Dominique Broccoli, Ph.D.
Fox Chase Cancer Center
Molecular Mechanisms of Telomere Dependent Senescence

Telomeres are specialized structures at the ends of chromosomes that are essential for stability. They are composed of several thousand base pairs of a repeating DNA sequence, (TTAGGG), and proteins that bind specifically to this sequence. In human cells, telomeric DNA is lost each time the cell divides due to the repression of a specialized enzyme, telomerase, which is responsible for the replication of telomeric DNA. Thus, telomeres become shorter as we age. In immortalized cells, for example tumor cells, telomerase is active and telomere length is stabilized. Recently, it has been demonstrated that expression of telomerase is sufficient to render human cells immortal while inhibition of telomerase in tumor derived cell lines leads to cell death. The current hypothesis developed from these data is that telomere length acts as a clock to limit the total number of divisions any cell is capable of achieving. This limitation on cell growth and renewal is believed to contribute to human aging. Activation of telomerase and concomitant stabilization of telomeric DNA circumvents this restraint on cell growth and permits unlimited numbers of cell division, a process required for tumor formation. Thus, telomeres are critically important in both aging and tumorigenesis in humans.

Although it is now clear that telomere length is a critical feature determining the number of divisions cells may attain, relatively little is known about how this process is regulated. Several components of the mammalian telomeric complex have been isolated in recent years including the DNA element, components of the telomerase enzyme, and two telomeric binding proteins called TRF1 and TRF2. Having these components in hand allows manipulation of the telomeric complex in tissue culture systems. For example, alterations in the levels of the telomeric binding proteins TRF1 and TRF2 can have profound effects on telomere length and cellular viability, respectively.

The work in my laboratory is focussed on determining how telomeres limit cellular proliferation. We will introduce a number of mutant forms of telomere proteins into human cells and establish the effect these mutations have on cellular lifespan. In addition, we are undertaking searches to uncover additional proteins that are associated with human telomeres to gain a deeper understanding of telomere structure and how the telomere functions to protect chromosome ends. The knowledge gained from these experiments will deepen our understanding of the processes underlying human aging and tumorigenesis.

Phillip Carpenter, Ph.D.
University. of Texas Medical School at Houston
Biochemical Characterization of a Putative p53-binding Protein from Xenopus

During the eukaryotic cell cycle, the genome is duplicated through the process of DNA replication and segregated to daughter cells during the process of mitosis. When the events that coordinate the cell cycle fail to properly operate, abnormal cellular conditions arise. Some of these conditions appear to be related to the biology of aging. For example, mutations in ATM, a gene known to be involved with the coordination of cell cycle events, causes Ataxia telangiectasia, a disease with numerous pathologies related to aging. Furthermore, defects in other cell cycle and/or related processes such as DNA replication and DNA repair, are suspected to be the major causes of progeroid syndromes such as Cockayne and Werner's syndrome. Therefore the study of cell cycle processes can greatly aid in our understanding of the molecular biology of aging.

We have previously designed a screen to isolate cDNA molecules that encode for important components of the cell cycle. Through the use of highly reliable biochemical assays, we can evaluate the roles that these gene products play in important cellular events, including DNA replication. One gene that we are currently evaluating is X535. The product of X535 appears to be a protein which can me modified by well known cell cycle kinases. Furthermore X535 contains "BRCT" motifs. These protein sequences were originally identified in the BRCA1 gene and appear to be involved with signaling events which control DNA repair processes. Because DNA repair is often compromised in various progeroid syndromes, X535 is a suitable candidate for studying cell cycle processes which impinge upon the aging process.

Stewart Frankel, Ph.D.
Yale University School of Medicine
The Regulation of Chromosomes and Longevity

Aging is often depicted as an inevitable grinding down of the bodily machine, but there is much evidence to suggest it is a biological process under some genetic control. In order to better understand this process, we are attempting to speed up and slow down aging experimentally. This is being done by testing a series of mutations for an effect upon longevity and aging in the fruit fly. The mutations affect genes that have close counterparts in man, and that appear to function similarly in both the fly and man. The genes help regulate chromosome structure. The rationale for our study lies in the properties of the chromosome apparatus. It does not follow the on/off, computer-program rules of some other biological systems, but instead follows "epigenetic" rules. This means that a small dose of randomness is part of its normal function. This element of randomness works well during the primary development of the organism and works well for the young adult, but it may not age well, literally and figuratively. If chromosomes do perform more poorly with age, their dysfunction could be one of the factors that cause aging and limit life span.

