Richard S. Miller - US grants

The long-range objectives of this project are to characterize the clinical and immunologic features of experimentally induced Cryptosporidium infection in the pigtailed macaque and to identify and define the immunologically significant surface antigens of the sporozoite stage and of the oocyst. Over the past three years, Cryptosporidium infection has been recognized as a common cause of severe diarrhea in patients with AIDS and as a significant contributant to diarrheal disease in preschool children. Research into the components of the host immune response in cryptosporidiosis has been hampered by the lack of a suitable animal model for human cryptosporidiosis. Infant macaques develop an enteritis following natural or experimental infection with Cryptosporidium which bears strong clinical resemblance to cryptosporidiosis in young children. Given the similarities between the human and primate immune systems, investigations into the natural history of the infection, time course for development of specific humoral immunity as measured by ELISA for sporozoite or oocyst specific IgG, IgM and IgA, and the clinical and immunologic response to rechallenge in this primate model may help to elucidate the corresponding aspects of the human disease. Rapid progress towards the characterization of a retrovirus-induced simian "AIDS" model at the Primate Center offers the opportunity to study experimentally induced cryptosporidiosis in an animal model with many immunologic similarities to human AIDS. Once the parameters (inoculum, incubation period, natural history) of these experimentally induced infections have been defined, the effects of modulation of the host immune system, or of specific passive immunotherapy, can be assessed. Results from these studies may have applicability to the therapy or prevention of cryptosporidiosis in humans, particularly in AIDS patients. Immunochemical analysis of the significant surface antigens will complement the above studies. Oocyst and sporozoite forms will be purified and analyzed by polyacrylamide gel electrophoresis and nitrocellulose immuno-blotting. Lactoperoxidase labeling techniques will be used to determine surface localization. Identification of specific antigens which are important in the protective host immune response may provide a basis for development of active immunotherapies.

The effects of opiates on ion transport across the intestinal epithelium will be further investigated. We shall a) examine the ability of opiates from different subclasses to reverse the secretory effects of a variety of agents in different parts of the intestine in vivo; b) examine the contribution of the central nervous system in mediating these intestinal effects of opiates; c) examine the local antisecretory actions of opiates in the guinea-pig ileum. This latter investigation will require a combination of neurophysiological and immunohistochemical techniques. Secondly, we shall investigate further the mechanisms by which kinins stimulate intestinal secretion. In particular, we shall investigate the icosanoid forming potential of the mucosa, the interactions between icosanoids and kinins and the regulation of intracellular enterocyte free calcium by these agents. This latter study will involve the use of the fluorescent dye quin-2. A third investigation will focus on the ability of muscarinic agonists to stimulate ion transport in the intestine. We shall try and elucidate the mechanism of agonist action and in particular the role of calcium in mediating agonist induced ion transport and receptor auto-desensitization. Finally, we shall further investigate the endocrine aspects of the mucosa by investigating the control of 5-HT synthesis and metabolism by mucosal enterochromaffin cells. These studies will help us to further understand the physiology of the intestinal mucosa and its regulation by opiate drugs and other factors. In addition, the studies will also increase our understanding of the molecular mechanisms underlying the action of these same agents.

Application for an ADAMHA RSDA award, Level II. This proposal describes aspects of research into the mechanism of action of opiates and opioid peptides at the cellular level and also their effects on the gastrointestinal system. Opiates have been shown to regulate electrolyte transport across the intestinal mucosa at both local sites and via the central nervous system. From a broader perspective, opioids are one of a family of neurotransmitters and hormones which have been shown to regulate mucosal electrolyte transport. Experiments are proposed to further investigate the mucosal actions of opiates and opioid peptides. The antisecretory actions of opiates against a variety of secretory stimuli will be examined. Electrophysiological studies examining the effects of opioids on submucous neurones are also proposed. The role of the central nervous system in mediating opioid effects on the gastrointestinal system will be investigated. Studies on the mechanism of action of several other regulators of electrolyte transport including, kinins, icosanoids and acetylcholine are also described. In a second series of studies, the ability of opiates to regulate neuronal (Ca+2)will be examined. This is part of a group of experiments investigating in Ca+2 regulation and voltage sensitive calcium channels in single cultured neuronal cells using a microspectrofluorometer designed for the purpose. The Ca+2 sensitive fluorescent probe, quin-2 will be used in these studies. The candidates future research plans are also described including a proposed period of training in molecular biophysics.

We propose a series of studies of T lymphocyte activation in old and young mice aimed at identifying in biochemical terms of defect(s) that prevent many T cells from old mice from responding to mitogens. We have previously shown age-related derangements in the generation of cytoplasmic calcium signals in T cells from old donors, and now propose to test two explanatory hypotheses: (a) that aged T cells have over-active calcium extrusion systems; and (b) that the low resting membrane potential of T cells from old mice interferes with calcium signal degeneration. Our studies of early signal transduction have hinted that pathways dependent on protein kinase C may be less impaired by aging than other kinase- dependent paths; we now propose more direct and systematic tests of protein kinase function and substrate specificity in T cells from old mice. We have previously documented age-related defects in Con A-induced expression of c-myc mRNA. We now propose to test for age-related changes in expression of selected cell cycle related genes (c-fos, c-jun IL-2, and IL-2R) in responses to mitogenic stimuli (Con A, anti-CD3). Nonmitogenic activators (PMA, ionomycin) will be used in studies of gene expression and protein kinase function to identify pathways to particularly susceptible to age-related dysfunction. Antibodies to c-myc and c-fos proteins will be used to look for altered gene expression at the single cell level and in T cell subsets thought to be particularly susceptible to senescent change. Finally we propose to look for evidence of age-related defects in splicing of primary RNA transcripts, defects that we have previously suggested may be responsible for age-related loss of myc mRNA accumulation. We hope to identify specific age-related defects in calcium signals, protein kinase function, and gene expression that might contribute to poor T lymphocyte function in old mice.

This application seeks to investigate the mechanisms by which opiates and other inhibitory neurotransmitters modulate synaptic transmission. in particular it has been shown that neuropeptide Y (NPY), a widely distributed neuropeptide, can inhibit neurotransmitter release throughout the central and peripheral nervous systems. We have demonstrated that NPY is a powerful inhibitor of Ca2+ currents in a variety of neurons. In the present application we shall propose experiments designed to investigate the way in which NPY, opiates and norepinephrine modulate Ca2+ signals in rat myenteric plexus neurons in vitro. We shall investigate whether inhibition of the Ca2+ current by these neurotransmitters involves a G-protein. The specificity of receptor/G-protein/Ca2+ channel interactions will be explored by reconstituting the system in pertussis toxin treated neurons and Xenopus oocytes using purified or recombinant G-protein alpha-subunits of various types, some of which will carry specific mutations. We shall also investigate whether the same neurotransmitters also modulate K+ channels in myenteric plexus neurons and whether the specificity of G-protein mediated coupling in this case is the same or different as that for receptor/Ca2+ channel interactions. We shall also use a combined patch clamp/microfluorimetry technique to investigate the effects of inhibitory neurotransmitters on [Ca2+](i) transients in myenteric plexus neurons. We shall attempt to elucidate those processes that are important for buffering [Ca2+](i) increases elicited by physiological stimuli and to analyze how these various processes can be regulated by inhibitory neurotransmitters. These investigations should be of importance in understanding the cellular basis of opiate action in the nervous system.

The major aim of my research is to understand the process of synaptic communication at a molecular cellular level and the way that it can be modulated by neurotransmitters and drugs. In particular I have been studying the mechanisms by which Ca2+ can enter nerve cells (calcium channels), factors that regulate the [Ca2+]i in neurons and the modulation of such processes by neurotransmitters. I am also particularly interested in the mechanism of action of opioid drugs which seem to inhibit neurotransmitter release in many instances by effects on neuronal Ca2+ channels. In this proposal I suggest further studies in several areas related to Ca2+ mediated processes in neurons. These include (a) the regulation of neuronal Ca2+ channels by receptors and G-proteins. (b) The regulation of neuronal K+ channels by receptors and G-proteins. (c) The regulation of synaptic transmission by receptors and G-proteins. (d) The regulation of neuronal [Ca2+]i under different circumstances in peripheral and central neurons. (e) The regulation of neuronal [Ca2+]i by excitatory amino acids, particularly the regulation of Ca2+ mobilization from intracellular stores and its role in synaptic transmission. (f) The regulation of [Ca2+]i in astrocytes by neurotransmitters. (g) The regulation of [Ca2+]i in identified anterior pituitary cells by hypothalamic factors and its role in the control of hormone secretion and (h) The construction of equipment for performing combined electrophysiology (patch-clamp) and [Ca2+]i measurements in thin slices from brain.

Our previous studies of T lymphocyte function in aging mice, using limiting dilution analyses (LSA), have suggested that a loss of antigen- and mitogen-reactive precursor cells for both helper and cytotoxic function may be an important component of age-associated immune dysfunction. We now propose to test the importance of precursor cell number for immune function by studies of mice in which the usual relationship between immune status and chronologic age has been altered by food restriction, parabiosis, or chimerism. Since the LDA technique permits repetitive, nondestructive testing of immune status using extremely small (5-50 ul) samples of peripheral blood, we will be able to use longitudinal designs to describe the time course of immune cell loss in normal and experimental mice. We also plan to use LDA techniques to study, at the clonal level, age-related changes in the production of memory T helper cells, and in the patterns of effector function -- e.g. lymphokine secretion and promotion of antibody synthesis -- generated by individual helper T cell precursors. Related methods will also allow us to classify precursor cells according to their fine specificity (in reactions to influenza virus), and in reactions to suboptimal stimulation at low antigen concentrations or in the presence of a limiting dose of blocking antibodies, e.g. L3T4. We plan as well to examine the kinetics and functional implications of IL-2 receptor expression in T cells from donors of different ages. Lastly, we plan to carry out pilot studies of age-related changes in T precursor cell number and subset distribution in humans. We hope that this research program will generate a coherent and detailed model of the cellular changes which underlie the age-associated decline in T cell immune function.

