- Email: [email protected]
Mitochondrial respiratory chain complex assembly and function during human fetal development
Available online at www.sciencedirect.com
Molecular Genetics and Metabolism 94 (2008) 120–126 www.elsevier.com/locate/ymgme
Mitochondrial respiratory chain complex assembly and function during human fetal development Limor Minai a, Jelena Martinovic b, Dominique Chretien a, Francßoise Dumez c, Fe´rechte´ Razavi b, Arnold Munnich a, Agne`s Ro¨tig a,* b
a INSERM U781 and Service de Ge´ne´tique, Hoˆpital Necker-Enfants Malades, 149 rue de Se`vres, 75015 Paris, France Service d’Histologie-Embryologie-Cytoge´ne´tique, Hoˆpital Necker-Enfants Malades, 149 rue de Se`vres, 75015 Paris, France c Service d’Obste´trique, Hoˆpital Necker-Enfants Malades, 149 rue de Se`vres, 75015 Paris, France
Received 16 November 2007; received in revised form 21 December 2007; accepted 21 December 2007 Available online 30 January 2008
Abstract Oxidative phosphorylation (OXPHOS) deficiency may have early antenatal manifestations, probably related to the time course and/ or tissue specificity of the disease gene expression during the embryo-fetal period. This feature hampers a fully reliable prenatal enzymological diagnosis of OXPHOS deficiency. We have studied OXPHOS in various human fetal tissues from 9 to 17 weeks of gestation. We found that the fetal respiratory chain complexes are fully assembled and functional at early stages of development in heart, liver, muscle, brain and kidney. We also observed a marked increase of respiratory chain activities and mitochondria content in postnatal compared to prenatal tissues. However, we were not able to detect obvious modification in the size, composition or activity of the various OXPHOS complexes during the second trimester of pregnancy that could account for the variations we observed in a pathological context. Therefore, we suggest that the time-dependent expression of respiratory chain deficiency either during fetal life or after birth could be related to the differential expression or regulation of the mutant proteins. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Mitochondria; Respiratory chain assembly; Enzymatic activity; Fetal development
Introduction Oxidative phosphorylation (OXPHOS)1 deficiency represents a group of early onset and severe disorders in infancy [1]. At variance with many metabolic diseases with a delayed onset and a symptom-free period, OXPHOS deficiency occasionally has an early antenatal expression, possibly related to the time course and/or tissue specificity of the disease gene expression in the embryo-fetal period [2]. Based on activities detected in fetal rat kidney [3] and human chorion villi, OXPHOS has long been known to function early in the *
Corresponding author. Fax: +33 (0) 147348514. E-mail address: [email protected] (A. Ro¨tig). 1 Abbreviations used: RC, respiratory chain; OXPHOS, oxidative phosphorylation; BN-PAGE, blue native-polyacrylamide gel electrophoresis; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. 1096-7192/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2007.12.007
embryo-fetal development. Yet, little is known regarding the kinetics of mitochondrial complex assembly during human embryonic and fetal life. However, at birth a spectacular activation of nuclear and mitochondrial gene expression occurs which is related to the adaptation to life at higher partial oxygen pressures [4–6]. Indeed, the early fetus develops in low oxygen environment (18 mm Hg in amniotic fluid between 11 and 16 weeks of gestation) [7]. When the principal stages of organogenesis are completed, oxygen concentration delivered to the embryo rises by the onset of blood flow into the placenta [8]. Upon birth, with high partial oxygen pressure (26 mm Hg), growing energetic demands and the need for OXPHOS, there is an up-regulation of mitochondrial biogenesis and activation of nuclear and mitochondrial gene expression occurs [4–6]. For these reasons, enzyme-based prenatal diagnosis on chorion villi or amniotic cells is hazardous and false negative or false positive results have been reported [9,10]. We
L. Minai et al. / Molecular Genetics and Metabolism 94 (2008) 120–126
have examined the onset of mitochondrial OXPHOS activities in various human fetal tissues from 9 to 17 weeks of gestation. These fetuses were obtained after termination of pregnancy for genetic diseases unrelated to OXPHOS deficiency. Our results show that the fetal OXPHOS complexes are fully assembled and enzymatically functional at least as from the end of the first trimester, although their absolute activity values are lower than those observed after birth. Materials and methods Pre- and postnatal tissues Fetal tissues (9–17 weeks of gestation) were obtained after termination of pregnancy for genetic disorders unrelated to OXPHOS deficiency (Table 1). Informed consents from parents were given. Termination of pregnancy was performed by aspiration. Fetuses were collected 5–10 min after abortion and the tissues were sampled in the following 30 min, immediately frozen and conserved in liquid nitrogen. They were usually analyzed 3 days later. Postnatal control tissues used for protein analysis were obtained from children who had died either from sudden death or genetic diseases unrelated to OXPHOS deficiency (a few days up to 5 years of age). The lag-period between death and tissue sampling was less than 4 h. Tissues were immediately frozen in liquid nitrogen. Respiratory chain enzyme analysis was normal for all the samples used in this study. Postnatal control tissues used for respiratory chain enzyme analysis were obtained from patients suspected of mitochondrial disease for whom RC analysis revealed normal activities.
Protein analysis For blue native-polyacrylamide gel electrophoresis (BN-PAGE), mitochondria and OXPHOS complexes were isolated as described [11–13] and proteins (40 lg) were analyzed on a 4–16% acrylamide non-denaturing gradient gel (Invitrogen). For SDS–PAGE, crude extracts from muscle, liver, brain, heart and kidney were prepared by homogenization of tissues (80 mg) in 0.1% Triton X-100, 0.01% digitonin and a Protease inhibitor cocktail (Sigma). The extracts were then filtered through a nylon mesh (100 lm). Total proteins (40 lg) were analyzed on 15% acrylamide gels [14]. For Western blot analysis, the gels were electroblotted onto PVDF filters and sequentially immunodecorated with specific antibodies. We used a cocktail of antibodies raised against the 20 kDa subunit of complex I, SDHB, core 2, COXII, F1a and monoclonal antibodies against GRIM 19, NDUFB6, NDUFA9, SDHA, core 1, COXIII and F1b (Mitosciences). The specific signal was detected using Western lightning chemilumi-
121
nescence reagent plus (PerkinElmer) with a charged-coupled-device camera (CCD) (Gene Gnome; Syngene, Cambridge, UK) and quantified using the GeneTools software (Syngene). For quantification, the signal obtained for an individual subunit was expressed relatively to the sum of signals obtained for all five subunits for the same tissue. Each calculation was done on at least three different samples. These arbitrary values allowed comparing the relative amount of the various complexes from one tissue to another and in pre- and postnatal period.
OXPHOS enzyme activities Spectrophotometric assays of OXPHOS enzymes were performed on muscle, liver, brain, heart and kidney as previously described [11]. Complex V activity was not measured in muscle as freezing this tissue results in a loss of sensitivity to oligomycin, probably related to the loss of oligomycin sensitivity conferring protein (OSCP). For each tissue, at least six different individuals were used and each enzyme activity was measured twice.
