Tamas L. Horvath - US grants

9728581 Horvath All animals studied to date possess endogenous biological clocks that mediate biological rhythms. In rats and other mammals, pulsatile hormone release from the anterior pituitary is essential for normal growth, metabolism, and reproduction. The interaction between visual, circadian and neuroendocrine systems underlies rhythmic anterior pituitary hormone secretions, including prolactin and luteinizing hormone (LH). The suprachiasmatic nucleus (SCN) is the critical brain structure underlying biological rhythms and a monosynaptic pathway between this hypothalamic nuclei and the principle neuronal populations regulating LH and prolactin release has been documented. Recently, it has become clear that pure visual signals may directly or indirectly via the intergeniculate leaflet of the lateral geniculate nucleus influence endocrine mechanisms and these pathways may be functioning in parallel with the biological clock that itself is under the influence of light. This indicates a more complex integration of visual signals into the neuroendocrine hypothalamus than previous suspected. Dr. Horvath, through a series of elegant neuroanatomical studies, will determine the morphological relationship between subcortical visual efferents and hypothalamic neurons responsible for the regulation of endocrine mechanisms. By using tract tracing methods in combination with degeneration and light and electron microscopic immunocytochemical approaches, and using biochemical assays, he will also assess the role of subcortical visual pathways in the regulation of pituitary gonadotrophs underlying ovulation. The delineation of the pathways via which visual and circadian signals are integrated into the neuroendocrine hypothalamus will enhance our understanding of how rhythmic anterior pituitary hormones secretions are regulated and this is critical for understanding reproduction and behavior.

The long-term goal of this research program is to examine the relationship between hormone levels, circadian function and behavior. The central aim of this proposal is to determine if hormones regulate the activity of the biological clock. In particular, we have focused on estrogen regulation of cells in the suprachiasmatic nucleus (SCN) and the intergeniculate leaflet (IGL) of the lateral geniculate body and their effects on circadian gonadotropin release. Our preliminary data revealed that subdivisions of the SCN and IGL of the lateral geniculate body are targets of gonadal steroid action during development and adulthood. We propose that disruption of these circuits by hormonal manipulation during development and/or adulthood interfere with the emergence and/or maintenance of the circadian positive gonadotropin feedback. To test this hypothesis we will address the following Specific Aims: 1) To reveal the interaction between gonadal steroid receptors and neuronal populations of the SCN and IGL. In this regard, a) determine if during perinatal development, neurons that express estrogen receptors contain estrogen synthetase, aromatase, b) reveal whether sexual dimorphism emerge in aromatase activity of the SCN and IGL; and c) in adult animals demonstrate whether estrogen- and/or progesterone receptor-containing neurons give rise to projections to hypothalamic regions where neuroendocrine cells are located. 2) To demonstrate whether the observed sex difference in the input of the neuroendocrine cells that arises from the biological clock is due to developmental effects of estradiol or is the consequence of the effects of gonadal steroids in adulthood. 3) To assess the effects of hormonal manipulations of the SCN and/or the IGL during adulthood on the gender specific gonadotropin secretion and the expression of GAD65 and GAD67 mRNAs in the SCN and IGL. The proposed experiments will elucidate a novel mechanism to support hormone-dependent endocrine mechanisms. Furthermore, our results revealing mechanisms via which hormonal signals can regulate components of the biological clock can provide a new insight into the etiology of discomforting symptoms of gonadal failure, including mood swings, sleep disorders and disturbances in thermoregulation (hot flushes), all of which are tightly coupled to the activity of the biological clock.

DESCRIPTION (provided by applicant): The critical importance of neuronal connectivity and its qualitative and quantitative aspects regarding neuronal activation have been recognized in the central regulation of energy balance. In non-human primates, and subsequently in transgenic mice, we revealed a rapid synaptic reorganization of the inputs of key hypothalamic peptidergic systems. The quantitative and qualitative reorganization of these synapses corresponded to the putative activity levels of the respective circuits in the face of changing metabolic state. The electrophysiological properties of these peptidergic systems closely reflected their qualitative and quantitative synaptic input organization and re-organization during various metabolic states. We conclude that the changing activity levels of hypothalamic peptidergic systems during changing metabolic states are determined, at least in part, by synaptic reorganization of their inputs triggered by the metabolic signal, leptin. Our central hypothesis is that leptin's effect on synaptic plasticity is mediated by the long form of leptin receptor (LRb) that activates the STAT-3 signaling pathway. We also predict that animals that develop diet-induced obesity exhibit altered synaptic organization of key peptidergic neuronal populations that will correspond to their altered electrophysiological characteristics. The following specific aims are proposed to test our hypotheses. Specific Aim 1: To reveal if (a) leptin-induced synaptic plasticity in the arcuate nucleus is impaired in mice with mutations in leptin signaling, (b) circulating leptin affects synaptic plasticity in a dose-dependent manner, and (c), acute leptin action on synaptic organization is long lasting. Specific Aim 2: To unmask molecular correlates of synaptic plasticity induced by leptin. Specific Aim 3: To determine if the synaptic organization and electrophysiological properties of the hypothalamic peptidergic neurons are altered in mice with diet-induced obesity. In all studies of Specific Aims 1-3, the parameters of synaptology and gene expression in each animal will be analyzed in conjunction with the animals' other metabolic characteristics, including feeding behavior, energy expenditure and blood hormonal and nutrient profiles. The understanding of the regulation and molecular mechanism of synaptic rearrangement caused by leptin will offer previously unsuspected targets for development of therapies against a variety of metabolic disorders, including obesity.

