Cynthia Kenyon - US grants

My research goal is to understand how spatial patterns arise within fields of developing cells: to identify the regulatory signals involved, to learn how they are localized, and to learn how they generate cell-specific responses. I am using C. elegans as an experimental system in order to analyze pattern formation at the level of single, identified cells with both genetic and molecular techniques. By analyzing the gene mab-5, I have discovered a control system in C. elegans that is responsible for antero-posterior differences in body pattern after hatching. mab-5 function cell-autonomously in ectodermal, neuronal, and mesodermal lineages located in a single posterior body region. It is required for posterior-specific patterns, including copulatory sensilla and sex muscles, cell fusion, cell death, and also patterns of cell migration. mab-5 appears to function as a regulator: In mab-5 null mutants, many posterior cells adopt anterior-specific fates. Conversely, when mab-5+ is overexpressed, anterior cells can adopt posterior-specific fates. Properties of this system suggest mab-5 is activated within posterior cells by extracellular signals. As a means of addressing the general problem of pattern formation within fields of cells, we will address three questions concerning mab-5: (1) What is the biochemical function of mab-5? (2) How is mab-5 activated in a position-specific fashion? and (3) How does mab-5 activity invoke cell-specific responses? To determine the biochemical function of mab-5 activity and its mode of regulation, we will initiate a long-term molecular analysis by cloning the mab-5 gene, and analyzing patterns of mab-5 RNA expression. To determine whether particular cells produce signals that activate mab-5 in posterior cells, we will effectively remove individual cells using laser microsurgery. We will also pursue observations suggesting that epidermal signals activate mab-5 in the medsoderm. To identify genes that activate or respond to mab-5, we will identify mutations that affect the positional specificity of mab-5 activity or the cell-specific responses to mab-5 activity. By standard genetic analysis, and also molecular approaches using cloned mab-5 DNA sequences, we will begin to determine how these genes are likely to interact with mab-5 and with one another.

Research on the nematode Caenorhabditis elegans ranges from such global problems as the functioning of an entire nervous system and the evolution of form to the molecular bases of the gene structure and function. C.l elegans has becomes an important experimental organism for the study of many aspects of animal biology, particularly the genetic and molecular bases of development and behavior. Dr. Anderson is requesting funds to help support two C. elegans meetings to be held at the university of Wisconsin at Madison, WI in June of 1991 and 1993. Previous C. elegans meeting shave led to the exchange of knowledge, methods, mutants, and clones and have been vital in establishing the sense of excitement and collegiality that characterizes this field.

During previous funding periods, we have learned that C. elegans uses a conserved system of homeotic genes (HOM-C genes) to generate anteroposterior (A/P) body pattern, and we have investigated the role of this complex in patterning several cell types. We now plan to focus on one biological process that involves the HOM-C genes: cell migration. The Q neuroblasts, QR and QL, are bilateral homologs that migrate in opposite directions. The QL cell migrates posteriorly into the domain of the HOM-C gene mab-5. As it does so, it begins to express mab-5, which functions within its descendants to cause them to migrate to posterior rather than anterior positions. QR instead migrates anteriorly into the domain of the HOM-C gene lin-39, which, in turn, allows the descendants of QR to migrate to specific anterior positions. Together the cells in the Q lineage migrate to positions that span the entire A/P body axis. Our findings suggest that, once programmed by the homeotic genes, the cells find their positions using a global system of guidance cues. We have identified genes required for specific steps in Q cell migration. These include (i) a gene required for left/right asymmetry, (ii) genes required to switch on mab-5 in the migrating QL cell, (iii) a candidate for a migration gene regulated by mab-5 (iv) a gene that may be part of a system of positional information that guides the cells; (iv) a gene required for certain cells to stop migrating, and (v) genes that probably function in the mechanics of migration itself. The mechanism of Q cell migration is probably evolutionarily conserved because it involves integrin receptors, which mediate migration in vertebrates. During this funding period, we will identify additional genes that affect Q cell migration, and we will carry out experiments with these and previously identified genes that are aimed at learning what signals trigger HOM-C gene expression in migrating cells, how the HOM-C genes program the cell's migratory behaviors, and what types of extracellular cues guide these cells to their destinations.

