Peter S. Rabinovitch - US grants

This is a proposal to purchase a Cell Sorter/Flow Cytometer to be shared by many investigators at the University of Washington. Twenty-two investigators participate in this proposal, describing their need for additional Cell Sorting Instrumentation. The present shared facility was established in 1980 and has utilized an Ortho Model 50 cell sorter during this entire period. Presently, needs for utilization of this instrument greatly exceed its availability, even with current operation 12hrs a day. The Ortho Sorter is unfortunately also becoming aged and less reliable; failure and needed repair is becoming more frequent and results in loss of experimental data and unavoidable failure to perform scheduled experiments. The only other cell sorter available on campus is rapidly about to become saturated by expansion of faculty and large new projects within that department. A newly designed Cell Sorter has been developed by Coulter Corp (one of the two U.S. manufacturers of these instruments). This new model represents a great improvement in software user-interaction and usage of economical and reliable low-power lasers. We supported investigators at the University of Washington.

This response to the RFA for Nathan Shock Centers of Excellence in Basic Biology of Aging is intended to provide shared resources in support of a large community of University of Washington faculty investigators with research on basic biology of aging. Proposed components of the Center include a Resources Core with 4 components: l) Transgenic Animal Model Development Resource Core; 2) Specific-Pathogen-Free Transgenic and Control Animal Maintenance Resource Core; 3) Flow Cytometry and Cell Sorting Resource Core; 4) Yeast Genetics Resource Core. The Research Development Core is composed of support for pilot study projects in the basic biology of aging, and junior faculty support. The Program Enrichment Core supports administrative management, an external advisory panel, and support for symposia and seminars. The Transgenic Animal Model Development Resource Core is a focal point of this proposal. This core seeks support for the necessary faculty, staff, and equipment to establish a resource utilizing state of the art technology for the development of genetically altered rodents in order to advance knowledge about the basic biology of aging. Faculty members whose ongoing research this core unit will enhance are among the leaders in aging research. However, access to, or the acquisition of equipment, technical expertise, and space for transgenic work is a major constraint for individual faculty members. Therefore, the purpose of this Transgenic Animal Model Development Core is to enable these investigators to utilize genetic engineering technology in mice for answering basic questions in various areas of the biology of aging and to develop new animal models for studying the mechanisms of the aging process. The Flow Cytometry and Cell Sorting Core will capitalize on existing expertise and instrumentation to make flow cytometry and cell sorting methodologies readily available to research projects in the basic biology of aging. The Yeast Genetics Core will allow Center investigators to have ready access to the yeast two- hybrid system for elucidating protein-protein interactions. A program for funding of pilot projects in the basic biology of aging will broadly benefit currently supported investigators in the basic biology of aging, and allow them to rapid pursue new and exciting findings. Partial salary support for a junior investigator will help to fill an existing gap in existing career development programs in Aging at the University of Washington. Support for symposia and seminars in the biology of aging will broadly enrich the environment for investigators in gerontologic research.

DESCRIPTION: The Research Development Core proposes to promote aging research at the University of Washington by (1) awarding 3 or 4 individual small grants to develop pilot studies in the biology of aging each year of funding and (2) providing 50% salary support for one junior faculty member in each budget year. A faculty award would be for a two year period. Infrastructure for announcement and review of the awards is described.

flow cytometry; biomedical equipment resource; biomedical facility; computer program /software; computer data analysis; computer system design /evaluation;

The Specific Aims of this Core are: 1) To make readily available flow and image cytometry and cell sorting instrumentation and techniques to Program Project research. 2) To assist investigators in finding appropriate ways to apply cytometric techniques to their research objectives. The Core operates 4 cytometers, providing instrumentation that best matches the needed application. These are: an ELITE cell sorter; an ORTHO 50/2150 cell sorter, an ICP-22 non-sorting cytometer and an ACAS (Adherent Cell Analysis and Sorting) 570 imaging and confocal cytometer, In addition, software in support of Core cytometry services is developed and applied. The Core will provide the Program Project both scientific expertise and access to advanced instrumentation and software. Cytometry and cell sorting will be essential parts of each of the Projects, providing both preparative (cell purification by sorting) and analytical services. Types of Cytometric analyses and sorting that will be performed include: 1) Sorting lymphocyte subsets; 2) Assays of cell proliferation and Cell Cycle; 3) Analysis of Heterokaryon and Cytoplast cell fusion products; 4) Alkaline unwinding DNA damage and repair assays. 5) Measurement of mitochondrial membrane potential and cellular redox status; 6) Measurement of Apoptosis; 7) Quantitating Intracellular Calcium and Glutathione or pH; 8) Sorting of transfected cells and characterization of transgenic mouse tissues.

DESCRIPTION: Five years of support are requested by the UW to provide in-depth training and research experiences for ten predoctoral and ten postdoctoral trainees in the area of genetic approaches to aging research. The goal of the program is to provide research training on the molecular genetic approaches to the biology and pathology of aging, with special emphasis upon a variety of model organisms and cell cultures that are amenable to genetic analysis (S. cerevisiae, C. elegans, M. domesticus, H. sapeins). Such materials are designed to permit trainees to address fundamental mechanisms relevant to aging and age-related diseases, including Alzheimer's disease (AD) and cancer. Didactic experiences will include courses in biochemistry, genetics, cell biology and pathology; journal clubs; review of on-going research (approximately 60 individual and six program projects, LEAD or center grants in aging); a course on "Molecular Genetic Approaches to Aging"; and bi-monthly meetings of the "Aging Journal Club". In addition, trainees will typically participate in weekly laboratory meetings and in individual conferences with their mentors. Research projects will include efforts to identify genes related to various forms of familial and sporadic AD, delineation of mechanisms of beta (B) amyloidosis and of its suppression, the role of Werner's syndrome helicase gene in aging and cancer, studies of free radical injury and defense in relation to aging, DNA damage and mutation in aging, and mechanisms underlying the limited replicative potential of somatic cells.

DESCRIPTION: (Applicant's Description) In this application, we propose to develop rapid and efficient methods to prepare purified populations of neoplastic epithelial cells for evaluation by new DNA and RNA array-based molecular detection technologies. Recent developments in high speed/high volume nucleic acid sequencing and hybridization arrays have enabled the analysis of human tissue specimens on an unprecedented scale. However, accuracy in nucleic acid sequencing, detection of loss of heterozygosity (LOH) and measurement of patterns of gene expression require that the cellular material submitted for molecular analysis be as free as possible from contaminating nontumor cells. While one approach may be to microdissect the desired cells, this method is laborious, not easily automated, yields small cell numbers, and is not always possible. We propose to optimize a combination of cell preparation and flow cytometric cell sorting technologies to address the need to rapidly isolate cells of interest in larger numbers. We will apply these methods and validate the results using array-based analysis of loss of heterozygosity, DNA sequencing and RNA expression hybridization.

The Specific Aim of this Core is to provide two categories of services to the Program Project: 1) flow cytometry and cell sorting methods and technologies, and 2) assays of glutathione and the activity of redox enzymes glutathione-s-transferase, superoxide dismutase (S.O.D.) and catalase. We will also assist investigators in finding the most appropriate ways to apply these techniques to their research objectives and to interpret the experimental results. Cytometry and cell sorting will be essential parts of each of the program projects, providing variety of analytical services and techniques. Assays of DNA content, cell cycle and cell proliferation, a novel cytometric assay of oxidative DNA damage and repair, and cytometric assays related to resistance to oxidative damage (glutathione content, cell viability and apoptosis) will constitute major uses of cytometry in this Program Project proposal. In addition, this core will provide non-cytometric assays that will be widely used in the Program Project: HPLC and spectrophotometric quantitative assays of the important intracellular antioxidant glutathione; assays of glutathione-s- transferase, a principal enzyme responsible for carcinogen detoxification; assays of GSH peroxidase and GSH reductase, important enzymes in the synthesis of glutathione, and assays of the oxygen radial scavenging enzymes superoxide dismutase (S.O.D.) and catalase.

