Richard O'Brien - US grants

health science research support; medical education;

The goal of this proposal is to investigate the mechanisms guiding synapse formation in the embryonic nervous system. In addition to its implications for neuronal development, this endeavor is likely to yield insight into many different facets of the adult CNS, such as short and long term synaptic modulation, with their implications for memory and learning respectively. One aspect of synapse formation amenable to analysis is neurotransmitter receptor accumulation. The recent cloning of multiple subunits for each of the two subtypes of excitatory neurotransmitter receptors, NMDA and non-NMDA receptors, has made it possible to study the regulation of these receptor subunits during synaptogenesis. A model system will be developed in which purified cultures of Purkinje cells, taken from the rat, can be grown in the presence and absence of their normal source of excitatory input, granule cells, taken from a different species. Using highly specific molecular techniques such as nuclease protection and Western blotting, combined with electrophysiology, changes in the expression of excitatory transmitter receptors by the post synaptic Purkinje cell during innervation will be quantified. Particular attention will be focused on changes in the number, distribution and phenotype of these receptors. An additional goal will be to correlate the expression of excitatory neurotransmitter receptor subunit mRNA and protein, with the electrophysiological properties of receptors recorded from Purkinje cells. Furthermore, the heterologous expression of subunit cDNA's in transfected cells will allow an estimation of the degree to which receptor phenotype is controlled by transcriptional regulation, and the role, if any, of post-translational modification. The mechanism by which excitatory transmitter receptors are regulated, such as phosphorylation, subunit synthesis, or desensitization, will be investigated. Additionally, the agents mediating these changes, and any correlative changes in cell morphology will be sought. The modulation of excitatory neurotransmitter receptors during synapse formation has important implications for many neurodegenerative processes. Excitatory transmission has been implicated as an etiologic agent of both acute and chronic neurological diseases. The alteration of post-synaptic receptor properties such as conductance and desensitization may increase the susceptibility of CNS neurons to the toxicity of excitatory agonists. The multidisciplinary training emphasized in this proposal will allow great flexibility in approaching this issue, and offer excellent preparation for a career integrating clinical investigation and molecular neuroscience.

DESCRIPTION (Adapted from applicant's abstract): Insulin is known to regulate the transcription of almost 100 genes indicating that this represents a major action of this hormone. For a few of these genes specific cis-acting elements, referred to as insulin response sequences/elements (IRSs/IREs), have been identified through which insulin regulates their transaction. This information is important since various investigators have speculated that a defect in insulin-regulated gene transcription may explain some aspects of non-insulin dependent diabetes mellitus (NIDDM). Moreover, it has recently been shown that a mutation in the apolipoprotein CIII (apo CIII) IRS, resulting in a loss of insulin-regulated apo CIII gene transcription, is one cause of hypertriglyceridemia in humans. A consensus IRS/IRE sequence has not emerged, instead most of the sequences identified to date appear unique. Based on what is known concerning the mechanisms by which other hormones regulate gene transcription, it seems very unlikely that insulin will use unique mechanisms to regulate every gene. Phorbol esters can regulate gene transcription through at least eight cis-acting elements and we believe that a similar situation will exist with respect to insulin-regulated gene transcription. Clearly this is a field still waiting for its general principles to emerge. A broad objective of this proposal is to use the gene encoding malic enzyme (ME) as a model system to further the understanding of the molecular mechanisms whereby insulin regulates gene transcription. The investigator's preliminary data suggests that, while insulin stimulates ME gene transcription and the second goal is to identify the trans-acting factor that this element binds. The relative basal expression of the ME gene has been genetically linked to the difference in the severity of diabetes seen in C57BL/KsJ-db/db as compared to C57BL/KsJ-db/db mice. The third objective of this proposal concerns the biochemical analysis of the basis for this variation in basal ME gene expression and the determination of whether an alteration in basal ME gene expression contributes directly to the pathophysiology of NIDDM. The investigator's preliminary data suggests that a transcription factor is differentially expressed in the mouse strains.

