|Senior Scholar Program|
|1999 Senior Scholar Awardees|
Bruce N. Ames, Ph.D.
Steven N. Austad, Ph.D.
Evidence that oxidative damage, a by-product of normal metabolism in virtually all animals, is causally involved in the process of aging and the development of degenerative disease has been steadily accumulating for more than forty years. An understanding of the exact nature of this damage to specific cell components and mechanisms by which organisms combat this damage via differential efficacy in the formation of damaging molecules (often called "reactive oxygen species" or ROS's), scavenging of these molecules by specialized enzymes or dietary products, or rapid and efficient repair or replacement of damaged parts is an active area of current aging research. However, all work on these basic mechanisms using animal models, utilize models that age quickly and are therefore demonstrably inefficient at combating oxidative damage. The unique approach of this particular project is to identify genetic mechanisms of oxidative damage resistance in a model bird species, the budgerigar, with exceptional resistance to oxidative damage. Indeed current evidence indicates that birds generally have superior oxidative damage resistance to any other known animal group, superior even to slowly-aging mammals such as humans. The logic of this approach is that by studying how nature has designed such an exquisitely effective system, we will ultimately learn something about how to effectively design interventions to slow aging in humans.
Two sorts of evidence indicate that birds indeed possess exceptional resistance to oxidative damage. First, despite a high metabolic rate, birds are exceptionally long-lived. Mouse-sized birds generally live 20+ years under captive conditions. During that life they process substantially more oxygen per cell than any other known group of organisms. Specifically, some bird species process as much as 5 times the oxygen per cell in a lifetime as humans (Holmes & Austad, 1995). Second, bird cells in culture, survive exposure to a number of ROS-producing agents better than mouse or human cells (Ogburn, et al., 1998).
The research supported by The Ellison Senior Scholar Award will use a multi-pronged approach with multiple collaborators to seek to identify and begin mapping the genes responsible for this resistance. This project will be the first gene mapping project of any organism of specific gerontological interest. Initially, we will seek to identify the chromosomal regions involved in oxidative damage resistance by creating microcell hybrid cell lines, which combine the genome of an oxidative damage-prone species with selected budgerigar genes or chromosomes. Hybrid cells are then exposed to oxidative stress and examined to see whether oxidative damage resistance has been conferred by the presence of specific bird genes. Second, gene expression profiling by three independent techniques (Serial Analysis of Gene Expression, cDNA microarray hybridization, subtractive hybridization) will compare gene expression patterns of cells before and after exposure to ROS-producing agents. By examining closely increased activity of specific gene products, we hope to identify those genes whose activation was causally involved in resistance to the oxidative challenge. Third, we will examine whether the dynamics of telomere length regulation is correlated with exceptional oxidative damage resistance by examining telomere length reulation is correlated with exceptional oxidative damage resistance by examining telomere length dynamics, telomerase activity, and cell proliferation ability in long-lived (budgerigar) versus short-lived (Japanese quail) bird species.
James E. Cleaver, Ph.D.
A relationship has often been suggested between the endogenous production of DNA damage, predominantly through reactive oxygen, and a wide range of chronic disorders such as cancer, neurodegeneration, cardiovascular disease and aging itself. These have been based in large part on epidemiological data and have entered the popular imagination as a cause of aging which can be intervened with by dietary supplements such as antioxidants. There have, however, been few experimental animal systems by which this theory of aging can be tested directly and the effectiveness of intervention schemes investigated. Our proposal describes the development of mouse strains by which the repair of endogenous DNA damage can be regulated to produce isogenic repair competent and deficient strains. These animals will permit precise investigation of the DNA damage theory of aging and define the most suitable dietary additives that can modify those effects of DNA damage that result in premature aging.
We have already demonstrated that the accumulation of unrepaired DNA single strand breaks, resulting from a knockout of base excision repair (BER), has a strong effect at the gastrulation stage of embryo development and animals which lack this repair die in utero. We wish to develop a mouse strain that can be maintained through this critical stage of development, and the role of DNA breakage investigated, postnatally. In our present mouse strain we have knocked out one of the genes that acts late on the BER pathway, XRCC1, which is a cofactor of DNA ligase III. This defect results in the spontaneous accumulation of unrepaired single strand DNA breaks in mouse tissues. The XRCC1 gene controlled by an inducible system will be introduced into the XRCC1 knockout background. This will allow us to express XRCC1 in a regulated manner, bypass the critical stage of embryogenesis, and so obtain viable animals. These animals can then be made BER-defective by reducing XRCC1 expression. Tissues in which spontaneous oxidative and other forms of DNA damage occur will accumulate DNA strand breaks. We would expect tissues such as liver, bone marrow, small intestine, brain and central nervous system to be most susceptible to degenerative age-related changes.
