Contribution of mitochondrial ND1 3394T>C mutation to the phenotypic manifestation of Leber’s hereditary optic neuropathy

Abstract

Mitochondrial DNA (mtDNA) mutations have been associated with Leber’s hereditary optic neuropathy (LHON) and their pathophysiology remains poorly understood. In this study, we investigated the pathophysiology of a LHON susceptibility allele (m.3394T>C, p.30Y>H) in the Mitochondrial (MT)-ND1 gene. The incidence of m.3394T>C mutation was 2.7% in the cohort of 1741 probands with LHON. Extremely low penetrances of LHON were observed in 26 pedigrees carrying only m.3394T>C mutation, while 21 families bearing m.3394T>C, together with m.11778G>A or m.14484T>C mutation, exhibited higher penetrance of LHON than those in families carrying single mtDNA mutation(s). The m.3394T>C mutation disrupted the specific electrostatic interactions between Y30 of p.MT-ND1 with the sidechain of E4 and backbone carbonyl group of M1 of NDUFA1 (NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 1) of complex I, thereby altering the structure and function of complex I. We demonstrated that these cybrids bearing only m.3394T>C mutation caused mild mitochondrial dysfunctions and those harboring both m.3394T>C and m.11778G>A mutations exhibited greater mitochondrial dysfunctions than cybrids carrying only m.11778G>A mutation. In particular, the m.3394T>C mutation altered the stability of p.MT-ND1 and complex I assembly. Furthermore, the m.3394T>C mutation decreased the activities of mitochondrial complexes I, diminished mitochondrial ATP levels and membrane potential and increased the production of reactive oxygen species in the cybrids. These m.3394T>C mutation-induced alterations aggravated mitochondrial dysfunctions associated with the m.11778G>A mutation. These resultant biochemical defects contributed to higher penetrance of LHON in these families carrying both mtDNA mutations. Our findings provide new insights into the pathophysiology of LHON arising from the synergy between mitochondrial ND1 and ND4 mutations.

Introduction

Leber’s hereditary optic neuropathy (LHON) is the most common maternally inherited eye disease (1–4). Mutations in the mitochondrial DNA (mtDNA) have been contributed to LHON, through to varying degrees (4–8). Human mtDNA encodes 2 rRNAs (ribosomal RNA), 22 tRNAs (transfer RNA) and 13 polypeptides (essential components of oxidative phosphorylation complexes) (9). Of these, MT-ND1, MT-ND4 and MT-ND6 encoding three subunits of NADH:ubiquinone oxidoreductase (complex I) are the hot spots for mutations associated with LHON (8,10). In particular, m.11778G>A, m.14484T>C and m.3460G>A mutations are the most prevalent primary LHON-associated mtDNA mutations worldwide (8,11–16). These mutations often occur nearly homoplasmy or homoplasmy. Functional characterization of cell lines derived from matrilineal relatives of Caucasian and Chinese families carrying one of these mtDNA mutations demonstrated that these LHON-associated mtDNA mutations conferred mild mitochondrial dysfunctions, especially the reduced activity of complex I (17–19). Typical features in LHON pedigrees carrying these mtDNA mutations are marked variations in penetrance and gender bias among affected subjects (20,21). These indicated that the LHON-associated mtDNA mutation(s) is the primary causative event, but nuclear and mitochondrial genetic modifiers are required for the phenotype expression of these LHON-associated mtDNA mutations (4,5,22,23). Our previous investigations showed that a LHON susceptibility allele (c.572G>T, p.191Gly>Val) in YARS2 gene coding for the mitochondrial tyrosyl-tRNA synthetase was a nuclear modifier for the phenotypic expression of m.11778G>A mutation (22). Furthermore, genetic evidences showed that the mitochondrial haplogroups influenced the phenotypic manifestation of LHON-associated mtDNA mutations (23–25). In particular, several mtDNA variants including tRNA^Met 4435A>G, tRNA^Thr 15951A>G, ND6 14502T>C and ND1 3394T>C mutations may act as mitochondrial genetic modifiers to enhance the phenotypic expression of the m.11778G>A mutation (26–30). However, the roles of these mitochondrial modifiers in the LHON expression remains poorly understood.

The m.3394T>C mutation resulted in the change of highly conserved tyrosine at position 30 with histidine (Y30H) in p.MT-ND1, the core component of 45 subunits of complex I (10,31). In fact, the hydroxyl group of the side chain of Y30 of p.MT-ND1 forms specific electrostatic interactions with the sidechain of E4 and backbone carbonyl group of M1 of NDUFA1 (31,32). Thus, the m.3394T>C mutation may perturb the interactions between p.MT-ND1 and NDUFA1, thereby altering the structure and function of complex I. The m.3394T>C mutation has been associated with LHON, either together with the m.11778G>A and m.14484T>C mutations or in isolation (11,29,30,33–36). However, the pathophysiology of this LHON-associated mtDNA mutation remains elusive. Thus, it is necessary to establish the direct link between LHON and the mtDNA mutation and their cause/effect relation. In this study, the mutational analysis of mitochondrial genomes showed the presence of m.3394T>C mutation in 47 probands (26 probands bearing only m.3394T>C mutation, 16 and 5 subjects carrying the m.3394T>C mutation, together with m.11778G>A, or m.14484T>C mutations, respectively) in large cohorts of 1683 Chinese and 58 European probands with LHON (16). These pedigrees carrying only m.3394T>C mutation exhibited extremely low penetrance of LHON, while the penetrances of LHON among these pedigrees bearing m.3394T>C mutation together with m.14484T>C and m.11778G>A mutations were higher than those in families carrying only single mtDNA mutation (16,29,30,37). We therefore hypothesized that the biochemical consequences caused by the m.3394T>C mutation may deteriorate the mitochondrial dysfunction associated with m.11778G>A mutation, thereby increasing the penetrance and risk of LHON. To test this hypothesis, we constructed the cybrid cell lines by fusing mtDNA-less ρ^o cells with nucleated cells from LHON patients carrying both m.11778G>A and m.3394T>C mutations, only m.3394T>C or m.11778G>A mutation and a control subject lacking these mtDNA mutations but belonging to the same mtDNA haplogroup (38,39). By using western blot and blue native polyacrylamide gel electrophoresis (BN-PAGE) analyses, we examined if the m.3394T>C mutation exerted on the stability of p.MT-ND1 and assembly of complex I. These cell lines were then assessed for effects of the mtDNA mutations on the enzymatic activities of electron transport chain complexes, oxygen consumption ratio (OCR), mitochondrial membrane potential, adenosine triphosphate (ATP) production and generation of reactive oxygen species (ROS).