This is an application for funds to support a Summer Training Course in Experimental Aging Research to be held at the University of Michigan's Institute of Gerontology in Ann Arbor, Michigan, from June 6th through June 10th, 1993. The Course is designed to provide an intensive exposure to modern research in experimental gerontology for a group of 15 - 20 researchers who are in the formative stages of their careers. Each trainee will be expected to have had at least two years of productive laboratory experience in some aspect of cell or molecular biology beyond the doctoral degree (PhD, MD, or DVM). Each day in the five day program will include three activities: (1) two "overview" lectures designed to introduce trainees to the main results and central concerns of one area of experimental gerontology; (2) a research development workshop in which each trainee will have an opportunity to present her or his own research ideas for critique; and (3) a research seminar presented by a faculty member of the host institution. Six experienced research gerontologists will serve as course faculty. Richard Miller, the Course Director, will provide overviews of immune function and of animal and human models for aging research. Judith Campisi will discuss cell cycle control, and the relationship between aging and cancer. Caleb Finch will discuss the comparative biology of aging and the biology of neurodegenerative diseases including Alzheimer's and Parkinson's diseases. Arlan Richardson will discuss transcription and translational control of gene expression, and the retardation of aging by calorie restriction. Phyllis Wise will discuss neuroendocrine models of aging. Michal Jazwinski will discuss invertebrate models for gerontology. In addition, five University of Michigan faculty members will present research seminars: Drs. Richard Adelman, Fred Bookstein, John Faulkner, Ari Gafni, and Jeffrey Halter. This new course will provide younger scientists with a solid foundation in modern experimental gerontology, and provide useful perspectives to established researchers who are developing new programs in biogerontology. Contacts among trainees and faculty are likely to lead to continuing interactions, and perhaps collaborations, as trainees develop research programs after the course. A successful course will serve as a model for similar intensive training sessions on an annual basis.

The central goal of this research program is to count and map polymorphic loci in the mouse that alter longevity via an effect on the aging process. The work will focus on a population of UM-HET3 mice, bred as the progeny of CB6F1 mothers mated to C3D2F1 fathers. This four-way cross breeding scheme will produce a group of 600 mice (400 females and 200 males) that are effectively full sibs. Each mouse will be genotyped using SSLP markers at each of 150 polymorphic loci, and allowed to live until natural death or severe morbidity, to search for quantitative trait locus (QTL) alleles that are preferentially associated with exceptional longevity. Each mouse in the main population will also be tested for a number of age-sensitive traits: T cell subset levels, antibody responses, a variety of collagen cross- links, and eye lens opacity, as well as body weight trajectories. Each of these traits is known to vary with genotype in mice, and each has been shown in at least one strain or species to predict subsequent longevity. QTL found to be associated both with unusually long life span and also with relatively "youthful" levels of one or more of the age-dependent traits will be taken as plausible candidates for loci that alter the aging rate. Each mouse will also receive a complete histopathological necropsy, both to search for QTL that modify the risk of specific late-life illnesses, and also to determine which longevity- associated QTL reduce risks of multiple terminal illnesses. An initial genome scan at an average resolution of 15 - 20 cM will be followed by higher resolution analyses (3 - 5 cM) of regions found in the initial scan to be of particular interest. The proposal includes two ancillary studies in addition to the main gene mapping program. One study involves the longitudinal characterization of mice selected for optimal combinations of longevity-related alleles, to see if inheritance of these combinations leads both to exceptional longevity and to decelerated aging in multiple phenotypic domains. The second ancillary study is designed to produce a series of additional four-way cross populations, using esoteric grandparental stocks likely to contain alleles rarely found in laboratory adapted mice. The goal of this part of the program is to produce mouse populations with exceptional longevity and/or very low spontaneous tumor risk because with these characteristics would be excellent tools for further genetic analysis in future work.

Innocuous, age-sensitive assays of immune and muscle function have been developed and tested in cross-sectional studies of mice. These assays provide valid measures of function and can be performed without harm to the mice. The overall goal is to develop reproducible and valid biomarkers of aging. Our working hypothesis is that the proposed assays of immune and muscle function can distinguish mice that are aging at different rates. The hypothesis will be supported if changes in immune and muscle function are highly correlated, and if mice that exhibit early declines in immune and muscle function die at an early age. Two strains of hybrid mice (B6D2F1 and B6C3F1) will be used to mitigate the possibility of strain-specific artifact. Half of the mice will be fed ad libitum and half a calorie restricted diet that retards the aging rate. The principal design is longitudinal, in which 30 male mice in each of the four groups (two strains x two diets) will be tested with assays of immune and muscle function at 6 month intervals until near the end of their lifespan. This design will provide data on relations among putative biomarkers and between the biomarkers and lifespan and pathologic processes. A separate group of mice will be tested at 6 month intervals with the innocuous assays and then be sacrificed at 21 months of age (ad libitum fed mice) or 30 months (calorie restricted mice). This cross-sectional component will provide material for tests of immune and muscle, function that require access to internal tissues, permit comparisons between the innocuous assays and more traditional measures, and identify biochemical correlates of functional changes. The data will be analyzed by an iterative, nonlinear partial least squares approach which has proven very useful for the study of longitudinal data in early life but has not yet been exploited for studies of aging. Each of the assays can be applied with only minor modifications to human beings. Consequently, several of the assays should provide useful biomarkers of age-specific physiologic decline suitable for interventional studies in human beings as well as for experimental investigations of rodents.

We shall utilize fura-2 based microspectrofluorimetry and imaging techniques to investigate the regulation of (Ca2+)i in vertebrate neurons from the central and peripheral nervous systems. In the first series of studies we shall investigate the properties of intracellular bound stores of Ca2+ within neurons. We shall ascertain the distribution of these stores and whether they can be regulated by methylxanthines such as caffeine or by inositol trisphosphate (IP3). Furthermore, we shall investigate the effect of various neurotransmitters on phospholipid metabolism in different types of central and peripheral neurons in vitro. Secondly, we shall also continue to investigate the different types of voltage sensitive Ca2+ channels found in vertebrate neurons. In particular we shall investigate the distribution of different types of Ca2+ channels in single neurons in vitro. We shall also continue to investigate the mechanism by which neuronal Ca2+ currents can be regulated by neurotransmitters and whether this modulation occurs in different parts of the neuron. We shall continue to analyze the molecular basis for Ca2+ current modulation in neurons and in particular the role of G-proteins and protein kinase C in this process. We shall also continue to investigate receptor operated Ca2+ channels in neurons, particularly those activated by the excitatory amino acid glutamate. We shall continue to characterize the ionic channels linked to these receptors and the way that they can be modulated by low concentrations of glycine. Finally, we shall attempt to measure changes in (Ca2+)i in neurons in slice preparations from the central nervous system. In such a situation the cells will be in a more "normal" environment. We hope to be able to characterize changes in (Ca2+)i occurring in CA1 pyramidal cells during the induction of long term potentiation and to assess whether such changes are sine quae non for the establishment of this phenomenon.

This is an application for funds to support a series of five Summer Training Courses in Experimental Aging Research to be held at the University of Texas (San Antonio), the University of Michigan (Ann Arbor) and The Buck Center for Aging Research (Novato, CA) each summer from 1995 - 1999. A similar course was held with great success in Ann Arbor in 1993, and the course scheduled for 1994 in Novato attracted more than 120 applicants for the 20 available positions. The Course is designed to provide an intensive exposure to modern research in experimental gerontology for a group of 20 researchers who are in the formative stages of their careers. Each trainee will be expected to have had at least two years of productive laboratory experience in some aspect of cell or molecular biology beyond the doctoral degree (phD, MD, or DVM). Each day in the five day program will include three activities: (1) two "overview" lectures designed to introduce trainees to the main results and central concerns of one area of experimental gerontology; (2) a research development workshop in which each trainee will have an opportunity to present her or his own research ideas for critique; and (3) a research seminar presented by a faculty member of the host institution. Five experienced research gerontologists will serve as Continuing Faculty. Richard Miller, the Course Director, will provide overviews of immune function and of animal and human models for aging research. Judith Campisi will discuss in vitro clonal senescence and aging effects on transcription and translation. Caleb Finch will discuss the comparative biology of aging and the biology of neurodegenerative diseases including Alzheimer's and Parkinson's diseases. Thomas Johnson will discuss invertebrate models of aging, and the genetics of aging and longevity. Phyllis Wise will discuss neurobiology and endocrinologic changes in aging. In addition, each program will include research seminars by five Local Faculty members. In San Antonio these will include Drs. Barbara Bowman, Ed Masoro, Jim Nelson, Arlan Richardson, and Byung Yu. At Michigan these will include Drs. David Burke, John Faulkner, John Fink, Ari Gafni, and Jeff Halter. At the Buck Center these will include Drs. Bruce Ames, Charles Epstein, Stan Prusiner, Robert Sapolsky, and Ed Schneider. These courses should provide younger scientists with a solid foundation in modern experimental gerontology, and provide useful perspectives to established researchers who are developing new programs in biogerontology. Contacts among trainees and faculty are likely to lead to continuing interactions, and perhaps collaborations, as trainees develop research programs after the course.

