Leber’s hereditary optic neuropathy (LHON)-associated ND5 12338T > C mutation altered the assembly and function of complex I, apoptosis and mitophagy

Leber’s hereditary optic neuropathy (LHON)-associated ND5 12338T > C mutation altered the... Abstract Mutations in mitochondrial DNA (mtDNA) have been associated with Leber’s hereditary optic neuropathy (LHON) and their pathophysiology remains poorly understood. In this study, we demonstrated that a missense mutation (m.12338T>C, p.1M>T) in the ND5 gene contributed to the pathogenesis of LHON. The m.12338T>C mutation affected the first methionine (Met1) with a threonine and shortened two amino acids of ND5. We therefore hypothesized that the mutated ND5 perturbed the structure and function of complex I. Using the cybrid cell models, generated by fusing mtDNA-less (ρ°) cells with enucleated cells from LHON patients carrying the m.12338T>C mutation and a control subject belonging to the same mtDNA haplogroup, we demonstrated that the m.12338T>C mutation caused the reduction of ND5 polypeptide, perturbed assemble and activity of complex I. Furthermore, the m.12338T>C mutation caused respiratory deficiency, diminished mitochondrial adenosine triphosphate levels and membrane potential and increased the production of reactive oxygen species. The m.12338T>C mutation promoted apoptosis, evidenced by elevated release of cytochrome c into cytosol and increased levels of apoptosis-activated proteins: caspases 9, 3, 7 and Poly ADP ribose polymerase in the cybrids carrying the m.12338T>C mutation, as compared with control cybrids. Moreover, we also document the involvement of m.12338T>C mutation in decreased mitophagy, as showed by reduced levels of autophagy protein light chain 3 and accumulation of autophagic substrate p62 in the in mutant cybrids as compared with control cybrids. These data demonstrated the direct link between mitochondrial dysfunction caused by complex I mutation and apoptosis or mitophagy. Our findings may provide new insights into the pathophysiology of LHON. Introduction Leber’s hereditary optic neuropathy (LHON) is the most common maternally inherited eye disease (1–4). LHON results from selective degeneration of retinal ganglion cells and their axons that leads to an acute or subacute loss of central vision (4,5). This disorder affects predominantly young adult males (6). A number of mitochondrial DNA (mtDNA) mutations have been identified that contributed to LHON, though to varying degree (3,7,8). The majority of LHON cases worldwide arise from three point mutations in mitochondrial genes encoding three subunits of Nicotinamide adenine dinucleotide (NADH): ubiquinone oxidoreductase (complex I): ND1 m.3460G>A, ND4 m.11778G>A and ND6 m.14484T>C (7–11). In fact, the complex I is a large protein complex made up of 46 different subunits, of seven subunits encoded by mtDNA (12). The primary defects in these mutations were the failures in the activity of complex I, thereby leading to the deficient function of oxidative phosphorylation, decrease in adenosine triphosphate (ATP) synthesis and increase in the production of reactive oxygen species (ROS) (13–15). These three mtDNA mutations accounted for ∼90% of LHON pedigrees in some countries (7–11), while these mutations are only responsible for 38.3 and 46.5% cases in two large cohorts of Chinese Han subjects with LHON (16–19). To further understand the pathophysiology of LHON, we performed the clinical, genetic and molecular analysis of 1281 Han Chinese probands with LHON (17–22). This analysis identified the known ND4 m.11778G>A, ND6 m.14484T>C, ND1 m.3460G>A, m.3635G>A and m.3866T>C mutations as well as ND5 m.12238T>C mutation (17–22). In particular, the m.12338T>C mutation was identified in six genetically related Han Chinese pedigrees with LHON (22). However, the pathophysiology of the m.12338T>C mutation remains poorly understood. Thus, it is necessary to establish the link between LHON and mitochondrial dysfunction and their cause/effect relation. As shown in Figure 1, the m.12338T>C mutation yielded the replacement of the translation-initiating methionine with a threonine, thereby shortening two amino acids of ND5 polypeptide (22). We therefore hypothesized that the mutated ND5 caused by the m.12338T>C mutation altered the structure and function of complex I. The m.12338T>C mutation is also located at 2 nt adjacent to the 3′ end of the tRNALeu(CUN). Thus, this mutation may affect the processing of tRNALeu(CUN) precursors. Functional significance of the m.12338T>C mutation was investigated through cell lines constructed by transferring mitochondria from lymphoblastoid cell lines derived from an affected matrilineal relative carrying the m.12238T>C mutation and from a control subject belonging to the same mtDNA haplogroup, into human mtDNA-less (ρo) cells (23,24). First, these cell lines were assessed for the effects of the m.12338T>C mutation on the stability of tRNALeu(CUN). We then examined if the m.12238T>C mutation perturbed the stability of ND1 and assembly of complex I by using western blot and blue native polyacrylamide gel electrophoresis (BN-PAGE) analysis. These cell lines were further evaluated for effects on enzymatic activities of electron transport chain complexes, respiration, production of ATP, mitochondrial membrane potential and generation of ROS. Finally, we examined if the m.12338T>C mutation affected the apoptosis and mitophagy. Figure 1. View largeDownload slide A schema of mtDNA sequence at position 12338 and adjacent sequence of ND5 and tRNALeu(CUN) from wild-type (WT) and mutant (MT). Arrow indicates the position of the m.12338T>C mutation. Figure 1. View largeDownload slide A schema of mtDNA sequence at position 12338 and adjacent sequence of ND5 and tRNALeu(CUN) from wild-type (WT) and mutant (MT). Arrow indicates the position of the m.12338T>C mutation. Results The Chinese pedigree carrying the m.12338T>C mutation and derived cybrid cell lines The WZ411 pedigree of the Chinese family carrying the m.12338T>C mutation was described previously (22). Immortalized lymphoblastoid cell lines were derived from the proband (II-10, female, 32 years) and one genetically unrelated control individual A60 belonging to the same mtDNA haplogroup (female, 28 years) (Supplementary Material, Table S1). These lymphoblastoid cells lines were enucleated and fused to a large excess of mtDNA-less human ρo206 cell line and cybrid clones were isolated by growing in selective Dulbecco's modified Eagle's medium (DMEM) medium (23). The cybrids derived from each donor cell line were analyzed for the presence and degrees of the m.12338T>C mutation and mtDNA copy numbers. The results confirmed the absence of the m.12338T>C mutation in the control clones and their presence in homoplasmy in all cybrids derived from the mutant cell lines (data not shown). Three mutant cybrids (II-10-1, II-10-2 and II-10-3) carrying the homoplasmic m.12338T>C mutation and three control cybrids (A60-1, A60-2 and A60-3) lacking the mutation with similar mtDNA copy numbers and the same karyotype as 143B.TK− cell lines were used for the biochemical characterization. Analysis of mitochondrial tRNALeu(CUN) The m.12338T>C mutation was located at two nt adjacent to the 3′ end of the tRNALeu(CUN) (22). To examine whether the m.12338T>C mutation affects the processing of tRNALeu(CUN) precursors, we subjected mitochondrial RNAs from cybrids cell lines to northern blots and hybridized them with digoxigenin (DIG)-labeled oligodeoxynucleotide probes for tRNALeu(CUN), tRNASer(UCN) and tRNAGlu as well as nuclear encoded 5S rRNA (25–27). As shown in Figure 2, the levels of tRNALeu(CUN), tRNASer(UCN) and tRNAGlu in three mutant cybrid cell lines carrying the m.12338T>C mutation were comparable with those in three control cell lines lacking the mtDNA mutation. These data indicated that the m.12338T>C mutation did not affect the tRNALeu(CUN) metabolism. Figure 2. View largeDownload slide Northern blot analysis of mitochondrial tRNA. (A) Equal amounts (2 μg) of total mitochondrial tRNA samples from the various cell lines were electrophoresed through a denaturing polyacrylamide gel, were electroblotted and were hybridized with DIG-labeled oligonucleotide probes specific for the tRNALeu(CUN), tRNASer(UCN), tRNAGln and 5S RNA, respectively. (B) Quantification of mitochondrial tRNA levels. Shown is the average relative tRNA content per cell, normalized to the average content per cell of 5S rRNA in the cells derived from three mutant cybrids carrying the m.12338T>C mutation and three cybrids lacking the mutation. The values for the latter are expressed as percentages of the average values for the control cell lines. The calculations were based on three independent determinations. The error bars indicate two standard errors of the mean (SEM). P indicates the significance, according to the t-test, of the difference between mutant mean and control mean. Figure 2. View largeDownload slide Northern blot analysis of mitochondrial tRNA. (A) Equal amounts (2 μg) of total mitochondrial tRNA samples from the various cell lines were electrophoresed through a denaturing polyacrylamide gel, were electroblotted and were hybridized with DIG-labeled oligonucleotide probes specific for the tRNALeu(CUN), tRNASer(UCN), tRNAGln and 5S RNA, respectively. (B) Quantification of mitochondrial tRNA levels. Shown is the average relative tRNA content per cell, normalized to the average content per cell of 5S rRNA in the cells derived from three mutant cybrids carrying the m.12338T>C mutation and three cybrids lacking the mutation. The values for the latter are expressed as percentages of the average values for the control cell lines. The calculations were based on three independent determinations. The error bars indicate two standard errors of the mean (SEM). P indicates the significance, according to the t-test, of the difference between mutant mean and control mean. The m.12338T>C mutation caused the reduction of ND5 protein To experimentally test the predicted effect of m.12338T>C mutation for ND5, we analyzed the levels of ND5 and other four mtDNA-encoding polypeptides by a western blotting in these mutant and control cell lines. Twenty micrograms of total cellular proteins from various cell lines were separated by PAGE, electroblotted. The blots were then hybridized with ND5 and other four mtDNA encoded polypeptides (ND1, CYTB, apocytochrome b; CO2, subunit 2 of cytochrome c oxidase and ATP6, subunit 6 of the H+-ATPase) in mutant and control cell lines as well as HSP60 (a nuclear-encoding mitochondrial protein) as a loading control. As shown in Figure 3, the levels of ND5 in three mutant cell ranged from ∼53 to 69%, with an average of 63% (P = 0.0021), relative to the mean value measured in three control cell lines. However, the levels of ND1, CYTB, CO2 and ATP6 in the mutant cell lines were comparable with those in control cell lines (Supplementary Material, Fig. S1). Figure 3. View largeDownload slide Western blot analysis of mitochondrial proteins. (A) Twenty micrograms of total cellular proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with ND5 and other three respiratory complex subunits in mutant and control cells with HSP60 as a loading control. ND1 and ND5, subunits 1 and 5 of the reduced nicotinamide–adenine dinucleotide dehydrogenase; ATP6, subunit 6 of the H+-ATPase and CYTB, apocytochrome b. (B) Quantification of mitochondrial protein levels. The levels of ND1 in three mutant cell lines and three control cell lines were determined as described elsewhere (36). The values for the mutant cell lines are expressed as percentages of the average values for the control cell lines. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to Figure 2. Figure 3. View largeDownload slide Western blot analysis of mitochondrial proteins. (A) Twenty micrograms of total cellular proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with ND5 and other three respiratory complex subunits in mutant and control cells with HSP60 as a loading control. ND1 and ND5, subunits 1 and 5 of the reduced nicotinamide–adenine dinucleotide dehydrogenase; ATP6, subunit 6 of the H+-ATPase and CYTB, apocytochrome b. (B) Quantification of mitochondrial protein levels. The levels of ND1 in three mutant cell lines and three control cell lines were determined as described elsewhere (36). The values for the mutant cell lines are expressed as percentages of the average values for the control cell lines. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to Figure 2. Perturbed the assembly of complex I To examine if the mutated ND5 affected the assembly of complex I, mitochondrial membrane proteins isolated from mutant and control cell lines were separated by BN-PAGE and electroblotting and hybridizing with NDUFA9 antibody (complex I subunit encoded by nuclear gene) and ATP5A as loading control (12,28,29). As shown in Figure 4, the altered assembly of intact complex I were observed in mutant cell lines carrying the m.12338T>C mutation. In particular, the levels of complex I in mutant cell lines carrying m.12338T>C mutation was 72 and 78%, with an average of 75% (P = 0.0013), relative to the average values in control cell lines. These data suggested that these cell lines carrying m.12338T>C mutation exhibited more unstable complex I than those in control cell lines. Figure 4. View largeDownload slide Analysis of complex I assembly. (A) Respiratory complex assembly and in-gel activity assay. Whole cells from mutant and controls were solubilized with digitonin and then subjected to BN-PAGE/immunoblot analysis. Blots were the hybridized with NDUFA9 antibody and ATP5A as internal control. (B) Quantification of complex I. The levels of complex I in mutant and control cell lines were determined as based on three independent determinations in each cell line. Graph details and symbols were explained in the legend of Figure 2. Figure 4. View largeDownload slide Analysis of complex I assembly. (A) Respiratory complex assembly and in-gel activity assay. Whole cells from mutant and controls were solubilized with digitonin and then subjected to BN-PAGE/immunoblot analysis. Blots were the hybridized with NDUFA9 antibody and ATP5A as internal control. (B) Quantification of complex I. The levels of complex I in mutant and control cell lines were determined as based on three independent determinations in each cell line. Graph details and symbols were explained in the legend of Figure 2. Reduced activity of complex I To evaluate the effect of the m.12338T>C mutation on the oxidative phosphorylation, we measured the activities of respiratory complexes by isolating mitochondria from three mutant cell lines and three control cell lines. Complex I activity was determined by following the oxidation of NADH with ubiquinone as the electron acceptor (30,31). The activity of complex II (succinate ubiquinone oxidoreductase) exclusively encoded by the nuclear DNA was examined by the artificial electron acceptor 2,6-Dichlorophenolindophenol (32). Complex III (ubiquinone cytochrome c oxidoreductase) activity was measured as the reduction of cytochrome c (III) using D-ubiquinol-2 as the electron donor. Complex IV (cytochrome c oxidase) activity was monitored by following the oxidation of cytochrome c (II). As shown in Figure 5, the activity of complex I in three mutant cell lines was 61.5%, relative to the mean value measured in the control cell lines (P = 0.0002). However, the activities of complexes II–IV in three mutant cell lines were comparable with those of three control cell lines. Figure 5. View largeDownload slide Enzymatic activities of respiratory chain complexes. The activities of respiratory complexes were measured by enzymatic assay on complexes I–IV in mitochondria isolated from mutant and control cell lines. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to Figure 2. Figure 5. View largeDownload slide Enzymatic activities of respiratory chain complexes. The activities of respiratory complexes were measured by enzymatic assay on complexes I–IV in mitochondria isolated from mutant and control cell lines. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to Figure 2. Respiration deficiency To assess if m.12338T>C mutation perturbed cellular bioenergetics, the oxygen consumption rates (OCRs) of mutant and control cell lines were measured with a Seahorse Bioscience XF-96 extracellular flux analyzer (Seahorse Bioscience) (33–35). As shown in Figure 6, the basal OCR in the mutant cell lines carrying m.12338T>C mutation was 60% (P = 0.0009), relative to the mean value measured in the control cell lines. To investigate which of the enzyme complexes of the respiratory chain was perturbed in the mutant cell lines, OCR were examined after the sequential additions of oligomycin (to inhibit the ATP synthase), Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) (to allow for maximum electron flux through the electron transport chains), rotenone (to inhibit complex I) and antimycin (to inhibit complex III). The difference between the basal OCR and the drug-insensitive OCR produced 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 bearing the m.12338T>C mutation were 68.3, 41.6, 69.2, 81.4 and 84.4% (P = 0.0008, 0.0193, 0.0096, 0.1882 and 0.0951), relative to the mean value measured in the control cell lines, respectively. Figure 6. View largeDownload slide 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 p-(trifluoromethoxy) phenylhydrazone (FCCP) (0.5 μM), rotenone (1.0 μM) and antimycin A (1.0 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 independent experiments for each cell line were shown, the horizontal dashed lines represent the average value for each group. Graph details and symbols are explained in the legend to Figure 2. Figure 6. View largeDownload slide 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 p-(trifluoromethoxy) phenylhydrazone (FCCP) (0.5 μM), rotenone (1.0 μM) and antimycin A (1.0 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 independent experiments for each cell line were shown, the horizontal dashed lines represent the average value for each group. Graph details and symbols are explained in the legend to Figure 2. Decrease in mitochondrial ATP generation The effect of m.12338T>C mutation on capacity of oxidative phosphorylation was further evaluated by measuring the levels of cellular and mitochondrial ATP production 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) (34–36). As shown in Figure 7A, the levels of total cellular ATP production in mutant cell lines were comparable to those in control cell lines. Conversely, the levels of mitochondrial ATP production in mutant cell lines ranged from 36 to 44.6%, with an average of 40.1%, relative to the mean value measured in the control cell lines (P < 0.0001) (Fig. 7B). Figure 7. View largeDownload slide Measurement of cellular and mitochondrial ATP levels. ATP levels from mutant and control cell lines were measure using a luciferin/luciferase assay. Cells 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 and are shown: (A) ATP level in total cells. (B) ATP level in mitochondria. Four independent experiments were made for each cell line. Graph details and symbols are explained in the legend to Figure 2. Figure 7. View largeDownload slide Measurement of cellular and mitochondrial ATP levels. ATP levels from mutant and control cell lines were measure using a luciferin/luciferase assay. Cells 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 and are shown: (A) ATP level in total cells. (B) ATP level in mitochondria. Four independent experiments were made for each cell line. Graph details and symbols are explained in the legend to Figure 2. Alterations in mitochondrial membrane potential To examine if the m.12338T>C mutation affected the mitochondrial membrane potential (ΔΨm), a fluorescence probe JC-10 assay system was used to examine the ΔΨm in mutant and control cell lines. The ratio of fluorescence intensities Ex/Em = 490/590 and 490/525 nm (FL590/FL525) were recorded to delineate the ΔΨm level of each sample. The relative ratios of FL590/FL525 geometric mean between mutants and controls were calculated to represent the level of ΔΨm. As shown in Figure 8, levels of the ΔΨm in the mutants carrying the m.12338T>C mutation were decreased, ranging from 72.4 to 80.2%, with an average 77.5% (P = 0.0285) of the mean value measured in the controls. In contrast, the levels of ΔΨm in mutant cell lines in the presence of FCCP were comparable with those measured in these control cell lines. Figure 8. View largeDownload slide Mitochondrial membrane potential analysis. The mitochondrial membrane potential (ΔΨm) was measured in mutant and control 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 ΔΨm 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 ΔΨm. Relative ratio of JC-10 fluorescence intensities at Ex/Em = 490/530 and 490/590 nm in absence (A) and presence (B) of 10 μM of carbonyl cyanide 3-chlorophenylhydrazone (FCCP). Three to five determinations were made for each cell line are shown. Graph details and symbols are explained in the legend to Figure 2. Figure 8. View largeDownload slide Mitochondrial membrane potential analysis. The mitochondrial membrane potential (ΔΨm) was measured in mutant and control 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 ΔΨm 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 ΔΨm. Relative ratio of JC-10 fluorescence intensities at Ex/Em = 490/530 and 490/590 nm in absence (A) and presence (B) of 10 μM of carbonyl cyanide 3-chlorophenylhydrazone (FCCP). Three to five determinations were