Shiv I. S. Grewal, Ph.D.
Cold Spring Harbor Laboratory
Heterochromatin Assembly and Replicative Life-Span in Fission Yeast

Heterochromatin is a description of transcriptionally 'silent,' condensed chromosomal regions which largely consist of repetitive sequences, located primarily at centromeres and telomeres. Recent studies have suggested that heterochromatin plays many important functions, including proper segregation of chromosomes during cell division and gene dosage compensation in mammals. Furthermore, it has been suggested that a gradual loss of heterochromatin structures might be associated with cellular and organismal aging. Despite its crucial functions in gene regulation and chromosome architecture, the underlying mechanisms for heterochromatin assembly remain largely unknown.

To understand how heterochromatin structures are established and maintained through many rounds of cell division, we are studying the gene-repression mechanism known as "silencing" in fission yeast. Earlier studies showed that silencing at the mating-type region, centromeres and telomeres of fission yeast is mediated through the assembly of repressed heterochromatin structures. Interestingly, heterochromatin assembly is controlled by an epigenetic mechanism whereby heritable changes other than a mutation in the DNA itself are passed on to the daughter cells. We found that nucleoprotein complexes provide cues for their own reassembly during cell division and help promote inheritance of the silenced state. Certain evolutionarily conserved proteins known as chromodomain proteins, as well as proteins involved in modification of histones play a crucial role in maintenance of heterochromatin-mediated silencing in fission yeast. Remarkably, mutation in one of these factors called Clr6, which shares homology to histone deacetylases, also reduces the replicative life-span of the cells providing a genetic link between heterochromatin assembly and cellular senescence. Our current research focuses on the analysis of a clr6 mutant for previously described aging related phenomena, such as telomere length, rDNA stability, chromosome segregation defects and redistribution of other know heterochromatin proteins. We are also purifying the Clr6 protein complex to identify its interacting partners, as well as characterizing additional mutants defective in maintenance of heterochromatic domains. These studies will test the hypothesis that dynamic reorganization of heterochromatin structures affects cellular aging.

Danesh Moazed, Ph.D.
Harvard Medical School
Connections Between the Nucleolus and Cellular Aging

Chromosomes, the carriers of the cell's genetic information, are composed of distinct functional domains that ensure their fateful inheritance during the process of cell division and chromosome duplication. One example of such functional organization is the presence of large stretches of DNA which are packaged into a repressed state where gene expression is silenced. Silencing of DNA domains is observed in a broad spectrum of organisms ranging from unicellular eukaryotes like yeast to complex multicellular organisms like us and appears to play a fundamental role in regulating chromosome behavior. For example, silencing has been shown to play a crucial role in both regulating gene expression and maintaining chromosome stability.

Studies of cellular aging in yeast have revealed a direct connection between life span and silencing in this organism. Loss of silencing at the chromosome locus that contains the genes for ribosomal RNA (components of cellular machines that synthesize protein) results in instability of this chromosome region. To make enough ribosomal RNA to sustain their rapid growth rate, cells need many copies of the genes that encode these RNAs. These genes, called rDNA, exist at about 150 tandem copies on one of the yeast chromosomes. This high copy number results in instability due to natural genetic exchange that occurs between identical DNA sequences. A byproduct of such DNA exchange events is the separation of DNA in the form of circles from the rest of the chromosome. Such separation removes the DNA from regulatory mechanisms that govern chromosome duplication and inheritance in the cell. The DNA circles can therefore duplicate and accumulate to very high numbers and cause cell death. By repressing genetic exchange between rDNA sequences, silencing slows the rate of accumulation of run away DNA circles and thereby slows cellular aging.

Our lab works on the factors that are responsible for packaging DNA into a silent state. In particular, we have identified a complex of proteins that acts specifically at the repetitive rDNA region. We have recently discovered that the Sir2 protein, a universally conserved component of the rDNA-silencing complex, possess an enzymatic activity that is essential for all gene silencing. Sir2 appears to covalently modify itself and other chromatin components in a step that is required for making DNA silent. The broad conservation of Sir2-like proteins suggests that Sir2-based silencing mechanisms also exist in human cells.

Henry L. Paulson, M.D., Ph.D.
University of Iowa
Mechanisms of Neuronal Dysfunction and Death in Neurodegenerative Proteinpathies

As we age, neurons in our brain tend to accumulate abnormal protein. The same process occurs in degenerative diseases of aging such as Alzheimer and Parkinson diseases, but at a much faster rate. Our research aims to identify the various ways that abnormal protein accumulation compromises the neuron in neurodegenerative diseases. Our long term objective is to extend these findings to the study of normal aging in the central nervous system.