The central hypothesis of this research program is that age-related changes in the distribution of T cell subsets revealed by differences in P- glycoprotein (PGP) can account for an important proportion of the senescent decline in T cell function. Aging leads to an increase in the fraction of mouse T cells that can extrude the fluorochrome R123. This translocation is mediated by PGP, the ATP-dependent plasma membrane pump that mediates multiple drug resistance in tumor cells. R123 efflux delineates age sensitive subpopulations in each of the four conventionally accepted T cell subsets: R123hi T cells are gradually replaced by R123lo cells within the naive and memory pools of CD4 and CD8 cells. Within the CD4 memory subset, only R123hi T cells can respond to anti-CD3 and IL-2 by proliferation and il-4 secretion. In the current proposal, Aim #1 is to determine if R123hi and R123lo CD4 memory T cells differ in the spectrum of lymphokines produced in responses to two infectious agents (influenza virus and schistomes). Aim #2 is to determine whether R123hi and R123lo cells differ in their requirements for activation using antibodies to CD3 and costimulatory signals (CD4, CD28, and PMA). The results will shed light on the functional significance of the age-related shift in R123 subsets, an on the cellular basis for age-related changes in memory T cell responsiveness. These two aims together will show whether the poor responses of the R123lo T cells to anti-CD3 reflect a generalized anergy, a need for specific costimuli, an altered pattern of lymphokine production, or a preference for specific classes of cognate antigen, and will also show which aspects of age-dependent memory T cell dysfunction are attributable to R123 subset shifts and/or to changes within the R123-delineated subsets. Aim #3 is to see whether the poor proliferative responses of R123lo T cells to Il-2 reflect defects in Il-2R expression or response (or both), and whether similar defects are seen after stimulation with CD3/IL-2 independent stimuli such as PMA an Il-4. Aims #4 and #5 compare R123hi and R123lo CD4 T cells from young and old mice in two other measures of T cell help: ability to stimulate Ig production from B cells, and ability to give rise to clones of antigen-responsive memory T cells. The studies of Aim #6 will provide additional information about the biochemical bases for the increases of PGP in the R123lo subsets, including measures of PGP mRNA and PK-C mediated PGP phosphorylation. The results of these studies should provide a comprehensive picture of the effects of aging on the generation, longevity, and functional abilities of CD4 memory T cells, and test the idea that differences between R123hi and R123lo T cells contribute to these aspects of immunosenescence, while also providing insights into the underlying functional and biochemical differences between the R123hi and R123lo in young and old animals.

DESCRIPTION: (adapted from Investigator's abstract) This project has two overall objectives: (a) to develop new stocks from wild-trapped animals that may retain genetic alleles for longer lifespan that have been lost in the laboratory adapted mouse stocks typically used for aging research; and (b) to determine whether genes that affect body size, litter size, corticosterone levels, and reproductive maturity have pleiotropic effects on age-sensitive traits such as immunity, collagen cross-linking, late-life disease, and longevity. The experimental design exploits four mouse stocks generated by this project in the preceding three years: Po and Ma, derived from tropical island populations; Id, derived from a mainland wild mouse population; and DC, a heterogeneous stock derived from four laboratory inbred mouse strains. Aim 1 tests the hypothesis that mice of the three wild-derived stocks will be longer lived than mice of the DC control stock, and the secondary hypothesis, based on evolutionary theories, that the island-derived mice (Po and Ma) may prove longer-lived than the mainland-derived Id stock. Aim 2 will characterize these mice using tests that provide information about early life history events (weight, reproductive maturation), stress resistance (corticosterone), or markers of aging (T cell subsets, antibody responses and collagen cross-linking). Differences among the four mouse stocks in the age-sensitive biomarkers will test the idea that differences in longevity (Aim 1) are mediated by an overall deceleration of aging-related changes. Differences among mice within each stock will test the ability of each of the traits to serve as validated biomarkers of aging, i.e. to identify mice with long lifespan and youthful levels of the other traits in the test battery. Aim 3 will provide necropsy data for all four stocks, to see whether differences in lifespan reflect diminished risk of a range of late-life illnesses. Aim 4 will produce and characterize F1 and F2 crosses between the wild-derived stocks and the laboratory-derived stock DC. These stocks are already known to differ in size, reproductive scheduling, immune responses, and corticosterone levels, and are hypothesized to differ in lifespan. Analysis of the segregating F2 populations will thus test the hypotheses that these life history and aging traits are controlled in part by genes with pleiotropic effects, and will also provide an estimate of the number of independently segregating loci with effects on each of the traits of interest (including lifespan) as a prelude to later QTL analyses.

The Core Facility for Aged Rodents (CFAR) provides animals and advice for research scientists who wish to use animal models to study the biology of aging, the relationship between aging and disease, or therapeutic approaches that could lead to the development of human intervention trials relevant to the independence and health care of the elderly. CFAR is directed by Dr. Richard A. Miller, Professor of Pathology and the Associate Director for Research of the Geriatrics Center. Veterinary consultation is provided by Dr. Robert Dysko, DVM, Assistant Professor of Laboratory Animal Medicine. Aged and control rats and mice are purchased from the NIA Contract Colonies and supplied to recipients of Pepper Center Pilot/Feasibility Grants, to junior faculty scientists who wish to acquire pilot data towards the preparation of an application for OAIC or extramural support, and to established scientists who wish to begin study of a problem in geriatrics or gerontology for which they do yet have external support. This facilitated access to old rodents serves to attract developing and established scientists to experimental geriatrics and to facilitate their initial forays in this field, while at the same time ensuring that they obtain advice (from the Core Director) on matters of choice of strain and species, appropriate selection of age groups, and possible influence of overt or latent diseases. Core resources are also devoted to the development of novel animal models that present unique advantages for studies of aging and geriatric diseases. Four such models are currently under development: (a) "HET" mice, bred by a four-way cross from inbred grandparents, to provide a degree of reproducible genetic and phenotypic heterogeneity that more closely resembles the variation present in aging human populations; (b) the WI/Hicks/Car rat whose lack of adult growth makes it particularly suited for studies of cross-age transplantation (e.g. of muscle and nerve grafts); (c) the mdx/mdx mouse that develops a mild form of muscular dystrophy in early adult life and that may provide a useful model of late life myopenia; and (d) the T(X;16)16H x SPE (Mus spretus) F1 hybrid mouse, a cross-species hybrid useful for investigations of X chromosome gene reactivation in aging. CFAR thus provides Pepper Center and other U/M researchers with consultation on experimental design, facilitated access to aged rats and mice, and assistance in producing and characterizing new rodent models that are particularly useful for studies of aging and not available elsewhere.

The goal of the Research Development Core is to promote the development of junior faculty members to research scientists who investigate the causes, consequences, and treatment of health care problems of the elderly. In accord with the goals of the OAIC program, special emphasis is given to career development activities that enhance the abilities of junior faculty members to study the relationship between disease mechanisms and independence-enhancing interventions. The Core is directed by Dr. Richard A. Miller, the Geriatrics Center's Associate Director for Research; Dr. Jeffrey Halter serves as Co-Director. The Core has three principal components. (1) An annual Pilot/Feasibility Grant program will fund junior faculty research projects in aging, age-related disease, and therapeutic interventions. Five such grants will be awarded each year, at a level of approximately $25,000 each. (2) Two research retreats will be held each year, on topics selected to that each of the Center's thematic areas (Cognitive Function, Homeostasis, Health and Well-Being, and Physical Function) is featured every second year. Each retreat will involve a 2 day program of scientific presentations, research development workshops, and career development sessions, and will bring to campus 2 senior geriatric investigators and 2-6 junior faculty members from other institutions to interact with Michigan junior and senior faculty members. UM participants will include all currently funded, and many former recipients of Pilot/Feasibility Awards. These retreats will emphasize the interactions between basic research, clinical research, and intervention development studies in the geriatric age group. Junior faculty members also acquire familiarity with a broad range of scientific problems and methods by participation in the Institute of Gerontology's weekly seminar series and (for physicians) the weekly conference of the Division of Geriatric Medicine. (3) A mentorship program pairs each UM Pepper Center junior faculty scientist with a senior faculty member who can provide advice on matters of research design, core utilization, and preparation of seminars, manuscripts, and grant applications. The mentor panel includes the directors of even Research Development Core (Drs. Ashton-Miller, Fries, Liang, and Miller), as well as other senior faculty scientists chosen for their training record and their expertise in specialized aspects of geriatrics research (Albin, Foster, Halter, Herzog, Park, Supiano, and Weissert). The Research Development Core also provides junior faculty members with subsidized access to the Medical Center's Molecular and Cell Biology ore laboratories, provides modest support for junior faculty travel to meetings in geriatrics, and coordinates a formal program of "pre-review" for nascent grant applications.

Interventions that could prevent or delay age related loss of T lymphocyte function might diminish the morbidity caused in elderly populations by neoplastic and infectious disease, and thus extend the number of years of independent life. The steroid hormone dehydroepiandrosterone sulfate (DHEA-S) has been shown to prevent or reverse important aspects of immunosenescence in mice. DHEA-S has also been reported to affect a wide range of non-immunologic aspects of aging and disease. The central goal of this study is to see if long-term administration of DHEA-S can delay the development of serious illness or disability in mice. The study will use genetically heterogeneous mice bred in the Pepper Center's Core Facility for Aged Rodents, since the range of pathophysiologic variation in this animal population may mimic human aging more closely than the traditional inbred animal models. Groups of mice will be treated with oral DHEA-S, and then followed until the development of serious morbidity (or death), to determine if the agent extends disease-free lifespan. Half of the treated mice will be treated from the 6th month of life, and half from the 15th month, to see if a therapeutic effect can be obtained when treatment is initiated only at mid-life. Necropsies will be carried out on each mouse found moribund or dead, to determine if the interventions alter the age-adjusted incidence of specific major illnesses. Each treated and control mouse will also be tested at 6 and 12 months after initiation of the intervention using several measures of age-sensitive immunological and virologic status, including (a) the proportion of memory T cells in the CD4 and CD8 subsets; (b) the proportion of T cells expressing high levels of P-glycoprotein function; (c) serum IL-6 levels; (d) production of IL-2 and IL-4 and IL-2 responsive cells; and (e) the presence of potentially tumorigenic retrovirus. The immunoassay data will be used to assess the hypothesis that the effects of DHEA-S on disease are attributable to preservation of vigorous immune function into old age, and may also allow the development of prognostic indices that can identify in youth or middle age individuals that are particularly prone to specific forms of late-life disease. The use of genetically heterogeneous animal models to test immunopharmacologic approaches to the prevention of disease is extremely cost effective compared to correspondingly powerful tests in aging humans, and could provide both a rationale for human intervention trials and insights into the pathophysiology of age-dependent disease.

