Abba J. Kastin - US grants

DESCRIPTION (provided by applicant): The interactions of leptin with the blood-brain barrier (BBB) play an important role in some forms of obesity. In this proposal, we will elucidate the novel aspects of how leptin regulates the permeation of some feeding-related peptides and cytokines across the BBB. (a) to test the hypothesis that leptin recruits receptors of selective satiety peptides and activates transport, we will examine how leptin increases the blood-to-brain permeation of urocortin, another potent satiety peptide. We will first determine whether receptors for both leptin and urocortin are involved in this process. We will then determine whether there is heterodimerization of these receptors by use of co-immunoprecipitation and ligand binding assays, after the receptors are expressed by co-transfection on mouse TM-BBB4 brain endothelial cells. Further, we will determine by immunofluorescent microscopy whether there is co-localization of these receptors on endocytotic vesicles after binding to leptin. (b) To test the hypothesis that leptin can enhance an existing transport system for a cytokine that also reduces feeding, we will examine how leptin upregulates the transport of tumor necrosis factor alpha (TNFalpha). We predict that leptin will crease TNFalpha influx by modulating phosphorylation of the p55-receptors, p75-receptors, and proteins related to TNFalpha transport, as shown by transport assays, immunoprecipitation, and Western blot. (c) To test the physiological relevance of leptin-mediated regulation of TNFalpha and urocortin transport, we will measure the transport of TNFalpha, urocortin, as well as leptin itself in mice that are fed normally and in mice that are food-deprived. We predict that leptin will increase urocortin and TNFalpha transport only in mice with free access to food, thereby providing additional satiety signals to the brain when leptin is already relatively high in the circulation. In contrast, we predict that in food-deprived mice, the transport systems for both leptin and TNFalpha will be down-regulated and that for urocortin will not be activated, as expected for anorectic agents under the severe condition in which no food is available. By completing the proposed studies, we will demonstrate the presence of novel protein-protein interactions at the BBB, the mechanisms involved in these interactions, and the additional functional role of the BBB in regulating feeding. This information will not only add to a better understanding of the mechanisms of BBB transport in general but also to the potential therapeutic use of ingestive peptides.

DESCRIPTION (provided by applicant): The interactions of leptin with the blood-brain barrier (BBB) play an important role in some forms of obesity. In this proposal, we will elucidate the novel aspects of how leptin regulates the permeation of some feeding-related peptides and cytokines across the BBB. (a) to test the hypothesis that leptin recruits receptors of selective satiety peptides and activates transport, we will examine how leptin increases the blood-to-brain permeation of urocortin, another potent satiety peptide. We will first determine whether receptors for both leptin and urocortin are involved in this process. We will then determine whether there is heterodimerization of these receptors by use of co-immunoprecipitation and ligand binding assays, after the receptors are expressed by co-transfection on mouse TM-BBB4 brain endothelial cells. Further, we will determine by immunofluorescent microscopy whether there is co-localization of these receptors on endocytotic vesicles after binding to leptin. (b) To test the hypothesis that leptin can enhance an existing transport system for a cytokine that also reduces feeding, we will examine how leptin upregulates the transport of tumor necrosis factor alpha (TNFalpha). We predict that leptin will crease TNFalpha influx by modulating phosphorylation of the p55-receptors, p75-receptors, and proteins related to TNFalpha transport, as shown by transport assays, immunoprecipitation, and Western blot. (c) To test the physiological relevance of leptin-mediated regulation of TNFalpha and urocortin transport, we will measure the transport of TNFalpha, urocortin, as well as leptin itself in mice that are fed normally and in mice that are food-deprived. We predict that leptin will increase urocortin and TNFalpha transport only in mice with free access to food, thereby providing additional satiety signals to the brain when leptin is already relatively high in the circulation. In contrast, we predict that in food-deprived mice, the transport systems for both leptin and TNFalpha will be down-regulated and that for urocortin will not be activated, as expected for anorectic agents under the severe condition in which no food is available. By completing the proposed studies, we will demonstrate the presence of novel protein-protein interactions at the BBB, the mechanisms involved in these interactions, and the additional functional role of the BBB in regulating feeding. This information will not only add to a better understanding of the mechanisms of BBB transport in general but also to the potential therapeutic use of ingestive peptides.