DESCRIPTION: (Applicant's Description) Defective function of the DNA helicase WRN in Werner syndrome (WS) is characterized by prolongation of S phase, chromosome translocations and an increased incidence of malignancies of unusual types. WS cells are sensitive to a restricted set of clastogens and mutagens and this sensitivity is very different from other known helicase-deficiency disorders. This suggests a different DNA substrate and/or a novel function for WRN. The model of the investigators proposes that WRN is required for the efficient and accurate repair or toleration in S phase of a specific set of blocking lesions and that failure to do so leads to aberrations in DNA replication. They believe that this defect is reflected in hypersensitivity of WRN-/- cells to a particular subset of DNA damaging agents that include 4NQO. Their model also proposes that the elevated incidence of certain uncommon tumors in WS reflects intrinsic differences in their progenitor cells to susceptibility to these DNA lesions or the capacity to repair them. Specifically, the applicants will test the following hypotheses: 1. The function of WRN is required for normal S phase progression. 2. The hypersensitivity of WS cells to the drug 4NQO is due to damage that is sustained in or carried into S phase. 3. That WRN is required to prevent blockage of S phase by specific blocking DNA adducts. 4. The unusual tissue distribution of cancer in Werner syndrome patients is related to differences in genotoxin sensitivity and genetic instability between specific cell lineages.

This application is for renewal of the Nathan Shock Center of Excellence in Basic Biology of Aging at the University of Washington. This Center has over the past 4 years provided resources in support of the large community of investigators that study the basic biology of aging in this region. We serve a broad spectrum of externally funded, peer-reviewed research programs, ranging from Centers to individual investigator grants. Proposed components of our renewal include a Resources Core with 3 components: 1.) Transgenic Animal Model Development Resource Core; 2) Cytometry Resource Core; 3) Gene Expression Resource Core. In this next funding period, the Transgenic Animal Model Development Resource Core will work to develop and apply constructs for developmental and exogenous regulation of transgenes. We will continue our philosophy of helping a large number of investigators to develop mouse models for aging studies. The Cytometry Core will in the future provide confocal microscopy services in addition to flow cytometry and cell sorting. The Gene Expression Core is an outgrowth of our former small core for protein-protein interactions; we propose to expand this core to provide expert assistance in the analysis of gene expression using cDNA arrays on glass and nylon membranes. The Research Development Core will continue to support junior faculty and pilot study projects in the basic biology of aging. The Program Enrichment Core supports administrative management, an external advisory panel, and a program of courses and seminars.

DESCRIPTION: This core is intended to provide investigators with DNA micro arrays formatted onto membranes or glass slides. Researchers would submit experimental plans and protocols to the core director for approval and prioritization, upon which RNA would be provided to a trained technician for probe preparation and hybridization. A full-time bioinformatics technician would process the data and aid in interpretation. The plan includes the purchase of nylon-based arrays from commercial suppliers for immediate use. The University is also developing a micro array core facility through which researchers could access high density glass arrays at a considerably reduced price, and it is anticipated that much of the throughput will switch to this format as the center comes on-line. A second, continuing activity of this core is the application of yeast 2-hybrid technologies for the identification of protein-protein interactions.

This application is a request to purchase a MoFLo high speed multilaser cell sorter in support of 33 users who are dependent upon longstanding shared resource cytometry facilities at the University of Washington. This user group is presently services by closely interacting resource facilities located in the Departments of Pathology and the Department of Environmental Health. The Department of Pathology facility, directed by Dr. Peter Rabinovitch has used a Coulter Elite Cell Sorter since 1990. The Department of Environmental Health facility, directed by Dr. Terrance Kavanagh, has utilized an Ortho SOH cytometer which has become inoperable over the past year. Cytometry usage is also facilitated by four separate Program Project and Center grant Cytometry Cores directed by Drs. Rabinovitch and Kavanagh. There is a great need for new instrumentation because 1) there is much greater demand for flow cytometry services than can be met with existing instrumentation; 2) there is a need for a more dependable and flexible instrument for multilaser analyses; 3) there is a need for higher speed analysis and sorting. The present excess demand for access to flow cytometry and the need for additional capabilities, including high speed sorting, are now limitations to the progress of many NIH funded research projects at the University of Washington.

DESCRIPTION (provided by applicant): This is an application for renewal of a program project that has, from its inception 1978, the persistent theme of elucidation of gene action capable of modulating the rate of aging in mammalian cells. For the present competitive renewal we are pursuing this theme by studying genetic models of enhanced longevity through augmented resistance to oxidative and DNA damage. Two projects capitalize on work in the past funding cycle that shows that over expression of catalase targeted to mitochondria leads to lifespan extension in mice, and that while over expression of wild type catalase (targeted to peroxisomes) and Cu-Zn superoxide dismutase (type 1) have little effect on murine lifespan by themselves, the combination of over expression of both produces a significant extension of mean lifespan in mice. Improved genetic models of lifespan extension through enhanced antioxidant defense in mice will be developed and characterized. Two projects study the mouse models of altered fidelity of DNA polymerases gamma and delta, respectively. This includes the study of aging in mice with "antimutator" polymerases with enhanced fidelity in the face of oxidative stress. This project requests support of Cores for administration, mitochondrial assays, animal assays and maintenance, and DNA damage assays.

DESCRIPTION: (Applicant's Description) The Seattle Cancer and Aging Program is a coordinated response between the Fred Hutchinson Cancer Research Center and the University of Washington Nathan Shock Center for aging research to explore the connection of cancer and aging. This proposal is a research planning and development grant application to support activities that will expand the capacity of our NCI-designated Cancer Center to engage in research that concentrates on aging- and age-related aspects of human cancer. Our goal is to support research and educational activities that will promote the establishment of a formal interdisciplinary aging/cancer research "Program" as a component of our NCI-funded Cancer Center Support Grant (CCSG). To achieve this goal we plan to fund a series of pilot projects that will foster new collaborations, yield promising research results, catalyze new independent research grants, and lay the foundation for a continuing unified research effort in cancer and aging. The consortium of the Fred Hutchinson Cancer Research Center and the University of Washington has an usually strong and diverse faculty with aging and cancer interest who will be able to take advantage of this support. In our initial series of pilot projects we first concentrate on the thematic area of the Biology of Aging and Cancer; as demonstrated in the proposal, future proposed pilots involve all of the seven thematic areas of support. We believe we are in an unique position to forge an interactive, top-rate cancer and aging program. We are blessed with top-rate researchers, state-of-the-art resources, and rock-solid institutional commitment to make this a successful program. We fully expect that the activities outlined in this proposal will lead to important discovery and lay the foundation for significant scientific effort in the future.

[unreadable] DESCRIPTION (provided by applicant): This competitive renewal application for a training grant requests funds for 9 predoctoral and 9 postdoctoral trainees. The goal is to provide opportunities for research training on molecular genetics approaches to biology and pathology of aging, with special emphasis upon a variety of model organisms and cell cultures that are amenable to genetic analysis (S. cerevisiae, C. elegans, M. domesticus, H. sapiens); such materials should permit trainees to address fundamentaI mechanisms highly relevant to aging and age-related diseases, including Alzheimer's disease (AD) and cancer. Didactic experiences will include courses in biochemistry, genetics, cell biology and pathology, research seminars, journal clubs, reviews of on-going research (approximately 60 individual and 6 program-projects, LEAD or center grants in aging), and a course on "Molecular Genetic Approaches to Aging," and bi-monthly "Aging Journal Club." In addition, trainees will typically participate in weekly lab meetings and in individual conferences with their mentors. Research projects will include efforts to identify genes related to various forms of familial and sporadic AD, delineations of mechanisms of p -amyloidogenesis and of its suppression, the role of the Wemer syndrome helicase gene in aging and cancer, studies of free radical injury and defense in relation to aging, DNA damage and mutation in aging, and mechanisms underlying the limited replicative potential of somatic cells. [unreadable] [unreadable]

The Flow Cytometry Laboratory functions as a shared resource for cell analysis and sorting. The laboratory assists a wide range of investigator-directed research from clinically related studies of primary malignant cells and their normal counterparts, stem cells and gene therapy, to fundamental studies of developmental biology, gene regulation, and neoplastic transformation. The resource supports population based studies of genomic instability and changes in human solid tumors. Since its inception in 1978, the resource has continued to enhance its instrumentation, sophistication of usage and overall level of use. The laboratory continues to maintain its College of American Pathology accreditation for clinically related investigation. In addition to the maintenance and upgrade of resource equipment, the Flow Cytometry Laboratory actively assists investigators and their laboratories in experimental design, sample preparation, development of novel techniques, analysis and interpretation of data, and training in the operation of the instrumentation. This application requests continued support for a resource which continues to fulfill an essential role for peer reviewed research.

SPECIFIC AIMS: 1) To make readily available flow cytometry, cell sorting, confocal image cytometry and laser capture microdissection instrumentation and techniques to research projects in the basic biology of aging. 2) To assist investigators in finding appropriate ways to apply these techniques to their research objectives. 3) To assist investigators in the application of computer software for flow and image cytometry data analysis and presentation, including customized software programming, as needed.

