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Cited by 102Autophagy in Disease and TherapyMitochondrial Function and PathologyHereditary Neurological Disorders

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Dysregulated mitophagy and mitochondrial organization in optic atrophy due to OPA1 mutations

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Liao, Chunyan, Ashley, Neil, Diot, Alan, Morten, Karl, Phadwal, Kanchan, Williams, Andrew, Fearnley, Ian, Rosser, Lyndon, Lowndes, Jo, Fratter, Carl, Ferguson, David J.P., Vay, Laura, Quaghebeur, Gerardine, Moroni, Isabella, Bianchi, Stefania, Lamperti, Costanza, Downes, Susan M., Sitarz, Kamil S., Flannery, Padraig J., Carver, Janet, Dombi, Eszter, East, Daniel, Laura, Matilde, Reilly, Mary M., Mortiboys, Heather, Prevo, Remko, Campanella, Michelangelo, Daniels, Matthew J., Zeviani, Massimo, Yu-Wai-Man, Patrick, Simon, Anna Katharina, Votruba, Marcela, Poulton, Joanna·2017·Neurology
DOI 10.1212/wnl.0000000000003491PMC PMC5224718
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Abstract

OBJECTIVE: To investigate mitophagy in 5 patients with severe dominantly inherited optic atrophy (DOA), caused by depletion of OPA1 (a protein that is essential for mitochondrial fusion), compared with healthy controls. METHODS: Patients with severe DOA (DOA plus) had peripheral neuropathy, cognitive regression, and epilepsy in addition to loss of vision. We quantified mitophagy in dermal fibroblasts, using 2 high throughput imaging systems, by visualizing colocalization of mitochondrial fragments with engulfing autophagosomes. RESULTS: Fibroblasts from 3 biallelic OPA1(-/-) patients with severe DOA had increased mitochondrial fragmentation and mitochondrial DNA (mtDNA)-depleted cells due to decreased levels of OPA1 protein. Similarly, in siRNA-treated control fibroblasts, profound OPA1 knockdown caused mitochondrial fragmentation, loss of mtDNA, impaired mitochondrial function, and mitochondrial mislocalization. Compared to controls, basal mitophagy (abundance of autophagosomes colocalizing with mitochondria) was increased in (1) biallelic patients, (2) monoallelic patients with DOA plus, and (3) OPA1 siRNA-treated control cultures. Mitophagic flux was also increased. Genetic knockdown of the mitophagy protein ATG7 confirmed this by eliminating differences between patient and control fibroblasts. CONCLUSIONS: We demonstrated increased mitophagy and excessive mitochondrial fragmentation in primary human cultures associated with DOA plus due to biallelic OPA1 mutations. We previously found that increased mitophagy (mitochondrial recycling) was associated with visual loss in another mitochondrial optic neuropathy, Leber hereditary optic neuropathy (LHON). Combined with our LHON findings, this implicates excessive mitochondrial fragmentation, dysregulated mitophagy, and impaired response to energetic stress in the pathogenesis of mitochondrial optic neuropathies, potentially linked with mitochondrial mislocalization and mtDNA depletion.

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Figure 1 Genetic analysis of a family with a very severe dominantly inherited optic atrophy (DOA) plus phenotype (A) Pedigrees of families 1 and 2. (B) OPA1 gene structure. Diagrammatic representation of the OPA1 gene. The diagram indicates the location both of mutations resulting in DOA plus syndromes as described8 (small symbols) and of the mutations reported in this study (large symbols; highlighting corresponds to pedigree). Mutation type: stars (missense); squares (nonsense); circles (splice site); triangles (deletion). CC = coiled-coil domain; GE = GTPase effector domain; UTR = untranslated region. (C) PicoGreen/tetramethyl rhodamine methyl ester (TMRM) costaining of live fibroblasts from biallelic DOA plus OPA1(−/−)1–3 patients, and their symptom-free mothers (N1 and OPA1[+/−]2; see A). PicoGreen stains DNA and TMRM is sensitive to mitochondrial membrane potential. Nuclei of cells exhibiting mitochondrial fragmentation are marked with an asterisk. PicoGreen panel shows the same field as the high-magnification TMRM panel. TMRM staining of cells from biallelic DOA plus patients with abnormal mitochondrial fragmentation were often also depleted of mtDNA (E), but this was more marked in the siRNA-treated cell cultures in figure 2. (D) We used IN Cell 1000 to measure the mean mitochondrial length in fibroblast cultures, stained either with TMRM and PicoGreen25,26 or with antibody to mitochondrial protein Tom20. Cultures were grown for 3 days in 96-well plates in triplicate. To quantify the degree of mitochondrial fragmentation, we measured the average mitochondrial length in each cell and plotted a frequency distribution. This shows that while the modal length was similar in both groups, the per cell average mitochondrial length was shorter in biallelic patients (D.a) than controls (D.b) (see also figure e-3). (E) Cells depleted of mtDNA are increased (E.a) and have a lower membrane potential by TMRM staining (E.b). Error bars are 1 standard error. Asterisks indicate p < 0.001 compared to controls (2-tailed t test). Each bar represents between 400 and 1,500 cells. mtDNA = mitochondrial DNA.