DESCRIPTION (applicant's abstract): We have identified the existence of mitochondrial uncoupling protein 2 (UCP2) in homeostatic circuits of healthy rodents and non-human primates. We also showed that ectopic expression of this uncoupling protein is induced in different models of neurodegeneration, including models of Parkinson's disease, hypoxia, epilepsy or trauma-induced brain injury. The expression of UCP2 in these experiments was associated with subpopulations of neurons and microglial cells at the site of the degenerative processes and predicted cells with the longest survival after the initial insult. In our preliminary studies, UCP2 overexpressing animals had diminished levels of free radical production in the brain and responded to transection of the entorhinal pathway with suppressed caspase 3 activation. We hypothesize that the induction of UCP2 in neurons and glial cells during pathological neurodegeneration is an attempt to protect and rescue injured neurons. Three Specific Aims are proposed to test this hypothesis: Specific Aim 1 To determine the role of the UCP2 gene product in intracellular calcium homeostasis and protection of cells in vitro by studying PC12 cells and primary cultures of retinal ganglion cells with and without UCP2 transfection and primary cultures of retinal ganglion cells taken from UCP2 transgenic, UCP2 knockout and wild type mice. The effects of oxygen and glucose deprivation and glutamate agonists will be assessed on cell death patterns and intracellular calcium metabolism in these cultures. Specific Aim 2 To determine the pattern of neurodegeneration, mitochondrial uncoupling activity, cytokine and ATP production in the brains of UCP2 knockout mice, UCP2 overexpressing transgenic mice and wild type mice undergoing hypoxia-, seizure- or 1-methyl-4-phenyl- 1,2,5,6 tetrahydropyridine (MPTP)-induced neurodegeneration. Specific Aim 3 To assess the effects on phenotype development of superoxide dismutase 2 knockout animals that are crossbred with either UCP2 knockout or UCP2 overexpressing mice. In these experiments, we will follow the phenotypic alterations by assessing neuronal loss, level of mitochondrial uncoupling activity, cytokine, free radical and ATP production and intracellular calcium levels using morphological, biochemical and molecular biological approaches. The results of the proposed studies will shed light on a novel mitochondrial mechanism that plays critical roles in the suppression of neurodegeneration regardless of the initial cause of disease. This will furnish one common target for the development of drugs against a variety of neurodegenerative pathologies, including those associated with hypoxia, epilepsy, Parkinson's, Alzheimer's and Huntington's Disease.

This is a revised competitive renewal application of our grant entitled "Ghrelin in Hypothalamic Regulation of Metabolism" (R01 DK-060711-04A1). The original application was planned for 5 years, but was cut to 2 years, 11 months, and the funds requested for each year were then further decreased due to budgetary constraints of NIDDK. Nevertheless, even with these funding and time constraints, we have made progress in the past two and a half years to better understand ghrelin's action within the brain. Of the newly acquired information, there are 2 critical findings which provide the basis for the request for continuation: 1) ghrelin induces synaptic remodeling of the melanocortin system while inducing positive energy balance;2) peripheral ghrelin administration activates ventral tegmental dopamine neurons elevating dopamine release in the nucleus accumbens, and, ghrelin administration selectively to the ventral tegmental area induces acute food intake. We make 2 inferences from these findings: 1) The action of ghrelin on the output of neuronal circuitry associated with metabolism regulation involves and partially relies on synaptic plasticity, and 2) ghrelin's effect on food intake is greatly augmented by direct activation of the basal forebrain reward circuitry. We propose the following Specific Aims to follow up on these premises: Specific Aim 1) To reveal a) whether circulating ghrelin affects synaptic plasticity in the arcuate nucleus in a dose-dependent manner, b) the time course of synaptic changes to emerge and to diminish after acute ghrelin administration, c) if the circadian fluctuation of the synaptic input organization of POMC neurons is ghrelin dependent, d) the interaction between ghrelin and leptin orcorticosterone (CORT) in the acute regulation of synaptic plasticity and feeding, and e) whether ghrelin-induced synaptic plasticity in the arcuate nucleus is impaired in mice with mutations in ghrelin signaling Specific Aim 2) To determine a) if ghrelin-induced synaptic plasticity also occurs in the major neuronal populations within the ventral tegmental area (VTA). In b) We will examine the role of GHS-R 1a in mediating synaptic changes in VTA neurons. Specific Aim 3) To determine that ghrelin targets the VTA to enhance motivational aspects of feeding and to promote the consumption of calorie rich diets, if the resistance of ghrelin knockout animals to diet-induced obesity is due to ghrelin's inaction upon reward circuitry. . The proposed studies will deliver new insights to metabolism regulation, likely revealing novel targets for obesity drug development.