DESCRIPTION (provided by applicant): This application is for renewal of the Nathan Shock Center of Excellence in the Basic Biology of Aging at the University of Washington. This Center has over the past 10 years provided resources in support of the large community of investigators that study the basic biology of aging in this region. We serve a broad spectrum of externally funded, peer-reviewed research programs, ranging from Centers to individual investigator grants. Proposed components of our renewal include a Resources Core with 3 components: 1) Transgenic Animal Model Development Resource Core; 2) Cytometry Resource Core; 3) Functional Genomics Resource Core. In this next funding period, the Transgenic Animal Model Development Resource Core will work to develop and apply constructs for developmental and exogenous regulation of transgenes. We will continue our philosophy of helping a large number of investigators to develop mouse models for aging studies. The Cytometry Core will provide confocal microscopy and laser capture microdissection services in addition to flow cytometry and cell sorting. The Functional Genomics Core is the evolution of our Gene Expression core and will provide expert assistance for transcriptomics, proteomics and metabolomics. The Research Development Core will continue to support pilot study projects in the basic biology of aging. The Program Enrichment Core supports administrative management, an external advisory panel, and a program of courses and seminars.

SPECIFIC AIMS The proposed Functional Genomics Core is an evolution of our Gene Expression Core. The past four years has seen an explosion of technology for genomics. The ability to survey expression of large parts of the genome is likely to be important for understanding the basic biology of aging. The specific aims of the core are to facilitate access by our biology of aging faculty to transcriptomics, metabolomics and proteomics technologies in a manner that will be most efficient and scientifically productive.

Genetic models of lifespan extension in mammals have not been previously known, and caloric restriction has been the only experimental manipulation shown to retard aging of mammals. We have now demonstrated that overexpression of catalase targeted to mitochondria (MCAT) leads to lifespan extension in mice. Also, we have shown that while overexpression of wild type catalase (targeted to peroxisomes) and Cu-Zn superoxide dismutase (SOD1) have little effect on murine lifespan by themselves, the combination of overexpression of these two produces a significant extension of mean lifespan. These are the first demonstrations that genetically augmented antioxidant defenses can appreciably extend lifespan in mammals. This proposal seeks to extend and better understand these findings. In Aim 1, we will study the physiologic, pathologic and molecular alterations that are responsible for the extended lifespan of MCAT mice. We will compare these changes to those associated with the extended lifespan of calorically restricted mice to determine whether these two mechanisms act through similar or different pathways. In Aim 2, we will build upon our initial observations by creating two improved models of augmented antioxidant protection. We will generate transgenic mice expressing both MCAT and SOD1 from the same promoter and with uniform tissue expression, and we will enhance the redox capacity of MCAT mice by expressing both MCAT and the glutamate-cysteine ligase modulatory subunit (the rate limiting step controlling glutathione synthesis) from the same promoter and with uniform tissue expression. Each model evaluates the benefit of protective mechanisms in different subcellular compartments and/or different antioxidant pathways, and complements the models studied elsewhere in this P01.

The goals of Core A are to provide cellular and molecular assays in support of all projects. The technologies and methods employed include flow and image cytometry; genetic analyses, cell and tissue culture support and development of common reagents. This Core and its personnel have had a long and productive history of application of specialized cell and molecular assays to the study of Werner Syndrome. These notably include flow and image cytometric assays of cell cycle, survival, DNA damage and telomere status, reagents and assays for Immunologic probes and gene silencing, as well as cell culture support for the broad variety of in vitro assays that is encompassed by this work. Both the characterization of WRN RecQ protein activities and the consequences of these activities on cellular phenotypes thus rely on the use of cell and molecular assays that are used in com man by all of the Projects within this P01 renewal application. The implementation, enhancement and where needed, development of these assays is a service will be most effectively and efficiently performed by this specialized Core in order to optimize their use by the P01 Projects.

DESCRIPTION (provided by applicant): We propose a response to Challenge 06-AG-104 that incorporates development of three novel tools and technologies that can alter or interrogate human mitochondrial function in vivo. We will then apply these tools to the study of aging heart and muscle in order to demonstrate that they can enable an integrated understanding of the role of mitochondrial ROS, mitochondrial energetics and changes in mitochondrial protein composition and turnover in age-related sarcopenia and heart disease. In Aim 1 we will manipulate in vivo mitochondrial structure and activity by the use of novel mitochondrial targeted antioxidant and protective peptides. This is important in human aging because data from our and other laboratories indicate that mitochondria and mitochondrial ROS play a critical role in age-related declines in organ function. In Aim 2 we will apply novel magnetic resonance and optical spectroscopy techniques to measure O2 and ATP fluxes simultaneously to create a totally non-invasive and more versatile measure of mitochondrial function. In Aim 3 w e will use novel proteomic methodologies to characterize the structural basis of changes in mitochondrial function in aging tissues in vivo and tissues protected by antioxidant peptides. This will include measurement of global mitochondrial protein abundance and mitochondrial proteome-wide differences in protein syntheses and turnover rates using in vivo heavy isotope labeling. In each of these aims we will apply and validate the tools developed using two tissues for which there is abundant evidence for the critical role of mitochondria and, mitochondrial ROS in aging: heart and skeletal muscle. We will examine healthy young and old muscle, plus young hearts stressed with angiotensin II and young muscle stressed by AZT treatment. The combined use of these three methods allows us to manipulate human mitochondrial structure and activity at the same time as we monitor and measure mitochondrial function and dysfunction in aging tissues. Thus, we can obtain an integrated assessment of the in vivo inter-relationships of mitochondrial, ROS, energetics and proteomics in aging and disease. PUBLIC HEALTH RELEVANCE: This response to Challenge 06-AG-104 incorporates three responsive elements that will be used together in concert to provide an integrated assessment of the in vivo inter-relationships of mitochondrial, ROS, energetics and proteomics in aging and disease: 1) we will manipulate in vivo mitochondrial structure and activity by the use of novel mitochondrial targeted antioxidant and protective peptides;2) we will use novel magnetic resonance and optical spectroscopy tools to create totally non-invasive and more versatile measures of mitochondrial energetics in vivo, and 3) we will use novel proteomic tools to characterize the structural basis of changes in mitochondrial function. The combined use of these methods allows us to manipulate human mitochondrial structure and activity at the same time as we monitor and measure mitochondrial function and dysfunction in aging tissues.

DESCRIPTION (provided by applicant): This application is for renewal of the Nathan Shock Center of Excellence in the Basic Biology of Aging at the University of Washington and affiliated institutions. This Center has over the past 15 years provided resources in support of our large community of investigators that are funded to study basic biological mechanisms of aging. We continue to build upon a scientific theme that became established in the past funding cycle, in which comparative gerontologic study informed us of conserved genetic pathways of longevity determination (the TOR pathway is an example of one such). The three proposed Research Resources Core components are well suited to assist in such efforts: 1) Transgenic Animal Model Development; 2) Functional Assessment; 3) Functional Genomics. The Transgenic Animal Model Development Resource Core continues to pursue a focus on inducible gene expression and knockout models, so that organ and age-specific effects of genetic alterations can be assessed. The Functional Assessment Core builds upon technical resources that have been gathered during the past funding period, such that we can now provide investigators with a broad and informative range of physiologic and cellular assays, including those that are most relevant to assessment of the healthspan of mouse models. The Functional Genomics Core provides expert assistance with state of the art technologies for transcriptomics and proteomics. Consistent with our theme, it includes a focus on translational state, protein turnover and post-translational modification. The Research Development Core will continue to support pilot study projects in the basic biology of aging. The Program Enrichment Core supports administrative management, an external advisory panel, a program of courses and seminars and data sharing and dissemination.