Figure 1 Genetic analysis of a family with a very severe dominantly inherited optic atrophy (DOA) plus phenotype (A) Pedigrees of families 1 and 2. (B) OPA1 gene structure. Diagrammatic representation of the OPA1 gene. The diagram indicates the location both of mutations resulting in DOA plus syndromes as described8 (small symbols) and of the mutations reported in this study (large symbols; highlighting corresponds to pedigree). Mutation type: stars (missense); squares (nonsense); circles (splice site); triangles (deletion). CC = coiled-coil domain; GE = GTPase effector domain; UTR = untranslated region. (C) PicoGreen/tetramethyl rhodamine methyl ester (TMRM) costaining of live fibroblasts from biallelic DOA plus OPA1(−/−)1–3 patients, and their symptom-free mothers (N1 and OPA1[+/−]2; see A). PicoGreen stains DNA and TMRM is sensitive to mitochondrial membrane potential. Nuclei of cells exhibiting mitochondrial fragmentation are marked with an asterisk. PicoGreen panel shows the same field as the high-magnification TMRM panel. TMRM staining of cells from biallelic DOA plus patients with abnormal mitochondrial fragmentation were often also depleted of mtDNA (E), but this was more marked in the siRNA-treated cell cultures in figure 2. (D) We used IN Cell 1000 to measure the mean mitochondrial length in fibroblast cultures, stained either with TMRM and PicoGreen25,26 or with antibody to mitochondrial protein Tom20. Cultures were grown for 3 days in 96-well plates in triplicate. To quantify the degree of mitochondrial fragmentation, we measured the average mitochondrial length in each cell and plotted a frequency distribution. This shows that while the modal length was similar in both groups, the per cell average mitochondrial length was shorter in biallelic patients (D.a) than controls (D.b) (see also figure e-3). (E) Cells depleted of mtDNA are increased (E.a) and have a lower membrane potential by TMRM staining (E.b). Error bars are 1 standard error. Asterisks indicate p < 0.001 compared to controls (2-tailed t test). Each bar represents between 400 and 1,500 cells. mtDNA = mitochondrial DNA.CC BY · PubMed Central
Figure 2 A reduction in OPA1 leads to rapid loss of mitochondrial DNA (mtDNA) Control fibroblasts were treated with OPA1 Pan OPA1 or scramble siRNA. (A) Western blot analysis confirms OPA1 levels were efficiently reduced following siRNA treatment (blot shows 3 days after knockdown). (B) PicoGreen/tetramethyl rhodamine methyl ester (TMRM) costaining of live fibroblasts treated with OPA1 siRNA shows a marked loss of mtDNA nucleoids (visible as green puncta) compared to treatment with scramble. In all OPA1 knockdown cells with mitochondrial fragmentation, a noticeable mitochondrial perinuclear clustering was observed (arrows). (C) Anti-DNA/MitoTracker colabeling of OPA1/scramble siRNA treated fibroblasts (note that this antibody is recognized to label mtDNA more strongly than nuclear DNA). This confirms the findings presented in B. (D) Cytochrome oxidase (COX) activity, demonstrated by routine histochemistry, is significantly reduced by day 14. In the panel showing COX at 14 days in the knockdown, the positions of nuclei are marked with asterisks. By day 5, the distribution of both COX and succinate dehydrogenase (SDH) activity reflect the mitochondrial perinuclear clustering. Figure e-6 shows that mitochondrial fragmentation occurred without loss of cytochrome c or alteration of cristae at 48 hours. (E) SiRNA to OPA1 significantly reduced the number of nucleoids visualized by PicoGreen (left panel, p < 0.001, 2-tailed t test) corresponding to mtDNA content of 8% ± 2% of the control. This was confirmed by quantitative PCR 14 days after treatment (right panel, p < 0.001, 2-tailed t test). (F) Cell biological basis for relocalization of mitochondria during OPA1 knockdown implies altered transport of fragmented mitochondria. OPA1 knockdown relocates mitochondria to the microtubule organizing center (MTOC), mediated by microtubules and actin. That this is prevented by overexpression of p50-dynamitin is shown in figure e-3B. (F.a) Anti-pericentrin (MTOC)/mitofillin (mitochondria) staining shows that siRNA to OPA1 causes relocation of mitochondria to the MTOC in over 90% of cells as opposed to the scrambled siRNA control (see chart). (F.b) The cell biological basis for relocalization of mitochondria during OPA1 knockdown