Results

Mutational screening of m.3394T>C mutation in the large cohorts of LHON patients

In our previous investigation, the mutational analysis of MT-ND1 gene in a cohort of 1281 Han Chinese probands with LHON led to the identification of 38 probands carrying the m.3394T>C mutation (16). In the present study, another 402 Han Chinese and 58 Caucasian probands with LHON underwent further mutational screening of m.3394T>C mutation. As a result, eight Chinese and one Caucasian probands harbored the m.3394T>C mutation. Therefore, there were 47 probands carrying the m.3394T>C mutation, including 25 Chinese and 1 Caucasian probands bearing only m.3394T>C mutation, 16 subjects carrying both m.3394T>C and m.11778G>A mutations and five individuals harboring both m.3394T>C and m.14484T>C mutations in these cohorts. This translated to the incidence of 2.73% and 1.73% in the cohorts of 1683 Chinese and 58 Caucasian probands with LHON, respectively.

Table 1

Summary of clinical and molecular data for 47 families carrying the m.3394T>C mutation

Pedigree	Ratio (affected male/female)	Age-at-onset (years)	No. of matrilineal relatives (affected)	Penetrance (%)	m.11778G>A mutation	m.14484T>C mutation	mtDNA haplogroup
WZ150	1:0	15	7(1)	14.29	No	No	M9a1
WZ233	1:1	18	7(2)	28.57	No	No	M9a3
WZ234	1:0	34	9(1)	11.11	No	No	M9a1
WZ277	1:0	16	9(1)	11.11	No	No	M
WZ470	1:0	5	5(1)	20	No	No	M9a
WZ1000	1:0	11	8(1)	12.5	No	No	M9a1
WZ1050	0:2	6	9(2)	22.22	No	No	M9
WZ1099	1:0	10	6(1)	16.67	No	No	M
WZ1122	1:0	22	9(1)	11.11	No	No	M9a1
WZ1181	2:1	24	7(2)	28.57	No	No	M9a’b
WZ1182	0:1	20	18(1)	5.56	No	No	D4
WZ1200	1:0	15	9(1)	11.11	No	No	M
WZ1229	1:0	32	5(1)	20	No	No	M9a1
WZ1251	0:1	21	9(1)	11.11	No	No	M9
WZ1262	2:1	13	11(3)	27.27	No	No	M9
WZ1320	1:0	6	10(1)	10	No	No	C4a4
WZ1394	1:0	28	19(1)	5.26	No	No	M9
WZ1421	1:0	41	20(1)	5	No	No	D4b
XT287	0:1	26	8(1)	12.5	No	No	M9a
XT302	1:0	5	13(1)	7.69	No	No	M9a
FJ021	1:0	21	6(1)	16.67	No	No	M9a
EUR023	1:0	28	13(1)	7.6	No	No	J1c
WZ98a	0:1	14	17(1)	5.88	No	No	D4b2
WZ99a	1:0	21	10(1)	10	No	No	M9a
WZ100a	1:0	14	11(1)	6.67	No	No	M9a
WZ101a	1:0	6	15(1)	6.67	No	No	M9a
WZ337	1:5	29	13(6)	46.15	Yes	No	M9a
WZ373	2:1	38	16(3)	18.75	Yes	No	B4c1b
WZ560	2:0	10	9(2)	22.22	Yes	No	M9a
WZ717	1:0	7	5(1)	20	Yes	No	B4c
WZ856	2:0	17	7(2)	28.57	Yes	No	M9a
WZ1075	1:0	16	6(1)	16.67	Yes	No	M8
WZ1366	3:0	18	11(3)	27.27	Yes	No	B4c
XT256	2:2	30	13(4)	30.77	Yes	No	M9a
XT273	7:0	32	14(7)	50	Yes	No	D5a
FJ043	5:3	36	17(8)	47.06	Yes	No	M9a
BJ053	1:1	24	8(2)	25	Yes	No	M9a
BJ108	5:2	33	16(7)	43.75	Yes	No	M9a
WZ102b	3:0	30	8(3)	37.5	Yes	No	M9
WZ103b	3:0	18	8(3)	37.5	Yes	No	M9a
WZ104b	3:1	24	9(4)	44.44	Yes	No	M9a
WZ105b	2:3	19	9(5)	55.56	Yes	No	M9a
WZ371	2:1	26	8(3)	37.5	No	Yes	M9
WZ1042	3:1	27	10(4)	40	No	Yes	M9a3
WZ1235	5:2	20	19(7)	36.84	No	Yes	R
WZ53c	4:2	18	13(6)	46.15	No	Yes	M9a
WZ54c	6:4	18	18(10)	55.56	No	Yes	M9a

^aLiang et al. (30),

^bZhang et al. (29);

^cZhang et al. (25)

