Judith Campisi - US grants

Cell proliferation in higher eukaryotes is a tightly regulated process that is largely governed by extracellular factors. These include hormones, growth factors, growth inhibitors and the cellular microenvironment, for example, the proximity of neighboring cells. Tumorigenic cells exhibit faulty or unregulated growth control, and they often fail to heed one or more of the environmental signals that limit the proliferation of normal cells. There is substantial evidence that growth-controlling environment signals have acute effects upon the expression of specific genes that are though important for the proliferative response. This proposal examines the biochemical mechanisms by which environmental signals alter the expression of certain genes in nontumorigenic murine fibroblasts, in tumorigenic derivatives derived by chemical or viral transformation, and in transfected derivatives bearing one or more known genetic change. The experiments will determine the intracellular pathways by which some environmental factors induce or suppress the transcription, mRNA accumulation or translation of certain growth-related genes, the role of certain protein kinases and other regulatory proteins in mediating changes in gene expression and the proliferative response, and they will explore the function of some of the genes in regulating proliferation. These studies will provide an important biochemical and molecular biological base for understanding the interactions between environmental signals and specific gene expression in the regulation of normal and tumor cell growth.

PROJECT SUMMARY Cellular senescence is a multi-faceted stress response that has become increasingly implicated as a driver of aging and many age-related pathologies. This proposal aims to renew a long-standing grant that has supported our work to understand the basic physiological and pathophysiological consequences of the senescence response. Through the use of simple and complex human and mouse cell cultures and a newly described mouse model developed by us, we propose a series of experiments that will provide a more in-depth understanding of the relationship between senescent cells, their presence and phenotypes during organismal aging, and aging phenotypes. We propose to provide answers to three crucial outstanding questions in aging research that have been unanswerable until recently. First, what are the occurrences, sub-tissue localization, cell type-specificities and phenotypic characteristics of senescent cells that accumulate during aging across tissues? Second, what are the phenotypes of senescent cells that accumulate in during aging compared to those that occur during wound healing? And finally, what is the impact of senescent cells on the apparent stochastic variation in aging phenotypes and life span among genetically identical mice? Together, these aims will fill crucial gaps in our knowledge about how senescent cells contribute to aging phenotypes and, consequently, organismal health span and ultimately life span.

Cell proliferation in higher eukaryotes is a tightly regulated process that is largely governed by extracellular factors. These include hormones, growth factors, growth inhibitors and the cellular microenvironment, for example, the proximity of neighboring cells. Tumorigenic cells exhibit faulty or unregulated growth control, and they often fail to heed one or more of the environmental signals that limit the proliferation of normal cells. There is substantial evidence that growth-controlling environment signals have acute effects upon the expression of specific genes that are though important for the proliferative response. This proposal examines the biochemical mechanisms by which environmental signals alter the expression of certain genes in nontumorigenic murine fibroblasts, in tumorigenic derivatives derived by chemical or viral transformation, and in transfected derivatives bearing one or more known genetic change. The experiments will determine the intracellular pathways by which some environmental factors induce or suppress the transcription, mRNA accumulation or translation of certain growth-related genes, the role of certain protein kinases and other regulatory proteins in mediating changes in gene expression and the proliferative response, and they will explore the function of some of the genes in regulating proliferation. These studies will provide an important biochemical and molecular biological base for understanding the interactions between environmental signals and specific gene expression in the regulation of normal and tumor cell growth.

Longevity, even in simple organisms, is undoubtedly regulated by multiple genetic loci. We propose to identify and functionally characterize genes that control one or more aspect of longevity in cultured human fibroblasts, the yeast Saccharomyces cerevisiae, and genetically heterogeneous and selectively bred mice. Genes that modulate the finite proliferative lifespan of human fibroblasts will be identified by a highly selective differential cloning strategy. Candidate senescence-modulating human genes will be cloned and tested for three functions: 1) inhibition of cell proliferation; 2) stimulation of DNA synthesis or mitosis; and 3) because it is unlikely that any single gene controls cellular senescence, cooperation with wild-type or mutant viral oncogenes in stimulating DNA synthesis or mitosis. In collaboration with Dr. Michal Jazwinski, S. cerevisiae will be used to 1) determine the effects of these genes on (yeast) longevity; 2) establish whether they act as dominant or recessive alleles; 3) isolate yeast genes that enhance or suppress their effects. In parallel, yeast genes shown by Jazwinski's laboratory to affect longevity will be tested for function in human fibroblasts. Yeast strains bearing or requiring viral oncogenes will be used to isolate additional yeast and human genes that control longevity or proliferative senescence. In collaboration with Dr. Richard Miller, genes that modulate senescence or longevity will be assessed for expression in young and old mice, either genetically heterogeneous or selectively bred for late reproduction or high or low immune function. mRNAs that are differentially expressed by pure cell types isolated from young, old or selectively bred animals will be identified, characterized for age- and tissue-specific expression, and a subset will be cloned and tested for function in human fibroblasts and S. cerevisiae. The long range goal is to identify strong candidates for human longevity assurance genes, and to know enough about their function to develop rational strategies for testing in transgenic animals and, ultimately, for therapeutic or interventional applications in humans.

Longevity, even in simple organisms, is undoubtedly regulated by multiple genetic loci. We propose to identify and functionally characterize genes that control one or more aspect of longevity in cultured human fibroblasts, the yeast Saccharomyces cerevisiae, and genetically heterogeneous and selectively bred mice. Genes that modulate the finite proliferative lifespan of human fibroblasts will be identified by a highly selective differential cloning strategy. Candidate senescence-modulating human genes will be cloned and tested for three functions: 1) inhibition of cell proliferation; 2) stimulation of DNA synthesis or mitosis; and 3) because it is unlikely that any single gene controls cellular senescence, cooperation with wild-type or mutant viral oncogenes in stimulating DNA synthesis or mitosis. In collaboration with Dr. Michal Jazwinski, S. cerevisiae will be used to 1) determine the effects of these genes on (yeast) longevity; 2) establish whether they act as dominant or recessive alleles; 3) isolate yeast genes that enhance or suppress their effects. In parallel, yeast genes shown by Jazwinski's laboratory to affect longevity will be tested for function in human fibroblasts. Yeast strains bearing or requiring viral oncogenes will be used to isolate additional yeast and human genes that control longevity or proliferative senescence. In collaboration with Dr. Richard Miller, genes that modulate senescence or longevity will be assessed for expression in young and old mice, either genetically heterogeneous or selectively bred for late reproduction or high or low immune function. mRNAs that are differentially expressed by pure cell types isolated from young, old or selectively bred animals will be identified, characterized for age- and tissue-specific expression, and a subset will be cloned and tested for function in human fibroblasts and S. cerevisiae. The long range goal is to identify strong candidates for human longevity assurance genes, and to know enough about their function to develop rational strategies for testing in transgenic animals and, ultimately, for therapeutic or interventional applications in humans.

