High aggregate burden of somatic mtDNA point mutations in aging and Alzheimer's disease brain.

The mitochondrial theory of aging proposes that mitochondrial DNA (mtDNA) accumulates mutations with age, and that these mutations contribute to physiological decline in aging and degenerative diseases. Although a great deal of indirect evidence supports this hypothesis, the aggregate burden of mtDNA mutations, particularly point mutations, has not been systematically quantified in aging or neurodegenerative disorders. Therefore, we directly assessed the aggregate burden of brain mtDNA point mutations in 17 subjects with Alzheimer's disease (AD), 10 elderly control subjects and 14 younger control subjects, using a PCR-cloning-sequencing strategy. We found that brain mtDNA from elderly subjects had a higher aggregate burden of mutations than brain mtDNA from younger subjects. The average aggregate mutational burden in elderly subjects was 2 x 10(-4) mutations/bp. The bulk of these mutations were individually rare point mutations, 60% of which changed an amino acid. Control experiments ensure that these results were not due to artifacts arising from PCR error, mistaken identification of nuclear pseudogenes or ex vivo oxidation. Cytochrome oxidase activity correlated negatively with increasing mutational burden. These findings significantly bolster the mitochondrial theory of aging.

[1]  F. Sanger,et al.  Sequence and organization of the human mitochondrial genome , 1981, Nature.

[2]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[3]  M. Wilson,et al.  Mitochondria: a practical approach. , 1987 .

[4]  Sangkot Marzuki,et al.  MITOCHONDRIAL DNA MUTATIONS AS AN IMPORTANT CONTRIBUTOR TO AGEING AND DEGENERATIVE DISEASES , 1989, The Lancet.

[5]  H G Claycamp,et al.  Phenol sensitization of DNA to subsequent oxidative damage in 8-hydroxyguanine assays. , 1992, Carcinogenesis.

[6]  J. Nobrega,et al.  Brain Cytochrome Oxidase in Alzheimer's Disease , 1992, Journal of neurochemistry.

[7]  H. Wiśniewski,et al.  Detection of point mutations in codon 331 of mitochondrial NADH dehydrogenase subunit 2 in Alzheimer's brains. , 1992, Biochemical and biophysical research communications.

[8]  L. Loeb,et al.  8-Hydroxyguanine, an abundant form of oxidative DNA damage, causes G----T and A----C substitutions. , 1992, The Journal of biological chemistry.

[9]  F. H. Lin,et al.  A comparison of single nucleotide primer extension with mispairing PCR-RFLP in detecting a point mutation. , 1992, Biochemical and biophysical research communications.

[10]  J. Cooper,et al.  Analyses of mitochondrial respiratory chain function and mitochondrial DNA deletion in human skeletal muscle: Effect of ageing , 1992, Journal of the Neurological Sciences.

[11]  D. Shibata,et al.  A pattern of accumulation of a somatic deletion of mitochondrial DNA in aging human tissues. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[12]  N. Arnheim,et al.  Deleterious mitochondrial DNA mutations accumulate in aging human tissues. , 1992, Mutation research.

[13]  M. Beal,et al.  Mitochondrial DNA deletions in human brain: regional variability and increase with advanced age , 1992, Nature Genetics.

[14]  E. Schon,et al.  Is a point mutation in the mitochondrial ND2 gene associated with Alzheimer's disease. , 1992, Biochemical and biophysical research communications.

[15]  B. Crain,et al.  Mitochondrial DNA variants observed in Alzheimer disease and Parkinson disease patients. , 1993, Genomics.

[16]  B. Ames,et al.  Oxidants, antioxidants, and the degenerative diseases of aging. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[17]  D. Price,et al.  Age‐Dependent Impairment of Mitochondrial Function in Primate Brain , 1993, Journal of neurochemistry.

[18]  C. Münscher,et al.  The point mutation of mitochondrial DNA characteristic for MERRF disease is found also in healthy people of different ages , 1993, FEBS letters.

[19]  K. Hirayasu,et al.  Purification of genomic DNA from human whole blood by isopropanol-fractionation with concentrated Nal and SDS. , 1994, Nucleic acids research.

[20]  Patrizia Mecocci,et al.  Oxidative damage to mitochondrial DNA is increased in Alzheimer's disease , 1994, Annals of neurology.