made for each cell line are shown. Graph details and symbols are explained in the legend to Figure 2. Increase of mitochondrial ROS production The levels of mitochondrial ROS (mitoROS) among the mutant and control cell lines were measured with flow cytometry using a MitoSOX assay (24,37). Geometric mean intensity was recorded to measure the rate of mitoROS of each sample. The relative levels of geometric mean intensity in each cell line were calculated to delineate the levels of mitoROS in mutant and control cells. As shown in Figure 9, the levels of mitoROS generation in the mutant cell lines carrying the m.12338T>C mutation ranged from 137.5 to 162.1%, with an average of 150.1% (P = 0.0027) of mean value measured in the control cell lines. Figure 9. View largeDownload slide Measurement of mitoROS. The levels of ROS generation by mitochondria in living cells from mutant and control cell lines were determined using the mitochondrial superoxide indicator MitoSOX-Red. Fluorescence was measured using a instrument (BD Biosciences), with excitation at 488 nm and emission at 580 nm. The data were analyzed with FlowJo software. (A) Flow cytometry histogram showing MitoSOX-Red fluorescence of A60-1 (Black) and II-10-1 91 (Red). (B) Relative ratios of MitoSOX-Red fluorescence intensity. The average of four determinations for each cell line is shown. Graph details and symbols are explained in the legend to Figure 2. Figure 9. View largeDownload slide Measurement of mitoROS. The levels of ROS generation by mitochondria in living cells from mutant and control cell lines were determined using the mitochondrial superoxide indicator MitoSOX-Red. Fluorescence was measured using a instrument (BD Biosciences), with excitation at 488 nm and emission at 580 nm. The data were analyzed with FlowJo software. (A) Flow cytometry histogram showing MitoSOX-Red fluorescence of A60-1 (Black) and II-10-1 91 (Red). (B) Relative ratios of MitoSOX-Red fluorescence intensity. The average of four determinations for each cell line is shown. Graph details and symbols are explained in the legend to Figure 2. Promoting apoptosis To evaluate if the m.12338T>C mutation affects the apoptotic processes, we examined the apoptotic state of mutant and control cybrids by immunofluorescence and western blot analyses. As shown in Figure 10A, the immunofluorescence patterns of double-labeled cells with rabbit monoclonal antibody specific for the cytochrome C and mouse monoclonal antibody to TOM20 revealed markedly increased levels of cytochrome c in the mutant cells, compared with control cells. The levels of cytochrome c in cytosol in mutant and control cell lines were further evaluated by western-blotting analysis. As shown in Figure 10B, the levels of cytochrome c in three mutant cell lines ranged from 224 to 243%, with an average of 235.4% (P = 0.0005), relative to the average values in three control cell lines. Furthermore, we examined the levels of four apoptosis-activated proteins [caspases 9, 3, 7 and Poly ADP ribose polymerase (PARP)] in mutant and control cell lines by western blot analysis (38). As showed in Figure 10C, the marked increasing levels of these proteins were observed in the mutant cell lines. In particular, the levels of caspases 9, 3, 7 and PARP in the mutant cell lines were 137.1, 158.2, 158.9 and 162.2% of the average values measured in the control cell lines, respectively (P = 0.0008, 0.0053, 0.0064 and 0.0026). Figure 10. View largeDownload slide Analysis of apoptosis. (A) The distributions of cytochrome c from cybrids (mutant II-10-1 and wild-A60-1) were visualized by immunofluorescent labeling with TOM20 antibody conjugated to Alex Fluor 488 (green) and cytochrome c antibody conjugated to Alex Fluor 594 (red) analyzed by confocal microscopy. DAPI-stained nuclei were identified by their blue fluorescence. (B) Measurement of cytochrome C levels in cytosol. Twenty micrograms of total cellular proteins from various cell lines were electrophoresed, electroblotted and hybridized with cytochrome c and with β-actin as a loading control. The levels of cytochrome c in mutant and control cell lines were determined as described elsewhere (36). (C) Western blot analysis of four apoptosis-activated proteins. Twenty micrograms of total proteins from various cell lines were electrophoresed, electroblotted and hybridized with caspases 9, 3, 7 and PARP antibodies and with β-actin as a loading control. (D) Quantification of four apoptosis-activated proteins. The levels of caspases 9, 3, 7 and PARP in various cell lines were determined as described elsewhere (36). Three independent determinations were done in each cell line. Graph details and symbols are explained in the legend to Figure 2. Figure 10. View largeDownload slide Analysis of apoptosis. (A) The distributions of cytochrome c from cybrids (mutant II-10-1 and wild-A60-1) were visualized by immunofluorescent labeling with TOM20 antibody conjugated to Alex Fluor 488 (green) and cytochrome c antibody conjugated to Alex Fluor 594 (red) analyzed by confocal microscopy. DAPI-stained nuclei were identified by their blue fluorescence. (B) Measurement of cytochrome C levels in cytosol. Twenty micrograms of total cellular proteins from various cell lines were electrophoresed, electroblotted and hybridized with cytochrome c and with β-actin as a loading control. The levels of cytochrome c in mutant and control cell lines were determined as described elsewhere (36). (C) Western blot analysis of four apoptosis-activated proteins. Twenty micrograms of total proteins from various cell lines were electrophoresed, electroblotted and hybridized with caspases 9, 3, 7 and PARP antibodies and with β-actin as a loading control. (D) Quantification of four apoptosis-activated proteins. The levels of caspases 9, 3, 7 and PARP in various cell lines were determined as described elsewhere (36). Three independent determinations were done in each cell line. Graph details and symbols are explained in the legend to Figure 2. Alteration in mitophagy Mitophagy is the selective removal of damaged mitochondria by autophagosomes and their subsequent catabolism by lysosomes (39). To investigate if the m.12338T>C mutation affected the mitophagy, we evaluated the mitophagic states of mutant and control cell lines using western-blotting and endogenous immunofluorescence assays. As shown in Figure 11A, the immunofluorescence patterns of cells lines double-labeled with the antibodies specific for lysosome-associated membrane glycoprotein 1 (LAMP1), and TOM20 indicated that the m.12338T>C mutation affected the autophagy process. The levels of mitophagy in mutant and control cell lines were then examined using two markers: microtubule-associated protein 1A/1B light chain 3B (LC3) and sequestosome 1 (SQSTM1/p62) (40). During autophagy, the cytoplasmic form (LC3-I) is processed into a cleaved and lipidated membrane-bound form (LC3-II), which is essential for membrane biogenesis and closure of the membrane. LC3-II is recleaved by cysteine protease (Atg4B) following completion of the autophagosome and recycled (41). SQSTM1/p62, one of the best-known autophagic substrates, interacts with LC3 to ensure the selective delivery of these proteins into the autophagosome (40). As shown in Figure 11B, the reduced levels of LC3 and increased level of p62 were observed in mutant cybrids carrying the m.12338T>C mutation, compared with those in control cybrids. In particular, the average levels of LC3-II/(LC3-I+II) and p62 in three mutant cell lines carrying the m.12338T>C mutation were 67.3% (P = 0.0308) and 140.2% (P = 0.0195) of the mean values measured in three control cell lines lacking the mutation, respectively. These data suggested that the m.12338T>C mutation led to the decreased mitophagy in cybrids. Figure 11. View largeDownload slide Analysis of mitophagy. (A) The distributions of LAMP1 from cybrids (II-10-1 and A60-1) were visualized by immunofluorescent labeling with TOM20 antibody conjugated to Alex Fluor 488 (green) and LAMP1 antibody conjugated to Alex Fluor 594 (red) analyzed by confocal microscopy. DAPI-stained nuclei were shown by the blue fluorescence. (B) Western blot analysis for mitophagic response proteins LC3-I/II and P62. Twenty micrograms of total cellular proteins from various cell lines were electrophoresed, electroblotted and hybridized with LC3, p62 and with β-actin as a loading control. (C) Quantification of autophagy markers LC3A/B and p62 in mutant and control cell lines were determined as described elsewhere (36). Figure 11. View largeDownload slide Analysis of mitophagy. (A) The distributions of LAMP1 from cybrids (II-10-1 and A60-1) were visualized by immunofluorescent labeling with TOM20 antibody conjugated to Alex Fluor 488 (green) and LAMP1 antibody conjugated to Alex Fluor 594 (red) analyzed by confocal microscopy. DAPI-stained nuclei were shown by the blue fluorescence. (B) Western blot analysis for mitophagic response proteins LC3-I/II and P62. Twenty micrograms of total cellular proteins from various cell lines were electrophoresed, electroblotted and hybridized with LC3, p62 and with β-actin as a loading control. (C) Quantification of autophagy markers LC3A/B and p62 in mutant and control cell lines were determined as described elsewhere (36). Discussion In this study, we investigated the molecular pathogenesis of LHON-associated ND5 12338T>C mutation. In particular, the m.12338T>C mutation has several deleterious effects that potentially contributed to the pathogenesis of LHON. These included the mitochondrial dysfunction, promoting apoptosis and decreased mitophagy due to the mtDNA mutation. Indeed, the occurrence of m.12338T>C mutation in several genetically unrelated families affected by optic neuropathy strongly indicated that this mutation is involved in the pathogenesis of LHON (21). The m.12338T>C mutation caused the replacement of the first methionine (Met1) with a threonine and the methionine at position 3 (Met3) acting as the initiation methionine and consequently shortened two amino acids of ND5 polypeptide (21,42). Therefore, it was anticipated that the m.12338T>C mutation results in the instability of mutant ND5 or perturbs the translation of ND5 mRNA by the replacement of the first methionine (Met1) with a threonine. These alterations led to 37% reduction in the levels of ND5 observed in mutant cybrids carrying the m.12338T>C mutation. In fact, ND5 is the essential subunit to be located at the periphery of NADH: ubiquinone oxidoreductase, which comprised ND5 and additional six subunits encoded by mtDNA and 39 subunits encoded by nuclear genes (43–45). We therefore hypothesized that the mutated ND5 altered the structure and function of complex I. In this investigation, we demonstrated that the mutant cybrids carrying the m.12338T>C mutation exhibited