[unreadable] DESCRIPTION (provided by applicant): As the U.S. population ages, it has become increasingly important to understand the fundamental bases of aging. Age is the largest single risk factor for a panoply of diseases, including cardiovascular dysfunction, cancer, type ll diabetes, osteoporosis, and neurodegeneradve disorders. If we could eliminate any one of these diseases, we would incrementally increase the average years of healthy life (health-span) the U.S. population might expect. If, however, we could postpone, or decrease the rate of, aging, we would retard the course of multiple age-related diseases and substantially increase average health-span. Thus, rational approaches to preventing or intervening in age-related disease will depend on an integrated strategy: understanding the specific etiologies of these diseases, and understanding the basic mechanisms of aging. [unreadable] [unreadable] In four years, this training program successfully brought together a nucleus of outstanding biologists from the Lawrence Berkeley National Laboratory (LBNL) and University of California at Berkeley (UCB) to train young scientists in basic aging research. We propose to continue and expand this thriving program. The proposed program consists of outstanding scientists from three Bay area institutions: LBNL, UCB and the Buck Institute for Age Research. These institutions now comprise a joint Center for Research and Education in Aging, and the Principal Investigator holds joint appointments at LBNL and the Buck Institute. The proposed preceptors are experienced investigators with diverse but complementary research interests and experimental approaches. The training program will emphasize collaborations among preceptor laboratories, and will be enhanced by the outstanding biomedical research community of the Bay area. [unreadable] [unreadable] The proposed program will train postdoctoral scientists for a career in aging research. It will offer laboratory experience in a variety of research projects, as well as education through seminars, research meetings, and special symposia, spanning diverse topics of importance and relevance to aging. It will also offer trainees access to outstanding facilities at the three participating institutions, and the opportunity to interact with leading investigators in experimental gerontology. [unreadable] [unreadable]

Aging is the ultimate expression of the interplay between an organism's genes and its environment and life history. It is now clear that multiple genes control the rate and manner in which organisms age. In addition, environmental and physiological stress strongly influence aging phenotypes. This conference will bring together scientists working on the genetics of aging and the ability of the aged organism to withstand environmental and physiological stress. It will also include selected scientists who are working in areas of importance to aging research, but not usually included in meetings on aging research. Thus, many of the speakers are leaders in molecular aging research, but the program also features prominent scientists working in important related areas. The conference is multi-disciplinary, bringing together molecular geneticists and epidemiologists, cell biologists and physiologists, and evolutionary biologists. It is designed to position the field of aging research to take advantage of the revolution in gene discovery and functional genomics research, and to integrate emerging information on molecular determinants of longevity with the organismal response to environmental influences and the phenotypes of aging organisms. The proposed meeting will bring scientists from diverse biological fields together to present new discoveries, and discuss new ideas, on the genetic on the molecular physiological basis of aging. The conference will begin with a Keynote address in which two opposing ideas on the control of complex phenotypes (genetic versus epigenetic) will be discussed. Subsequent sessions will address the evolution of aging; model systems, longevity-determining genes and their effects on stress responses; cellular aging in humans; genetic and biochemical manipulation of longevity and stress responses in mammals; mammalian age-related disease genes and polymorphisms; and current approaches for mining the human genome for candidate longevity determination genes.

Normal somatic cells of higher eukaryotic organisms do not proliferate indefinitely due to a process termed cellular or replicative senescence, or cellular aging. Several lines of evidence suggest that cellular senescence is a tumor suppressive mechanism that has the unselected, deleterious effect of contributing to organismic aging. Upon reaching the end of their replicative life span, cells undergo three phenotypic changes: 1) they irreversibly arrest proliferation (used here interchangeably with growth) with a G1 DNA content; 2) they become resistant to apoptotic death; 3) they show sometimes striking, cell type specific changes in differentiated functions. The growth arrest of senescent cells has been attributed to one or more critically short telomere, acquired as an inevitable consequence of multiple rounds of DNA replication by cells that lack telomerase. How a critically short telomere leads to the complex, senescent phenotype is unknown. One possibility is that a short telomere triggers a DNA damage response, in and of itself or in conjunction with intragenomic damage acquired during multiple rounds of DNA replication and cell division. The idea that DNA damage, and subsequent mutations, cause or contribute to replicative senescence and organismic aging has been long debated. This proposal aims to test this idea in ways that have not previously been possible. First, in collaboration with the Vijg Project, we will establish a new method to test mutation frequency and spectra in normal human cell cultures. We will combine viral transfer technology with plasmid-based receptor systems to deliver and evaluate mutation-reported vectors in normal and transformed human cells, cells derived from human donors with defined defects in DNA repair or age-related phenotypes, and human cells with perturbations in the establishment or maintenance of the senescent phenotype. Second, in collaboration with the Hoeijmakers Project, we will ask whether and to what extent embryo fibroblasts cultured from mice deficient or transgenic for genes known to be critical for DNA repair capacity exhibit signs of premature replicative senescence. We will establish a panel of senescent cell markers or endpoints that will enable us to compare the phenotype of mouse and human cells in culture, and relate this comparison to the phenotypes of human and mouse organisms. Third, in collaboration with both the Hoeijmakers and Vijg Projects, we will provide transgenic mice bearing a senescence-responsive element (SnRE)-reporter vector for interbreeding with DNA repair-deficient mice and mutation-reporter mice, and evaluate the senescence response of embryo fibroblasts. Together, these experiments will provide for the first time a comparison of human and mouse cells, and to a limited extent human and mouse organisms, with respect to mutation frequency and spectra, DNA repair capacity and replicative senescence. They will help critically test the relationship between DNA repair capacity, somatic mutations, replicative senescence and organismic aging.

DESCRIPTION (provided by applicant): Genomic maintenance systems are important determinants of longevity in all species. In mammals, DNA damage and faulty repair are major causes of cancer and non-cancer aging phenotypes. Normal cells respond to unrepaired DNA damage by apoptosis or cellular senescence, which suppress tumorigenesis but may also contribute to age-related pathology. WRN and BLM are related members of the mammalian RECQ-like family of DNA helicases. Current evidence suggests that both proteins participate in repairing DNA damage, among other possible functions. The phenotypes of humans deficient in these helicases suggest that both proteins prevent certain age-related diseases, including cancer. Werner syndrome (WS) and Bloom syndrome (BS) are caused by a deficiency in WRN and BLM, respectively. WS and BS share several features, but also show striking differences, the bases for which are largely unknown. Our overall goal is to better understand the cellular functions of the human WRN and BLM proteins. We propose to focus primarily on human cells, and determine how WRN and BLM are regulated, and how they regulate responses to DNA damage. We propose to determine effects of wild type and mutant WRN and BLM proteins on the senescence and apoptotic responses, telomere dynamics and genomic integrity of human cells. We will also determine how selected damage-sensing protein kinases regulate the subnuclear localization of WRN and BLM and its response to DNA damage. Our studies will provide important insights into how WRN and BLM postpone the development of aging phenotypes and the development of cancer in humans.