Mitochondrial DNA quantification Total DNA was extracted from tissues by standard phenol/chloroform extraction and ethanol precipitation, followed by column purification (Macherey-Nagel kit). Quantitative PCR analyses were performed on a Taqman 7300 (Applied Biosystems). The mitochondrial 12S rRNA and the nuclear MutL protein homolog 1 (MLH1) genes were individually amplified using primers 12S rRNA-F/12S rRNA-R and primers MLH1F/MLH1-R, respectively, and mtDNA-A and MLH1-A as hybridization probes (Table 2). The efficiency of the two probes was tested for their compatibility according to the manufacturer’s instruction (Applied Biosystems). A standard curve composed of a series of 3 dilutions (1/10, 1/ 100, 1/1000) in triplicate, was carried out for each individual. The amount of mtDNA was normalized according to the nuclear DNA quantity using the DDCT method provided that the efficiencies of these two PCRs were compatible. To compensate for any eventual inaccuracies due to different PCR efficiencies, we have used the equation R0,T/R0,R = (1+ER)CT,R/ (1+ET)CT,T as developed by [15]. (R, reporter fluorescence; R0, initial reporter; T, target gene; R, reference gene; E, efficiency; CT, threshold cycle). For each DNA, the average CT of the second dilution triplicate of each standard curve was taken. Standard deviation (SD) was calculated for each triplicate of CT to assure that the average is correct.
Statistics Non-parametric Wilcoxon rank test for small samples with non-Gaussian distribution was carried out to evaluate whether the difference between pre- and postnatal mtDNA quantity was significantly different.
Results
Table 1 Genetic diseases leading to the various terminations of pregnancy
Respiratory chain complex assembly
Genetic disease
Weeks of gestation
Exencephaly X-linked hydrocephaly Alpert syndrome Angelman syndrome Spinal muscular atrophy Stickler syndrome Pierre Robin syndrome Fragile X Encephalocele Fragile X X-linked retinitis pigmentosa Fragile X
9 11 11 12 12 12 13 13 14 14 15 17
To asses the assembly state of the fetal OXPHOS complexes, complexes were studied in fetal (11–17 weeks of gestation) and postnatal liver and heart (6 and 36 weeks). Table 2 Oligonucleotide primers 12S rRNA-F 12S rRNA-R mtDNA-A MLH1-F MLH1-R MLH1-A
50 -TAGAGGAGCCTGTTCTGTAAT-30 50 -TGCGCTTACTTTGTAGCCTTCAT-30 6FAM-50 -AAACCCCGATCAACC-30 -MGB 50 -GTAGTCTGTGATCTCCGTTT-30 50 -ATGTATGAGGTCCTGTCCTA-30 6FAM-50 -TGGTGATGCACACTGGCACC-30 -TAMRA
122
L. Minai et al. / Molecular Genetics and Metabolism 94 (2008) 120–126
They were analyzed under native conditions using a blue native-polyacrylamide gel electrophoresis system (BNPAGE). Western blots were successively incubated with five different antibodies raised against one subunit of each of the five OXPHOS complexes. Similar size and steadystate levels of all complexes were observed in the six fetal liver tissues tested and in the postnatal livers (Fig. 1) suggesting that all five OXPHOS complexes are fully assembled at least as from 11 weeks of gestation. The same results were obtained in heart tissues of five different fetuses aged from 12 to 15 weeks (Fig. 1). Subunit composition of the fetal OXPHOS complexes Crude extracts of brain, heart, liver and kidney tissues of a 17 weeks old fetus and postnatal heart and liver tissues (36 weeks) were analyzed by SDS–PAGE followed by Western blot. We used a cocktail of antibodies raised against the 20 kDa subunit of complex I, SDHB of complex II, core protein 2 of complex III, COXII of complex IV and F1a of complex V. The presence of most of them, with the exception of F1a, reflects a fully assembled complex as these subunits are either essential for assembly of their corresponding complex or labile if the complex is not fully assembled [16–20]. In fetal heart, liver and kidney the protein profile was similar to that obtained in postnatal heart and liver tissues (Fig. 2). However, a slightly faster migrating F1a subunit of complex V was detected in fetal brain compared to other pre- and postnatal tissues. These results were repeatedly observed at various developmental stages in fetal brain (11, 15, 17 weeks), heart (9, 11, 14, 15, 17 weeks), liver (11, 14, 15, 17 weeks) and kidney (14, 15, 17 weeks, not shown). To further characterize the fetal protein content, we studied additional subunits of the OXPHOS complexes. Fetal brain showed different protein migration patterns compared to postnatal brain. Indeed, NDUFA9 and to a lesser extent NDUFB6 subunits migrated slower, whereas
Fig. 2. SDS–PAGE and Western blot analysis of respiratory chain proteins in prenatal brain, heart, liver, kidney and postnatal heart and liver using a cocktail of five antibodies against one subunit of each of the OXPHOS complexes (20 kDa subunit for complex I, SDHB for complex II, core protein 2 for complex III, COXII for complex IV and F1a for complex V).
the F1b subunit of a 15 weeks fetus migrated faster than in postnatal tissues (Fig. 3). The anti-Ip antibody of CIII (Rieske protein) revealed two closely migrating bands in prenatal kidney and postnatal brain, heart, liver and kidney. The signal for COXIII subunit of complex IV was lower in prenatal tissues. The other subunits showed similar migration patterns in the various pre- and postnatal tissues. In order to compare the relative amount of the various complexes from one tissue to another and in pre- and postnatal period, the signal obtained for an individual subunit observed in Fig. 2 was expressed relatively to the sum of signals obtained for all five subunits for the same tissue. This showed that similar relative amounts of the various OXPHOS complexes were present in pre- and postnatal heart and liver (Fig. 4). Moreover, it is worth noting that complex II was relatively more abundant in liver than in
Fig. 1. BN-PAGE and Western blot analysis of mitochondria from fetus liver and heart at various ages of gestation (11–17 weeks of gestation) and from controls (6 and 36 weeks) using antibodies against GRIM19 for complex I (CI), SDHA for complex II (CII), core 2 for complex III (CIII), COXII for complex IV (CIV) and F1b subunits for complex V (CV).