For the past few years my laboratory has studied a group of inherited neurodegenerative diseases known as polyglutamine diseases. Eight polyglutamine diseases have already been identified, the most well known being Huntington disease. Polyglutamine diseases are so named because they are due to an abnormal protein that contains an enlarged, or expanded, polyglutamine stretch. We have discovered that expanded polyglutamine protein aggregates within neurons and causes the cells to undergo a stress response. In research funded by the Ellison Medical Foundation New Scholar award, my laboratory will identify the cellular players that mediate this stress response and determine whether their presence is good or bad for the neuron. Similar stress probably occurs in other neurodegenerative diseases, and perhaps even as part of normal aging. We are thus interested in applying our findings on polyglutamine disease to more common age-related degenerative diseases. Toward this goal, we recently developed cellular models of Parkinson disease that will allow us to define the broader role of the stress response in neurodegeneration. We hope that our research will identify components of the stress response which can then be modulated in ways to develop novel therapies for these incurable disorders.

Zhou Songyang, Ph.D.
Baylor College of Medicine
Functional Analysis of Aging and Cell Survival Signal Pathways in Mammalian Cells

One intriguing puzzle in modern biology is how different life-spans and aging rates are determined in different species. Answers to such questions will help us battle various aging-related diseases, and ultimately improve the health and quality of life in humans. Aging rates are believed to be primarily controlled genetically, although environmental factors may contribute as well. The life span of an organism is intricately controlled through the network of signaling pathways mediated by cellular gene products. It is believed that coordinated gene expression and modulation by extracellular factors may be crucial. Several diseases that affect the aging process in humans have been identified. And multiple candidates that may regulate human aging have been proposed based on studies of these diseases. In Werner's syndrome, a mutation in the WRN gene results in inheritable premature aging. In Alzheimer's disease, neuronal degeneration and apoptosis may be responsible for the onset of neurological disorders. However, the exact mechanisms that lead to these aging-related diseases remain poorly understood.

In the past few years, my interest has been to identify and study genes that regulate cell survival and senescence, two pathways that are important for aging. A number of aging-related genes have been identified in lower organisms. Aging regulatory genes are likely to be conserved throughout evolution, therefore mammalian homologues of these genes are likely to exist. The short-term goal of our research is to (1) study the role of a protein kinase named Akt in mammalian aging and (2) identify mammalian homologues of DAF-16, a gene that has been shown to regulate cell survival and longevity in lower organisms.

Our long-term goal is to isolate new cellular factors that modulate the aging process in mammals. We have begun to use new genetic approaches developed in the lab to clone genes that prevent programmed cell death in mammalian cells. These genes will be expressed in human fibroblast cells to study their effects on the cellular aging process. In addition, transgenic mice that overexpress these genes will be generated to determine if they prolong or lessen the survival and life-span of aging mice.

David Q.-H. Wang, M.D., Ph.D.
Brigham and Women's Hospital, Harvard Medical School
Aging and Cholesterol Gallstone Formation: Molecular Mechanisms of Gallstone (Lith) Genes

Cholesterol gallstone disease occurs rarely in childhood and adolescence. As epidemiological observations have suggested and as clinical studies have confirmed, the prevalence of cholesterol gallstone disease increases linearly with advancing age and approaches 50% at age 80. Elderly individuals are at high risk for developing gallstone complications and the death rate from surgery is often unacceptably high in patients older than 65. It has been known that cholesterol-supersaturated bile due to excess hepatic secretion of cholesterol into bile is a essential metabolic condition for the formation of cholesterol gallstones. Although it was observed that cholesterol concentration of bile is significantly higher in elderly people, and age is positively correlated with hepatic cholesterol secretion rate, as well as negatively correlated with bile salt synthesis and bile salt pool size in the liver, it has not been established at the molecular level whether aging modifies biliary cholesterol metabolism or how aging influences biliary lipid secretion.

Recently, I studied 9 strains of inbred mice fed a diet containing high fat and cholesterol (plus cholic acid) for 8 weeks, and found that differences in gallstone susceptibility between C57L mice and AKR mice are determined by at least two gallstone (Lith) genes. The first gallstone gene (Lith1) maps to mouse chromosome 2 by a powerful genetic analysis (quantitative trail locus mapping). Compared to gallstone-resistant AKR mice, gallstone-susceptible C57L mice with gallstone (Lith) genes display significantly higher secretion rate of biliary cholesterol, greater cholesterol concentration of gallbladder bile, more rapid cholesterol crystallization, and higher prevalence of gallstones. In response to the diet with high fat and cholesterol (plus cholic acid), AKR mice decrease HMG-CoA reductase activity (hepatic cholesterol biosynthesis), but C57L mice fail to down-regulate this enzyme activity. Down-regulation of cholesterol 7a-hydroxylase and sterol 27-hydroxylase activities (hepatic bile salt synthesis) are significantly more pronounced in C57L mice than in AKR mice. Whereas, acyl-CoA:cholesterol acyltransferase (cholesterol ester synthesis) decreases significantly in C57L mice than in AKR mice. My results suggest that the observed changes in the lipid regulatory enzyme activities are secondary effects of gallstone genes, and are, in part, an integrated response that ensures the continuous supply of hepatic cholesterol for secretion into bile. Furthermore, I found that the sister of P-glycoprotein, a bile salt export pump (bile salt transporter) in the liver, has a high function in C57L mice, as well as is a strong candidate gene for the first gallstone gene (Lith1) because the gene for the sister of P-glycoprotein is localized on mouse chromosome 2 within the similar region for the first gallstone gene.