[unreadable] DESCRIPTION (provided by applicant): Type 2 diabetes is characterized by defects in insulin secretion, peripheral glucose utilization (PGU) and hepatic glucose production (HGP). The ability of insulin to stimulate PGU and repress HGP in patients with type 2 diabetes is reduced as a consequence of insulin resistance. In addition, in Type 1 diabetics, HGP can increase if circulating insulin levels are low, a particular problem when poor glycemic control has led to the development of insulin resistance. In both type 1 and type 2 diabetes this increased HGP was thought to be a consequence of an increased rate of gluconeogenesis, rather than glycogenolysis, but more recent data suggest that the latter may also be important. In either case, the final reaction in both metabolic pathways, the hydrolysis of glucose-6-phosphate to glucose, is catalyzed by glucose-6-phosphatase. In animal models of both type 1 and type 2 diabetes glucose-6-phosphatase catalytic subunit (G6Pase) gene expression is elevated. Moreover, hepatic overexpression of G6Pase is sufficient to induce an increased rate of HGP. These observations suggest that the suppression of G6Pase gene expression may represent a potential strategy for reducing HGP in diabetic patients. The rationale development of a pharmaceutical agent that suppresses G6Pase gene transcription will require a detailed knowledge of the cis-acting elements and trans-acting factors through which transcription of the gene is regulated. We have shown that the inhibitory action of insulin on basal G6Pase gene transcription requires two promoter regions designated A and B. Region A binds hepatocyte nuclear factor-1 (HNF-1) but does not directly mediate the action of insulin. Instead, HNF-1 enhances the action of insulin mediated through Region B. Region B contains three insulin response sequences (IRSs) designated IRS 1-3. IRS 1 and 2 bind the insulin-responsive transcription factor FKHR (FOXO1a) whereas IRS 3 binds an unidentified insulin-responsive factor. Aim 1 of this grant application seeks to address several questions, namely (i) how does HNF-1 enhance the inhibitory action of insulin on G6Pase gene transcription? (ii) how does insulin inhibit glucocorticoid stimulated G6Pase gene transcription? (iii) how does insulin inhibit cAMP-stimulated G6Pase gene transcription? and (iv) what is the unidentified insulin-responsive factor binding IRS 3? In Aims 2 and 3 we propose characterizing the cis-acting elements and trans-acting factors that mediate the inhibitory effect of phorbol esters and the stimulatory effect of PGC-1 on G6Pase gene transcription, respectively. We have shown that the G6Pase promoter region between -484 and +66 is sufficient to confer maximal hormonally regulated G6Pase fusion gene transcription in situ. In Aim 4 we propose generating transgenic mice to determine whether this same promoter region can confer a developmental, tissue-specific and hormonally regulated pattern of G6Pase fusion gene expression in vivo that mimics that of the endogenous gene. [unreadable] [unreadable]

DESCRIPTION: (from applicant's abstract) The amino acid glutamate mediates excitatory neurotransmission in the brain, through the activation of a recently identified family of receptors which are concentrated on dendrites at sites opposite excitatory nerve terminals. The processes that control the development of excitatory synapses, including the aggregation of postsynaptic receptors, are likely to be key to understanding plasticity inherent in learning, memory, and regeneration in the adult brain. In addition, the degeneration of excitatory synapses seen in Alzheimer's disease, and the glutamate-mediated toxicity seen in ALS may be examples of alterations in those mechanisms. Our present understanding of excitatory synaptogenesis is rudimentary. In cultured rat spinal neurons, AMPA type glutamate receptors are initially expressed throughout the dendritic surface, but over time, become concentrated at synapses, and disappear from non-synaptic sites. This alteration in distribution coincides with the synthesis of the AMPA receptor subunits GluR2 and/or GluR3 (hereafter referred to as GluR2/3). In addition, overexpression of the intracellular C-terminus shared by GluR2 and GluR3 disrupts the accumulation of synaptic AMPA receptors. In this grant, I outline an approach to understanding the mechanisms and molecules involved in the targeting of glutamate receptors to excitatory synapses. Using a sensitive, high resolution, in situ hybridization technique, the role of GluR2/3 in regulating the synaptic aggregation of AMPA receptors will be investigated by determining whether the delayed synaptic appearance of GluR2/3 is due solely to a delay in the synthesis of these subunits, or to a tight regulation of their distribution at the protein level. In addition, the synthesis of GluR2/3 will be examined in isolated neurons, revealing any inductive effect of synapse formation on the synthesis of these subunits. Once synthesized, GluR2 is transported to and stabilized at excitatory synapses through signals present in its C-terminus. To identify the precise sites necessary for the synaptic targeting of AMPA receptors, a mutagenesis strategy will be employed in which epitope tagged GluR2, cloned into an expression vector, will be subjected to both deletion and point mutations, and the resultant product transfected into cultured spinal neurons. The ability of the transfected receptor, as well as chimeric molecules created between GluR2 and non-synaptic molecules such as CD8, to target to synapses will be assessed. Finally, we will investigate the role of a novel immediate early gene NARP (neuronal activity regulated pentraxin) in the development and stabilization of excitatory synapses in cultured spinal neurons. NARP is expressed exclusively at excitatory synapses where it is secreted by the presynaptic terminal. In HEK-293 cells, NARP induces the aggregation of coexpressed AMPA receptor subunits through a direct interaction. Moreover, NARP expressing 293 cells can aggregate AMPA receptors in co-cultured spinal neurons.