more unstable complex I than those in control cell lines lacking the mutation. These data were consistent with the previous observations that mutations in mtDNA-encoding subunits of complex I including ND1, ND4 and ND6 perturbed the assembly of NADH: ubiquinone oxidoreductase (46–49). Both instability of ND5 and altered assembly of complex I were responsible for a decline of complex I activity observed in cybrids harboring the m.12338T>C mutation. However, cybrids bearing the m.11338T>C mutation exhibited less reductions in the complex I activity than those in cybrids carrying the m.11778G>A mutation (13,35). Furthermore, less reductions in the basal OCR, or ATP-linked OCR, proton leak OCR and maximal OCR were observed in cybrids carrying the m.12338T>C mutation than those in cybrids carrying the m.11778G>A mutation (35). All these data strongly supported the functional significance of m.12338T>C mutation leading to mitochondrial dysfunction. The respiratory deficiency caused by the m.12338T>C mutation may lead to the alterations on ATP synthesis, oxidative stress and subsequent failure of cellular energetic process (50,51). In this investigation, we demonstrated significant decreases in mitochondrial ATP production in these cybrids carrying m.12338T>C mutation, as in the case of cell lines bearing the m.11778G>A mutation (15,34,35,52). Furthermore, the deficient activities of respiratory chain complexes caused by mtDNA mutations often perturbed mitochondrial membrane potentials, which is a key indicator of cellular viability (24,36,53). Significant reductions in mitochondrial membrane potential in mutant cybrids bearing the m.12338T>C mutation revealed the impaired pumping ability of hydrogen ions across the inner membrane and more electron leakage during the process of electron transport and oxidative phosphorylation (24,53). Alterations in both oxidative phosphorylation and mitochondrial membrane potential resulted in the ROS overproduction and the subsequent bioenergetic failure in mutant cybrids carrying the m.12338T>C mutation. In turn, the increased production of ROS may produce the damage of proteins, lipids and nuclear acids and subsequently alter the apoptosis and mitophage processes (54–56). Mitochondrial dysfunction affected the apoptotic sensitivity of cells carrying the LHON-associated mtDNA mutations (57–59). The cybrids carrying one of three primary mtDNA mutations such as m.11778G>A underwent apoptotic cell death, when exposed to the media containing the galactose, which renders the cells more dependent on mitochondrial ATP production (58). After FAS-stimulation, the enhanced apoptosis were observed in the mutant cybrids bearing the m.3460G>A or m.11778G>A mutation, as compared with control cybrids (60). In the present investigation, mutant cybrids bearing the m.12338T>C mutation exhibited more apoptotic susceptibility than control cybrids lacking the mutation. These were evidenced by the elevated release of cytochrome c into cytosol and increased levels of apoptosis-activated proteins: caspases 9, 3, 7 and PARP in the cybrids carrying the m.12338T>C mutation, as compared with control cybrids. These data demonstrated that mitochondrial dysfunction caused by several LHON-associated mtDNA mutations promoted the apoptosis (60–62). Mitophagy regulates mitochondrial energy metabolism by controlling the amount and efficiency of the mitochondrial metabolic machinery (63). In particular, the impairment of OXPHOS and alteration of mitochondrial membrane potential affected the mitophagic removal of damaged mitochondria (64–66). The activation of mitophagic machinery is crucial to the complete degradation of mitochondrial components. The p62 can both aggregate ubiquitinated proteins by polymerizing with other p62 molecules and recruit ubiquitinated cargo into autophagosomes by binding to LC3 (67). The p62 mediates clumping of mitochondria and links ubiquitinated substrates to LC3 to facilitate the autophagic degradation of ubiquitinated proteins. Here, the increased levels of p62 in cybrids carrying the m.12338T>C mutation indicated the accumulation of autophagic substrates such as misfolded proteins, leading to the deleterious effects. Moreover, reduced levels of LC3 were observed in mutant cybrids carrying the m.12338T>C mutation. The reductions in LC3 level in mutant cybrids suggested a general decrease in the capacity of the mutant cells to generate autophagoeomes, thereby perturbing the autophagic degradation of ubiquitinated proteins. These results provided the evidences that the m.12338T>C mutation may mediate mitophagy in cybrids. Therefore, our findings established the association between mitochondrial dysfunction caused by complex I mutations and apoptosis or mitophagy. In summary, our results suggested the pathogenic mechanism leading to an impaired oxidative phosphorylation in cybrid cell lines carrying the m.12338T>C mutation. The m.12338T>C (p.1M>T) mutation in the ND5 gene perturbed the assembly and activity of NADH: ubiquinone oxidoreductase. As a result, this respiratory deficiency resulted in the decrease of mitochondrial ATP production, mitochondrial membrane potential and the increasing production of oxidative reactive species. All those alterations consequently elevated the apoptotic cell death and decreased the mitophagy in cells carrying the m.12338T>C mutation, thereby leading to visual loss. Therefore, our findings may provide new insights into pathophysiology of LHON. Materials and Methods Cell lines and culture condition Lymphoblastoid cell lines derived from one affected matrilineal relative WZ411(II-10) carrying the m.12338T>C mutation (22) and from one genetically unrelated control individuals belonging to the same mtDNA haplogroup (A60) were immortalized by transformation with the Epstein–Barr virus, as described previously (68). Lymphoblastoid cell lines were grown in Roswell Park Memorial Institute medium 1640 medium (Invitrogen), supplemented with 10% fetal bovine serum (FBS). 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 ρo206 cell line, derived from 143B.TK− (23,69) 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 ρo206 cells was performed by using two immortalized lymphoblastoid cell lines, as detailed elsewhere (23,69). All cybrid cell lines constructed with enucleated lymphoblastoid cell lines were maintained in the same medium as the 143B.TK− cell line. An analysis for the presence and level of the m.12338T>C mutation was carried out as described previously (22). The quantifications of mtDNA copy number from different cybrids were performed as detailed elsewhere (37). Three mutant cybrids carrying the m.12338T>C mutation and three control cybrids lacking the mutation with similar mtDNA copy numbers and the same karyotype were used for the biochemical characterization described later. Mitochondrial tRNA northern analysis Total mitochondrial RNA were obtained using TOTALLY RNATM kit (Ambion) from mitochondria isolated from the cybrid cell lines (∼2.0 × 108 cells), as described previously (70). Oligodeoxynucleosides used for DIG-labeled probes of tRNALeu(CUN), tRNASer(UCN), tRNAGlu and 5S RNA were described as elsewhere (34,36,52). DIG-labeled oligodeoxynucleotides were generated by using DIG oligonucleotide Tailing kit (Roche). Two micrograms of total mitochondrial RNA were electrophoresed through a 10% polyacrylamide/7M urea gel in Tris-borate-ethylenediaminetetraacetic acid buffer (after heating the sample at 65°C for 10 min) and then electroblotted onto a positively charged nylon membrane (Roche) for the hybridization. Quantification of density in each band was made as detailed previously (34,36). Western blot analysis Western-blotting analysis was performed as detailed previously (34–36). Twenty micrograms of total cellular proteins obtained from lysed mutant and control cell lines were electrophoresed through sodium dodecyl sulfate polyacrylamide gels, electroblotted onto a polyvinylidene difluoride membrane and hybridized with the following antibodies, respectively. Antibodies were obtained from different companies including LC3A/B (#4108), LAMP1 (#9091), caspase-3 (#9664), caspase-7 (#8438), caspase-9 (#7237) and PARP (#5625) from Cell Signaling Technology, CYTB (55090-1-AP) from Proteintech, CO2 (ab110258), ND5 (ab92624), ND1 (ab74257), ATP6 (ab101908), Cytochrome c (ab13575), HSP60 (ab46798), β-actin (ab8226), p62 (ab56416), TOM20 (ab56783) and GAPDH (ab8245) from Abcam. Peroxidase Affini Pure goat anti-mouse IgG and goat anti-rabbit IgG (Jackson) were used as a secondary antibody and protein signals were detected using the electrochemiluminescence system (CWBIO). Quantification of density in each band was performed as detailed previously (34–36). BN gel electrophoresis BN gel electrophoresis were carried out using mitochondrial proteins isolated from mutant and control cell lines, as detailed elsewhere (28,35,71). The antibodies used for this investigation were NDUFA9 (ab14713) from Abcam and ATP5A (14676-1-AP) from Proteintech. Peroxidase Affini Pure goat anti-mouse IgG and goat anti-rabbit IgG (Cell Signaling) were used as a secondary antibody. Protein signals were detected using Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA) or 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) substrate (Promega, Madison, WI, USA). Quantification of density in each band was performed as detailed previously (34,35). Enzymatic assays The enzymatic activities of complexes I–IV were assayed as detailed elsewhere (28,31,32). 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 (33,36). 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 (24,36). ROS measurements ROS measurements were performed following the procedures detailed previously (24,35,72). Immunofluorescence analysis Immunofluorescence experiments were performed as described elsewhere (73). Cells were cultured on cover glass slips (Thermo Fisher), fixed in 4% formaldehyde for 15 min, permeabilized with 0.2% Triton X-100, blocked with 5% FBS for 1 h and immunostained with TOM20, cytochrome C and LAMP1 antibodies overnight at 4°C, respectively. The cells were then incubated with Alex Fluor 594 goat anti-rabbit IgG (H+L) and Alex Fluor 488 goat anti-mouse IgG (H+L) (Thermo Fisher), stained with 4′, 6-diamidino-2-phenylindole (DAPI; Invitrogen) for 15 min and mounted with Fluoromount (Sigma-Aldrich). Cells were examined using a confocal fluorescence microscope (Olympus Fluoview FV1000, Japan) with three lasers (Ex/Em = 550/570, 492/520 and 358/461 nm). 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( 2016 ) Effects of genetic correction on the differentiation of hair cell-like cells from iPSCs with MYO15A mutation . Cell Death Differ ., 23 , 1347 – 1157 . Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Human Molecular Genetics Oxford University Press

