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Konstantin Khrapko

Publications and source records attributed to Konstantin Khrapko.

16 recordsLinked to original sources
PubMed2006

Mitochondrial DNA deletions are abundant and cause functional impairment in aged human substantia nigra neurons.

Using a novel single-molecule PCR approach to quantify the total burden of mitochondrial DNA (mtDNA) molecules with deletions, we show that a high proportion of individual pigmented neurons in the aged human substantia nigra contain very high levels of mtDNA deletions. Molecules with deletions are largely clonal within each neuron; that is, they originate from a single deleted mtDNA molecule that has expanded clonally. The fraction of mtDNA deletions is significantly higher in cytochrome c oxidase (COX)-deficient neurons than in COX-positive neurons, suggesting that mtDNA deletions may be directly responsible for impaired cellular respiration.

Aging↗
PubMed2006

Origins of human mitochondrial point mutations as DNA polymerase gamma-mediated errors.

Mitochondrial mutational spectra in human cells, tissues and derived tumors for bp 10,030-10,130 are essentially identical, suggesting a predominant mutagenic role for endogenous processes. We hypothesized that errors mediated by mitochondrial DNA polymerase gamma were the primary sources of mutations. Point mutations created in this sequence by human DNA pol gamma in vitro were thus compared to the eighteen mutational hotspots, all single base substitutions, previously found in human tissues. The set of concordant hotspots accounted for 83% of these in vivo mutational events. About half of these mutations are insensitive to prolonged heating of DNA during PCR and half increase proportionally with heating time at 98 degrees C. Primary misincorporation errors and miscopying errors past thermal denaturing products such as deaminated cytosines (uracils) thus appear to be of approximately equal importance. For the sequence studied, these data support the conclusion that, endogenous error mediated by DNA pol gamma constitutes the primary source of mitochondrial point mutations in human tissues.

Base Sequence↗
PubMed2006

Does premature aging of the mtDNA mutator mouse prove that mtDNA mutations are involved in natural aging?

Recent studies have demonstrated that transgenic mice with an increased rate of somatic point mutations in mitochondrial DNA (mtDNA mutator mice) display a premature aging phenotype reminiscent of human aging. These results are widely interpreted as implying that mtDNA mutations may be a central mechanism in mammalian aging. However, the levels of mutations in the mutator mice typically are more than an order of magnitude higher than typical levels in aged humans. Furthermore, most of the aging-like features are not specific to the mtDNA mutator mice, but are shared with several other premature aging mouse models, where no mtDNA mutations are involved. We conclude that, although mtDNA mutator mouse is a very useful model for studies of phenotypes associated with mtDNA mutations, the aging-like phenotypes of the mouse do not imply that mtDNA mutations are necessarily involved in natural mammalian aging. On the other hand, the fact that point mutations in aged human tissues are much less abundant than those causing premature aging in mutator mice does not mean that mtDNA mutations are not involved in human aging. Thus, mtDNA mutations may indeed be relevant to human aging, but they probably differ by origin, type, distribution, and spectra of affected tissues from those observed in mutator mice.

Adult↗
PubMed2005

Recombination of mitochondrial DNA in skeletal muscle of individuals with multiple mitochondrial DNA heteroplasmy.

Experimental evidence for human mitochondrial DNA (mtDNA) recombination was recently obtained in an individual with paternal inheritance of mtDNA and in an in vitro cell culture system. Whether mtDNA recombination is a common event in humans remained to be determined. To detect mtDNA recombination in human skeletal muscle, we analyzed the distribution of alleles in individuals with multiple mtDNA heteroplasmy using single-cell PCR and allele-specific PCR. In all ten individuals who carried a heteroplasmic D-loop mutation and a distantly located tRNA point mutation or a large deletion, we observed a mixture of four allelic combinations (tetraplasmy), a hallmark of recombination. Twelve of 14 individuals with closely located heteroplasmic D-loop mutation pairs contained a mixture of only three types of mitochondrial genomes (triplasmy), consistent with the absence of recombination between adjacent markers. These findings indicate that mtDNA recombination is common in human skeletal muscle.

