Title: Nuclear and Mitochondrial Interaction Involving mt-Nd2 Leads to Increased Mitochondrial Reactive Oxygen Species Production
Abstract: NADH dehydrogenase subunit 2, encoded by the mtDNA, has been associated with resistance to autoimmune type I diabetes (T1D) in a case control study. Recently, we confirmed a role for the mouse ortholog of the protective allele (mt-Nd2a) in resistance to T1D using genetic analysis of outcrosses between T1D-resistant ALR and T1D-susceptible NOD mice. We sought to determine the mechanism of disease protection by elucidating whether mt-Nd2a affects basal mitochondrial function or mitochondrial function in the presence of oxidative stress. Two lines of reciprocal conplastic mouse strains were generated: one with ALR nuclear DNA and NOD mtDNA (ALR.mtNOD) and the reciprocal with NOD nuclear DNA and ALR mtDNA (NOD.mtALR). Basal mitochondrial respiration, transmembrane potential, and electron transport system enzymatic activities showed no difference among the strains. However, ALR.mtNOD mitochondria supported by either complex I or complex II substrates produced significantly more reactive oxygen species when compared with both parental strains, NOD.mtALR or C57BL/6 controls. Nitric oxide inhibited respiration to a similar extent for mitochondria from the five strains due to competitive antagonism with molecular oxygen at complex IV. Superoxide and hydrogen peroxide generated by xanthine oxidase did not significantly decrease complex I function. The protein nitrating agents peroxynitrite or nitrogen dioxide radicals significantly decreased complex I function but with no significant difference among the five strains. In summary, mt-Nd2a does not confer elevated resistance to oxidative stress; however, it plays a critical role in the control of the mitochondrial reactive oxygen species production. NADH dehydrogenase subunit 2, encoded by the mtDNA, has been associated with resistance to autoimmune type I diabetes (T1D) in a case control study. Recently, we confirmed a role for the mouse ortholog of the protective allele (mt-Nd2a) in resistance to T1D using genetic analysis of outcrosses between T1D-resistant ALR and T1D-susceptible NOD mice. We sought to determine the mechanism of disease protection by elucidating whether mt-Nd2a affects basal mitochondrial function or mitochondrial function in the presence of oxidative stress. Two lines of reciprocal conplastic mouse strains were generated: one with ALR nuclear DNA and NOD mtDNA (ALR.mtNOD) and the reciprocal with NOD nuclear DNA and ALR mtDNA (NOD.mtALR). Basal mitochondrial respiration, transmembrane potential, and electron transport system enzymatic activities showed no difference among the strains. However, ALR.mtNOD mitochondria supported by either complex I or complex II substrates produced significantly more reactive oxygen species when compared with both parental strains, NOD.mtALR or C57BL/6 controls. Nitric oxide inhibited respiration to a similar extent for mitochondria from the five strains due to competitive antagonism with molecular oxygen at complex IV. Superoxide and hydrogen peroxide generated by xanthine oxidase did not significantly decrease complex I function. The protein nitrating agents peroxynitrite or nitrogen dioxide radicals significantly decreased complex I function but with no significant difference among the five strains. In summary, mt-Nd2a does not confer elevated resistance to oxidative stress; however, it plays a critical role in the control of the mitochondrial reactive oxygen species production. Single nucleotide polymorphisms in the mtDNA have been associated with degenerative diseases and various cancers. Yet sequence changes may also result in resistance to disease. Indeed, a cytosine to adenine transversion (C5178A) resulting in a leucine to methionine substitution in the human NADH dehydrogenase subunit 2 gene (mt-ND2) encoded in the mtDNA has been associated with increased longevity (1Yao Y.G. Kong Q.P. Zhang Y.P. Hum. Genet. 2002; 111: 462-463Crossref PubMed Scopus (41) Google Scholar, 2Tanaka M. Gong J.S. Zhang J. Yoneda M. Yagi K. Lancet. 