Leber’s hereditary optic neuropathy (LHON)-associated ND5 12338T > C mutation altered the assembly and function of complex I, apoptosis and mitophagy

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Abstract

Abstract Mutations in mitochondrial DNA (mtDNA) have been associated with Leber’s hereditary optic neuropathy (LHON) and their pathophysiology remains poorly understood. In this study, we demonstrated that a missense mutation (m.12338T>C, p.1M>T) in the ND5 gene contributed to the pathogenesis of LHON. The m.12338T>C mutation affected the first methionine (Met1) with a threonine and shortened two amino acids of ND5. We therefore hypothesized that the mutated ND5 perturbed the structure and function of complex I. Using the cybrid cell models, generated by fusing mtDNA-less (ρ°) cells with enucleated cells from LHON patients carrying the m.12338T>C mutation and a control subject belonging to the same mtDNA haplogroup, we demonstrated that the m.12338T>C mutation caused the reduction of ND5 polypeptide, perturbed assemble and activity of complex I. Furthermore, the m.12338T>C mutation caused respiratory deficiency, diminished mitochondrial adenosine triphosphate levels and membrane potential and increased the production of reactive oxygen species. The m.12338T>C mutation promoted apoptosis, evidenced by elevated release of cytochrome c into cytosol and increased levels of apoptosis-activated proteins: caspases 9, 3, 7 and Poly ADP ribose polymerase in the cybrids carrying the m.12338T>C mutation, as compared with control cybrids. Moreover, we also document the involvement of m.12338T>C mutation in decreased mitophagy, as showed by reduced levels of autophagy protein light chain 3 and accumulation of autophagic substrate p62 in the in mutant cybrids as compared with control cybrids. These data demonstrated the direct link between mitochondrial dysfunction caused by complex I mutation and apoptosis or mitophagy. Our findings may provide new insights into the pathophysiology of LHON. Introduction Leber’s hereditary optic neuropathy (LHON) is the most common maternally inherited eye disease (1–4). LHON results from selective degeneration of retinal ganglion cells and their axons that leads to an acute or subacute loss of central vision (4,5). This disorder affects predominantly young adult males (6). A number of mitochondrial DNA (mtDNA) mutations have been identified that contributed to LHON, though to varying degree (3,7,8). The majority of LHON cases worldwide arise from three point mutations in mitochondrial genes encoding three subunits of Nicotinamide adenine dinucleotide (NADH): ubiquinone oxidoreductase (complex I): ND1 m.3460G>A, ND4 m.11778G>A and ND6 m.14484T>C (7–11). In fact, the complex I is a large protein complex made up of 46 different subunits, of seven subunits encoded by mtDNA (12). The primary defects in these mutations were the failures in the activity of complex I, thereby leading to the deficient function of oxidative phosphorylation, decrease in adenosine triphosphate (ATP) synthesis and increase in the production of reactive oxygen species (ROS) (13–15). These three mtDNA mutations accounted for ∼90% of LHON pedigrees in some countries (7–11), while these mutations are only responsible for 38.3 and 46.5% cases in two large cohorts of Chinese Han subjects with LHON (16–19). To further understand the pathophysiology of LHON, we performed the clinical, genetic and molecular analysis of 1281 Han Chinese probands with LHON (17–22). This analysis identified the known ND4 m.11778G>A, ND6 m.14484T>C, ND1 m.3460G>A, m.3635G>A and m.3866T>C mutations as well as ND5 m.12238T>C mutation (17–22). In particular, the m.12338T>C mutation was identified in six genetically related Han Chinese pedigrees with LHON (22). However, the pathophysiology of the m.12338T>C mutation remains poorly understood. Thus, it is necessary to establish the link between LHON and mitochondrial dysfunction and their cause/effect relation. As shown in Figure 1, the m.12338T>C mutation yielded the replacement of the translation-initiating methionine with a threonine, thereby shortening two amino acids of ND5 polypeptide (22). We therefore hypothesized that the mutated ND5 caused by the m.12338T>C mutation altered the structure and function of complex I. The m.12338T>C mutation is also located at 2 nt adjacent to the 3′ end of the tRNALeu(CUN). Thus, this mutation may affect the processing of tRNALeu(CUN) precursors. Functional significance of the m.12338T>C mutation was investigated through cell lines constructed by transferring mitochondria from lymphoblastoid cell lines derived from an affected matrilineal relative carrying the m.12238T>C mutation and from a control subject belonging to the same mtDNA haplogroup, into human mtDNA-less (ρo) cells (23,24). First, these cell lines were assessed for the effects of the m.12338T>C mutation on the stability of tRNALeu(CUN). We then examined if the m.12238T>C mutation perturbed the stability of ND1 and assembly of complex I by using western blot and blue native polyacrylamide gel electrophoresis (BN-PAGE) analysis. These cell lines were further evaluated for effects on enzymatic activities of electron transport chain complexes, respiration, production of ATP, mitochondrial membrane potential and generation of ROS. Finally, we examined if the m.12338T>C mutation affected the apoptosis and mitophagy. Figure 1. View largeDownload slide A schema of mtDNA sequence at position 12338 and adjacent sequence of ND5 and tRNALeu(CUN) from wild-type (WT) and mutant (MT). Arrow indicates the position of the m.12338T>C mutation. Figure 1. View largeDownload slide A schema of mtDNA sequence at position 12338 and adjacent sequence of ND5 and tRNALeu(CUN) from wild-type (WT) and mutant (MT). Arrow indicates the position of the m.12338T>C mutation. Results The Chinese pedigree carrying the m.12338T>C mutation and derived cybrid cell lines The WZ411 pedigree of the Chinese family carrying the m.12338T>C mutation was described previously (22). Immortalized lymphoblastoid cell lines were derived from the proband (II-10, female, 32 years) and one genetically unrelated control individual A60 belonging to the same mtDNA haplogroup (female, 28 years) (Supplementary Material, Table S1). These lymphoblastoid cells lines were enucleated and fused to a large excess of mtDNA-less human ρo206 cell line and cybrid clones were isolated by growing in selective Dulbecco's modified Eagle's medium (DMEM) medium (23). The cybrids derived from each donor cell line were analyzed for the presence and degrees of the m.12338T>C mutation and mtDNA copy numbers. The results confirmed the absence of the m.12338T>C mutation in the control clones and their presence in homoplasmy in all cybrids derived from the mutant cell lines (data not shown). Three mutant cybrids (II-10-1, II-10-2 and II-10-3) carrying the homoplasmic m.12338T>C mutation and three control cybrids (A60-1, A60-2 and A60-3) lacking the mutation with similar mtDNA copy numbers and the same karyotype as 143B.TK− cell lines were used for the biochemical characterization. Analysis of mitochondrial tRNALeu(CUN) The m.12338T>C mutation was located at two nt adjacent to the 3′ end of the tRNALeu(CUN) (22). To examine whether the m.12338T>C mutation affects the processing of tRNALeu(CUN) precursors, we subjected mitochondrial RNAs from cybrids cell lines to northern blots and hybridized them with digoxigenin (DIG)-labeled oligodeoxynucleotide probes for tRNALeu(CUN), tRNASer(UCN) and tRNAGlu as well as nuclear encoded 5S rRNA (25–27). As shown in Figure 2, the levels of tRNALeu(CUN), tRNASer(UCN) and tRNAGlu in three mutant cybrid cell lines carrying the m.12338T>C mutation were comparable with those in three control cell lines lacking the mtDNA mutation. These data indicated that the m.12338T>C mutation did not affect the tRNALeu(CUN) metabolism. Figure 2. View largeDownload slide Northern blot analysis of mitochondrial tRNA. (A) Equal amounts (2 μg) of total mitochondrial tRNA samples from the various cell lines were electrophoresed through a denaturing polyacrylamide gel, were electroblotted and were hybridized with DIG-labeled oligonucleotide probes specific for the tRNALeu(CUN), tRNASer(UCN), tRNAGln and 5S RNA, respectively. (B) Quantification of mitochondrial tRNA levels. Shown is the average relative tRNA content per cell, normalized to the average content per cell of 5S rRNA in the cells derived from three mutant cybrids carrying the m.12338T>C mutation and three cybrids lacking the mutation. The values for the latter are expressed as percentages of the average values for the control cell lines. The calculations were based on three independent determinations. The error bars indicate two standard errors of the mean (SEM). P indicates the significance, according to the t-test, of the difference between mutant mean and control mean. Figure 2. View largeDownload slide Northern blot analysis of mitochondrial tRNA. (A) Equal amounts (2 μg) of total mitochondrial tRNA samples from the various cell lines were electrophoresed through a denaturing polyacrylamide gel, were electroblotted and were hybridized with DIG-labeled oligonucleotide probes specific for the tRNALeu(CUN), tRNASer(UCN), tRNAGln and 5S RNA, respectively. (B) Quantification of mitochondrial tRNA levels. Shown is the average relative tRNA content per cell, normalized to the average content per cell of 5S rRNA in the cells derived from three mutant cybrids carrying the m.12338T>C mutation and three cybrids lacking the mutation. The values for the latter are expressed as percentages of the average values for the control cell lines. The calculations were based on three independent determinations. The error bars indicate two standard errors of the mean (SEM). P indicates the significance, according to the t-test, of the difference between mutant mean and control mean. The m.12338T>C mutation caused the reduction of ND5 protein To experimentally test the predicted effect of m.12338T>C mutation for ND5, we analyzed the levels of ND5 and other four mtDNA-encoding polypeptides by a western blotting in these mutant and control cell lines. Twenty micrograms of total cellular proteins from various cell lines were separated by PAGE, electroblotted. The blots were then hybridized with ND5 and other four mtDNA encoded polypeptides (ND1, CYTB, apocytochrome b; CO2, subunit 2 of cytochrome c oxidase and ATP6, subunit 6 of the H+-ATPase) in mutant and control cell lines as well as HSP60 (a nuclear-encoding mitochondrial protein) as a loading control. As shown in Figure 3, the levels of ND5 in three mutant cell ranged from ∼53 to 69%, with an average of 63% (P = 0.0021), relative to the mean value measured in three control cell lines. However, the levels of ND1, CYTB, CO2 and ATP6 in the mutant cell lines were comparable with those in control cell lines (Supplementary Material, Fig. S1). Figure 3. View largeDownload slide Western blot analysis of mitochondrial proteins. (A) Twenty micrograms of total cellular proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with ND5 and other three respiratory complex subunits in mutant and control cells with HSP60 as a loading control. ND1 and ND5, subunits 1 and 5 of the reduced nicotinamide–adenine dinucleotide dehydrogenase; ATP6, subunit 6 of the H+-ATPase and CYTB, apocytochrome b. (B) Quantification of mitochondrial protein levels. The levels of ND1 in three mutant cell lines and three control cell lines were determined as described elsewhere (36). The values for the mutant cell lines are expressed as percentages of the average values for the control cell lines. