The connection between COX activity and mtDNA mutations is contradictory
Since most cellular energy is generated in mitochondria by OXPHOS, a correlation between aging and mitochondrial functions is strongly suggested and has been shown for respiratory chain activity in different tissues (77,78). Post-mitotic tissues develop a bioenergy mosaic during the process of normal aging that eventually can culminate into a bioenergetically diverse tissue containing cells differing in their OXPHOS capacity from normal to grossly defective. Using a single-cell extra long PCR (XLPCR), it was shown that COX-deficient muscle fibers extracted from different individuals, regardless of age, were accompanied by extensive mtDNA rearrangements and reduced levels of full-length mtDNA (79-81). These observations indicate evidence linking mtDNA mutations to COX activity decline in skeletal muscle.
It is possible that accumulation of mutations of mtDNA, probably induced by continued exposure to ROS, leads to errors in the mtDNA-encoded polypeptide chains. These errors are then stochastic and randomly transmitted during mitochondrial division and cell division. The consequence would be defective electron transfer and OXPHOS, which could lead to a higher production of ROS, leading to more damage to the mtDNA creating a vicious circle of mtDNA mutations and oxidative stress (82), reviewed in (2). On the other hand, experiments with human fibroblasts showed that mutations of the mtDNA are not responsible for age-related respiratory deficiencies of the cell. The authors suggest nuclear recessive mutations are involved in mitochondrial translation and so are responsible for mitochondrial respiratory deficiencies (83). Tengan et al. also criticize the idea of a vicious cycle in mitochondria by comparing the amounts of the 4977-bp deletion in normal controls and in patients with genetically characterized mitochondrial disorders associated with pathogenic mtDNA point mutations or deletions other than the common deletion and by amplification of the mitochondrial genome in those samples to detect every possible deletion. They found a positive correlation between age and the 4977-bp deletion but no co-segregation of pathogenic point-mutated mtDNA with the common deletion and no increased number of age-related deletions in the patients (84).
Oxidative stress may play a role in the aging process
The idea that oxidative stress can be important for the aging process (37) is strongly supported by the findings that amelioration of oxidative stress by the overexpression of Cu, Zn superoxide dismutase, and catalase significantly lowers the level of oxidative damage and extends the life span of transgenic Drosophila melanogaster (85). Other evidences for this hypothesis are the high level of oxidative damage and its accumulation with age, the correlation between oxidative damage and maximum life span potential, and the increased oxidative damage and premature aging found in people with Down syndrome (86).
mtDNA is affected by multiple alterations
Alterations to the structure of mtDNA are identified in a variety of tissues in rats (16,87), mice (88), rhesus monkeys (89), nematodes (90), D. melanogaster (91), and also in almost all human tissues, be it post-mitotic differentiated tissues such as skeletal muscle (17), heart muscle (19), and brain (18,92), or in highly replicative tissues such as skin (21,93) or blood (22). Even in hair follicles (94), oocytes (95), or sperm cells (96) mutated mtDNA was found. Alterations to mtDNA include large deletions (97), point mutations (98), insertions, and short duplications (reviewed in (2,99)). By using a special PCR covering the whole mitochondrial genome, fragmentation of mtDNA into various sizes of deleted molecules up to more than 100 types could be observed (20,100).
Point mutations of mtDNA seem to accumulate randomly
There are several reports showing contradictory results concerning the increase of point mutations with age. In 1998, one working group showed a correlation between three different mtDNA mutations and age, including the probably most frequent 3243 A to G point mutation with a correlation coefficient of r = 0.57 (101) and confirmed earlier results (98,102). By using a QPCR method, a different occurrence of point mutations in mtDNA of human muscle was presented (103). Point mutations were detectable at a variety of positions at the mitochondrial genome of both young and old individuals indicating random occurrences at the level of base substitutions. They seemed to be primarily spontaneous in origin and arise either from DNA replication error or from reactions of DNA with endogenous metabolites (104). The finding, that the original tissue samples displayed a spectrum similar to that observed in human cell culture, suggests a common pathway and seems to disprove the hypothesis that environmental mutagens are important contributors to mitochondrial point mutagenesis. Pallotti et al. used a modified PCR/RFLP, a so-called "double PCR and digestion (DPD)" method, in which a minority of mutated mtDNA sequences can be enriched. They detected levels of point mutations between 0.002 and 0.040% without any correlation to the age of the subject (105). The discrepancy of these reports concerning age dependence may be due to several factors, including the specific mutations, the authors investigated, or the method of detection, the tissue, or the age of the sample itself.
