Richard Elton Miller - US grants

During the insulin-mediated adipocyte conversion of confluent 3T3-L1 cells, glutamine synthetase (GS) specific activity increases more than 100-fold. Incubation of the adipocytes for 24 h with dibutyryl cAMP (Bt2cAMP) plus theophylline decreases GS specific activity by greater then 70 percent. Incubation of 3T3 adiopocytes with hydrocortisone increases GS activity by 1.5-to 2-fold. Immunotitration of GS activity from 3T3 adipocytes indicates that observed changes are due to changes in the cellular content of GS molecules. The Bt2cAMP-mediated decrease in glutamine synthetase activity results from a decrease in the synthesis rate and an increase in the degradation rate of the enzyme. Our current effects are directed toward determining the effects of insulin, glucocorticoids and other effectors on the rate of GS synthesis and degradation. In addition, we will attempt to determine whether GS in 3T3-L1 adipocytes might be subject to regulation by covalent modification.

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.

Recently radiolabeled dihydropyridines (DHP's) such as 3H-nitrendipine have been used to identify voltage sensitive calcium channels (VSCC) in many tissues including the brain. Howver, to date VSCC in the brain have been found to be insensitive to such drugs. Thus, the function of the brain DHP receptor is in question at this time. We have identified several neuronal cell lines that do possess VSCC that are blocked by DHP's. We have utilized these cells to identify novel DHP's and toxins (e.g., maitotoxin) that activate VSCC. We propose to examine the properties of VSCC in primary cultures of authentic neurones from the central and peripheral nervous systems. In order to do this, we have devised a specialized microspectrofluorometer capable of analyzing fluorescent signals from single neurones. When cultured neurones such as dorsal root ganglia cells are loaded with the Ca+2 chelating dye, quin-2 they emit a signal which is proportional to the intracellular free [Ca+2]. Thus, on stimulation of the neurone, the influx of Ca+2 via VSCC can be detected as a fluorescent signal. Using this system, we shall analyze the interaction of drugs with VSCC in authentic neurones. In addition, we shall analyze the ability of opiates to mobilize [Ca+2]in in certain neurones.

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.

The broad aim of this proposal is to understand the electrophysiological consequence of polyphosphatidylinositol (PPI) turnover in mammalian central neurons. It has become clear that the hydrolysis of membrane inositol phospholipids is an important component of signal transduction for many neurotransmitters. It is implicated in secretion and muscle contraction and may initiate relatively long term effects such as growth and memory. Most Of our knowledge of the PPI system and its significance comes from non-neuronal cells, yet the brain has the highest levels of the receptors for the second messengers produced. The PPI system is composed of two major branches. This proposal will focus on the inositol phosphate limb, since there is almost nothing known of the effects of the inositol phosphates in mammalian central neurons. Preliminary studies with inositol trisphosphates indicate that they exert dramatic inhibitory influences on neuronal excitability, apparently activating two distinct potassium-dependent afterhyperpolarizations. Similar afterhyperpolarizations seen in normal cells function to limit burst duration and repetitive firing. Thus, inositol phosphates may play an important regulatory role in neuronal communication, particularly in epilepsy and memory. The rodent hippocampal slice will be used as a model system to investigate the neurophysiology of the inositol phosphates by studying the activity of single neurons. A unique strength of this proposal is that metabolically stable analogues of the inositol phosphates, as well as the natural compounds, will be utilized. Thus, ambiguities related to metabolism are avoided and observed actions can be ascribed to a specific inositol phosphate. The effects of applied inositol phosphates will be related to the actions of neurotransmitters thought to be coupled to PPI. At present, there are few cases in central neurons in which particular electro- physiological effects of neurotransmitters have been attributed to these second messengers. The influence of inositol phosphates on neuronal activity thought to be important in epilepsy and memory will also be evaluated. The studies proposed can advance our understanding of the neuronal PPI system by identifying electrophysiological consequences of increases in inositol phosphates. As alterations in PPI metabolism have been found in epilepsy and memory models, the hippocampus is particularly relevant for studies of this kind, as it is a structure thought to play an important role in these neuronal activities.

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 work detailed in this proposal is designed to elucidate the mechanisms by which opioid receptors regulate ion channels and synaptic communication.An associated aim is to understand the regulation of neuronal Ca signals in general and the role of Ca in determining the viability of neurons under normal and pathological conditions. The major areas of research will be: 1) What are the structures and properties of neuronal voltage sensitive Ca channels? How can these channels be regulated by opioid and other receptors? How do G-proteins participate in this process? How does this process contribute to the phenomenon of presynaptic inhibition? 2) How do opioid and other receptors regulate neuronal K channels? How are G-proteins involved in this process? How do these details differ from the regulation of Ca channels? 3)How are neuronal Ca signals regulated by opioid and other receptors? How do Ca and other intracellular mediators such as free radicals contribute to the death of neurons in different circumstances ? What are the relative roles of necrosis and apoptosis in these processes? These studies will involve the use of biophysical,biochemical and molecular biological techniques. It is hoped that this research will provide us with further information on the way that opioid drugs can regulate the activity of nerve cells.

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.

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.

