Changes in Mitochondrial Morphology and Bioenergetics in Human Lymphoblastoid Cells With Four Novel OPA1 Mutations.
OBJECTIVE
Mutations in the optic atrophy 1 gene (OPA1) have been reported in patients with autosomal dominant optic atrophy (ADOA). OPA1 plays important roles in mitochondrial dynamics and cell apoptosis. The link between OPA1 mutations and changes in bioenergetics is still not fully resolved. The aim of this study was to investigate the effects of OPA1 mutations on the mitochondrial tubular network and bioenergetics.


METHODS
We established lymphoblastoid cell lines from four ADOA families harboring different OPA1 mutations, unaffected relatives (internal control cell lines), and unrelated normal controls (normal control cell lines). OPA1 splice variants and mRNA were analyzed by reverse transcription-PCR and quantitative real-time PCR. Protein isoforms were examined by Western blotting. The mitochondrial network was visualized by confocal microscopy. Mitochondrial bioenergetics were assessed using a Seahorse XF24 flux analyzer. Mitochondrial membrane potential and oxidative damage were analyzed by flow cytometry.


RESULTS
OPA1 mutant cell lines showed significant decreases in OPA1 mRNA and protein expression, mitochondrial membrane potential, and ATP synthesis. A marked deficiency of the long isoform of OPA1 was observed in cells with OPA1 mutations in the middle domain and GTPase effector domain. Confocal microscopy revealed increased mitochondrial fragmentation in OPA1 mutant cells. OPA1 mutant cells also displayed reduced oxygen consumption and underwent glycolysis to produce ATP. Moreover, OPA1 mutations caused the accumulation of oxidative damage.


CONCLUSIONS
Our experiments demonstrated that OPA1 mutations induced mitochondrial fragmentation, uncoupled mitochondrial respiration, and elicited dysfunctional bioenergetics. However, there were no significant differences among the various OPA1 mutations.
School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.
Shu-Huei
SH
Kao
Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan 3School of Medicine, National Yang-Ming University, Taipei, Taiwan.
May-Yung
MY
Yen
Department of Ophthalmology, Taipei Veterans General Hospital, Taipei, Taiwan 3School of Medicine, National Yang-Ming University, Taipei, Taiwan.
An-Guor
AG
Wang
School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan.
Yi-Ling
YL
Yeh
School of Medical Laboratory Science and Biotechnology, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan 4Graduate Institute of Medical Science, College of Medicine, Taipei Medical University, Taipei, Taiwan.
An-Lo
AL
Lin
D017209:Apoptosis; D015153:Blotting, Western; D002478:Cells, Cultured; D004734:Energy Metabolism; D005260:Female; D005434:Flow Cytometry; D020558:GTP Phosphohydrolases / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008297:Male; D053078:Membrane Potential, Mitochondrial; D008875:Middle Aged; D008928:Mitochondria / Q000378:metabolism* / Q000473:pathology; D009154:Mutation; D029241:Optic Atrophy, Autosomal Dominant / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D010375:Pedigree; D012333:RNA, Messenger / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012333:RNA, Messenger; D020558:GTP Phosphohydrolases; C415198:OPA1 protein, human