DESCRIPTION (provided by applicant): Genome maintenance is an important mechanism for assuring organismal longevity. In complex organisms, DNA repair systems postpone aging by preventing mutations, which ensures cell and tissue function and suppresses cancer, and by promoting rapid repair, which prevents apoptosis and senescence, cellular responses to DNA damage that suppress cancer but are thought to contribute to aging. Werner syndrome (WS) is hereditary progeroid syndrome in humans caused by loss of WRN, a DNA helicase related to a bacterial DNA repair protein. WS manifests after puberty with multiple age-related phenotypes and pathologies, including an aged appearance, atherosclerosis, type II diabetes, cataracts, osteoporosis and cancer. About half the cancers are mesenchymal, compared to <10% in the general population. WS individuals typically die of cardiovascular disease or cancer in the fifth decade of life. WRN deficient mice do not show WS phenotypes. WRN has been shown to modulate the repair of DNA double strand breaks (DSBs), and may play a role in telomere maintenance. Thus, WRN is a human longevity assurance gene that participates in multiple genome maintenance processes with cell-type and species specificity. To understand how WRN functions to postpone aging, we propose to determine the role of WRN and its catalytic activities in the responses to DNA damage, including senescence, apoptosis, replicative life span, telomere dynamics, and DSB repair. We also propose to explore the cell type specificity of WRN action and test the hypothesis that diverse cell types respond differently to WRN deficiency. Finally we propose to explore the species specificity of WRN action by determining whether there are differences between mouse and human cells in WRN regulation, localization or function. Understanding WRN function will provide a unique opportunity to understand an important human longevity assurance mechanism with a level of sophistication that includes cell type and species specificity, which are at present poorly understood.

An important accomplishment of the Program Project was to show that mutations in genes that participate in selected DNA repair pathways accelerate aging and/or cancer phenotypes in mice. In some cases, accelerated aging and/or increased cancer susceptibility were accompanied by an increase in somatic mutations. In other cases, accelerated aging occurred without an increase in mutations, suggesting other processes are responsible for the aging phenotypes. In all cases, however, we do not yet fully understand the cellular bases for the organismal phenotypes, which will be crucial for understanding how premature aging develops and how aging phenotypes are postponed by genome maintenance systems. Specifically, how do defects in genome maintenance impact the behavior and fate of cells, and, ultimately, how do the cellular responses cause the organismal phenotypes? This project aims to link the organismal phenotypes resulting from changes in genome maintenance systems with specific cellular phenotypes, using cells cultured from mice and humans. Our overall goals are to 1) understand the cellular consequences of intact or impaired genome maintenance; 2) determine how mouse and human cells differ in their responses to genotoxic stress; and 3) test specific hypotheses and strategies for manipulating cellular responses to genotoxic stress, in anticipation of creating additional mouse models within the Program Project. To achieve these goals, we propose to test cells, primarily embryo fibroblasts (MEFs), from wild type and mutant mice for proliferative and apoptotic responses under physiological oxygen and oxidative stress. Cellular endpoints will include replicative and stress-induced cellular senescence, cell death, growth in semisolid medium, and oxidative DNA damage and DNA double strand breaks. For mice carrying the lacZ mutation reporter, we will also determine mutation frequencies and , where feasible, compare the phenotypes of human fibroblasts with those of MEFs. We will also manipulate the expression level or function of selected mediators of the mammalian stress response and/or anti-oxidant defense systems, and determine whether these manipulations alter the behavior or fate of the cells. The overall goal of these experiments will be to identify potential interventions that can alter cellular endpoints that correlate with aging, and ultimately might postpone aging phenotypes in vivo. Finally, we will determine the feasibility of identifying senescence-responsive genes that can be used to detect and follow senescent cells in vivo, and eventually eliminate senescent cells in mice, with the expectation that this manipulation will delay or ameliorate selected aging phenotypes in the intact organism.

ABSTRACT NOT PROVIDED

[unreadable] DESCRIPTION (provided by applicant): As the U.S. population ages, it has become increasingly important to understand the fundamental basis of aging and age-related disease. Age is the largest single risk factor for a panoply of diseases, including neurodegeneration, cancer, type II diabetes, osteoporosis and cardiovascular disease. Eliminating any one of these diseases will increase average years of healthy life (health span). Postponing, or decreasing the rate of, aging will retard the course of multiple age-related diseases and thus provide even greater increases in average health span. Thus, rational approaches to preventing or intervening in age-related disease will require an integrated basic research strategy: understanding the specific etiologies of these diseases, and understanding the basic mechanism of aging. Crucial to this strategy is our ability to train a cohort of scientists, knowledgeable in modern cell, molecular and physiological sciences, and committed and prepared to apply that knowledge to the problem of aging and age-related disease. This application proposes to create a unique and exciting new training program in aging research based on two ongoing programs, one at the Buck Institute for Age Research (PI: Dale Bredesen) and the other at the Lawrence Berkeley National Laboratory (LBNL)/University of California Berkeley (UCB) (PI: Judith Campisi). In the past four and seven years, these programs provided postdoctoral fellows with excellent training in basic aging and age-related disease research. We now propose to continue and expand these thriving programs by assembling one cohesive training program from outstanding scientists from these three Bay area institutions. As a recently designated NIA Nathan Shock Center for Aging and growing Institute focused on aging and age-related disease research, the Buck Institute is well-poised to lead this combined effort. The proposed new program will offer postdoctoral scientists unique and outstanding training for a career in aging research. In addition to benefiting from diverse faculty expertise in topics relevant to aging, age-dependent diseases, the program will provide trainees with access to superb facilities in genomics, proteomics and model systems. Trainees will gain skills in research design, critical thinking, and evaluation of new research findings through participation in courses, basic science and clinical research seminars, journal clubs and focused research meetings, in which their results will be discussed from both basic and applied perspectives. In addition, trainees will receive instruction in research ethics, as well as written and verbal skills to enrich their publications, grant proposals and oral presentations. Trainees will also have opportunities to meet and interact with current leaders in aging and age-related disease research through organized trainee-sponsored seminars and Institute sponsored symposia and courses. Thus, fellows in this program will gain state-of the-art training and knowledge in modern aging research, enabling them to embark on careers dedicated to understanding the bases for aging and age-related disease and devise strategies to improve the health span of the aging U.S. population. [unreadable] [unreadable] [unreadable]