L. Minai et al. / Molecular Genetics and Metabolism 94 (2008) 120–126
123
(Fig. 5C–F). The higher activities were consistently observed for complex III and the lower ones for complex I in pre- and postnatal stages. The activity of citrate synthase, a TCA cycle enzyme that reflects the mitochondrial mass was similar at the different stages (see Fig. 5A for a representative example). Finally, prenatal values in liver, heart and muscle were constantly lower than postnatal ones (Fig. 5B, D and F). Only citrate synthase activity did not significantly increase after birth in liver. It is worth noting that muscle complex IV activities were relatively lower in the prenatal than in the postnatal period, as compared to other complexes. Mitochondrial DNA content
Fig. 3. SDS–PAGE and Western blot analysis of respiratory chain proteins in prenatal (15 weeks) and postnatal brain, heart, liver, kidney (12 weeks) using antibodies against various subunits of RC complexes (CI– CV: complexes I–V).
heart. Fetal kidney also presented a similar distribution of the relative amounts of the five OXPHOS complexes. However, a slight increase of the relative content of complex V was observed in fetal brain. OXPHOS enzyme activities The activities of the OXPHOS complexes were measured in fetal liver, kidney, heart, brain and muscle for fetuses aged between 9 and 17 weeks of gestation. The absolute activity of each complex was plotted for the various ages of gestation for liver samples (Fig. 5A) and also expressed as mean ± SD (Fig. 5B). For kidney, heart, brain and muscle the results were only expressed as mean ± SD
We then studied the mitochondrial DNA (mtDNA) content in various pre- and postnatal tissues by real-time quantitative PCR. The results showed a wide range of values for both pre- and postnatal tissues (Fig. 6). Nevertheless, no correlation with the age of gestation could be observed for prenatal tissues. Yet, mtDNA content was higher in fetal heart tissue compared to fetal liver and muscle. Finally, we observed an increase of mtDNA content after birth in liver (W = 11, p-value = 0.002985) and muscle (W = 92, p-value = 0.002536, Wilcoxon test), despite a wide range of values in postnatal tissues (Fig. 6). However, no significant modification of mtDNA content could be noticed in muscle after birth (W = 68, p-value = 0.06525). Discussion The present study reports on the mitochondrial respiratory chain assembly and function during fetal development from 9 to 17 weeks of gestation. The various criteria studied show that the different fetal OXPHOS complexes are
Fig. 4. Relative ratios of RC complexes in pre- and postnatal liver (A) and heart (C) and in prenatal kidney (B) and brain (D). The results (means ± SD) were expressed as described in Materials and methods.
124
L. Minai et al. / Molecular Genetics and Metabolism 94 (2008) 120–126
Fig. 5. Enzyme activities of RC complexes in fetal and postnatal tissues. (A) Enzyme activities in liver of fetuses between 11 and 17 weeks of gestation. Enzyme activities (means ± SD) obtained from (B) liver (11, 13, 14, 15, 17 weeks of gestation), (C) kidney (11, 12, 14, 15, 17 weeks of gestation), (D) heart (9, 11, 12, 14, 15, 17 weeks of gestation), (E) brain (9, 11, 12, 14, 15, 17 weeks of gestation) and (F) muscle (11, 12, 13, 14, 15 weeks of gestation) and in postnatal liver, heart and muscle (0–5 years) (CI–CV: complexes I–V, CS: citrate synthase).
Fig. 6. Mitochondrial DNA quantification in fetal and postnatal tissues. Black bars: mtDNA content in fetal liver (11, 13, 13 14, 14, 14, 15, 17 weeks of gestation; from left to right), heart (9, 11, 12, 12, 13, 14, 14, 15, 17 weeks of gestation; from left to right) and muscle (11, 12, 12, 13, 14, 14, 14, 15; from left to right). White bars: mtDNA content obtained from postnatal liver, heart and muscle (0–5 years).
fully assembled and functional in heart, liver, muscle, brain and kidney as early as 11 weeks of gestation. Only coenzyme Q10 content could not be investigated due to limited amount of material. These results are consistent with previous studies showing an already functional respiratory chain in peri-implantation mouse embryos [21], muscle of premature neonates (23–29 weeks) [22] and heart of 6–16 weeks human fetuses [23]. No major difference in the size of each complex could be observed by BN-PAGE analysis during fetal development. Moreover the fetal complexes showed the same size as the ones observed after birth suggesting a full co-ordination of gene expression between nuclear and mitochondrial genomes early in the fetal development. Examination of the subunits that are essential for OXPHOS complex assembly demonstrated that the relative abundance of the different OXPHOS complexes resembles between pre- and postnatal tissues. A further study of the subunit composition of the OXPHOS complexes in
L. Minai et al. / Molecular Genetics and Metabolism 94 (2008) 120–126
pre- and postnatal tissues revealed that some of the subunits examined have different patterns of migration, notably NDUFA9 and NDUFB6 subunits of complex I, and F1a and F1b subunits of complex V. These differences might be the result of different post-translational modifications or processing of these subunits. It could also reflect tissue-specific properties of the corresponding complexes at this stage of development. The additional bands observed for Ip subunit of complex III and COXIII of complex IV could suggest the existence of tissue- and/or time-specific isoforms. Another explanation for the additional bands can be time-dependent expression of crossreacting proteins other than those examined. Nevertheless, these differences do not seem to have an impact at the level of the enzyme activity as all complexes showed similar relative activities in brain and other fetal tissues. When looking at the relative activity of each OXPHOS complex out of the total enzymatic activity, no major changes could be observed in the course of fetal development between 9 and 17 weeks. It should be mentioned that complex II activity and amount were higher in liver than in other tissues as previously reported for postnatal liver [11,24]. Comparing pre- and postnatal OXPHOS complexes absolute activities we observed a significant increase in the activities of postnatal complexes regardless of the tissue examined. In addition, a significant increase in mtDNA content was observed for postnatal liver and muscle tissues. This observation is consistent with several previous reports [3,6,23] and is related to the switch from fetal mostly anaerobic glycolysis to neonatal oxidative phosphorylation [4,25]. This change has been shown to be associated with an induction of mitochondrial biogenesis and occurs within 1 h after birth in rat liver [6]. Antenatal manifestations such as intrauterine growth retardation, polyhydroamnios and cardiac abnormalities are often observed in OXPHOS disorders [2]. Moreover, we have observed that normal OXPHOS activities, measured during early pregnancy, turned out to be abnormal at later stages of gestation or after birth [9]. These data could be related to modifications in the assembly, composition or regulation of the respiratory chain complexes that could occur during human fetal development. Yet, we were not able to detect obvious modifications in the size, composition or activity of the various OXPHOS complexes during the second trimester of pregnancy that could account for the variations we observed in a pathological context. This does not preclude however that major modifications could occur later during fetal development as we could only study fetuses between 9 and 17 weeks of gestation. Moreover, evolution of the mutant load in case of mtDNA mutations is unlikely as it seems that mtDNA does not much segregate during embryogenesis and that the proportion of mutant mtDNA does not markedly change from embryo to fetus [26–28]. Therefore, we hypothesize that the time-dependent expression of OXPHOS deficiency during either fetal life or after birth could be related to the differential expres-
