AAVS1 locus is always protected from silencing when driven by various promoters, especially after differentiation and transplantation from hiPS cells. In this paper, we describe a pair of transcription activator-like effector nucleases (TALENs) that enable more efficient genome editing than the commercially available zinc finger nuclease at the AAVS1 site. Using these TALENs for targeted gene addition, we find that the cytomegalovirus-immediate early enhancer/chicken ß-actin/rabbit ß-globin (CAG) promoter is better than cytomegalovirus 7 and elongation factor 1α short promoters in driving strong expression of the transgene. The two independent AAVS1, CAG, and enhanced green fluorescent protein (EGFP) hiPS cell reporter lines that we have developed do not show silencing of EGFP either in undifferentiated hiPS cells or in randomly and lineage-specifically differentiated cells or in teratomas. Transplanting cardiomyocytes from an engineered AAVS1-CAG-EGFP hiPS cell line in a myocardial infarcted mouse model showed persistent expression of the transgene for at least 7 weeks in vivo. Our results show that high-efficiency targeting can be obtained with open-source TALENs and that careful optimization of the reporter and transgene constructs results in stable and persistent expression in vitro and in vivo.

TNFα-induced lysosomal membrane permeability is downstream of MOMP and triggered by caspase-mediated NDUFS1 cleavage and ROS formation.

When NF-κB activation or protein synthesis is inhibited, tumor necrosis factor alpha (TNFα) can induce apoptosis through Bax- and Bak-mediated mitochondrial outer membrane permeabilization (MOMP) leading to caspase-3 activation. Additionally, previous studies have implicated lysosomal membrane permeability (LMP) and formation of reactive oxygen species (ROS) as early steps of TNFα-induced apoptosis. However, how these two events connect to MOMP and caspase-3 activation has been largely debated. Here, we present the novel finding that LMP induced by the addition of TNFα plus cycloheximide (CHX), the release of lysosomal cathepsins and ROS formation do not occur upstream but downstream of MOMP and require the caspase-3-mediated cleavage of the p75 NDUFS1 subunit of respiratory complex I. Both a caspase non-cleavable p75 mutant and the mitochondrially localized antioxidant MitoQ prevent LMP mediated by TNFα plus CHX and partially interfere with apoptosis induction. Moreover, LMP is completely blocked in cells deficient in both Bax and Bak, Apaf-1, caspase-9 or both caspase-3 and -7. Thus, after MOMP, active caspase-3 exerts a feedback action on complex I to produce ROS. ROS then provoke LMP, cathepsin release and further caspase activation to amplify TNFα apoptosis signaling.

Downregulation of the expression of mitochondrial electron transport complex genes in autism brains.

Mitochondrial dysfunction (MtD) and abnormal brain bioenergetics have been implicated in autism, suggesting possible candidate genes in the electron transport chain (ETC). We compared the expression of 84 ETC genes in the post-mortem brains of autism patients and controls. Brain tissues from the anterior cingulate gyrus, motor cortex, and thalamus of autism patients (n = 8) and controls (n = 10) were obtained from Autism Tissue Program, USA. Quantitative real-time PCR arrays were used to quantify gene expression. We observed reduced expression of several ETC genes in autism brains compared to controls. Eleven genes of Complex I, five genes each of Complex III and Complex IV, and seven genes of Complex V showed brain region-specific reduced expression in autism. ATP5A1 (Complex V), ATP5G3 (Complex V) and NDUFA5 (Complex I) showed consistently reduced expression in all the brain regions of autism patients. Upon silencing ATP5A1, the expression of mitogen-activated protein kinase 13 (MAPK13), a p38 MAPK responsive to stress stimuli, was upregulated in HEK 293 cells. This could have been induced by oxidative stress due to impaired ATP synthesis. We report new candidate genes involved in abnormal brain bioenergetics in autism, supporting the hypothesis that mitochondria, critical for neurodevelopment, may play a role in autism.

Increased ATP generation in the host cell is required for efficient vaccinia virus production.

To search for cellular genes up-regulated by vaccinia virus (VV) infection, differential display-reverse transcription-polymerase chain reaction (ddRT-PCR) assays were used to examine the expression of mRNAs from mock-infected and VV-infected HeLa cells. Two mitochondrial genes for proteins that are part of the electron transport chain that generates ATP, ND4 and CO II, were up-regulated after VV infection. Up-regulation of ND4 level by VV infection was confirmed by Western blotting analysis. Up-regulation of ND4 was reduced by the MAPK inhibitor, apigenin, which has been demonstrated elsewhere to inhibit VV replication. The induction of ND4 expression occurred after viral DNA replication since ara C, an inhibitor of poxviral DNA replication, could block this induction. ATP production was increased in the host cells after VV infection. Moreover, 4.5 microM oligomycin, an inhibitor of ATP production, reduced the ATP level 13 hr after virus infection to that of mock-infected cells and inhibited viral protein expression and virus production, suggesting that increased ATP production is required for efficient VV production. Our results further suggest that induction of ND4 expression is through a Bcl-2 independent pathway.