DESCRIPTION (provided by applicant): The formation and maintenance of excitatory synapses in the central nervous system is crucial to the development of the brain as well as to the ongoing plasticity that underlies learning, memory and adaptation. The neuronal pentraxin Narp (Neuronal Activity Regulated Pentraxin) has been implicated in the aggregation of AMPA type glutamate receptors at excitatory synapses in the brain and spinal cord because exogenously applied Narp can cluster AMPA type glutamate receptors on cultured neurons. Narp is especially intriguing for neuronal function because its expression is tightly regulated by electrical (synaptic) activity. Therefore Narp serves as a potential link between brain activity and synapse formation. Such a link may well be impaired in diseases that affect the formation of new memories such as Alzheimer's disease. In the present proposal, we examine the role and mode of action of endogenous Narp and its related molecule NP1 in excitatory synapse formation by utilizing novel, dominant negative Narp mutants (dnNarp) that selectively bind endogenous Narp and prevent its accumulation at synapses. Neurons transfected with these mutants are deficient in excitatory but not inhibitory synapse formation. In addition we have developed techniques to isolate and characterize the receptor to which Narp binds at excitatory synapses and localizes its function. One possible candidate for this receptor is another activity regulated molecule termed Neuritin. The molecular basis for the interaction between Narp and its receptor and Narp and the AMPA class of glutamate receptors will be investigated using a series of deletion and point mutations in both HEK 293 cells and neurons. Finally the mechanism whereby the NMDA class of glutamate receptors is aggregated at excitatory synapses will be investigated. Our experiments indicate that both Narp and an as yet uncharacterized molecule are involved in this process. The mechanism of Narp's involvement in NMDA receptor aggregation and the identity of any additional molecules involved will be sought.

DESCRIPTION (provided by applicant): One of the most promising approaches to curing type I diabetes is pancreatic transplantation. However, major problems with this strategy are the need to use immunosuppressive drugs to prevent rejection and a limited donor supply. Therefore, as a variation on this approach, there is considerable interest in the growth, differentiation and modification of stem cells with a view to converting them into non-immunogenic cells that secrete insulin in response to changes in plasma glucose concentrations. The identification of islet-enriched transcription factors is critical to achieving this goal because the controlled expression of these transcription factors in stem cells may circumvent one of the difficulties facing investigators who are studying such cells, namely that the developmental cues that promote stem cell growth and differentiation are not all known. The identification of these transcription factors is most expeditiously achieved by analyzing the promoters of genes whose expression are islet-specific. We have recently cloned one such gene that encodes an islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP). Our published studies have demonstrated that multiple cis-acting elements are required for maximal IGRP gene transcription and strongly suggest that novel transcription factors will be identified through studying the IGRP promoter. In addition, since IGRP is an autoantigen in human type 1 diabetes, understanding the mechanisms that regulate IGRP gene expression should not only lead to the identification of novel transcription factors but this information may also have clinical significance. This application proposes four Specific Aims. In Aim 1 we will characterize five cis-acting elements and their associated trans-acting factors. This Aim will be achieved using a fusion gene strategy, in conjunction with the transfection of tissue culture cell lines and primary islet cells. We have already found that one other cis-acting element in the IGRP promoter binds a novel, islet-enriched transcription factor so in Aim 2 we will clone a cDNA that encodes this protein. Our preliminary data suggest that during the remodeling of the islet that occurs after birth there is a switch in the nature of the promoter elements that are required for IGRP gene expression. Thus, the -306 to +3 IGRP promoter region is sufficient to direct IGRP-beta galactosidase transgene expression to newborn mice islets but the -911 to +3 IGRP promoter region is required for the maintenance of transgene expression in adult animals. In Aim 3 we will perform an initial characterization of the factors binding the -911 to -307 IGRP promoter region by in situ foot-printing. And in Aim 4 we will compare the developmental expression of the endogenous IGRP gene with that of the -911 IGRP-betaa galactosidase transgene.