Clinical and genetic evaluation of 47 families carrying the m.3394T>C mutation

Comprehensive physical and ophthalmologic examinations were performed to identify personal or family medical histories of visual impairments and other clinical abnormalities in all available members of 47 pedigrees including 26 harboring only m.3394T>C mutation, 16 bearing both m.3394T>C and m.11778G>A mutations as well as 5 harboring both m.3394T>C and m.14484T>C mutations. As shown in Supplementary Material, Figure S1, ophthalmologic and genetic examinations showed that matrilineal relatives exhibited the variable penetrance and expressivity of optic neuropathy among and within families. The severity of visual impairment varied from profound visual loss to normal vision. As illustrated in Table 1, the age-at-onset of optic neuropathy of 26 pedigrees bearing only m.3394T>C mutation ranged from 5 to 41 years, with an average of 18.1 years, whereas the average age-at-onset of visual impairment of 16 pedigrees harboring both m.3394T>C and m.11778G>A mutations and 5 pedigrees harboring both m.3394T>C and m.14484T>C mutations were 23.8 and 22.2 years, respectively. Strikingly, 26 pedigrees harboring only m.3394T>C mutation exhibited extremely low penetrance of optic neuropathy, ranged from 5% to 28.6%, with an average of 13.5%. In contrast, the average penetrances of optic neuropathy of 16 pedigrees harboring both m.3394T>C and m.11778G>A mutations and 5 pedigrees harboring both m.3394T>C and m.14484T>C mutations were 34.5% and 43.2%, respectively. To assess whether the differences in the penetrance and age-at-onset of optic neuropathy differ based on the presence and absence of additional mtDNA variants, a statistical analysis was performed using the unpaired, two-tailed Student’s t-test contained in the Microsoft Excel program. The penetrance of optic neuropathy among these Chinese pedigrees bearing both m.3394T>C and m.11778G>A or m.14484T>C mutations showed significantly higher than those pedigrees bearing only m.3394T>C, m.11778G>A or m.14484T>C mutation (P < 0.01). In contrast, there was no significant difference in the average age-at-onset of LHON between these Chinese pedigrees carrying both m.3394T>C and m.11778G>A or m.14484T>C mutations and those pedigrees bearing only m.3394T>C, m.11778G>A or m.14484T>C mutation. Furthermore, all affected matrilineal relatives of these families showed no other clinical abnormalities, including diabetes, muscular disorders and neurological diseases.

mtDNA analysis

The entire sequence of mtDNA in four Chinese families harboring only m.3394T>C mutation, four Chinese pedigrees bearing both m.3394T>C and m.11778G>A mutations as well as two Chinese pedigrees carrying both m.3394T>C and m.14484T>C mutations were determined previously (25,29,30). In this study, we further analyzed the entire sequence of mtDNAs among additional 22 probands carrying only m.3394T>C mutation, 12 individuals harboring both m.3394T>C and m.11778G>A mutations and 3 subjects bearing both m.3394T>C and m.14484T>C mutations. In addition to the known m.3394T>C, m.11778G>A and m.14484T>C mutations, these probands displayed the distinct sets of polymorphisms including 251 known and 12 novel variants, as shown in the Supplementary Material, Table S1. The mtDNAs from 47 pedigrees (26 harboring only m.3394T>C mutation, 16 having both m.3394T>C and m.11778G>A mutations and 5 having both m.3394T>C and m.14484T>C mutations) resided at the mtDNA haplogroups B4c (3 pedigrees), C4a (1 pedigree), D (4 pedigrees), J1c (1 pedigree), M (3 pedigrees), M8 (1 pedigree), M9 (33 pedigrees) and R (1 pedigree), respectively (40).

These mtDNA variants included 82 in the D-loop region, 13 in the 12S rRNA gene, 8 in the 16S rRNA gene, 7 in the tRNA genes, 9 bp deletion in the NC7 region and 97 silent and 55 missense variants in the genes encoding polypeptides (7,9). These variants in RNAs and polypeptides were evaluated by phylogenetic analysis of these variants and sequences from 17 vertebrates, as shown in Supplementary Material, Table S1. These variants were further assessed for the presence of 376 control subjects and potential structural and functional alterations. Of these, two variants may have potential structural and functional alterations. As shown in the Supplementary Material, Table S1, m.14927A>G or m.15317G>A mutation were coexisted with m.3394T>C mutation in the pedigrees WZ1181 and WZ1262, respectively. These indicated that these mtDNA variants may also play a role in the phenotypic manifestation of m.3394T>C mutation.

Generation of cell lines from three Chinese families with LHON

To obtain the functional evidences that the m.3394T>C mutation contributed to the pathogenesis of LHON, we utilized three representative Chinese pedigrees with LHON (WZ234 family bearing only m.3394T>C mutation, WZ351 family carrying only m.11778G>A mutation, WZ337 family having both m.3394T>C and m.11778G>A mutations) used for the biochemical characterization (Fig. 1). Immortalized lymphoblastoid cell lines were generated from three individuals (WZ234-IV-3 harboring only m.3394T>C mutation, WZ351-IV-2 bearing only m.11778G>A mutation, WZ337-III-7 carrying both m.11778G>A and m.3394T>C mutations, all three probands between 25–35 years) and one genetically unrelated control individual WZC241 (male, 30 years) belonging to the same mtDNA haplogroup M9. These lymphoblastoid cells line were enucleated, and subsequently fused to a large excess of mtDNA-less human ρ⁰206 cells and the resultant cybrid clones were isolated by growing in selective DMEM medium (38,39,41,42). The cybrids derived from each donor cell line were analyzed for the presences and levels of the m.3394T>C or m.11778G>A mutation and mtDNA copy numbers. The results confirmed the absence of these mtDNA mutations in the control cybrids and their presence in homoplasmy in all cybrids derived from the mutant cell line (data not shown). Three cybrids derived from each donor cell line with similar mtDNA copy numbers were used for the following biochemical characterization.