125
sion or regulation of the mutant protein and/or its partners. This is illustrated for example by the diverging expression pattern of BCS1L during mouse embryo development compared to other complex III subunits [21]. Acknowledgments We wish to thank the service d’Obste´trique and the service d’Histologie-Embryologie-Cytoge´ne´tique in the Hoˆpital Necker-Enfants Malades for their cooperation. We are grateful to Dr. Leo G.J. Nijtmans and Marı¨el A.M. van den Brand for their support. This research was supported in part by the Association Francßaise contre les Myopathies and Mitocircle European Union Project (contract number LSHB-CT-2004-005260) and the French Agence Nationale pour la Recherche. LM is a recipient of a scholarship from Mitocircle contract. References [1] A. Munnich, P. Rustin, Clinical spectrum and diagnosis of mitochondrial disorders, Am. J. Med. Genet. 106 (2001) 4–17. [2] J.C. von Kleist-Retzow, V. Cormier-Daire, G. Viot, A. Goldenberg, B. Mardach, J. Amiel, P. Saada, Y. Dumez, F. Brunelle, J.M. Saudubray, D. Chretien, A. Rotig, P. Rustin, A. Munnich, P. De Lonlay, Antenatal manifestations of mitochondrial respiratory chain deficiency, J. Pediatr. 143 (2003) 208–212. [3] B. Prieur, L. Cordeau-Lossouarn, A. Rotig, J. Bismuth, J.P. Geloso, E. Delaval, Perinatal maturation of rat kidney mitochondria, Biochem. J. 305 (Pt. 2) (1995) 675–680. [4] J.M. Izquierdo, A.M. Luis, J.M. Cuezva, Postnatal mitochondrial differentiation in rat liver. Regulation by thyroid hormones of the beta-subunit of the mitochondrial F1-ATPase complex, J. Biol. Chem. 265 (1990) 9090–9097. [5] K. Kim, A. Lecordier, L.H. Bowman, Both nuclear and mitochondrial cytochrome c oxidase mRNA levels increase dramatically during mouse postnatal development, Biochem. J. 306 (Pt 2) (1995) 353–358. [6] S. Papa, Mitochondrial oxidative phosphorylation changes in the life span. Molecular aspects and physiopathological implications, Biochim. Biophys. Acta 1276 (1996) 87–105. [7] E. Jauniaux, A. Watson, G. Burton, Evaluation of respiratory gases and acid–base gradients in human fetal fluids and uteroplacental tissue between 7 and 16 weeks’ gestation, Am. J. Obstet. Gynecol. 184 (2001) 998–1003. [8] E. Jauniaux, A.L. Watson, J. Hempstock, Y.P. Bao, J.N. Skepper, G.J. Burton, Onset of maternal arterial blood flow and placental oxidative stress. A possible factor in human early pregnancy failure, Am. J. Pathol. 157 (2000) 2111–2122. [9] L. Faivre, V. Cormier-Daire, D. Chretien, J. Christoph von KleistRetzow, J. Amiel, M. Dommergues, J.M. Saudubray, Y. Dumez, A. Rotig, P. Rustin, A. Munnich, Determination of enzyme activities for prenatal diagnosis of respiratory chain deficiency, Prenat. Diagn. 20 (2000) 732–737. [10] M. Schuelke, A. Detjen, L. van den Heuvel, C. Korenke, A. Janssen, A. Smits, F. Trijbels, J. Smeitink, New nuclear encoded mitochondrial mutation illustrates pitfalls in prenatal diagnosis by biochemical methods, Clin. Chem. 48 (2002) 772–775. [11] P. Rustin, D. Chretien, T. Bourgeron, B. Gerard, A. Rotig, J.M. Saudubray, A. Munnich, Biochemical and molecular investigations in respiratory chain deficiencies, Clin. Chim. Acta 228 (1994) 35–51. [12] H. Schagger, G. von Jagow, Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form, Anal. Biochem. 199 (1991) 223–231.
126
L. Minai et al. / Molecular Genetics and Metabolism 94 (2008) 120–126
[13] L.G. Nijtmans, N.S. Henderson, I.J. Holt, Blue native electrophoresis to study mitochondrial and other protein complexes, Methods 26 (2002) 327–334. [14] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [15] W. Liu, D.A. Saint, A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics, Anal. Biochem. 302 (2002) 52–59. [16] Y. Bai, G. Attardi, The mtDNA-encoded ND6 subunit of mitochondrial NADH dehydrogenase is essential for the assembly of the membrane arm and the respiratory function of the enzyme, EMBO J. 17 (1998) 4848–4858. [17] S. Rahman, J.W. Taanman, J.M. Cooper, I. Nelson, I. Hargreaves, B. Meunier, M.G. Hanna, J.J. Garcia, R.A. Capaldi, B.D. Lake, J.V. Leonard, A.H. Schapira, A missense mutation of cytochrome oxidase subunit II causes defective assembly and myopathy, Am. J. Hum. Genet. 65 (1999) 1030–1039. [18] Z.G. Wang, D. Sheluho, D.L. Gatti, S.H. Ackerman, The alphasubunit of the mitochondrial F(1) ATPase interacts directly with the assembly factor Atp12p, EMBO J. 19 (2000) 1486–1493. [19] H. Schagger, U. Brandt, S. Gencic, G. von Jagow, Ubiquinolcytochrome-c reductase from human and bovine mitochondria, Methods Enzymol. 260 (1995) 82–96. [20] M.A. Birch-Machin, C. Marsac, G. Ponsot, B. Parfait, R.W. Taylor, P. Rustin, A. Munnich, Biochemical investigations and immunoblot analyses of two unrelated patients with an isolated deficiency in complex II of the mitochondrial respiratory chain, Biochem. Biophys. Res. Commun. 220 (1996) 57–62.
[21] H. Kotarsky, I. Tabasum, S. Mannisto, M. Heikinheimo, S. Hansson, V. Fellman, BCS1L is expressed in critical regions for neural development during ontogenesis in mice, Gene Expr. Patterns 7 (2007) 266–273. [22] L. Wenchich, J. Zeman, H. Hansikova, R. Plavka, W. Sperl, J. Houstek, Mitochondrial energy metabolism in very premature neonates, Biol. Neonate 81 (2002) 229–235. [23] J. Marin-Garcia, R. Ananthakrishnan, M.J. Goldenthal, Heart mitochondrial DNA and enzyme changes during early human development, Mol. Cell. Biochem. 210 (2000) 47–52. [24] H. Antonicka, F. Sasarman, N.G. Kennaway, E.A. Shoubridge, The molecular basis for tissue specificity of the oxidative phosphorylation deficiencies in patients with mutations in the mitochondrial translation factor EFG1, Hum. Mol. Genet. 15 (2006) 1835–1846. [25] A. Nakai, Y. Taniuchi, H. Asakura, A. Oya, A. Yokota, T. Koshino, T. Araki, Developmental changes in mitochondrial activity and energy metabolism in fetal and neonatal rat brain, Brain Res. Dev. Brain Res. 121 (2000) 67–72. [26] P.M. Matthews, J. Hopkin, R.M. Brown, J.B. Stephenson, D. HiltonJones, G.K. Brown, Comparison of the relative levels of the 3243 (A ? G) mtDNA mutation in heteroplasmic adult and fetal tissues, J. Med. Genet. 31 (1994) 41–44. [27] J. Poulton, D.R. Marchington, Progress in genetic counselling and prenatal diagnosis of maternally inherited mtDNA diseases, Neuromuscul. Disord. 10 (2000) 484–487. [28] S.L. White, V.R. Collins, R. Wolfe, M.A. Cleary, S. Shanske, S. DiMauro, H.H. Dahl, D.R. Thorburn, Genetic counseling and prenatal diagnosis for the mitochondrial DNA mutations at nucleotide 8993, Am. J. Hum. Genet. 65 (1999) 474–482.