How the rate of aging is determined is a fundamental problem in biology. Recently, tremendous progress has been made by identifying and characterizing genes that influence the aging of nematode C. elegans. This organism has a very rapid rate of aging and lives just a little over two weeks, making it very easy for investigators to identify and characterize mutations affecting aging. Studies in a number of labs, including ours, have shown that a pathway that is similar to the vertebrate insulin and IGF-1 signaling pathways controls the rate of aging in C. elegans. When a homolog of the insulin/IGF-1 receptor, DAF-2, is partially disabled, the animals live twice as long as normal, remaining active and healthy when normal worms are decrepit and still (nursing-home appearance). Since many biological processes in C. elegans have been shown to be conserved with higher organisms, studies of this pathway may ultimately allow us to devise strategies for improving the quality of old age in humans. My lab originally discovered that daf-2 mutations increase lifespan. During this funding period, we have shown that DAF-2 acts in signaling cells, which, in turn, must produce a second signal that directly controls the rate of aging. In addition, we have identified and cloned a transcription factor whose activity is required to extend the lifespans of daf-2 mutants. Here we propose to learn which cells in the animal require the activity of this transcription factor, and whether its activity can be sufficient to extend the lifespans of otherwise normal animals. Many mysteries remain. How DAF-2 influences the production of the secondary signal(s), the identity of this signal(s), the signal transduction pathway that must operate in target tissues, and the mechanism by which this pathway controls aging are all unknown. To find the missing genes, we carried out a large-scale mutant screen and isolated twenty new long- lived mutants. During this funding period, we will begin to learn what genes these mutations affect, and how these genes influence the aging process. We will also assemble a set of fluorescent molecular biomarkers of aging that can be observed in living animals. These markers will revolutionize the study of aging because they will allow us to describe the aging process more accurately in normal and long- lived animals, to identify new aging mutants extremely rapidly, and also to analyze mutants with accelerated aging. Understanding this pathway in detail will ultimately answer the two most important questions in the aging field: how the aging process itself takes place, and how it can be regulated by endocrine signaling.

The Q cell migrations in C elegans provide a wonderful opportunity to investigate fundamental aspects of cell patterning and positioning. The QL and QR neuroblasts are bilateral homologs that migrate in opposite directions. QR and its descendants migrate anteriorly, whereas QL and its descendants migrate posteriorly. During its migration, QL switches on the Hox gene mab-5, which, in turn, causes QL's descendants to migrate posteriorly instead of anteriorly. Ultimately, each Q-cell descendant migrates to a unique anteroposterior (A/P) position that does not correspond to any obvious landmarks. Thus, by studying Q cell migration, we can ask how left-right asymmetry is generated, how Hox gene regulation and function are integrated into a biological process, how guidance information can direct these complex migrations, and how guidance cues polarize the cytoskeleton and control cell movement. During this past funding period, we have found that these migrations are regulated by many interesting signaling proteins, both novel and conserved. These include Wnt pathway members, a netrin receptor, novel transmembrane proteins required for cell positioning and for left-right asymmetry, and a Rho family member, exchange factor, and integrin receptors required for migration itself. Many of the conserved signaling molecules behave in unexpected ways, making them especially interesting to investigate. During this funding period, we will identify missing components of this system by exploiting a rapid new screening procedure. In addition, by manipulating expression of genes we have characterized, we will ask how Q migration is regulated. For example, we will ask whether particular proteins specify left-right asymmetry, and whether extracellular guidance cues have permissive or instructive roles. In addition, we will investigate the functions of proteins that appear to polarize and move the Q cells by using GFP fusions to visualize intracellular events. Together these studies should help to define the molecular mechanisms that regulate and execute these migrations.