Mitochondrially-generated reactive oxygen species (ROS) are thought to be a major cause of aging and many age-related diseases. ROS can be beneficial or detrimental, depending on their level, as well as the species, differentiation state and genotype of the cell in which they are generated. The major source of intracellular ROSis mitochondria, and several enzymes mitigate their damaging effects, including superoxide dismutases and enzymes that maintain glutathione pools. Several lines of evidence support the hypothesis that oxidative stress caused by mitochondrial ROS drives aging and age-related disease. Nonetheless, there are many gaps and uncertainties in our knowledge regarding the relevant ROS targets and how their modification might influence aging phenotypes. In complex organisms such as mammals, ROS can elicit any of a variety of cellular responses, depending on the level and cell and tissue context. These include stimulation of repair pathways (particularly genome maintenance pathways) or loss of genomic integrity, cell proliferation (growth) or growth arrest (transient or permanent), and cell death or survival. Strikingly, many of these responses are controlled by the tumor suppressor protein p53, a multifunctional redox-sensitive nuclear protein having DNA binding, transactivation and transrepression activities. p53 also functions at the mitochondria to promote damage- induced apoptosis, and was recently shown capable of modulating aging phenotypes and life span in mice. Our preliminary data suggest that mitochondrially-generated oxidative stress can alter biochemical functions of p53, and, conversely, that p53 status can influence mitochondrial function. We propose to test three hypotheses regarding the interplay between mitochondrial ROS and p53 in mammalian cells: 1) p53 is a crucial mediator of cellular responses to mitochondrially-generated oxidative stress;2) mitochondrially-generated oxidative stress alters p53 structure and functions;3) p53 modulates mitochondrial functions, independent of apoptosis. Accordingly, our specific aims are: 1) test the idea that p53 determines the fate of rodent and human cells that experience elevated endogenous ROS owing to mitochondrial dysfunction (Aim 1);2) explore the idea that mitochondrially-generated oxidative stress alters p53 structure and functions, facilitating a switch from nuclear to mitochondrial responses (Aim 2);3) test the idea that p53 can modulate mammalian mitochondrial function, directly or indirectly, by altering specific mitochondrial reactions complexes (Aim 3). Our proposed experiments will require the use of all the Cores, and will require close interactions with Projects 1 and3.

DESCRIPTION (provided by applicant): The Buck Institute for Age Research is the only free-standing institute in the United States that is devoted exclusively to basic research on the biology of aging and age-related diseases. Its intensive, interdisciplinary, institution-wide approach to aging research represents a new model, distinct from the traditional university-based approach in which the study of aging is one of numerous disciplines, spread throughout an institution, and competing with other disciplines for scarce scientific resources. In the five years since the Buck Institute began operation, it has made a substantial investment in and commitment to aging research. In the process, Buck Institute investigators have made important contributions to research on the biology of aging and age-related diseases, including Alzheimer's disease, Parkinson's disease, stroke and cancer, and the Institute has established itself as a major aging research center. We believe that the Buck Institute is ideally suited to fulfill the goal of the Nathan Shock Centers of Excellence in Basic Biology of Aging program - to enhance the ability of institutions with well-developed research programs in basic research on aging to utilize state-of-the-art research resources and provide the strongest environment for the conduct of research on aging by: (a) enhancing the quality of research in the basic biology of aging (b) facilitating the planning and coordination of aging research activities (c) providing support and a suitable environment for investigators new to aging research and (d) developing potential regional or national resource centers. To achieve these objectives, we propose to establish a Shock Center at the Buck Institute with the following Cores: An Animal/Transgenics Core offering animal care and procedures and transgenic/knockout production, an Imaging Core in which existing morphology resources will be supplemented with functional imaging technology, a Genomics Core that will produce cDNA and oligo microarray chips and offer microarray analysis services, and a Proteomics Core using mass spectrometry and new chemical methods to identify posttranslational modifications and protein-protein interaction networks associated with aging and age-related diseases. Finally, a Research Development Core will foster the development of investigators and projects in aging research through postdoctoral training programs, Research Development Research Seminars, Pilot Project awards to junior investigators and investigators new to the aging field, and a Summer Scholars program for high school and college students from underrepresented minority backgrounds.

SUMMER TRAINING COURSE ABSTRACT Support is requested to continue a series of Summer Training Courses in Experimental Aging Research to be held in June from 2008 through 2012. The 5-day Course will be directed from the Buck Institute for Age Research in Novato, CA and the conference site will rotate among three host institutions: The Buck Institute for Age Research, the University of Texas Health Science Center at San Antonio, and the University of Washington in Seattle. Similar courses have been held with great success every summer since 1993. The Course is designed to provide trainees with intensive exposure to modern research in experimental biogerontology and individualized guidance regarding research ideas and plans. Each year's enrollment will be limited to 20 researchers. Most trainees will be in formative stages of their careers, but the Course will also consider senior investigators who wish to enter or redirect their efforts to an area of aging research. Each trainee is expected to have at least 2 years of productive laboratory experience in some aspect of cell or molecular biology beyond the doctoral degree (MD, PhD, or DVM). Each day during the 5-day program will include 3 activities: (a) 2 overview lectures designed to introduce trainees to the context, latest findings and main unanswered questions in a major area of gerontology; (b) a research development workshop, at which each trainee will have an opportunity to present his or her own research ideas and plans for critique; and (c) a research seminar presented by a prominent faculty member of the host or nearby institution, who will generally cover a topic or disease aspect not covered in the overview lectures. This Summer Training Course will provide younger researchers with a solid foundation in modern experimental gerontology, and provide a useful perspective to more senior scientists who are developing new programs in aging research. Four scientists will serve as the Course Steering Committee and will participate in the course on a regular basis: Judith Campisi, the Course Director, will discuss cancer, stem cells and the cell biology of aging; Jan Vijg will lecture on mouse models of aging and genome instability; Arlan Richardson will discuss on caloric restriction and oxidative stress; and Peter Rabinovich will lecture on free radicals and mitochondria. Fourteen other researchers will serve as Participating Faculty, attending the course approximately every third year. They include: Andrzej Bartke (endocrinology of aging), Brian Kennedy (invertebrate models, caloric restriction), Caleb Finch (neurodegeneration, stochasticity), George Martin (progerias, neurodegeneration, age-related disease), Gordon Lithgow (invertebrate models, stress), Jim Nelson (endocrinology, stress), John Tower (invertebrate models, sex determination), Marc Tatar (demography, evolution), Nir Barzilai (genetic variation), Richard Miller (vertebrate models, immunology), Rita Effros (immunology, cell biology), Steve Austad (comparative biology, evolution), Tom Rando (stem cells, cell biology), Virginia Lee (neurodegeneration).

