Eric P. Zorrilla - US grants

DESCRIPTION (provided by applicant): The prevalence of overweight and obesity in the United States is epidemic. Obesity and overweight are major public health problems, annually responsible for approximately 300,000 deaths and health costs of $117 billion nationally. Long-term administration of anorectics is one proven approach to weight control. To identify drug targets, a promising approach is to understand better the neurochemistry of feeding behavior. While it has long been known that peptides of the corticotropin-releasing factor (CRF) family reduce food intake when given centrally, their mechanisms of action remain poorly understood. CRF and urocortin were the first and second mammalian members of the CRF peptide family to be identified. Each of these peptides is non-selective, binding both mammalian CRF receptors with similar affinities. Because of the overlapping distribution of CRF receptors and the non-selectivity of available peptide ligands, the relative roles of CRF1 and CRF2_ receptors in the regulation of feeding have remained elusive. Brain CRF1 receptors mediate bodily stress-like responses. As such, CRFl-mediated anorexia may simply reflect non-specific feeding suppression secondary to a stress-like state. This concern limits the viability of the CRF1 receptor as a drug target for obesity. Several findings, however, have spurred interest in the role of the CRF2 receptor in appetite regulation. Unfortunately, selective ligands for the CRF2 receptor have not been available, precluding pharmacological characterization of its role in feeding behavior. Recently, two groups independently identified genes encoding mammalian CRF/Ucn-related peptides that are evolutionarily distinct from one another as well as from CRF and Ucn. They are the first known direct, highly selective CRF2 receptor agonists. Of them, murine Ucn III is the most selectively potent. Contemporaneously, the first potent, highly selective, and long acting CRF2 receptor antagonist -- astressin2-B -- has been developed. The present proposal uses these tools to probe the ingestive functions of brain CRF2 receptors. In addition, it is not clear whether the CRF2 receptor shares the adverse consequences of CRF1 receptor activation. The central sites of action and physiologic role for CRF2-mediated anorexia also remain unknown. The proposed studies will address these questions as well to understand better the role of the CRF2 receptor in feeding behavior.

[unreadable] DESCRIPTION (provided by applicant): Early nutritional environment is hypothesized to "program" adult risk for overeating, adiposity, and associated medical complication. However, distinguishing genetic, intrauterine, and postnatal effects has been hampered by the lack of appropriate models. The current application uses a new rodent model that separates "imprinting" effects of early postnatal life on appetite, from genomic and prenatal effects. In Specific Aim 1, behavioral and neurochemical mechanisms by which being reared by an obese dam results in life-long hyperphagia will be studied. Experiments will determine the ontogeny of hyperphagia; the behavioral basis of excess intake, with increased positive drive rather than diminished negative feedback hypothesized to underlies the overeating; and the mediating role of functional neuroadaptations in interrelated systems that subserve the "positive drive" control of feeding, namely neuropeptide Y1, mu/kappa opioid and cannabinoid type 1 receptor systems. The studies combine the following features: 1) highly innovative microstructure analysis of behavior, 2) powerful sibpair split-adoption research designs, 3) detailed dose-response analysis with novel, highly selective pharmacological ligands, 4) recently developed polygenic models of obesity risk, 5) instrumental measures of the reinforcing efficacy of food, and 6) well- matched, controlled diet exposure. In addition, the hormonal and proximate molecular brain mechanisms by which prenatal or postnatal nutritional factors developmentally program adult appetite neurocircuitry are not well understood. Therefore, Specific Aim 2 will determine the effects of postnatal nutrition on early ontogeny of 3 humoral factors - leptin, adiponectin, or corticosterone - hypothesized to program long-term effects of obese adoption on appetite. Laser capture microdissection of discrete hypothalamic nuclei will be combined with microarray analysis to identify genes that are differentially expressed between neonates in relation to postnatal deviations in these "programming" hormones. Microarray analysis also will be used to identify genes that are differentially expressed at birth in the arcuate nucleus in relation to differential genetic or intrauterine overnutrition risk for overeating and obesity in adulthood. The results may identify hormones or hypothalamic genes that program lifelong hyperphagia in developmentally overnourished animals as well as the behavioral and neurochemical mechanisms that proximately mediate the overeating in adulthood. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Type 2 urocortins, novel ligands for corticotropin-releasing factor type 2 (CRF2) systems, retain their central anorectic properties in rats fed an energy dense "cafeteria" diet composed of palatable high fat and refined carbohydrate foods. This finding is promising because developing insensitivity to anorectics is an obstacle to obesity treatment. The present application hypothesizes that brain corticotropin- releasing factor type 2 (CRF2) systems are downstream components of the endogenous counter-regulatory system that curbs body weight gain. Acute and chronic effects of intracranial infusion of selective CRF2 ligands will be studied in rat lines selectively bred for differential vulnerability to diet-induced obesity. Neuropharmacologic studies will be performed under different dietary conditions, some of which promote obesity, to determine whether the anorectic properties of type 2 urocortins are retained despite obesity risk, high-fat diet history or manifest obesity. Indirect calorimetry and pair-feeding studies will examine metabolic components of the body weight-altering effects of CRF2 ligands in relation to genotype and diet with brain- site specificity. Long-term effects of continuous, intra-parenchymal CRF2 ligand treatment on adiposity and glucose regulation will be compared in treatment-free states. The role of CRF receptors in the energy balance-related effects of central leptin and insulin infusion will be tested using subtype-specific antagonists. Innovative forms of microstructure analysis and progressive schedules of operant responding will be used to identify the behavioral construct through which type 2 urocortins control food intake. Alternative explanations, such as malaise, will be considered. The proposed studies would identify general and brain CRF receptor- related differences in feeding patterns and metabolism that are associated with obesity and diet history. Relevance: Effective obesity treatments are few in part because obese individuals become resistant to biological signals that otherwise help regulate body weight. Brain mechanisms that can still promote weight loss in obese individuals and those with access to palatable foods are of great public health interest. The present application uses new pharmacological tools to study the role of a recently identified neuropeptide system in the control of body weight, via changes in appetite and metabolism, findings which may identify a therapeutic target for the treatment of obesity and its related medical complications.