Figure 1

Three Chinese pedigrees with LHON. Vision-impaired individuals are indicated by blackened symbols.

The m.3394T>C mutation caused the reduction of p.MT-ND1 protein

To experimentally test the predicted effect of m.3394T>C mutation on p.MT-ND1, we examined the level of p.MT-ND1 by a western blotting in these mutant cell lines carrying only m.3394T>C mutation, only m.11778G>A mutation or both m.3394T>C and m.11778G>A mutations and control cell lines. The 20 μg of total cellular proteins from various cell lines were separated by polyacrylamide gel electrophoresis, electroblotted onto a polyvinylidene difluoride membrane. These blots were then hybridized with p.MT-ND1, p.MT-ND4 and p.MT-ND6 as well as VDAC (voltage-dependent anion channels) (a nuclear gene encoding mitochondrial protein) antibodies as a loading control, respectively. As shown in Figure 2, the levels of p.MT-ND1 in mutant cell lines carrying only m.3394T>C, m.11778G>A or both m.3394T>C and m.11778G>A mutations were 91.1%, 99.1% and 59.7%, relative to the average values of control cell lines. Furthermore, the levels of p.MT-ND4 in mutant cell lines carrying only m.3394T>C, m.11778G>A, both m.3394T>C and m.11778G>A mutations were 99.8%, 49.2% and 47.1%, relative to the average control values, respectively. In contrast, the levels of p.MT-ND6 in mutant cell lines were comparable with those in control cell lines.

Figure 2

Western blot analysis of mitochondrial proteins. (A) The 5 μg of mitochondrial proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with three respiratory complex subunits (p.MT-ND1, p.MT-ND4 and p.MT-ND6) in mutant and control cell lines with VDAC (voltage-dependent anion channels) as a loading control. p.MT-ND1, p.MT-ND4 and p.MT-ND6; subunits 1, 4 and 6 of complex I. (B) Quantification of three complex I subunits. The levels of p.MT-ND1, p.MT-ND4 and p.MT-ND6 in mutant and control cell lines were determined as described elsewhere (28). The calculations were based on three independent determinations in each cell line. The error bars indicate two standard errors of the means. P indicates the significance, according to the t-test, of the differences between mutant and control cell lines.

Altered assembly of complex I

Based on the crystal structure of the mammalian complex I (31), the hydroxyl group of the side chain of Y30 of p.MT-ND1 forms specific electrostatic interactions with the sidechain of E4 and backbone carbonyl group of M1 of NDUFA1 (nuclear encoding complex I subunit) (Fig. 3A). Indeed, these interactions are the only electrostatic interactions formed between p.MT-ND1 and the N-terminal part of NDUFA1. Therefore, the anticipated disrupted these interactions caused by the m.3394T>C mutation may perturb the assemble of complex I. To test this hypothesis, mitochondrial membrane proteins isolated from mutant and control cell lines were separated by BN-PAGE, electroblotting and hybridizing with NDUFA9 antibody (complex I subunit encoded by nuclear gene) and VDAC as loading control. As shown in Figure 3B, the altered assembly of intact complex I was observed in mutant cell lines bearing the only m.3394T>C, only m.11778G>A mutation and both m.3394T>C and m.11778G>A mutations, respectively. In particular, the levels of complex I in mutant cell lines carrying only m.3394T>C, only m.11778G>A and both m.3394T>C and m.11778G>A mutations were 79.2%, 47.7% and 39.7%, relative to the average values of control cell lines. These data suggested that these cell lines carrying both m.3394T>C and m.11778G>A mutations displayed more unstable complex I than those cell lines carrying only single mutation.

Figure 3

Altered assembly of complex I. (A) The electrostatic interactions between Y30 of p.MT-ND1 (shown in yellow) and the N-terminal part of NDUFA1 (shown in blue). Based on the crystal structure of mammalian complex I (protein data bank entry: 5XTD) (31), the hydroxyl group of the side chain of Y30 forms specific electrostatic interactions with the sidechain of E4 and backbone carbonyl group of M1 of NDUFA1. (B). Respiratory complex assembly. Whole cells were solubilized with n-dodecyl-β-D-maltoside and then subjected to BN-PAGE/immunoblot analysis. Blots were hybridized with anti-NDUFA9 antibody and VDAC as internal control. The levels of complex I in mutant and control cell lines were determined as described elsewhere (28). The calculations were based on three independent determinations in each cell line. The error bars indicate two standard errors of the means. P indicates the significance, according to the t-test, of the differences between mutant and control cell lines.

Decreased activity of complex I

To investigate the effect of the m.3394T>C mutation on oxidative phosphorylation, we measured the activities of respiratory complexes in isolating mitochondria from various mutant and control cell lines. Complex I (NADH ubiquinone oxidoreductase) activity was determined by following the oxidation of NADH with ubiquinone as the electron acceptor (43,44). Complex III (ubiquinone cytochrome c oxidoreductase) activity was measured as the reduction of cytochrome c (III) using D-ubiquinol-2 as the electron donor. The activities of complex IV (cytochrome c oxidase) were monitored by following the oxidation of cytochrome c (II). As shown in Figure 4, the activities of complex I in mutant cell lines carrying only m.3394T>C, only m.11778G>A and both m.3394T>C and m.11778G>A mutations were 79.2%, 55.7% and 45.6%, relative to the average values of control cell lines, respectively. However, the average activities of complex II, III and IV in mutant cell lines were comparable with those of three control cell lines, as shown in Supplementary Material, Figure S2.

Figure 4

Enzymatic activity of respiratory chain complex I. The activities of respiratory complex I were investigated by enzymatic assay on complex I in mitochondria isolated from various cell lines. The calculations were based on three independent determinations. The error bars indicate two standard errors of the means. P indicates the significance, according to the t-test, of the differences between mutant and control cell lines.