DESCRIPTION (From the application): Aging and neurodegenerative disease inflict great suffering and financial cost on individuals and society. Although these processes are poorly understood, recent advances including genetic analysis in small organisms such as C. elegans and D. melanogaster, the identification of human disease genes, the construction of models by genetic manipulation in mice and developments in cell biology have opened up new and highly fruitful entry points for molecular analysis. For example, the process of aging has long been considered passive in nature; however, studies in C. elegans demonstrate the hormonal regulation of aging by genes with human homologs. Telomere loss causes cell senescence in vitro, and gene knock-out studies suggest that telomere loss in vivo may contribute to tissue decline. The study of prions has led to a new paradigm for neurodegenerative disease involving changes in protein conformation. Human genetics has identified proteins that participate in the pathogenesis of Alzheimer's and Parkinson's diseases. In general, many previously mysterious disorders are now known to have a specific molecular basis that can be analyzed using genetic, cellular and molecular approaches. These apparently intractable problems have become ripe for analysis, and extremely talented young investigators are now showing an increased interest in them. Demographic shifts towards an aging population increase the magnitude of the social problem and make the training of first-rate researchers all the more urgent. As a center of excellence committed to the training of students and postdoctoral fellows as well as the study of aging and neurodegenerative disease, UCSF is in a unique position to help solve these major biomedical problems. Here we propose to create a training program that takes advantage of UCSF's commitment to aging and neurodegenerative disease and interactive environment. The program will facilitate training and collaborations and focus the interests of students and faculty on aging and neurodegenerative disorders, with the goal of spawning new research initiatives that will lead to better understanding and more effective intervention.

DESCRIPTION (provided by applicant): In C. elegans, the germline stem cells; that is, the cells that give rise to sperm and oocytes, influence the aging process. Killing the germline precursors extends lifespan approximately 60%. This lifespan extension is not simply due to sterility, because killing the precursors of the entire reproductive system (the germline as well as the somatic gonad) has no effect on lifespan. In order for germline-ablation to extend lifespan, the DAF-16 protein, a forkhead-family transcription factor, is required. Thus, lifespan extension requires changes in transcription. In addition, the animals also require a functional DAF-12 steroid hormone receptor homologue. Thus, germline stem cells may exert their effects on aging by regulating asteroid hormone. By using mutations to eliminate specific subsets of germ cells (sperm, oocytes, germline stem cells), we have found that the germline stem cells regulate aging in adult animals. If the germline stem cells are forced to exit mitosis and enter meiosis in adult animals, lifespan is extended. In this study, we will investigate the mechanism by which germline stem cells influence the aging process. We will determine the sites of action of DAF-16 and DAF-12 activity, and we will identify downstream targets of these genes that extend lifespan using microarray analysis. In addition, we will screen for new genes that function in this pathway, and we will begin to determine their molecular activities and their times and sites of action. These studies could define pathways that regulate aging not only in C. elegans, but also in higher organisms, including humans. Using this information, it may be possible to improve the quality of old age, and to delay the onset of age-related diseases, such as cancer and diabetes. In addition, our findings may yield insights into the ways that stem cells can influence endocrine signaling in vertebrates.