Molecular Genetics and Metabolism 94 (2008) 120–126 www.elsevier.com/locate/ymgme
Mitochondrial respiratory chain complex assembly and function during human fetal development Limor Minai a, Jelena Martinovic b, Dominique Chretien a, Francßoise Dumez c, Fe´rechte´ Razavi b, Arnold Munnich a, Agne`s Ro¨tig a,* b
a INSERM U781 and Service de Ge´ne´tique, Hoˆpital Necker-Enfants Malades, 149 rue de Se`vres, 75015 Paris, France Service d’Histologie-Embryologie-Cytoge´ne´tique, Hoˆpital Necker-Enfants Malades, 149 rue de Se`vres, 75015 Paris, France c Service d’Obste´trique, Hoˆpital Necker-Enfants Malades, 149 rue de Se`vres, 75015 Paris, France
Received 16 November 2007; received in revised form 21 December 2007; accepted 21 December 2007 Available online 30 January 2008
Abstract Oxidative phosphorylation (OXPHOS) deficiency may have early antenatal manifestations, probably related to the time course and/ or tissue specificity of the disease gene expression during the embryo-fetal period. This feature hampers a fully reliable prenatal enzymological diagnosis of OXPHOS deficiency. We have studied OXPHOS in various human fetal tissues from 9 to 17 weeks of gestation. We found that the fetal respiratory chain complexes are fully assembled and functional at early stages of development in heart, liver, muscle, brain and kidney. We also observed a marked increase of respiratory chain activities and mitochondria content in postnatal compared to prenatal tissues. However, we were not able to detect obvious modification in the size, composition or activity of the various OXPHOS complexes during the second trimester of pregnancy that could account for the variations we observed in a pathological context. Therefore, we suggest that the time-dependent expression of respiratory chain deficiency either during fetal life or after birth could be related to the differential expression or regulation of the mutant proteins. Ó 2008 Elsevier Inc. All rights reserved. Keywords: Mitochondria; Respiratory chain assembly; Enzymatic activity; Fetal development
Introduction Oxidative phosphorylation (OXPHOS)1 deficiency represents a group of early onset and severe disorders in infancy [1]. At variance with many metabolic diseases with a delayed onset and a symptom-free period, OXPHOS deficiency occasionally has an early antenatal expression, possibly related to the time course and/or tissue specificity of the disease gene expression in the embryo-fetal period [2]. Based on activities detected in fetal rat kidney [3] and human chorion villi, OXPHOS has long been known to function early in the *
Corresponding author. Fax: +33 (0) 147348514. E-mail address: [email protected] (A. Ro¨tig). 1 Abbreviations used: RC, respiratory chain; OXPHOS, oxidative phosphorylation; BN-PAGE, blue native-polyacrylamide gel electrophoresis; SDS–PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. 1096-7192/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2007.12.007
embryo-fetal development. Yet, little is known regarding the kinetics of mitochondrial complex assembly during human embryonic and fetal life. However, at birth a spectacular activation of nuclear and mitochondrial gene expression occurs which is related to the adaptation to life at higher partial oxygen pressures [4–6]. Indeed, the early fetus develops in low oxygen environment (18 mm Hg in amniotic fluid between 11 and 16 weeks of gestation) [7]. When the principal stages of organogenesis are completed, oxygen concentration delivered to the embryo rises by the onset of blood flow into the placenta [8]. Upon birth, with high partial oxygen pressure (26 mm Hg), growing energetic demands and the need for OXPHOS, there is an up-regulation of mitochondrial biogenesis and activation of nuclear and mitochondrial gene expression occurs [4–6]. For these reasons, enzyme-based prenatal diagnosis on chorion villi or amniotic cells is hazardous and false negative or false positive results have been reported [9,10]. We
L. Minai et al. / Molecular Genetics and Metabolism 94 (2008) 120–126
have examined the onset of mitochondrial OXPHOS activities in various human fetal tissues from 9 to 17 weeks of gestation. These fetuses were obtained after termination of pregnancy for genetic diseases unrelated to OXPHOS deficiency. Our results show that the fetal OXPHOS complexes are fully assembled and enzymatically functional at least as from the end of the first trimester, although their absolute activity values are lower than those observed after birth. Materials and methods Pre- and postnatal tissues Fetal tissues (9–17 weeks of gestation) were obtained after termination of pregnancy for genetic disorders unrelated to OXPHOS deficiency (Table 1). Informed consents from parents were given. Termination of pregnancy was performed by aspiration. Fetuses were collected 5–10 min after abortion and the tissues were sampled in the following 30 min, immediately frozen and conserved in liquid nitrogen. They were usually analyzed 3 days later. Postnatal control tissues used for protein analysis were obtained from children who had died either from sudden death or genetic diseases unrelated to OXPHOS deficiency (a few days up to 5 years of age). The lag-period between death and tissue sampling was less than 4 h. Tissues were immediately frozen in liquid nitrogen. Respiratory chain enzyme analysis was normal for all the samples used in this study. Postnatal control tissues used for respiratory chain enzyme analysis were obtained from patients suspected of mitochondrial disease for whom RC analysis revealed normal activities.
Protein analysis For blue native-polyacrylamide gel electrophoresis (BN-PAGE), mitochondria and OXPHOS complexes were isolated as described [11–13] and proteins (40 lg) were analyzed on a 4–16% acrylamide non-denaturing gradient gel (Invitrogen). For SDS–PAGE, crude extracts from muscle, liver, brain, heart and kidney were prepared by homogenization of tissues (80 mg) in 0.1% Triton X-100, 0.01% digitonin and a Protease inhibitor cocktail (Sigma). The extracts were then filtered through a nylon mesh (100 lm). Total proteins (40 lg) were analyzed on 15% acrylamide gels [14]. For Western blot analysis, the gels were electroblotted onto PVDF filters and sequentially immunodecorated with specific antibodies. We used a cocktail of antibodies raised against the 20 kDa subunit of complex I, SDHB, core 2, COXII, F1a and monoclonal antibodies against GRIM 19, NDUFB6, NDUFA9, SDHA, core 1, COXIII and F1b (Mitosciences). The specific signal was detected using Western lightning chemilumi-
121
nescence reagent plus (PerkinElmer) with a charged-coupled-device camera (CCD) (Gene Gnome; Syngene, Cambridge, UK) and quantified using the GeneTools software (Syngene). For quantification, the signal obtained for an individual subunit was expressed relatively to the sum of signals obtained for all five subunits for the same tissue. Each calculation was done on at least three different samples. These arbitrary values allowed comparing the relative amount of the various complexes from one tissue to another and in pre- and postnatal period.
OXPHOS enzyme activities Spectrophotometric assays of OXPHOS enzymes were performed on muscle, liver, brain, heart and kidney as previously described [11]. Complex V activity was not measured in muscle as freezing this tissue results in a loss of sensitivity to oligomycin, probably related to the loss of oligomycin sensitivity conferring protein (OSCP). For each tissue, at least six different individuals were used and each enzyme activity was measured twice.