1998; 351: 185-186Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar) as well as reductions in atherosclerosis (3Matsunaga H. Tanaka Y. Tanaka M. Gong J.S. Zhang J. Nomiyama T. Ogawa O. Ogihara T. Yamada Y. Yagi K. Kawamori R. Diabetes Care. 2001; 24: 500-503Crossref PubMed Scopus (63) Google Scholar), blood pressure (4Kokaze A. Ishikawa M. Matsunaga N. Yoshida M. Sekine Y. Sekiguchi K. Harada M. Satoh M. Teruya K. Takeda N. Fukazawa S. Uchida Y. Takashima Y. J. Hum. Hypertens. 2004; 18: 41-45Crossref PubMed Scopus (31) Google Scholar), myocardial infarction (5Takagi K. Yamada Y. Gong J.S. 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Diabetes Care. 2002; 25: 2106Crossref PubMed Scopus (30) Google Scholar) as many of the diseases against which mt-ND2a imparts resistance, such as aging and T1D, have been associated with ROS damage. In support of the hypothesis that mt-Nd2a results in elevated resistance to oxidative stress, Turko et al. (38Turko I.V. Li L. Aulak K.S. Stuehr D.J. Chang J.Y. Murad F. J. Biol. Chem. 2003; 278: 33972-33977Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar) have reported that complex I enzymatic activity of ALR was not affected by free radicals, although the same treatments reduced complex I activity in ALS by ∼50%. The aim of this study was to determine the effect of mt-Nd2a on basal mitochondrial function as well as on mitochondrial function in the presence of oxidative stress to further characterize its role in the protection against T1D. To study the specific role of mt-Nd2a, two lines of reciprocal conplastic mouse strains (CS) were developed, one with ALR nuclear DNA and NOD mtDNA (ALR.mtNOD) and one with NOD nuclear DNA and ALR mtDNA (NOD.mtALR). By combining mt-Nd2a with NOD nuclear DNA, the effects of this allele could be considered separately from the protective effects of the nuclear genome of ALR. Here we assess basal mitochondrial functions and determine the resistance of mitochondria to free radicals. We find that mt-Nd2a does not confer elevated resistance to oxidative stress. However, mt-Nd2a suppresses mitochondrial ROS production. Mice—ALR/LtJ, NOD/LtJ, and C57BL/6J (B6) mice were bred and maintained in the animal research facility at the Rangos Research Center, Pittsburgh, PA. Conplastic strains of mice, NOD/LtJ-mtALR/LtJ/Mx (NOD.mtALR) and ALR/LtJ-mtNOD/LtDvs/Mx (ALR.mtNOD), were generated as described below. All mice were bred and maintained in a specific pathogen-free vivarium and allowed free access to food (autoclaved diet NIH-31, 6% fat, PMI, St. Louis, MO) and acidified drinking water. All procedures involving animals were approved by the Children's Hospital of Pittsburgh and were in compliance with "Principles of Laboratory Animal Care" and the current laws of the United States. Reagents—All reagents were obtained from Sigma unless otherwise noted. Generation of Reciprocal Conplastic Strains of Mice—Lines of reciprocal CS mice were generated to determine the role of mt-Nd2a in resistance to T1D and for any possible effects on mitochondrial function. Strains with ALR nuclear DNA and NOD mtDNA (ALR.mtNOD) or with NOD nuclear DNA and ALR mtDNA (NOD.mtALR) were generated as described previously (39Pomerleau D.P. Bagley R.J. Serreze D.V. Mathews C.E. Leiter E.H. Diabetes. 2005; 54: 1603-1606Crossref PubMed Scopus (27) Google Scholar) with minor modifications. Because mtDNA is inherited exclusively from the egg, only female breeders with the appropriate mtDNA were employed. The generation of ALR.mtNOD CS utilized an F1 outcross of ALR males to NOD females resulting in F1 progeny with NOD mtDNA. Females of this outcross were then backcrossed to ALR males for 10 generations, allowing for continued inheritance of the NOD mtDNA. Conversely, to generate NOD.mtALR CS, NOD males were outcrossed to ALR females resulting in F1 progeny with ALR mtDNA. At each generation females were backcrossed to NOD males until the 10th backcross generation and then intercrossed. Single nucleotide polymorphism typing was conducted to determine the mt-Nd2 allele in the CS as described (23Mathews C.E. Leiter E.H. Spirina O. Bykhovskaya Y. Gusdon A.M. Ringquist S. Fischel-Ghodsian N. Diabetologia. 