DNA, Mitochondrial↗
PubMed2005

Clustering of mutant mitochondrial DNA copies suggests stem cells are common in human bronchial epithelium.

Tissue maintenance stem cells, as opposed to transition and/or terminal cells in the epithelium, are possible progenitor cells for human tumors, but little is known about their frequency in human tissues. It occurred to us that the colonies of mutants that should be created when a stem cell mutates and transmits the rare mutation to its descendent transition and terminal cells should, given a quantitative mutation assay, define the average number of cells in a maintenance turnover unit and permit calculation of stem cell number. To test this concept we used a combination of high fidelity PCR and constant denaturant capillary electrophoresis to enumerate mitochondrial point mutations and define their number and distribution among multiple small samples of approximately one million cells containing about 400 million copies of mitochondrial DNA. The bulk of the data were best explained by a model in which most stem cells, defined here as long-lived cells, give rise to colonies of approximately 8-128 cells. In addition, we found that about 1.5% of colonies contained hundreds or even thousands of homoplasmic mutant cells. These expanded turnover units suggest the bronchial epithelium may contain large clusters of cells with mutations, and possibly phenotypic alterations as well.

Aged, 80 and over↗
PubMed2005

Mitochondrial DNA gene therapy: a gene therapy for aging?

Mutations in mitochondrial DNA cause a group of diverse diseases that affect an estimated half a million people worldwide. These disorders are remarkably resistant to conventional treatments, and thus several gene therapy approaches are being explored. As some of these approaches develop towards maturity, one can't help thinking that some day they may be used against a much more common health problem currently affecting about 6 billion people- aging, which also has been quite resistant to treatment. Unfortunately, we still do not know whether mtDNA mutations significantly contribute to the aging process or not. The prospect of success in mtDNA gene therapy makes getting the answer a high priority.

Aging↗
PubMed2005

Single-molecule PCR: an artifact-free PCR approach for the analysis of somatic mutations.

A critical review of the clone-by-clone approach to the analysis of complex spectra of somatic mutations is presented. The study of a priori unknown somatic mutations requires painstaking analysis of complex mixtures of multiple mutant and non-mutant DNA molecules. If mutant fractions are sufficiently high, these mixtures can be dissected by the cloning of individual DNA molecules and scanning of the individual clones for mutations (e.g., by sequencing). Currently, the majority of such cloning is performed using PCR fragments. However, post-PCR cloning may result in various PCR artifacts - PCR errors and jumping PCR - and preferential amplification of certain mutations. This review argues that single-molecule PCR is a simple alternative that promises to evade the disadvantages inherent to post-PCR cloning and enhance mutational analysis in the future.

Alleles↗
PubMed2004

Where and when do somatic mtDNA mutations occur?

It is generally assumed that somatic mtDNA mutations are originally created in the cells where these mutations are currently found. Accumulating data indicate, however, that cells with a particular mtDNA mutation tend to "cluster," that is, occur repeatedly within a given sample, but not in the others. Clusters likely are clonal, which implies that mtDNA mutations do not originate in the cells that currently carry them, but rather in those cells' progenitors, such as stem or satellite cells, or even earlier in the development. Importantly, a majority of mtDNA mutations appear to belong to such clusters, and thus mutational events in progenitor cells may be one of the major sources of mtDNA mutations in healthy aging tissue. More research including the analysis of multiple samples per individual is needed to confirm the existence of clustering and to distinguish between the possible clustering mechanisms.

Aging↗
PubMed2003

Clonal expansions of mitochondrial genomes: implications for in vivo mutational spectra.