DESCRIPTION (provided by applicant): This application is in direct response to a Request For Application by the National Institute of Aging (NIA), RFA-AG-03-003 "COMPARATIVE BIOLOGY: MECHANISMS OF AGING." We have discovered that subtle and sustained reduction of mitochondrial membrane potential by the means of increased proton transport through its inner membrane by uncoupling proteins (UCPs) diminish age associated declines in certain metabolic parameters and increase longevity in mice. We have collected preliminary evidence that baseline level of mitochondrial uncoupling is significantly elevated in the brains of longer-lived (30-40 years) non-human primates compared to that of rats and mice, and, that certain UCPs, for example UCP2, are more widespread in the brains of non-human primates than that of rodents and proptect agains neuronal degeneration. Together these observations gave impetus to our central hypothesis that differential level of mitochondrial uncoupling contributes to species differences in longevity. In our application we propose to provide further evidence for the role of mitochondrial uncoupling proteins in the aging process with particular emphasis on species differences. Specific Aim 1: We hypothesize that species with greater longevity have elevated basal mitochondrial uncoupling levels in different tissue types, including brain, heart, muscle, liver and kidney. Specific Aim 2: We propose that key mitochondrial enzymes controlling inner mitochondrial membrane potential are differentially expressed in species with longer versus shorter longevity. Specific Aim 3: We predict that the growth hormone-dependent changes in age related processes are directly associated with uncoupling proteins and changes in inner mitochondrial membrane potential. Our attempts to provide comparative analyses of age related molecular mechanisms in rodents and nonhuman primates, will provide novel therapeutic targets, UCPs, to increase longevity without compromising tissue function.

DESCRIPTION (provided by applicant): In adult, reproductively active females, there is a continued increase in prevalence and propagation of metabolic disorders, such as obesity and type II diabetes. Beyond the harmful effects of these disorders on the adult population, there is increasing awareness of the potential long lasting negative influence of metabolic disorders of pregnant mothers on their offspring. Indeed, arguments have been made that chronic disease development maybe dramatically accelerated in children who were nursed in utero and early post-natally by obese and/or diabetic mothers. The underlying molecular mechanism of this early programming remains ill- defined. The central nervous system, and the hypothalamic arcuate nucleus in particular, has emerged as one of the key sites from which both behavioral and endocrine aspects of metabolism are governed. Our preliminary observations revealed the disruption of certain genes in specific subpopulation of arcuate nucleus neurons underlie the emergence of altered metabolic phenotypes not dissimilar to those emerging in offspring of obese or diabetic mothers. Our central hypothesis stem from these investigations and propose that altered intracellular signaling in the arcuate nucleus melanocortin system triggered by insulin is a major component in the etiology of subsequent metabolic disturbances. We will further strengthen this hypothesis by investigations proposed here on wild type and genetically altered mice. We will address the following specific aims: Aim 1 Determine the cellular changes in AgRP and POMC neurons in the offspring of obese/diabetic mothers. Aim 2 Unveil the responses of AgRP and POMC neurons to feeding, fasting, leptin, insulin and glucose in the offspring of obese/diabetic mothers. Aim 3 Assess the effect of targeted mutations of insulin and leptin signaling in subpopulations of arcuate nucleus neurons on circuit development and metabolic phenotype of offspring derived from obese/diabetic mothers. The execution of the above aims will proved molecular explanations for the better understanding of the etiology of metabolic disorders in association with in utero environment of obese and diabetic mothers. PUBLIC HEALTH RELEVANCE: The proposed project will analyze the role of pregnancy on the development of obesity and diabetes of the offspring. This is a highly relevant area of medical research as the majority of women are overweight during pregnancy, which may be a main reason for the increasing obesity epidemic and the increased prevalence of type II diabetes.

Chronic hypoxia in children that survive premature birth leads to brain atrophy, presumably due to death of neurons and glia and altered proliferation and differenfiation of their progenitors. However, structural MRI studies also indicate that there is progressive recovery of cortical gray matter volume in these children as well as an overgrowth of gray matter in specific cortical regions. In addition, funcfional MRI studies also suggest intriguing changes in cortical representafion of language. Both results suggest that the developing brain can undergo extensive reorganization in cortical connectivity, possibly adaptive in nature. To investigate the mechanisms and the implicafions of these events, we used our hypoxic mouse model, which reproduces the initial brain atrophy and the subsequent recovery, and reared these mice in enriched environment. We will use a multidisciplinary approach to investigate whether cortical connections change after an eariy postnatal hypoxic insult and the contribution of adaptive changes in mitochondrial bioenergefics to these events. We identified and characterized mitochondrial uncoupling protein 2 (UCP2), as a crifical mitochondrial protein that enables adaptation and survival of neurons under cellular stress, including that triggered by eariy postnatal hypoxia. UCP2 promotes mitochondrial biogenesis, suppresses intracellular free radical levels, increases synaptogenesis and promotes survival of cell under hypoxia and ischemia. UCP2 was also found crifical for the metabolic adaptafion of neurons during environmental enrichment, whereby shifts in mitochondrial bioenergetics favored cellular survival and synaptic plasticity. Our central hypothesis is that improved mitochondrial bioenergetics regulated by UCP2 is a key determinant of adaptive changes in neuronal development and connectivity enhancing recovery after a hypoxic event. In this proposal. Specific Aim 1 will elucidate the profile of mitochondrial bioenergefics in the brain of pups after eariy postnatal hypoxic insult. Specific Aim 2 will determine whether neuronal connecfions change after eariy postnatal hypoxia, and how these connections are altered by rearing the mice in enriched environment. The execution of the above specific aims will provide new insights into the pathogenesis of neuronal abnormalifies triggered by developmental hypoxia.

