Chunfang Zhang

Multiple mitochondrial DNA deletions in an elderly human individual.

We have used the polymerase chain reaction (PCR) to study deletions in the mitochondrial DNA (mtDNA) of an elderly human individual. An extended set of PCR primers has been utilised to identify 10 mitochondrial DNA deletions in a 69‐year‐old female subject with no known mitochondrial disease. The particular deletions visualised as PCR products depended on the primer pairs used, such that the more distantly separated PCR primers enabled visualisation of larger deletions. Some deletions were common to the heart, brain and skeletal muscle, whereas others were apparently specific to individual tissues. DNA sequencing analysis of PCR products showed that short direct repeat sequences (5 to 13 bp) flanked all deletion breakpoints; in most cases one copy of the repeat was deleted. It is proposed that the accumulation of such multiple deletions is a general phenomenon during the ageing process.

Mitochondrial DNA mutation and the ageing process: bioenergy and pharmacological intervention

A comprehensive hypothesis concerning the contribution of mitochondrial DNA (mtDNA) mutations to the human ageing process is reviewed and the implications for cellular bioenergy loss and pharmacological therapy are considered. The central idea is that random mutations in the population of mtDNA molecules of each cell occur throughout life, and that this is a major contributor to the gradual loss of cellular bioenergy capacity within tissues and organs, associated with general senescence and diseases of ageing. An elaboration of four major aspects of the general proposition, together with relevant supporting data, is presented. (1) An extensive array of deletions in mtDNA of many tissues of humans and other mammals has been observed to occur in an age-related manner. (2) The preservation and selection of fully functional mtDNA molecules in the female germ line cells is proposed to occur via a human mtDNA cycle, in which selective amplification of a limited number of mtDNA templates occurs during oocyte development. This proposal explains the endowment of normal neonates with a mtDNA complement minimally contaminated by damaged mtDNA molecules. The phenomena of maternal inheritance and rapid fixation of sequence variants of mtDNA in mammals, as well as selection of cells based on mitochondrial function, are taken into account. (3) Tissue bioenergy mosaics result from accumulated mtDNA damage during ageing, representing different rates of cellular bioenergy loss within individual cells of a tissue. The random segregation of mtDNA during cell division will also further contribute to the tissue energy mosaic. Cells unable to meet their particular bioenergy demand will become non-functional, leading to cell death; the bioenergy threshold is different for the various cell types in the tissues of the body. (4) In order to bioenergetically resuscitate cells and tissues suffering from impaired mitochondrial functions as a result of the ageing process, we propose that redox compounds may be used therapeutically in the pharmacological configurations of a by-pass strategy or as a redox sink therapy. The role of these compounds is to maintain at least part of the mitochondrial respiratory chain function (by-pass) as well as to maintain adequate levels of cellular NAD+ (redox sink) for ATP synthesis, predominantly by the cytosolic glycolytic pathway, with some contribution from mitochondrial oxidative phosphorylation.

Cellular redox activity of coenzyme Q10: effect of CoQ10 supplementation on human skeletal muscle.

In this paper, we report results obtained from a continuing clinical trial on the effect of coenzyme Q 10 (CoQ 10 ) administration on human vastus lateralis (quadriceps) skeletal muscle. Muscle samples, obtained from aged individuals receiving placebo or CoQ 10 supplementation (300 mg per day for four weeks prior to hip replacement surgery) were analysed for changes in gene and protein expression and in muscle fibre type composition. Microarray analysis (Affymetrix U95A human oligonucleotide array) using a change in gene expression of 1.8-fold or greater as a cutoff point, demonstrated that a total of 115 genes were differentially expressed in six subject comparisons. In the CoQ 10 -treated subjects, 47 genes were up-regulated and 68 down-regulated in comparison with placebo-treated subjects. Restriction fragment differential display analysis showed that over 600 fragments were differentially expressed using a 2.0-fold or greater change in expression as a cutoff point. Proteome analysis revealed that, of the high abundance muscle proteins detected (2086 - 115), the expression of 174 proteins was induced by CoQ 10 while 77 proteins were repressed by CoQ 10 supplementation. Muscle fibre types were also affected by CoQ 10 treatment; CoQ 10 -treated individuals showed a lower proportion of type I (slow twitch) fibres and a higher proportion of type IIb (fast twitch) fibres, compared to age-matched placebo-treated subjects. The data suggests that CoQ 10 treatment can act to influence the fibre type composition towards the fibre type profile generally found in younger individuals. Our results led us to the conclusion that coenzyme Q 10 is a gene regulator and consequently has wide-ranging effects on over-all tissue metabolism. We develop a comprehensive hypothesis that CoQ 10 plays a major role in the determination of membrane potential of many, if not all, sub-cellular membrane systems and that H 2 O 2 arising from the activities of CoQ 10 acts as a second messenger for the modulation of gene expression and cellular metabolism.