DESCRIPTION (provided by applicant): This is a proposal from a multidisciplinary team with expertise in cardiac physiology, mitochondrial biology, advanced proteomics technologies, biostatistics and bioinformatics to work together to improve understanding of the role of cardiomyocyte mitochondrial dysfunction and adaptation in cardiac aging and failure. This proposal builds upon our novel observations that overexpression of mitochondrial catalase (mCAT) can delay cardiac aging and prevent cardiac hypertrophy and failure and that mitochondrial targeted peptide antioxidants can be cardioprotective. We will study the mechanisms responsible for cardioprotection conferred by mCAT and we will test the translational potential of newly developed mitochondrially targeted antioxidant and protective peptide drugs. Aim 1 (Mechanism) will determine the mechanism(s) of this protection and will elucidate how the changes in the mitochondrial proteome contribute to the functional and biochemical causes of cardiac hypertrophy and failure. To do this we will combine state-of-the art quantitative label-free differential proteomics to measure differences in abundance of the cardiac mitochondrial proteome with in vivo heavy labeling to measure proteome-wide differences in cardiac mitochondrial protein syntheses and turnover rates. The proposed proteomic approach will provide a unique insight into the relationship between mitochondrial function, biology and protein turnover, and abundance. Aim 2 (Translation) will test whether and how newly developed mitochondrially targeted antioxidant and protective drugs have the potential to translate mitochondrial protection into a clinical intervention. This comprehensive approach will provide an integrated assessment of the role of mitochondria in cardiac health, disease and disease-resistance. (End of Abstract)

DESCRIPTION (provided by applicant) Nutrient signaling is central and evolutionarily conserved in pathways that modulate aging and lifespan, as our Program Project investigators have demonstrated in preliminary studies of yeast and worms. The mTOR (mammalian target of rapamycin) signaling pathway monitors Intracellular amino add levels and regulates protein synthesis, cell growth, and ribosomal biogenesis. We hypothesize that reduced signaling through the mTOR pathway has positive effects on aging and lifespan, which are mediated by translational regulation. Furthermore, signaling through mTOR may be intimately linked to mitochondrial metabolism and reactive oxygen species (ROS), thus providing a mechanistic connection between the paradigms of ROS and dietary restriction in aging. Evidence, including our preliminary data, suggests that these effects may be particularly important in cardiac aging, an important cause of human morbidity and mortality. The focus of the research in Project 2 derives from these key findings. We will use knockout, conditional knockout and transgenic mice to delineate the effects of reduced signaling through the TORCl arm of the mTOR pathway and the interactions of this signaling with dietary restriction and reactive oxygen species (ROS). Specific Aim 1 is to establish whether reduced TORCl signaling enhances cardiac resistance to aging. We will determine whether this mechanism can account for the cardiac benefits of dietary restriction, whether it is mediated by alterations in protein translation and whether reduced levels of ROS are a significant part of this mechanism. In Aim 2, we will extend these studies to effects on mouse lifespan to confirm that reduced TORCl signaling Increases mouse longevity. These genetic approaches will lead to fundamental insights into key regulators of longevity and determinants of health span, and that with this knowledge, pharmacologic interventions can be designed to confer similar health benefits to humans. Cardiac aging Is a significant cause of late-life mortality and diastolic dysfunction is a major contributor to the physiological declines in the aging heart. The proposed studies may reveal novel therapeutic interventions for diastolic dysfunction which currently are sorely lacking.

DESCRIPTION (provided by applicant): This application for renewal of our longstanding Program Project is based on a highly focused, integrated and interactive effort to examine the hypothesis that mitochondrial antioxidants are capable of resisting age-related disease and improving health and function in multiple organ systems in mammals. This builds on past progress, including observations that mice overexpressing mitochondrially targeted catalase have extended lifespan, improved cardiac, and muscle health and resistance to epithelial cancers. We therefore have developed an increasing focus on health and healthspan, including study of acute models of decline in organ function that can serve as surrogate assays for similar disorders in aging. In this Program Project we propose four Projects to apply this approach to disorders of aging in which mitochondrial reactive oxygen species (ROS) and ROS-induced damage play an important role: 1) Mitochondrial ROS and cardiac aging;2) Mitochondrial ROS and neurodegenerative disease;3) Mitochondrial ROS and protection from epithelial cancers in aging;4) Mitochondrial-targeted antioxidants, aging and AZT in skeletal muscle dysfunction. The Projects are supported by four Cores: 1) Administrative;2) Mouse pathobiology;3) Proteomics;4) Mitochondrial protective chemistry. In each of these Projects and Cores we seek to understand the mechanisms underlying the role of mitochondrial ROS in aging and healthspan, as well as pursuing the translational goal of identifying mitochondrial protective drugs to deliver these healthspan benefits to humans. PUBLIC HEALTH RELEVANCE: By working with mouse models of protection from mitochondrial oxidation and damage, and translating from genetic models to pharmacologic agents, this Program Project hopes to deliver significant health benefits in muscle, heart, brain, and cancer protection to the aging human population. REVIEW OF INDIVUDUAL COMPONENTS OF THE PROGRAM PROJECT CORE A: ADMINISTRATION CORE;Dr. Peter S. Rabinovitch, Core Leader (CL) DESCRIPTION (provided by applicant): Specific Aims 1. Provision of an organizational structure to expedite research and promote communication between Program Project components and investigators. 2. Monitor and regularly review the quality and progress of research 3. Insure adherence to rigorous statistical considerations in experimental design and data analysis 4. Management of fiscal components of the Program Project including reallocation of funds to optimize overall function. 5. Short-range and long-range planning for the enhancement and integration of Program Project facilities. 6. Provide data sharing and data dissemination facilities for the P01. PUBLIC HEALTH RELEVANCE: Statistical rigor in experimental design and data analysis is an important administrative coordinating goal to ensure that all experimental designs meet the highest statistical rigor. Ensuring that all Cores and Projects are coordinated is the main goal of this Core.

This application is for renewal of the Nathan Shock Center of Excellence in the Basic Biology of Aging at the University of Washington and affiliated institutions. This Center has over the past 15 years provided resources in support of our large community of investigators that are funded to study basic biological mechanisms of aging. We continue to build upon a scientific theme that became established in the past funding cycle, in which comparative gerontologic study informed us of conserved genetic pathways of longevity determination (the TOR pathway is an example of one such). The three proposed Research Resources Core components are well suited to assist in such efforts: 1) Transgenic Animal Model Development; 2) Functional Assessment; 3) Functional Genomics. The Transgenic Animal Model Development Resource Core continues to pursue a focus on inducible gene expression and knockout models, so that organ and age-specific effects of genetic alterations can be assessed. The Functional Assessment Core builds upon technical resources that have been gathered during the past funding period, such that we can now provide investigators with a broad and informative range of physiologic and cellular assays, including those that are most relevant to assessment of the healthspan of mouse models. The Functional Genomics Core provides expert assistance with state of the art technologies for transcriptomics and proteomics. Consistent with our theme, it includes a focus on translational state, protein turnover and post-translational modification. The Research Development Core will continue to support pilot study projects in the basic biology of aging. The Program Enrichment Core supports administrative management, an external advisory panel, a program of courses and seminars and data sharing and dissemination.

This application is for renewal of the Nathan Shock Center of Excellence in the Basic Biology of Aging at the University of Washington and affiliated institutions. This Center has over the past 15 years provided resources in support of our large community of investigators that are funded to study basic biological mechanisms of aging. We continue to build upon a scientific theme that became established in the past funding cycle, in which comparative gerontologic study informed us of conserved genetic pathways of longevity determination (the TOR pathway is an example of one such). The three proposed Research Resources Core components are well suited to assist in such efforts: 1) Transgenic Animal Model Development; 2) Functional Assessment; 3) Functional Genomics. The Transgenic Animal Model Development Resource Core continues to pursue a focus on inducible gene expression and knockout models, so that organ and age-specific effects of genetic alterations can be assessed. The Functional Assessment Core builds upon technical resources that have been gathered during the past funding period, such that we can now provide investigators with a broad and informative range of physiologic and cellular assays, including those that are most relevant to assessment of the healthspan of mouse models. The Functional Genomics Core provides expert assistance with state of the art technologies for transcriptomics and proteomics. Consistent with our theme, it includes a focus on translational state, protein turnover and post-translational modification. The Research Development Core will continue to support pilot study projects in the basic biology of aging. The Program Enrichment Core supports administrative management, an external advisory panel, a program of courses and seminars and data sharing and dissemination.

This application is for renewal of the Nathan Shock Center of Excellence in the Basic Biology of Aging at the University of Washington and affiliated institutions. This Center has over the past 15 years provided resources in support of our large community of investigators that are funded to study basic biological mechanisms of aging. We continue to build upon a scientific theme that became established in the past funding cycle, in which comparative gerontologic study informed us of conserved genetic pathways of longevity determination (the TOR pathway is an example of one such). The three proposed Research Resources Core components are well suited to assist in such efforts: 1) Transgenic Animal Model Development; 2) Functional Assessment; 3) Functional Genomics. The Transgenic Animal Model Development Resource Core continues to pursue a focus on inducible gene expression and knockout models, so that organ and age-specific effects of genetic alterations can be assessed. The Functional Assessment Core builds upon technical resources that have been gathered during the past funding period, such that we can now provide investigators with a broad and informative range of physiologic and cellular assays, including those that are most relevant to assessment of the healthspan of mouse models. The Functional Genomics Core provides expert assistance with state of the art technologies for transcriptomics and proteomics. Consistent with our theme, it includes a focus on translational state, protein turnover and post-translational modification. The Research Development Core will continue to support pilot study projects in the basic biology of aging. The Program Enrichment Core supports administrative management, an external advisory panel, a program of courses and seminars and data sharing and dissemination.