[21]  H. C. Lee,et al.  Differential accumulations of 4,977 bp deletion in mitochondrial DNA of various tissues in human ageing. , 1994, Biochimica et biophysica acta.

[22]  B. Ames,et al.  Oxidative damage and mitochondrial decay in aging. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[23]  C. Filley,et al.  Electron transport chain defects in Alzheimer's disease brain , 1994, Neurology.

[24]  M. Graeber,et al.  No association of mutations at nucleotide 5460 of mitochondrial NADH dehydrogenase with Alzheimer's disease. , 1994, Biochemical and biophysical research communications.

[25]  M. Beal,et al.  Cortical Cytochrome Oxidase Activity Is Reduced in Alzheimer's Disease , 1994, Journal of neurochemistry.

[26]  E. Wang,et al.  Modelling the effects of age-related mtDNA mutation accumulation; complex I deficiency, superoxide and cell death. , 1995, Biochimica et biophysica acta.

[27]  H. Reichmann,et al.  Electron transport chain defects in Alzheimer's disease. , 1994, Neurology.

[28]  Y. Konishi,et al.  Improved genomic/nuclear DNA extraction for 8-hydroxydeoxyguanosine analysis of small amounts of rat liver tissue. , 1995, Cancer letters.

[29]  R. Weindruch,et al.  High levels of mitochondrial DNA deletions in skeletal muscle of old rhesus monkeys , 1995, Mechanisms of Ageing and Development.

[30]  J. Morris,et al.  No association found between Alzheimer's disease and a mitochondrial tRNA glutamine gene variant , 1995, Neuroscience Letters.

[31]  Rappold,et al.  Human Molecular Genetics , 1996, Nature Medicine.

[32]  W. Parker,et al.  Creation and Characterization of Mitochondrial DNA‐Depleted Cell Lines with “Neuronal‐Like” Properties , 1996, Journal of neurochemistry.

[33]  E. Schon,et al.  Evidence that specific mtDNA point mutations may not accumulate in skeletal muscle during normal human aging. , 1996, American journal of human genetics.

[34]  C. Brayne,et al.  The tRNA(Gln) 4336 mitochondrial DNA variant is not a high penetrance mutation which predisposes to dementia before the age of 75 years. , 1996, Journal of medical genetics.

[35]  L. Cavelier,et al.  Human brain contains high levels of heteroplasmy in the noncoding regions of mitochondrial DNA. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[36]  M. Hayakawa,et al.  Accumulation of Deletions and Point Mutations in Mitochondrial Genome in Degenerative Diseases , 1996, Annals of the New York Academy of Sciences.

[37]  N. Bresolin,et al.  Aging-dependent Functional Alterations of Mitochondrial DNA (mtDNA) from Human Fibroblasts Transferred into mtDNA-less Cells* , 1996, The Journal of Biological Chemistry.

[38]  Accumulation of somatic nucleotide substitutions in mitochondrial DNA associated with the 3243 A-to-G tRNA(leu)(UUR) mutation in encephalomyopathy and cardiomyopathy. , 1996, Biochemical and biophysical research communications.

[39]  J. Brandt,et al.  Regional hypometabolism in Alzheimer's disease as measured by positron emission tomography after correction for effects of partial volume averaging , 1996, Neurology.

[40]  N Howell,et al.  Mutations in mitochondrial cytochrome c oxidase genes segregate with late-onset Alzheimer disease. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[41]  Quantitative allele‐specific PCR: Demonstration of age‐associated accumulation in human tissues of the A→G mutation at nucleotide 3243 in mitochondrial DNA , 1997, Human mutation.

[42]  A. Sinclair,et al.  Mitochondrial DNA mutations in Alzheimer's disease. , 1997, Biochemical and biophysical research communications.

[43]  S. Dimauro,et al.  Apparent mtDNA heteroplasmy in Alzheimer's disease patients and in normals due to PCR amplification of nucleus-embedded mtDNA pseudogenes. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[44]  M. Graeber,et al.  Association of the mitochondrial tRNA(A4336G) mutation with Alzheimer's and Parkinson's diseases. , 1997, Neuropathology and applied neurobiology.

[45]  M. Graeber,et al.  Association of the mitochondrial tRNAA4336G mutation with Alzheimer's and Parkinson's diseases , 1997 .