Differential occurrence of mutations in mitochondrial DNA of human skeletal muscle during aging

Seven mtDNA mutations (five base substitutions and two deletions) were studied in skeletal muscle samples of 18 human subjects aged 1 hr to 90 years. Quantitative PCR procedures were applied to determine the incidence (frequency of occurrence) and abundance (percentage of mutant mtDNA out of total mtDNA). The base substitutions, in general, showed a very early onset, three such mutations being detectable in the muscles of infants aged 1 hr and 5 weeks. Of two disease‐associated point mutations studied, 3243 A→G showed significant accumulation with age (P < 0.05), while 8993 T→G showed no significant age accumulation (P > 0.1). Moreover, three arbitrarily chosen mutations (not disease‐associated) showed no age‐associated accumulation: two (7029 C→T and 7920 A→G) showed little change over the years (P > 0.1), while the other (13167 A→G) showed a significant decrease (P < 0.05). Both the 4,977‐bp and 7,436‐bp deletions showed a significant age‐associated occurrence (P < 0.01 and P < 0.05, respectively). The age of onset of detectable deletions is about 20–40 years; thereafter, the incidence and abundance of deletions tend to increase as a function of advancing age. The seven specific mutations were found to occur independent of each other, indicating the random nature of mtDNA mutations in skeletal muscle. Moreover, the age‐associated accumulation of multiple deletions was observed in the same set of muscle tissues, each extract displaying a unique set of multiple PCR products. Thus, mutations in mtDNA occur differentially in human skeletal muscle during aging. Hum Mutat 11:360–371, 1998.

Human Aging and Global Function of Coenzyme Q10

In this paper, we review two parts of our recent work on human skeletal muscle. The first part mainly describes changes occurring during aging, whereas the second part discusses the functions of coenzyme Q10 (CoQ10), particularly in relation to the aging process. During the lifetime of an individual, mtDNA undergoes a variety of mutation events and rearrangements. These mutations and their consequent bioenergenic decline, together with nuclear DNA damage, contribute to the reduced function of cells and organs, especially in postmitotic tissues. In skeletal muscle, this functional decline can be observed by means of changes with age in fiber type profile and the reduction in the number and size of the muscle fibers. In addition to the functions of coenzyme Q10 as an electron carrier in the respiratory chain and as an antioxidant, CoQ10 has been shown to regulate global gene expression in skeletal muscle. We hypothesize that this regulation is achieved via superoxide formation with H2O2 as a second messenger to the nucleus.

Quantitative allele‐specific PCR: Demonstration of age‐associated accumulation in human tissues of the A→G mutation at nucleotide 3243 in mitochondrial DNA