The rationale of this Program Project is that spontaneous DMA damage drives major components of the aging process, through direct adverse effects, but more likely by inducing genome maintenance responses, resulting in senescence, apoptosis and/or genomic and epigenomic errors. The long-term objectives of Project 2 are to test the hypothesis that somatic DMA alterations, including genome rearrangements and epigenomic changes, causally contribute to aging by gradually dysregulating gene expression leading to cell functional decline and degeneration and eventually to age-related pathologies, including but not limited to cancer. In Specific Aim 1 of the renewal application we first plan to significantly broaden the scope of the molecular endpoints thus far analyzed. For that purpose, in collaboration with Project 1, we will measure spontaneous DMA damage, changes in CpG island methylation and transcriptional noise levels in tissues of normal and DMA repair-deficient, prematurely aging mice. In Specific Aim 2 we will further study DMA double-strand breaks, as a potentially important intermediate in generating genome instability, dysregulated gene expression and cellular senescence in mouse and human primary fibroblast cultures (with projects 3, 4 and 5). In Specific Aim 3, we propose to combine functional assessment of a single cell with genome-wide analyses of its transcriptome, epigenome and genome. Successful pursuit of these Specific Aims should provide new insight into the role of genome maintenance as a determinant of aging, with a focus on the relationships among various molecular and cellular end points.

PROJECT SUMMARY (See instructions): A challenge of biomedical research is to compress the period of frailty and disability as people reach advanced age. Cellular senescence, the growth arrest that occurs when cells experience potentially oncogenic insults, has been proposed to contribute to age-related dysfunction. There is as yet no definitive evidence for this. To understand the role of senescent cells in age-related dysfunction, we created a mouse model from which p16-expressing senescent cells can be removed selectively. We devised mechanismbased interventions that interfere with the inflammatory senescence-associated secretory phenotype (SASP), which may be the basis of the inflammation that underlies many age-related diseases and frailty. We discovered a link between the SASP and immune system dysfunction, and found potential ways to break this link. Our unifying hypotliesis is that preventing the accumulation of senescent cells or their effects can restore age-related decrements in function. We propose the following Specific Aims. Aim 1 Eliminate senescent cells. We will use an innovative animal model from which we can selectively remove senescent, potentially cancerous cells to determine if this intervention attenuates development of age-related functional decrements and frailty and enhances healthspan. Aim 2 Inhibit the SASP by manipulating Jak/Stat. We found the SASP is attenuated by inhibiting Jak/Stat, an intervention that dramatically reduces the frailty associated with cancer and hematological disorders. We will test the impact of this intervention onage-related dysfunction. Aim 3 Inhibit the SASP by manipulating mTOR. We also found that inhibiting components of the mTOR pathway inhibits the SASP without interfering with the senescence-associate replicative arrest that defends against cancer. We will determine whether and how this inhibition reduces age-related dysfunction. Aim 4 Break the link between the SASP and inflammasomal activation. We found the SASP activates the inflammasome, while inhibiting it restored immune function in old animals. We will test if inflammasome inhibitors reduce age-related senescent cell accumulation and dysfunction. These Aims will be tested in four Subprojects supported by Administrative, Mouse Phenotyping and Pathological Assessment (MPPA), and Systems Biology/ Bioinformatics Cores. We will use innovative culture systems, novel animal models, and comprehensive healthspan phenotyping to test our hypothesis, focusing on frailty/muscle, metabolic/fat, skin, and immune function initially. Our approach will provide timely, innovative, and clinically relevant interventional results based on addressing the fundamental question of the role of cellular senescence that has remained unanswered for many years.

DESCRIPTION (provided by applicant): The largest risk factor for developing cancer is age. Age also poses the largest risk for developing many of the degenerative pathologies of aging. This exploratory application will test the idea that cellular senescence - a tumor suppressive response - is a basic aging process that links the development of cancer with the development of age-related degenerative disease. We propose to critically test the idea that senescent cells are important determinants of cancer risk and contribute to the age-dependent rise in cancer incidence. We will use two newly developed mouse models to: 1) induce senescence in a tissue-specific and temporally-controlled manner, and 2) eliminate senescent cells at will. We will use these models in conjunction with a well-described two-stage skin carcinogenesis protocol that will allow us to distinguish the effects of senescent cells on cancer initiation from effects on cancer promotion. We will also determine the extent to which senescent cells mediate the effects of natural aging on cancer development. The results will pave the way to applying the principles we uncover in skin carcinogenesis to a wide variety of other cancers. They will also provide a strong rationale for developing novel preventive or interventional strategies aimed at eliminating senescent cells, or selectively ameliorating their deleterious effects, from tissues that are at risk for developing malignancies. PUBLIC HEALTH RELEVANCE: Age is the largest single risk factor for developing cancer, but the reasons for the steep rise in cancer with age are incompletely understood. We will use two novel mouse models to critically test the idea that senescent cells, which accumulate with age as a consequence of oncogenic stress, are crucial determinants of the development of cancer in older organisms. Our proposed studies will provide crucial insights into the link between cancer and aging, and identify novel strategies for cancer prevention and interventions.

DESCRIPTION (provided by applicant): Age is the largest single risk factor for the majority of diseases seen in clinics throughout the U.S. Demographic calculations predict that eliminating any single age-related disease would produce only a modest increase in human health span (years of healthy life) or life span. However, postponing or decreasing the rate of aging would retard the course of multiple age-related diseases and thus substantially increase health span and likely life span. Our ability to develop rational approaches to preventing or intervening in th debilitating and costly consequences of aging depends crucially on a thorough understanding of the causes of aging and how they interact with the etiology of specific age-related diseases. Training young scientists to integrate research on basic aging mechanisms with mechanisms of specific age-related diseases is a critical objective of this application. The long-term goal of ths training program is to provide exceptional young scientists with the broad knowledge, skills and interactions they will need to mitigate, through research, the enormous human and financial burdens caused by aging and age-related diseases. Each year, the program will train 12 talented postdoctoral scientists who will conduct research for a 2-year period in one or more of 32 laboratories headed by outstanding preceptors at the Buck Institute for Research on Aging, Lawrence Berkeley National Laboratory, University of California, Berkeley and Stanford University. Trainees will participate in research projects that include basic mechanisms of cellular stress responses, protein homeostasis, genomic and epigenomic stability, stem cell maintenance, bioenergetics and energy metabolism and hormonal, growth factor and nutrient signaling path- ways. They will utilize a variety of model systems including yeast, round and flat worms, fruit flies, fish, mice and human cells and tissues. And they will focus on an array of age-related diseases including Alzheimer's, Parkinson's and Huntington's diseases, stroke, cardiac and vascular dysfunction, cancer, diabetes, osteoporosis and sarcopenia. Trainees will be instructed in state-of-the-art techniques in genomics, epigenomics, drug screening, proteomics and metabolomics, as well as genetics, biochemistry, structural biology, cell biology, and cell and organismal imaging. They will receive the benefits of diverse seminar series and other scientific events and frequent networking opportunities. They will also attend courses in specialized scientific topics, as well as courses in ethics, presentation skills, proposal and manuscript writing, and laboratory management skills. The program will fill an important national and international need for high-quality advanced training that integrates basic aging research with research on age-related disease.