implies altered transport mediated by microtubules and actin. Upper left: confirms the presence of perinuclear mitochondrial clusters following OPA1 knockdown. Upper right: Disassembly of microtubules by nocodazole treatment (5 μM, 24 hours) rescues the mitochondrial clustering (p < 0.001). Lower left: Taxotere (5 μM, 24 hours) causes assembly and stabilization of microtubules and hence randomly distributed mitochondrial clusters (3 clusters in one cell each marked with an arrow, p < 0.01). Lower right: Treatment with cytochalasin D (5 μM, 24 hours), which depolymerizes actin, also redistributes mitochondria away from the MTOC, although a weak perinuclear clustering was still present (p < 0.01). Each of these experiments was carried out at least 3 times.
Figure 2 A reduction in OPA1 leads to rapid loss of mitochondrial DNA (mtDNA) Control fibroblasts were treated with OPA1 Pan OPA1 or scramble siRNA. (A) Western blot analysis confirms OPA1 levels were efficiently reduced following siRNA treatment (blot shows 3 days after knockdown). (B) PicoGreen/tetramethyl rhodamine methyl ester (TMRM) costaining of live fibroblasts treated with OPA1 siRNA shows a marked loss of mtDNA nucleoids (visible as green puncta) compared to treatment with scramble. In all OPA1 knockdown cells with mitochondrial fragmentation, a noticeable mitochondrial perinuclear clustering was observed (arrows). (C) Anti-DNA/MitoTracker colabeling of OPA1/scramble siRNA treated fibroblasts (note that this antibody is recognized to label mtDNA more strongly than nuclear DNA). This confirms the findings presented in B. (D) Cytochrome oxidase (COX) activity, demonstrated by routine histochemistry, is significantly reduced by day 14. In the panel showing COX at 14 days in the knockdown, the positions of nuclei are marked with asterisks. By day 5, the distribution of both COX and succinate dehydrogenase (SDH) activity reflect the mitochondrial perinuclear clustering. Figure e-6 shows that mitochondrial fragmentation occurred without loss of cytochrome c or alteration of cristae at 48 hours. (E) SiRNA to OPA1 significantly reduced the number of nucleoids visualized by PicoGreen (left panel, p < 0.001, 2-tailed t test) corresponding to mtDNA content of 8% ± 2% of the control. This was confirmed by quantitative PCR 14 days after treatment (right panel, p < 0.001, 2-tailed t test). (F) Cell biological basis for relocalization of mitochondria during OPA1 knockdown implies altered transport of fragmented mitochondria. OPA1 knockdown relocates mitochondria to the microtubule organizing center (MTOC), mediated by microtubules and actin. That this is prevented by overexpression of p50-dynamitin is shown in figure e-3B. (F.a) Anti-pericentrin (MTOC)/mitofillin (mitochondria) staining shows that siRNA to OPA1 causes relocation of mitochondria to the MTOC in over 90% of cells as opposed to the scrambled siRNA control (see chart). (F.b) The cell biological basis for relocalization of mitochondria during OPA1 knockdown implies altered transport mediated by microtubules and actin. Upper left: confirms the presence of perinuclear mitochondrial clusters following OPA1 knockdown. Upper right: Disassembly of microtubules by nocodazole treatment (5 μM, 24 hours) rescues the mitochondrial clustering (p < 0.001). Lower left: Taxotere (5 μM, 24 hours) causes assembly and stabilization of microtubules and hence randomly distributed mitochondrial clusters (3 clusters in one cell each marked with an arrow, p < 0.01). Lower right: Treatment with cytochalasin D (5 μM, 24 hours), which depolymerizes actin, also redistributes mitochondria away from the MTOC, although a weak perinuclear clustering was still present (p < 0.01). Each of these experiments was carried out at least 3 times.CC BY · PubMed Central