Therefore, I propose three specific aims to explore the molecular mechanisms responsible for the high incidence of gallstone formation with age using genetically gallstone-susceptible C57L mice compared to gallstone-resistant AKR mice. Aim 1: To examine the influence of aging on cholesterol levels of gallbladder and hepatic biles and the formation of cholesterol gallstones. Aim 2: To explore molecular mechanisms how aging induces hypersecretion of cholesterol and the formation of lithogenic bile. Aim 3: To study whether aging modifies activities of hepatic lipid regulatory enzymes that alter biliary lipid metabolism. The elucidation of the molecular mechanisms underlying influence of aging on gallstone formation, biliary lipid secretion and hepatic lipid regulatory enzyme activities will shed more light on the pathogenesis of cholesterol gallstone disease, as well as open a door for the gene therapy in the human.

Weidong Wang, Ph.D.
National Institute on Aging, National Institutes of Health
Characterization of a Novel Protein Complex Involved in the Human Premature Aging Disease - Werner Syndrome

Werner Syndrome (WS) is a rare human genetic disease with many features of premature aging. The patients usually appear normal during their teenage years. But later they prematurely develop several age-related diseases, including artherosclerosis, osteoporosis, diabetes, malignant neoplasm and cataracts. They also exhibit an aged appearance when they are still young, with features such as skin atrophy and early graying of hair. Cells obtained from WS patients also have a shortened life-span compared to those taken from normal human beings of similar age, and they are more similar to those from older people. The disease has therefore been considered by the research community as a model that can be compared to normal human aging.

Other groups have previously identified the gene defective in WS patients. However, it is unclear how it causes the age-related symptoms. Dr. Wang's group has now purified the WS gene product from normal human cells and found that it is one component of a large protein complex. They subsequently identified most of the other components within the complex, which enabled them to determine the exact cellular process in which the WS gene product functions. Their work links aging to a specific cellular process and provides a molecular mechanism for how premature aging may occur in WS patients.

Huaxi Xu, Ph.D.
The Rockefeller University
Gonadal Steroid Regulation of ß-Amyloid Generation: Cellular Mechanisms and Animal Models

Alzheimer's disease (AD) occurs when large numbers of neurons - the cells which make up the brain, die over many years. These cells are in the areas of the brain which are responsible for forming and storing memories and for performing higher thought processes. This is why AD affects memory and the ability to think in patients afflicted with the disease. The neurons are believed to die as the result of the toxic accumulation of deposits in the brain called amyloid. This amyloid is normally produced and secreted by neurons. However, in AD, neurons seem to make greater quantities, and thus larger toxic amounts are deposited in the brains of patients. The deposits are referred to as plaques and appear to be directly responsible for the death of the neurons in their vicinity.

The best hope researchers may have to slow or prevent AD is to interfere with the molecules which are responsible for forming amyloid inside neurons. Unfortunately, these molecules have not yet been discovered, so scientists have to pursue other leads. One lead was provided when it was discovered that women, who had been taking estrogen as part of a long term program of postmenopausal estrogen replacement, were less likely to get AD, and those who did eventually get AD, got it at much older ages. Our group was the first to show that estrogen may actually prevent or delay AD by slowing the secretion of amyloid from neurons. In order to understand how estrogen exerts these anti-amyloid effects we have proposed to study how estrogen regulates the precursor protein from which amyloid is derived. It is clear from previous work that estrogen exerts many effects in the brain and we would like to further pursue the link that we and others have established between estrogen treatment and the reduction in toxic secreted amyloid mice which have been genetically modified to produce high levels of human amyloid. Our preliminary results showed that estrogen may reduce brain amyloid production in these animals. In addition, we recently found that the male gonadal hormone, testosterone, may have a similar amyloid-reducing effect, thereby impacting on AD risk in elderly men who have declining testosterone levels. Our long term goal is to understand the detailed mechanism by which estrogen/testosterone exerts this effect, scientists may be able to design even more effective drugs to halt and possibly reverse the effects of AD.

Richard L. Sprott, Ph. D.
Executive Director
The Ellison Medical Foundation
4710 Bethesda Avenue
Suite 204
Bethesda, MD 20814
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