DESCRIPTION (provided by applicant): Cellular senescence prevents the proliferation of mitotically-competent cells in the periphery in response to various stressors, including endogenous oxidative stress. Senescent cells express a 'senescence- associated secretory phenotype' or SASP, involving the secretion of pro-inflammatory factors that can cause degeneration of neighboring cells. While cell senescence in peripheral tissues has recently been shown to result in a number of age-related pathologies, whether it plays a causal role in brain aging and neurodegenerative disease is currently unknown. Preliminary data from our laboratory suggests that induction of astrocytic senescence may contributes significantly to neurodegeneration associated with Parkinson's disease (PD) and that this can be induced by candidate environmental toxicants previously linked to increased risk for PD. We propose to use two available environmental toxicant libraries to perform a small molecule screen in order to identify additional senescence-inducing agents, paying particular attention to bioavailable compounds previously associated with risk for PD. This will form the basis of future studies assessing the ability of environmental toxins identified in the screen to induce astrocytic senescence in vivo and its contribution to PD neuropathology. Based on this data, we plan to test the hypothesis that environmental agents may increase risk for PD in part via induction of astrocytic senescence that can in turn impact on PD-related neurodegeneration. If correct, our hypothesis has the potential to transform how we think about and treat PD and other related neurological disorders.

SUMMARY Dysfunctional mitochondria and mitochondrial DNA (mtDNA) mutations are hypothesized to drive aging phenotypes, but the mechanisms by which these age-related defects act are poorly understood. This proposal focuses on the mechanisms linking mitochondrial dysfunction, including mtDNA mutations, to aging phenotypes through the pleiotropic stress response known as cellular senescence. The senescence response is a permanent arrest of cell proliferation, accompanied by phenotypic changes, including the development of a senescence-associated secretory phenotype (SASP). The SASP entails the secretion of a myriad of pro- inflammatory cytokines, angiogenic factors, growth factors and proteases. However, in the case of mitochondrial dysfunction, the secretory and mitotic arrest phenotypes can be partially uncoupled. Depending on the extracellular environment, mitochondrial dysfunction can either drive SASP-like secretion in the absence of mitotic arrest, or can drive a senescence arrest that is notably free of SASP inflammatory components. We propose to use cultured human and mouse cells, and mouse models to critically determine the role of cellular senescence phenotypes in mitochondrial dysfunction-induced aging. Our specific aims are designed to answer the following questions: How does loss of mitochondrial function drive cellular senescence phenotypes? Does the accumulation of mtDNA mutations drive cellular senescence phenotypes in a mouse model, and can the elimination of senescent cells prevent mtDNA-induced tissue dysfunction? Finally, is mitochondrial dysfunction an inducer of cellular senescence phenotypes during normal aging and does a mouse model of delayed aging accumulate fewer senescent cells with age? If successful, our proposed experiments will provide an important link between mitochondrial function and physiological manifestations of aging. In addition to generating important basic knowledge, the experiments will also provide a new targets (dysfunctional mitochondria and senescent cells) for the development of potential interventions into aging and age-related diseases.

? DESCRIPTION (provided by applicant): Senescent cells as a source of pro-geronic factors several classic experiments using heterochronic parabiosis - surgically joining the circulatory systems of young and old animals - indicate the existence of at least two classes of circulating factors that influence aging. One class is abundant in young animals, declines with age, and can rejuvenate tissue homeostasis when given to old animals (anti-geronic factors). A second class is scarce in young animals, increases with age and can reduce tissue homeostasis when supplied to young animals (pro-geronic factors). Pro-geronic factors have attracted less attention than anti-geronic factors, yet are equally critical targets for therapies aimed at rejuvenating the aged systemic milieu. This proposal focuses on circulating pro-geronic factors secreted by senescent cells. Cellular senescence is a stress response that results in an arrest of cell proliferation and the secretion of many inflammatory cytokines, chemokines, growth factors and proteases, termed the senescence- associated secretory phenotype (SASP). Senescent cells increase with age in many vertebrate tissues, but the reason for this increase is not clear. Recent findings show that senescent cells can confer senescent phenotypes on neighboring cells, and our preliminary data suggest one or more circulating factor is responsible for spreading senescent phenotypes to non-senescent cells in vivo. We propose to study this secondary senescence and its role in creating an aged systemic milieu. Two laboratories will join forces to use heterochronic parabiosis and a novel transgenic mouse model to determine the kinetics, extent and cell type or tissue specificity of the ability of factors in the circulatin of aged mice to induce a senescence response in cells in young mice. We will phenotype secondary senescent cells induced by circulating factors present in old animals with regard to cell type and senescence markers, including the SASP. We will also test the idea that parabiosis-induced secondary senescence and increased circulating HMGB1 promote aging phenotypes in several tissues. In parallel, we will characterize the factors produced by secondary senescent cells to identify possible additional pro-geronic proteins. Finally, we will interfere with circulating HMGB1 activity to test the idea that interference can blunt the ability f the old circulation to induce secondary senescence and aging phenotypes. These experiments will provide much needed insights into pro-geronic factors, the neutralization of which will be essential for strategies aimed at rejuvenating the systemic milieu.

ABSTRACT This proposal requests partial support for an innovative conference on ?Cell Fate Diversity in Aging?. The conference will discuss the molecular damage, developmental cascades and pleiotropic effects that drive different cell fates, including cell death and cellular senescence and the emerging areas of adaptive reprogramming and cell fitness. It will feature innovative experimental and conceptual approaches, with participation by accomplished investigators new to the field of aging, as well as a core of leaders established in aging research to provide context, from the US, Europe and Asia. The program will also feature numerous short talks by young investigators, and two interactive sessions on emerging tools -- one focused on quantitative measurements of the drivers and cellular outcomes of aging to facilitate predictions of tissue- specific consequences, and another focused on computational and experimental models to test hypotheses. The program will also include poster sessions, ample time for discussion and opportunities for senior and junior investigators to interact. The conference aims to 1) highlight recent findings regarding how cell fate decisions are regulated, how they contribute to aging, and how understanding cell fate decisions can be used to prevent or postpone aging in humans; 2) provide a forum for integrating results from different disciplines, emerging technologies, and diverse experimental paradigms; 3) stimulate discussions on future directions for the field, identify gaps in our knowledge and experimental approaches, both basic and translational, for filling those gaps; 4) foster collaborations among biologists with different research emphases; 5) provide a stimulating environment and platform for young investigators, or investigators new to the field, to learn about how cell fates contribute to aging, present their work to a diverse audience, and establish professional and collaborative networks. The conference is sponsored by the not-for-profit Zing Conferences, which will provide only partial support. This proposal requests partial support for US-based speakers (citizens or permanent residents). It also requests partial support for young US-based students, postdoctoral fellows and new investigators to attend the conference and present a short talk and/or poster. The conference venue, Hotel Croatia Cavtat in Dubrovnik (Croatia), is readily accessible from the Dubrovnik airport (10-15 minutes by automotive ground transportation), which in turn is readily accessible to US, European and Asian speakers by many major airlines with European hubs. The conference organizers (Campisi, PI of the proposal, and Vijg) are experienced conference organizers, long-time collaborators and established aging research investigators. The conference is designed to highlight -- and stimulate research into ? a relatively underexplored area in aging research: how cell fate decisions result in aging phenotypes and pathologies.