DESCRIPTION Abstract: Guided by the notion that late onset chronic diseases are the consequence of prolonged overworking of various tissues that have genetic and epigenetic vulnerabilities, I assert that it is the cellular energy metabolism of the different tissues that determines their health and longevity and consequently that of the entire organism. Based on my previous and ongoing work, I hypothesize that a small set of neurons in the hypothalamus, which produce Agouti-related protein (AgRP), act as the master regulator of energy utilization by all tissues, and hence, these hypothalamic neurons determine healthy tissue function and longevity. We will selectively up- or down-regulate the activity of hypothalamic AgRP neurons and test the effect of these perturbations on normal physiology of peripheral tissues and that of the brain. I suggest that these changes in peripheral tissue function by altered AgRP neuronal functioning will have critical impact on higher brain functions as well, including learning and memory and the ability of the brain to withstand stress during neurodegeneration induced either by normal aging or by pathological processes, such as Alzheimer's and Parkinson's disease. This project is uniquely suited for the NDPA program because it is unconventional and represents an approach in biomedical research that is nonexistent. It is a high risk avenue, but, if successful, would have great benefits in that it could immediately lead to novel treatments for various chronic diseases. Public Health Relevance: Late onset chronic diseases, such as dementias, Alzheimer's and Parkinson's disease, diabetes, cardiovascular disorders and tissue malignancies, are the leading causes of morbidity and mortality in the U.S., creating the greatest emotional and financial burden on the individual and society. As the size of the aging population continues to grow, late onset chronic diseases are predicted to further dominate the attention of biomedicine and society at large. This pr

DESCRIPTION (provided by applicant): Over the past decade it has became evident that the central nervous system plays a major role in mediating the actions of certain peripheral tissue-derived hormones on the skeleton. A key finding resulting from work on our lab and our collaborative work with others was that the adipose hormone, leptin, selectively targets hypothalamic neuronal populations and alters bone mass and energy metabolism via the raphae serotoninergic system (Fernandez-Galaz et al., 2002 Yadav et al., 2009), However despite the fact that leptin acts in the hind brain, the hypothalamic melanocortin system remains a key downstream effector for bone homeostasis. In particular, we found that the hindbrain action of leptin results in alteration in melanocortin tone in a manner entirely consistent with the previously described action of leptin to induce bone loss (Yadav et al., 2009). The melanocortin system consists of two distinct populations of hypothalamic neurons. One population produces proopiomelanocortin (POMC) and its key derivate, 1-melanocyte stimulating hormone (1MSH), which is the agonist for the melanocortin 4 receptor (MC4R). Activation of MC4R receptors leads to satiety and increased sympathetic tone. It is thought that this increased sympathetic tone, in turn, suppresses osteoblast function and increases osteoclast activity resulting in bone loss. The other "arm" of the melanocortin system is the population of neurons that produce neuropeptide Y (NPY), the inhibitory neurotransmitter, GABA, and Agouti- related protein (AgRP). AgRP is an inverse agonist of the MC4R, and, the AgRP/NPY/GABA neurons and tonically inhibit POMC neuronal activity (Horvath et al., 1992a,b;Cowley et al., 2001) leading to the suppression of the POMC effect on many outputs including the sympathetic nervous system. While not all hormones that affect skeletal metabolims act by modulating the melanocortin system and sympathetic outflow to the skeleton, it is our hypothesis that the activity of the melanocortin system has a critical role in regulating skeletal homeostasis. Specifically, we hypothesize that increased NPY/AgRP tone, suppresses POMC neuronal activity, reduces skeletal sympathetic tone and thereby promotes bone anabolism. Based on preliminary data which demonstrate a detrimental effect of reactive oxygen species (ROS) on NPY/AgRP neuronal function but a permissive effect of ROS on POMC neuronal firing (Andrews et al., 2008), we also predict that increasing ROS exposure of the melanocortin system during aging is a critical contributor to aging-associated bone loss and the pathogenesis of osteoporosis. PUBLIC HEALTH RELEVANCE: This grant aims to provide a novel explanation for osteoporosis in natural aging suggesting new ways of interfering with this debilitating condition To understand the etiology of osteoporosis, a devastating impairment of the bone associated with aging, it is essential that we gain better insight into the integrative regulation of bone homeostasis. We and others have contributed to the emerging critical role of various brain regions in this process. Thus, the critical analyzes of specific brain neuronal circuits in these mechanisms is important and may lead to the development of better strategies to combat impaired bone functions.

DESCRIPTION Abstract: Guided by the notion that late onset chronic diseases are the consequence of prolonged overworking of various tissues that have genetic and epigenetic vulnerabilities, I assert that it is the cellular energy metabolism of the different tissues that determines their health and longevity and consequently that of the entire organism. Based on my previous and ongoing work, I hypothesize that a small set of neurons in the hypothalamus, which produce Agouti-related protein (AgRP), act as the master regulator of energy utilization by all tissues, and hence, these hypothalamic neurons determine healthy tissue function and longevity. We will selectively up- or down-regulate the activity of hypothalamic AgRP neurons and test the effect of these perturbations on normal physiology of peripheral tissues and that of the brain. I suggest that these changes in peripheral tissue function by altered AgRP neuronal functioning will have critical impact on higher brain functions as well, including learning and memory and the ability of the brain to withstand stress during neurodegeneration induced either by normal aging or by pathological processes, such as Alzheimer's and Parkinson's disease. This project is uniquely suited for the NDPA program because it is unconventional and represents an approach in biomedical research that is nonexistent. It is a high risk avenue, but, if successful, would have great benefits in that it could immediately lead to novel treatments for various chronic diseases.

