Cheng-Feng Lee

Increases of Mitochondrial Mass and Mitochondrial Genome in Association with Enhanced Oxidative Stress in Human Cells Harboring 4,977 BP‐Deleted Mitochondrial DNA

Abstract: In order to investigate the effect of aging‐ and disease‐associated deletion of mtDNA on cellular functions, we used cytoplasm fusion to construct a series of the cybrids harboring varying proportions of mtDNA with 4,977 bp deletion from skin fibroblasts of a patient with chronic progressive external ophthalmoplegia. The cybrids were grown in the Dulbeccos modified Eagle medium supplemented with 5% fetal bovine serum, 100 μg/ml pyruvate and 50 μg/ml uridine. The population doubling time was longer for the cybrids containing higher proportions of 4,977 bp‐deleted mtDNA. In addition, we found that the respiratory function was decreased with the increase of mtDNA with 4,977 bp deletion in the cybrids. Since impairment of the respiratory system of mitochondria increases the electron leak of the respiratory chain, we further determined the oxidative stress in these cybrids. The results showed that the specific contents of 8‐hydroxy 2′‐deoxyguanosine and lipid peroxides of the cybrids harboring > 65% of the 4,977 bp‐deleted mtDNA were significantly increased as compared with those of the cybrids containing undetectable mutant mtDNA. On the other hand, we found that the mitochondrial mass and the relative content of the mitochondrial genome in the cybrids harboring 4,977 bp‐deleted mtDNA were higher than those of the cybrids containing only wild type mtDNA. The relative content of mtDNA was increased 17% and 30%, respectively, in the cybrids harboring 17% and 56% of mtDNA with 4,977 bp deletion. Moreover, both mitochondrial mass and mtDNA content were concurrently increased by treatment of the cybrids with 180 μM of hydrogen peroxide. Taken these findings together, we conclude that increase of mitochondrial mass and mtDNA are the molecular events associated with enhanced oxidative stress in human cells with impaired respiratory function caused by mtDNA deletion.

Role of Reactive Oxygen Species-elicited Apoptosis in the Pathophysiology of Mitochondrial and Neurodegenerative Diseases Associated with Mitochondrial DNA Mutations

A wide spectrum of pathogenic mutations of mitochondrial DNA (mtDNA) has been demonstrated to cause mitochondrial dysfunction and overproduction of reactive oxygen species (ROS), in relation to mitochondrial and neurodegenerative diseases. Our previous studies have shown that large-scale deletions of mtDNA not only serve as an indicator of oxidative damage, but also result in greater susceptibility of human cells to apoptosis triggered by UV irradiation and other apoptotic stimuli. In this review, we focus on the involvement of mtDNA-mutation-associated oxidative stress and susceptibility to apoptosis in the pathophysiology of mitochondrial and neurodegenerative diseases. Different lines of research have provided concordant data to suggest that the mtDNA-mutation-elicited energy insufficiency and enhanced oxidative stress and damage lead to cell dysfunction, and increase the susceptibility of affected cells to apoptosis in patients with these diseases. Moreover, accumulating experimental evidence has shown that antioxidant therapy is a good strategy for decreasing intracellular ROS and alleviating oxidative-stress-induced apoptosis in cells of patients that harbor pathogenic mtDNA mutations.

Alteration in the copy number of mitochondrial DNA in leukocytes of patients with mitochondrial encephalomyopathies.

Objectives –  We investigated whether mutation of mitochondrial DNA (mtDNA) affects the copy number of the mitochondrial genome in patients with mitochondrial myopathy encephalopathy with lactic acidosis and stroke‐like episodes (MELAS) and those with myoclonic epilepsy with ragged‐red fiber (MERRF) syndromes.

Increased Oxidative Damage with Altered Antioxidative Status in Type 2 Diabetic Patients Harboring the 16189 T to C Variant of Mitochondrial DNA

Abstract: A transition of T to C at nucleotide position 16189 in mitochondrial DNA (mtDNA) has attracted biomedical researchers for its probable correlation with the development of diabetes mellitus in adult life. In diabetes, persistent hyperglycemia may cause high production of free radicals. Reactive oxygen species are thought to play a role in a variety of physiologic and pathophysiologic processes in which increased oxidative stress may play an important role in disease mechanisms. The aim of the present study was to clarify the degree of oxidative damage and plasma antioxidant status in diabetic patients and to see the potential influence of the 16189 variant of mtDNA on the oxidative status in these patients. An indicative parameter of lipid peroxidation, malondialdehyde (MDA), and total free thiols were measured from plasma samples of 165 type 2 diabetic patients with or without this variant and 168 normal subjects. Here we report an increase in the plasma levels of MDA and total thiols in type 2 diabetic patients compared with control subjects. The levels of plasma thiols in diabetic patients with the 16189 variant of mtDNA were not different from those in controls. These results suggest an increase in the oxidative damage and a compensatory higher antioxidative status in patients with type 2 diabetes. Harboring the 16189 mtDNA variant may impair the ability of a cell to respond properly to oxidative stress and oxidative damage.