The overall goal of this project is to identify and characterize abnormalities in the electrophysiological function of beta-cells associated with diabetes or induced by exposure to high concentrations of glucose. We will test the hypothesis that these conditions are associated with specific defects in the ion channels which regulate depolarization and repolarization of the beta-cell membrane or of Ca2+ homeostatic processes within the beta-cell. The techniques of fura-2 fluorimetry and digital image analysis will be applied in combination with whole cell and perforated patch recordings and single channel measurements to study the patterns of Ca2+ signalling within individual insulin secreting cells and cell groups. Experiments will be conducted in dispersed islet beta-cells isolated from normal animals or from animals with beta-cell dysfunction induced by infusion of glucose or with spontaneous diabetes resulting from a reduction in beta-cell mass (the GK rat) or autoimmune beta-cell destruction (the diabetes prone BB/Wor rat). The various components of the normal beta-cell and beta-cell derived lines which are concerned in the regulation of Ca2+ signalling will be studied including Ca2+ channels, K+ channels and various types of intracellular Ca2+ homeostatic mechanisms such as Ca2+ stores, pumps and exchange mechanisms. We shall attempt to determine how these factors contribute to the production of Ca2+ signals in beta-cells in response to various regulators including glucose, amino acids, sulfonuylureas and neurotransmitters. An insulin secreting beta- cell line, the betaTC3 cell line which retains many of the secretory properties of normal beta-cells has been demonstrated to increase its secretory response to a secretory stimulus after incubation in low glucose compared with high glucose. The electrophysiologic correlates of this increase in beta-cell function will be studied. Finally the electrophysiologic properties of novel ion channel genes expressed in the beta-cell will be characterized using heterologous expression systems. Using antisense oligonucleotides and antibodies the effects of these genes on cellular electrophysiology and Ca2+ signalling will be determined. It is anticipated that these studies will provide insights into the role of the ion channels in the regulation of insulin secretion in normal beta-cell physiology and in the secretory dysfunction of early IDDM.

We have recently cloned a novel Ca channel alpha1 subunit which we have named alpha1c. The transcript for this channel is widely distributed in the nervous system. However, expression of alpha1c produces Ca currents that are mysterious in several respects. The biophysical and pharmacological profile of the currents are not really similar to those that have generally been reported with a number of exceptions. We now wish to investigate the situation further with a view to establishing the characteristics of alpha1c based currents in normal cells and their physiological functions. In order to do this we shall take a varied approach which will include the following types of experiments. 1) Preparation of antibodies against alpha1c to be used for examining the distribution of the protein and its expression. 2) Expression of different splice variants of alpha1c in combination with different ancillary subunits in order to provide information on how alpha1c based Ca currents may normal appear. 3) Investigations of whether alpha1c currents can be regulated by diverse second messengers and receptors. 4) Removal of alpha1c transcripts from cells in which they normally occur and examination of the consequences of this for cell function. 5) Examination of the distribution of the different forms of alpha1c. These experiments will utilize a combination of biophysical and molecular biological paradigms. It is hope that these studies will provide us with important information about the functions of this novel neuronal Ca channel.

The central hypothesis of this research program is that age-related changes in the distribution of T cell subsets revealed by differences in P- glycoprotein (PGP) can account for an important proportion of the senescent decline in T cell function. Aging leads to an increase in the fraction of mouse T cells that can extrude the fluorochrome R123. This translocation is mediated by PGP, the ATP-dependent plasma membrane pump that mediates multiple drug resistance in tumor cells. R123 efflux delineates age sensitive subpopulations in each of the four conventionally accepted T cell subsets: R123hi T cells are gradually replaced by R123lo cells within the naive and memory pools of CD4 and CD8 cells. Within the CD4 memory subset, only R123hi T cells can respond to anti-CD3 and IL-2 by proliferation and il-4 secretion. In the current proposal, Aim #1 is to determine if R123hi and R123lo CD4 memory T cells differ in the spectrum of lymphokines produced in responses to two infectious agents (influenza virus and schistomes). Aim #2 is to determine whether R123hi and R123lo cells differ in their requirements for activation using antibodies to CD3 and costimulatory signals (CD4, CD28, and PMA). The results will shed light on the functional significance of the age-related shift in R123 subsets, an on the cellular basis for age-related changes in memory T cell responsiveness. These two aims together will show whether the poor responses of the R123lo T cells to anti-CD3 reflect a generalized anergy, a need for specific costimuli, an altered pattern of lymphokine production, or a preference for specific classes of cognate antigen, and will also show which aspects of age-dependent memory T cell dysfunction are attributable to R123 subset shifts and/or to changes within the R123-delineated subsets. Aim #3 is to see whether the poor proliferative responses of R123lo T cells to Il-2 reflect defects in Il-2R expression or response (or both), and whether similar defects are seen after stimulation with CD3/IL-2 independent stimuli such as PMA an Il-4. Aims #4 and #5 compare R123hi and R123lo CD4 T cells from young and old mice in two other measures of T cell help: ability to stimulate Ig production from B cells, and ability to give rise to clones of antigen-responsive memory T cells. The studies of Aim #6 will provide additional information about the biochemical bases for the increases of PGP in the R123lo subsets, including measures of PGP mRNA and PK-C mediated PGP phosphorylation. The results of these studies should provide a comprehensive picture of the effects of aging on the generation, longevity, and functional abilities of CD4 memory T cells, and test the idea that differences between R123hi and R123lo T cells contribute to these aspects of immunosenescence, while also providing insights into the underlying functional and biochemical differences between the R123hi and R123lo in young and old animals.

DESCRIPTION: Support is requested for the continuation of a series of Summer Training Courses (STC) in the Biology of Aging to be held each summer from 1998 through 2002. The course will be directed from the University of Michigan and the conference site will rotate among three host institutions: the University of Michigan, the University of Texas at San Antonio, and the Berkeley National Laboratory in Berkeley, CA. Similar courses have been held with great success every summer since 1993. The Course is designed to provide an intensive exposure to modern research in experimental biogerontology for a group of 20 researchers who are in the formative stages of their careers. Each trainee is expected to have had at least two years of productive laboratory experience in some aspect of cell or molecular biology beyond the doctoral degree. Each day in the five day program includes three activities: a) Two overview lectures; b) a research development workshop; and c) a research seminar presented by a senior faculty member of the host or nearby institution. Four scientists will serve as the Course Steering committee and participate in the course on a regular basis. Richard Miller, the Course Director, will provide overviews of aging and immune function and animal models for aging. Judith Campisi will discuss clonal senescence and aging/cancer interactions. James Nelson will cover endocrine aging and caloric restriction. Phyllis Wise will discuss brain aging and regulation of the menopause. Six other researchers will serve as continuing faculty, attending the course approximately every other year. These include Steven Austad, James Curtsinger, Caleb Finch, Olivia Pereira-Smith, Arlan Richardson and Rudy Tanzi. It is believed that this series of courses should 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. Contacts among trainees and faculty are likely to lead to continuing interactions and perhaps collaborations, as trainees develop their research programs after the course.

The rate of growth in the first tenth of the life span has a strong influence on the risks of late life disease in a variety of animal models, whether the growth retardation is produced by dietary manipulation (e.g. calorie or methionine restriction) or by genetic changes (such as the Pit-1 mutants or by selective breeding for slow growth rate). This project will attempt to discriminate among several hypotheses that might explain the linkages between slow early life growth rates and increased disease resistance in old age. Aim 1 will measure lifespan of dw/dw mutant mice treated with growth hormone (GH), thyroid hormone, both, or neither, to determine if the exceptionally long lifespan of these mutants is due to hormonal deficiencies or to some unknown concomitant of small body size per se. Aim 2 will measure lifespan in another mouse life, the Atchley LS2 stock, which has been selected for diminished weight gain in early life, to see if the exceptional longevity of the LS2 mice is maintained when these mice are subjected to a brief course of GH injections sufficient to restore them to normal size. Aim 3 will determine whether lifespan of the LS2 mice can be extended still further by constructing a congenic line carrying the dw allele on a background 97% identical to the LS2 stock. Lifespan data on these congenic mice will show whether the dw allele and the genes that lead to LS2 longevity are at least partly complementary. Aim 4 will test whether a dietary protocol involving low methionine levels, shown previously to extend lifespan in one inbred rat strain, will also promote longevity and disease resistance in a long-lived genetically heterogeneous mouse stock, and show further whether the intervention works when first imposed in mid-adult life. Aim 5 will determine whether each of these long-lived populations-dw/dw, LS2, and methionine deprivation-is accompanied by a parallel retardation in immune senescence, as an indication of whether the effect is mediated by a deceleration of aging per se. Lastly, Aim 6 will provide measures of key metabolic and hormonal endpoints-leptin, IGF-1, corticosterone-glucose and insulin, body temperature, and fat/lean rations and cause of death-to support or refute a series of mechanistic theories about the way in which retardation of aging, with concomitant delay in late-life illness, is linked to trajectories of weight gain in early life. By sorting among various explanations for the association of small adult body size and disease-free lifespan, the results should set the stage for intervention protocols to develop new ways to prevent the premature onset of late-life illness.

Core C: Animal Production and Specimen Preparation The goal of this core is to produce all of the mice needed in the Program Project, to assess their health status, and to generate the biopsy and necropsy specimens needed by each of the component projects within the program. Specific Aims include: 1. Breeding the 600 UM-HET3 mice of Population 1. 2. Providing tail tip DNA specimen for genotyping by Genotyping Core. 3. Providing biopsy specimens (blood cells, plasma, skin, etc.) needed at defined ages by Projects 1 and 5. 4. Providing body weight data at 3 month intervals to the Analysis ore. 5. Sacrificing each mouse in Population 1 at 18 months of age, and providing tissue specimens needed by each of the projects for physiological and/or biochemical analysis. Data on pathological lesions will be provided to each investigation and to the Analysis Core for use as a co-variate or exclusion criterion in statistical tests. 6. Producing the 180 mice in Population 2 (half of which are selected for genetic alleles associated with prolonged lifespan), and providing biopsy and necropsy specimens for these mice to each project. 7. Breeding UM-HET3 mice needed at various ages as controls by several of the component projects. The Core will be directed by Dr. Richard Miller, who will be assisted by the effort of a half-time research technician. All mice will be housed in the specific pathogen free vivarium of the newly opened Cancer Center and Geriatrics Center Building, and will be protected from infectious illness by use of individual micro-isolator caging. Husbandry procedures in use by the Geriatrics animal colonies have been able to maintain documented specific-pathogen free status since 1993, and should ensure a supply of high quality research animals for use in this program.