As shown in Figure 5, the m.3394T>C mutation-induced alterations in complex I were further verified by the in-gel activity assays. The in-gel activities of complex I in mutant cell lines carrying only m.3394T>C, only m.11778G>A and both m.3394T>C and m.11778G>A mutations were 72.5%, 71.1% and 66.8%, relative to the average values of control cell lines, respectively. In contrast, the average in-gel activities of complex II and IV in mutant cell lines were comparable with those of three control cell lines.

Figure 5

In-gel activity of respiratory chain complex I, II and IV. A total of 20 μg of mitochondrial proteins (8 g/g digitonin/protein ratio) from various mutant and control cell lines were used for BN-PAGE and the activities of complexes were measured in the presence of specific substrates (NADH and NTB for complex I, sodium succinate, phenazine methosulfate and NTB for complex II, DAB and cytochrome c for complex IV). The activities of complex I (A) and complex II (B) were shown in violet. (C) Complex IV in brown. VDAC was used as an internal control. Quantification of in-gel activities of respiratory chain complexes I (D), II (E) and IV (F). The calculations were based on three independent determinations in each cell line. The error bars indicate two standard errors of the means. P indicates the significance, according to the t-test, of the differences between mutant and control cell lines.

Respiration defects

To examine if m.3394T>C mutation altered cellular bioenergetics, we measured the OCR of various mutant and control cell lines with a Seahorse Bioscience XF-96 extracellular flux analyzer (Seahorse Bioscience) (45,46). In fact, OCR can be assigned to oxidative phosphorylation. In this system, all major aspects of mitochondrial coupling and respiratory control including basal respiration, O₂ consumption attributed to ATP production, proton leak, maximum respiratory rate, reserve capacity and non-mitochondrial respiration can be measured in a single experiment. As shown in Figure 6, the basal OCR in the mutant cell lines carrying only m.3394T>C, only m.11778G>A or both m.3394T>C and m.11778G>A mutations were 82.8%, 59.2% and 52.6%, relative to the mean value measured in the control cell lines, respectively. To assess which of the enzyme complexes of the respiratory chain was perturbed in the mutant cell lines, OCR were measured after the sequential addition of oligomycin (inhibit the ATP synthase), FCCP (to uncouple the mitochondrial inner membrane and allow for maximum electron flux through the electron transport chain), rotenone (to inhibit complex I) and antimycin (to inhibit complex III). The difference between the basal OCR and the drug-insensitive OCR yields the amount of ATP-linked OCR, proton leak OCR, maximal OCR, reserve capacity and non-mitochondrial OCR. As shown in Figure 6, the ATP-linked OCR, proton leak OCR, maximal OCR, reserve capacity and non-mitochondrial OCR in mutant cell lines carrying both m.3394T>C and m.11778G>A mutations were 41.8%, 102.3%, 68.8%, 153.7% and 51.2%, those cell lines carrying only m.3394T>C were 79.4%, 87.6%, 100.1%, 197.6% and 76.5%, cell lines carrying only m.11778G>A mutations were 45.6%, 82.4%, 69.7%, 162.8% and 76.5%, relative to the mean values measured in the control cell lines, respectively.

Figure 6

Respiration assays. (A) An analysis of O₂ consumption in the various cell lines using different inhibitors. The rates of O₂ (OCR) were first measured on 2 × 10⁴ cells of each cell line under basal condition and then sequentially added to oligomycin (1.5 μm), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (0.5 μm), rotenone (1 μm) and antimycin A (1 μm) at indicated times to determine different parameters of mitochondrial functions. (B) Graphs presented the ATP-linked OCR, proton leak OCR, maximal OCR, reserve capacity and non-mitochondrial OCR in mutant and control cell lines. Non-mitochondrial OCR was determined as the OCR after rotenone/antimycin A treatment. Basal OCR was determined as OCR before oligomycin minus OCR after rotenone/antimycin A. ATP-lined OCR was determined as OCR before oligomycin minus OCR after oligomycin. Proton leak was determined as basal OCR minus ATP-linked OCR. Maximal was determined as the OCR after FCCP minus non-mitochondrial OCR. Reserve capacity was defined as the difference between maximal OCR after FCCP minus basal OCR. The average values of four determinations for each cell line were shown; the horizontal dashed lines represent the average value for each group. The error bars indicate two standard errors of the means. P indicates the significance, according to the t-test, of the differences between mutant and control cell lines.

Reductions in mitochondrial ATP production

The impact of m.3394T>C mutation on capacity of oxidative phosphorylation was further examined by measuring the levels of cellular and mitochondrial ATP using a luciferin/luciferase assay. Populations of cells were incubated in the media in the presence of glucose (total cellular ATP production), and 2-deoxy-D-glucose with pyruvate (mitochondrial ATP production) (46,47). As shown in Figure 7A, the levels of mitochondrial ATP production in the mutant cell lines bearing only m.3394T>C, only m.11778G>A, both m.3394T>C and m.11778G>A mutations were 84.5%, 62.8% and 51.5% of average values of control cell lines, respectively. In contrast, the levels of total cellular ATP production in mutant cell lines were comparable with those measured in the control cell lines (Figure 7B).

Figure 7

Measurement of cellular and mitochondrial ATP levels. ATP levels from mutant and control cell lines were measure using a luciferin/luciferase assay. Mutant and control cell lines were incubated with 10 mm glucose or 5 mm 2-deoxy-d-glucose plus 5 mm pyruvate to determine ATP generation under mitochondrial ATP synthesis. Average rates of ATP level per cell line in mitochondria are show. Six to seven determinations were made for each cell line. The error bars indicate two standard errors of the means. P indicates the significance, according to the t-test, of the differences between mutant and control cell lines.