Figure 3 Analysis of primary cultures from biallelic dominantly inherited optic atrophy (DOA) plus OPA1(−/−) patients demonstrates increased mitophagy compared to an age-matched control (A) ImageStream analysis of cultured fibroblasts shows that basal mitophagy is significantly increased in DOA plus OPA1(−/−)2 and DOA plus OPA1(−/−)3 compared with control both at baseline and following treatment with CQ (all p < 0.01, 2-tailed t test). (A.a) The number of puncta per cell that were positive for light chain 3 (LC3) (representing both autophagosomes and autolysosomes) and Tom20 (representing mitochondria). These were counted at baseline in control cells, for comparison with patients DOA plus OPA1(−/−)2 and 3. Exposure to chloroquine (CQ; 25 μM) overnight blocks mitophagy at this stage by preventing lysosomal acidification increasing the signal, more so in patients than controls. (A.b, A.c) The counts of puncta that are positive for both LC3 and LysoID (representing only autolysosomes), counted in control cells, for comparison with patient DOA plus OPA1(−/−)1. (A.b) The total number of puncta per cell that were positive for the autolysosome markers. (A.c) The counts of these autolysosomes that colocalized with mitochondria (hence autolysosomes involved in mitophagy) for the same dataset. In all cases there were more counts in the patient than the control. Galactose-based starvation medium increased the number of LC3/LysoID-positive puncta above baseline. Exposure to 25 μM CQ overnight did not increase the signal, because it prevents progression of autophagosomes to autolysosomes. Error bars are standard errors (SEs) (technical replicates). Patient values all significantly greater than control p < 0.01 (2-tailed t test). All are representative of 1 out of 3 independent replicates. (B) A statistical analysis of 4 consecutive ImageStream runs on all the patients listed in the table along with 6 controls. The output shows increased mitophagy in patients with severe OPA1 mutations (both biallelic and monoallelic, that is, DOA plus OPA1[−/−] and DOA plus OPA1[+/−]) compared with normal controls (p = 0.035). We show one bar per patient group, with each bar's height (y axis) representing the estimated difference between a particular patient group and controls. The whiskers on a bar represent the SE of the estimated difference (±1 SE is shown); an approximate 95% confidence interval for the patient-control difference could be calculated as the bar height ± 2 SEs. The p values in the figure are from the test of the null hypothesis that there is no actual difference between a patient group and controls. Useful intuition connecting the hypothesis test with the estimated difference is that a p value < 0.05 corresponds to a 95% confidence interval not overlapping zero. Uncomplicated symptomatic DOA OPA1(+/−) and asymptomatic OPA1(+/−) were not different from controls (n = 1, 2, and 6, respectively). Chloroquine 25 μM overnight CQ significantly increased the number of LC3 puncta colocalizing with the mitochondrial signal in all individuals in all experiments (p < 0.001). (C) OPA1 knockdown by siRNA also increases mitophagy. (C.a) Bar chart of ImageStream output shows that siRNA to OPA1 increases mitophagy. The summed area of LC3 puncta that colocalize with mitochondria (PDH signal) in fibroblasts treated with OPA1 siRNA is greater than in scramble siRNA and the untreated controls (p = 0.05 and p < 0.01, respectively, both 2-tailed t tests). Mitochondrial mean intensity was also reduced by 5% (not shown). Error bars are SEs (technical replicates). (C.b) In OPA1 knockdown fibroblasts (OPA1 siRNA) compared to untreated (No siRNA) and siRNA scramble (Scr siRNA) controls, OPA1 levels are reduced and LC3-II levels are substantially increased relative to actin by the Western blot analysis. (D) Validation of the increased mitophagic flux, using IN Cell 1000 quantitative fluorescence microscopy. The number of LC3-positive puncta per cell was quantitated in fibroblasts from biallelic OPA1 patients at baseline and after 2 hours in the presence of lysosomal protease inhibitors (E64D and pepstatin A, labeled E+P) or after 2 hours starvation in minimal medium compared with age-matched controls. (D.a) Per cell count of LC3-positive puncta colocalizing with mitochondria (p < 0.0001 and p < 0.005 for baseline compared to lysosomal inhibitors or starvation, respectively). (D.b) Mean number of total LC3-positive puncta per cell. Each patient had significantly higher counts than control (p < 0.02) in all conditions, except baseline patient DOA plus OPA1(−/−)3 autophagy and baseline patient DOA plus OPA1(−/−)1 mitophagy. (D.c) The mitophagic flux is increased in biallelic patients relative to controls (the increase in colocalization during starvation or lysosomal inhibitors, relative to baseline). Error bars are SEs of technical replicates, all p values 2-tailed t tests. For further evidence of increased mitophagic flux, see figure e-4A.