DESCRIPTION (provided by applicant): During the past decade, it has become clear that the rate of aging, like many other processes in biology, is subject to regulation. In many animals, including mammals, longevity is regulated by a conserved insulin- and IGF-l-like signaling pathway. This longevity system was discovered in the genetically-tractable organism C. elegans, and a great deal about it has been learned by studying this animal. In C. elegans, DAF-2, an insulin/IGF-l-like receptor, activates a conserved signal transduction pathway that inhibits longevity, at least in part, by inhibiting the activity of the transcription factor DAF-16. In mutants defective in this signaling pathway, DAF-16 accumulates in the nucleus and coordinates expression of a battery of diverse downstream genes that together produce dramatic extensions of lifespan. This study addresses key, unsolved questions about this signaling pathway. Sensory neurons regulate the DAF-2 pathway, possibly in response to environmental cues, and this study tests the hypothesis that sensory neurons influence lifespan by controlling the production or release of specific insulin-like peptides. The C. elegans heat-shock transcription factor, HSF-1, which regulates the highly-conserved heat-shock response, acts with DAF-16 to regulate the expression of genes required for youthfulness and longevity. This study will determine how HSF-1 activity is regulated and how it functions in this system. Finally, a large number of genes with unknown functions act in this system to influence longevity. This study will better define the biochemical roles that these genes play in the aging process. Together these studies may ultimately define new therapeutic strategies for combating age-related disease and increasing the quality of old age.

[unreadable] DESCRIPTION (provided by applicant): Several years ago, the Kenyon lab discovered that the reproductive tissues of C. elegans profoundly affect lifespan. This is intriguing, as the link between aging and reproduction is central to life history. When the germline is removed, lifespan is extended ~60%. Thus somehow the germline shortens lifespan. Conversely, the somatic reproductive tissues extend lifespan, because if they are removed in animals lacking a germline, no lifespan extension occurs. Recently, the Kenyon lab discovered that signaling from the reproductive system to the intestine, which is also the animal's adipose tissue, is required for lifespan extension. A lipophilic-hormone signaling pathway triggers the nuclear localization of DAF-16/FOXO, a lifespan-extending transcription factor, within the intestine. A second, yet undefined, pathway up-regulates a new, essential transcription factor in the intestine. The Kenyon lab has found that the somatic reproductive tissues are required for DAF-16 to activate some but not all of its target genes, and it has identified several genes that may be required for this somatic-gonad activity. Autophagy, microRNA processing, innate immunity and regulated proteolysis all appear to play a role in this lifespan-extending system, as do additional signaling proteins and transcription factors. During this funding period, the Kenyon lab will use genetics, laser microsurgery and molecular approaches to investigate how these and new genes act at the molecular level to execute and coordinate an extension in lifespan when the germline is removed. When the reproductive tissues are perturbed in long-lived insulin/IGF-1 -pathway mutants, the animals remain healthy and vigorous and live six times as long as normal. This spectacular lifespan extension provides a wonderful opportunity to address, using genetics and molecular biology, the question of how dramatic differences in lifespan can be produced. Are the same genes that are up-regulated in the long-lived insulin/IGF-1-pathway mutants further stimulated, or are new genes activated? How different species in nature evolved striking differences in lifespan is a profound and fundamental question. This study, which takes place within a single, genetically-tractable, species, provides a fantastic opportunity to identify mechanisms that can produce extreme differences in lifespan [unreadable] [unreadable] [unreadable] [unreadable]

During the past decade, it has become clear that the rate of aging, like many other processes in biology, is subject to regulation. In many animals, including mammals, longevity is regulated by a conserved insulin- and IGF-l-like signaling pathway. This longevity system was discovered in the genetically-tractable organism C. elegans, and a great deal about it has been learned by studying this animal. In C. elegans, DAF-2, an insulin/IGF-l-like receptor, activates a conserved signal transduction pathway that inhibits longevity, at least in part, by inhibiting the activity of the transcription factor DAF-16. In mutants defective in this signaling pathway, DAF-16 accumulates in the nucleus and coordinates expression of a battery of diverse downstream genes that together produce dramatic extensions of lifespan. This study addresses key, unsolved questions about this signaling pathway. Sensory neurons regulate the DAF-2 pathway, possibly in response to environmental cues, and this study tests the hypothesis that sensory neurons influence lifespan by controlling the production or release of specific insulin-like peptides. The C. elegans heat-shock transcription factor, HSF-1, which regulates the highly-conserved heat-shock response, acts with DAF-16 to regulate the expression of genes required for youthfulness and longevity. This study will determine how HSF-1 activity is regulated and how it functions in this system. Finally, a large number of genes with unknown functions act in this system to influence longevity. This study will better define the biochemical roles that these genes play in the aging process. Together these studies may ultimately define new therapeutic strategies for combating age-related disease and increasing the quality of old age.