This application is for renewal of the Nathan Shock Center of Excellence in the Basic Biology of Aging at the University of Washington and affiliated institutions. This Center has over the past 15 years provided resources in support of our large community of investigators that are funded to study basic biological mechanisms of aging. We continue to build upon a scientific theme that became established in the past funding cycle, in which comparative gerontologic study informed us of conserved genetic pathways of longevity determination (the TOR pathway is an example of one such). The three proposed Research Resources Core components are well suited to assist in such efforts: 1) Transgenic Animal Model Development; 2) Functional Assessment; 3) Functional Genomics. The Transgenic Animal Model Development Resource Core continues to pursue a focus on inducible gene expression and knockout models, so that organ and age-specific effects of genetic alterations can be assessed. The Functional Assessment Core builds upon technical resources that have been gathered during the past funding period, such that we can now provide investigators with a broad and informative range of physiologic and cellular assays, including those that are most relevant to assessment of the healthspan of mouse models. The Functional Genomics Core provides expert assistance with state of the art technologies for transcriptomics and proteomics. Consistent with our theme, it includes a focus on translational state, protein turnover and post-translational modification. The Research Development Core will continue to support pilot study projects in the basic biology of aging. The Program Enrichment Core supports administrative management, an external advisory panel, a program of courses and seminars and data sharing and dissemination.

This application is for renewal of the Nathan Shock Center of Excellence in the Basic Biology of Aging at the University of Washington and affiliated institutions. This Center has over the past 15 years provided resources in support of our large community of investigators that are funded to study basic biological mechanisms of aging. We continue to build upon a scientific theme that became established in the past funding cycle, in which comparative gerontologic study informed us of conserved genetic pathways of longevity determination (the TOR pathway is an example of one such). The three proposed Research Resources Core components are well suited to assist in such efforts: 1) Transgenic Animal Model Development; 2) Functional Assessment; 3) Functional Genomics. The Transgenic Animal Model Development Resource Core continues to pursue a focus on inducible gene expression and knockout models, so that organ and age-specific effects of genetic alterations can be assessed. The Functional Assessment Core builds upon technical resources that have been gathered during the past funding period, such that we can now provide investigators with a broad and informative range of physiologic and cellular assays, including those that are most relevant to assessment of the healthspan of mouse models. The Functional Genomics Core provides expert assistance with state of the art technologies for transcriptomics and proteomics. Consistent with our theme, it includes a focus on translational state, protein turnover and post-translational modification. The Research Development Core will continue to support pilot study projects in the basic biology of aging. The Program Enrichment Core supports administrative management, an external advisory panel, a program of courses and seminars and data sharing and dissemination.

Core C will provide a state of the art proteomics core to improve the understanding of the molecular changes in the mitochondria associated with reactive oxygen species (ROS) and aging. The four research projects seek to understand the role of mitochondrial catalase and mimetics on health and lifespan in mouse models. Integral to the success of the program project grant is the measurement of changes in relative mitochondrial protein abundance and half-life in health, disease, and aging. To support the needs of the individual research projects, the Proteomics Core has three specific aims: 1)to perform perform the unbiased detection of differences in peptide/protein abundance between complex mixtures. To understand the effect of ROS on mitochondrial function we will provide the capability to comprehensively detect differences in A) protein abundance between mitochondrial enriched fractions and B) peptide abundance between samples following enrichment of peptides with oxidative modifications. 2) to perform targeted analysis of protein abundance for mitochondrial proteins of interest. 3) to perform global measurement of individual mitochondrial protein halflife. An active area of technology development within the core will be refining the capability for the measurement of individual protein half-life in a rodent system. We have the capability to measure mitochondrial protein half-life for hundreds of proteins in a single measurement.

Specific Aims 1. Provision of an organizational structure to expedite research and promote communication between Program Project components and investigators. 2. Monitor and regulariy review the quality and progress of research 3. Insure adherence to rigorous statistical considerations in experimental design and data analysis 4. Management of fiscal components of the Program Project including reallocation of funds to optimize overall function. 5. Short-range and long-range planning for the enhancement and integration of Program Project facilities. 6. Provide data sharing and data dissemination facilities for the POl.

Aging is inevitable. It is governed by both inheritance and environmental factors. A deeper understanding of the aging process might allow us to slow its progression or at least delay the onset of age-associated diseases, and thus extend the well being of individuals. The universality in the decline of energy with age highlights energy metabolism and the role of role mitochondria (mt) in aging. A widely accepted???but still unproven theory of aging centers on the accumulation of cellular damage by the generafion of reacfive oxygen species. In cells, reactive oxygen species are generated in mitochondria and have been demonstrated to damage mitochondrial DNA. Since DNA repair is limited in mitochondria, some of this damage may go on to cause mutafions when the DNA is replicated. We have developed, established, and validated excepfionally sensifive assays to quantify mutafions in nuclear and mitochondrial DNA. Our specific aims will be focused in two direcfions: 1) We will determine the frequency and types of mutations that increase in different fissues during aging in humans. We will analyze the mechanism by which specific mitochondrial mutations are be selective amplified. 2) Our focus will be on Parkinson syndrome, one of the most prevalent age-dependent neurological diseases. Using cell culture and mouse models, we will examine the contribufion of reactive oxygen species to mitochondrial mutagenesis. Most importanfiy, we will ascertain if selectively amplified mitochondrial mutafions can provide a marker for diagnosis of Parkinson syndrome and or monitoring of disease progression and response to treatment. RELEVANCE (See instmctions): The goal of this project is to determine the mechanism for the generafion ofmitochondial DNA mutations during normal aging and in age-associated diseases. If these mutafions are generated by oxygen metabolism, it should be feasible to decrease their production by specific anfi-oxidants that target mitochondria, and thus the progression of age-associated diseases. We will investigate the generafion of mitochondrial mutations in one of the most prevalent aae-associated neurolnaioal diseases. Parkinson