It is often assumed mutant frequencies, as measured in a DNA sample, faithfully represent basic mutation rates associated with these mutations. This paradigm was extremely helpful for in vitro studies of the mechanisms of mutagenesis/repair and causes of mutations. However, in vivo, mutant fractions appear to vary dramatically and randomly from sample to sample. It's unlikely that basic mutational rates vary so much. Such variations are probably caused by clonal expansions of mutants within tissue. Whether a particular tissue sample includes an expansion or not, is a matter of chance, which explains the observed random fluctuations of mutant fractions. Well-known examples of clonal expansions involve pathological conditions such as cancer or mitochondrial disease. It is less appreciated that even in normal tissue, expansions of somatic mutants create local deviations from the "expected" mutant frequencies. The sizes of clonal expansions appear to span a wide range and thus, may affect samples of various sizes, from individual cells to individuals. In conclusion, human body appears to be a sort of a "gambling ground" for clonally expanding mutants. We speculate that expansion of early mutants rather than de novo mutation at old age may be the major source of at least some aging-specific mutants in our bodies.

Animals↗
PubMed2003

Mutation and intracellular clonal expansion of mitochondrial genomes: two synergistic components of the aging process?

The foundations of the Mitochondrial mutational theory of aging include two assumptions: the high abundance of mitochondrial mutations and their ability to clonally expand within individual cells. The up-to-date data pertinent to these assumptions is reviewed and semi-quantitative estimates of the frequencies of mutants and intracellular expansions are offered. The incidence of mutations in various aged tissues may be on the order of one mutant per mitochondrial genome copy, and most of the cells are likely to be affected by intracellular clonal expansions of mitochondrial genomes. Thus aged tissue may be considered a mosaic of cells with different mutant mitochondrial genotypes. Interestingly, independent studies show that a wide range of aged tissues presents with a mosaic of cells with different mitochondrial phenotypes. The necessary methodologies are available to explore whether the two mosaics are causally related. The answer apparently is positive in muscle; other tissues, brain in particular, await exploration.

Aging↗
PubMed2003

A microRNA array reveals extensive regulation of microRNAs during brain development.

Several hundred microRNAs (miRNAs) have recently been cloned from a wide range of organisms across phylogeny. Despite the high degree of conservation of miRNAs, their functions in general, and in mammals particularly, are just beginning to be defined. Here we show that an oligonucleotide DNA array can be successfully used for the simultaneous analysis of miRNA expression profiles from tissues or cells. From a subset of miRNAs expressed in the brain we designed an oligonucleotide array spotted with probes specific for 44 mature miRNAs. These arrays demonstrated precise regulation of miRNA expression at mammalian brain developmental epochs. About 20% of the probed miRNAs changed significantly in their expression during normal brain development, and two of them, miR-9 and miR-131, were dysregulated in presenilin-1 null mice exhibiting severe brain developmental defects. Transcripts with regulated expression patterns on the arrays were validated by Northern blots. Additionally, a bioinformatic analysis of developmentally regulated miRNAs suggested potential mRNA targets. The arrays also revealed miRNAs distributed to translating polyribosomes in primary neurons where they are likely to modulate translation. Therefore, oligonucleotide arrays provide a new tool for studying miRNA expression in a variety of biological and pathobiological settings. Creating clusters of coexpressed miRNAs will contribute to understanding their regulation, functions, and discovery of mRNA targets.

Animals↗
PubMed2002

Gene expression profiling of the aging mouse cardiac myocytes.

Heart disease remains the most frequent cause of death in the general population with increasing incidence in the elderly population. The pathologic failure of the aging heart may be related to structural and functional alterations in cardiac muscle cells. However, the molecular mechanisms underlying the aging-related decline in cardiac muscle function are largely unknown. To provide the first analysis of cardiac aging at the level of gene expression, we established and compared cDNA libraries from apparently healthy young and aged mouse ventricular cardiac muscle cells. We report the identification of genes that exhibit aging-related changes of mRNA levels. Aging expression profiles in aged hearts indicate decreased cellular adaptation and protection against stress-induced injury together with the development of contractile dysfunction. The data suggest reduced activity of the mitochondrial electron transport system and reduced levels of cardiac-specific transcription regulators. The cardiomyocyte aging profile of gene expression displays similarities with known heart disorders. Genes whose mRNA levels change with aging in cardiomyocytes might profoundly affect pathological changes in the heart.