DESCRIPTION (provided by applicant): This proposal requests continued support for the Molecular Endocrinology Training Program (METP) at Vanderbilt University. Progress towards understanding and curing obesity, diabetes and many other diseases requires the training of the next generation of scientists with expertise in molecular endocrinology, the goal of this program. The METP comprises 30 faculty members in 6 basic science departments. Of this group 27 are established faculty with stable, well-funded programs and substantial training experience and 3 are new investigators; 5 of these preceptors are female, 1 is a minority and 1 is disabled. This preceptor group constitutes a unusually diverse and talented group of individuals whose work covers the spectrum of molecular endocrinology. These preceptors conduct research in the general areas of: 1) signal transduction 2) the hormonal regulation of gene expression, 3) metabolic regulation and 4) beta-cell development and function. The request for a steady state level of 8 predoctoral and 4 postdoctoral trainees is justified on the basis of the number, size and quality of the research programs directed by the preceptors and the Institutional commitment to continue the same level of trainee recruitment despite the tough economic climate. All METP trainees are appointed upon the advice of an Admissions Committee after being nominated by a preceptor. Postdoctoral trainees have a Ph.D. degree. Rigorous in depth research training is the focus of both the pre and postdoctoral training programs. However, the METP also ensures that all trainees receive a broad didactic education. Predoctoral training in the METP follows that received in the Interdisciplinary Graduate Program (IGP). The IGP recruits almost all predoctoral trainees in the biomedical sciences at Vanderbilt, provides a first year core curriculum, safety training and formal evaluation and career counseling programs. This centralized recruitment has considerably increased the number and quality of predoctoral students that enter Vanderbilt. After four laboratory rotations predoctoral students choose a preceptor for their thesis project and compete for METP support. The IGP and METP have been very successful in promoting diversity and both provide ongoing training in the Responsible Conduct of Research. All METP trainees attend an annual METP Day retreat and the Vanderbilt Diabetes Center (VDC) seminar series where they meet with visiting scientists. In conjunction with the Juvenile Diabetes Research Foundation the METP has recently initiated a novel strategy to increase the recruitment of disabled individuals, specifically undergraduates with type 1 diabetes, through the creation of a VDC funded Summer Diabetes Research Program. The METP has already successfully trained 153 scientists of whom 54 have already gone onto assume academic/pharmaceutical positions with another 34 trainees still in training.