Abundant circumstantial evidence indicates that oxidative stress contributes to many consequences of normal aging and several major diseases, including cardiovascular diseases, diabetes, neurodegenerative diseases, and cancer. Oxidative stress is generally defined as an imbalance of prooxidants and antioxidants. However, despite a wealth of scientific evidence to support increased oxidative tissue damage, large-scale clinical studies with antioxidants have not demonstrated significant health benefits in these diseases. One of the reasons may be due to the inability of the available antioxidants to reach the site of prooxidant production. The mitochondrial (mt) electron transport chain (ETC) is the primary intracellular producer of ROS, and mt themselves are most vulnerable to oxidative stress. Protecting mt function would therefore be a prerequisite to preventing cell death caused by mt oxidative stress. The benefits of overexpressing catalase targeted to mt (mCAT), but not peroxisomes (pCAT)(see Project 1), provided proof-of-concept that mt-targeted antioxidants would be necessary to overcome the detrimental effects of aging (9-12). However, adequate delivery of chemical antioxidants to the IMM remains a challenge. The common approach for mt targeting makes use of the potential gradient across the IMM. The triphenylalkyl-phosphonium cation (TPP*) has been conjugated to lipophilic antioxidants such as Vit E (MitoE) and coenzyme Q (MitoQ)(13), and plastoquinone (SkQI) (14) to facilitate their delivery into the mt matrix. However, it is uncertain whether these TPP+ -conjugated antioxidants will be adequately taken up by stressed or aged mt. Furthermore, accumulation of these lipophilic cations in the mt matrix can disrupt mt potential and inhibit mt respiration and ATP production (15). In addition, mt redox cycling of MitoQ can lead to superoxide production and cellular apoptosis (16,17). Thus it is essential to develop alternate mechanisms for targeting therapeutic agents to mt. In a chance discovery, Szeto and Schiller found that a series of cell-permeable aromatic-cationic tetrapeptides (SS peptides) selectively target mt and concentrate 1000-fold in the IMM(4). Although the exact mechanism by which these peptides target the IMM is not understood, it is clear that their uptake is not dependent on mt potential(4). Mt targeting does appear to be dependent on the alternating aromatic-cationic amino acid motif in these peptides, and this has been confirmed by other investigators(8). Significantly, the SS peptides represent the first class of chemical agents to target the IMM rather than mt matrix. These aromatic-cationic peptides can be used to facilitate the delivery of other cargoes to mt (18), and can therefore be conjugated to available antioxidants. More importantly, antioxidant properties can be incorporated into this aromatic-cationic motif simply by the selection of appropriate aromatic and cationic amino acid residues. One of the SS peptide analogs, SS-31, possesses intrinsic antioxidant ability because the modified tyrosine residue is redox-active and can undergo one-electron oxidation(19). We have shown that SS-31 can neutralize H2O2, hydroxyl radical, and peroxynitrite, and inhibit lipid peroxidation (4,6). SS-31 is at least 100-fold more potent than the TPP+ -conjugated antioxidants in protecting cultured cells from proxidants such as t-butyl-hydroperoxide (20) and hypochlorous acid (5), and can even confer protection after mt depolarization(5). The SS peptides were designed to be stable against peptidase degradation and have excellent pharmacokinetic properties(21). SS-31 has demonstrated remarkable efficacy in animal models of ischemia-reperfusion injury(22,23), neurodegenerative diseases (24,25), and metabolic syndrome(11). SS-31 can readily cross the BBB and toxicology studies have revealed an excellent safety profile for SS-31 {Stealth Peptides Inc, personal communication). The therapeutic potential of the SS peptides has been discussed in several reviews(26-28) (N.B. Cornell Research Foundation has licensed the SS peptide technology platform to Stealth Peptides for clinical development). Significantly, SS-31 provided comparable protection as mt catalase overexpression in three animal models of mt oxidative stress (see Approach below), thus supporting our proposal that this small molecule chemical approach can be used to complement our genetic approach in studying the role of mt oxidative stress in aging and health span. The proposed Chemistry Core will carry out large-scale synthesis of SS-31 and SS-20 to provide to all projects for the initial proof-of-concept studies. At the same time, we will proceed to design an orally-active analog of SS-31 for the chronic aging studies. In addition to providing SS-31, we also propose to further investigate the use of this novel IMM-targeting strategy to develop other antioxidants with different mechanisms of action. Mt oxidative stress is commonly defined as a disturbance in the prooxidant-antioxidant balance, implying that interventions should be based on either reducing the level of prooxidants or the addition of appropriate antioxidants. Alternatively, it was recently proposed that mt oxidative stress may be defined as a disruption of electron transfer reactions leading to an oxidant/antioxidant imbalance...(29). The ETC can be viewed as a high-flux electron transfer pathway, and inhibition of electron transfer in the ETC or redox cycling agents greatly simulate ROS generation(30). The view that mt oxidative stress is a disruption of redox circuitry would imply that it may be more fruitful to develop strategies to improve electron flow by facilitating electron transfer. The proposed Chemistry Core will extend the design of the mt-targeted peptides to incorporate and enhance one or more of the following modes of action: (i) scavenging excess ROS, (ii) reducing ROS production by facilitating electron transfer, or (iii) increasing mt reductive capacity. The advantage of peptide molecules is that it is possible to incorporate natural or unnatural amino acids that can serve as redox centers, facilitate electron transfer, or increase sulfydryl groups while retaining the aromatic-cationic motif required for mt targeting. The proposed design strategies are supported by known electron chemistry and will be confirmed by chemical, biochemical, cell culture, and animal studies. State-of-the-art physical, chemical and molecular biology approaches will be used to screen the new analogs for mt ROS production and redox regulation, testing and validating the hypothesized molecular modes of action. The most promising analogs will be provided to the various projects for evaluation in mt, cellular, and tissue models. The proposed studies represent a novel integrated approach to the design of mt-targeted antioxidants that is significantly different from other efforts in the field.

Core B will provide mouse model resources, including extensive breeding, colony management and record keeping, intensive monitoring of mice on experimental and lifespan studies, and physiological and pathological assessment. The need for an animal core is paramount in that all projects use mice in a focused manner. Consolidation of animal resources focused on maximizing efficiency and minimizing costs is essential to the success of this program project. The Mouse Core is central to the programmatic efforts of the investigators. Specific aim 1 is designed to maintain mouse lines relevant for the program project and generate stock mice for experimental procedures for research projects. Specific aim 2 will provide the resources to conduct physiological and pathological studies in mouse lines relevant to specific research project aims and objectives. These will include a special focus on the physiological and pathological assessment of cardiovascular, skeletal muscle, behavioral and cancer phenotypes.

We have previously shown that genefic overexpression of catalase targeted to mitochondria (mCAT) prolongs murine median lifespan by 17-21%. To better define the health impact of reducing mitochondrial reacfive oxygen species (ROS), we have now focused carefully on cardiac contribufions to aging. We have demonstrated that cardiac aging in the mouse closely recapitulates human aging, with cardiac hypertrophy and decline in diastolic and systolic functions in the absence of cardiac-extrinsic risk factors, and accompanied by the same molecular and biochemical changes that are seen in the aging human heart. Most significanfiy, mCAT substantially delays and attenuates both the functional and biochemical changes of cardiac aging. Furthermore, we have also found that mCAT protects from acute models of both cardiac hypertrophy and heart failure in the mouse, and that the hypertrophy model recapitulate much of the pathology of cardiac aging. Understanding the mechanisms by which reduced mitochondrial ROS and improved mitochondrial funcfion attenuates intrinsic cardiac aging and signaling pathways (Aim 1) is central to understanding this effect. Intrinsic cardiac aging is also believed to increase the susceptibility of the heart to failure. As heart failure associated with aging is likely to become the major cause of hospital admissions and mortality in North America, we will study the role of mitochondria in this cardiac aging-heart failure interaction (Aim 2). Finally, in order to better translate our findings to human health, we will determine the capacity of mitochondrially targeted antioxidant and protecfive daigs to recapitulate the MCAT benefits to cardiac aging and heart failure (Aim 3), RELEVANCE (See instructions): Project 1: This project is studying a genefic mouse model of protecfion from mitochondrial oxidation and damage that appears to have significant protecfion from heart aging and failure. We wish to understand how these benefits are caused and then apply them using pharmacologic agents so that these same cardiac health benefits can be translated to the aging human populafion.

We are addressing the question of whether age-associated oncogenesis, and more specifically, tumor progression, is associated with mitochondrial (mt) ROS. We hypothesize that age-associated cancers are driven by increasing levels of mitochondrial-mediated ROS. Our preliminary data suggests that ROS is associated with tumor progression, and provides the rafionale for three specific aims to better understand the mechanisms whereby protection of mitochondria reduces oncogenesis. Aim 1 is designed to determine what cellular processes are involved in the mCAT suppression of metastatic tumor progression in the lungs of young and old mice, primary skin tumor progression in young and old mice. We will use primary skin tumor and pulmonary metastatic mammary tumor models in the presence and absence of mitochondrial-targeted catalase (mCAT) to analyze neoplastic progression and tumor metastasis. We will compare the host response and the protecfive effects of mCAT in young and old mice. Aim 2 is designed to determine the contribution of specific cell types in the mCAT suppression of tumor progression. There is increasing evidence that the microenvironment plays a crifical role in oncogeneis. Our preliminary results showing attenuation of tumor progression via expression of mCAT could be explained, in part, by mCAT expression within specific cell types within the microenvironment of the neoplastic cells. We will therefore assess the roles of mCAT expression in several different stromal cell types and compare their putative suppressive effects with epithelial cells (both neoplastic and non-neoplastic) that express mCAT. Specific mCAT expression will be driven by cell-specific Cre transgenesis. Aim 3 is designed to evaluate the efficacy of mitochondrial antioxidant and protective drugs for intervention in tumor progression and metastasis. We will correlate the differences in modes of acfion of the drugs with differences in effects on tumor progression, cell proliferafion and survival in order to better understand the mechanisms whereby protection of mitochondria reduces neoplasia and enhances an anti-aging phenotype. The experimental approach is designed to determine if specific mitochondrial targeted anfioxidant mimefics are effective in suppressing tumor progression in young and old animals with cancer or in aged wild type mice that develop multiple tumor types.