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to Figure 2. Figure 3. View largeDownload slide Western blot analysis of mitochondrial proteins. (A) Twenty micrograms of total cellular proteins from various cell lines were electrophoresed through a denaturing polyacrylamide gel, electroblotted and hybridized with ND5 and other three respiratory complex subunits in mutant and control cells with HSP60 as a loading control. ND1 and ND5, subunits 1 and 5 of the reduced nicotinamide–adenine dinucleotide dehydrogenase; ATP6, subunit 6 of the H+-ATPase and CYTB, apocytochrome b. (B) Quantification of mitochondrial protein levels. The levels of ND1 in three mutant cell lines and three control cell lines were determined as described elsewhere (36). The values for the mutant cell lines are expressed as percentages of the average values for the control cell lines. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to Figure 2. Perturbed the assembly of complex I To examine if the mutated ND5 affected the assembly of complex I, mitochondrial membrane proteins isolated from mutant and control cell lines were separated by BN-PAGE and electroblotting and hybridizing with NDUFA9 antibody (complex I subunit encoded by nuclear gene) and ATP5A as loading control (12,28,29). As shown in Figure 4, the altered assembly of intact complex I were observed in mutant cell lines carrying the m.12338T>C mutation. In particular, the levels of complex I in mutant cell lines carrying m.12338T>C mutation was 72 and 78%, with an average of 75% (P = 0.0013), relative to the average values in control cell lines. These data suggested that these cell lines carrying m.12338T>C mutation exhibited more unstable complex I than those in control cell lines. Figure 4. View largeDownload slide Analysis of complex I assembly. (A) Respiratory complex assembly and in-gel activity assay. Whole cells from mutant and controls were solubilized with digitonin and then subjected to BN-PAGE/immunoblot analysis. Blots were the hybridized with NDUFA9 antibody and ATP5A as internal control. (B) Quantification of complex I. The levels of complex I in mutant and control cell lines were determined as based on three independent determinations in each cell line. Graph details and symbols were explained in the legend of Figure 2. Figure 4. View largeDownload slide Analysis of complex I assembly. (A) Respiratory complex assembly and in-gel activity assay. Whole cells from mutant and controls were solubilized with digitonin and then subjected to BN-PAGE/immunoblot analysis. Blots were the hybridized with NDUFA9 antibody and ATP5A as internal control. (B) Quantification of complex I. The levels of complex I in mutant and control cell lines were determined as based on three independent determinations in each cell line. Graph details and symbols were explained in the legend of Figure 2. Reduced activity of complex I To evaluate the effect of the m.12338T>C mutation on the oxidative phosphorylation, we measured the activities of respiratory complexes by isolating mitochondria from three mutant cell lines and three control cell lines. Complex I activity was determined by following the oxidation of NADH with ubiquinone as the electron acceptor (30,31). The activity of complex II (succinate ubiquinone oxidoreductase) exclusively encoded by the nuclear DNA was examined by the artificial electron acceptor 2,6-Dichlorophenolindophenol (32). Complex III (ubiquinone cytochrome c oxidoreductase) activity was measured as the reduction of cytochrome c (III) using D-ubiquinol-2 as the electron donor. Complex IV (cytochrome c oxidase) activity was monitored by following the oxidation of cytochrome c (II). As shown in Figure 5, the activity of complex I in three mutant cell lines was 61.5%, relative to the mean value measured in the control cell lines (P = 0.0002). However, the activities of complexes II–IV in three mutant cell lines were comparable with those of three control cell lines. Figure 5. View largeDownload slide Enzymatic activities of respiratory chain complexes. The activities of respiratory complexes were measured by enzymatic assay on complexes I–IV in mitochondria isolated from mutant and control cell lines. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to Figure 2. Figure 5. View largeDownload slide Enzymatic activities of respiratory chain complexes. The activities of respiratory complexes were measured by enzymatic assay on complexes I–IV in mitochondria isolated from mutant and control cell lines. The calculations were based on three independent determinations. Graph details and symbols are explained in the legend to Figure 2. Respiration deficiency To assess if m.12338T>C mutation perturbed cellular bioenergetics, the oxygen consumption rates (OCRs) of mutant and control cell lines were measured with a Seahorse Bioscience XF-96 extracellular flux analyzer (Seahorse Bioscience) (33–35). As shown in Figure 6, the basal OCR in the mutant cell lines carrying m.12338T>C mutation was 60% (P = 0.0009), relative to the mean value measured in the control cell lines. To investigate which of the enzyme complexes of the respiratory chain was perturbed in the mutant cell lines, OCR were examined after the sequential additions of oligomycin (to inhibit the ATP synthase), Carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) (to allow for maximum electron flux through the electron transport chains), rotenone (to inhibit complex I) and antimycin (to inhibit complex III). The difference between the basal OCR and the drug-insensitive OCR produced 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 bearing the m.12338T>C mutation were 68.3, 41.6, 69.2, 81.4 and 84.4% (P = 0.0008, 0.0193, 0.0096, 0.1882 and 0.0951), relative to the mean value measured in the control cell lines, respectively. Figure 6. View largeDownload slide 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 p-(trifluoromethoxy) phenylhydrazone (FCCP) (0.5 μM), rotenone (1.0 μM) and antimycin A (1.0 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 independent experiments for each cell line were shown, the horizontal dashed lines represent the average value for each group. Graph details and symbols are explained in the legend to Figure 2. Figure 6. View largeDownload slide 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 p-(trifluoromethoxy) phenylhydrazone (FCCP) (0.5 μM), rotenone (1.0 μM) and antimycin A (1.0 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 independent experiments for each cell line were shown, the horizontal dashed lines represent the average value for each group. Graph details and symbols are explained in the legend to Figure 2. Decrease in mitochondrial ATP generation The effect of m.12338T>C mutation on capacity of oxidative phosphorylation was further evaluated by measuring the levels of cellular and mitochondrial ATP production 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) (34–36). As shown in Figure 7A, the levels of total cellular ATP production in mutant cell lines were comparable to those in control cell lines. Conversely, the levels of mitochondrial ATP production in mutant cell lines ranged from 36 to 44.6%, with an average of 40.1%, relative to the mean value measured in the control cell lines (P < 0.0001) (Fig. 7B). Figure 7. View largeDownload slide Measurement of cellular and mitochondrial ATP levels. ATP levels from mutant and control cell lines were measure using a luciferin/luciferase assay. Cells 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 and are shown: (A) ATP level in total cells. (B) ATP level in mitochondria. Four independent experiments were made for each cell line. Graph details and symbols are explained in the legend to Figure 2. Figure 7. View largeDownload slide Measurement of cellular and mitochondrial ATP levels. ATP levels from mutant and control cell lines were measure using a luciferin/luciferase assay. Cells 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 and are shown: (A) ATP level in total cells. (B) ATP level in mitochondria. Four independent experiments were made for each cell line. Graph details and symbols are explained in the legend to Figure 2. Alterations in mitochondrial membrane potential To examine if the m.12338T>C mutation affected the mitochondrial membrane potential (ΔΨm), a fluorescence probe JC-10 assay system was used to examine the ΔΨm in mutant and control cell lines. The ratio of fluorescence intensities Ex/Em = 490/590 and 490/525 nm (FL590/FL525) were recorded to delineate the ΔΨm level of each sample. The relative ratios of FL590/FL525 geometric mean between mutants and controls were calculated to represent the level of ΔΨm. As shown in Figure 8, levels of the ΔΨm in the mutants carrying the m.12338T>C mutation were decreased, ranging from 72.4 to 80.2%, with an average 77.5% (P = 0.0285) of the mean value measured in the controls. In contrast, the levels of ΔΨm in mutant cell lines in the presence of FCCP were comparable with those measured in these control cell lines. Figure 8. View largeDownload slide Mitochondrial membrane potential analysis. The mitochondrial membrane potential (ΔΨm) was measured in mutant and control 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 ΔΨm 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 ΔΨm. Relative ratio of JC-10 fluorescence intensities at Ex/Em = 490/530 and 490/590 nm in absence (A) and presence (B) of 10 μM of carbonyl cyanide 3-chlorophenylhydrazone (FCCP). Three to five determinations were made for each cell line are shown. Graph details and symbols are explained in the legend to Figure 2. Figure 8. View largeDownload slide Mitochondrial membrane potential analysis. The mitochondrial membrane potential (ΔΨm) was measured in mutant and control 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 ΔΨm 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 ΔΨm. Relative ratio of JC-10 fluorescence intensities at Ex/Em = 490/530 and 490/590 nm in absence (A) and presence (B) of 10 μM of carbonyl cyanide 3-chlorophenylhydrazone (FCCP). Three to five determinations were made for each cell line are shown. Graph details and symbols are explained in the legend to Figure 2. Increase of mitochondrial ROS production The levels of mitochondrial ROS (mitoROS) among the mutant and control cell lines were measured with flow cytometry using a MitoSOX assay (24,37). Geometric mean intensity was recorded to measure the rate of mitoROS of each sample. The relative levels of geometric mean intensity in each cell line were calculated to delineate the levels of mitoROS in mutant and control cells. As shown in Figure 9, the levels of mitoROS generation in the mutant cell lines carrying the m.12338T>C mutation ranged from 137.5 to 162.1%, with an average of 150.1% (P = 0.0027) of mean value measured in the control cell lines. Figure 9. View largeDownload slide Measurement of mitoROS. The levels of ROS generation by mitochondria in living cells from mutant and control cell lines were determined using the mitochondrial superoxide indicator MitoSOX-Red. Fluorescence was measured using a instrument (BD Biosciences), with excitation at 488 nm and emission at 580 nm. The data were analyzed with FlowJo software. (A) Flow cytometry histogram showing MitoSOX-Red fluorescence of A60-1 (Black) and II-10-1 91 (Red). (B) Relative ratios of MitoSOX-Red fluorescence intensity. The average of four determinations for each cell line is shown. Graph details and symbols are explained in the legend to Figure 2. Figure 9. View largeDownload slide Measurement of mitoROS. The levels of ROS generation by mitochondria in living cells from mutant and control cell lines were determined using the mitochondrial superoxide indicator MitoSOX-Red. Fluorescence was measured using a instrument (BD Biosciences), with excitation at 488 nm and emission at 580 nm. The data were analyzed with FlowJo software. (A) Flow cytometry histogram showing MitoSOX-Red fluorescence of A60-1 (Black) and II-10-1 91 (Red). (B) Relative ratios of MitoSOX-Red fluorescence intensity. The average of four determinations for each cell line is shown. Graph details and symbols are explained in the legend to Figure 2. Promoting apoptosis To evaluate if the m.12338T>C mutation affects the apoptotic processes, we examined the apoptotic state of mutant and control cybrids by immunofluorescence and western blot analyses. As shown in Figure 10A, the immunofluorescence patterns of double-labeled cells with rabbit monoclonal antibody specific for the cytochrome C and mouse monoclonal antibody to TOM20 revealed markedly increased levels of cytochrome c in the mutant cells, compared with control cells. The levels of cytochrome c in cytosol in mutant and control cell lines were further evaluated by western-blotting analysis. As shown in Figure 10B, the levels of cytochrome c in three mutant cell lines ranged from 224 to 243%, with an average of 235.4% (P = 0.0005), relative to the average values in three control cell lines. Furthermore, we examined the levels of four apoptosis-activated proteins [caspases 9, 3, 7 and Poly ADP ribose polymerase (PARP)] in mutant and control cell lines by western blot analysis (38). As showed in Figure 10C, the marked increasing levels of these proteins were observed in the mutant cell lines. In particular, the levels of caspases 9, 3, 7 and PARP in the mutant cell lines were 137.1, 158.2, 158.9 and 162.2% of the average values measured in the control cell lines, respectively (P = 0.0008, 0.0053, 0.0064 and 0.0026). Figure 10. View largeDownload slide Analysis of apoptosis. (A) The distributions of cytochrome c from cybrids (mutant II-10-1 and wild-A60-1) were visualized by immunofluorescent labeling with TOM20 antibody conjugated to Alex Fluor 488 (green) and cytochrome c antibody conjugated to Alex Fluor 594 (red) analyzed by confocal microscopy. DAPI-stained nuclei were identified by their blue fluorescence. (B) Measurement of cytochrome C levels in cytosol. Twenty micrograms of total cellular proteins from various cell lines were electrophoresed, electroblotted and hybridized with cytochrome c and with β-actin as a loading control. The levels of cytochrome c in mutant and control cell lines were determined as described elsewhere (36). (C) Western blot analysis of four apoptosis-activated proteins. Twenty micrograms of total proteins from various cell lines were electrophoresed, electroblotted and hybridized with caspases 9, 3, 7 and PARP antibodies and with β-actin as a loading control. (D) Quantification of four apoptosis-activated proteins. The levels of caspases 9, 3, 7 and PARP in various cell lines were determined as described elsewhere (36). Three independent determinations were done in each cell line. Graph details and symbols are explained in the legend to Figure 2. Figure 10. View largeDownload slide Analysis of apoptosis. (A) The distributions of cytochrome c from cybrids (mutant II-10-1 and wild-A60-1) were visualized by immunofluorescent labeling with TOM20 antibody conjugated to Alex Fluor 488 (green) and cytochrome c antibody conjugated to Alex Fluor 594 (red) analyzed by confocal microscopy. DAPI-stained nuclei were identified by their blue fluorescence. (B) Measurement of cytochrome C levels in cytosol. Twenty micrograms of total cellular proteins from various cell lines were electrophoresed, electroblotted and hybridized with cytochrome c and with β-actin as a loading control. The levels of cytochrome c in mutant and control cell lines were determined as described elsewhere (36). (C) Western blot analysis of four apoptosis-activated proteins. Twenty micrograms of total proteins from various cell lines were electrophoresed, electroblotted and hybridized with caspases 9, 3, 7 and PARP antibodies and with β-actin as a loading control. (D) Quantification of four apoptosis-activated proteins. The levels of caspases 9, 3, 7 and PARP in various cell lines were determined as described elsewhere (36). Three independent determinations were done in each cell line. Graph details and symbols are explained in the legend to Figure 2. Alteration in mitophagy Mitophagy is the selective removal of damaged mitochondria by autophagosomes and their subsequent catabolism by lysosomes (39). To investigate if the m.12338T>C mutation affected the mitophagy, we evaluated the mitophagic states of mutant and control cell lines using western-blotting and endogenous immunofluorescence assays. As shown in Figure 11A, the immunofluorescence patterns of cells lines double-labeled with the antibodies specific for lysosome-associated membrane glycoprotein 1 (LAMP1), and TOM20 indicated that the m.12338T>C mutation affected the autophagy process. The levels of mitophagy in mutant and control cell lines were then examined using two markers: microtubule-associated protein 1A/1B light chain 3B (LC3) and sequestosome 1 (SQSTM1/p62) (40). During autophagy, the cytoplasmic form (LC3-I) is processed into a cleaved and lipidated membrane-bound form (LC3-II), which is essential for membrane biogenesis and closure of the membrane. LC3-II is recleaved by cysteine protease (Atg4B) following completion of the autophagosome and recycled (41). SQSTM1/p62, one of the best-known autophagic substrates, interacts with LC3 to ensure the selective delivery of these proteins into the autophagosome (40). As shown in Figure 11B, the reduced levels of LC3 and increased level of p62 were observed in mutant cybrids carrying the m.12338T>C mutation, compared with those in control cybrids. In particular, the average levels of LC3-II/(LC3-I+II) and p62 in three mutant cell lines carrying the m.12338T>C mutation were 67.3% (P = 0.0308) and 140.2% (P = 0.0195) of the mean values measured in three control cell lines lacking the mutation, respectively. These data suggested that the m.12338T>C mutation led to the decreased mitophagy in cybrids. Figure 11. View largeDownload slide Analysis of mitophagy. (A) The distributions of LAMP1 from cybrids (II-10-1 and A60-1) were visualized by immunofluorescent labeling with TOM20 antibody conjugated to Alex Fluor 488 (green) and LAMP1 antibody conjugated to Alex Fluor 594 (red) analyzed by confocal microscopy. DAPI-stained nuclei were shown by the blue fluorescence. (B) Western blot analysis for mitophagic response proteins LC3-I/II and P62. Twenty micrograms of total cellular proteins from various cell lines were electrophoresed, electroblotted and hybridized with LC3, p62 and with β-actin as a loading control. (C) Quantification of autophagy markers LC3A/B and p62 in mutant and control cell lines were determined as described elsewhere (36). Figure 11. View largeDownload slide Analysis of mitophagy. (A) The distributions of LAMP1 from cybrids (II-10-1 and A60-1) were visualized by immunofluorescent labeling with TOM20 antibody conjugated to Alex Fluor 488 (green) and LAMP1 antibody conjugated to Alex Fluor 594 (red) analyzed by confocal microscopy. DAPI-stained nuclei were shown by the blue fluorescence. (B) Western blot analysis for mitophagic response proteins LC3-I/II and P62. Twenty micrograms of total cellular proteins from various cell lines were electrophoresed, electroblotted and hybridized with LC3, p62 and with β-actin as a loading control. (C) Quantification of autophagy markers LC3A/B and p62 in mutant and control cell lines were determined as described elsewhere (36). Discussion In this study, we investigated the molecular pathogenesis of LHON-associated ND5 12338T>C mutation. In particular, the m.12338T>C mutation has several deleterious effects that potentially contributed to the pathogenesis of LHON. These included the mitochondrial dysfunction, promoting apoptosis and decreased mitophagy due to the mtDNA mutation. Indeed, the occurrence of m.12338T>C mutation in several genetically unrelated families affected by optic neuropathy strongly indicated that this mutation is involved in the pathogenesis of LHON (21). The m.12338T>C mutation caused the replacement of the first methionine (Met1) with a threonine and the methionine at position 3 (Met3) acting as the initiation methionine and consequently shortened two amino acids of ND5 polypeptide (21,42). Therefore, it was anticipated that the m.12338T>C mutation results in the instability of mutant ND5 or perturbs the translation of ND5 mRNA by the replacement of the first methionine (Met1) with a threonine. These alterations led to 37% reduction in the levels of ND5 observed in mutant cybrids carrying the m.12338T>C mutation. In fact, ND5 is the essential subunit to be located at the periphery of NADH: ubiquinone oxidoreductase, which comprised ND5 and additional six subunits encoded by mtDNA and 39 subunits encoded by nuclear genes (43–45). We therefore hypothesized that the mutated ND5 altered the structure and function