The question arises why the mtDNA is affected by so many induced sequence alterations but can still be investigated for identification purposes. One explanation could be that no point mutation exceeds the amount which can be detected by sequencing analysis, i.e. about 5-10%. Nevertheless, it should be stressed that reports exist on heteroplasmy of mtDNA sequences in the D-loop within one individual (106) even though other investigations did not detect point mutations in the control region in normal aging and neurodegenerative human brains, suggesting that mutations in the D-loop region did not contribute to the aging and degenerative process in vivo (107) and thus obviously would not be a disturbing factor for forensic analysis. Other reports of an age-related accumulation of sequence alterations in the D-loop have been contradictory since both possibilities, accumulation (101) and no accumulation (105) have been reported.
The 4977-bp deletion might be a marker molecule for the aging process
The most common deletion of mtDNA is the 4977-bp deletion, which has been observed in biopsy (108) and autopsy material from individuals aged 20 years and above (109-112). It occurs in the highest percentage and with the closest correlation to age in well-differentiated tissues such as brain and muscle tissue (17). The reason for this relatively high frequency of the common deletion is probably the structure of the DNA flanking their breakpoints. Hou et al. showed a retarded and increased mobility in this special region and suggested that these frequencies are rendered to assume a more distorted structure than B-DNA by the two flanking bent-inducing DNA sequences in organelles and thereby render this region to be more vulnerable to attacks by ROS and free radicals (113). Using a kinetic PCR, we detected levels starting from 0.00049 to 0.14% for the 4977-bp deleted mtDNA in skeletal muscle of aged individuals older than 20 years (111). The findings summarized here confirm many other investigations regarding the occurrence of the common deletion in skeletal muscle, its accumulation with age up to a physiological amount of less than 1% deleted mtDNA (101,103,108,114-116).
Thus, the common 4977-bp deletion with its strong correlation to the age of an individual might be a tool for the estimation of the age of an unknown individual based only on soft tissue. However, the confidence interval is rather wide (111,116,117). Therefore, the method is not as reliable as an age estimation based on the racemization of aspartic acid in bones or teeth (118,119). But the 4977-bp deletion works well as a biomarker of skin photo aging pointing up the correlation between mtDNA damage and exogenous stressors (120).
mtDNA mutations are not distributed equally in different tissues
The distribution of the mutated mtDNA molecules differs widely between tissues, e.g. in different regions of the brain (92), even between cells and can result in either focal or mosaic effects of the organism.
To answer the unequal distribution of mtDNA alterations and their effects on mito-chondrial function, in situ investigations seem to be an useful method as presented by Kovalenko et al. in 1997 and could be useful for studying the localization of mtDNA mutations in individual cells of the tissues. This could then lead to more insights into the correlation between mutations and bioenergetic effects in single cells (67).
mtDNA copy number may also increase with advanced age
In addition to the qualitative changes of mtDNA mentioned above, the copy number of mtDNA in animal tissue has also been shown to change with age. Petruzella et al. detected a significant decrease of the D-loop number in rat brain tissues, using southern hybridization (120,121). The mtDNA is apparently affected by multiple deletions which also occur in an age-dependent correlation (101) as it increases the copy number of the total mitochondrial genome in human tissue (122). This phenomenon, e.g. the amount of total mtDNA in human dentin, was already investigated under the aspect of age dependency for a forensic approach, unfortunately for the forensic community without any chances of its successful employment (123). mtDNA content seems to depend - similar to the mtDNA alterations -on many other factors. Our studies showed a threefold transient increase of total mtDNA in NT2 cells after the first 13 days of treatment with 10 ^M EtBr. This elevation might be due to compensation effects of the cell (70). Other authors showed that cells, e.g. from brain tissue, can exhibit an age-related increase in total mtDNA content, that coincided with a decrease in mtRNA levels. This was proposed as an inefficient compensatory mechanism to maintain the normal levels of RNA transcripts (124). In experiments with cybrids from human fibroblasts, it was shown that these cells answered a treatment with hydrogen peroxide with an increase of the relative mtDNA content by 17-30% and additionally with an increase of 4977-bp deleted mtDNA (125) supporting the compensation theory. Using real-time PCR, we could not find any correlation between mtDNA copy number and the age of an individual in blood from male individuals. The oldest individual investigated in this study was in his 50s. Thus, it might be possible that alterations in mtDNA content only occur in later years. Regarding the literature, findings are still controversial. Some authors confirm our results, finding no age-dependent specific differences in the mtDNA: nDNA ratio in different tissues (126,127). Others detected a steady increase of mtDNA in skeletal muscle and a decrease in blood with age, investigating 300 skeletal muscle samples and 200 blood samples from patients with a broad range of age suspected of having a mitochondrial disorder (128). It could be possible that such a steady age-dependent alteration is only detectable in patients suffering from mitochondrial diseases and not in controls with normal mitochondria. However, this possible correlation did not seem striking since we did not find any hints for age dependence.
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