Late onset chronic diseases associated with aging, including cardiovascular disorders, Alzheimer's and Parkinson's disease, diabetes and tissue malignancies, are the leading causes of morbidity and mortality creating the greatest emotional and financial burden on the individual and society. As the aging population continues to expand, late onset chronic diseases will further dominate the attention of biomedicine. We have been pursuing the role of the brain circuits and humoral factors in health and aging. We have identified the hypothalamus as an intersection point between CNS and peripheral tissue communications, defined serum proteins differentially expressed in young and old animals, and recognized cardiovascular health as a principal determinant of lifespan. We and others have established that hypothalamic neurons sense the changing peripheral milieu and also send out signals to control complex behaviors and organ system and peripheral tissue functions. Our preliminary data and the work of others identified the same hypothalamic circuits to control the aging process. For example, we found that neurons of the hypothalamus that control hunger have significant impact on survival and lifespan. We also identified that these neurons mediate the action of peripheral hormones implicated in the lifespan extending impact of calorie restriction. We hypothesize, that action of circulating geronic substances on age related central and peripheral processes are mediated, at least in part, by the hypothalamus. We will interrogate this question using mouse models in Specific Aim 1. We will analyze complex behaviors, cardiac, muscle, bone and immune functions in control and experimental animals, including animals with different age and genotype in a state of parabiosis. From a translational perspective, it is crucial that the presence and relevance of geronic peptides are confirmed in higher species such as primates, including humans. In an effort to address this issue, we have analyzed plasma from young and aged nonhuman primates. Proteomics analysis identified candidate molecules in primates that have not been identified in mice. In Specific Aim 2, we propose to utilize our newly established, high throughput in vitro system to assess the cellular effects of putative geronic targets identified in our screen using nonhuman primate fibroblasts. We will focus on intracellular events that are associated with aging cells. In Specific Aim 3, we will test primates to evaluate anti- and pro-geronic interventions on CNS and cardiovascular systems. We will analyze the effects of young and aged plasma treatment of animals, known anti- and pro-geronic peptides and those newly defined by in vivo analyses on cognitive brain functions of young, middle aged and old nonhuman primates. Overall, execution of our aims will deliver new insights regarding the mechanisms of action of geronic substances, new circulating geronic molecules and exploit robust test systems that will lend themselves for collaborative work with other projects and investigators and the advance of defined biology to clinically relevant application.

Calorie restriction and the peripherally-derived growth factor, FGF21, have been correlated with increased lifespan. The hypothalamus plays an important role in the adjustment of whole body metabolism in calorie restriction. We uncovered that cell-selective impairment of hypothalamic Agouti-related peptide (AgRP)- expressing neuronal circuitry, part of the hypothalamic melanocortin system, in ad libitum fed mice, results in accelerated aging phenotype of many tissues, including the immune system and bone. In addition, we found that males of mice strains with impaired AgRP neuronal circuitry have shorter mean life-span when ad libitum fed. Our central hypothesis is that the AgRP system functions as a flip-flop switch for of fuel utilization for all tissues, and, that proper adjustment in the functioning of this central pathway is key in the aging process. We will test our hypothesis through the following specific aims: Aim 1. To test the hypothesis that calorie restriction-induced changes in the activity and synaptic input organization of the melanocortin system is mimicked by FGF21. Aim 2. To unmask if hypothalamic AgRP neurons are critical for behavioral- and peripheral tissue adaptations to calorie restriction and FGF21. Aim 3. To determine the role of the AgRP neurons in the effect of calorie restriction and FGF21 on hippocampal and cortical circuit integrity. Studies of this project will offer conceptual interface with Projects 1-3 as well as cellular- and behavioral analyzes for studies proposed in those projects.

Calorie restriction has been show to extend lifespan. A key aspect of calorie restriction is a shift in systemic metabolism from carbohydrate to lipid metabolism. The hypothalamus is a crucial regulator of systemic metabolism and has been directly implicated in the aging process of mice. We uncovered that cell-selective impairment of hypothalamic Agouti-related peptide (AgRP)-expressing neuronal circuitry, part of the hypothalamic melanocortin system, in ad libitum fed mice, results in accelerated aging phenotype of many tissues, including the immune system and bone. We also found that males of mice strains with impaired AgRP neuronal circuitry have shorter mean lifespan when ad libitum fed. Taken together these results gave impetus to the central hypothesis of this proposal, which is that the AgRP system is key mediator in calorie restriction-induced extension of lifespan. We will test our hypothesis through the following specific aims: Specific Aim 1. To test the hypothesis that calorie restriction prevents declining functioning of hypothalamic AgRP neurons in chronological aging. In our preliminary studies we found that AgRP neurons manifest aging associated decline in mitochondrial integrity in ad libitum fed mice. We also observed that calorie restriction promotes AgRP mRNA expression and suppression of POMC mRNA levels. We showed that the input organization of the melanocortin system is shifted by calorie restriction to a constellation that enhances AgRP neuronal activity and suppresses POMC cells. We hypothesize that calorie restriction suppresses deterioration of AgRP neurons during chronological aging, and that this effect is mediated by intracellular pathways regulating mitochondrial dynamics and ROS generation. We will test this hypothesis by analyzing the effect of calorie restriction on AgRP neuronal activity, mitochondrial dynamics and ROS production in control and transgenic mice, in which specific processes of mitochondrial fission, fusion or ROS control is cell-selectively down or up-regulated. Specific Aim 2. To unmask if hypothalamic AgRP neurons are critical for lifespan promotion by calorie restriction. We will utilize multiple lines of transgenic animals in which AgRP neuronal function is selectively up- or down-regulated. Groups of animals will be maintained for lifespan assessment and whole body necropsy will be carried out at the time of death in each. In other control and experimental cohorts, we will analyze the effect of calorie restriction on systemic metabolism and behavior of mice. We will also assess pancreatic beta cell-, adipose-, liver, muscle and immune system parameters of control and experimental animals. The execution of these studies will shad new light on integrative physiology and molecular principles of calorie restriction-induced alterations in heath- and lifespan.