Aging is the primary risk factor for cardiovascular disease (CVD), and by 2030, 40% of Americans will have some form of CVD incurring a huge economic and human toll on society. Much remains unknown about why cardiovascular dysfunction increases with age. One potential cause of cardiovascular aging is cellular senescence. This complex stress response can be beneficial or detrimental, depending on the physiological context. Two hallmarks of the response are: 1) a permanent arrest of cell proliferation; and 2) development of a senescence-associated secretory phenotype (SASP) -- the transcriptional upregulation and secretion of numerous inflammatory cytokines, chemokines, growth factors and proteases. Senescent cells increase with age in many tissues, including the cardiovascular (CV) system, and the SASP is associated with a variety of age-related pathologies, including cardiac and vascular dysfunction. Senescent cells accumulate with age in the hearts and vasculature of mice and humans, but it is not known if cellular senescence is a cause or consequence of cardiovascular aging. We developed a novel mouse model that permits the visualization and elimination of senescent cells in vivo and their isolation from tissues, making it possible to study the function of integrated systems -- such as the heart and vasculature -- with and without senescent cells. We propose to test the novel hypothesis that senescent cells, and particularly the SASP, are an important mechanistic process driving CV aging. To test this hypothesis, we will develop three specific aims. Aim 1: Determine the extent to which senescent cells cause CV system dysfunction in settings of both experimental senescence and natural aging. This aim will determine when and where senescent cells arise in the cardiovasculature, using an acute model, as well as naturally aged mice. We will employ our novel mouse model, the 3MR mouse in which we can eliminate cells to contrast CV function with and without senescent cells; Aim 2: Determine the cellular and molecular mechanisms by which senescent cells negatively impact cardiovascular tissue. This aim will determine the sensitivity of critical cell types in the CV to senescence, including fibroblasts, endothelial cells, and vascular smooth muscle cells isolated from both arteries and intact hearts. We will determine how secreted factors from senescent cells influence function of other cell types using co-cultures and genetic strategies; Aim 3: To identify novel therapeutic targets for age-related CV dysfunction in humans. Having established specific protein and gene expression signatures for different cell types as a result of senescence in aims 1 and 2, in this final aim we will use a novel translational model ? biopsies of endothelial cells from humans to validate our predictions about senescence in different aged humans. Overall our program will provide novel insights into the role of senescence as a major mediator of age- related CVD, and potentially provide new targets of opportunity to combat this devastating disorder.

PROJECT ABSTRACT ? Cellular senescence, astrocytes and Alzheimer's disease (Parent Grant: R01 AG051729, Cellular senescence as a mediator of mitochondrial dysfunction) Alzheimer's disease (AD) is a devastating neurodegenerative disease for which the largest single risk factor is age. At present, there are no efficacious treatments, and new approaches to understanding and managing the disease are desperately needed. AD research has focused largely on understanding how and why neurons lose function and ultimately die. A relatively understudied cell type, however, is the astrocyte, which is crucial for neuronal survival and function. Aging results in a progressive accumulation of astrocytes that have undergone cellular senescence ? a complex stress response that can radically alter cell function and, importantly, the ability of cells to interact and communicate with neighboring cells. Senescent cells increase with age in most mammalian tissues, including the brain, and many of their pathophysiological effects are thought to be a consequence of the complex senescence-associated secretory phenotype (SASP) -- the increased expression and secretion of numerous pro-inflammatory cytokines, chemokines, growth factors, proteases, bioactive lipids and damage-associated molecular patterns (DAMPs). Our preliminary data show a decline in the expression of genes that encode proteins responsible for the uptake of glutamate, an astrocytic function that is essential for preventing glutamate toxicity to neurons. We propose to explore this and other aspects of cellular senescence in astrocytes using state-of-the-art single cell sequencing techniques. Our aim is to understand in unprecedented depth the heterogeneity of the senescence responses of astrocytes, and to uncover vulnerabilities that have the potential to open new possibilities for interventions in AD.

ABSTRACT - PROJECT 3 DNA damage and its sequelae are now recognized as major drivers of aging. However, many questions remain regarding the nature of age-promoting DNA damage, the role of mutations in non-cancer age-related pathology, and how genomic damage and its sequelae drive age-related phenotypes and pathologies. Project 3 will determine -- in depth, breadth and primarily at the level of cellular responses -- how genes that participate in genome maintenance assure tissue health and ultimately longevity. The primary (but not the only) consequence-generating cell fate responses are cellular senescence and apoptosis. The extent to which cells in aged organisms acquire mutations and/or experience these cellular consequences due to DNA damage are poorly understood. We will explore the relationship between DNA damage-induced cell fates and mutations (with Project 2), molecular consequences of DNA damage (with Project 1), and human gene variants implicated in longevity (with Project 4). Specifically, we will determine cell fate responses of cells carrying genome maintenance genotypes associated with premature and delayed aging identified by Projects 1 and 4, focusing on multifunctional repair genes that participate in repair processes important for relieving replication or transcriptional stress. We will interrogate cellular senescence and apoptosis (and other forms of cell death), and markers of cell function. With Project 2, we will determine whether some genomic changes (e.g., aneuploidy, INDELS) preferentially elicit one cell fate over another. We will use simple and complex culture systems, focusing primarily on the skin, brain and liver and use single cell analyses to determine how different types of DNA damage affect cellular heterogeneity and variability. We will also determine the cellular responses to genotoxic stress in the backgrounds of genome maintenance genotypes and in response to genomic damage in wild-type and mutant mouse models, and naturally aged mice. We will exploit our mouse model in which senescent cells can be eliminated, or use pharmacological means to eliminate senescent cells, to determine how modifying this cell fate affects hallmarks of aging. Finally, once we have a candidate list of human gene variants associated with aging and longevity from Project 4, we will use human embryonic stem cells harboring these variants to assess the cells and their differentiated progeny for their cell fate responses to genomics stressors. Together, this Project will allow us to integrate the genomic and cellular responses to DNA damage during natural, accelerated and delayed aging in mouse and human cells, and provide mechanistic bases for predicting the efficacy of interventions into aging phenotypes and pathologies.