This Program Project on Genetics of Age-Sensitive Traits in Mice takes an integrated, multi-system in mice takes an integrated, multi-system approach to two related, fundamental questions in basic gerontology: (1) whether major aspects of the aging process are coordinately regulated at rates that differ among individuals; and (2) whether the pace of age- related change is under the control of polymorphic genetic loci. Each of the six projects is devoted mainly to assessing a set of age-sensitive traits in a specific physiological or biochemical domain-immunity, muscle, bone, protein structure, collagen modification, and somatic mutation-in a population of 600 female mice bred using a four-way cross procedure. Each of these 600 mice will also be genotyped at approximately 150 polymorphic loci. The combination of genetic and phenotypic data will allow the Analysis Core to map genes that influence age-sensitive traits in each domain and to see if some of these polymorphisms influence multiple traits in different domains. The data will also help to identify sets of traits that covary among mice-a key test of their usefulness as biomarkers of aging- and make it possible to determine the extent to which this co-variation is attributable to genetic and non-genetic factors. A second population of 180 test mice will be produced by genotypic selection, so that 50% of the mice bear allele combination shown to be associated with extended longevity influence age-dependent traits in a range of physiological and biochemical areas. Dr. Richard A. Miller will direct the Administrative Core and serve as overall Program Director. The Genotyping, Animal, and Analysis cores will be directed respectively by David Burke, Richard Miller, and Andrzej Galecki. The six research projects focus on Immunity (Miller), Muscles (Susan Brooks), Bones (Steven Goldstein), Proteins (Ari Gafni), Collagen Adducts (Vincent Monnier, Case Western Reserve) and Somatic Mutations (Eric Radany). This Program represents a collaboration among a set of biogerontologists who will collaborate towards the solution of problems that no one of the laboratories can address by itself. At worst, the Program should help to map genes that influence traits relevant to age-associated changes in tissue structure and function, and will test the idea that changes with age in protein and DNA structure may influence muscles, bones, and immune cells. At best, the program may permit the localization of loci that influence multiple age-sensitive domains and are thus plausible candidates for genes that regulate the rate of aging per se.

The goal of Project 1 is to assess the immune status of the mice that form the focus of this Program Project, i.e. the 600 UM-HET3 mice in Population 1 and the 180 genotype-selected mice in Population 2. The test battery includes the following components: . Production of anti-erythrocyte antibodies at 4 and again at 15 months of age. . Quantitation of six T cell subsets (CD4 and CD8, CD4 and CD8 memory, and the CD4P and CD8P subsets that express cell surface P-glycoprotein) in blood and in spleen of mice at 18 months of age. . Tests of in vitro proliferation by cultured splenocytes activated by Con A or by anti-CD3 antibodies with or without anti-CD28 co- stimulation. In addition to providing assessments of immune phenotypes for tests of the three Program Aims shared by all of the projects, the data generated by Project 1 will provide answers to several questions about the relationships among T cell subsets, in vitro proliferation, and in vivo immunity in aging mice. These include; 1. Do age-sensitive T cell subset levels in peripheral blood provide a useful index of subset levels in internal lymphoid tissues? 2. Do the factors that lead to age-atypical values of any given T cell subset also regulate the levels of other age-sensitive subsets? For example, do middle-aged mice with high levels of CD4 memory cells, characteristic of advanced age, also exhibit relatively high levels of CD4M, CD4P, CD8P cells? 3. Do mice with relatively "old" levels of T cell subsets show low levels of antibody production and poor responses in tests for in vitro proliferation? In conjunction with other Projects and Cores, these tests for immune status will provide a comprehensive picture of genetic effects on age- sensitive immune traits, and will help test the idea that the pace of immune change in adult life is regulated by factors that also time age- dependent changes in other physiological domains.

DNA methylation; leukocyte activation /transformation; gene expression; T lymphocyte; cellular immunity; biological signal transduction; aging; immunosenescence; chemokine; cell senescence; methyltransferase; enzyme substrate; helper T lymphocyte; T cell receptor; protein kinase; methionine; genetic screening; laboratory mouse; enzyme linked immunosorbent assay; polymerase chain reaction; genetically modified animals; gene targeting;

neuroregulation; apoptosis; membrane proteins; cell migration; neurogenesis; cell differentiation; biological signal transduction; telencephalon; phenotype; neurons; neurotrophic factors; genetic promoter element; neural plate /tube; glia; olfactory nerve; tissue /cell culture; early embryonic stage; laboratory mouse; genetically modified animals; transfection; neuroblastoma; embryo /fetus;

[unreadable] DESCRIPTION (provided by applicant): Infection with the HIV-1 virus is associated with effects on both the central and peripheral nervous systems including the HIV-1 cognitive/motor syndrome and HIV-1 -related sensory neuropathies. In this grant proposal we shall investigate the mechanisms by which the HIV-1 virus can compromise the function of sensory neurons associated with pain. In our preliminary data we demonstrate that sensory (dorsal root ganglion, [DRG]) neurons express the CXCR4 as well as other chemokine receptors. Activation of these receptors by chemokines or by the HIV-1 coat protein gpl20 activates intracellular signaling pathways that produce neuronal excitation and pain. Gp120 also activates the JNK signaling pathway that ultimately produces neuronal apoptotic death. In the experiments discussed in this proposal we shall (1) further examine the role of the CXCR4 and other receptors in mediating the effects of gp120 on DRG neurons; (2) examine the interactions between chemokines, gpl20, anti-retroviral drugs and members of the JNK signaling pathway in compromising the function of sensory neurons; and (3) examine the mechanisms by which gpl20 produces activation of JNK and induces the death of DRG neurons. These results will help us to understand how the HIV-1 virus can compromise the functions of sensory neurons and suggest novel targets for therapeutic interventions in the treatment of HIV-1 -associated sensory neuropathies.

Project STEP, involves graduate and undergraduate Fellows, secondary science and mathematics teachers, University of Cincinnati faculty and a graphics/web developer, working in teams to design, develop, and implement hands-on activities and technology-driven inquiry-based projects which relate to the students' community issues, as vehicles to teach science and math skills. Activities will be incorporated into lessons, demonstrations, laboratory exercises, individual and group projects, and field experiences to enable middle and high school students to directly experience authentic learning practices that requires them to use higher-order thinking skills; encourage creative problem-solving skills that require collaborative learning, teamwork, writing, and presentation; cultivate an interest in service learning in which students are active participants, achieve outcomes that show a perceptible impact, and engage in evaluative reflection; and better motivate and prepare secondary school students for advanced education. The Fellows will be trained to create and implement these activities by taking an educational methods course, an advanced course in instructional technology, and by serving as teaching assistants and tutors in guiding summer academies for middle and high school students. Quantitative formative and summative evaluation will be conducted to assess the project's effectiveness on Fellows' teaching skills, its impact on middle and high school science and mathematics education, and to continually improve the program as it develops. This project is receiving partial support from the Directorate for Engineering.

DESCRIPTION (provided by applicant): Laboratory for Anti-Geric Testing, Evaluation and Research Funding is sought for a series of investigations of interventions thought likely to extend life span in a population of genetically heterogeneous mice. The protocol will test five new interventions each year in Phase I screening studies, as well as conducting more comprehensive Phase II studies for selected interventions in Years 3, 4, and 5. Key features of the Phase I protocol include: (a) use of asymmetric group assignment to maximize statistical power by over sampling control animals; (b) use of separate monitor mice to document drug distribution and effectiveness; and (c) complete assessment of life table for all Phase I interventions. Phase I studies will include assessments of several age-sensitive traits as indirect indices of biological age, including (a) spontaneous activity; (b) IGF-I, glucocorticoid, and glycated hemoglobin levels, (c) five T cell subsets; (d) cataract severity; and (e) tests of learning and memory. Limited necropsy analyses will be done on all Phase I mice, and comprehensive necropsy analysis will be available for all Phase II mice and for those Phase I mice exposed to interventions found either to increase or to decrease longevity to a significant extent. Phase II studies will, in addition to replication of tests used in Phase I, add more comprehensive examinations of immune function, liver enzyme heat stability, eye lens protein extraction rates, tail tendon break time, and array-based analysis of age-sensitive liver and muscle mRNAs. Four interventions are suggested for initial exploration: (a) piaglitazone, an enhancer of insulin sensitivity, (b) pyridoxamine, which inhibits glycation-based cross links, (c) a-phenyl-N-tert-butyl nitrone (PBN), a scavenger of free radicals, and (d) pegvisomant, an inhibitor of GH action. The project as a whole should serve two important functions: providing critical tests of hypotheses that specific varieties of intervention will delay aging or late life illnesses in mammals; and, perhaps more importantly, provide important new leads to biochemical or hormonal pathways that can indeed modulate aging and delay late life illnesses and disabilities.

This is a proposal to acquire a High Performance Computing Cluster (HPCC) to facilitate the study of solar physics, solar-terrestrial physics, high-energy astrophysics, and astrophysics instrumentation development at the University of Alabama in Huntsville (UAH). The proposed facility would consist of a high-performance computational engine, data storage, and archiving hardware, as well as visualization hardware and software. The goal would be to develop a significant facility that is freely available to senior scientists, postdocs, and graduate students in the participating research groups.

Specifically, the proposed HPCC would support the proposers' primary science goals by enhancing efforts to simulate and model solar and astrophysical processes, facilitating the analysis of large volume datasets from multiple experimental programs, and aiding the characterization of existing (and the development of future) instrumentation by providing a platform for generating instrument simulations with high statistics.

