| Summary: | 碩士 === 長庚大學 === 基礎醫學研究所 === 93 === Pathogenic mutation of mitochondrial DNA (mtDNA) is often fatal to cells and can cause a large variety of human mitochondrial dysfunction and cell death. In this study, I focused on one of the most commonly seen 4977 bp mtDNA deletion, so called common deletion (CD), which clinically results in chronic progressive external ophthalmoplegia (CPEO) symdrome and Kearns-sayre syndrome (KSS). This study aims to investigate how CD links to its final pathological mitochondrial dysfunction and apoptosis as well as to seek for possible treatment for CD. For the treatment, a potent mitochondrial antioxidant, melatonin, and the photodynamic effect (PDE) of a mitochondrial targeted photosensitizer, Benzoporphyrin derivative monoacid ring A (BPD-MA), were used. In particular, an established array of CD cybrids created by cytoplasmic fusion a skin fibroblasts from a CPEO patients with mtDNA-deleted (ρ0 ) osteosarcoma line were conducted for study. Mitochondrial pathology at single cell level were visualized by the application of fluorescent probes coupled with conventional and advanced multiphoton imaging microscopy.
The pathological mechanistic investigation of CD reveals that CD significantly augmented mROS formation. CD-augmented mROS formation was followed with an enhanced ΔΨm depolarization, opening of the mitochondrial permeability transition pore (MPTP) and apoptosis. In addition, heterogeneous mitochondrial dysfunction was visualized in two subtypes of CD cybrids, namely the super sensitive-D (SS-D) and the low sensitive-D (LS-D) cybrids. In SS-D cybrids, higher resting mROS level and faster H2O2 stress-induced mROS generation and ΔΨm depolarization were observed. Moreover, H2O2-induced mROS generation solely was able to induce the opening of the MPTP and cell deterioration. In the part of treatment of CD-induced mitochondrial dysfunction, I demonstrated melatonin significantly prevents CD-augmented mROS generation and delta si depolarization in H2O2-treated D cybrids. In particular, melatonin protected CD cybrid CL depletion, s-mac release and apoptosis upon H2O2 treatment. I also demonstrated that PDE of BPD-MA significantly reverse the CD-augmented mitochondrial dysfunction including mROS formation and delta si depolarization. After the PDE of BPD-MA, the mutant mitochondrial complexes recovered significantly. The activity of antioxidative enzymes including catalase and glutathione peroxidase, however, remained unchanged. This reverse effect CD-augmented mitochondrial dysfunction was not observed when entire mtDNA was 100% removed (in zho zero cells). These results, thus, suggest PDE of BPD-MA may significantly and specifically enhance the removal of mutant mtDNA and allow normal mtDNA to gradually recover.
As a conclusion, this study demonstrated for the first time CD-augmented mROS formation play a pivotal role in the pathological outcome and apoptosis in CD contained cybrids. Precise detection of spatial-temporal profiles of mROS generation at single cell level, thus, may provide a crucial clinical index for an early diagnosis and prediction of CD-mediated pathological consequences. In addition, mitochondrial antioxidant melatonin may prevent CD-induced mitochondrial dysfunction and apoptosis for short term. Finally, the effective reversion of mutant CD back to normal by PDE of BPD-MA may provide a potential therapeutic treatment for CD-related mitochondrial diseases.
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