Complex I mutations synergize to worsen the phenotypic expression of Leber's hereditary optic neuropathy
2020; Elsevier BV; Volume: 295; Issue: 38 Linguagem: Inglês
10.1074/jbc.ra120.014603
ISSN1083-351X
AutoresYanchun Ji, Juanjuan Zhang, Yuanyuan Lu, Qiuzi Yi, Mengquan Chen, Shipeng Xie, Xiaoting Mao, Yun Xiao, Feilong Meng, Minglian Zhang, Rulai Yang, Min‐Xin Guan,
Tópico(s)Trace Elements in Health
ResumoLeber's hereditary optic neuropathy (LHON) is a maternal inheritance of eye disease because of the mitochondrial DNA (mtDNA) mutations. We previously discovered a 3866T>C mutation within the gene for the ND1 subunit of complex I as possibly amplifying disease progression for patients bearing the disease-causing 11778G>A mutation within the gene for the ND4 subunit of complex I. However, whether and how the ND1 mutation exacerbates the ND4 mutation were unknown. In this report, we showed that four Chinese families bearing both m.3866T>C and m.11778G>A mutations exhibited higher penetrances of LHON than 6 Chinese pedigrees carrying only the m.3866T>C mutation or families harboring only the m.11778G>A mutation. The protein structure analysis revealed that the m.3866T>C (I187T) and m.11778G>A (R340H) mutations destabilized the specific interactions with other residues of ND1 and ND4, thereby altering the structure and function of complex I. Cellular data obtained using cybrids, constructed by transferring mitochondria from the Chinese families into mtDNA-less (ρ°) cells, demonstrated that the mutations perturbed the stability, assembly, and activity of complex I, leading to changes in mitochondrial ATP levels and membrane potential and increasing the production of reactive oxygen species. These mitochondrial dysfunctions promoted the apoptotic sensitivity of cells and decreased mitophagy. Cybrids bearing only the m.3866T>C mutation displayed mild mitochondrial dysfunctions, whereas those harboring both m.3866T>C and m.11778G>A mutations exhibited greater mitochondrial dysfunctions. These suggested that the m.3866T>C mutation acted in synergy with the m.11778G>A mutation, aggravating mitochondrial dysfunctions and contributing to higher penetrance of LHON in these families carrying both mtDNA mutations. Leber's hereditary optic neuropathy (LHON) is a maternal inheritance of eye disease because of the mitochondrial DNA (mtDNA) mutations. We previously discovered a 3866T>C mutation within the gene for the ND1 subunit of complex I as possibly amplifying disease progression for patients bearing the disease-causing 11778G>A mutation within the gene for the ND4 subunit of complex I. However, whether and how the ND1 mutation exacerbates the ND4 mutation were unknown. In this report, we showed that four Chinese families bearing both m.3866T>C and m.11778G>A mutations exhibited higher penetrances of LHON than 6 Chinese pedigrees carrying only the m.3866T>C mutation or families harboring only the m.11778G>A mutation. The protein structure analysis revealed that the m.3866T>C (I187T) and m.11778G>A (R340H) mutations destabilized the specific interactions with other residues of ND1 and ND4, thereby altering the structure and function of complex I. Cellular data obtained using cybrids, constructed by transferring mitochondria from the Chinese families into mtDNA-less (ρ°) cells, demonstrated that the mutations perturbed the stability, assembly, and activity of complex I, leading to changes in mitochondrial ATP levels and membrane potential and increasing the production of reactive oxygen species. These mitochondrial dysfunctions promoted the apoptotic sensitivity of cells and decreased mitophagy. Cybrids bearing only the m.3866T>C mutation displayed mild mitochondrial dysfunctions, whereas those harboring both m.3866T>C and m.11778G>A