of complex I. In this investigation, we demonstrated that the mutant cybrids carrying the m.12338T>C mutation exhibited more unstable complex I than those in control cell lines lacking the mutation. These data were consistent with the previous observations that mutations in mtDNA-encoding subunits of complex I including ND1, ND4 and ND6 perturbed the assembly of NADH: ubiquinone oxidoreductase (46–49). Both instability of ND5 and altered assembly of complex I were responsible for a decline of complex I activity observed in cybrids harboring the m.12338T>C mutation. However, cybrids bearing the m.11338T>C mutation exhibited less reductions in the complex I activity than those in cybrids carrying the m.11778G>A mutation (13,35). Furthermore, less reductions in the basal OCR, or ATP-linked OCR, proton leak OCR and maximal OCR were observed in cybrids carrying the m.12338T>C mutation than those in cybrids carrying the m.11778G>A mutation (35). All these data strongly supported the functional significance of m.12338T>C mutation leading to mitochondrial dysfunction. The respiratory deficiency caused by the m.12338T>C mutation may lead to the alterations on ATP synthesis, oxidative stress and subsequent failure of cellular energetic process (50,51). In this investigation, we demonstrated significant decreases in mitochondrial ATP production in these cybrids carrying m.12338T>C mutation, as in the case of cell lines bearing the m.11778G>A mutation (15,34,35,52). Furthermore, the deficient activities of respiratory chain complexes caused by mtDNA mutations often perturbed mitochondrial membrane potentials, which is a key indicator of cellular viability (24,36,53). Significant reductions in mitochondrial membrane potential in mutant cybrids bearing the m.12338T>C mutation revealed the impaired pumping ability of hydrogen ions across the inner membrane and more electron leakage during the process of electron transport and oxidative phosphorylation (24,53). Alterations in both oxidative phosphorylation and mitochondrial membrane potential resulted in the ROS overproduction and the subsequent bioenergetic failure in mutant cybrids carrying the m.12338T>C mutation. In turn, the increased production of ROS may produce the damage of proteins, lipids and nuclear acids and subsequently alter the apoptosis and mitophage processes (54–56). Mitochondrial dysfunction affected the apoptotic sensitivity of cells carrying the LHON-associated mtDNA mutations (57–59). The cybrids carrying one of three primary mtDNA mutations such as m.11778G>A underwent apoptotic cell death, when exposed to the media containing the galactose, which renders the cells more dependent on mitochondrial ATP production (58). After FAS-stimulation, the enhanced apoptosis were observed in the mutant cybrids bearing the m.3460G>A or m.11778G>A mutation, as compared with control cybrids (60). In the present investigation, mutant cybrids bearing the m.12338T>C mutation exhibited more apoptotic susceptibility than control cybrids lacking the mutation. These were evidenced by the elevated release of cytochrome c into cytosol and increased levels of apoptosis-activated proteins: caspases 9, 3, 7 and PARP in the cybrids carrying the m.12338T>C mutation, as compared with control cybrids. These data demonstrated that mitochondrial dysfunction caused by several LHON-associated mtDNA mutations promoted the apoptosis (60–62). Mitophagy regulates mitochondrial energy metabolism by controlling the amount and efficiency of the mitochondrial metabolic machinery (63). In particular, the impairment of OXPHOS and alteration of mitochondrial membrane potential affected the mitophagic removal of damaged mitochondria (64–66). The activation of mitophagic machinery is crucial to the complete degradation of mitochondrial components. The p62 can both aggregate ubiquitinated proteins by polymerizing with other p62 molecules and recruit ubiquitinated cargo into autophagosomes by binding to LC3 (67). The p62 mediates clumping of mitochondria and links ubiquitinated substrates to LC3 to facilitate the autophagic degradation of ubiquitinated proteins. Here, the increased levels of p62 in cybrids carrying the m.12338T>C mutation indicated the accumulation of autophagic substrates such as misfolded proteins, leading to the deleterious effects. Moreover, reduced levels of LC3 were observed in mutant cybrids carrying the m.12338T>C mutation. The reductions in LC3 level in mutant cybrids suggested a general decrease in the capacity of the mutant cells to generate autophagoeomes, thereby perturbing the autophagic degradation of ubiquitinated proteins. These results provided the evidences that the m.12338T>C mutation may mediate mitophagy in cybrids. Therefore, our findings established the association between mitochondrial dysfunction caused by complex I mutations and apoptosis or mitophagy. In summary, our results suggested the pathogenic mechanism leading to an impaired oxidative phosphorylation in cybrid cell lines carrying the m.12338T>C mutation. The m.12338T>C (p.1M>T) mutation in the ND5 gene perturbed the assembly and activity of NADH: ubiquinone oxidoreductase. As a result, this respiratory deficiency resulted in the decrease of mitochondrial ATP production, mitochondrial membrane potential and the increasing production of oxidative reactive species. All those alterations consequently elevated the apoptotic cell death and decreased the mitophagy in cells carrying the m.12338T>C mutation, thereby leading to visual loss. Therefore, our findings may provide new insights into pathophysiology of LHON. Materials and Methods Cell lines and culture condition Lymphoblastoid cell lines derived from one affected matrilineal relative WZ411(II-10) carrying the m.12338T>C mutation (22) and from one genetically unrelated control individuals belonging to the same mtDNA haplogroup (A60) were immortalized by transformation with the Epstein–Barr virus, as described previously (68). Lymphoblastoid cell lines were grown in Roswell Park Memorial Institute medium 1640 medium (Invitrogen), supplemented with 10% fetal bovine serum (FBS). 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 ρo206 cell line, derived from 143B.TK− (23,69) 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 ρo206 cells was performed by using two immortalized lymphoblastoid cell lines, as detailed elsewhere (23,69). All cybrid cell lines constructed with enucleated lymphoblastoid cell lines were maintained in the same medium as the 143B.TK− cell line. An analysis for the presence and level of the m.12338T>C mutation was carried out as described previously (22). The quantifications of mtDNA copy number from different cybrids were performed as detailed elsewhere (37). Three mutant cybrids carrying the m.12338T>C mutation and three control cybrids lacking the mutation with similar mtDNA copy numbers and the same karyotype were used for the biochemical characterization described later. Mitochondrial tRNA northern analysis Total mitochondrial RNA were obtained using TOTALLY RNATM kit (Ambion) from mitochondria isolated from the cybrid cell lines (∼2.0 × 108 cells), as described previously (70). Oligodeoxynucleosides used for DIG-labeled probes of tRNALeu(CUN), tRNASer(UCN), tRNAGlu and 5S RNA were described as elsewhere (34,36,52). DIG-labeled oligodeoxynucleotides were generated by using DIG oligonucleotide Tailing kit (Roche). Two micrograms of total mitochondrial RNA were electrophoresed through a 10% polyacrylamide/7M urea gel in Tris-borate-ethylenediaminetetraacetic acid buffer (after heating the sample at 65°C for 10 min) and then electroblotted onto a positively charged nylon membrane (Roche) for the hybridization. Quantification of density in each band was made as detailed previously (34,36). Western blot analysis Western-blotting analysis was performed as detailed previously (34–36). Twenty micrograms of total cellular proteins obtained from lysed mutant and control cell lines were electrophoresed through sodium dodecyl sulfate polyacrylamide gels, electroblotted onto a polyvinylidene difluoride membrane and hybridized with the following antibodies, respectively. Antibodies were obtained from different companies including LC3A/B (#4108), LAMP1 (#9091), caspase-3 (#9664), caspase-7 (#8438), caspase-9 (#7237) and PARP (#5625) from Cell Signaling Technology, CYTB (55090-1-AP) from Proteintech, CO2 (ab110258), ND5 (ab92624), ND1 (ab74257), ATP6 (ab101908), Cytochrome c (ab13575), HSP60 (ab46798), β-actin (ab8226), p62 (ab56416), TOM20 (ab56783) and GAPDH (ab8245) from Abcam. Peroxidase Affini Pure goat anti-mouse IgG and goat anti-rabbit IgG (Jackson) were used as a secondary antibody and protein signals were detected using the electrochemiluminescence system (CWBIO). Quantification of density in each band was performed as detailed previously (34–36). BN gel electrophoresis BN gel electrophoresis were carried out using mitochondrial proteins isolated from mutant and control cell lines, as detailed elsewhere (28,35,71). The antibodies used for this investigation were NDUFA9 (ab14713) from Abcam and ATP5A (14676-1-AP) from Proteintech. Peroxidase Affini Pure goat anti-mouse IgG and goat anti-rabbit IgG (Cell Signaling) were used as a secondary antibody. Protein signals were detected using Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA) or 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT) substrate (Promega, Madison, WI, USA). Quantification of density in each band was performed as detailed previously (34,35). Enzymatic assays The enzymatic activities of complexes I–IV were assayed as detailed elsewhere (28,31,32). 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 (33,36). 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 (24,36). ROS measurements ROS measurements were performed following the procedures detailed previously (24,35,72). Immunofluorescence analysis Immunofluorescence experiments were performed as described elsewhere (73). Cells were cultured on cover glass slips (Thermo Fisher), fixed in 4% formaldehyde for 15 min, permeabilized with 0.2% Triton X-100, blocked with 5% FBS for 1 h and immunostained with TOM20, cytochrome C and LAMP1 antibodies overnight at 4°C, respectively. The cells were then incubated with Alex Fluor 594 goat anti-rabbit IgG (H+L) and Alex Fluor 488 goat anti-mouse IgG (H+L) (Thermo Fisher), stained with 4′, 6-diamidino-2-phenylindole (DAPI; Invitrogen) for 15 min and mounted with Fluoromount (Sigma-Aldrich). Cells were examined using a confocal fluorescence microscope (Olympus Fluoview FV1000, Japan) with three lasers (Ex/Em = 550/570, 492/520 and 358/461 nm). 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Human Molecular GeneticsOxford University Press

Published: Mar 22, 2018

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