Ching-You Lu

Oxidative Damage and Mutation to Mitochondrial DNA and Age‐dependent Decline of Mitochondrial Respiratory Functiona

ABSTRACT: Mitochondrial respiration and oxidative phosphorylation are gradually uncoupled, and the activities of the respiratory enzymes are concomitantly decreased in various human tissues upon aging. An immediate consequence of such gradual impairment of the respiratory function is the increase in the production of the reactive oxygen species (ROS) and free radicals in the mitochondria through the increased electron leak of the electron transport chain. Moreover, the intracellular levels of antioxidants and free radical scavenging enzymes are gradually altered. These two compounding factors lead to an age‐dependent increase in the fraction of the ROS and free radical that may escape the defense mechanism and cause oxidative damage to various biomolecules in tissue cells. A growing body of evidence has established that the levels of ROS and oxidative damage to lipids, proteins, and nucleic acids are significantly increased with age in animal and human tissues. The mitochondrial DNA (mtDNA), although not protected by histones or DNA‐binding proteins, is susceptible to oxidative damage by the ever‐increasing levels of ROS and free radicals in the mitochondrial matrix. In the past few years, oxidative modification (formation of 8‐hydroxy‐2′‐deoxyguanosine) and large‐scale deletion and point mutation of mtDNA have been found to increase exponentially with age in various human tissues. The respiratory enzymes containing the mutant mtDNA‐encoded defective protein subunits inevitably exhibit impaired respiratory function and thereby increase electron leak and ROS production, which in turn elevates the oxidative stress and oxidative damage of the mitochondria. This vicious cycle operates in different tissue cells at different rates and thereby leads to the differential accumulation of mutation and oxidative damage to mtDNA in human aging. This may also play some role in the pathogenesis of degenerative diseases and the age‐dependent progression of the clinical course of mitochondrial diseases.

Aging- and smoking-associated alteration in the relative content of mitochondrial DNA in human lung.

mtDNA mutations and oxidative DNA damage has been observed to accumulate in the lung and other tissues in human aging. Thus, it is of interest to know whether the content of mtDNA is changed in aging tissues of the human. Using a competitive PCR method, we determined the relative content of mtDNA in the lung tissues of 49 subjects aged 16–85 years. The results showed that the relative content of mtDNA (with respect to the β‐actin gene) in the lung tissues was significantly increased with age (P<0.005). The average mtDNA content in the lung tissues of the subjects over 80 years of age was found to be about 2.6‐fold higher than that of the subjects below age 20. However, the relative content of mtDNA was slightly increased in the lung tissues of light smokers but significantly decreased in heavy smokers. Moreover, we found a significant increase with age in the level of oxidative damage to DNA as indicated by the ratio of 8‐OH‐dG/dG in total DNA (P<0.0005). These results together with our previous findings suggest that the increase in mtDNA content of aging tissues may be effected through a feedback mechanism to compensate for the functional decline of mitochondria in human aging and that smoking may modulate the mechanism.

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.

Upregulation of Matrix Metalloproteinase 1 and Disruption of Mitochondrial Network in Skin Fibroblasts of Patients with MERRF Syndrome

Abstract: By using cDNA microarray and RT‐PCR techniques, we investigated the genome‐wide alteration of gene expression in skin fibroblasts from patients with myoclonic epilepsy and ragged‐red fibers (MERRF) syndrome. By screening for the genes with altered levels of expression, we first discovered that matrix metalloproteinase 1 (MMP1) was highly induced in the primary culture of skin fibroblasts of a female patient in a four‐generation family with MERRF syndrome. This phenomenon was confirmed in skin fibroblasts from three other MERRF patients harboring about 85% of mtDNA with A8344G mutation. A further study revealed that the expression of MMP1 could be further induced by treatment of the skin fibroblasts with 200 μM hydrogen peroxide (H2O2) and inhibited by 1 mM N‐acetylcysteine. Moreover, the intracellular level of H2O2 in skin fibroblasts of the female MERRF patient was higher than those of the asymptomatic family members and age‐matched healthy controls. These findings imply that the increase in the expression of MMP1 may represent one of the responses to the increased oxidative stress in the skin fibroblasts of MERRF patients. We suggest that in affected tissues the oxidative stress‐elicited overexpression of MMP1, and probably other matrix metalloproteinases involved in cytoskeleton remodeling, may play an important role in the pathogenesis and progression of mitochondrial encephalomyopathies such as MERRF syndrome.

INDEPENDENT OCCURRENCE OF SOMATIC MUTATIONS IN MITOCHONDRIAL DNA OF HUMAN SKIN FROM SUBJECTS OF VARIOUS AGES

The incidence (frequency of occurrence) and abundance (percentage of mutant out of total mtDNA population) of two different somatic mtDNA mutations in human skin were investigated in 44 subjects ranging from 19 to 87 years of age. Using quantitative allele‐specific polymerase chain reaction (AS‐PCR) to analyse the A→G base substitution at nucleotide 3243, 50% of the samples showed detectable levels of that particular mutation, with abundances ranging from 0.01% to 0.12%. In the same set of skin samples, the overall incidence of the 4977 bp “common” deletion was also ˜ 50%. Where detected, the abundance of this deletion ranged from 0.0002% to 0.1%. Comparative analyses of the incidence and abundance of these two mutations, collectively and in individual skin samples, led to these two conclusions: (1) there is independent occurrence of these two mtDNA mutations in human skin, and (2) whereas the 4977 bp deletion shows an age‐associated accumulation in human skin, no age association is apparent for the 3243 A→G base substitution. Furthermore, in general, there is a much lower incidence of somatic mutations in mtDNA of human skin as compared to that in postmitotic tissues such as skeletal muscle. Hum Mutat 11:191–196, 1998.