Description (adapted from applicant's abstract): Chemokines are a family of small proteins that are known to play an important role as messengers in the immune system. Chemokines exert their effects through the activation of a family of G-protein coupled receptors (GPCRs). It is also known that some of these GPCRs can act as receptors for the HIV-1 virus and are key elements in the ability of the virus to infect cells of the immune system. Chemokine receptors have also been found in the brain, however nothing is known about their cellular functions or how neuronal chemokine receptors may contribute to HIV-1 associated neuropathology or interact with other endogenous systems such as those for opioids. We have recently shown that neuronal chemokine receptors can produce effects on neurons typical of other GPCRs, including rapid changes in synaptic communication. Moreover, some chemokines can block neuronal apoptosis induced by the HIV-1 coat glycoprotein gp120. In the present proposal we shall extend these findings and ask the following questions: 1. What are the mechanisms underlying chemokine induced changes inn synaptic communication? How does neuronal chemokine receptor activation produce changes in neuronal Ca and other ion channels? 2. What is the range of chemokine effects in the nervous system? Which neurons are chemokine sensitive and to which chemokines? 3. What is the mechanism of the anti-apoptotic effects of chemokines? How do chemokines activate the anti-apoptotic enzyme Akt? 4. What is the range of survival effects mediated by chemokines? Are chemokines effective in different models of neurodegenerative disease? In order to carry out these studies we shall utilize a variety of electrophysiological, imaging, biochemical and molecular biological techniques. These studies will help us ascertain how chemokines regulate neuronal communication and excitability and how chemokine receptors may contribute to the neuropathological correlates of HIV-1 infection.

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.

DESCRIPTION (provided by applicant): The specific Aims of the Nathan Shock Center (NSC) at the University of Michigan (UM) are to: (i) facilitate and stimulate research programs in the cellular and molecular biology of aging;(ii) encourage the career development of biogerontologists, particularly junior faculty, and enrich the training environment for trainees;(iii) attract established UM faculty who are not working in biogerontology to initiate studies in this area;and (iv) utilize resources to develop collaborative relationships with other NSC's and with other universities world-wide. These aims will be achieved through the following initiatives: (i) an Administration/Enrichment Core (Faulkner) with an Internal Advisory &Core Directors'Committees;(ii) Research Development Core (Carlson) that supports workshops, pilot grants, and transitional salary support for junior faculty;(iii) five research resource cores;Transgenic Animal Models Core Camper), Aging Transgenic Rodent/Pathology Core (Keller), In Vivo Functionality Core (Metzger), and Contractility Core (Faulkner);(iv) an annual meeting of 15 senior NSC investigators to provide input to the Director and the two Committees;and (v) an External Advisory Committee. During the 9 years of NIA support, the NSC has done much to facilitate and stimulate research on aging since the participants have grown from 22 to 62 scientists at the UM and include 25 scientists at USA and foreign universities. The NSC at UM has also developed collaborative programs with the NSC at UTHSC at San Antonio and -other universities in the US and world-wide. The 62 NSC scientists investigate the molecular and cellular mechanisms of aging through multidisciplinary research in a dozen or more diverse research themes. The 15 senior scientists provide leadership to the NSC through directing cores, serving on committees, directing workshops, mentoring junior faculty and trainees, and organizing multidisciplinary collaborative research groups. The NSC has enhanced the quality and quantity of research on aging for scientists and trainees at UM and at universities world-wide.

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.

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 receptor CXCR4 and its endogenous ligand SDF-1 are expressed in the neurogenic regions of the adult brain and are critical for guiding neurogenesis and migration of neural stem and progenitor cells. Recent evidence indicates that chemokines also regulate the proliferative and restorative response of the brain following focal cell death in brain injuries and neurological diseases. Activated chemokine receptors stimulate several cellular effectors, of which the mobilization of Ca2+ is a key signaling process necessary for its biological effects. As a multifunctional second messenger, Ca2+ activates distinct genetic programs that control many processes such as the proliferation of neural progenitor cells (NPCs), NPC migration, and differentiation of NPCs into mature neurons and glia. However, how NPCs generate Ca2+ signals in response to chemokines and other regulatory factors remains unknown. Our preliminary studies indicate that store-operated Ca2+ release-activated Ca2+ channels (CRAC channels) are a major mechanism for chemokine-driven Ca2+ signals in NPCs. We further find that ablation of CRAC channel expression or pharmacological blockade suppresses chemokine-mediated Ca2+ signaling, NPC proliferation, and chemokine-mediated migration of NPCs to their terminal destinations. Based on this evidence, we hypothesize that CRAC channels are essential regulators of chemokine-mediated Ca2+ signaling and directed migration of NPCs. We propose the three specific aims to address this question: 1) Define the expression of STIM1/Orai1 proteins in the neurogenic regions of the brain. How does this correlate with the known expression of the CXCR4 receptor? 2) Elucidate the contribution of CRAC channels for the generation of complex Ca2+ signals by the chemokine, SDF1. 3) Determine the role of CRAC channels for chemokine-mediated migration of NPCs and neuroblasts. We will approach these questions using a multidisciplinary approach that combines in-depth protein expression studies using confocal microscopy, electrophysiology and Ca2+ imaging using 2-photon laser scanning microscopy, and in vitro and in vivo cell migration assays. Collectively, results from these studies will advance our understanding of the physiological role of CRAC channels for the directed migration of neural progenitors and aid the quest for developing new therapies for brain repair following injuries and in neurodegenerative diseases.

? 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.