These mouse strains will allow us to observe, directly, whether normal cell metabolism produces premature aging in repair-deficient animals. Therapeutic intervention with antioxidants, vitamin E and N-acetyl cysteine and other supplements, can then be used as protective agents to discover the best diets for effectively counter-acting chronic health effects of aging.
Gretchen J. Darlington, Ph.D.
Titia de Lange, M.D., Ph.D.
In collaboration with Jack Griffith (UNC) we have found that human telomeres form large duplex loops (t-loops). We propose that t-loops represent the mechanism by which telomere ends are masked from the cellular machinery that detects DNA breaks. Our working hypothesis is that telomere shortening in aging human cells results in chromosome ends that no longer form t-loops. Such t-loop deficient termini are proposed to constitute the main signal leading to cell cycle arrest, senescence, and apoptosis in aging cells.
We have previously identified a key player at mammalian telomeres, the telomeric DNA binding factor, TRF2. Loss of TRF2 from chromosome ends results in immediate deprotection of telomeres. Cells respond to this insult with the activation of a DNA damage pathway that includes the ATM kinase and p53, resulting in cell cycle arrest. Depending on the cellular context, such cells undergo apoptosis or display a phenotype similar to senescence. The unprotected telomeres lose the 3' protrusion of TTAGGG repeats typical of mammalian chromosome ends and eventually become ligated, forming dicentric and multicentric chromosomes. Thus, the removal of TRF2 from chromosome ends mimics events observed in aging human cells in which the telomeres have become critically shortened.
A likely mechanism for TRF2-mediated telomere protection has now been revealed in a study on the structure of the telomeric complex. Using electron microscopy, we have found that telomeres can exist in a specific higher order conformation (t-loops). EM analysis of psoralen crosslinked telomeric DNA demonstrated frequent large t-loops at natural chromosome ends. Molecular studies indicate that t-loops are formed through invasion of the 3' telomeric overhang into the duplex telomeric repeat array. We propose that t-loops are the main mechanism by which cells sequesters telomere ends from DNA damage checkpoints. In agreement with its protective role at telomeres, TRF2 has the ability to promote t-loop formation in vitro, suggesting that its main function in vivo is to facilitate remodeling of telomeric DNA into the t-loop configuration.
The presence of t-loops at human chromosome ends suggests a mechanism by which telomere shortening could induce cellular senescence and apoptosis. We propose that critically shortened telomeres fail to form t-loops, resulting in unfolded, exposed chromosome ends that activate a senescence or apoptotic signalling pathway, possibly involving ATM and p53. This hypothesis will be tested by determining the relationship between t-loop loss, telomere shortening, and cellular aging phenotypes. In addition, we will aim to manipulate the presence of t-loops at chromosome ends in order to test whether loss of t-loops can induce premature senescence and apoptosis. Conversely, we will test whether improved t-loop formation can extend the in vitro life-span of human cells.
Michael E. Greenberg, Ph.D.
We propose to study the importance of a cellular signaling pathway, termed the P13K/Akt/Daf16 pathway, for determining the life span of cells and multi-cellular organisms. Disruption of the P13K/Akt pathway has previously been shown to affect the life span of the invertebrate organism C. elegans, a nematode. We plan to extend these studies to mammals by developing strains of mice that bear mutations in components of this signaling cascade. The mutant mice will be analyzed to determine if disruption of the P13K/Akt/Daf16 pathway affects cell or organismal life-span. The focus of our research will be on the last protein in the signaling cascade-Daf16. The mammalian version of DAF16 is called FKHRL1. Our first objective will be to engineer mutations into FKHRL1 that will render it either highly active all the time, or alternatively mutations that will inhibit FKHRL1 function. Once mice are generated that bear these specific mutations we will examine how the mutations affect the life span of the mice. Since the P13K/Akt/Daf16 pathway has also been shown to control the proliferation and survival of cells we will also examine the mutant mice for alterations in cell proliferation and survival during development. One might expect that the mutations in FKHRL1 might lead to diseases such as cancer or cell death since the disruption of other components of the P13K/Akt pathway have previously been found to lead to these disorders. The second objective of our research will be to determine how FKHRL1 acts within cells. We have obtained evidence that FKHRL1 controls the expression of genes that may regulate cellular responses. Our goal will be to determine which of the 100,000 or more genes in the mammalian genome are targets of FKHRL1. Once the FKHRL1 targets are identified we plan to study their function and to determine how they contribute to aging as well as disease states such as cancer and neurological disorders.