2005; 48: 261-267Crossref PubMed Scopus (37) Google Scholar). To preclude nuclear DNA contamination in the CS mice, genotyping was performed by PCR amplification of 94 polymorphic microsatellite primers (Invitrogen) covering all 19 autosomes (24Mathews C.E. Graser R.T. Bagley R.J. Caldwell J.W. Li R. Churchill G.A. Serreze D.V. Leiter E.H. Immunogenetics. 2003; 55: 491-496Crossref PubMed Scopus (33) Google Scholar) (supplemental Table). Liver Mitochondrial Isolation—Livers were removed and homogenized in ice-cold isolation buffer (IB) I (225 mm mannitol, 75 mm sucrose, 10 mm HEPES potassium salt, 0.10% bovine serum albumin, fatty acid-free, and 1 mm EDTA, pH 7.4). The homogenate was centrifuged at 1,300 × g for 10 min. The supernatant was transferred into new tubes, diluted with IB I, and centrifuged at 10,000 × g for 10 min. The supernatant was discarded, and the pellet was resuspended in IB II (225 mm mannitol, 75 mm sucrose, 10 mm HEPES potassium salt, and 100 μm EDTA, pH 7.4) and spun at 10,000 × g for 10 min. The resulting pellet was resuspended in ∼100 μl of IBII. Brain Mitochondrial Isolation—Brains were removed, homogenized in ice-cold 12% Percoll in IB I, layered on top of a gradient of 24 and 42% Percoll, and centrifuged at 27,000 × g for 10 min. The mitochondrial fraction was removed with a syringe, diluted with IB I, and centrifuged at 10,000 × g for 10 min. The supernatant was discarded, and the pellet was resuspended in IB II and spun at 10,000 × g for 5 min. The supernatant was again discarded, and the pellet was resuspended in ∼100 μl of IB II. Protein concentration of both liver and brain mitochondria was determined using the BCA protein assay (Pierce). Mitochondrial Respiration—Mitochondria (1.6 mg/ml) were incubated in media containing 125 mm KCl, 2 mm K2HPO4, 5 mm MgCl2, 10 mm HEPES, and 10 μm EGTA, pH 7.1 (IM). To assay respiration through complex I, the assay medium was supplemented with 5 mml-glutamate and 5 mml-malate. To assay respiration through complex II, the assay medium was supplemented with 5 mm succinate. State 3 respiration was stimulated by the addition of 1.81 mm ADP. The respiratory control ratio was calculated by dividing state 3 respiration rates by state 4 respiration rates. Mitochondrial respiration was determined using a Clark-type oxygen electrode (Hansatech Instruments Ltd., Norfolk, UK). Assays were performed at 37 °C with constant stirring. Individual Mitochondrial Electron Transport Chain Complex Enzymatic Activity Assays—For each assay, mitochondria samples were subjected to membrane disruption by freeze-thawing. All assays were run at 30 °C. The activity of complex I (NADH:ubiquinone oxidoreductase) was determined by monitoring the oxidation of NADH at 340 nm. The assay medium contained potassium phosphate (25 mm, pH 7.2 at 20 °C), 5 mm MgCl2, 2.5 mg/ml bovine serum albumin (fraction V), and 2 mm KCN. A base line was established for 1 min after the addition of 0.13 mm NADH, 65 μm ubiquinone1, and 2 μg/ml antimycin A. The reaction was initiated by the addition of mitochondria (200 μg/ml), and the rate of oxidation of NADH was recorded for 3 min. Rotenone (2 μg/ml) was then added, and the rate of change in absorbance was measured for an additional 3 min. Complex I activity was determined by subtracting the rotenone insensitive activity from the total activity. The activity of complex II (succinate:ubiquinone oxidoreductace) was determined by monitoring the reduction of 2,6-dichloroindophenolate at 600 nm. The assay medium contained potassium phosphate (25 mm, pH 7.2 at 20 °C), 5 mm MgCl2, and 20 mm sodium succinate. Mitochondria (40 μg/ml) were incubated in the assay medium at 30 °C for 10 min. A base line was recorded for 1 min after the addition of 2 μg/ml antimycin A, 2 μg/ml rotenone, 2 mm KCN, and 50 μm 2,6-dichloroindophenolate. The reaction was initiated by the addition of 65 μm ubiquinone1, and the rate of reduction of 2,6-dichloroindophenolate was recorded for 3 min (40Birch-Machin M. Turnbull D.M. Pon L. Schon E.A. Mitochondria. 