We have developed an improved allele‐specific polymerase chain reaction (AS‐PCR) procedure that can selectively amplify mutant DNA sequences (which differ from the normal sequences by a single base pair) in the presence of large excess of normal sequences. We applied this procedure to quantification of mutant molecules of human mitochondrial DNA (mtDNA). Conditions for AS‐PCR have been systematically varied, encompassing DNA template input, annealing temperature, and PCR cycle number. Adjustment of these three reaction parameters to optimal conditions, using plasmids containing cloned segments of mutant and normal mtDNA, enabled the reliable detection of as little as 0.01% of mutant mtDNA. By standardising the DNA input for AS‐PCR, the percentage of mutant molecules can be accurately quantified. This improved procedure was used here to detect and quantify the base substitution at nucleotide position 3243 (A→G) in mtDNA from total cellular DNA isolated from various tissues of both infants and adults. We observed a 5‐ to 10‐fold higher mutant mtDNA (3243 A→G) frequency in adult tissues than in infant tissues. The results are consistent with the hypothesis that the accumulation of mtDNA mutations is an important feature of the human aging process. The quantitative and sensitive allele‐specific amplification system described here is applicable to the quantification of low levels of somatic mutations in oncogenes and tumour suppressor genes in the context of human mutation, and could be extended to any biological situation in which only a small proportion of a DNA molecular population is subjected to a particular base substitution. Hum Mutat 9: 265–271, 1997.

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.

Stochastic mitochondrial dna changes : Bioenergy decline in type I skeletal muscle fibres correlates with a decline in the amount of amplifiable full-length mtDNA

Extra-long PCR (XL-PCR) was used to assess the relative concentration of functional full-length mitochondrial DNA (mtDNA) in single type I human vastus lateralis muscle fibres of defined cytochrome c oxidase (COX)activity. Type I muscle fibres rely more on mitochondrial oxidative phosphorylation for their energy demands, compared to the other common fibre types (IIa, IIab and IIb) that principally depend on glycolysis for their energy requirements. A total of 60 single type I fibres were analyzed from 15 individuals (8males and 7 females) of various ages. COX positive muscle fibres were shown to contain amplifiable full-length mtDNA together with a small number of mtDNA rearrangements. By contrast, COX negative fibres did not contain detectable full-length mtDNA, but did contain aheterogeneous mixture of rearranged mtDNA species with the frequency and occurrence of each deletion varying considerably from fibre to fibre. These data lead us to the conclusion that the level of COX activity in type I muscle fibres is reflected by the amount of amplifiable full-length mtDNA. It is proposed that the amount of amplifiable full-length mtDNA constitutes the functional fraction of the total mtDNA. A comprehensive hypothesis that relates the dynamics of mtDNA turnover, mtDNA mutations, mtDNA damage and repair to the ageing process is discussed.

Mitochondrial DNA Mutations in Aging

Human aging is characterized by the progressive decline in function at the levels of cells, tissues and organs. Various proposals have been put forward to explain the basis of aging, but the different processes envisaged should not be considered as mutually exclusive. The most prominent mechanisms proposed include genetic programming of senescence, damage to macromolecules by free radicals, molecular crosslinking, changes in immunological function, telomere shortening and somatic genetic changes in DNA.1’2 Among these mechanisms, the contribution of mitochondrial DNA (mtDNA) mutations to the aging process, with consequential changes in cellular bioenergetic functions, has attracted wide attention in recent years (for reviews, see refs. 3–8).

Mitochondrial DNA deletions parallel age-linked decline in rat sensory nerve function

In rats, the function of sensory nerves in the hind limb declines significantly with age. Normally aging rats and rats treated neonatally with capsaicin were studied here. Quantification of vascular response and substance P in young (3 months) and old (24 months) rats showed additive effects of age and capsaicin treatment. The levels in dorsal root ganglion of a particular deletion in mitochondrial DNA (mtDNA(4834)) were about 300-fold higher in old compared to young rats. Capsaicin treatment had no significant effect on mtDNA(4834) abundance. Dorsal root ganglia of old (but not young) rats were found to contain a spectrum of multiple deletions. The abundance of mtDNA(4834) in dorsal root ganglia from individual rats correlated strongly with their decline in vascular function, even where vascular responses were systematically depressed due to prior capsaicin treatment. One possibility is that mitochondrial DNA mutations directly lead to functional decline at mitochondrial and tissue levels. Alternatively, loss of mitochondrial DNA integrity and physiological decline may be consequences of the same factor, such as oxidative stress.