This revised proposal builds on our exciting preliminary findings demonstrating the power of a mitochondrial targeted antioxidant to protect skeletal muscle from zidovudine (AZT)-induced dysfunction. Due to the success of HIV treatments, which often includes AZT, over 35% of all HIV patients in the US are 50 years and older. New data demonstrates that AZT-induced changes are very different in young and old skeletal muscle. However, the interactions between these life-saving therapies and aging on mitochondria remain poorly understood. AZT and other nucleoside reverse transcriptase inhibitors (NRTl) have significant mitochondrial toxicity that shares many characteristics with aging muscle, including increased mitochondrial mutations, reduced mitochondrial content, impaired energy production, and increased oxidative stress leading to muscle loss and frailty. Despite these similarities, there have been no studies examining the potential synergistic effects of aging and NRTl treatment on mitochondrial function in skeletal muscle. In this proposal, we use a common NRTl combination, AZT/3TC (combivir), to test whether aging exacerbates NRTI-induced mitochondrial toxicity and whether mitochondrial targeted antioxidants can prevent this mitochondrial dysfunction. Aim 1 (Function) uses state of the art spectroscopic and traditional approaches to test whether aging worsens the functional decline in AZT/3TC treated muscle and whether mitochondrial targeted catalase (mCAT) is able to prevent this decline. Aim 2 (Mechanism) elucidates the cellular mechanisms underlying AZT/3TC toxicity and mCAT protection in young and old mouse muscle. We combine methods developed during the last P01 grant cycle for measuring changes in the mitochondrial proteome, damage, and quality control processes. Aim 3 (Translation) tests the potential for translating the protective effects of the transgenic mCAT model to human application. We test whether newly developed mitochondrial targeted antioxidant peptides (SS peptides) can protect against AZT/3TC toxicity in young and old mouse muscles. We combine methods from Aims 1 and 2 to measure in vivo functional outcomes and underlying mechanisms of protection of the SS peptides.

DESCRIPTION (provided by applicant): This application is for renewal of the Nathan Shock Center of Excellence in the Basic Biology of Aging at the University of Washington and affiliated institutions. This Center has over the past 20 years provided key resources in support of investigators who study the biology of aging. This application shifts to a theme that emphases outreach and service to the broadest community of investigators in the gerosciences. Of proximal relevance is the focus on characterizing aging-related phenotypes of longevity and healthspan. As our Center services must be easily accessible to outside users, our Longevity and Healthspan Core C focuses on invertebrate assays, many of them novel. Two other Research Resources focus on the high dimensional assessments that are most directly related to aging phenotypes, Protein Phenotypes of Aging (Core A) and Metabolite Phenotypes of Aging (Core B). Each of these three Resource Cores is led by a highly respected expert in that field, Michael MacCoss (Core A), Daniel Promislow (Core B) and Matt Kaeberlein (Core C). Each will push the envelope of appropriate technologies, developing new state-of-the art approaches for assessments that are the most applicable to gerontology and making them accessible to the aging community. The Research Development Core will support pilot projects, with an emphasis on support and career development of junior investigators, focusing on projects that can capitalize on the strengths of our Research Resource Core services. To broaden our outreach, the majority of awards will be to investigators outside our region. The Program Enrichment Core supports administrative management, an external advisory panel, a program of courses and seminars, data sharing and dissemination and advanced informatics and biostatistical support. A program of workshops will be focused on the fields of our three Research Resource Cores, to most fully integrate and disseminate the strengths of our Center.

SUMMARY ? Core A The goal of Core A is to provide centralized clinical, analytical and cell-based services and materials to facilitate AML and GBM research across all three Projects. The integration of services and materials in a single Core ensures efficient, standardized and cost-effective handling, characterization and distribution of clinical samples, and provides a common focus for coordinating all Program AML and GBM research. Core A also provides disease-specific research support and important services such as flow cytometry, imaging, cell culture and pathology. These services and materials play critical roles in facilitating Program research to test our Program-wide organizing hypothesis: that the mechanisms driving genetic and epigenetic heterogeneity identify, and may serve as targets for, new and highly effective cancer therapies. Core services: The Core A service plan was developed from the prior period service plan and substantially expanded to provide disease-specific support. Core A will provide centralized support for: ? culture, cryopreservation, and characterization of cells, cell lines and tissue from AML and GBM patients. ? newly developed, tumor-specific Experimental Hematology and Experimental Neurosurgery Core lab support services for Program investigators using these materials to pursue Project Aims. ? mutation typing of recurrent AML/GBM mutations, and for genes of high interest to Program investigators, e.g., the replicative DNA polymerase catalytic subunit genes POLD1 and POLE in Project 1. ? centralized support for drug and RNAi profiling, histopathology, IHC and imaging. The 4 Core Services we will provide to facilitate and support Program research by providing the above are: Core Service 1 ? Integrated clinical and laboratory support services for Program AML research. Core Service 2 ? Integrated clinical and laboratory support services for Program GBM research. Core Service 3 ? Mutation typing and genomic characterization of AML and GBM samples. Core Service 4 ? Centralized support for Program drug/RNAi profiling, imaging and histopathology. Summary: Core A has developed and will continue to support a sophisticated array of support services in addition to supplying high quality, well-characterized AML, GBM and GSC material to support Project research.

CORE D: RESEARCH DEVELOPMENT - SUMMARY/ABSTRACT The Research Development Core D provides support for Pilot Projects for junior faculty, or more rarely, more senior faculty who wish to enter the field of aging. Pilot Project Support fulfills two main objectives. First, as shown from our record of past performance, they can be an important part of the support for junior faculty who are embarking on new projects that may not yet have achieved outside funding. Second, and less often, they can play a similar role for more senior faculty who are changing career objectives in order to study the basic biology of aging for the first time. The total award budget is $90,000/yr, distributed as (typically) 6 individual awards. The large majority of awards will be targeted to candidates outside our region. These awards will provide fully subsidized access to our Research Resource Core services, necessary reagents and supplies, mentoring by a senior faculty member, and for external recipients, travel expenses for at least one visit to our Center. This activity is directed by the Core Leader in concert with the Executive Committee of our Nathan Shock Center. We present 1) a plan for the advertising, review and selection of investigators to receive support, 2) a plan for administration of awards, 3) a plan for the career development of individuals who will be selected for these positions, 4) a list of senior faculty who will participate in research career development. We will demonstrate that our institutions are able to provide adequate resources for the support of the research efforts of junior investigators, and a plan for monitoring their progress and development towards career objectives.

CORE E: PROGRAM ENRICHMENT/ADMINISTRATIVE ? SUMMARY/ABSTRACT Core E provides leadership, management, and support for education, outreach and collaboration, all necessary to ensure success of the Center. More specifically: 1) The Center Co-PIs, Drs. Rabinovitch and Kaeberlein jointly direct the Core activities, providing effective leadership, administration and management of the Center; 2) the Core supports an External Advisory Panel of scientists who will provide review and recommendations to the executive leadership of the Center; 3) The Core directs outreach activities, including workshops, courses, lectures and symposia; 4) The Core provides dissemination of Center resources and research findings to the broader Gerontological community; 5) the Core facilitates collaboration with other funded Nathan Shock Centers. Finally, 6) the Core insures that Center and Pilot Project investigators have expert informatics and biostatistical support by providing the services of our longstanding and highly skilled bioinformatician, Dr. Richard Beyer. An Executive Committee assists the Co-PIs in making policy decisions and is responsible for selection of Pilot Proposals and resolution of any Resource Core accessibility issues. An External Advisory Panel of nationally and internationally respected gerontologists provides external review and advice.

OVERALL: MITOCHONDRIAL PROTECTIVE INTERVENTIONS, AGING AND HEALTHSPAN P01 - SUMMARY/ABSTRACT We propose a highly focused, integrated and interactive effort to examine the hypothesis that mitochondrial targeted therapeutics are capable of resisting age-related disease and improving health and function in multiple organ systems in aging mammals. We are focused on heart, skeletal muscle and retina (vision) in aging; the vital function of each of these organs is highly reliant on mitochondrial energetics; each declines reproducibly with age and each contributes to frailty in the elderly human population. Most importantly, we show evidence that each of these age related healthspan limitations can be not just attenuated, but substantially reversed in old animals treated with a mitochondrial therapeutic agent, the tetrapeptide drug SS- 31. Thus, we have developed a strong translational goal of applying mitochondrial protective agents to achieve substantial health benefits in the aging mammalian models of mice and rats. The potential translational impact of this approach is high, as SS-31 is already in multiple phase II clinical trials, including three inspired by the prior animal studies performed by our P01 investigators. We propose three Projects: 1) Mitochondrial ROS and Cardiac Aging (Rabinovitch); 2) Mitochondrial Function and Skeletal Muscle Aging (Marcinek) and 3) Mitochondrial Function and Vision Aging (Prusky/Szeto). While each of these thee projects has their specific organ focus, each also shares a common third Aim of working jointly to establish the healthspan and lifespan benefits of SS-31 when it is continuously delivered to mice beginning at middle age, with and without the added stress of a high fat diet. We propose 5 Cores to provide vital support and services that are used by all three projects to accomplish their aims: A: Administration (Rabinovitch); B: Murine Healthspan and Lifespan, (Ladiges); C: Proteomics (MacCoss); D: Mitochondrial Protective Chemistry (Szeto) and E: Magnetic Resonance & Optics Spectroscopy (Conley).