Alteration of the Copy Number of Mitochondrial DNA in Leukocytes of Patients with Hyperlipidemia

Abstract: Lipid metabolism in leukocytes may be disturbed by mitochondrial dysfunction caused by depletion of mitochondrial DNA (mtDNA) in response to an increase of oxidative stress in blood circulation. It is possible that alteration in mtDNA copy number of the leukocyte is involved in the impairment of the scavenging of oxidatively modified plasma proteins such as oxidized low‐density lipoprotein (oxLDL). To test this hypothesis, we recruited 91 healthy subjects and 63 patients with hyperlipidemia (LDL >130 mg/dL) for this study. The copy number of mtDNA in the leukocyte and the titer of oxLDL IgG autoantibody (oLAB) were determined as indices of the oxidative stress response of immune cells. The results revealed a significant higher level of plasma oxLDL, lower titer of oLAB, and decreased copy number of mtDNA in patients with hyperlipidemia (P <0.05). In the analysis of partial correlations under age control, we found that an increase in the copy number of mtDNA was positively correlated with an increase in the level of oLAB (P <0.005, r= 0.3002) and a decrease in the oxLDL level (P <0.05, r=−0.2654) in healthy subjects but not in patients. Based on the results obtained from this case‐control study, we conclude that the increase of mtDNA copy number might provide the leukocyte an increased capability of scavenging oxLDL, possibly by enhanced generation of oLAB in healthy subjects, but not in hyperlipidemic patients who had lower mtDNA copy numbers in their leukocytes. Taken together, these findings suggest that an alteration of mtDNA copy number in the leukocyte may be one of the risk factors for hyperlipidemia.

Attenuation of UV-induced apoptosis by coenzyme Q10 in human cells harboring large-scale deletion of mitochondrial DNA

Abstract: Chronic progressive external ophthalmoplegia (CPEO) syndrome is one of the mitochondrial diseases caused by large‐scale deletions in mitochondrial DNA (mtDNA) that impair the respiratory function of mitochondria and result in decreased production of ATP in affected tissues. In order to investigate whether CPEO‐associated mtDNA mutations (i.e., 4,366‐bp and 4,977‐ bp large‐scale deletions) render human cells more vulnerable to apoptosis, we constructed cybrids carrying the deleted mtDNA. Assays for cell viability, DNA fragmentation, cytochrome c release, and caspase 3 activation revealed that UV irradiation at 20 J/m2 triggered apoptosis in all the cybrids. This treatment also produced elevated intracellular levels of reactive oxygen species (ROS). The rate of UV‐induced cell death was more pronounced in the cybrids harboring mtDNA deletions than in the control cybrid with wild‐type mtDNA. Subsequently, we evaluated the effect of coenzyme Q10 on the UV‐triggered apoptosis. The results showed that after pretreatment of the cybrids with 100 μM coenzyme Q10 the UV‐induced cell damage (i.e., ROS production and activation of caspase 3) was significantly reduced. Taken together, these findings suggest that large‐scale deletions of mtDNA increased the susceptibility of human cells to the UV‐triggered apoptosis and that coenzyme Q10 mitigated the damage; hence, it might potentially serve as a therapeutic agent to treat mitochondrial diseases resulting from mtDNA deletions.

Association of the mitochondrial DNA 16189 T to C variant with lacunar cerebral infarction: evidence from a hospital-based case-control study.

Abstract: A transition of T to C at nucleotide position 16189 in the hypervariable D‐loop region of mitochondrial DNA (mtDNA) has attracted research interest for its probable correlation with increasing insulin resistance and development of diabetes mellitus (DM) in adult life. In this article, we present our observations of the positive relationship between this variant and cerebral infarction. Six hundred and one subjects in two groups—one with cerebral infarction (307 cases), the other with no cerebral infarction (294 cases)—were recruited. Their clinical features, fasting blood sugar and insulin levels, and insulin resistance index, were recorded. Patients with cerebral infarction were further categorized into four different subgroups according to the TOAST criteria for stroke classification. The results showed the occurrence of the mtDNA 16189 variant in 34.2% of patients with cerebral infarction and in 26.5% of normal controls. The difference in the occurrence rates between the two groups was statistically significant (P= 0.041). Further studies of the occurrence rate in each stroke subgroup revealed that the variant occurred at the highest frequency in the small vessel subgroup (41.5%). The difference in occurrence rate between this subgroup and the normal controls is highly significant (P= 0.006). These results correlated well with the findings of significantly increased levels of average fasting blood insulin and a higher index of average insulin resistance in the small vessel subgroup of patients harboring this mtDNA variant. Taken together, we suggest that the mtDNA 16189 variant is a predisposing genetic factor for the development of insulin resistance and may be related to various phenotypic expressions in adult life such as development of DM and vascular pathologies involved in stroke and cardiovascular diseases.