The proposers have outlined a model system to achieve these goals that includes a multi-CPU design based on commercially available equipment, high-speed interconnects between CPUs, large data storage capability, archiving hardware and software, data analysis and visualization hardware and software, and software development workstations for students and postdocs.

The increasing volumes of experimental data, as well as the complexity of ongoing and future analyses, pose significant data analysis challenges. These factors have the potential to severely restrict data processing throughput, instrument characterization studies, and theoretical modeling programs. A modest investment in computing resources would make a major impact on the scientific return of both existing and future research programs at UAH. The proposed HPCC system is designed to address these issues and to provide a resource that would ensure UAH's continued leadership in the study of the Sun and the Cosmos. This new computational infrastructure would also complement the ongoing efforts of UAH's newly established High Energy Photonics Laboratory (HEPL) by enabling the simulation, design, calibration, and analysis of instrumentation developed in that laboratory.

CFAR, the Core Facility for Aged Rodents, has been a major feature of the University of Michigan Claude Pepper Center since its inception in 1989. CFAR will continue to serve the needs of Pepper Center investigators through four Specific Aims. (1) CFAR will provide advice to all OAIC investigators, from student through faculty levels, in the use of rodents for research into the biology of aging and its role in late life disease. (2) CFAR will support specialized colonies of mice particularly well suited for research on the biology of aging and its relationship to late-life disease. These include (a) genetically heterogeneous mice of the UM-HET3 stock; (b) calorically restricted UM-HET3 mice; and (c) mice of the long-lived Snell dwarf (dw/dw) stock, carrying the Pitl dw mutation. Mice from these colonies will be provided to faculty members working on PESC and RCDC research projects, as well as to Geriatrics Center faculty members who wish to conduct pilot studies on mouse aging supported by other sources of NIA funds. (3) CFAR funds will support the development of new animal models for specific purposes. In the first year, these will include a new fourway cross suitable for studies of late-life hearing loss, and a CAG-repeat knock-in mouse to study the timing of age-dependent motor neuron disease. (4) To nurture new rodent-based research initiatives through Core Development Projects. In the first year two such projects will be supported, one to develop instrumentation for assessment of age-dependent changes in balance and vestibular function, and the other to use calorically restricted and dwarf mice to evaluate the role of aging and anti-aging interventions in skin cell proliferation and pro-neoplastic transformation. The CFAR director will also play a role in helping to develop and design RCDC and PESC projects that make use of rodent models.

The goal of the Pilot and Exploratory Studies Core (PESC) is to provide support for studies that will develop and test new research ideas of high relevance to the Center's overall theme, the predictors and modulators of the aging phenotype and the ways in which genetic, environmental, and social factors influence late life disease through effects on the aging process. The PESC will thus fund pilot research studies over a wide range of disciplines, ranging from basic genetics and physiology through behavioral and health services research. Each submitted proposal will first receive an evaluation for scientific merit by at least two external reviewers, and then be further evaluated by OAIC leaders for its relevance to Center objectives. The Pepper Center's former Research Development Core (REIC) has made 61 pilot grant awards since its inception in 1989, mostly to junior faculty members, many of whom have now established themselves as productive independent researchers in geriatric medicine and cognate disciplines. Four projects have been selected for funding in the 2004 - 2005 academic year, including studies of (a) the genetics of bone chemistry and fragility; (b) a new system for investigation of insulin sensitivity using muscle explants; (c) factors modifying risk of falls in frail and healthy elderly subjects; and (d) the influences of gender and ethnic group on trajectories of alcohol consumption. The PESC will be directed by Dr. Richard Miller, who has served as director of the OAIC's RDC since 1993, and will be co-directed by Dr. Jeffrey Halter. Drs. Miller and Halter will work closely with the Directors of the RCDC to assist RCDC junior faculty as they develop plans for competitive Pilot Grant applications for PESC consideration.

DESCRIPTION (provided by applicant): Genetically heterogeneous (UM-HET3) mice produced as the progeny of (BALB x B6)F1 dams and (C3H x DBA/2)F1 sires will be used to map quantitative trait loci (QTL) that modulate longevity, age-sensitive traits, and early-life predictors of late-life events. Aim 1 will exploit an existing database, accumulated in the first 10 years of this project, which contains genetic data and phenotypic measures (of bones, hormones, immunity, weight, life span, and cause of death) for 1200 to 1800 mice, to evaluate new statistical methods for finding (a) epistatic interactions among QTL; (b) QTL that modulate relationships among traits, and (c) QTL that modulate variance in one or more traits. Aim 2 will use high resolution SNP genotyping to provide more accurate localization of QTL already localized by genome-wide scans. Aim 3 wilt produce and evaluate a new group of 600 UM-HET3 mice, first using a 300 - 400 SNP genome scan, and then, for selected traits, at higher resolution. Each mouse will be tested for a wide range of traits, including growth and maturation rates, T cell subsets, geometry and fragility of femur and vertebrae, multiple hormones, cataract severity, cause of death, and non-lethal pathological findings, as well as longevity. Most of these traits will be measured at two or more times in adult life, to allow searches for (a) QTL whose actions are restricted to specific times of the life span, and (b) QTL which influence the rate of change in age-sensitive outcomes. The data produced in Aim 3 will also be evaluated using the biostatistical methods developed in Aim 1, and QTL of special interest localized more precisely using the high resolution SNP methods of Aim 2. Aim 4 will ask which traits, measured in young or in middle-aged adults, best predict longevity and the timing of late life declines in immunity, endocrine pattern, bone status, and lens clarity. Early life predictors will provide physiological clues to the developmental events that influence aging in mammals, and late life predictors can be used as biomarkers to track the pace of aging in individual mice. This program should produce a detailed map of the genes that modulate individual aspects of aging in genetically heterogeneous mice, and allow a search for QTL that influence rate of change in multiple age-sensitive domains as well as life span and disease susceptibility.

DESCRIPTION (provided by applicant): The proposed program, "Biomedical Research Training in Aging," represents a five-year continuation (-21 to -25) of the current award, which was originally funded in 1985 and is supported until April 30, 2005. Funds to support 10 predoctoral and 6 postdoctoral trainees are requested. This Training Program constitutes part of a larger Institute of Gerontology (loG) Research Training Program, with about 45% female and 22% members of under-represented racial/ethnic groups. The principal goal of the Training Program is to select, train and prepare both graduate students and postdoctoral fellows for careers as leaders in biomedically oriented gerontological research. An important underlying theme of the Training Program is the importance and power of collaborative research programs in the biology of aging and understanding the broadly interdisciplinary nature of the field of gerontology as a whole. The Training Program involves 21 well-funded faculty members from 17 different departments and units on campus. The principal activity of each trainee is the development of a faculty-supervised research project. One of the defining features of our Training Program is the intensity of training faculty collaboration and the consequent team mentorship provided to our trainees. Another important principle of our Training Program is the desire for all of our trainees to receive a solid grounding in some biomedical discipline in addition to the emphasis on understanding their research problem in the context of the overall biology of aging. Important group aspects of our Training Program are monthly trainee meetings, a weekly interdisciplinary seminar series, an annual joint poster session with the loG at Wayne State University and the Michigan Alzheimer's Disease Research consortium, trainee attendance and presentations at relevant scientific meetings and participation in formal research responsibility programs at both the University and loG levels. Predoctoral trainees are admitted to graduate studies through individual departmental programs or the common admissions program in the Medical School and are admitted to this Training Program after they have completed preliminary examinations administered by their academic departments and have attained candidacy. Postdoctoral trainees must have satisfactorily completed all requirements for a doctoral degree and have demonstrated outstanding potential to conduct research with an loG Training Program faculty member. The training resources and facilities available through both the loG and the University are outstanding. These include the laboratories within the 21,000 sq ft loG itself, as well as a large series of University-supported computer and technical core facilities and libraries.

This project focuses on analysis of the genetic controls and physiological regulators that modulate hearing loss in a population of genetically heterogeneous mice. The test population will consist of 600 animals bred as the progeny of (CAST/Ei X 129S1/SvImJ)F1 females and (C3H/HeJ X FVB/NJ)F1 males. Each mouse generated in this four-way cross is genetically unique, and a full sib to each other animal in the population. Each mouse will be tested for hearing acuity by auditory brainstem response at ages 2, 8, 14, 18 and 22 months. Half of the 600 mice will be exposed to noise-induced cochlear injury at age 14.5 months. SNP-based genotyping at each of 300 loci will provide a genomic map of loci modulating hearing acuity, its change over age, and its resistance to noise-induced damage (Aim 1). Aim 2 will test the hypothesis that hearing loss is related to, and predictable by, individual differences in cellular resistance to oxidative cytotoxic stress, using in vitro analyses of fibroblast cell lines from each tested mouse. This strategy reflects recent evidence that mutations that retard aging and extend life span in mice may do so by alterations of stress-sensitivity in multiple cell types, including fibroblasts. Aim 3 will test the hypothesis that the genes, and potentially the non-genetic factors, that modulate functional hearing loss and cellular stress resistance also affect cochlear hair cell loss and oxidative damage to cochlear structures. Aim 4 tests a series of related hypothesis concerning the effects of early-life growth, maturation, and hormone patterns on mid- and late-life stress resistance and functional outcome, including hearing loss. Aim 5 will measure a range of age-sensitive traits, including indices of immunity and visual function, to test the hypothesis that inter-individual differences in age-related hearing loss are linked to differences among mice in multiple aspects of aging. This strategy will also yield, as a useful byproduct, a great deal of genetic information about loci that modulate growth, maturation, stress resistance, cataracts, hormones, and immunity in young and older mice. The approach chosen will permit a comprehensive assessment of the extent to which age-depending alterations in hearing are molded by genetic, cellular and hormonal factors that time the aging process in mice.

