Kynurenic acid is a nutritional cue that enables behavioral plasticity.

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 refeeding, kynurenic acid levels are eventually replenished, ending the elevated feeding period. Because 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.

Glucagon-like peptide 1 and glucose-dependent insulinotropic polypeptide stimulate release of substance P from TRPV1- and TRPA1-expressing sensory nerves.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are released from enteroendocrine cells (EECs) in response to nutrient ingestion and lower blood glucose levels by stimulation of insulin secretion and thus are defined as incretins. GLP-1 receptor (GLP-1R) expression has been identified on enteric neurons that include intrinsic afferent neurons, extrinsic spinal, and vagal sensory afferents but has not been shown to have an incretin effect through these nerves. GLP-1 and GIP enter the mesenteric lymphatic fluid (MLF) after a meal via the interstitial fluid (IF) from local tissue secretion and/or blood capillaries. We tested if MLF could induce diet-dependent intransient increases in intracellular calcium ([Ca2+]i) in cultured sensory neurons. Postprandial rat MLF, collected from the superior mesenteric lymphatic duct, induced a significant twofold higher intransient increase in [Ca2+]i in primary-cultured sensory neurons than MLF from fasted rats. Inhibition of transient receptor potential vanilloid 1 (TRPV1) and TRPV1 and ankyrin 1 cation channels (TRPA1) with ruthenium red eliminated the difference. Substance P (SP) (a peptide that stimulates insulin secretion) sensor cells cocultured with sensory neurons showed both the GLP-1R agonist exendin-4 (Ex-4) and GIP induced transient increases in [Ca2+]i directly coupled to SP secretion in the sensory nerves. Ex-4-induced release of SP required expression of either TRPA1 or TRPV1. These data identify unrecognized actions of GLP-1 and GIP as incretins by acting as neurolymphocrines and suggest a mechanism for sensory nerves to respond to the postprandial state through MLF.NEW & NOTEWORTHY 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.

In silico molecular comparisons of C. elegans and mammalian pharmacology identify distinct targets that regulate feeding.

Phenotypic screens can identify molecules that are at once penetrant and active on the integrated circuitry of a whole cell or organism. These advantages are offset by the need to identify the targets underlying the phenotypes. Additionally, logistical considerations limit screening for certain physiological and behavioral phenotypes to organisms such as zebrafish and C. elegans. This further raises the challenge of elucidating whether compound-target relationships found in model organisms are preserved in humans. To address these challenges we searched for compounds that affect feeding behavior in C. elegans and sought to identify their molecular mechanisms of action. Here, we applied predictive chemoinformatics to small molecules previously identified in a C. elegans phenotypic screen likely to be enriched for feeding regulatory compounds. Based on the predictions, 16 of these compounds were tested in vitro against 20 mammalian targets. Of these, nine were active, with affinities ranging from 9 nM to 10 µM. Four of these nine compounds were found to alter feeding. We then verified the in vitro findings in vivo through genetic knockdowns, the use of previously characterized compounds with high affinity for the four targets, and chemical genetic epistasis, which is the effect of combined chemical and genetic perturbations on a phenotype relative to that of each perturbation in isolation. Our findings reveal four previously unrecognized pathways that regulate feeding in C. elegans with strong parallels in mammals. Together, our study addresses three inherent challenges in phenotypic screening: the identification of the molecular targets from a phenotypic screen, the confirmation of the in vivo relevance of these targets, and the evolutionary conservation and relevance of these targets to their human orthologs.

A new trauma frontier: Exploratory pilot study of platelet transcriptomics in trauma patients.

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.

Physiologic and anatomic characterization of the brain surface glia barrier of Drosophila.

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. 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 many unique BBB physiologies are conserved across phyla and suggest new methods for modeling CNS physiology and disease.

Evolutionary conservation of vertebrate blood-brain barrier chemoprotective mechanisms in Drosophila.

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.

Novel models for studying the blood-brain and blood-eye barriers in Drosophila.

In 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 endothelial cells, only partially reconstitute the anatomy and physiology of a fully intact blood-brain barrier (BBB). To address these problems, we and others continue to develop in vivo assays for studying the complex physiologic function of central nervous system (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 provide detailed methods for examining BBB biology in the fly, including a Dm visual assay to screen for novel modulators of the BBB.