mutations exhibited greater mitochondrial dysfunctions. These suggested that the m.3866T>C mutation acted in synergy with the m.11778G>A mutation, aggravating mitochondrial dysfunctions and contributing to higher penetrance of LHON in these families carrying both mtDNA mutations. Leber's hereditary optic neuropathy (LHON) is the most common maternally transmitted eye disorder, characterized by the degeneration of retinal ganglion cells (RGC) and loss of central vision (1Wallace D.C. Lott M.T. Leber hereditary optic neuropathy: exemplar of an mtDNA disease.Handb. Exp. Pharmacol. 2017; 240 (28233183): 339-37610.1007/164_2017_2Crossref PubMed Scopus (36) Google Scholar, 2Newman N.J. Leber's hereditary optic neuropathy.Ophthalmol. Clin. N. Am. 1993; 50 (8489411): 540-54810.1001/archneur.1993.00540050082021Google Scholar, 3Nikoskelainen E.K. Clinical picture of LHON.Clin. Neurosci. 1994; 2: 115-120Google Scholar, 4Sadun A.A. La Morgia C. Carelli V. Leber's hereditary optic neuropathy.Curr. Treat Options Neurol. 2011; 13 (21063922): 109-11710.1007/s11940-010-0100-yCrossref PubMed Scopus (95) Google Scholar, 5Carelli V. La Morgia C. Valentino M.L. Barboni P. Ross-Cisneros F.N. Sadun A.A. 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Sci. 2012; 53 (22577081): 4586-459410.1167/iovs.11-9109Crossref PubMed Scopus (30) Google Scholar). These Chinese pedigrees carrying only the m.3866T>C mutation exhibited extremely low penetrance of LHON, whereas the penetrances of LHON among those pedigrees bearing the m.3866T>C mutation together with m.11778G>A mutations were higher than those in families carrying only single mtDNA mutation (15Ji Y. Liang M. Zhang J. Zhu L. Zhang Z. Fu R. Liu X. Zhang M. Fu Q. Zhao F. Tong Y. Sun Y. Jiang P. Guan M.-X. Mitochondrial ND1 variants in 1281 Chinese subjects with Leber's hereditary optic neuropathy.Invest. Ophthalmol. Vis. Sci. 2016; 57 (27177320): 2377-238910.1167/iovs.16-19243Crossref PubMed Scopus (33) Google Scholar, 29Zhou X. Qian Y. Zhang J. Tong Y. Jiang P. Liang M. Dai X. Zhou H. Zhao F. Ji Y. Mo J.Q. Qu J. Guan M.-X. Leber's hereditary optic neuropathy is associated with the T3866C mutation in mitochondrial ND1 gene in three Han Chinese families.Invest. Ophthalmol. Vis. Sci. 2012; 53 (22577081): 4586-459410.1167/iovs.11-9109Crossref PubMed Scopus (30) Google Scholar). These data suggested the role of m.3866T>C mutation in the pathogenesis of LHON. However, the pathogenic mechanisms underlying these LHON-associated mutations remain elusive. The m.3866T>C mutation resulted in the change of highly conserved isoleucine at position 187 with threonine (I187T) in ND1, the core component of 45 subunits of complex I (29Zhou X. Qian Y. Zhang J. Tong Y. Jiang P. Liang M. Dai X. Zhou H. Zhao F. Ji Y. Mo J.Q. Qu J. Guan M.-X. Leber's hereditary optic neuropathy is associated with the T3866C mutation in mitochondrial ND1 gene in three Han Chinese families.Invest. Ophthalmol. Vis. Sci. 2012; 53 (22577081): 4586-459410.1167/iovs.11-9109Crossref PubMed Scopus (30) Google Scholar, 30Scheffler I.E. Mitochondrial disease associated with complex I (NADH-CoQ oxidoreductase) deficiency.J. Inherit. Metab. Dis. 2015; 38: 405-41510.1007/s10545-014-9768-6Crossref PubMed Scopus (45) Google Scholar, 31Zhu J. Vinothkumar K.R. Hirst J. Structure of mammalian respiratory complex I.Nature. 2016; 536 (27509854): 354-35810.1038/nature19095Crossref PubMed Scopus (357) Google Scholar). Thus, the m.3866T>C mutation may perturb both the structure and function of complex I, thereby causing mitochondrial dysfunction. The higher penetrance of LHON in the families carrying both m.11778G>A and m.3866T>C mutations implied that biochemical consequences caused by the m.3866T>C mutation deteriorate the mitochondrial dysfunction associated with m.11778G>A mutation, thereby increasing the penetrance and risk of LHON. The structural consequences of m.11778G>A and m.3866T>C mutations were evaluated by the tertiary structure analysis of complex I from Homo sapiens and Mus musculus (31Zhu J. Vinothkumar K.R. Hirst J. Structure of mammalian respiratory complex I.Nature. 