ABSTRACT Cell fates are important intermediates between age-related molecular events, including damage and stress, and the phenotypes and pathologies that are hallmarks of aging organisms. Cell fates can also offer unique -- and broadly applicable -- opportunities for interventions into many age-related diseases. The Cellular Senescence and Beyond Core (CSB) in this Nathan Shock Center (NSC) application will help academic researchers determine whether and to what extent specific cell fates play a role in their aging and/or intervention models. Depending on the project, the Core will characterize cell autonomous and non- autonomous features of selected cell fates, advise investigators of their possible physiological consequences, and test proposed intervention strategies. Aim 1 will establish and offer standardized methods to detect cell- associated markers of the cell fate decisions of cellular senescence, cell death and cell competition. Aim 2 will establish standardized methods to detect and assess the impact of the cell non-autonomous effects resulting from the cell fate decisions of cellular senescence, cell death and cell competition. The core will employ a variety of techniques, including ELISAs, immunocytochemistry, western analyses and, of particular utility, mass spectrometry to provide comprehensive, unbiased assessments. The Core will also advise investigators on strategies to identify the most promising cell autonomous and non-autonomous acting candidates and perform relevant assays to test these candidates for biological activity. In summary, the proposed Cellular Senescence and Beyond Core will provide investigators in aging research with a broad array of experimental assays and advice on how to determine whether and to what extent selected cell fate decisions are important contributors to the age-related phenotypes and pathologies of interest to the investigators.

OVERALL TMC - PROJECT SUMMARY Cellular senescence is a multi-faceted cell fate that arrests cell proliferation and activates the synthesis and secretion of numerous cytokines, chemokines, growth factors, proteases and lipids, termed the Senescence Associated Secretory Phenotype (SASP). The SASP can influence tissue microenvironments, locally and distally, and thus senescent cells can strongly affect tissue function. Senescent cells (SnCs) increase with age in most vertebrate organisms, including mice and humans, and it is increasingly clear through both genetic and pharmacological manipulations that they can drive a growing list of age-related pathologies, ranging from neurodegeneration to cancer. At present, there are no invariant biomarkers of SnCs and the molecular characteristics of SnCs are remarkably heterogeneous and variable, depending on cell and tissue type, microenvironment, senescence inducer, and timing. Our overall goal is to determine, molecularly and spatially, when and where senescent cells occur in humans, and also how their patterns of gene expression and SASPs vary with tissue physiology and age. These goals are also the goals of the SenNet Consortium. We therefore propose to establish a SenNet tissue mapping center (TMC) comprised of an Administrative Core, Biospecimen Core, Biological Analysis Core and Data Analysis Core. Our proposed TMC will focus on three human tissues (ovary, breast and skeletal muscle) and three biofluids (follicular fluid, plasma and urine). These samples have distinct biological characteristics and cell types (somatic and reproductive; stromal, epithelial and vasculature) that show significant changes with age. All materials are available through established collaborations, subcontractors and/or biobanks (through the Biospecimen Core). Their analyses will include cutting-edge transcriptomic and proteomic techniques that will take advantage of the expertise of several Buck Institute investigators. Our preliminary data show that these tissues and biofluids are amenable to the state-of-the-art technologies proposed in the Biological Analysis and Data Analysis Cores, as well as new technologies proposed in our accompanying Technology Development application (RFA-RM- 21-009). Importantly, our findings will be applicable to many other human tissues and biofluids in order to encompass and complement the overall goals of the larger SenNet Consortium. The Administrative Core will coordinate all aspects of the TMC, from specimen acquisition to analysis, and will oversee and facilitate frequent interactions between the proposed TMC and other investigators, Cores and components of the SenNet Consortium.

OVERALL PROJECT SUMMARY Aging is by far the most important driver and risk factor for developing a variety of neurodegenerative diseases, including Alzheimer?s disease (AD) and related dementias. These devastating diseases exact an enormous emotional, social and economic toll on patients and their families, yet to date there are no effective treatments that delay, much less reverse, the onset or progression of these diseases. Clearly, new approaches to understanding and treating age-related neurodegeneration are needed. This Program Project Grant (PPG) proposal aims to fill this serious gap in our knowledge and treatment approaches. The proposed PPG consists of three research projects, each focused on an aspect of brain aging that is known to be crucial for brain function: 1) cell fate decisions, particularly cell death and cellular senescence; 2) metabolism, particularly responses leading to metabolic reprogramming and inflammation; and 3) cell-cell interactions, particularly interactions between neurons and non-neuronal cells in the brain. We propose to support the projects by an administrative core, which will also provide statistical and bioinformatics support, and three scientific cores: 1) an iPSC/Organoid core; 2) a Proteomics and Metabolism core; and 3) a Single Cell and Spatial Transcriptomics core. The PPG benefits from the exceptionally diverse expertise of the Project and Core leaders and co-leaders, all of whom are acknowledged leaders in contemporary aging research. Each of the projects is a close collaboration among several PPG members, many of whom have a history of productive collaboration. Each of the scientific cores will provide state-of-the art support to the projects, enabling conceptual and technical advances that would be difficult to achieve in isolation. Together, the Projects and Cores have the potential to uncover new mechanisms of AD and related dementias, which will be tested in human cells and organoids and mice. Importantly, these mechanisms can be developed into interventions that can be used treat human patients.

PROJECT SUMMARY A critical level at which aging is regulated is the cellular responses to molecular events, which in turn alter the tissue and systemic milieu. Understanding cell fate decisions that occur in response to endogenous and exogenous insults that cause aging offers opportunities to develop novel therapies for age-related phenotypes and pathologies. One important cell fate that drives aging is cellular senescence: an essentially irreversible arrest of cell proliferation coupled to a complex senescence-associated secretory phenotype (SASP). Senescent cells increases with age in most tissues. These cells can drive a surprisingly diverse array of aging phenotypes and diseases, largely through the SASP. Moreover, eliminating senescent cells, using either transgenic mouse models or a new class of drugs termed senolytics, can help maintain homeostasis in aged or damaged tissues, and postpone or ameliorate many age-related pathologies. In this proposal, we will characterize a novel aspect of cellular senescence, cell competition, to determine how it contributes to aging. Cell competition is a process by which neighboring (winner) cells detect and remove suboptimal (loser) cells by killing and engulfing them. Our preliminary data show that several senescence-inducing stimuli allow a subset of senescent cells to acquire winner status, allowing them to actively kill and engulf a subset of neighboring non-senescent cells. This killing is transient and coincides with the onset of the pro-inflammatory SASP. We propose that this early transient killing of non-senescent neighbors is required for the long-term survival of pro- inflammatory senescent cells during aging. It is possible that a subset of senescent cells acquires winner status to avoid elimination and/or acquire nutrients (by engulfment) for survival. Deciphering the mechanisms of cell competition in the context of cellular senescence is essential to understand many processes in biology, ranging from embryogenesis to cancer. Using unbiased high throughput techniques such as proteomics and single cell transcriptomics, we plan to determine the molecular events that regulate cell competition by early senescent cells. Further, investigating these processes will open new opportunities for eliminating pro- inflammatory senescent cells before they can drive pathology.