Mitochondrial DNA quantification Total DNA was extracted from tissues by standard phenol/chloroform extraction and ethanol precipitation, followed by column purification (Macherey-Nagel kit). Quantitative PCR analyses were performed on a Taqman 7300 (Applied Biosystems). The mitochondrial 12S rRNA and the nuclear MutL protein homolog 1 (MLH1) genes were individually amplified using primers 12S rRNA-F/12S rRNA-R and primers MLH1F/MLH1-R, respectively, and mtDNA-A and MLH1-A as hybridization probes (Table 2). The efficiency of the two probes was tested for their compatibility according to the manufacturer’s instruction (Applied Biosystems). A standard curve composed of a series of 3 dilutions (1/10, 1/ 100, 1/1000) in triplicate, was carried out for each individual. The amount of mtDNA was normalized according to the nuclear DNA quantity using the DDCT method provided that the efficiencies of these two PCRs were compatible. To compensate for any eventual inaccuracies due to different PCR efficiencies, we have used the equation R0,T/R0,R = (1+ER)CT,R/ (1+ET)CT,T as developed by [15]. (R, reporter fluorescence; R0, initial reporter; T, target gene; R, reference gene; E, efficiency; CT, threshold cycle). For each DNA, the average CT of the second dilution triplicate of each standard curve was taken. Standard deviation (SD) was calculated for each triplicate of CT to assure that the average is correct.
Statistics Non-parametric Wilcoxon rank test for small samples with non-Gaussian distribution was carried out to evaluate whether the difference between pre- and postnatal mtDNA quantity was significantly different.
Results
Table 1 Genetic diseases leading to the various terminations of pregnancy
Respiratory chain complex assembly
Genetic disease
Weeks of gestation
Exencephaly X-linked hydrocephaly Alpert syndrome Angelman syndrome Spinal muscular atrophy Stickler syndrome Pierre Robin syndrome Fragile X Encephalocele Fragile X X-linked retinitis pigmentosa Fragile X
9 11 11 12 12 12 13 13 14 14 15 17
To asses the assembly state of the fetal OXPHOS complexes, complexes were studied in fetal (11–17 weeks of gestation) and postnatal liver and heart (6 and 36 weeks). Table 2 Oligonucleotide primers 12S rRNA-F 12S rRNA-R mtDNA-A MLH1-F MLH1-R MLH1-A
50 -TAGAGGAGCCTGTTCTGTAAT-30 50 -TGCGCTTACTTTGTAGCCTTCAT-30 6FAM-50 -AAACCCCGATCAACC-30 -MGB 50 -GTAGTCTGTGATCTCCGTTT-30 50 -ATGTATGAGGTCCTGTCCTA-30 6FAM-50 -TGGTGATGCACACTGGCACC-30 -TAMRA
122
L. Minai et al. / Molecular Genetics and Metabolism 94 (2008) 120–126
They were analyzed under native conditions using a blue native-polyacrylamide gel electrophoresis system (BNPAGE). Western blots were successively incubated with five different antibodies raised against one subunit of each of the five OXPHOS complexes. Similar size and steadystate levels of all complexes were observed in the six fetal liver tissues tested and in the postnatal livers (Fig. 1) suggesting that all five OXPHOS complexes are fully assembled at least as from 11 weeks of gestation. The same results were obtained in heart tissues of five different fetuses aged from 12 to 15 weeks (Fig. 1). Subunit composition of the fetal OXPHOS complexes Crude extracts of brain, heart, liver and kidney tissues of a 17 weeks old fetus and postnatal heart and liver tissues (36 weeks) were analyzed by SDS–PAGE followed by Western blot. We used a cocktail of antibodies raised against the 20 kDa subunit of complex I, SDHB of complex II, core protein 2 of complex III, COXII of complex IV and F1a of complex V. The presence of most of them, with the exception of F1a, reflects a fully assembled complex as these subunits are either essential for assembly of their corresponding complex or labile if the complex is not fully assembled [16–20]. In fetal heart, liver and kidney the protein profile was similar to that obtained in postnatal heart and liver tissues (Fig. 2). However, a slightly faster migrating F1a subunit of complex V was detected in fetal brain compared to other pre- and postnatal tissues. These results were repeatedly observed at various developmental stages in fetal brain (11, 15, 17 weeks), heart (9, 11, 14, 15, 17 weeks), liver (11, 14, 15, 17 weeks) and kidney (14, 15, 17 weeks, not shown). To further characterize the fetal protein content, we studied additional subunits of the OXPHOS complexes. Fetal brain showed different protein migration patterns compared to postnatal brain. Indeed, NDUFA9 and to a lesser extent NDUFB6 subunits migrated slower, whereas
Fig. 2. SDS–PAGE and Western blot analysis of respiratory chain proteins in prenatal brain, heart, liver, kidney and postnatal heart and liver using a cocktail of five antibodies against one subunit of each of the OXPHOS complexes (20 kDa subunit for complex I, SDHB for complex II, core protein 2 for complex III, COXII for complex IV and F1a for complex V).
the F1b subunit of a 15 weeks fetus migrated faster than in postnatal tissues (Fig. 3). The anti-Ip antibody of CIII (Rieske protein) revealed two closely migrating bands in prenatal kidney and postnatal brain, heart, liver and kidney. The signal for COXIII subunit of complex IV was lower in prenatal tissues. The other subunits showed similar migration patterns in the various pre- and postnatal tissues. In order to compare the relative amount of the various complexes from one tissue to another and in pre- and postnatal period, the signal obtained for an individual subunit observed in Fig. 2 was expressed relatively to the sum of signals obtained for all five subunits for the same tissue. This showed that similar relative amounts of the various OXPHOS complexes were present in pre- and postnatal heart and liver (Fig. 4). Moreover, it is worth noting that complex II was relatively more abundant in liver than in
Fig. 1. BN-PAGE and Western blot analysis of mitochondria from fetus liver and heart at various ages of gestation (11–17 weeks of gestation) and from controls (6 and 36 weeks) using antibodies against GRIM19 for complex I (CI), SDHA for complex II (CII), core 2 for complex III (CIII), COXII for complex IV (CIV) and F1b subunits for complex V (CV).