2016; 536 (27509854): 354-35810.1038/nature19095Crossref PubMed Scopus (357) Google Scholar, 32Guo R. Zong S. Wu M. Gu J. Yang M. Architecture of human mitochondrial respiratory megacomplex I2III2IV2.Cell. 2017; 170 (28844695): 1247-125710.1016/j.cell.2017.07.050Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 33Agip A.-N.A. Blaza J.N. Bridges H.R. Viscomi C. Rawson S. Muench S.P. Hirst J. Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states.Nat. Struct. Mol. Biol. 2018; 25 (29915388): 548-55610.1038/s41594-018-0073-1Crossref PubMed Scopus (133) Google Scholar). Functional consequences of m.3866T>C mutation was further investigated through the cybrid cell lines constructed by transferring mitochondria from lymphoblastoid cell lines derived from an affected matrilineal relative carrying the m.11778G>A and m.3866T>C mutation, individuals bearing only m.3866T>C or m.11778G>A mutation, and a control subject belonging to the same mtDNA haplogroup, all into human mtDNA-less (ρ°) cells (19Zhang J. Ji Y. Lu Y. Fu R. Xu M. Liu X. Guan M.X. Leber's hereditary optic neuropathy (LHON)-associated ND5 12338T > C mutation altered the assembly and function of complex I, apoptosis and mitophagy.Hum. Mol. Genet. 2018; 27 (29579248): 1999-201110.1093/hmg/ddy107Crossref PubMed Scopus (44) Google Scholar, 34King M.P. Attadi G. Mitochondria-mediated transformation of human rhoo cells.Methods Enzymol. 1996; 264: 313-334Crossref PubMed Google Scholar). Using Western blotting and blue native PAGE (BN-PAGE) analyses, we examined if the m.3866T>C and m.11778G>A mutations exerted an effect on the stability of ND1 and ND4 as well as assembly of complex I. These cell lines were then assessed for effects of the mtDNA mutations on the enzymatic activities of respiratory chain complexes, the rate of O2 consumption, mitochondrial ATP production, mitochondrial membrane potential, and generation of reactive oxygen species (ROS). These cell lines were further evaluated for the effect of these mtDNA mutations on apoptotic state and mitophagy. Ten Han Chinese pedigrees bearing the m.3866T>C mutation (6 bearing only the m.3866T>C mutation, 4 carrying m.3866T>C mutation together with m.11778G>A mutation) were identified in a large cohort of 1793 Chinese probands with LHON (15Ji Y. Liang M. Zhang J. Zhu L. Zhang Z. Fu R. Liu X. Zhang M. Fu Q. Zhao F. Tong Y. Sun Y. Jiang P. Guan M.-X. Mitochondrial ND1 variants in 1281 Chinese subjects with Leber's hereditary optic neuropathy.Invest. Ophthalmol. Vis. Sci. 2016; 57 (27177320): 2377-238910.1167/iovs.16-19243Crossref PubMed Scopus (33) Google Scholar). This translated to an incidence of 0.6% in this cohort. All available members of 10 pedigrees underwent comprehensive physical and ophthalmologic examinations to identify personal or family medical histories of visual impairments and other clinical abnormalities. As shown in Table S1 and Fig. S1, matrilineal relatives exhibited variable penetrance and expressivity of optic neuropathy among and within families. In particular, the severity of visual loss ranged from profound visual loss to normal vision. The age at onset of optic neuropathy of 6 pedigrees bearing only m.3866T>C mutation ranged from 5 to 39 years, with an average of 24.8 years, whereas the average age at onset of visual impairment of 4 pedigrees harboring both m.3866T>C and m.11778G>A mutations were 22.2 years, respectively. Strikingly, 6 pedigrees