Lawrence A. Loeb, M.D., Ph.D.
Accurate copying of DNA in cells is carried out by DNA polymerases. These enzymes polymerize nulceotides that are complementary to the nucleotides in the cellular DNA template, yielding daughter DNA molecules that preserve the nucleotide sequence of the parental molecules. Errors in this process, if not repaired, result in mutations. Mutations in DNA polymerases themselves can lead to inaccurate copying of template DNA and to a general mutator phenotype. We are now in a position to determine if alterations in DNA polymerases can cause mutations throughout the genomes of animal cells, and whether these mutations accumulate in somatic cells during aging. We propose to create a series of mice in which a wild type DNA polymerase(s) is replaced by a mutant DNA polymerase that exhibits either a decrease or an increase in fidelity. The construction of such a series of mice should allow us to determine the relationship of the fidelity of DNA synthesis to the aging phenotype, and to document an association of mutations produced by DNA polymerases with the pathologies associated with aging.
David S. Thaler, Ph.D.
Mitochondria have been proposed by others to be both initiators and targets of cellular degeneration associated with aging. The proposed work is designed to test specific hypotheses in which cellular degradation and aging are related to mitochondrial mutation. Measurement and implications of reversible intermediates in mitochondrial mutation.
This proposal builds on a concept originally put forward by FW Stahl. Most mutation and recombination occur, via reversible intermediates.
Consider a gene, G, mutating to a new allele, G', via an intermediate state, G*. Suppose state G* is a reversible state. In fact it usually is: The double helix has two copies of genetic information, so a modification of one chain leaves intact the information on the other chain and allows for conservative reversal of G*. In addition to the non-canonical base pair, which is a substrate for repair, many G* states are due to chemical modification of bases into non-standard forms that are recognized as substrates for restorative repair. G -k1---> G*-k3---> G' and G<--k2--- G*---k5-->cell dies
Suppose further that G*, the reversible intermediate state, is responsible for (a) phenotype(s). A particular example of interest is to suppose G* encodes mRNA of the new allele, i.e. G*-k4>mRNAG'. This would follow if a particular lesion codes in an informationally similar way, whether the template containing it is a substrate for transcription or for replication. Several reversible mutational intermediates have this property of informational miscoding. These include DNA-DNA mismatches, DNA-ribonucleotide mismatches (my own work, part published and part in progress), alkylated bases (especially O-6methyl Guanine), and oxidized bases (especially 8-Oxy Guanine). The phenotypes of G*s also may engender repair. A particular G* may thus make its own fixation either more or less probable depending on its own phenotypes.
Consider the consequences if the phenotype(s) of intermediates- e.g. mRNAG'- alters the rate of allele fixation, k3. k3 becomes a function of the phenotype of G* (e.g. mRNAG'). The [mRNAG'] is a function of k4, therefore, k3 is a function of k4 and k1, and an inversely related function of k2 and k5. Further complications exist. For example, G* may transit to other states G** each with its own vectors of transition. A particular G* or G** allele or set of G* alleles in a single cell or in the population may alter the global intracellular environment and the k's of other cells through cell-cell communication.
Mitochondria are an especially likely site for the modulation of mutation fixation inherent in this model. Mitochondrial information is multicopy and potentially selectable at two levels not available to the nuclear genome: within each organelle, and by multiple organelles in each cell.
We will develop both the theory and the experimental analysis of this Reversible-Intermediate model with reference to mitochondria. This work and related studies of mutation in mitochondria, and elsewhere in the cell but initiated by mitochondria 'gone bad' will be undertaken in collaboration with Dr. Marcelo Magnasco (Institute for the Study of Physics and Biology at Rockefeller University) and Dr. Zeena Nackerdian who is joining our group.