Alterations in mitochondrial membrane potential

It was hypothesized that deficient respiration impaired mitochondrial membrane potential. To assess if the m.3394T>C mutation affected the mitochondrial membrane potential (ΔΨ_m), a fluorescence probe JC-10 assay system was utilized to examine the ΔΨ_m in various mutant and control cell lines (46). The ratios of fluorescence intensities Ex/Em = 490/590 and 490/530 nm (FL₅₉₀/FL₅₃₀) were recorded to delineate the ΔΨ_m level of each cell line. The relative ratios of FL₅₉₀/FL₅₃₀ geometric mean between mutant and control cell lines were calculated to represent the level of ΔΨ_m. As shown in Fig. 8A and C, the levels of the ΔΨ_m in the mutant cell lines carrying only m.3394T>C, only m.11778G>A or both m.3394T>C and m.11778G>A mutations were 85.1%, 64.6% and 55% of the mean values measured in the control cell lines, respectively. Conversely, the levels of ΔΨ_m in mutant cell lines in the presence of FCCP were comparable with those measured in the control cell lines (Fig. 8B and D).

Figure 8

Mitochondrial membrane potential analysis. The mitochondrial membrane potential (ΔΨ_m) was measured in mutant and control cybrids cell lines using a fluorescence probe JC-10 assay system. The ratio of fluorescence intensities Ex/Em = 490/590 nm and 490/530 nm (FL₅₉₀/FL₅₃₀) were recorded to delineate the ΔΨ_m level of each sample. The relative ratios of FL₅₉₀/FL₅₃₀ geometric mean between mutant and control cell lines were calculated to reflect the level of ΔΨ_m. Flow cytometry images of a control cell line WZC241—T4, WZ23-IV-3-T3 carrying only m.3394 T > C mutation and WZ351-IV-2.T8 carrying only m.11778G>A mutation, and WZ337-III-7-T7 bearing both m.3394T>C and m.11778G>A mutations without (A) and with (B) FCCP. Relative ratio of JC-10 fluorescence intensities at Ex/Em = 490/530 nm and 490/590 nm in absence (C) and presence (D) of 10 μm of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP). The average of three to five determinations for each cell line is shown. The error bars indicate two standard errors of the means. P indicates the significance, according to the t-test, of the differences between mutant and control cell lines.

The increase of ROS production

It was anticipated that the alterations in oxidative phosphorylation and mitochondrial membrane potential elevated the production of ROS. The levels of the ROS generation in the vital cells derived from mutant and control cell lines were measured with a flow cytometry under normal and H₂O₂ stimulation (48,49). Geometric mean intensity was recorded to measure the rate of ROS of each cell line. The ratio of geometric mean intensity between unstimulated and stimulated with H₂O₂ in each cell line was calculated to delineate the reaction upon increasing level of ROS under oxidative stress. As shown in Figure 9, the levels of ROS generation in the mutant cell lines carrying only m.3394T>C, only m.11778G>A mutation, both m.11778G>A and m.3394T>C mutations were 110.8%, 125% and 128.1% of the mean values measured in three control cell lines.

Figure 9

Measurement of ROS production. The rates of production in ROS from mutant and control cell lines were analyzed by BD-LSR II flow cytometer system with or without H₂O₂ stimulation. (A) Flow cytometry histogram showing the fluorescence of representative mutant and control cell lines (WZ234-IV3-T3 carrying only m.3394T>C mutation, WZ351-IV2.T8 carrying only m.11778G>A mutation and WZ337-III7-T7 bearing both m.3394T>C and m.11778G>A mutations and control WZC241-T4). (B) Relative ratios of intensity. The relative ratio of intensity (stimulated versus unstimulated with H₂O₂) was calculated in three cybrid cell lines derived from one Chinese control subject (WZC241) and three cybrid cell lines for each genotype derived from three mutant individuals (WZ234-IV3 carrying only m.3394T>C mutation, WZ351-IV2 carrying only m.11778G>A mutation and WZ337-III7 bearing both m.3394T>C and m.11778G>A mutations). The values for the latter are expressed as percentages of the average values for the control cell lines. The average of three determinations for each cell line is shown. The error bars indicate two standard errors of the means. P indicates the significance, according to the t-test, of the differences between mutant and control cell lines.

Discussion

The pathogenicity of m.3394T>C mutation

In this present study, we investigated the pathophysiology of LHON-associated m.3394T>C mutation. The m.3394T>C mutation has been associated with LHON, either together with the m.11778G>A and m.14484T>C mutations or in isolation among Caucasian and Asian families (11,29,30,33–36). The incidences of the m.3394T>C mutation were 2.73% and 1.73% in the cohorts of 1683 Chinese and 58 Caucasian probands with LHON, respectively. The occurrence of m.3394T>C mutation in these genetically unrelated European and Asian pedigrees affected by LHON and differing considerably in their mtDNA haplotypes (B4, C4, D4, D5, F1, J1, M, M9, R) strongly indicated that this mutation is involved in the pathogenesis of this disorder (11,29,30,33–36). However, 22 pedigrees carrying only m.3394T>C and 2 families bearing both m.3394T>C and m.11778G>A mutations did not have a family history of optic neuropathy. The extremely lower penetrance of LHON in these families carrying only m.3394T>C mutation was consistent of those in Chinese families bearing only m.3460G>A, m.11778G>A, m.14484T>C, m.12338T>C, m.14502T>C or m.11696G>A mutations (28,37,50–54).