harboring only the m.3866T>C mutation exhibited extremely low penetrance of optic neuropathy, ranging from 11.1% to 29.2%, with an average of 17.0%. In contrast, the average penetrances of optic neuropathy of 4 pedigrees harboring both m.3866T>C and m.11778G>A mutations were 41.9%. Entire sequence analysis of mtDNAs among these Chinese pedigrees revealed that these probands showed the presence of m.3866T>C or m.11778G>A mutations and distinct sets of mtDNA polymorphisms, including 129 known variants, as shown in Table S2. As shown in Table S1, the mtDNAs from 10 pedigrees resided at mtDNA haplogroups C4a (1), D4 (2), G2 (1), M 10 (4), H2 (1), and R (1), respectively (35Kong Q.-P. Bandelt H.-J. Sun C. Yao Y.-G. Salas A. Achilli A. Wang C.-Y. Zhong L. Zhu C.-L. Wu S.-F. Torroni A. Zhang Y.-P. Updating the East Asian mtDNA phylogeny: a prerequisite for the identification of pathogenic mutations.Hum. Mol. Genet. 2006; 15 (16714301): 2076-208610.1093/hmg/ddl130Crossref PubMed Scopus (323) Google Scholar). These mtDNA variants included 35 in the D-loop region, 4 in the 12S rRNA gene, 3 in the 16S rRNA gene, 5 in the tRNA gene, and 53 silent and 29 missense variants in the genes encoding polypeptides (36Andrews R.M. Kubacka I. Chinnery P.F. Lightowlers R.N. Turnbull D.M. Howell N. Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA.Nat. Genet. 1999; 23 (10508508): 14710.1038/13779Crossref PubMed Scopus (2551) Google Scholar). These variants in RNAs and polypeptides were evaluated by phylogenetic analysis of these variants and sequences from 17 vertebrates, as shown in Table S2. These variants were further evaluated for the presence of 485 control subjects and potential structural and functional alterations. Of these, these variants may not have potential structural and functional alterations. These findings indicated that these mtDNA variants do not play an important role in the phenotypic manifestation of m.3866T>C mutations. To test the effect of m.3866T>C (I187T) and m.11778G>A (R340H) mutations on the structure and function of ND1 and ND4, we performed the tertiary structure analysis of complex I from H. sapiens and M. musculus. Based on the Cryo-EM structure of mammalian complex I (PDB entry 5XTD and 6G2J) (32Guo R. Zong S. Wu M. Gu J. Yang M. Architecture of human mitochondrial respiratory megacomplex I2III2IV2.Cell. 2017; 170 (28844695): 1247-125710.1016/j.cell.2017.07.050Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 33Agip A.-N.A. Blaza J.N. Bridges H.R. Viscomi C. Rawson S. Muench S.P. Hirst J. Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states.Nat. Struct. Mol. Biol. 2018; 25 (29915388): 548-55610.1038/s41594-018-0073-1Crossref PubMed Scopus (133) Google Scholar), the hydrophobic side chain of I187 forms the hydrophobic interaction with phosphatidylcholine in ND1 (Fig. 1A), whereas the side chain guanidino group of R340 forms specific electrostatic interactions with the backbone hydroxyl group of V221 in ND4, which is located at the matrix side and water-membrane interface (Fig. 1B). Hence, the replacement of hydrophobic isoleucine at position 187 with the hydrophilic threonine by m.3866T>C mutation in ND1 or arginine at position 340 with histidine by m.11778G>A mutation in ND4 may destabilize these interactions inside ND1 or ND4, thereby perturbing the structure and stability of ND1 and ND4 proteins, respectively. To experimentally test this hypothesis, we examined the levels of ND1 and ND4 proteins by Western blotting in these mutant cell lines carrying only m.3866T>C mutation, only m.11778G>A mutation, or both m.3866T>C and m.11778G>A mutations, as well as control cell lines lacking these mutations. As shown in Fig. 1, C and D, the levels of ND1 in mutant cell lines carrying only