Douglas C. Wallace, Ph.D.
Evidence continues to accumulate that aging is associated with a decline in mitochondrial function, suggesting that mitochondrial dysfunction maybe a major factor in the pathophysiology of aging and senescence. The mitochondria provide most of the cellular energy through oxidative phosphorylation (OXPHOS), and generate most of the endogenous oxygen radicals (reactive oxygen species, ROS) as a toxic by-product. The mitochondria are also the primary regulators of apoptosis, which is initiated by the opening of the mitochondrial permeability transition pore (mtPTP). This releases cytochrome c, procaspases, and the apoptosis initiating factor (AIF) from the mitochondrial inter-membrane space, causing degradation of cytosolic proteins and chromosomal DNA. The opening of the mtPTP is stimulated by a decline in mitochondrial energy and an increase in mitochondrial oxidative stress.
The mitochondria are assembled from the genes of both the nuclear DNA (nDNA) and the mitochondrial DNA (mtDNA). The mtDNA codes for 13 proteins essential for OXPHOS as well as the structural RNAs for mitochondrial protein synthesis. In humans, maternally-inherited mtDNA disease mutations cause many of the same symptoms as seen in aging, and mitochondrial OXPHOS declines with age in association with the accumulation of somatic mtDNA mutations. This suggests that aging is a mitochondrial disease.
To further explore the mitochondrial hypothesis of aging, we propose to study the chimpanzee, our closest relative. The chimp genome is 98.5% homologous to our own, but the chimp's life span is half of our own. Moreover, Emory's Yerkes Primate Center houses a colony of over 200 chimps, which are regulary monitored for health and available for detailed autopsy at death.
First, we will characterize the physiolgical and biochemical changes that occur in chimp mitochondria with age. Muscle physiology will be monitored using non-invasive Near Infra Red (NIR) Spectroscopy, and correlated with muscle biopsy mitochondrial OXPHOS enzymes, respiration, ROS production, and excitability of the mtPTP. These studies will be extended to the other major organs using fresh autopsy tissues.
Second, we will correlate the decline in mitochondrial function with the accumulation of somatic mtDNA mutations in muscle biopsy and autopsy tissues. Rearrangements in the mtDNA will be monitored by long extension-PCR (LX-PCR) and mtDNA point mutations will be analyzed by direct mtDNA sequencing and by Protein Nucleic Acid (PNA)-competitive PCR (PNA-PCR). The regional distribution of mtDNA mutations will be assessed by in situ PCR. We will then determine the functional significance of the somatic mtDNA mutations by recovering mutant mtDNAs from the brains of post-mortem animals by synaptosome fusions to human mtDNA-deficient (ro) cells. Synaptosome cybrids, harboring mutant mtDNAs, will then be characterized for the biochemical consequences of the mutation.
Finally, we will analyze the age-related changes in mitochondrial gene expression using our human mitochondrial gene DNA microarray (mitochondrial gene chip). Initially, we will profile the age-related changes in nDNA and mtDNA mitochondrial gene expression in muscle, and then compare these changes with those seen in the post-mortem tissues from older chimps. These mitochondrial gene aging profiles will then be compared to profiles that we obtain from the muscle biopsies and autopsy tissues of human patients harboring known pathogenic mtDNA mutations. If the chimpanzee aging profiles approximate the patient mitochondrial disease profiles, then this will provide strong evidence that aging is a mitochondrial disease.
Sherman M. Weissman, M.D.
Werner disease is remarkable in that all of the single gene mutations and diseases known in man, it is the one that produces a syndrome most closely resembling the premature onset of normal aging. As such, there is the hope that understanding the mechanisms by which defects in the Werner protein (SRN) produce these symptoms might tell us what pathways are the "weak links" in normal aging. The WRN protein has been cloned and identified as a DNA helicase. We have established an assay for one WRN function based on its ability to increase resistance of a Werner disease cell line to genotoxic agents. In addition, we have identified other proteins that interact with WRN including a subunit of a DNA polymerase. We are currently investigating whether WRN can re-direct DNA polymerase to specific sites in genomic DNA.
| For further information, contact:
Richard L. Sprott, Ph. D.