The m.3394T>C (Y30H) mutation changed a conserved tyrosine at position 30 with histidine in p.MT-ND1, which is an essential subunit of complex I (10). The complex I comprised of 7 subunits including p.MT-ND1 and p.MT-ND4 encoded by mtDNA, and 38 subunits, including NDUFA1, encoded by nuclear genes (10). The structural data showed that the hydroxyl group of the side chain of Y30 of p.MT-ND1 forms specific electrostatic interactions with the sidechain of E4 and backbone carbonyl group of M1 of NDUFA1 (31,32). Thus, the substitution of tyrosine 30 with histidine by m.3394T>C mutation may abolish these interactions between p.MT-ND1 and NDUFA1, thereby altering the structure and function of complex I. In this study, the instability of mutated p.MT-ND1 was evidenced by the reduced levels of p.MT-ND1 observed in cybrids bearing only m.3394T>C mutation or both m.3394T>C and m.11778G>A mutations. The mutated p.MT-ND1 indeed perturbed the assembly of complex I, as in the cases of m.11778G>A and m.14502T>C mutations (28). Both instability of p.MT-ND1 and altered assembly of complex I were responsible for 20% reductions in the activity of complex I observed in cybrids carrying only m.3394T>C on the M9 background. The m.3394T>C mutation-induced alterations in complex I were further verified by 27% reductions of complex I activity in-gel. In the previous study, cybrids carrying only m.3394T>C mutation on haplogroups B4c and F1 exhibited 28% and 15% reductions in complex I activity, respectively (33). Furthermore, the m.3394T>C mutation led to the reduced rates in the basal OCR and ATP-linked OCR in mutant cybrid cell lines. The respiratory deficiency can perturb the ATP synthesis, oxidative stress and subsequent cellular energetic process (55,56). In the present study, there was a very significant correlation between the reduced levels of complex I activity and mitochondrial ATP production, mitochondrial membrane potentials or the increasing rates of ROS production (P < 0.01) in the control and mutant cybrids harboring the m.3394T>C mutation. This correlation is clearly consistent with the importance that the defective activity of complex I in the mutant cell lines carrying m.3394T>C mutation plays a critical role in producing their mitochondrial dysfunction for the development of LHON. However, the extremely low penetrance of LHON and mild mitochondrial dysfunction strongly suggested that the m.3394T>C mutation is the primary factor underlying the development of LHON, but insufficient to produce a clinical phenotype, as in the cases of m.12338T>C, 14052T>C and m.4317A>G mutations (28,42,57). The additional hereditary or environmental factors are necessary for the phenotypic expression of m.3394T>C mutation.

The m.3394T>C mutation enhanced the phenotypic expression of the primary LHON-associated m.11778G>A mutation

Mitochondrial genetic background such as specific mtDNA variants may act as genetic modifiers to increase the phenotypic expression of the m.11778G>A mutation (23–28,58). The average penetrances of optic neuropathy of 21 pedigrees harboring both m.3394T>C and m.11778G>A or m.14484T>C mutations were significantly higher than those in 26 families carrying only m.3394T>C mutation and other pedigrees harboring only m.11778G>A or m.14484T>C mutation (28,37,50–54). These observations strongly suggested that the m.3394T>C mutation may increase the expression of optic neuropathy in these families carrying the primary mtDNA mutations, as in the cases of m.4216T>C and m.13708G>A mutations in European families (11,23) and m.4435A>G, m.15951A>G and m.14502T>C mutations in other Chinese pedigrees (26–28).

To assess the biochemical consequences due to specific mtDNA mutations, we used these cybrid cell models under the identical nuclear background and mtDNA haplogroup (28). We demonstrated that cybrids cell lines bearing both m.3394T>C and m.11778G>A mutations exhibited more severe mitochondrial dysfunctions than those carrying only m.11778G>A or m.3394T>C mutation. In particular, the cell lines carrying both m.3394T>C and m.11778G>A mutations exhibited more unstable complex I than those bearing only m.3394T>C or m.11778G>A mutation. As a result, the m.3394T>C mutation worsened the deficient activity of complex I, associated with m.11778G>A mutation. Furthermore, more drastic decreases of mitochondrial ATP production and mitochondrial membrane potentials were observed in the cybrid cell lines carrying both m.3394T>C and m.11778G>A mutations than those in cybrid cell lines carrying only m.3394T>C or m.11778G>A mutation. The retinal ganglion cells carrying both m.3394T>C and m.11778G>A mutations may be preferentially involved because they are high ATP demand cells and somehow exquisitely sensitive to subtle imbalance in cellular redox state or increased level of free radicals (59–62). The significant correlation between severe biochemical phenotypes in these mutants cybrids and higher penetrance of LHON in these pedigrees harboring both m.11778G>A and m.3394T>C mutation demonstrated that the m.3394T>C mutation increased the penetrance and occurrence of optic neuropathy in these Chinese families carrying both m.3394T>C and m.11778G>A mutations. Therefore, our findings provided new insights into the pathophysiology of LHON that were manifested by the synergy between the m.11778G>A and m.3394T>C mutations.

Materials and Methods

Families and subjects

A total of 1683 genetically unrelated Han Chinese subjects with LHON were recruited from the eye clinics across the China, as described previously (14–16), while 58 European probands with LHON were recruited from Cincinnati Children’s Hospital Medical Center. This study was following the Declaration of Helsinki. Informed consent, blood samples and clinical evaluations were obtained from all participating family members under protocols approved by the Ethic Committees of Zhejiang University and Wenzhou Medical University and Internal Review Board of Cincinnati Children’s Hospital Medical Center. A comprehensive history and physical examination for these participating subjects were performed at length to identify both personal or family medical histories of visual impairment and other clinical abnormalities. The ophthalmic examinations of probands and other members of these families carrying the m.3394T>C mutation were conducted as detailed previously (14–16). A total of 376 control DNA samples were derived from adult Han Chinese from same area.

mtDNA analysis

The mutational screening of m.3394T>C mutation was performed as previously (29,39). The entire mitochondrial genomes of additional 22 probands carrying only m.3394T>C mutation, 12 individuals harboring both m.3394T>C and m.11778G>A mutations and 3 subjects bearing both m.3394T>C and m.14484T>C mutations (63). The resulting sequence data were compared with the updated consensus Cambridge sequence (GenBank accession number: NC_012920) (9). The entire mtDNA sequences of 47 pedigrees and 1 control subject were assigned to the mitochondrial haplogroups using the nomenclature as described previously (40). An analysis for the presence and levels of m.11778G>A and m.3394T>C mutations in mutant and control cell lines were carried out as described previously (26,29). The quantification of mtDNA copy numbers from different cell lines was performed as detailed elsewhere (64).