Over the past 20 years, the arcuate nucleus melanocortin system emerged as a crucial regulator of feeding and energy metabolism. A yin-yang relationship between orexigenic AgRP and anorexigenic POMC neurons has been considered a primum movens in control of whole body metabolism. Strikingly, however, we recently discovered that activation of POMC neurons by cannabinoid receptor 1 (CB1R) is associated with increased feeding, and, that POMC neuronal activation is crucial for cannabinoid-induced feeding. Our observations suggest that the orexigenic tone of POMC neurons rely on switching vesicular release of ?-MSH to ?-endorphin. Indeed, inhibition of opiate receptors in the brain diminished cannabinoid-induced feeding. We also found that CB1R activation-induced feeding is associated whit uncoupling protein 2 (UCP2)-dependent mitochondrial dynamics in the hypothalamus, of which selective impairment by knocking down UCP2 abolished CB1R-induced feeding. These observations gave impetus to the hypothesis that cell autonomous expression of CB1R and UCP2 in POMC neurons are critical for CB1R-induced feeding and that this behavioral response relies on POMC-released ?-endorphin. We also predict that POMC-driven feeding occurs in physiological circumstances. For example, elevated endogenous cannabinoid levels coincide with activation of POMC neurons after feeding initiation subsequent to a prolonged fast as well as in diet-induced obese mice. We hypothesize that an orexigenic POMC tone, utilizing the mechanisms described above, is an important element in rebound feeding after food deprivation and in high fat diet-induced obesity. We will test our hypotheses by utilization of unique transgenic mice lines in combination with state-of- the art neurobiological, cellular biological, biochemical and physiological approaches. With the execution of these studies, we will deliver an entirely novel aspect of feeding regulation by the POMC system with immediate implications to physiology and disease states of metabolism.

This is an administrative supplement request (PA-18-591) in response to NOT-AG-18-008 Disease focused (Alzheimer's and its related Dementias (AD/ADRD)-focused Administrative supplements for NIH grants that are not on Alzheimer's disease) to our ongoing NIH grant1R01AG052986-01A1 entitled ?In vivo and in vitro systems to validate geronic proteins and their mechanisms of action?. This project aims to deliver new insights into the possibility of rejuvenation by circulating substances using animal models. There are no Alzheimer's Disease-related studies proposed in this grant. circulating development that and of hippocampal response to NOT-AG-18-008, we request funds test the hypothesis that geronic peptides, substances with contrasting levels in young and aged animals, impact the onset of phenotype in a murine model of AD (directly relevant to Aim 1 of the parent grant). We recently published changes in brain oscillations (EEG) in this rat model of AD ( precedes brain pathology behavioral changes in rats carrying human mutations predictive of AD [1; see below for details] . The goal our proposed studies is to determine whether the geronic peptide, oxytocin, alters the onset of cortical and oscillatory abnormalities, histological changes and behaviors identified in In to TgF344-AD Rats) TgF344-AD rats.

This is an administrative supplement request (PA-18-591) in response to NOT-AG-18-008 Disease focused (Alzheimer's and its related Dementias (AD/ADRD)-focused Administrative supplements for NIH grants that are not on Alzheimer's disease) to our ongoing NIH grant1R01AG052986-01A1 entitled ?In vivo and in vitro systems to validate geronic proteins and their mechanisms of action?. This project aims to deliver new insights into the possibility of rejuvenation by circulating substances using animal models. There are no Alzheimer's Disease-related studies proposed in this grant. In cohorts grant. We AD/ADRD structurally pursue Macaque response to NOT-AG-18-008, we request funds to scan of aged and younger non-human primates currently studied at Yale with the support of the parent We have l ongitudinal cognitive testing data on these Rhesus Macaques going back to the late 1990s. suggest that these animals represent a unique resource that can enhance therapy development for because age, cognition and the proposed imaging can be used to establish a unique, functional and analogous model of AD/ADRD. If successful, these animals will provide a unique platform to mechanistic studies on AD/ADRD by the research community at large and validate the Rhesus as an NHP model in AD/ADRD research. We propose the application of 3 Positron Emission Tomography (PET) tracers to image the evolution of dementia-like brain biomarkers in aging nonhuman primates (NHP). Specifically, we will image the development of amyloid plaque with 11C-PIB and tau protein with 18F-MK6240; these 2 tracers have been in widespread clinical use. In addition, we will use the novel synaptic density marker (11C-UCB-J) for the synaptic vesicle glycoprotein 2A (SV2A), which is expressed ubiquitously in synaptic vesicles, and is thus a marker of synaptic density. This is an entirely unique combination of in vivo imaging biomarkers and can uniquely be performed at Yale. When combined with the unique nonhuman primate (NHP) population at Yale, it opens the possibility to follow the evolution of dementia biomarkers in a way not possible with human subjects.