Figure 3 Analysis of primary cultures from biallelic dominantly inherited optic atrophy (DOA) plus OPA1(−/−) patients demonstrates increased mitophagy compared to an age-matched control (A) ImageStream analysis of cultured fibroblasts shows that basal mitophagy is significantly increased in DOA plus OPA1(−/−)2 and DOA plus OPA1(−/−)3 compared with control both at baseline and following treatment with CQ (all p < 0.01, 2-tailed t test). (A.a) The number of puncta per cell that were positive for light chain 3 (LC3) (representing both autophagosomes and autolysosomes) and Tom20 (representing mitochondria). These were counted at baseline in control cells, for comparison with patients DOA plus OPA1(−/−)2 and 3. Exposure to chloroquine (CQ; 25 μM) overnight blocks mitophagy at this stage by preventing lysosomal acidification increasing the signal, more so in patients than controls. (A.b, A.c) The counts of puncta that are positive for both LC3 and LysoID (representing only autolysosomes), counted in control cells, for comparison with patient DOA plus OPA1(−/−)1. (A.b) The total number of puncta per cell that were positive for the autolysosome markers. (A.c) The counts of these autolysosomes that colocalized with mitochondria (hence autolysosomes involved in mitophagy) for the same dataset. In all cases there were more counts in the patient than the control. Galactose-based starvation medium increased the number of LC3/LysoID-positive puncta above baseline. Exposure to 25 μM CQ overnight did not increase the signal, because it prevents progression of autophagosomes to autolysosomes. Error bars are standard errors (SEs) (technical replicates). Patient values all significantly greater than control p < 0.01 (2-tailed t test). All are representative of 1 out of 3 independent replicates. (B) A statistical analysis of 4 consecutive ImageStream runs on all the patients listed in the table along with 6 controls. The output shows increased mitophagy in patients with severe OPA1 mutations (both biallelic and monoallelic, that is, DOA plus OPA1[−/−] and DOA plus OPA1[+/−]) compared with normal controls (p = 0.035). We show one bar per patient group, with each bar's height (y axis) representing the estimated difference between a particular patient group and controls. The whiskers on a bar represent the SE of the estimated difference (±1 SE is shown); an approximate 95% confidence interval for the patient-control difference could be calculated as the bar height ± 2 SEs. The p values in the figure are from the test of the null hypothesis that there is no actual difference between a patient group and controls. Useful intuition connecting the hypothesis test with the estimated difference is that a p value < 0.05 corresponds to a 95% confidence interval not overlapping zero. Uncomplicated symptomatic DOA OPA1(+/−) and asymptomatic OPA1(+/−) were not different from controls (n = 1, 2, and 6, respectively). Chloroquine 25 μM overnight CQ significantly increased the number of LC3 puncta colocalizing with the mitochondrial signal in all individuals in all experiments (p < 0.001). (C) OPA1 knockdown by siRNA also increases mitophagy. (C.a) Bar chart of ImageStream output shows that siRNA to OPA1 increases mitophagy. The summed area of LC3 puncta that colocalize with mitochondria (PDH signal) in fibroblasts treated with OPA1 siRNA is greater than in scramble siRNA and the untreated controls (p = 0.05 and p < 0.01, respectively, both 2-tailed t tests). Mitochondrial mean intensity was also reduced by 5% (not shown). Error bars are SEs (technical replicates). (C.b) In OPA1 knockdown fibroblasts (OPA1 siRNA) compared to untreated (No siRNA) and siRNA scramble (Scr siRNA) controls, OPA1 levels are reduced and LC3-II levels are substantially increased relative to actin by the Western blot analysis. (D) Validation of the increased mitophagic flux, using IN Cell 1000 quantitative fluorescence microscopy. The number of LC3-positive puncta per cell was quantitated in fibroblasts from biallelic OPA1 patients at baseline and after 2 hours in the presence of lysosomal protease inhibitors (E64D and pepstatin A, labeled E+P) or after 2 hours starvation in minimal medium compared with age-matched controls. (D.a) Per cell count of LC3-positive puncta colocalizing with mitochondria (p < 0.0001 and p < 0.005 for baseline compared to lysosomal inhibitors or starvation, respectively). (D.b) Mean number of total LC3-positive puncta per cell. Each patient had significantly higher counts than control (p < 0.02) in all conditions, except baseline patient DOA plus OPA1(−/−)3 autophagy and baseline patient DOA plus OPA1(−/−)1 mitophagy. (D.c) The mitophagic flux is increased in biallelic patients relative to controls (the increase in colocalization during starvation or lysosomal inhibitors, relative to baseline). Error bars are SEs of technical replicates, all p values 2-tailed t tests. For further evidence of increased mitophagic flux, see figure e-4A.CC BY · PubMed Central