Actinin↗
PubMed2002

mtLOH (mitochondrial loss of heteroplasmy), aging, and 'surrogate self'.

In tribute to Dr Strehler, an attempt is made to use a style of reasoning found in some of his later papers as an outline of this article. First, general arguments in favor of the involvement of somatic mutations in mtDNA in the aging process are presented. Second, evidence is provided in support of a general tendency of mitochondrial genomes to reach homoplasmic state at the cellular level, for which we propose the term mitochondrial loss of heteroplasmy (mtLOH). This process is likely to facilitate the involvement of mtDNA mutations in the aging process by streamlining the phenotypic expression of the mutant genotype. Third, preliminary evidence of the very high incidence of clonal deletions in pigmented neurons of substantia nigra is reported. This observation highlights the possibility that accumulation of mtDNA mutations specific in certain cell types of a complex tissue may account for the involvement of mtDNA mutations in the aging process despite the relatively low average incidence of these mutations in the tissue as a whole. High incidence of mtDNA deletions in pigmented neurons evokes Strehler's idea that efforts to delay aging may not be the most cost-efficient way of preserving 'self awareness and a joyful sense of life', as he put it. A potential alternative suggested by Strehler, i.e. creation of a 'surrogate self' by computer simulation may deserve more attention than it currently enjoys.

Aging↗
PubMed2002

Clonally expanded mtDNA point mutations are abundant in individual cells of human tissues.

Using single-cell sequence analysis, we discovered that a high proportion of cells in tissues as diverse as buccal epithelium and heart muscle contain high proportions of clonal mutant mtDNA expanded from single initial mutant mtDNA molecules. We demonstrate that intracellular clonal expansion of somatic point mutations is a common event in normal human tissues. This finding implies efficient homogenization of mitochondrial genomes within individual cells. Significant qualitative differences observed between the spectra of clonally expanded mutations in proliferating epithelial cells and postmitotic cardiomyocytes suggest, however, that either the processes generating these mutations or mechanisms driving them to homoplasmy are likely to be fundamentally different between the two tissues. Furthermore, the ability of somatic mtDNA mutations to expand (required for their phenotypic expression), as well as their apparently high incidence, reinforces the possibility that these mutations may be involved actively in various physiological processes such as aging and degenerative disease. The abundance of clonally expanded point mutations in individual cells of normal tissues also suggests that the recently discovered accumulation of mtDNA mutations in tumors may be explained by processes that are similar or identical to those operating in the normal tissue.

Adult↗
PubMed2002

Frequent intracellular clonal expansions of somatic mtDNA mutations: significance and mechanisms.

It has been proposed that age-dependent accumulation of somatic mutations in mtDNA is responsible for some aspects of the aging process. However, most cells contain hundreds to thousands of mtDNA molecules. Any nascent somatic mutant therefore appears as a single copy among a majority of wild-type species. A single mutant molecule is unlikely to influence the physiology of the cell and thus cannot play a role in the aging process. To affect cellular physiology, the nascent somatic mutants must somehow accumulate clonally in the cell to significant levels. The evidence supporting the view that, indeed, clonal expansion of mtDNA mutations is a widespread process in various human tissues, and the mechanisms by which clonal expansions may affect the aging process, are reviewed. Originally, clonal expansion was demonstrated for mtDNA with large deletions in muscle. Cell-by-cell analysis of human cardiomyocytes and buccal epithelial cells revealed that clonal expansion affects point mtDNA mutations as well as deletions. Expansions are not limited to muscle, but likely are present in most tissues, and almost every cell of an aged tissue is likely to be affected by an expansion. While the very existence of clonal expansion is beyond doubt, the mechanisms driving this process are largely controversial. The hypotheses explaining expansion includes random or various selective mechanisms, or both. We show that the spectra of expanded point mutations are drastically different in cardiomyocytes and epithelial cells. This suggests that the mechanisms of expansion in these tissues are different. In particular, we propose random segregation and positive selection models for epithelial and muscle cells, respectively.

Aging↗