The primary goal of the DRC Enrichment Program is to orchestrate a wide range of diabetes related activities within the Yale scientific community that promotes the open exchange of information and ideas among Yale DRC faculty, DRC trainees and students working in member labs as well as ?cutting edge? visiting scientists, thereby fostering interdisciplinary diabetes-related research collaborations and the training of the next generation of diabetes researchers. The Enrichment Program consists of 1) a weekly DRC-Endocrinology Seminar Series that is a collaboration of both the Sections of Adult and Pediatric Endocrinology and Metabolism; 2) diabetes- related Special Lectureships incorporated into the seminar programs or grand rounds of Internal Medicine, Comparative Medicine, Immunobiology, Cell Biology, OB-GYN and other basic science and clinical programs at Yale School of Medicine; 3) a yearly half-day DRC retreat to allow junior investigators to present their most recent research work, and 4) a Diabetes Research Day at the School of Medicine in which DRC supported scientists and a visiting scientist present their work. An important complementary aim of the program is through exposure to interdepartmental research activities and DRC cores to stimulate junior and senior faculty interest in diabetes- related scientific issues by investigators who are not currently engaged in the field. The explosion of knowledge in the basic biomedical sciences over the past two decades has created unparalleled opportunities for the advancement of diabetes treatment and prevention. To take maximal advantage of these opportunities, it is essential to attract the best and the brightest students, fellows, and junior faculty members to careers in diabetes research. The DRC has worked with major support from the CTSA-supported Yale Center for Clinical Investigation (YCCI) and T32 diabetes research training grants in medicine, pediatrics, and immunology, and other programs to accomplish this goal. In short, the mission of the DRC Enrichment Program is to create an educational infrastructure that will serve as a breeding ground for future academic leaders in diabetes.

Drug addiction has been considered a chronic disease and a risk factor for many other diseases and disorders. To better treat addiction and prevent future abuse of illicit drugs, it is essential to understand the mechanisms underlying addictive behaviors. Clinical and animal studies have established that the metabolic status contributes to the determination of reward threshold in humans and animals. Food restriction increases the sensitivity to drugs of abuse, while over-nutrition decreases the sensitivity to drugs. However, it is still elusive how the brain circuitry regulating the metabolic status interacts with the reward circuitry. The lateral hypothalamus (LH), a central hub integrating a wide range of inputs from various brain regions encoding metabolic, behavioral and environmental cues, is a critical brain area to regulate both energy homeostasis and food/drug reward. Specifically, a selective group of neurons exclusively synthesizing the neuropeptide hypocretin (Hcrt, also called orexin) affect food intake and play a prominent role in food award and drug addiction. It is not entirely clear what role the Hcrt system plays in the hierarchy of circuitry responsible for food reward and drug addiction. Recent studies by others and us indicate that the Hcrt system undergoes experience-dependent synaptic plasticity in animals exposed to cocaine, which leads to our overall hypothesis that the expression of experience-dependent synaptic plasticity in Hcrt cells contributes to the development of addictive behaviors in animals. If this is true, the ability to establish synaptic plasticity in Hcrt neurons may contribute to the susceptibility of animals to addictive behaviors. Based on our previous studies, we hypothesize that metabolic/energy status may determine the sensitivity to reward reinforcers through modulating activity and plasticity in Hcrt neurons in animals. In this application stemmed from an R21 grant, we will begin to address this hypothesis by determining whether either over- nutrition or chronic energy deficiency alters the ability of cocaine to trigger plasticity in the Hcrt system with molecular (Hcrt-IRES-Cre mice and DREADDs), cellular (electrophysiological and EM studies) and behavioral (cocaine conditioned place preference) approaches. Three specific aims are: 1) To determine whether over-nutrition causes adaptive changes in Hcrt neurons, which is required in the impairment of drug- seeking behaviors in animals. 2) To test whether chronic energy deficiency leads to adaptation in Hcrt neurons, which facilitates the expression of drug reward in animals. 3) To interrogate whether over-nutrition and energy deficiency-induced adaptations in Hcrt neurons lead to altered responses of target areas of the Hcrt system when animals exposed to cocaine. Our long-term goal is to bridge the knowledge gap in our current understanding of addiction and to bridge the gap between clinical studies and basic research on the role of the Hcrt system in addictive behaviors, an area of study that has not been well explored thus far.

This application responds to RFA-AG-20-013 entitled Worldwide aging-associated Approaches to Alzheimer's increase in life expectancy has produced a dramatic rise in the prevalence, and thus impact of diseases, including Alzheimer's disease (AD). There is no effective treatment to halt or slow ?Geroscience Disease?. AD, and progress made in the development of new therapies is disappointing since many new compounds, despite initial promise at the preclinical level, failed in clinical trials (Cummings et al., 2018). The biggest risk factor for AD is aging. Therefore, novel approaches detecting mechanistic link between aging and AD at the cellular level, that leads to prodromal neuronal dysfunctions associated with later cognitive impairment and dementia, core features of AD, are crucial for identifying therapeutic interventions that have potential to modify course of disease. Our precedes promoting relevant the pharmacological manifestation are Specific Specific recent published and preliminary results showed that dyssynchronous activity of neuronal ensembles onset of symptoms in rat and mouse models of AD. We also identified hypothalamic hunger- neurons expressing Agouti-related peptide (AgRP) as crucial determinant of systemic metabolism to calorie restriction, aging and higher brain functions. These observations collectively gave impetus to central hypothesis of this proposal, which is that suppression of aging by nutrient (calorie restriction) or (FGF21) interventions will delay t he onse of electrophysiological, behavioral and pathological of impairments in animal models of AD. We also hypothesize that hypothalamic AgRP neurons central to the beneficial effects of these interventions. To test these hypotheses, we propose the following Aims: Aim 1 t will test the hypothesis that calorie restriction and FGF21 treatment suppress subclinical and clinical symptom development and brain pathologies in a transgenic rat model (TgF344-AD) of AD expressing mutant human amyloid precursor protein (APPswe) and presenilin 1 (PS1?E9) genes. Specific Aim 2 will of pathologies interrogate the hypothesis that hypothalamic AgRP neurons are critical for mediating effects calorie restriction and FGF21 in modulation of subclinical and clinical symptoms development and brain in transgenic mouse models (5xFAD and Tg2576) of AD. To execute these Specific Aims we will utilize rat and mouse models of AD with nutrient and pharmacological interventions, the combination of in vivo electrophysiology, behavioral analyses, biochemistry and pathological evaluation of control and experimental animals. Our studies will directly and forcefully analyze the relationship between aging-related systemic and cellular processes using electrophysiological (EEG), behavioral, histological and biochemical methods to track symptoms development of AD in known animal models. The outcome of these studies will immediately suggest possible interventions to alter AD development in primates, including humans.