Figure 4 Effect of OPA1 mutations on mitophagy is impaired by knockdown of ATG7 and recapitulated by a mitofusin 2 (MFN2 )–dominant negative genotype (A) Western blot analysis confirms that knockdown of ATG7 by siRNA reduced protein abundance relative to actin in both patient and control cells. ATG7 = siRNA to ATG7; DOA = dominantly inherited optic atrophy; scr = scramble; siRNA = no treatment. (B) The number of colocalizing mitochondrial puncta (mitophagy events per cell, measured by IN Cell 1000) is significantly increased in fibroblasts from biallelic patients compared to controls at baseline (B.a) (p < 0.001) and after blocking autophagy with lysosomal inhibitors E64D and pepstatin A (E+P, p = 0.01). (B.b) Adding E64D and pepstatin A significantly increased the numbers of colocalizing puncta (p < 0.001) compared to baseline. Knockdown of ATG7 protein by siRNA significantly reduced the colocalizing puncta (p < 0.001) and eliminated the increase in patients over controls. Cells were treated with siRNA against ATG7 for 48 hours, then the medium was changed and either solvent or lysosomal inhibitors, E64D, and pepstatin A were added for 3 days. Error bars are standard errors of technical replicates, all p values 2-tailed t tests. (C) The number of mitophagy events per cell (measured by IN Cell 1000) at baseline (C.a) is significantly increased in fibroblasts from biallelic DOA plus OPA1(−/−) patients (n = 3) and in a patient with a dominant negative MFN2 mutation compared to control (p < 0.001 and p < 0.02, respectively). After adding chloroquine (C.b), this was significantly increased in both the DOA plus OPA1(−/−) patients (n = 3) and the MFN2 fibroblasts compared to control (both p < 0.001). In neither case was the MFN2 significantly different from biallelic DOA plus OPA1(−/−) patients. Error bars are standard errors of technical replicates, all p values 2-tailed t tests.
Figure 4 Effect of OPA1 mutations on mitophagy is impaired by knockdown of ATG7 and recapitulated by a mitofusin 2 (MFN2 )–dominant negative genotype (A) Western blot analysis confirms that knockdown of ATG7 by siRNA reduced protein abundance relative to actin in both patient and control cells. ATG7 = siRNA to ATG7; DOA = dominantly inherited optic atrophy; scr = scramble; siRNA = no treatment. (B) The number of colocalizing mitochondrial puncta (mitophagy events per cell, measured by IN Cell 1000) is significantly increased in fibroblasts from biallelic patients compared to controls at baseline (B.a) (p < 0.001) and after blocking autophagy with lysosomal inhibitors E64D and pepstatin A (E+P, p = 0.01). (B.b) Adding E64D and pepstatin A significantly increased the numbers of colocalizing puncta (p < 0.001) compared to baseline. Knockdown of ATG7 protein by siRNA significantly reduced the colocalizing puncta (p < 0.001) and eliminated the increase in patients over controls. Cells were treated with siRNA against ATG7 for 48 hours, then the medium was changed and either solvent or lysosomal inhibitors, E64D, and pepstatin A were added for 3 days. Error bars are standard errors of technical replicates, all p values 2-tailed t tests. (C) The number of mitophagy events per cell (measured by IN Cell 1000) at baseline (C.a) is significantly increased in fibroblasts from biallelic DOA plus OPA1(−/−) patients (n = 3) and in a patient with a dominant negative MFN2 mutation compared to control (p < 0.001 and p < 0.02, respectively). After adding chloroquine (C.b), this was significantly increased in both the DOA plus OPA1(−/−) patients (n = 3) and the MFN2 fibroblasts compared to control (both p < 0.001). In neither case was the MFN2 significantly different from biallelic DOA plus OPA1(−/−) patients. Error bars are standard errors of technical replicates, all p values 2-tailed t tests.CC BY · PubMed Central

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Authors
Liao, C., Ashley, N., Diot, A., Morten, K., Phadwal, K., Williams, A., Fearnley, I., Rosser, L., Lowndes, J., Fratter, C., Ferguson, D. J. P., Vay, L., Quaghebeur, G., Moroni, I., Bianchi, S., Lamperti, C., Downes, S. M., Sitarz, K. S., Flannery, P. J., Carver, J., Dombi, E., East, D., Laura, M., Reilly, M. M., Mortiboys, H., Prevo, R., Campanella, M., Daniels, M. J., Zeviani, M., Yu-Wai-Man, P., Simon, A. K., Votruba, M., & Poulton, J.