[46]  D. Murdock,et al.  Ancient mtDNA sequences in the human nuclear genome: a potential source of errors in identifying pathogenic mutations. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[47]  R. Davis,et al.  Cybrids in Alzheimer's disease: A cellular model of the disease? , 1997, Neurology.

[48]  H. Coller,et al.  Mitochondrial mutational spectra in human cells and tissues. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[49]  S. Papa,et al.  Low Reserve of Cytochrome c Oxidase Capacity in Vivo in the Respiratory Chain of a Variety of Human Cell Types* , 1998, The Journal of Biological Chemistry.

[50]  W. Parker,et al.  Evidence that two reports of mtDNA cytochrome c oxidase "mutations" in Alzheimer's disease are based on nDNA pseudogenes of recent evolutionary origin. , 1998, Biochemical and biophysical research communications.

[51]  H. C. Yeo,et al.  DNA oxidation matters: the HPLC-electrochemical detection assay of 8-oxo-deoxyguanosine and 8-oxo-guanine. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[52]  Y. Kagawa,et al.  Nuclear-recessive Mutations of Factors Involved in Mitochondrial Translation Are Responsible for Age-related Respiration Deficiency of Human Skin Fibroblasts* , 1998, The Journal of Biological Chemistry.

[53]  P. Nagley,et al.  Differential occurrence of mutations in mitochondrial DNA of human skeletal muscle during aging , 1998, Human mutation.

[54]  P. Sheard,et al.  Bioenergetic consequences of accumulating the common 4977-bp mitochondrial DNA deletion. , 1998, European journal of biochemistry.

[55]  Z. Janka,et al.  No mitochondrial haplotype was found to increase risk for alzheimer’s disease , 1998, Biological Psychiatry.

[56]  K. Okuizumi,et al.  mtDNA Polymorphisms in Japanese Sporadic Alzheimer’s Disease , 1998, Neurobiology of Aging.

[57]  N. Bresolin,et al.  Aging-dependent large accumulation of point mutations in the human mtDNA control region for replication. , 1999, Science.

[58]  Y. Kagawa,et al.  Functional integrity of mitochondrial genomes in human platelets and autopsied brain tissues from elderly patients with Alzheimer's disease. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[59]  B. Clark,et al.  N(6)-Furfuryladenine, kinetin, protects against Fenton reaction-mediated oxidative damage to DNA. , 1999, Biochemical and biophysical research communications.

[60]  D. Turnbull,et al.  Reanalysis and revision of the Cambridge reference sequence for human mitochondrial DNA , 1999, Nature Genetics.

[61]  A E Aust,et al.  Mechanisms of DNA oxidation. , 1999, Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine.

[62]  H. Chung,et al.  The frequency of point mutations in mitochondrial DNA is elevated in the Alzheimer's brain. , 2000, Biochemical and biophysical research communications.

[63]  C. Elger,et al.  Flux control of cytochrome c oxidase in human skeletal muscle. , 2000, The Journal of biological chemistry.

[64]  I. McKeith,et al.  Mitochondrial DNA haplogroups and susceptibility to AD and dementia with Lewy bodies , 2000, Neurology.

[65]  G Parmigiani,et al.  Detection of mitochondrial DNA mutations in pancreatic cancer offers a "mass"-ive advantage over detection of nuclear DNA mutations. , 2001, Cancer research.

[66]  I. Thompson,et al.  A reliable assessment of 8-oxo-2-deoxyguanosine levels in nuclear and mitochondrial DNA using the sodium iodide method to isolate DNA. , 2001, Nucleic acids research.

[67]  J. Hayashi,et al.  Human cells are protected from mitochondrial dysfunction by complementation of DNA products in fused mitochondria , 2001, Nature Genetics.

[68]  H. Coller,et al.  High frequency of homoplasmic mitochondrial DNA mutations in human tumors can be explained without selection , 2001, Nature Genetics.

[69]  Gary E. Gibson,et al.  Low mutational burden of individual acquired mitochondrial DNA mutations in brain. , 2001, Genomics.

[70]  T. Parsons,et al.  Point mutations of the mtDNA control region in normal and neurodegenerative human brains. , 2001, American Journal of Human Genetics.

[71]  A. Suomalainen,et al.  Diseases caused by nuclear genes affecting mtDNA stability. , 2001, American journal of medical genetics.