[unreadable] DESCRIPTION (provided by applicant): The islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is approximately 50% identical at the amino acid level to the glucose-6-phosphatase catalytic subunit and has recently been identified as a major autoantigen in the mouse Non-Obese Diabetic (NOD) model of type 1 diabetes. In Aim 1 we propose cross- breeding NOD mice and IGRP knockout mice to determine whether the absence of the IGRP gene in the NOD background is sufficient to prevent the onset of type 1 diabetes. If diabetes is prevented, we will perform (i) a detailed analysis of immune system function in the NOD/LtJ IGRP-/- mice to evaluate whether IGRP reactive T-cells persist in these animals and the impact on the autoimmune response directed at other islet autoantigens (ii) gene rescue experiments to determine whether re-introduction of IGRP as a BAG or cDNA restores diabetes susceptibility; only the former will be spliced and preliminary data suggest that differential splicing of IGRP RNA in thymus and islets may explain how IGRP escapes central tolerance. Alternatively, if diabetes is not prevented, we will (iii) perform a detailed analysis of cellular and humoral autoreactivity targeted at IGRP and other islet autoantigens, especially insulin and (iv) generate combined NOD/LtJ IGRP-/- and insulin I -/- mice to determine whether the absence of IGRP expression combined with a reduction in insulin expression is now sufficient to prevent the development of diabetes. Preliminary data show that deletion of the IGRP gene in mice only results in mild hypoglycemia. In Aim 2 we propose investigating the physiological basis for this observation. Since IGRP catalyzes glucose-6-phosphate hydrolysis and is expressed exclusively in pancreatic islet beta cells, we hypothesize that IGRP deletion alters the Km of glucose-stimulated insulin secretion. Therefore, oral glucose tolerance tests and hyperglycemic clamps will be used to compare insulin secretion in IGRP knockout mice and wild type littermates in vivo. In addition, insulin secretion from wild type and IGRP knockout mouse perfused pancreata will be compared in situ. Finally, insulin secretion from isolated wild type and IGRP knockout mouse islets will be compared in vitro. Relevance: A protein called IGRP has been implicated in the development of type 1 diabetes. This project will assess whether the absence of IGRP in mice is sufficient to preven the onset of diabetes. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This continuation proposal requests support for the Molecular Endocrinology Training Program (METP) at Vanderbilt. Twenty nine faculty members from five basic science departments-constitute the preceptors of the METP. Of this group 25 are established faculty with stable, well-funded programs and training experience and 4 are new investigators. The preceptor group constitutes an unusually diverse and talented group of individuals whose work covers the spectrum of molecular endocrinology. These preceptors conduct research in the general areas of: 1) signal transduction 2) the hormonal regulation of gene expression, 3) metabolic regulation and 4) Stem cells, ( cell development and function. The request for maintaining the current level of 8 predoctoral and 4 postdoctoral trainees is justified on the basis of the number, size and quality of the research programs directed by the preceptors. All METP trainees are appointed upon the advice of an Advisory Committee after being nominated by a preceptor. Postdoctoral trainees have a Ph.D. degree. Rigorous in-depth research training is the focus of both the pre- and postdoctoral training programs. However, the METP also ensures that all trainees receive a broad didactic education. In addition, all METP trainees attend the NIDDK-funded Vanderbilt Diabetes Research and Training Center seminar series and meet with the visiting scientists. New additions to the program include (i) a didactic course focusing on the molecular endocrinology of obesity and diabetes, (ii) an Annual METP Day and weekly data clubs to foster interactions between trainees and preceptors, (iii) a post-doctoral trainee mentoring program, and (iv) training in both grant writing as well as laboratory &project management. The Program also provides formal training in the proper use of radioisotopes, in appropriate procedures of dealing with toxic and dangerous materials, and in the responsible conduct in research. METP trainees also have access to a formal career-counseling program. The METP has been successful in attracting and supporting the training of individuals from diverse backgrounds. Relevance: The field of Molecular Endocrinology is of central relevance to multiple human diseases, most notably obesity and diabetes. Continued progress towards understanding and curing these and many other diseases requires the training of the next generation of scientists, which is the goal of this program.

DESCRIPTION (provided by applicant): The glucose-6-phosphatase catalytic subunit gene family comprises three members, G6PC, G6PC2 and G6PC3. G6PC is predominantly expressed in liver where it catalyses the terminal step in the gluconeogenic and glycogenolytic pathways, namely the hydrolysis of glucose-6-phosphate (G6P) to glucose and inorganic phosphate. G6PC2, initially known as IGRP, is ~50% identical at the amino acid level to G6PC but it is expressed specifically in pancreatic islets. G6PC2 also hydrolyses G6P, though at a much lower rate than G6PC. This is consistent with the observation that deletion of the G6PC2 gene in mice results in decreased fasting blood glucose levels, suggesting that G6PC2 normally opposes the action of glucokinase in islets and lowers intracellular G6P concentrations and consequently glucose-stimulated insulin secretion. Two recent genome wide association (GWA) studies have linked single nucleotide polymorphisms (SNPs) in the G6PC2 gene to variations in fasting blood glucose levels in humans, a parameter that is linearly correlated with cardiovascular-associated mortality. Our preliminary data strongly support the hypothesis that sequence variations in the G6PC2 gene, rather than surrounding genes, contribute to elevated fasting blood glucose levels and hence cardiovascular-associated mortality. This project is therefore at a more advanced stage relative to many other GWA studies where disease-associated SNPs have been reported but the disease-associated genes remain to be identified. The goal of the experiments proposed in this application is to extend the GWA studies by functionally characterizing the effects of G6PC2 gene variants on transcription (Aim 1) and splicing (Aim 2). Our initial experiments have shown that two SNPs in the G6PC2 promoter affect G6PC2 fusion gene transcription. In addition, our sequence analyses suggest that the G6PC2 SNP that was first linked to variations in fasting blood glucose is located in a branch point, a key cis-acting element controlling pre-mRNA splicing.