DESCRIPTION (provided by applicant): The University of Michigan requests funding for Years 16 - 20 of our Nathan Shock Center. UM receives more NIA funding than any other institution, and 48 grants in the biology of aging provide $10.4m/year in annual funding. Our Center's 2006 move to the new BSR building provided 21,000 square feet for Biogerontology, and allowed recruitment of four new tenure track researchers studying aging in mice, flies, and worms. The NSC Administrative Core will be led by Richard A. Miller, who will also serve as the Center Director. This core will facilitate communication among biogerontologists at UM and at other institutions, and take responsibility for advisory committees, interaction with UM and NIH officials, and supervision of an animal resource sharing website. The Research Development Core, headed by Susan Brooks, will administer a pilot grants program, organize an annual conference on a topic in aging research, and provide mentoring and financial support for a select group of junior faculty scientists. The Aging Rodent Core, led by Evan Keller, will support production of new transgenic and knockout mice, pay per diem costs to allow scientists to raise mice to old ages, and contribute to the costs of histopathologic analyses in the context of lifespan studies. The Drosophila Aging Core, directed by Scott Pletcher, will provide specialized equipment and validated protocols to support studies of aging, in flies, by experienced Drosophila geneticists new to aging, and by gerontologists who are just starting to incorporate Drosophila into their program. The Comparative Biogerontology Core, headed by Richard Miller, will create and characterize short-term primary fibroblast cells from a wide range of short-lived and long-lived rodents, primates, bats, birds, and dogs, and stimulate research at UM and elsewhere into cellular traits correlated with longevity across species. The Functional Assessment Core, directed by Greg Cartee, will provide advice and financial assistance to UM scientists who wish to make use of UM's exceptionally rich set of biomedical service core laboratories, to help Shock Center scientists, especially junior faculty members, introduce advanced methodologies into their research programs, including work on aims that might otherwise be deemed too risky or ambitious to tackle.

This project focuses on analysis of the genetic controls and physiological regulators that modulate hearing loss in a population of genetically heterogeneous mice. The test population will consist of 600 animals bred as the progeny of (CAST/Ei X 129S1/SvImJ)F1 females and (C3H/HeJ X FVB/NJ)F1 males. Each mouse generated in this four-way cross is genetically unique, and a full sib to each other animal in the population. Each mouse will be tested for hearing acuity by auditory brainstem response at ages 2, 8, 14, 18 and 22 months. Half of the 600 mice will be exposed to noise-induced cochlear injury at age 14.5 months. SNP-based genotyping at each of 300 loci will provide a genomic map of loci modulating hearing acuity, its change over age, and its resistance to noise-induced damage (Aim 1). Aim 2 will test the hypothesis that hearing loss is related to, and predictable by, individual differences in cellular resistance to oxidative cytotoxic stress, using in vitro analyses of fibroblast cell lines from each tested mouse. This strategy reflects recent evidence that mutations that retard aging and extend life span in mice may do so by alterations of stress-sensitivity in multiple cell types, including fibroblasts. Aim 3 will test the hypothesis that the genes, and potentially the non-genetic factors, that modulate functional hearing loss and cellular stress resistance also affect cochlear hair cell loss and oxidative damage to cochlear structures. Aim 4 tests a series of related hypothesis concerning the effects of early-life growth, maturation, and hormone patterns on mid- and late-life stress resistance and functional outcome, including hearing loss. Aim 5 will measure a range of age-sensitive traits, including indices of immunity and visual function, to test the hypothesis that inter-individual differences in age-related hearing loss are linked to differences among mice in multiple aspects of aging. This strategy will also yield, as a useful byproduct, a great deal of genetic information about loci that modulate growth, maturation, stress resistance, cataracts, hormones, and immunity in young and older mice. The approach chosen will permit a comprehensive assessment of the extent to which age-depending alterations in hearing are molded by genetic, cellular and hormonal factors that time the aging process in mice.

[unreadable] DESCRIPTION (provided by applicant): This grant proposal seeks to determine the mechanisms underlying the development of neuropathic pain in association with HIV-1 infection and its treatment with Nucleoside Reverse Transcriptase Inhibitors (NRTIs). We observed that receptors for CHEMOtactic cytoKINES (chemokines) are expressed by cells in the Dorsal Root Ganglia under different circumstances. Under normal conditions CXCR4, the receptors for the chemokine SDF-1/CXCL12, are expressed by DRG neurons and glia. SDF-1 is also constitutively expressed. Other types of chemokines and their receptors are not normally expressed by these cells. We observed that in several rodent models of neuropathic pain the expression of certain chemokines such as MCP- 1/CCL2 and their receptors such as CCR2, is greatly increased by DRG neurons and glia. Following their upregulation by DRG neurons, chemokines are packaged into synaptic vesicles and can be released from DRG neurons by depolarizing stimuli and from glial cells in a Ca dependent fashion. DRG neurons from animals with neuropathic pain are strongly depolarized by chemokines such as MCP-1. We have observed that treatment of peripheral nerves with the HIV-1 envelope protein gp120 produces increased expression of MCP-1/CCR2 in the DRG in association with neuropathic pain. We also observed that treatment of rodents with NRTIs produces neuropathic pain and that this is associated with upregulation of CXCR4 receptors in DRG neurons and glia. Inhibition of CXCR4 function inhibits NRTI associated pain hypersensitivity. Narrative: In this grant proposal we shall determine- 1) The relative roles of glial and neuronal CXCR4 expression in the development of NRTI induced pain hypersensitivity. 2) The role of CXCR4 receptor upregulation in the DRG in the synergistic effects of NRTIs and HIV-1 infection in producing pain hypersensitivity. 3) The role of chemokines as novel neurotransmitters in the DRG in producing neuropathic pain. The proposed studies will help us to understand how infection with HIV-1 and treatment with anti-HIV-1 drugs produces chronic pain syndromes. The resulting data will suggest novel approaches to the treatment of these disorders. [unreadable] [unreadable] [unreadable]

Abstract The NIA Interventions Testing Program represents a multi-site translational research program to evaluate agents hypothesized to extend mouse lifespan by retardation of aging or postponement of late life diseases. Interventions proposed by multiple collaborating scientists from the research community are initially tested, in parallel, at three sites (Jackson Laboratories, Michigan and Texas), using identical, standardized protocols, and using sufficient numbers of genetically heterogeneous mice to provide 80% power for detecting changes in lifespan of 10%, for either sex, after pooling data from any two of the test sites. Forty such lifespan experiments, involving various doses of 25 distinct agents, have been initiated in the first nine years of the ITP. Significant effects on longevity, in one or both sexes, have been documented for 5 of the tested agents: aspirin, NDGA, rapamycin, Acarbose, and 17-?-estradiol. Initial lifespan trials are now underway for 8 agents, as well as comprehensive analyses of the effects of rapamycin, Acarbose, and NDGA on health and on cellular and physiological traits thought likely to mediate the beneficial effects seen. Plans for the next five year period include additional lifespan ("Stage I") trials, detailed analyses ("Stage II") of agents found to increase lifespan, and some increased emphasis on collaborations with other scientists skilled at evaluating traits related to health and disease or at testing ideas about mechanisms of drug action on aging. Each of the three ITP laboratories will bring special expertise to the effort: measures of age-sensitive traits at the Jackson Laboratory, pathology and statistical analysis at Michigan, and pharmacology at the University of Texas.

Abstract The principal goal of the Core Facility for Aged Rodents (CFAR) is to develop new mouse models that can be used to evaluate the physiological and cellular factors that control the rate of aging and tie aging to late-life diseases. New mouse models are often suggested by other scientists, at UM or at other institutions, and these are evaluated as potential collaborations. CFAR resources are used for initial characterizations, often including studies of lifespan, age-sensitive indices of health, and pathology, with a view towards producing sufficient data to allow the new mouse models to be used in competitive applications for extramural support. In keeping with the overall theme of the UM OAIC, many of the mouse systems developed depend on alterations of metabolic and/or inflammatory pathways, whether by genetic changes, dietary interventions, or, more recently, drugs. The specific mutants, diets, or drugs chosen each year depend on scientific discoveries and interactions in each preceding year. Specific Aims: Aim 1 will support six externally funded projects (EPs) in year 1, including work on serotonin receptor mutants, drug effects on gene expression patterns, effects of anti- aging drugs on cancer cells, work on cap-independent translation of specific mRNAs, studies of adipose tissue inflammation, and an analysis of over-expression of an enzyme that protect mitochondria from oxidative damage. Aim 2 uses CFAR resources to maintain breeding colonies of special interest to mouse aging research, including mutants with tissue-specific alterations in Growth Hormone receptor, the IGF1 binding protein protease PAPPA, the mitochondrial redox protecting enzyme thioredoxin reductase 2, and two proteins that promote cap-independent translation of mRNA subsets. Aim 3 focuses on training, consultation, and professional education, so that CFAR can provide sophisticated perspectives on mouse aging models to PES and REC applications, and to UM students, fellows and faculty, and contribute to development of national standards for optimal use of rodents in biogerontology. Aim 4 involves selection of one or two new innovative mouse models for development in each year of the award. CFAR has, since its inception in 1989, provided both local and national leadership in the development of new mouse models for research on the control of aging and its links to late-life diseases, and hopes to maintain this role in the next award period.

