| Summary: | 碩士 === 長庚大學 === 基礎醫學研究所 === 93 === Mitochondria, not only act as ATP producers through oxidative phosphorylation but also as regulators of intracellular Ca2+ homeostasis and endogenous producers of reactive oxygen species (ROS). Pathogenic mutation of mitochondrial DNA (mtDNA) is often fatal to cells and can cause a large variety of human mitochondrial diseases. A specific mitochondrial DNA (mtDNA) point mutation T > G at nucleotide position 8993 affects gene of the subunit 6 of ATP synthase and is clinically closely associated with neurological muscle weakness, ataxia, and retinitis pigmentosa syndrome, so called NARP mutation. Precise mechanistic investigation on how NARP mutation links to cellular and mitochondrial dysfunction, an established NARP cybrids harboring 98% T > G mutation and its control wild type (143B) were conducted. Single cell level of measurement of mitochondrial dysfunction including mitochondrial reactive oxygen species (mROS) and mitochondrial membrane potential (m) and the mitochondrial permeability transition (MPTP) opening were performed by the application of fluorescent probes coupled with conventional and single photon imaging microscopy. Results of this study are described in three parts:
Part I: NARP point mutation-augmented mitochondrial dysfunction and apoptosis upon Ca2+ stress
Morphologically NARP cybrids is very similar to its control wild cybrids. However, NARP significantly increase the sensitivity to various stresses. For instance, NARP enhanced H2O2-induced cell blabbing, nuclear condensation and apoptosis. In addition, NARP enhanced high Ca2+ (10 mM), ionomycin-induced apoptosis. Apoptosis induced by high Ca2+ was evidenced by TUNEL staining. Intriguingly, NARP cybrids are particularly sensitive to Ca2+ stress as compared to oxidative stress-induced by H2O2.
Part II: Role of NARP mutation induced mitochondrial dysfunction upon oxidative stress induced by arachidonic acid
Dual fluorescence single photon imaging measurement of mROS and delta si shows that NARP enhanced significantly mROS formation and m depolarization upon AA stress. A mitochondrial specific antioxidant, mito Q, inhibits completely mROS formation, however, did not protect m depolarization in NARP cybrids. Cyclosporin A (Cs A), a MPTP inhibitor, partially prevented m depolarization but enhanced significantly mROS formation in NARP cybrids treated with AA. RU360 and Cs A together significantly prevent mROS formation and m depolarization. NARP also significantly decreased mitochondrial movement at resting and upon AA treatment. Mito Q reversed the effect of AA on mitochondrial movement. RU360 and Cs A show similar effect as mito Q on the mitochondrial movement.
Part III: Role of NARP mutation induced-mitochondrial dysfunction upon oxidative stress induced by visible light irradiation
My conventional microscopic study shows that NARP enhances significantly resting level of mROS and mCa2+. NARP also enhanced visible light induced mROS formation and mCa2+ increases. Detail mitochondrial level investigation using confocal imagining microscopy, I demonstrated that NARP enhanced significantly resting level of mROS formation and also induced a much hyperpolarized m. Upon stress induced 488 nm local irradiation, NARP enhanced mROS and m depolarization. CCCP (0.1 nM) significantly inhibited NARP enhanced light irradiation-induced mROS formation indicating a m originated mechanism probably via an enhanced electron flow. Localized mROS formation induced by 488 nm irradiation propagated to the entire cell as well as adjacent cells. Heterogeneous propagation of mROS and m depolarization within the same cell as well as to adjacent cell were observed. Wile mROS level and the opening of the MPTP were carefully analyzed, I was found that the MPTP in mitochondrion containing higher level of ROS did not always open earlier than that in those containing lower mROS. Similar to the effect on AA-induced mROS formation, Cs A 10-20 M partially prevented m depolarization but enhanced mROS formation. Cs A also enhanced significantly mROS propagation to the adjacent cells indicating opening of the MPTP may be protective to the mitochondria. In the presence of mito Q, light-induced mROS formation was completely inhibited and m depolarization was greatly reduced. Mito Q completely prevented mROS propagation to the adjacent cells. Possible role of Ca2+ involved in light irradiation-induced mROS formation and its propagation was further investigated by removal of endoplasmic reticulum Ca2+ pool and extracellular Ca2+ as well as by plasma membrane Ca2+ channel blockers and blocker of mitochondrial uniporter, RU360. Intriguingly, light-induced mCa2+ increased preceded mROS formation and was enhanced by NARP mutation. Mito Q prevented mROS formation but not mCa2+ increase indicating light irradiation alone can induce an increase in mCa2+. However, time to the peak in light-induced mCa2+ increase was much prolonged indicating involved mROS in mCa2+ regulation. Propagation of mCa2+ was observed within the same cells as well as to the adjacent cells. Mito Q, by preventing mROS formation, slightly inhibited light-induced mCa2+ increase in the adjacent cells but not the mCa2+ propagation to the adjacent cells. mROS increase and propagation can be partially inhibited by removal of extracellular mCa2+ and can be completely inhibited by removal of cellular plus extracellular mCa2+ indicating a potential role of the ER involved. RU360, by inhibiting mCa2+ uptake, partially inhibited mROS formation and propagation. These results indicate mCa2+ is crucially involved in light irradiation-induced mROS formation and propagation. Dual measurement of Fluo-4 and Rhod-2 further confirmed that light irradiation induced an oscillation of cytosolic and mitochondrial Ca2+. Mitochondrial Ca2+ oscillation was preceded by the cytosolic Ca2+ oscillation. The light-induced cytosolic Ca2+ oscillation is dependent on Ca2+ influx via a Nimodipine sensitive L-type Ca2+ channel as well as on Ca2+ release from the thapsigargin sensitive ER Ca2+ pool.
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