Project 2 in the Johns Hopkins Alzheimer's Disease Research Center (ADRC) is entitled "The roles of AB, tau and synaptic loss in early AD". The overarching goal of this project is to understand the mechanisms that allow some individuals to tolerate substantial Alzheimer's disease (AD) pathology, whereas others with similar brain abnormalities develop MCI or dementia. We will use a collection of brains from prospectively followed subjects from the ADRC, known as the Johns Hopkins ADRC Autopsy Cohort (JHAAC). The JHAAC includes brain tissue from a substantial number of subjects who were cognitively normal shortly before death, but were found to have substantial AD pathology on autopsy, referred to as 'asymptomatic AD'. The JHAAC also includes brain tissue from controls, subjects with MCI and patients with AD. We will examine three hypotheses in this project. Aim 1: We will test the hypothesis that amyloid-beta (AP) oligomers, not AB deposits, are responsible for cognitive decline. We will determine whether AP40, AP42 and AB oligomers distinguish the cognitive phenotypes of subjects with similar levels of AD pathology, as measured by the standard Braak and CERAD scales. In addition, we will examine whether the significant AB accumulation seen in the brains of the subset of cognitively normal subjects with substantial AD pathology is due to quantitative differences in the amount, bioactivity or distribution of enzymes purported to degrade or transport AB in vivo. Aim 2: We will test the hypothesis that the process that couples AB deposition with neuronal/synaptic abnormalities is associated with Tau phosphorylation or cleavage. We propose to quantitate the amount of Tau phosphorylation and fragmentation in JHAAC brain specimens to determine the strength of the relationship between these biochemical changes and cognitive status. We will also examine whether quantitative differences in the regional distribution of AB monomers, AB oligomers or glycogen synthetase kinase (GSK) 3a and 3B are associated with Tau phosphorylation or cleavage. Aim 3: On the assumption that synaptic dysfunction and degeneration underlies the cognitive impairment in AD, we will test the hypothesis that enhanced synaptic plasticity allows for normal cognition in the face of significant AD pathology.

DESCRIPTION (provided by applicant): Genome wide association (GWA) studies have linked the G6PC2 gene to variations in fasting blood glucose (FBG) and hemoglobin A1C levels in humans, parameters that are associated with both the risk of type 2 diabetes and cardiovascular-associated mortality. The overall objective of this application is to build on the results of these GWA studies by determining the function of G6PC2. Our preliminary data show that the human G6PC2 gene is selectively expressed in pancreatic islet beta cells and that G6PC2 hydrolyzes glucose-6-phosphate (G6P). Based on these data our first hypothesis is that the glucose-6-phosphatase activity of G6PC2 opposes the action of glucokinase (GCK), which catalyses the conversion of glucose to G6P. Glycolytic flux has been shown to determine the S {0.5} of glucose-stimulated insulin secretion (GSIS) and the existing paradigm in the islet field proposes that GCK alone is the beta cell glucose sensor. The significance of our observations is that they challenge this paradigm and suggest that G6PC2 is a fundamental inhibitory component of that sensor. Instead we propose that a GCK/G6PC2 futile cycle acts as the beta cell glucose sensor determining glycolytic flux and the S{0.5} of GSIS. Additional preliminary data show that the mouse G6pc2 gene is also selectively expressed in pancreatic islet beta cells and that G6pc2 also hydrolyzes G6P. This suggests that the use of G6pc2 knockout (KO) mice represents an innovative and appropriate tool to study the function of G6PC2. Deletion of the mouse G6pc2 gene results in reduced FBG levels, consistent with the human GW A study data. But in addition we have found that deletion of the G6pc2 gene also results in exercise intolerance, characterized by hypoglycemia and inappropriately high GSIS. Based on these data our second hypothesis is that the GCK/G6PC2 futile cycle is physiologically important for the attenuation of insulin secretion during exercise. Neural inputs to the islet are activated during exercise and the existing paradigm in the islet field proposes that these inputs inhibit insulin secretion by hyperpolarizing the beta cell and also directly inhibiting the exocytotic machinery. The significance of our observations is that they challenge this paradigm and suggest that G6PC2 is a fundamental component of the machinery through which GSIS is inhibited during exercise. As with the control of FBG and hemoglobin AIC, this topic is also clinically important because exercise induced hypoglycemia is a major problem in individuals with diabetes that limits the duration and hence the beneficial effects of exercise. The goal of this proposal is to test our two hypotheses. The application is divided into two matching Specific Aims. Aim I explores the function of G6pc2 at a molecular level whereas Aim 2 explores the physiological importance of the Gck/G6pc2 futile cycle for the attenuation of insulin secretion during exercise. PUBLIC HEALTH RELEVANCE: Impaired insulin secretion that results in elevated fasting blood glucose and hemoglobin A (1C) levels in humans is associated with increased risk for the development of type 2 diabetes and cardiovascular-associated mortality. In contrast, an inability to suppress insulin secretion results in exercise induced hypoglycemia, which is a major problem in individuals with diabetes. The experiments proposed in this application aim to elucidate the function of a protein called G6PC2 that we hypothesize plays a critical role in the control of fasting blood glucose and hemoglobin A (1C) levels as well as the termination of insulin secretion during exercise.