L. Minai et al. / Molecular Genetics and Metabolism 94 (2008) 120–126
123
(Fig. 5C–F). The higher activities were consistently observed for complex III and the lower ones for complex I in pre- and postnatal stages. The activity of citrate synthase, a TCA cycle enzyme that reflects the mitochondrial mass was similar at the different stages (see Fig. 5A for a representative example). Finally, prenatal values in liver, heart and muscle were constantly lower than postnatal ones (Fig. 5B, D and F). Only citrate synthase activity did not significantly increase after birth in liver. It is worth noting that muscle complex IV activities were relatively lower in the prenatal than in the postnatal period, as compared to other complexes. Mitochondrial DNA content
Fig. 3. SDS–PAGE and Western blot analysis of respiratory chain proteins in prenatal (15 weeks) and postnatal brain, heart, liver, kidney (12 weeks) using antibodies against various subunits of RC complexes (CI– CV: complexes I–V).
heart. Fetal kidney also presented a similar distribution of the relative amounts of the five OXPHOS complexes. However, a slight increase of the relative content of complex V was observed in fetal brain. OXPHOS enzyme activities The activities of the OXPHOS complexes were measured in fetal liver, kidney, heart, brain and muscle for fetuses aged between 9 and 17 weeks of gestation. The absolute activity of each complex was plotted for the various ages of gestation for liver samples (Fig. 5A) and also expressed as mean ± SD (Fig. 5B). For kidney, heart, brain and muscle the results were only expressed as mean ± SD
We then studied the mitochondrial DNA (mtDNA) content in various pre- and postnatal tissues by real-time quantitative PCR. The results showed a wide range of values for both pre- and postnatal tissues (Fig. 6). Nevertheless, no correlation with the age of gestation could be observed for prenatal tissues. Yet, mtDNA content was higher in fetal heart tissue compared to fetal liver and muscle. Finally, we observed an increase of mtDNA content after birth in liver (W = 11, p-value = 0.002985) and muscle (W = 92, p-value = 0.002536, Wilcoxon test), despite a wide range of values in postnatal tissues (Fig. 6). However, no significant modification of mtDNA content could be noticed in muscle after birth (W = 68, p-value = 0.06525). Discussion The present study reports on the mitochondrial respiratory chain assembly and function during fetal development from 9 to 17 weeks of gestation. The various criteria studied show that the different fetal OXPHOS complexes are
Fig. 4. Relative ratios of RC complexes in pre- and postnatal liver (A) and heart (C) and in prenatal kidney (B) and brain (D). The results (means ± SD) were expressed as described in Materials and methods.
124
L. Minai et al. / Molecular Genetics and Metabolism 94 (2008) 120–126
Fig. 5. Enzyme activities of RC complexes in fetal and postnatal tissues. (A) Enzyme activities in liver of fetuses between 11 and 17 weeks of gestation. Enzyme activities (means ± SD) obtained from (B) liver (11, 13, 14, 15, 17 weeks of gestation), (C) kidney (11, 12, 14, 15, 17 weeks of gestation), (D) heart (9, 11, 12, 14, 15, 17 weeks of gestation), (E) brain (9, 11, 12, 14, 15, 17 weeks of gestation) and (F) muscle (11, 12, 13, 14, 15 weeks of gestation) and in postnatal liver, heart and muscle (0–5 years) (CI–CV: complexes I–V, CS: citrate synthase).
Fig. 6. Mitochondrial DNA quantification in fetal and postnatal tissues. Black bars: mtDNA content in fetal liver (11, 13, 13 14, 14, 14, 15, 17 weeks of gestation; from left to right), heart (9, 11, 12, 12, 13, 14, 14, 15, 17 weeks of gestation; from left to right) and muscle (11, 12, 12, 13, 14, 14, 14, 15; from left to right). White bars: mtDNA content obtained from postnatal liver, heart and muscle (0–5 years).
fully assembled and functional in heart, liver, muscle, brain and kidney as early as 11 weeks of gestation. Only coenzyme Q10 content could not be investigated due to limited amount of material. These results are consistent with previous studies showing an already functional respiratory chain in peri-implantation mouse embryos [21], muscle of premature neonates (23–29 weeks) [22] and heart of 6–16 weeks human fetuses [23]. No major difference in the size of each complex could be observed by BN-PAGE analysis during fetal development. Moreover the fetal complexes showed the same size as the ones observed after birth suggesting a full co-ordination of gene expression between nuclear and mitochondrial genomes early in the fetal development. Examination of the subunits that are essential for OXPHOS complex assembly demonstrated that the relative abundance of the different OXPHOS complexes resembles between pre- and postnatal tissues. A further study of the subunit composition of the OXPHOS complexes in
L. Minai et al. / Molecular Genetics and Metabolism 94 (2008) 120–126
pre- and postnatal tissues revealed that some of the subunits examined have different patterns of migration, notably NDUFA9 and NDUFB6 subunits of complex I, and F1a and F1b subunits of complex V. These differences might be the result of different post-translational modifications or processing of these subunits. It could also reflect tissue-specific properties of the corresponding complexes at this stage of development. The additional bands observed for Ip subunit of complex III and COXIII of complex IV could suggest the existence of tissue- and/or time-specific isoforms. Another explanation for the additional bands can be time-dependent expression of crossreacting proteins other than those examined. Nevertheless, these differences do not seem to have an impact at the level of the enzyme activity as all complexes showed similar relative activities in brain and other fetal tissues. When looking at the relative activity of each OXPHOS complex out of the total enzymatic activity, no major changes could be observed in the course of fetal development between 9 and 17 weeks. It should be mentioned that complex II activity and amount were higher in liver than in other tissues as previously reported for postnatal liver [11,24]. Comparing pre- and postnatal OXPHOS complexes absolute activities we observed a significant increase in the activities of postnatal complexes regardless of the tissue examined. In addition, a significant increase in mtDNA content was observed for postnatal liver and muscle tissues. This observation is consistent with several previous reports [3,6,23] and is related to the switch from fetal mostly anaerobic glycolysis to neonatal oxidative phosphorylation [4,25]. This change has been shown to be associated with an induction of mitochondrial biogenesis and occurs within 1 h after birth in rat liver [6]. Antenatal manifestations such as intrauterine growth retardation, polyhydroamnios and cardiac abnormalities are often observed in OXPHOS disorders [2]. Moreover, we have observed that normal OXPHOS activities, measured during early pregnancy, turned out to be abnormal at later stages of gestation or after birth [9]. These data could be related to modifications in the assembly, composition or regulation of the respiratory chain complexes that could occur during human fetal development. Yet, we were not able to detect obvious modifications in the size, composition or activity of the various OXPHOS complexes during the second trimester of pregnancy that could account for the variations we observed in a pathological context. This does not preclude however that major modifications could occur later during fetal development as we could only study fetuses between 9 and 17 weeks of gestation. Moreover, evolution of the mutant load in case of mtDNA mutations is unlikely as it seems that mtDNA does not much segregate during embryogenesis and that the proportion of mutant mtDNA does not markedly change from embryo to fetus [26–28]. Therefore, we hypothesize that the time-dependent expression of OXPHOS deficiency during either fetal life or after birth could be related to the differential expres-