At environmental doses, dietary methylmercury inhibits mitochondrial energy metabolism in skeletal muscles of the zebra fish (Danio rerio).

The neurotoxic compound methylmercury (MeHg) is a commonly encountered pollutant in the environment, and constitutes a hazard for human health through fish eating. To study the impact of MeHg on mitochondrial structure and function, we contaminated the model fish species Danio rerio with food containing 13 microg of MeHg per gram, an environmentally relevant dose. Mitochondria from contaminated zebrafish muscles presented structural abnormalities under electron microscopy observation. In permeabilized muscle fibers, we observed, a strong inhibition of both state 3 mitochondrial respiration and functionally isolated maximal cytochrome c oxidase (COX) activity after 49 days of MeHg exposure. However, the state 4 respiratory rate remained essentially unchanged. This suggested a defect at the level of ATP synthesis. Accordingly, we measured a dramatic decrease in the rate of ATP release by skinned muscle fibers using either pyruvate and malate or succinate as respiratory substrates. However, the amount and the assembly of the ATP synthase were identical in both control and contaminated muscle mitochondrial fractions. This suggests that MeHg induced a decoupling of mitochondrial oxidative phosphorylation in the skeletal muscle of zebrafish. Western blot analysis showed a 30% decrease of COX subunit IV levels, a 50% increase of ATP synthase subunit alpha, and a 40% increase of the succinate dehydrogenase Fe/S protein subunit in the contaminated muscles. This was confirmed by the analysis of gene expression levels, using RT-PCR. Our study provides a basis for further analysis of the deleterious effect of MeHg on fish health via mitochondrial impairment.

Optimized allotopic expression of the human mitochondrial ND4 prevents blindness in a rat model of mitochondrial dysfunction.

Mitochondrial diseases due to mutations in mitochondrial DNA can no longer be ignored in most medical areas. With prevalence certainly higher than one in 6000, they probably represent the most common form of metabolic disorders. Despite progress in identification of their molecular mechanisms, little has been done with regard to therapy. We have recently optimized the allotopic expression for the mitochondrial genes ATP6, ND1, and ND4 and obtained a complete and long-lasting rescue of mitochondrial dysfunction in the human fibroblasts in which these genes were mutated. However, biosafety and benefit to mitochondrial function must be validated in animal models prior to clinical applications. To create an animal model of Leber Hereditary Optic Neuropathy (LHON), we introduced the human ND4 gene harboring the G11778A mutation, responsible of 60% of LHON cases, to rat eyes by in vivo electroporation. The treatment induced the degeneration of retinal ganglion cells (RGCs), which were 40% less abundant in treated eyes than in control eyes. This deleterious effect was also confirmed in primary cell culture, in which both RGC survival and neurite outgrowth were compromised. Importantly, RGC loss was clearly associated with a decline in visual performance. A subsequent electroporation with wild-type ND4 prevented both RGC loss and the impairment of visual function. Hence, these data provide the proof-of-principle that optimized allotopic expression can be an effective treatment for LHON, and they open the way to clinical studies on other devastating mitochondrial disorders.

A critical phosphate concentration in the stationary phase maintains ndh gene expression and aerobic respiratory chain activity in Escherichia coli.

Escherichia coli NADH dehydrogenase-2 (NDH-2) is a primary dehydrogenase in aerobic respiration that shows cupric-reductase activity. The enzyme is encoded by ndh, which is highly regulated by global transcription factors. It was described that the gene is expressed in the exponential growth phase and repressed in late stationary phase. We report the maintenance of NDH-2 activity and ndh expression in the stationary phase when cells were grown in media containing at least 37 mM phosphate. Gene regulation was independent of RpoS and other transcription factors described to interact with the ndh promoter. At this critical phosphate concentration, cell viability, oxygen consumption rate, and NADH/NAD+ ratio were maintained in the stationary phase. These physiological parameters gradually changed, but NDH-2 activity remained high for up to 94 h. Phosphate seems to trigger an internal signal in the stationary phase mediated by systems not yet described.