Abstract In the third grant cycle of [unreadable]leptin transport across the BBB[unreadable], we will focus on the role of leptin receptor (ObR)- positive astrocytes in relaying leptin from blood to the CNS by crossing the blood-brain barrier (BBB). We hypothesize that astrocytes not only regulate leptin transport as vital components of the BBB, but also modulate neuronal leptin signaling by enabling a more rapid onset and faster termination of leptin action in neurons. The cellular studies will use a co-culture model of primary cerebral microvascular endothelial cells (PBMEC) and astrocytes. We will determine the effects of astrocytes on the permeability and integrity of fluorescently or radioactively labeled leptin and the permeability markers dextran and albumin across the tight monolayer of PBMEC. The mouse studies will test the effects of obesity-induced increase of astrocytic ObR expression, inhibition of astrocyte metabolic activity, and specific deletion of ObR from astrocytes on leptin distribution and neuronal activation in response to leptin. In Aim 1, we will test the hypothesis that astrocytes thwart leptin transport across the PBMEC monolayer by slowing down permeation kinetics. The transcellular rather than paracellular pathways will be shown by morphological examination of the location of fluorescently labeled leptin, as well as the permeation kinetic studies to test the specificity of transport. The effect the level of astrocytic ObR expression will be tested. The transport of leptin will be compared with the distribution of co-administered paracellular permeability markers and immunocytochemistry of tight junction proteins. In Aim 2, we will test the hypothesis that astrocytes and astrocytic ObR participate in the degradation of leptin within the CNS, modulate the half-life of leptin in neurons, and influence neuronal leptin signaling by generation of soluble glial transmitters. Aim 3 will focus on regulatory changes in adult mice with diet-induced obesity or the Avy mutation, both of which have been shown to have regional specific increases of astrocytic ObR. By use of glial metabolic inhibitors and newly generated astrocyte-specific ObR knockout mice, we will show that these ObR(+) astrocytes play an essential role in the regulation of neuronal leptin signaling. The results will provide the first evidence of the functions of ObR(+) astrocytes in linking BBB transport to the CNS response to leptin. An understanding of the consequence of astrogliosis and upregulation of astrocytic ObR in obesity should enable the targeting of astrocytes to counteract the neuroendocrine dysregulation in obese subjects.

DESCRIPTION (provided by applicant): The proposed study aims to determine how astrocytic leptin receptors (ObR, or LR) affect obesity regulation. Obesity, like neural injury, results in regional astrogliosis. We observed that knockout of ObR in astrocytes leads to partial resistance of the mice to diet-induced obesity, a finding opposite to the obesity found in mice with knockout of ObR in neurons. Moreover, mice with adult-onset obesity show both astrogliosis and robust upregulation of astrocytic ObR in selective nuclei of the hypothalamus. These ObR are functional, as leptin treatment activates multiple signaling pathways in primary astrocytes. We, therefore, hypothesize that the ObR (+) astrocytes provide negative regulation to facilitate obesity in two main ways: (a) reducing availability of leptin to neurons by accelerating leptin turnover; (b) modulating neuronal function by alterations of the profile of glial transmitters released. Such a role for astrocytes provides a direct mechanism linking neuroinflammatory processes and body weight control. We will test the hypotheses in three specific aims. (1) To show that reactive astrocytes facilitate the turnover of leptin and attenuate neuronal leptin signaling in the brain, Aim 1 will use an established model of reactive astrogliosis - adult-onset obesity - to determine the distribution of fluorescently conjugated leptin and pSTAT3 signaling after intracerebroventricular delivery of leptin. The results will be compared with those from astrocyte specific leptin receptor knockout mice, and after pretreatment with the astrocyte inhibitor fluorocitrate. (2) In Aim 2, we will test the hypothesis that leptin modulates the production profile of gliotransmitters, including glutamate and ATP in the acute phase and cytokines at a later time. Primary astrocytes and neurons will be used for calcium imaging, and the effects of astrocyte conditioned medium and mixed neuron-glial culture will be tested. (3) Aim 3 will identify functional consequences of astrocyte specific leptin receptor knockout on the metabolic phenotype and correlate this with serum biomarkers of neuroinflammation. Overall, the results will demonstrate an essential role of ObR(+) astrocytes in the regulation of neuronal leptin signaling. As astrocytes are crucially involved in neural injury and an integral part of the neuroimmune axis, these studies will provide tangible mechanisms by which neuroinflammation can regulate body weight.