Cell lines and culture conditions

Lymphoblastoid cell lines come from three probands (WZ234-IV-3 bearing only m.3394T>C mutation, WZ351-IV-2 harboring only m.11778G>A mutation, WZ337-III-7 carrying both m.11778G>A and m.3394T>C mutations) and one control subject WZC241 lacking these mtDNA mutations were immortalized by transformation with the Epstein–Barr virus, as described elsewhere (65). Lymphoblastoid cell lines were grown in RPMI 1640 medium (Corning), supplemented with 10% fetal bovine serum. The 143B.TK⁻ cell line was grown in DMEM (containing 4.5 mg of glucose and 0.11 mg pyruvate per ml), supplemented with 100 μg of BrdU per ml and 5% FBS. The mtDNA-less ρ^o206 cell line, derived from 143B.TK⁻ was grown under the same conditions as the parental line, except for the addition of 50 μg of uridine/ml. Transformation by cytoplasts of mtDNA-less ρ^o206 cells was performed by using four immortalized lymphoblastoid cell lines, as detailed previously (38,39). All cybrid cell lines were maintained in the same medium as the 143B.TK⁻ cell line.

Western blot analysis

Mitochondria were isolated from mutant and control cell lines according to the protocol described previously (66). Western blotting analysis was carried out by using total mitochondrial proteins isolated from mutant and control cell lines, as detailed elsewhere (46,67). The antibodies used for this investigation were from Proteintech Group [anti-VDAC1/2(10866-1-AP), p.MT-ND1 (19703-1-AP)], Santa Cruz Biotechnology [p.MT-ND4 (sc-20499-R) and p.MT-ND6 (sc-20667)] and the secondary antibodies were from Beyotime Biotechnology [Peroxidase AffiniPure goat anti-rabbit IgG (A0208) and goat anti-mouse IgG (A0216)]. The protein signals were detected using the ECL system (CoWin Biosciences). Quantification of density in each band was performed as detailed previously (46,67).

BN-PAGE and in-gel activity assays

BN-PAGE was performed by using mitochondrial proteins, as detailed previously (68,69). The antibodies applied for this experiment were from Sigma-Aldrich [anti-NDUFA9 (SAB1100073)] and Proteintech Group [anti-VDAC1/2(10866-1-AP)] and the secondary antibodies were from Beyotime Biotechnology [Peroxidase AffiniPure goat anti-rabbit IgG (A0208) and goat anti-mouse IgG (A0216)].

For the in-gel activity assay, 20 μg of mitochondrial proteins (8 g/g digitonin/protein ratio) from cultured cells were used for BN-PAGE (70). The native gels were prewashed in cold water, and then incubated with the substrates of complex I [2 mm Tris–HCl, pH 7.4, 0.1 mg/ml NADH, 2.5 mg/ml nitrotetrazolium blue chloride (NTB)], complex II (20 mm sodium succinate, 2.5 mg/ml NTB, 0.2 mm phenazine methosulfate, 5 mm Tris–HCl) and complex IV [0.5 mg/ml diaminobenzidine (DAB), 1 mg/ml cytochrome c, 45 mm phosphate buffer, pH 7.4] at room temperature. After stopping reaction with 10% acetic acid, gels were washed with water and scanned to visualize the activities of respiratory chain complexes.

Enzymatic assays

The enzymatic activities of complex I, II, III and IV were assayed as detailed elsewhere (41,43,44).

Measurements of oxygen consumption

The rates of oxygen consumption in cybrid cell lines were measured with a Seahorse Bioscience XF-96 extracellular flux analyzer (Seahorse Bioscience), as detailed previously (45,46,47).

ATP measurements

The Cell Titer-Glo Luminescent Cell Viability Assay kit (Promega) was used for the measurement of cellular and mitochondrial ATP levels, according to the modified manufacturer’s instructions (46,47,71).

Assessment of mitochondrial membrane potential

The JC-10 Mitochondrial Membrane Potential Microplate Assay Kit (Abcam, ab112134) was used for measurement of mitochondrial membrane potential, based on the modified manufacturer’s instructions detailed previously (67,72).

ROS Measurements

ROS measurements were performed following the procedures detailed previously (48,49).

Statistical analysis

All statistical analysis was computerized using the unpaired, two-tailed Student’s t-test contained in the Microsoft Excel program (version 2016). A P value <0.05 was considered statistically significant.

Acknowledgements

We are grateful to patients and their family members for their participation.

Conflict of Interest statement. All authors have no proprietary or commercial interest in any of materials discussed in this article.

Funding

Natural Science Foundation of China [grants 31471191, 81400434, 81600769, 81372116 and 81671287 to M.X.G., Y.J., M. L., P.J. and T.Z., respectively]; Ministry of Science and Technology of China [National Key Technologies R&D Program grant 2012BAI09B03 to M.X.G. and P.J.]; Zhejiang Provincial Medical and Health Research Project [grants 2019325380 and 2019313572 to X.J. and J.Y., respectively].

Author notes

The first two authors equally contributed to the work.