m.3866T>C, m.11778G>A, or both m.3866T>C and m.11778G>A mutations were 74.1%, 90.3%, and 56.4% relative to the average values of control cell lines. Furthermore, the levels of ND4 in mutant cell lines carrying only m.3866T>C, m.11778G>A, and both m.3866T>C and m.11778G>A mutations were 94.5%, 66.0%, and 51.9%, respectively, relative to the average control values. The complex I of human and mice is composed of 45 subunits, including 7 subunits encoded by mtDNA and 38 subunits encoded by nuclear genes. These subunits are organized into six modules, Q, ND1, ND2, ND4, ND5, and N, with the help of specific assembly factors that begin to assemble separately and ultimately combine to form the complex I holocomplex in the inner membrane of mitochondria (37Guerrero-Castillo S. Baertling F. Kownatzki D. Wessels H.J. Arnold S. Brandt U. Nijtmans L. The assembly pathway of mitochondrial respiratory chain complex I.Cell Metab. 2017; 25 (27720676): 128-13910.1016/j.cmet.2016.09.002Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar). As shown in Fig. 2A, ND1 interacts with NDUFA1, NDUFA3, NDUFA8, and NDUFA13, whereas ND4 interacts with ND5, NDUFS2, NDUFB1, NDUFB4, NDUFB5, NDUFB8, and NDUFB11 (32Guo R. Zong S. Wu M. Gu J. Yang M. Architecture of human mitochondrial respiratory megacomplex I2III2IV2.Cell. 2017; 170 (28844695): 1247-125710.1016/j.cell.2017.07.050Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 33Agip A.-N.A. Blaza J.N. Bridges H.R. Viscomi C. Rawson S. Muench S.P. Hirst J. Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states.Nat. Struct. Mol. Biol. 2018; 25 (29915388): 548-55610.1038/s41594-018-0073-1Crossref PubMed Scopus (133) Google Scholar). To examine whether the m.3866T>C or m.11778G>A mutation affected the expression of other subunits of complex I, we measured the levels of NDUFA3, NDUFA8, NDUFA13, ND5, NDUFS2, NDUFB4, NDUFB5, NDUFB8, and NDUFB11 by Western blot analysis among mutant and control cell lines, respectively. As shown in Fig. 1, C and D, and 2, B–D, the levels of these subunits in mutant cell lines were comparable with those in control cell lines. We analyzed the consequence of m.3866T>C and m.11778G>A mutations on the stability and activity of complex I using the in-gel activity assay. Mitochondrial membrane proteins isolated from mutant and control cell lines were separated by BN-PAGE and stained with specific substrates of complexes I, II, and IV (38Jha P. Wang X. Auwerx J. Analysis of mitochondrial respiratory chain super-complexes using blue native polyacrylamide gel electrophoresis (BN-PAGE).Curr. Protoc. Mouse Biol. 2016; 6: 1-1410.1002/9780470942390.mo150182Crossref PubMed Scopus (127) Google Scholar, 39Wittig I. Braun H.P. Schagger H. Blue native PAGE.Nat. Protoc. 2006; 1: 418-42810.1038/nprot.2006.62Crossref PubMed Scopus (1261) Google Scholar, 40Li Y. D'Aurelio M. Deng J.H. Park J.S. Manfredi G. Hu P. Lu J. Bai Y. An assembled complex IV maintains the stability and activity of complex I in mammalian mitochondria.J. Biol. Chem. 2007; 282 (17452320): 17557-1756210.1074/jbc.M701056200Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). As illustrated in Fig. 3A, mutant cell lines carrying only m.3866T>C, only m.11778G>A, or both m.3866T>C and m.11778G>A mutations exhibited altered assembly of intact supercomplexes and complex I. As shown in Fig. 3B, the in-gel activities of complex I in mutant cell lines carrying only m.3866T>C, only m.11778G>A, and both m.3866T>C and m.11778G>A mutations were 72.7%, 66.7%, and 51.5%, respectively, relative to the average values of control cell lines. In contrast, the average in-gel activities of complexes II and IV in mutant cell lines were comparable with those of the control cell lines. We th
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