2001Google Scholar). The activity of complex III (cytochrome c reductase) was determined by monitoring the reduction of ferricytochrome c at 550 nm. The assay medium contained potassium phosphate (25 mm, pH 7.2 at 20 °C), 5 mm MgCl2, 2.5 mg/ml bovine serum albumin (fraction V), and 2 mm KCN. KCN was included in the assay media to prevent the reoxidation of the product, ferrocytochrome c, by cytochrome c oxidase. Nonenzymatic activity was recorded for 1 min after the addition of 15 μm ferricytochrome c, 2 μg/ml rotenone, 0.6 mm dodecyl-β-d-maltoside, and 35 μm ubiquionol. Ubuiquinol was prepared by dissolving 8 μg of ubiquinone in 1 ml of ethanol; the solution was adjusted to pH 2 with 6 m HCl. Ubiquinone was reduced using excess sodium borohydride. Ubiquinol was extracted into 2:1 (v/v) diethyl ether/cyclohexane, evaporated under nitrogen gas, dissolved in 1 ml of ethanol, and acidified to pH 2 with 6 m HCl. The complex III activity assay was initiated by the addition of mitochondria (100 μg/ml), and the rate of reduction of ferricytochrome c to ferrocytochrome c was recorded for 1 min. The activity quickly became nonlinear, and the rate was calculated based on the linear first 10 s. In replicate wells, 2 μg/ml antimycin A was added, and the complex III specific activity was calculated by subtracting the antimycin A insensitive activity from the total activity (40Birch-Machin M. Turnbull D.M. Pon L. Schon E.A. Mitochondria. 2001Google Scholar). Complex IV (cytochrome c oxidase) activity was determined by monitoring the oxidation of ferrocytochrome c at 550 nm. The assay medium contained 10 mm Tris-HCl and 120 mm KCl, pH 7.0. The nonenzymatic rate was recorded for 1 min after the addition of 2 μg/ml antimycin A, 0.45 mm dodecyl-β-d-maltoside, and mitochondria (2.5 μg/ml). The reaction was initiated by the addition of 11 μm ferrocytochrome c, and the rate of oxidation of ferrocytochrome c to ferricytochrome c was measured for 3 min. The activity quickly became nonlinear, and the rate was calculated based on the linear first 30s. In replicate wells, 2 μg/ml KCN was added, and the complex IV specific activity was calculated by subtracting the KCN insensitive activity from the total activity. Ferrocytochrome c was prepared by reducing ferricytochrome c with 0.5 mm dithiothreitol (40Birch-Machin M. Turnbull D.M. Pon L. Schon E.A. Mitochondria. 2001Google Scholar). Mitochondrial Membrane Potential and ROS Production—Membrane potential and free radical production were measured by fluorescence using a Shimadzu RF-5301 spectrofluorimeter (Kyoto, Japan) as described previously (41Votyakova T.V. Reynolds I.J. J. Neurochem. 2005; 93: 526-537Crossref PubMed Scopus (93) Google Scholar). All assays were performed with 350 μg of mitochondrial protein suspended in IM plus 5 mml-glutamate and 5 mml-malate or 5 mm succinate with constant stirring at 37 °C. ROS production was measured using 2 μm fluorescent Amplex Red dye (Molecular Probes, Eugene, OR) in the presence of 1 unit/ml horseradish peroxidase. The excitation wavelength was 560 nm (slit 1.5 nm), and the emission wavelength was 590 nm (slit 3 nm). Mitochondrial transmembrane potential (Δψm) was measured using the fluorescence quenching of the cationic dye safranin O (2.5 μm). The excitation wavelength was 495 nm (slit 3 nm), and the emission wavelength was 586 nm (slit 10 nm). Free Radical Treatment for Mitochondrial Respiration—Mitochondrial respiration was assayed as described above. Approximately 30 s after the addition of ADP, 15 μm nitric oxide (NO) was added using the NO donor diethylamine NONOate (Cayman Chemical, Ann Arbor, MI). Percent oxygen consumption was calculated by comparing chamber oxygen content at the time of NO addition to the chamber oxygen content 1.5 min after the addition of NO. Calculations were based on the percent of control oxygen consumption. To test whether the effect of NO was reversible, bovine hemoglobin (1.32 mg/ml) was added after 5 min of incubation with 15 μm NO. Free Radical Treatment for Complex I Enzymatic Activity Assay—Mitochondria were treated with either a 3 mm bolus of NO donated by diethylamine NONOate or a 1 μm steady-state level of NO donated by DETA-NONOate (Cayman