DESCRIPTION (provided by applicant): Aging and reproduction are central aspects of life history. We have found that in C. elegans, signals from the reproductive system regulate lifespan. When the germ cells are removed, lifespan is increased approximately sixty percent. This regulation may be evolutionarily conserved, as germline-stem cell removal in flies also extends lifespan. It is possible that the control of aging by reproductive tissues provides a way for the animal to coordinate its rate of aging with its timing of reproduction. In C. elegans, signals from the reproductive system affect lifespan by controlling the FOXO-family transcription factor DAF-16. DAF-16/FOXO-dependent transcription is known to be stimulated when the level of insulin/IGF-1 hormone signaling is reduced, but the genes needed to stimulate DAF-16's activity when the germline is removed are not needed to stimulate DAF-16 when insulin/IGF-1 signaling is reduced. Therefore, in essence, we are studying a new signaling pathway that conveys information about reproductive status to evolutionarily-conserved, core longevity mechanisms. We have learned that germ-cell loss triggers a molecular response in another tissue, the intestine, which includes the nuclear localization of DAF-16/FOXO and increased expression of gos-1, which encodes another transcription factor. (In C. elegans, the intestine behaves as the entire endoderm, including the adipose tissue.). Both DAF-16 and GOS-1 are required for lifespan extension, and both are required for new patterns of gene expression in animals that lack germ cells. We have identified several genes, including components of a Wnt signaling pathway, that allow the reproductive system to control DAF-16 and GOS-1, and we have developed powerful tools for their analysis. Using these tools, we will ask whether a Wnt signal conveys information about the reproductive system to the intestine, and we will ask what events occur in the intestine to cause DAF-16 and GOS-1 to stimulate new patterns of gene expression. This is exciting, because understanding this system well at the molecular level may suggest ways to artificially activate conserved longevity mechanisms in humans, with great health and longevity benefits. PUBLIC HEALTH RELEVANCE: Mutations that increase lifespan also confer resistance to age-related disease. We have found that the reproductive system of C. elegans, a simple roundworm, influences lifespan by signaling to core, evolutionarily-conserved longevity pathways. We have identified new genes that convey information from the reproductive tissues to these conserved pathways. We propose to learn how signals from the reproductive system influence lifespan, in hopes of finding new ways to increase youthfulness and health, and to combat age-related disease, in humans.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The aging process is not an immutable phenomenon as we used to believe, but a highly regulated process. Major advances in research on aging have been achieved using simple model organisms such as the roundworm, Caenorhabditis elegans and the fruit fly, Drosophila melanogaster. Until now age-related modifications in the proteome have been only summarily characterized with low-resolution two-dimensional electrophoresis, which precluded the identification of all but the most abundant proteins. In this project we will ask how the C. elegans proteome changes with normal aging. In collaboration with the UCSF Mass Spectrometry Facility, we will use a gel-free method by combining liquid chromatography (LC) mass spectrometry and a tag-based quantification to identify and quantify differences between protein extracts from young and old C. elegans. To perform the quantification we will take advantage of the iTRAQ labeling technology. This method consists of 4 isobaric tagging reagents allowing the quantification of four different samples at the same time. After trypsin digestion both old and young worm extracts will be labeled with iTRAQ and combined. We will further reduce the sample complexity by separating the peptides with strong cation exchange chromatography. We will analyze the fractions obtained with the nano-LC-electrospray ionization-quadrupole-time of flight mass spectrometer (nano-LC-ESI-Qq-TOF MS). This proteomics overview of aging should create a valuable database for the whole aging field. We expect to identify groups of functionally related proteins that are modified with aging, for example, proteins involved in the proteasome-mediated degradation or the unfolded-protein response. Some of the changes we see may cause, rather than reflect, aging. Therefore we will up- and down-regulate interesting components of these systems to measure how they affect lifespan.

