| Summary: | 碩士 === 國立陽明大學 === 生化暨分子生物研究所 === 94 === About 85% of the patients with myoclonic epilepsy and ragged-red fibers (MERRF) syndrome have been found to carry the A8344G mutation in the tRNALys gene of mitochondrial DNA (mtDNA). The cells harboring this mtDNA mutation show reduced activities of respiratory chain enzymes and generate more reactive oxygen species (ROS). Histochemical analysis of skeletal muscle biopsies of MERRF patients often reveal the presence of ragged-red fibers, which represent subsarcolemmal accumulation of abnormal mitochondria. We have suggested that excess ROS may play an important role in mitochondrial biogenesis. In this study, experiments have been designed to investigate the mechanisms underlying the changes of mtDNA content under different levels of oxidative stress in 143B cells induced by H2O2. We then conducted similar studies using skin fibroblasts from normal subjects (N1-N3) and patients with MERRF syndrome (M1-M3) to unravel possible role of oxidative stress response in the pathogenesis of MERRF syndrome. The results showed that the intracellular ROS and mtDNA oxidative damages were respectively increased 1.6, 1.9, 2.1, 2.1, 2.4 fold and 1.9, 3.1, 4.8, 6.0, 6.9 fold in the concentration- and time-dependent manner after 0-500 �嵱 H2O2 treatment for 48 hours. Moreover, the mtDNA content and the protein expression of DNA polymerase gamma (Pol ��) were increased, 1.5 and 2.0 fold respectively, by treatment with 200 �嵱 H2O2. However, the mtDNA content was dramatically decreased to about 50% of that of control, while Pol �� was decreased to the basal level after 500 �嵱 H2O2 treatment. These findings suggest that ROS might induce mtDNA replication and transcription to compensate for the oxidative damage caused by ROS under mild oxidative stress. Under higher oxidative stress, ROS may interrupt transcription and translation of the genes required for the maintenance of mtDNA, resulting in the reduction of protein expression or activity of Pol ��, which in return may cause a decrease of the copy number of mtDNA. On the other hand, we found that the endogenous levels of H2O2, mtDNA copy number and mtDNA oxidative damage in MERRF skin fibroblasts were 1.8, 1.6 and 2.9 fold higher than those of normal subjects, but the protein expression level of Pol ���nwas inversely correlated with the intracellular ROS in MERRF skin fibroblasts. Thus, we suggest that the intracellular ROS produced by defective respiratory enzymes of the damaged mtDNA may initiate an increase of mtDNA replication to compensate for the oxidative damage. Consquently, accumulation of mutated mtDNA may be a result of the replication of mutated mtDNA by Pol ���n. The protein expression or enzyme activity of Pol ���nmay be decreased by oxidative damage under low oxidative stress over a long period of time in the affected tissues of patients. These molecular changes cause an increase of intracellular ROS and mtDNA copy number, damage of mtDNA, and a decrease in the expression of Pol ��. Based on these findings, we suggest that an oxidative stress-elicited vicious cycle may play an important role in the pathophysiology of mitochondrial diseases such as MERRF syndrome through the regulation of Pol ���nand mtDNA content in affected tissues of the patients.
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