Obesity, referring to having an abnormally high proportion of body fat, has become a health and economic problem in wealthy countries such as the United States. Therefore, intensive investigations have been conducted to determine the factors leading to the positive energy balance between energy intake and energy expenditure. The hypothalamus plays a substantial role in the regulation of energy intake and expenditure. However, it is still not clear how various neuronal systems in the hypothalamus interact to maintain normal body weight and how the dys-regulated interactions may compromise the balance between energy intake and expenditure. In this application we propose to identify interactions between melanin-concentrating hormone- synthesizing (MCH) neurons and hypocretin (Hcrt) neurons in the lateral hypothalamus in the regulation of energy balance and body weight at molecular, cellular and whole animal levels. Both MCH and Hcrt systems are critical players in the regulation of energy balance in animals and humans. The MCH system promotes energy intake and decreases energy expenditure while the Hcrt system promotes both energy intake and expenditure. There has been morphological and functional evidence on the possible interactions between these two systems. However, it is still not entirely clear whether the interaction between these two systems is required in the regulation of energy balance in animals and humans and whether changes in the interaction may lead to altered responses of the brain to the energy status of organisms and compromised energy metabolism. Based on current data we hypothesize that a crosstalk between MCH and Hcrt systems is required to maintain normal body weight. Specifically, we propose that the MCH system requires activation from Hcrt cells to promote energy intake, while MCH neurons may provide a feedback pathway to limit the activity in the Hcrt system to curb energy expenditure and maintain the normal body weight in animals. Since neuronal plasticity plays a critical role in the formation and modification of homeostatic and behavioral functions in animals, it is very likely that the modulation of synaptic function in MCH and Hcrt neurons could be one of the mechanisms underlying the interaction between these two systems. Therefore, three goals of this application are: 1) To test the hypothesis that MCH-mediated food intake requires activation from the Hcrt system in animals.; 2) To interrogate the hypothesis that the MCH system is required to limit activity in the Hcrt system to curb energy expenditure; and 3) To examine whether the imbalance between the activities in MCH and Hcrt cells underlie dys-regulated metabolic status (DIO and anorexia) in animals. The answers to these questions will help address the crosstalk between homeostatic centers in the hypothalamus and the role of the crosstalk in the determination of energy metabolism. We hope that this project will benefit people suffering from diseases/conditions (such as obesity and anorexia) resulting from impaired regulation of energy metabolism.

We have identified a role for mitochondrial dynamics (fission and fusion) in central regulation of feeding, energy- and glucose metabolism. We showed that mitochondrial fission is important for proper promotion of feeding and body weight gain by hypothalamic AgRP neurons, while mitochondrial fusion is critical for hypothalamic POMC neurons to support satiety and related adjustment of systemic glucose metabolism. In our preliminary studies we also found that interference with mitochondrial dynamics selectively in adult adipocytes has a robust impact on systemic metabolism, in which knockdown of the mitochondrial fusion protein, mitofusin 2 (Mfn2), resulted in rapid weight gain and elevated feeding of mice with concomitant elevations in hypothalamic transcripts for AgRP. These observations indicate weight gain is supported both centrally and peripherally by mitochondrial fission, and, that mitochondrial dynamics in either of the hypothalamus or adipocytes reciprocally impacts mitochondrial function in these tissues to affect behavior and systemic energy and glucose metabolism. In support of this, we revealed in an in vitro system that elevated fatty acid levels, which are critical for weight gain do promote mitochondrial fission. We observed that different fatty acids species have different effects on mitochondrial dynamics, and that altering dietary fat composition alone results in elevated in food intake and body weight gain. Taken together our observations gave impetus to the central hypothesis of this grant proposal that mitochondrial fission is a key dietary-influenced mechanism both in the hypothalamus and adipocytes that regulates body weight and adiposity. We propose the following Specific Aims to test our hypothesis: Specific aim 1 will assess the role of mitochondrial dynamics in food intake and energy expenditure by assessing the effects of both altered mitochondrial fission and fusion in mature adipocytes and in central feeding circuitry neurons. In addition, aim 1 will explore the afferent signaling from adipocytes that impacts the function of feeding circuitry neurons. Specific Aim 2 will use both in vitro and in vivo approaches to establish the role of hypothalamic and adipocyte mitochondrial dynamics on dietary fat-influenced food intake.