Pharmacologic remedy of many brain diseases is difficult because of the powerful drug exclusion properties of the blood-brain barrier (BBB). Chemical isolation of the vertebrate brain is achieved through the highly integrated, anatomically compact and functionally overlapping chemical isolation processes of the BBB. These include functions that need to be coordinated between tight diffusion junctions and unidirectionally acting xenobiotic transporters. Understanding of many of these processes has been hampered, because they are not well mimicked by ex vivo models of the BBB and have been experimentally difficult and expensive to disentangle in intact rodent models. Here we show that the Drosophila melanogaster (Dm) humoral/CNS barrier conserves the xenobiotic exclusion properties found in the vertebrate vascular endothelium. We characterize a fly ATP binding cassette (ABC) transporter, Mdr65, that functions similarly to mammalian xenobiotic BBB transporters and show that varying its levels solely in the Dm BBB changes the inherent sensitivity of the barrier to cytotoxic pharmaceuticals. Furthermore, we demonstrate orthologous function between Mdr65 and vertebrate ABC transporters by rescuing chemical protection of the Dm brain with human MDR1/Pgp. These data indicate that the ancient origins of CNS chemoprotection extend to both conserved molecular means and functionally analogous anatomic spaces that together promote CNS selective drug partition. Thus, Dm presents an experimentally tractable system for analyzing physiological properties of the BBB in an intact organism.

Summary The kynurenine pathway of tryptophan metabolism is involved in the pathogenesis of several brain diseases but its physiological functions remain unclear. We report that kynurenic acid, a metabolite in this pathway, functions as a regulator of food-dependent behavioral plasticity in C. elegans. The experience of fasting in C. elegans alters a variety of behaviors, including feeding rate, when food is encountered post-fast. Levels of neurally produced kynurenic acid are depleted by fasting leading to activation of NMDA-receptor-expressing interneurons and initiation of a neuropeptide-y like signaling axis that promotes elevated feeding through enhanced serotonin release when animals re-encounter food. Upon re-feeding, kynurenic acid levels are eventually replenished ending the elevated feeding period. Since tryptophan is an essential amino acid, these findings suggest that a physiological role of kynurenic acid is in directly linking metabolism to activity of NMDA and serotonergic circuits, which regulate a broad range of behaviors and physiologies.

Central nervous system (CNS) physiology requires special chemical, metabolic and cellular privileges for normal function, and blood brain barrier (BBB) structures are the anatomic and physiologic constructs that arbitrate communication between the brain and body. In the vertebrate BBB two primary cell types create CNS exclusion biology, a polarized vascular endothelium (VE) and a tightly associated single layer of astrocytic glia (AG). Examples of direct action by the BBB in CNS disease are constantly expanding, including key pathophysiologic roles in multiple sclerosis, stroke and cancer. In addition, its role as a pharmacologic treatment obstacle to the brain is long standing, thus molecular model systems that can parse BBB functions and understand the complex integration of sophisticated cellular anatomy and highly polarized chemical protection physiology are desperately needed. Compound barrier structures that use two primary cell types (i.e. functional bicellularity) are common to other humoral/CNS barrier structures. For example, invertebrates use two cell layers of glia, perineurial and subperineurial, to control chemical access to the brain, and analogous glial layers, fenestrated and pseudocartridge, to maintain the blood-eye barrier (BEB). In this article we summarize our current understanding of brain-barrier glial anatomy in Drosophila, demonstrate the power of live imaging as a screening methodology for identifying physiologic characteristics of BBB glia, and compare the physiologies of Drosophila barrier layers to the VE/AG interface of vertebrates. We conclude that the many unique BBB physiologies are conserved across phyla and suggest new methods for modeling CNS physiology and disease.

SummaryIn species as varied as humans and flies, humoral/central nervous system barrier structures are a major obstacle to the passive penetration of small molecules including endogenous compounds, environmental toxins and drugs. In vivo measurement of blood brain physiologic function in vertebrate animal models is difficult and current ex vivo models for more rapid experimentation using, for example, cultured brain epithelial cells, only partially reconstitute the anatomy and physiology of a fully intact BBB. To address these problems, we and others continue to develop in vivo assays for studying the complex physiologic function of CNS barriers using the fruit fly Drosophila melanogaster (Dm). These methods involve the introduction of small molecule reporters of BBB physiology into the fly humoral compartment by direct injection. Since these reporters must cross the Dm BBB in order to be visible in the eye, we can directly assess genetic or chemical modulators of BBB function by monitoring retinal fluorescence. This assay has the advantage of utilizing a physiologically intact BBB in a model organism that is economical and highly amenable to genetic manipulation. In combination with other approaches outlined here, such as brain dissection and behavioral assessment, one can produce a fuller picture of BBB biology and physiology. In this chapter, we will provide detailed methods for examining BBB biology in the fly, including a Dm visual assay to screen for novel modulators of the BBB.

This paper takes advantage of similarities between the C. elegans and human pharmacopeia to identify and validate pharmacological targets that regulate C. elegans feeding rates.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are secreted upon eating to lower blood sugar. GLP-1 and GIP were found to induce the secretion of substance P (SP) from cultured sensory nerves. SP enhances insulin secretion. Mesenteric lymphatic fluid (MLF) also stimulates sensory neurons in a diet-dependent manner. These studies identify new actions of GLP-1 and GIP as incretins and suggest a mechanism for sensory nerves to respond to diet through MLF.

BACKGROUND:The earliest measurable changes to postinjury platelet biology may be in the platelet transcriptome, as platelets are known to carry messenger ribonucleic acids (RNAs), and there is evidence in other inflammatory and infectious disease states of differential and alternative platelet RNA splicing in response to changing physiology. Thus, the aim of this exploratory pilot study was to examine the platelet transcriptome and platelet RNA splicing signatures in trauma patients compared with healthy donors. METHODS:Preresuscitation platelets purified from trauma patients (n = 9) and healthy donors (n = 5) were assayed using deep RNA sequencing. Differential gene expression analysis, weighted gene coexpression network analysis, and differential alternative splicing analyses were performed. In parallel samples, platelet function was measured with platelet aggregometry, and clot formation was measured with thromboelastography. RESULTS:Differential gene expression analysis identified 49 platelet RNAs to have differing abundance between trauma patients and healthy donors.Weighted gene coexpression network analysis identified coexpressed platelet RNAs that correlated with platelet aggregation. Differential alternative splicing analyses revealed 1,188 splicing events across 462 platelet RNAs that were highly statistically significant (false discovery rate <0.001) in trauma patients compared with healthy donors. Unsupervised principal component analysis of these platelet RNA splicing signatures segregated trauma patients in two main clusters separate from healthy controls. CONCLUSION: Our findings provide evidence of finetuning of the platelet transcriptome through differential alternative splicing of platelet RNA in trauma patients and that this finetuning may have relevance to downstream platelet signaling. Additional investigations of the trauma platelet transcriptome should be pursued to improve our understanding of the platelet functional responses to trauma on a molecular level. (

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