BIOSPECIMEN CORE - CORE D: ABSTRACT The Biospecimen Core (Core D) is responsible for all aspects of cerebrospinal fluid (CSF) and blood collection, storage, and analysis. The aims of the Biospecimen Core are: The specific aims of Biospecimen Core are: (1) Collect, catalog, and store CSF and blood specimens from participants in the BIOCARD study and from patients in the Johns Hopkins CSF Disorders Center (the latter to be used for exploratory analyses). CSF collection will be reinitiated biannually in the BIOCARD cohort, to add to the existing specimens. (2) Perform AlzBio3 assays for CSF A?1-42, total tau, and ptau-181 and an MSD assay for A?1-42 on all newly collected CSF specimens from BIOCARD participants and from well characterized patients in the Johns Hopkins CSF Disorders Center, and compare the validity of the two assays for A?1-42. (3) Examine the utility of new CSF assays as Alzheimer's disease (AD) biomarkers, using samples from patients in the Johns Hopkins CSF Disorders Center for discovery, and from participants in the BIOCARD cohort for validation. These assays will include: (a) a multiplexed assay for inflammatory markers, (b) a proteomics assay for synaptic markers and (c) a metabolomic assay for small molecules.

ABSTRACT - Clinical Core The Clinical Core is critical to the mission of the Duke/UNC Alzheimer?s Disease Research Center, given its role in recruiting, clinically characterizing, and following a diverse group of individuals that will provide participants, biomarker data and brain tissue to investigators working on Alzheimer?s disease (AD) at Duke and UNC. Although we support investigators pursuing any research on AD or Alzheimer?s disease-related dementias (AD+ADRD), the particular focus of the Core relates to the Center?s theme of identifying changes across the lifespan, including the onset of comorbidities and cellular and molecular changes associated with aging, that influence the development, progression, or experience of AD. A related goal is to assist national efforts to understand the increased prevalence of memory disorders in minority and rural populations. The Clinical Core will develop and maintain a prospectively followed cohort of 420 research participants, ages 45 to 80, supplemented with a one-time evaluation of 120 younger participants, allowing our center to investigate age- related drivers of AD (Aim 1); the cohort will have substantial rural and African-American enrollment, allowing us to address known disparities related to AD. The Longitudinal Cohort (n=420) will consist of 320 participants who are cognitively normal at entry and roughly equal in distribution between ages 45 to 80, and with at least 100 participants symptomatic at entry (MCI or dementia). This cohort will enable us to observe key transition periods related to AD risk, including menopause, onset of comorbidities, and the transition to MCI and dementia. This cohort design will also allow us to supply Duke and UNC investigators with symptomatic and normal participants from the outset of the study. The second, Young Cohort (n=120) will consist of participants ages 25-44 who will undergo a one-time evaluation and biomarker collection. The availability of this unique comparison group, which is supported almost entirely by institutional funding, will enable investigators to evaluate the degree to which novel biomarkers or signatures associated with AD in older cohorts are linked to other contributing factors, such as age or genetic risk. The Clinical Core will perform UDS-based medical, neurologic, psychiatric, and neuropsychological evaluations (Aim 2). In addition, working with the Biomarker Core, the Clinical Core will facilitate the collection of blood and cerebrospinal fluid specimens, brain MRI and PET scans, and motor-sensory assessments including retinal imaging to enable discovery of novel biomarkers (Aim 3). The Core will also develop and utilize novel and portable testing modalities. The Clinical Core, with the assistance of the Outreach, Recruitment, and Engagement Core (autopsy consent) and Neuropathology Core (brain recovery), will facilitate collection of brain tissue obtained at the time of death (Aim 4). The Core will interact with the Data Management and Statistics Core to ensure all datasets, biological samples and participants are accessible to qualified investigators (at Duke and UNC for investigator-initiated studies or more broadly) and to share data in a timely fashion with the National Alzheimer?s Coordinating Center (NACC).