Blood-brain barrier regulates leptin transport in obesity

PI: Kastin AJ
Funding: NIDDK R01
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.

Neuroimmune modulation across the BBB: Regulation of IL15 trafficking by TNF

PI: Pan W

Funding: NINDS R01

The goals of the proposed study are to determine the mechanisms by which TNF, an important component of neuroinflammation, upregulates IL15 production in the endothelial cells of the blood-brain barrier (BBB), the transport of IL15 across the BBB, and the resulting pathophysiological implications for neuroinflammatory and autoimmune disorders. The proinflammatory cytokine TNF not only crosses the BBB to exert its multipotent effects, but it also modulates the endothelial signaling and permeation of other cytokines. IL15 and its receptors show specific and robust upregulation in cerebral microvessel endothelial cells challenged by TNF or lipopolysaccharide, shown by microarray, qPCR and immunohistochemistry in our preliminary results. We will address the overall hypothesis that IL15 potentiates the effects of TNF in the CNS with four aims. (1) Test the hypothesis that TNF upregulates IL15 transport. We will identify the roles of IL15 receptors in transporting IL15 by use of in-situ brain perfusion studies in receptor knockout and wildtype mice, determine the effects of TNF on the transcellular permeability of IL15 in primary cerebral microvessel endothelial cells cultured in the Transwell system, and determine the differential roles and interactions of IL15R1, IL2R2, and IL2R3 receptors in TNF-facilitated IL15 transcytosis by immunofluorescent trafficking studies. (2) Test the hypothesis that TNF stimulates protein synthesis and basolateral secretion of IL15 from the endothelial cells of the BBB. This will be achieved by protein translation and turnover assays, confocal analyses of time-dependent colocalization of IL15, its receptors, and intracellular organelles, and quantification by enzyme-linked immunosorbent assay. We will also test the mediating effect of prostaglandins in the TNF-induced increase of the basolateral secretion of IL15. (3) Test the hypothesis that an upregulated endothelial IL15 system enhances TNF-induced neuroinflammation, shown by fever, sickness behavior, reactive gliosis, and cerebral production of proinflammatory cytokines. Apart from studies on mice to test the effects of soluble IL15 receptors in blocking these effects, the inflammatory changes in cultured cerebral microvessel endothelial cells obtained from knockout and wildtype mice will also be identified. (4) Test the hypothesis that an upregulated IL15 system exacerbates experimental autoimmune encephalomyelitis (EAE), a disorder of neuroimmune modulation and a model for multiple sclerosis. The regulatory changes of IL15 permeation across the BBB of the EAE mice will be determined, and the effects of soluble receptors in reducing the symptoms and disease burden of EAE will be determined. The feasibility of the studies is supported by preliminary results, our past experience, and the literature. By completion of the proposed studies, we will acquire novel and essential understanding of IL15 and IL15 receptor trafficking in cerebral endothelial cells, and identify new therapeutic targets for intervention in neuroinflammation and CNS autoimmune diseases.

Transport of TNFα across the BBB
PI: Pan W

Funding: NINDS R01
The blood-brain and blood-spinal cord barriers (BBB/BSCB), lying between the CNS (brain and spinal cord) and its supplying capillary blood vessels, provide an immense interface for interaction and exchange of information between the CNS and the rest of the body. We have shown that the BSCB is not static or physically passive, but undergoes dynamic changes, to regulate the availability of the cytokine tumor necrosis factor a (TNFa) from blood to the CNS. In the proposed studies, we will test the hypothesis that TNFa, after being transported across the BBB/BSCB, can facilitate functional recovery after spinal cord injury (SCI). We will also characterize the mechanisms by which this specific transport can be regulated. To determine the functional implications of TNFe transport, we will first show that spinal cord uptake of radioactively labeled TNFe will increase specifically in a modified mouse model of thoracic hemisection, and that this increase will not coincide with passive disruption of the barrier (measured by increased paracellular permeability of albumin). We will then determine the effects of TNFa treatment by evaluating locomotor behavior, intraspinal conduction of evoked potentials, and histological evidence of long tract regeneration. We expect that small doses of TNFa will facilitate recovery whereas large doses will worsen the deficits resulting from SCI. To identify the roles of p55- and p75-receptors, we will study TNFa transport in the presence of receptor antibodies and in the receptor knockout mice. We predict that increased spinal cord uptake of TNFe after SCI will be dependent upon upregulation of the receptors, as determined by Western blot and quantitative PCR analysis. Since transcytosis of a cytokine across the BBB/BSCB is a complicated process involving not only the receptors but also other regulatory proteins, we will identify novel transport regulatory proteins by co-immunoprecipitation, comparative proteomics, transfection and transport assays. We predict that the transport process will be regulated by the receptors, vesicular and cytoskeletal proteins, and chaperons in different phosphorylation states. By completing the proposed study, we will better understand the dual roles of TNFa in SCI and in its transport process involving the regulatory proteins. These transporters could be novel drug targets and therefore provide promising therapeutic potential.