DESCRIPTION (provided by applicant): FOXO transcription factors extend lifespan and delay age-related disease in animals, and many studies have now linked FOXO3A DNA variants to exceptional longevity in humans. Thus, the time seems right to look for human genes that are likely to regulate FOXO-dependent, or other, longevity pathways. FOXO proteins can be activated in many ways to extend animal lifespan. For example, C. elegans FOXO can promote longevity in response to reduced insulin/IGF-1 signaling, altered serotonin signaling, and elevated AMP kinase activity, elevated heat-shock factor activity, elevated lin-4 microRNA activity, elevated Jun kinase activity and other inputs. Thus, there could be many gene perturbations that can extend healthy lifespan in humans; and some of these perturbations may be safer and more effective than others. Because it is not possible to do genetic screens for long-lived humans, we are doing genetic screens in human cells instead. Our experimental strategy is based on the observation that all FOXO- dependent life-extending pathways tested so far (as well as many other life-extension pathways) increase resistance to oxidative stress. Although the role of oxidative stress resistance in life extension is not clear, the correlation is tight enough that in many model organisms, screens for oxidative stress resistance have yielded long-lived mutants. Therefore, to obtain a set of potential human longevity genes, the Kenyon lab has carried out a genome-wide siRNA screen for oxidative stress resistance in a human primary cell line. The gene hits include known C. elegans FOXO regulators, regulators of other longevity proteins such as TOR and NRF2, and new genes as well. From this set, the Kenyon lab will identify good candidates for new human longevity and healthspan genes. To do this, they will determine which knockdowns trigger other correlates of longevity, such as xenobiotic resistance or autophagy. In addition, they will ask which knockdowns perturb the activities of FOXO3A, TOR or NRF2. Finally, to link these genes to longevity, they will test for their ability to influence lifespan in C. elegans and for their altered expression in centenarian families. This fresh approach will define new potential drug targets for extending the youthful and productive years of human life, and for delaying age-related diseases such as cancer, heart disease and/or neurodegenerative disease.

DESCRIPTION (provided by applicant): This project employs a new strategy, suggested by basic research in the science of aging, to find small molecules (precursors of drugs) that extend healthy, youthful lifespan and combat age-related diseases such as cancer. In animals, mutations in many nutrient, energy and stress-sensing genes extend youthfulness and lifespan and prevent age-related disease. A shared dream of aging researchers is that these remarkable findings will lead to new ways to keep us more youthful and disease-free as we age. My lab has carried out a small-molecule screen in hopes of bringing this dream closer to reality. Instead of targeting a single aging regulator, we cast a wide net by screening first for a cellular phenotype that is shared by cells from many long-lived animal mutants, as well as cells from long-lived species: increased resistance to multiple forms of environmental stress. When similar screens have been done in animals, a fraction of the hits have proven to extend lifespan. From a screen of 104,000 compounds, we found ~ 50 that make human primary cells in culture resistant to oxidative stress. Some confer resistance to other stressors as well. We are now asking which of these compounds might activate human longevity pathways. So far, at least some small molecules activate pathways that extend lifespan in animals, and at least some appear to increase C. elegans stress resistance and lifespan. A hallmark of many long-lived mutants is cancer resistance, and several of our top hits are predicted to have anticancer activity. Thus this screening strategy may lead to new ways to treat or prevent cancer. We will explore this hypothesis by testing our small molecules for anticancer activity. Relevance: Cancer is an age-related disease, and many gene perturbations that extend lifespan in animals also delay cancer. In this study, we use our knowledge of the basic biology of aging to develop new ways to find drugs against cancer. These drugs should also increase youthfulness and general health.