Affiliations
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Corresponding author
Poulton, Joanna
ORCID
Venue
Neurology
Publisher
Ovid Technologies (Wolters Kluwer Health)
Published
2017-01-10
Volume
88
Issue
2
Pages
131-142
ISSN
  • 0028-3878 (ISSN-L)
  • 1526-632X
DOI
10.1212/wnl.0000000000003491
PMC
PMC5224718
OpenAlex
W2549272530
Cited by
97
References
41
Citations by year
2026: 9 · 2025: 7 · 2024: 5 · 2023: 5 · 2022: 16 · 2021: 17 · 2020: 15 · 2019: 5 · 2018: 11 · 2017: 7
FWCI
7.19
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Neurology (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • MDC Repository (Max-Delbrueck-Center for Molecular Medicine) (acceptedVersion)
  • White Rose Research Online (University of Leeds, The University of Sheffield, University of York) (acceptedVersion)
  • ORCA Online Research @Cardiff (Cardiff University) (acceptedVersion)
  • RVC Research Online (Royal Veterinary College) (submittedVersion, cc-by)
  • Cineca Institutional Research Information System (Tor Vergata University) (submittedVersion)
  • DIGITAL.CSIC (Spanish National Research Council (CSIC)) (submittedVersion, cc-by)
  • UCL Discovery (University College London) (submittedVersion)
  • Research Explorer (The University of Manchester) (submittedVersion, cc-by)
  • Padua Research Archive (University of Padova) (submittedVersion)
Funders
  • Wellcome Trust (WT098519MA, 098519/Z/12/Z)
  • Newlife the Charity for Disabled Children
  • Kennedy Trust for Rheumatology Research (KENN161708)
  • Cancer Research UK
  • National Institute for Health and Care Research
  • Parkinson's UK (F1301)
  • University of Oxford
  • Newcastle University
  • Cardiff University
  • Instituo Cajal
  • Moorfields Eye Hospital NHS Foundation Trust
  • Institute for Translational Neuroscience
  • Royal Veterinary College
  • Medical Research Council (MR/J010448/1)
  • Biotechnology and Biological Sciences Research Council (BB/I013695/1, BB/N007042/1)
  • Wellcome Trust Centre for Mitochondrial Research
  • NIHR Oxford Biomedical Research Centre
  • National Institute of Neurological Disorders and Stroke (U54NS065712)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Autophagy in Disease and Therapy
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Mitochondrial Function and Pathology
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Hereditary Neurological Disorders
Keywords
mitophagy, OPA1 mutations, optic atrophy, mitochondrial fragmentation, mitochondrial DNA depletion, ATG7, mitochondrial fusion, mitochondrial optic neuropathy, Leber's hereditary optic neuropathy, autophagosomes, dermal fibroblasts
MeSH terms
PTEN-Induced Putative Kinase, Antioxidants, Cells, Cultured, Cognition Disorders, DNA Mutational Analysis, DNA, Mitochondrial, Family Health, Female, Fibroblasts, Humans, Male, Mutation, Optic Atrophy, Pedigree, Protein Kinases, Transfection, Ubiquinone, GTP Phosphohydrolases, Mitochondrial Proteins, RNA, Small Interfering, Ubiquitin-Protein Ligases, Membrane Potential, Mitochondrial, Mitophagy

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