PROJECT 3 focuses on the idea that diminished exposure to GH and/or IGF-1 signals in early life leads both to lifespan extension and to a spectrum of cellular abnormalities that we have documented in fibroblasts from long-lived Snell dwarf mutant mice, including sensitivity to inducers of ER stress, resistance to a broad range of lethal agents, and resistance to inhibition of the plasma membrane redox system (PMRS). Eight varieties of mice will be compared in the project, including: (a) Snell dwarf mice;(b) LID mice that lack IGF-1 expression in liver;(c) IGF-1 midi mice, with abnormally low levels of IGF-1 and high GH levels;(d) IGF-1 R het mice, with abnormally low response to IGF-1, (e) GHRKO mice, which lack GH receptors in all tissues; and (f) three new varieties of tissue-specific GHR mutants, which lack GH receptors, respectively, in liver, adipose tissue, or skeletal muscle. Aim 1will test skin-derived fibroblast cell lines from these mice, evaluating resistance to lethal oxidative and non-oxidative stresses (peroxide, paraquat, cadmium, and UV), to which Snell dwarf cells are resistant, and inducers of the unfolded protein response (tunicamycin, thapsigargin), to which Snell cells are sensitive. PMRS reactivity will be tested using non-lethal doses of rotenone. Fibroblasts from week-old mice will be tested to see if stress patterns require post-natal maturation, and from middle-aged mice to see if the stress-resistance profile lasts into midlife. Pre- adipocytes will be tested to see if they, too, show stress resistance when taken from long-lived donor stocks. Aim 2, using biochemical approaches, will test in vitro fibrobtasts, and tissues from intact and UV-exposed mice, for four pathways involved in stress resistance: Erk-family MAP kinase signals, activation of mTOR, repair and apoptotic pathways of the unfolded protein response, and mRNA levels for heat shock proteins and HSFs. Aim 3 will measure lifespan in the three tissue-specific GHRKO models, and will measure three age-sensitive traits (immune status, cataracts, and activity) as indices of delayed aging. Project 3 will provide resources to other parts of the program: tissues from terminal necropsies to Core B, fibroblast lysates for gene expression analyses to Project 1, and adipose tissue depots to Project 4. Project 3 will provide tests of the hypothesis that endocrine manipulations that modulate stress resistance lead to extended longevity, and in collaboration with the program as a whole will shed new light on the connections linking cellular stress resistance to genetic and pharmacologic modulators of endocrine and adipose tissues. RELEVANCE (See instructions): This project is intended to suggest clues about the biology of aging and late-life illness, provide models for investigation of the aging process, and confirm or refute ideas about proposed anti-aging drugs. Positive findings could, potentially, suggest new strategies in preventive medicine.

Administrative Core: Richard A. Miller, Director The Administrative Core (AC) will take responsibility for overall coordination of Center activities, communications with NIA personnel and administrative offices at UIVI, and interactions with scientists at other NSCs and in the broader biogerontology community. These responsibilities will include (a) coordination of the Center's scientific program;(b) meetings with the Center's Internal Advisory Committee;(c) meetings of the Center's External Advisory Committee;(d) coordination of NSC programs with those of UM's Aging Training Grant and Claude Pepper Center;(e) oversight of budgetary matters through the Geriatrics Center's research management office;(f) interactions with Other Nathan Shock Centers on matters of mutual interest;and (g) administration of our new Animal Resource website. In addition, the AC Director will serve on the ARC and FAC project review panels, and help the RDC in the coordination of pilot grant and research retreat programs.

Comparative Biogerontology Core: Richard Miller, Director The goal of the Comparative Biogerontology Core is to establish cryopreserved cell lines from multiple species of long-lived and short-lived birds and mammals, including long-lived and short-lived breeds of dogs, and to use these to learn more about the biochemical, biophysical, and cellular properties that are associated with exceptionally long lifespans as these have evolved under natural selection. The CBC cell archives are already being exploited by scientists at UM and at other institutions in studies of stress resistance, protein oxidation, lipid profiles, metal chelation, and sulfur amino acid metabolites as these relate to species and breed differences in aging rate and longevity.

DESCRIPTION (provided by applicant): The proposed program, "Research Training in Biogerontology," seeks a five-year continuation (-26 to -30) of our current award, a grant originally funded in 1985 and supported through April of 2010. Funds to support 6 predoctoral and 3 postdoctoral trainees are requested, matching current program size. This Program is situated within a Geriatrics Center that provides an exceptionally rich intellectual environment for research and training in the biology of aging, through its dedicated research space, Pepper Center, Nathan Shock Center, and GRECC core grants, multiple NIA-funded R01, U01, and P01 grants, and recent recruitment of new faculty. The Preceptor group includes 23 well-funded faculty members from 15 departments. The main goal of the Training Program is to select, train and prepare graduate students and postdoctoral fellows for careers as leaders in biological and biomedical aging research. Predoctoral trainees are accepted into the program only after they have completed departmental course requirements and embarked on full-time research programs. The main activity of each predoctoral and postdoctoral trainee is the development of a faculty-supervised research project leading to discoveries and peer-reviewed publications on important questions in the biology of aging. Trainee research projects are also expected to meet the highest professional standards in cognate disciplines of neuroscience, genetics, cell biology, biochemistry, immunology and physiology. In addition to discipline-specific training provided by the mentor and department, each trainee also benefits from Training Program activities that provide deep and broad background in modern aging research. These include a biweekly research series in which faculty presentations alternate with trainee research-in-progress talks;a monthly journal club;participation in Shock and Pepper Center annual research retreats;presentations at the annual Geriatrics Center research symposium;and opportunities to interact with guest speakers who visit each year to discuss topics in aging and geriatrics. Trainees also benefit from the University's well-established resources for training in the responsible conduct of research. The physical resources available to trainees through the Geriatrics Center and the University are outstanding, and include over 17,000 sq. ft. of wet lab space in the newly opened Biomedical Sciences Research Building, as well as the Medical Center's many sophisticated technical core facilities. PUBLIC HEALTH RELEVANCE: High quality training in the biology of aging will prepare students and trainees for outstanding careers at the forefront of Biogerontology, helping them make discoveries in the relation of aging to the diseases that afflict people in the second half of their lifespan.

? DESCRIPTION (provided by applicant): The chemokine CXCL12 (SDF-1) and its cognate receptor CXCR4 are involved in diverse physiological and pathological processes such as HIV infectivity, inflammation, tumorigenesis, stem cell migration, and autoimmune diseases. Although the CXCR4 receptor and its unique ligand SDF-1 have been widely studied, all small molecule modulators of the SDF-1/CXCR4 axis have been antagonists. The lack of available small molecule agonists constitutes a substantial gap in the ability to probe the biology of CXCR4. Using new in silico screening strategies, we have recently discovered the first series of small molecule CXCR4 agonists and have demonstrated their unique behavior in a variety of biological settings. Notably, our small molecules cause internalization of the CXCR4 receptor, a strong chemotactic response, and chemosensitization of tumor cell lines. Development of these small molecule agonists and structurally related antagonists will provide a unique and powerful means to study the function of the CXCR4 receptor and how this relates to disease processes. Acute myeloid leukemia (AML) is a group of myeloid leukemias with a very aggressive and fatal course if left untreated. The bone marrow (BM) microenvironment provides an important protective effect against chemotherapy and disruption of this interaction renders AML cells sensitive to chemotherapy in vitro and in vivo. The SDF-1/CXCR4 axis plays a key role in regulating stem cell mobilization and trafficking and its expression has been shown to negatively correlate to the prognosis of many cancers. Our lead agonist significantly enhances chemosensitivity of multiple leukemic cell lines to several chemotherapies, suggesting that CXCR4 agonists may provide a novel therapeutic approach for the treatment of AML. The overall goal of this project is to optimize small molecule CXCR4 agonist probes and characterize their activity against the CXCR4 receptor and AML in vitro and in vivo. Our unique small molecule CXCR4 agonists and antagonists give us a set of unique molecular tools to understand how CXCR4 receptor pharmacology impacts AML. In Aim 1 we will use rational medicinal chemistry to optimize our lead series for potency and drug-like properties, incorporating in silico design and robust biological testing into an iterative process. We will characterize new CXCR4 modulators in Aim 2 by evaluating their behavior against a number of in vitro systems including calcium mobilization, receptor binding, receptor internalization, chemotaxis, and signaling through various pathways. Aim 3 will evaluate the effects of CXCR4 modulation on AML using leukemia cell lines and primary human tumor cells as well as in vivo using patient-derived xenografts models of AML. The proposed studies will generate new molecular probes to investigate the pharmacology of CXCR4 and understand how this important receptor is involved in AML. These results will provide new insights into AML and potentially open up new avenues for therapeutic development.

Identification of small molecules that extend mouse lifespan provides new insights into mechanisms of longevity determination in mammals, and may lay the groundwork for eventual anti-aging therapies in humans. The NIA Interventions Testing Program (ITP) evaluates agents proposed to extend mouse lifespan by retardation of aging or postponement of late life diseases. Interventions proposed by multiple collaborating scientists from the research community are tested, in parallel, at three sites (Jackson Laboratories, University of Michigan and University of Texas), using identical, standardized protocols, and using sufficient numbers of genetically heterogeneous mice to provide 80% power for detecting changes in lifespan of 10%, for either sex, after pooling data from any two of the test sites. Seventy-two such lifespan experiments, involving various doses of 44 distinct agents, have been initiated in the first fifteen years of the ITP. Thirty-seven experiments have involved comparative tests of multiple doses of effective agents, variable starting ages, or alternative dosing schedules. Significant effects on longevity, in one or both sexes, have been documented and then confirmed for NDGA, rapamycin, acarbose, and 17-?-estradiol (17aE2), with significant (but currently unconfirmed) effects also noted for Protandim, glycine and, in an interim analysis, canagliflozin. Lifespan trials are now underway for 18 new agents. ITP survival results have also documented longevity benefits from three agents started in middle-age: rapamycin, acarbose, and 17aE2. The previous five year period has introduced three new features to the ITP: increased emphasis on health outcomes (functional tests relevant to human health not necessarily linked to lifespan), a Collaborative Interactions Program to provide tissues from ITP drug-treated mice to an open, growing, international network of scientific collaborators, and a publicly accessible data repository and display engine hosted by the Mouse Phenome Database at the Jackson Laboratory. Plans for the next five-year period include additional lifespan (Stage I) trials, detailed analyses (Stage II) of agents found to increase lifespan, continued growth in data on health outcomes, and collaborative work with scientists to study drug effects on postulated aging mechanisms and links to disease. Studies at Michigan will follow up our analyses of cellular pathways relevant to stress resistance and inflammation, by continuing ongoing studies of cap-independent protein translation, chaperone mediated autophagy, and browning of white adipose cells. The work proposed should allow the ITP to continue to make major contributions to mammalian aging biology.