PREVENTING SKELETAL AND CARDIAC MUSCLE AGING BY RESTORING MITOCHONDRIAL FUNCTION SUMMARY Aging is accompanied by slowly progressive and irreversible structural changes and functional declines in both heart and skeletal muscle that combine to contribute to exercise intolerance and frailty in the elderly. The increased rates of nursing home placement and hospitalization make the loss of muscle function with age a growing public health crisis in terms of both quality of life and economic costs to society. Despite this, there are few treatment options to reverse either skeletal or cardiac muscle degeneration in the elderly, due in large part to the poor understanding of the mechanisms that underlie these dysfunctions. Our previous work has demonstrated that treatment with the mitochondrial targeted peptide SS-31 improves skeletal and cardiac muscle performance, mitochondrial function, and reduces redox stress. These surprising results demonstrate that mitochondrial dysfunction with age is a more dynamic process than previously thought and can be reversed by late-life treatment to improve healthspan. Recent data indicates that SS-31 does not act as a traditional antioxidant by scavenging reactive oxygen species. Instead SS-31 appears to interact with mitochondrial cardiolipin to improve mitochondrial electron transport system (ETS) function and reduce mitochondrial oxidative stress. We propose that improved ETS function with short-term treatment reduces redox and energy stress which improves function and stress response of the aged heart and skeletal muscle. With long-term treatment this improved stress signaling restores mitochondrial and tissue structure, leading to further improvements in muscle performance. This proposal will define the redox and energy dependent signaling mechanisms by which SS-31 treatment reverses cardiac and skeletal muscle energetic dysfunction at late age (Aim 1), as well as the mechanisms by which these changes subsequently rejuvenates cardiac and skeletal muscle structure to improve performance (Aim 2). The final Aim 3 will test whether reducing mitochondrial oxidative stress by treating mice beginning in middle age can preserve muscle healthspan and exercise tolerance. We believe that the combined study of both heart and skeletal muscle will provide key insights into similarities and differences in how their functional impairments respond to enhanced energetics and redox signaling and how improvements in both will combine to enhance healthspan and exercise tolerance. The end result will be new insights into the mechanistic basis of this new paradigm for improving muscle health with potential for direct translation to elderly humans.

PROJECT 1: MITOCHONDRIAL ROS AND CARDIAC AGING ? PROJECT SUMMARY/ABSTRACT Aging is accompanied by slowly progressive and irreversible structural changes and functional declines in the heart that include increased prevalence of left ventricular hypertrophy, decline in diastolic function, and a decline in exercise capacity that contributes to frailty in the elderly. Extending work begun with mitochondrial targeted catalase, mCAT, we have recently demonstrated that short term (8 week) treatment with the mitochondrial protective drug SS-31 ?rejuvenates? cardiac function in old mice, reducing hypertrophy, improving diastolic function and remodeling the cardiac structure and proteome to a more youthful state. We hypothesize that SS-31 enhanced mitochondrial energetics and redox signaling subsequently result in remodeling of the cardiomyocyte and extracellular matrix to a more youthful state. This proposal will define the mechanisms that mediate both acute mitochondrial (Aim 1) and subacute cardiomyocyte and extracellular matrix (Aim 2) rejuvenating effects. As this approach offers the promise of substantial improvement in cardiac health of older humans, Aim 3 will help establish the potential longer-term benefits of these changes to murine healthspan and lifespan. Specifically, in Aim 1 In order to test the hypothesis that treatment with SS-31 restores redox and energy dependent signaling that results in improved mitochondrial structure and function we will measure mitochondrial and cardiac function, comparing and contrasting the mechanisms of mCAT, SS- 20 and SS-31 effects and their ability reverse cardiac aging in old mice and to protect mice challenged with doxorubicin to disrupt electron transport chain function. In Aim 2 we will determine the mechanisms by which SS-31 treatment of old mice rejuvenates cardiomyocytes and extracellular matrix (ECM) to improve aging diastolic function and cardiac performance. Aim 3 is shared across the entire P01, and will establish the translational benefits of SS-31 by determining whether SS31 can attenuate the decline of murine healthspan and extend lifespan after SS-31 is continuously delivered to mice beginning at middle age on regular and high fat diets. In this aim Project 1 will focus on cardiac healthspan.

CORE A: ADMINISTRATION ? SUMMARY/ABSTRACT An effective, proactive and efficient Administrative Core is essential to the productive planning, operation and support of the P01. Aging is a complex phenomenon and we believe it is through the coordinated efforts of motivated scientists with complementary expertise that real progress in unraveling underlying mechanisms and developing strategies to extend healthspan will occur. The Administrative Core of the P01 provides overall direction, internal and external review, administrative and statistical support. It 1) Provides an effective organizational structure to expedite research and promote communication between Program Project components and investigators. 2) Monitors and regularly review the quality and progress of research; 3) Insures adherence to rigorous statistical considerations in experimental design and data analysis. 4) Manages fiscal components of the Program Project including reallocation of funds to optimize overall function, and resolve any conflicts that arise over resources in a fair and equitable manner to maximize research findings. 5) Provides short-range and long-range planning, including internal meetings and external advisory meetings, for the enhancement and integration of Program Project facilities. 6) Provides data sharing and data dissemination facilities for the P01.

CORE B: RESEARCH DEVELOPMENT - SUMMARY/ABSTRACT The Research Development Core B provides support for Pilot Projects for junior faculty, or more rarely, more senior faculty who wish to enter the field of aging. Pilot Project Support fulfills two main objectives. First, as shown from our record of past performance, they can be an important part of the support for junior faculty who are embarking on new projects that may not yet have achieved outside funding. Second, and less often, they can play a similar role for more senior faculty who are changing career objectives in order to study the basic biology of aging for the first time. The vast majority of awards will be targeted to candidates outside our region. These awards will provide fully subsidized access to our Resource Core services, necessary reagents and supplies, statistical support at every stage of the project, and mentoring by a senior faculty member. This activity is directed by the Core Leaders in concert with the Executive Committee of our Nathan Shock Center. We present 1) a plan for the advertising, review and selection of investigators to receive support; 2) a plan for administration of award; 3) a plan for the mentorship and monitoring of individuals who will be selected for these positions.

OVERALL PROJECT SUMMARY This application is for renewal of the Nathan Shock Center of Excellence in the Basic Biology of Aging at the University of Washington and affiliated institutions. This Center has over the past 25 years provided key resources in support of investigators who study the biology of aging. This application continues a theme that emphasizes outreach and service to the broadest community of investigators in the gerosciences. Of proximal relevance is the characterization of aging-related phenotypes of longevity and healthspan. As our Center services must be easily accessible to outside users, our Longevity and Healthspan Core (Core E) focuses on invertebrate assays, many of them novel. Two other Resources Cores focus on the high dimensional assessments that are closely related to aging phenotypes: Protein Phenotypes of Aging (Core C) and Metabolite Phenotypes of Aging (Core D). Sophisticated computational and bioinformatic tools for data analysis and optimal insight are provided by the Artificial Intelligence and Bioinformatics Core F. Each of these four Resource Cores is led by highly respected experts in that field, including Michael MacCoss and Judit Villen (Core C), Daniel Promislow (Core D), Matt Kaeberlein and Maitreya Dunham (Core E) and Su-In Lee (Core F). Each will push the envelope of appropriate technologies, developing new state-of-the art approaches for assessments that are the most applicable to gerontology and making them accessible to the national aging community. The Research Development Core (Core B) will continue to support pilot and junior faculty studies, with a firm focus on outreach of service to the national geroscience constituency. The Administrative and Program Enrichment Core (Core A) supports administrative management, an external advisory panel, courses, and data sharing and dissemination. Core A?s program of seminars and symposia will continue a focus on sponsorship and organization of national courses, meetings and pre-meetings, as well as workshops in the fields allied to our Resource Core Services. In coordination with other Nathan Shock Centers, we will support a new Geropathology Research initiative.