125
sion or regulation of the mutant protein and/or its partners. This is illustrated for example by the diverging expression pattern of BCS1L during mouse embryo development compared to other complex III subunits [21]. Acknowledgments We wish to thank the service d’Obste´trique and the service d’Histologie-Embryologie-Cytoge´ne´tique in the Hoˆpital Necker-Enfants Malades for their cooperation. We are grateful to Dr. Leo G.J. Nijtmans and Marı¨el A.M. van den Brand for their support. This research was supported in part by the Association Francßaise contre les Myopathies and Mitocircle European Union Project (contract number LSHB-CT-2004-005260) and the French Agence Nationale pour la Recherche. LM is a recipient of a scholarship from Mitocircle contract. References [1] A. Munnich, P. Rustin, Clinical spectrum and diagnosis of mitochondrial disorders, Am. J. Med. Genet. 106 (2001) 4–17. [2] J.C. von Kleist-Retzow, V. Cormier-Daire, G. Viot, A. Goldenberg, B. Mardach, J. Amiel, P. Saada, Y. Dumez, F. Brunelle, J.M. Saudubray, D. Chretien, A. Rotig, P. Rustin, A. Munnich, P. De Lonlay, Antenatal manifestations of mitochondrial respiratory chain deficiency, J. Pediatr. 143 (2003) 208–212. [3] B. Prieur, L. Cordeau-Lossouarn, A. Rotig, J. Bismuth, J.P. Geloso, E. Delaval, Perinatal maturation of rat kidney mitochondria, Biochem. J. 305 (Pt. 2) (1995) 675–680. [4] J.M. Izquierdo, A.M. Luis, J.M. Cuezva, Postnatal mitochondrial differentiation in rat liver. Regulation by thyroid hormones of the beta-subunit of the mitochondrial F1-ATPase complex, J. Biol. Chem. 265 (1990) 9090–9097. [5] K. Kim, A. Lecordier, L.H. Bowman, Both nuclear and mitochondrial cytochrome c oxidase mRNA levels increase dramatically during mouse postnatal development, Biochem. J. 306 (Pt 2) (1995) 353–358. [6] S. Papa, Mitochondrial oxidative phosphorylation changes in the life span. Molecular aspects and physiopathological implications, Biochim. Biophys. Acta 1276 (1996) 87–105. [7] E. Jauniaux, A. Watson, G. Burton, Evaluation of respiratory gases and acid–base gradients in human fetal fluids and uteroplacental tissue between 7 and 16 weeks’ gestation, Am. J. Obstet. Gynecol. 184 (2001) 998–1003. [8] E. Jauniaux, A.L. Watson, J. Hempstock, Y.P. Bao, J.N. Skepper, G.J. Burton, Onset of maternal arterial blood flow and placental oxidative stress. A possible factor in human early pregnancy failure, Am. J. Pathol. 157 (2000) 2111–2122. [9] L. Faivre, V. Cormier-Daire, D. Chretien, J. Christoph von KleistRetzow, J. Amiel, M. Dommergues, J.M. Saudubray, Y. Dumez, A. Rotig, P. Rustin, A. Munnich, Determination of enzyme activities for prenatal diagnosis of respiratory chain deficiency, Prenat. Diagn. 20 (2000) 732–737. [10] M. Schuelke, A. Detjen, L. van den Heuvel, C. Korenke, A. Janssen, A. Smits, F. Trijbels, J. Smeitink, New nuclear encoded mitochondrial mutation illustrates pitfalls in prenatal diagnosis by biochemical methods, Clin. Chem. 48 (2002) 772–775. [11] P. Rustin, D. Chretien, T. Bourgeron, B. Gerard, A. Rotig, J.M. Saudubray, A. Munnich, Biochemical and molecular investigations in respiratory chain deficiencies, Clin. Chim. Acta 228 (1994) 35–51. [12] H. Schagger, G. von Jagow, Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form, Anal. Biochem. 199 (1991) 223–231.
126
L. Minai et al. / Molecular Genetics and Metabolism 94 (2008) 120–126
[13] L.G. Nijtmans, N.S. Henderson, I.J. Holt, Blue native electrophoresis to study mitochondrial and other protein complexes, Methods 26 (2002) 327–334. [14] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [15] W. Liu, D.A. Saint, A new quantitative method of real time reverse transcription polymerase chain reaction assay based on simulation of polymerase chain reaction kinetics, Anal. Biochem. 302 (2002) 52–59. [16] Y. Bai, G. Attardi, The mtDNA-encoded ND6 subunit of mitochondrial NADH dehydrogenase is essential for the assembly of the membrane arm and the respiratory function of the enzyme, EMBO J. 17 (1998) 4848–4858. [17] S. Rahman, J.W. Taanman, J.M. Cooper, I. Nelson, I. Hargreaves, B. Meunier, M.G. Hanna, J.J. Garcia, R.A. Capaldi, B.D. Lake, J.V. Leonard, A.H. Schapira, A missense mutation of cytochrome oxidase subunit II causes defective assembly and myopathy, Am. J. Hum. Genet. 65 (1999) 1030–1039. [18] Z.G. Wang, D. Sheluho, D.L. Gatti, S.H. Ackerman, The alphasubunit of the mitochondrial F(1) ATPase interacts directly with the assembly factor Atp12p, EMBO J. 19 (2000) 1486–1493. [19] H. Schagger, U. Brandt, S. Gencic, G. von Jagow, Ubiquinolcytochrome-c reductase from human and bovine mitochondria, Methods Enzymol. 260 (1995) 82–96. [20] M.A. Birch-Machin, C. Marsac, G. Ponsot, B. Parfait, R.W. Taylor, P. Rustin, A. Munnich, Biochemical investigations and immunoblot analyses of two unrelated patients with an isolated deficiency in complex II of the mitochondrial respiratory chain, Biochem. Biophys. Res. Commun. 220 (1996) 57–62.
[21] H. Kotarsky, I. Tabasum, S. Mannisto, M. Heikinheimo, S. Hansson, V. Fellman, BCS1L is expressed in critical regions for neural development during ontogenesis in mice, Gene Expr. Patterns 7 (2007) 266–273. [22] L. Wenchich, J. Zeman, H. Hansikova, R. Plavka, W. Sperl, J. Houstek, Mitochondrial energy metabolism in very premature neonates, Biol. Neonate 81 (2002) 229–235. [23] J. Marin-Garcia, R. Ananthakrishnan, M.J. Goldenthal, Heart mitochondrial DNA and enzyme changes during early human development, Mol. Cell. Biochem. 210 (2000) 47–52. [24] H. Antonicka, F. Sasarman, N.G. Kennaway, E.A. Shoubridge, The molecular basis for tissue specificity of the oxidative phosphorylation deficiencies in patients with mutations in the mitochondrial translation factor EFG1, Hum. Mol. Genet. 15 (2006) 1835–1846. [25] A. Nakai, Y. Taniuchi, H. Asakura, A. Oya, A. Yokota, T. Koshino, T. Araki, Developmental changes in mitochondrial activity and energy metabolism in fetal and neonatal rat brain, Brain Res. Dev. Brain Res. 121 (2000) 67–72. [26] P.M. Matthews, J. Hopkin, R.M. Brown, J.B. Stephenson, D. HiltonJones, G.K. Brown, Comparison of the relative levels of the 3243 (A ? G) mtDNA mutation in heteroplasmic adult and fetal tissues, J. Med. Genet. 31 (1994) 41–44. [27] J. Poulton, D.R. Marchington, Progress in genetic counselling and prenatal diagnosis of maternally inherited mtDNA diseases, Neuromuscul. Disord. 10 (2000) 484–487. [28] S.L. White, V.R. Collins, R. Wolfe, M.A. Cleary, S. Shanske, S. DiMauro, H.H. Dahl, D.R. Thorburn, Genetic counseling and prenatal diagnosis for the mitochondrial DNA mutations at nucleotide 8993, Am. J. Hum. Genet. 65 (1999) 474–482.