PdhR (pyruvate dehydrogenase complex regulator) controls the respiratory electron transport system in Escherichia coli.

The pyruvate dehydrogenase (PDH) multienzyme complex plays a key role in the metabolic interconnection between glycolysis and the citric acid cycle. Transcription of the Escherichia coli genes for all three components of the PDH complex in the pdhR-aceEF-lpdA operon is repressed by the pyruvate-sensing PdhR, a GntR family transcription regulator, and derepressed by pyruvate. After a systematic search for the regulation targets of PdhR using genomic systematic evolution of ligands by exponential enrichment (SELEX), we have identified two novel targets, ndh, encoding NADH dehydrogenase II, and cyoABCDE, encoding the cytochrome bo-type oxidase, both together forming the pathway of respiratory electron transport downstream from the PDH cycle. PDH generates NADH, while Ndh and CyoABCDE together transport electrons from NADH to oxygen. Using gel shift and DNase I footprinting assays, the PdhR-binding site (PdhR box) was defined, which includes a palindromic consensus sequence, ATTGGTNNNACCAAT. The binding in vitro of PdhR to the PdhR box decreased in the presence of pyruvate. Promoter assays in vivo using a two-fluorescent-protein vector also indicated that the newly identified operons are repressed by PdhR and derepressed by the addition of pyruvate. Taken together, we propose that PdhR is a master regulator for controlling the formation of not only the PDH complex but also the respiratory electron transport system.

Obligatory role for complex I inhibition in the dopaminergic neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mice and nonhuman primates causes a parkinsonian disorder characterized by a loss of dopamine-producing neurons in the substantia nigra and corresponding motor deficits. MPTP has been proposed to exert its neurotoxic effects through a variety of mechanisms, including inhibition of complex I of the mitochondrial respiratory chain, displacement of dopamine from vesicular stores, and formation of reactive oxygen species from mitochondrial or cytosolic sources. However, the mechanism of MPTP-induced neurotoxicity is still a matter of debate. Recently, we reported that the yeast single-subunit nicotinamide adenine dinucleotide (reduced) dehydrogenase (NDI1) is resistant to rotenone, a complex I inhibitor that produces a parkinsonian syndrome in rats, and that overexpression of NDI1 in SK-N-MC cells prevents the toxicity of rotenone. In this study, we used viral-mediated overexpression of NDI1 in SK-N-MC cells and animals to determine the relative contribution of complex I inhibition in the toxicity of MPTP. In cell culture, NDI1 overexpression abolished the toxicity of 1-methyl-4-phenylpyridinium, the active metabolite of MPTP. Overexpression of NDI1 through stereotactic administration of a viral vector harboring the NDI1 gene into the substantia nigra protected mice from both the neurochemical and behavioral deficits elicited by MPTP. These data identify inhibition of complex I as a requirement for dopaminergic neurodegeneration and subsequent behavioral deficits produced by MPTP. Furthermore, combined with reports of a complex I defect in Parkinson's disease (PD) patients, the present study affirms the utility of MPTP in understanding the molecular mechanisms underlying dopaminergic neurodegeneration in PD.

Activation of mitochondrial promoter P(H)-binding protein in a radio-resistant Chinese hamster cell strain associated with Bcl-2.

The cellular response to ionizing radiation is mediated by a complex interaction of number of proteins involving different pathways. Previously, we have shown that up regulation of mitochondrial genes ND1, ND4, and COX1 transcribed from the heavy strand promoter (P(H)) has been increased in a radio-resistant cell strain designated as M5 in comparison with the parental Chinese hamster V79 cells. These genes are also up regulated in Chinese hamster V79 cells VB13 that express exogenous human Bcl2. In the present study, the expression of the gene ND6 that is expressed from the light strand promoter (P(L)) was found to be similar in both the cell lines, as determined by RT-PCR. To test the possibility that this differential expression of mitochondrial genes under these two promoters was mediated by differences in proteins' affinity to interact with these promoters, we have carried out electrophoretic mobility shift assay (EMSA) using mitochondrial cell extracts from these two cell lines. Our result of these experiments revealed that two different proteins formed complex with the synthetic promoters and higher amount of protein from M5 cell extracts interacted with the P(H) promoter in comparison to that observed with cell extracts from Chinese hamster V79 cells. The promoter-specific differential binding of proteins was also observed in VB13. These results showed that differential mitochondrial gene expression observed earlier in the radio-resistant M5 cells was due to enhanced interaction proteins with the promoters P(H) and mediated by the expression of Bcl2.