Activation of PKCδ and ERK1/2 in the sensitivity to UV-induced apoptosis of human cells harboring 4977 bp deletion of mitochondrial DNA

The 4977 bp deletion of mitochondrial DNA (mtDNA), often found in patients with chronic progressive external ophthalmoplegia (CPEO), has been demonstrated to increase the susceptibility to apoptosis of human cells. We investigated the mechanism underlying the apoptotic susceptibility of the Delta4977 cybrid harboring about 80% 4977 bp-deleted mtDNA. The production of hydrogen peroxide (H(2)O(2)) and phosphorylation of PKCdelta and ERK1/2 were increased in the Delta4977 cybrid, which was more susceptible to UV-induced apoptosis. Moreover, treatment with N-acetyl-l-cysteine (NAC) or blocking of activation of PKCdelta by rottlerin or PKCdelta-siRNA, and inhibition of ERK1/2 by PD98059 or ERK1/2-siRNA significantly attenuated the susceptibility of the Delta4977 cybrid to apoptosis. Furthermore, the increase of PKCdelta expression in the Delta4977 cybrid also amplified the apoptotic signal through caspase 3-mediated proteolytic activation of PKCdelta. In addition, PKCdelta and ERK1/2 were hyperphosphorylated in skin fibroblasts of CPEO patients harboring 4977 bp-deleted mtDNA. We suggest that the activation of PKCdelta and ERK1/2 elicited by 4977 bp-deleted mtDNA-induced oxidative stress plays a role in the susceptibility of the mutant cells to apoptosis. This may explain, at least in part, the degenerative manifestation of brain and muscle in patients with mitochondrial encephalomyopathies such as CPEO syndrome.

Low Antioxidant Content and Mutation Load in Mitochondrial DNA A3243G Mutation-Related Diabetes Mellitus

BACKGROUND AND PURPOSE Diabetes mellitus (DM) is a common clinical manifestation in patients harboring mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes (MELAS)-specific A3243G mitochondrial DNA (mtDNA) mutation. However, in some MELAS family members, the presence of mtDNA mutation in the blood is not always associated with DM, and the relationship between development of DM and A3243G mtDNA mutation is not fully understood. This study evaluated the relationship between A3243G mtDNA mutation and DM in a Taiwanese family. METHODS We analyzed the relation of genotypic and phenotypic characteristics in a 2-generation DM family associated with the A3243G mtDNA mutation. The contents of mutant mtDNA in various tissue samples of 11 family members and their serum levels of antioxidants, including protein thiols and alpha-tocopherol (vitamin E) were determined and correlated with their past history and various clinical manifestations. RESULTS DM in 4 members of the first and second generations was associated with age and decreased serum levels of antioxidant protein thiols. In a series of studies of mutant mtDNA content in various tissues, a relatively low proportion of A3243G mutant mtDNA was noted in the elderly proband and her elderly symptomatic siblings. A low proportion of mutant mtDNA was also noted in a younger family member presenting with DM. Moreover, a significantly lower average level of protein thiols was found in the symptomatic family members compared to the asymptomatic members (2.3 +/- 0.2 vs 3.5 +/- 0.54 nmol/mg protein, p < 0.05). CONCLUSIONS The finding of relatively lower levels of mutant mtDNA in the elderly proband and her elderly symptomatic family members indicates that DM may be a late phenotypic expression in patients harboring MELAS-specific mtDNA mutation. Decreased serum protein thiols, suggestive of increased oxidative stress, also appear to be an early sign associated with subsequent development of DM.

Association of the Mitochondrial DNA 16189 T to C Variant with Lacunar Cerebral Infarction

A transition of T to C at nucleotide position 16189 in the hypervariable D-loop region of mitochondrial DNA (mtDNA) has attracted research interest for its probable correlation with increasing insulin resistance and development of diabetes mellitus (DM) in adult life. In this article, we present our observations of the positive relationship between this variant and cerebral infarction. Six hundred and one subjects in two groups-one with cerebral infarction (307 cases), the other with no cerebral infarction (294 cases)-were recruited. Their clinical features, fasting blood sugar and insulin levels, and insulin resistance index, were recorded. Patients with cerebral infarction were further categorized into four different subgroups according to the TOAST criteria for stroke classification. The results showed the occurrence of the mtDNA 16189 variant in 34.2% of patients with cerebral infarction and in 26.5% of normal controls. The difference in the occurrence rates between the two groups was statistically significant (P = 0.041). Further studies of the occurrence rate in each stroke subgroup revealed that the variant occurred at the highest frequency in the small vessel subgroup (41.5%). The difference in occurrence rate between this subgroup and the normal controls is highly significant (P = 0.006). These results correlated well with the findings of significantly increased levels of average fasting blood insulin and a higher index of average insulin resistance in the small vessel subgroup of patients harboring this mtDNA variant. Taken together, we suggest that the mtDNA 16189 variant is a predisposing genetic factor for the development of insulin resistance and may be related to various phenotypic expressions in adult life such as development of DM and vascular pathologies involved in stroke and cardiovascular diseases.