Chemical, Ann Arbor, MI) for 2 h. Mitochondria were treated with hydrogen peroxide and superoxide generated by 100 μm xanthine and 20 milliunits of xanthine oxidase and incubated for 2 h. Control samples were treated with xanthine without xanthine oxidase. Peroxynitrite or heme peroxidase-dependent reactions were employed to facilitate protein nitration. Mitochondria were treated with 0.25, 0.50, or 1.00 mm peroxynitrite (Upstate Biotechnology, Inc., Lake Placid, NY) and incubated for 1 h. Controls were treated with the equivalent concentration of degraded peroxynitrite. Mitochondria were treated with 200 μg/ml glucose and 60 μg/ml or 40 ng/ml glucose oxidase (to generate H2O2) in the presence of 0.5 mm sodium nitrite and 100 nm myeloperoxidase and incubated for 1 h. In the presence of hydrogen peroxide, myeloperoxidase has been shown previously to oxidize nitrite to the nitrogen dioxide radical, which is capable of nitrating phenolic protein residues (42van der Vliet A. Eiserich J.P. Halliwell B. Cross C.E. J. Biol. Chem. 1997; 272: 7617-7625Abstract Full Text Full Text PDF PubMed Scopus (721) Google Scholar). Controls were treated with glucose, sodium nitrite, and myeloperoxidase without glucose oxidase. Complex I enzymatic activity was assayed as described above following incubations with each free radical generator. Free Radical Treatment for Complex IV Enzymatic Activity Assay—Mitochondria were incubated with 15 μm NO donated by diethylamine NONOate and incubated for 1 h. Complex IV enzymatic activity was assayed as described above following this incubation period. Statistical Analysis—All values reported are of at least n = 3. Significance was determined by one-way analysis of variance using Graphpad Prism 4 for Windows (GraphPad Software, Inc., San Diego, CA). Differences were considered significant at p < 0.05. Single Nucleotide Polymorphism Typing for Conplastic Strains—Pyrosequencing was performed for the A4738C single nucleotide polymorphism to confirm the presence of the mt-Nd2 allele in both CS. The pyrograms from ALR (supplemental Fig. A) and NOD.mtALR (supplemental Fig. C) were equivalent and demonstrated the presence of the mt-Nd2a allele. NOD (supplemental Fig. B) and ALR.mtNOD (supplemental Fig. D) both contained the mt-Ndc allele. Typing with a panel of microsatellite markers that discriminate ALR from NOD DNA (24Mathews C.E. Graser R.T. Bagley R.J. Caldwell J.W. Li R. Churchill G.A. Serreze D.V. Leiter E.H. Immunogenetics. 2003; 55: 491-496Crossref PubMed Scopus (33) Google Scholar) was performed from N1 to N7 to confirm the elimination of nuclear DNA contamination in both CS. We did not detect NOD nuclear DNA contamination in the ALR.mtNOD after generation N6. Likewise, ALR nuclear DNA contamination in the NOD.mtALR in mice after the N5 generation was undetectable. Basal Mitochondrial Enzymatic Activities, Respiration, and Transmembrane Potential—To discern effects of the two mt-Nd2 alleles on basal mitochondrial function, the mitochondrial ETS was assayed. As shown in Table 1, no differences were measured in enzymatic activities of complexes I–IV when comparing ALR, NOD, B6, NOD.mtALR, and ALR.mtNOD. Basal mitochondrial respiration supported by complex I or complex II substrates was also assayed (Table 2). State 4 respiration was measured after the addition of mitochondria to the chamber, and state 3 respiration was measured after the addition of ADP. Among the five strains, no differences were detected in either state 4 respiration, state 3 respiration, or the respiratory control ratio (rate of state 3 respiration divided by rate of state 4 respiration) while respiring via complex I or complex II (Table 2). No differences in Δψm values supported by complex I or II substrates among the five strains were observed (data not shown).TABLE 1Comparison of the individual enzymatic activities of ETS complexes I–IV for ALR, NOD, B6, NOD.mtALR, and ALR.mtNODStrainComplex I (nmol of NADH oxidized)Complex II (nmol of DCIP reduced)Complex III (nmol of Cyt c reduced)Complex IV (nmol of Cyt c oxidized)min × mg proteinmin × mg proteinmin × mg proteinmin × mg proteinALR35.83