DESCRIPTION (provided by applicant): Galanin is a neuropeptide believed to promote food intake and fat deposition via a dietary fat-dependent positive feed-forward mechanism, a salient property given the spread of obesity with the rise of Western diets. However, the physiologic and therapeutic relevance of galanin systems for overeating and fat accrual has remained uncertain because the proper tools to define its endogenous role and mediating receptor subtype, behavioral mechanism and neuroanatomical sites of action were not available. The present application seizes upon a convergence of molecular, pharmacological and behavioral advances to determine how galanin systems increase food intake and promote adiposity when palatable diets are present. Aim 1 identifies the galanin receptor (GalR) subtype that mediates orexigenic and metabolic actions of galanin through complementary approaches: acute intracranial infusion of galanin in GalR deficient mice and acute intracranial infusion of recently available subtype-preferring agonists and antagonists. The physiologic role of endogenous galanin signaling in facilitating hyperphagia and obesity will be studied in mutant GalR knockout mice with long-term access to diets high or low in saturated fat. Aim 2 uses concentration-response analysis of intake/preference curves, microstructure and progressive ratio analyses, and experimental manipulation of post-oral feedback to identify the behavioral mechanism underlying galanin system modulation of intake. Aim 3 combines functional mapping approaches and local short hairpin RNA (shRNA) knockdown of GalR gene expression to identify, with molecular and neuroanatomical specificity, brain targets through which galanin has orexigenic action. Persistent, local knockdown of GalR expression also will test the physiologic influence of endogenous hypothalamic GalR systems on food intake, metabolism, and adiposity with both brain region- and molecular-specificity. Results from the project will help define the physiologic role and molecular, behavioral and neuroanatomical modes of action by which galanin promotes obesity in the presence of palatable, high-fat food. Targeted therapies against these activities could then potentially be devised. PUBLIC HEALTH RELEVANCE About 1 billion people worldwide are overweight or obese, cutting across age, race, ethnicity and gender, and these conditions increase mortality, morbidity, and economic costs. Galanin is a neuropeptide that promotes food intake and fat deposition in rodents and which is associated with obesity risk. In this proposal, we begin to identify the behavioral, anatomical, and receptor mechanisms of action by which galanin promotes food intake. The functional significance of galanin receptor signaling in obesity risk and overeating in the presence of palatable diets also will be determined. By increasing our understanding of this understudied molecular control of energy homeostasis, the results may lead to new preventative or therapeutic options for obesity and will increase our understanding of the neurobiology of appetite.

As ethanol dependence develops, intake transitions from controlled drinking to uncontrolled, compulsive, and excessive intake. Neuroadaptations that result from chronic, heavy ethanol use (binge-like drinking) are hypothesized to drive this progression, leading to the emergence of relapse-motivating negative affect upon cessation of intake. Ethanol intake thereby becomes compelled for its negative reinforcing properties -- to ward off or relieve affective withdrawal symptoms. Time course studies suggest that acute and late protracted abstinence are distinct stages of withdrawal that motivate drinking and whose neurobiological underpinnings remain unclear. Behavioral and neurochemical data implicate roles for dysregulated extrahypothalamic corticotropin-releasing factor (CRF) and neuropeptide Y (NPY) function in affective withdrawal symptoms. The overarching hypothesis of the present application is that chronic heavy ethanol use leads to increases in anxiogenic extrahypothalamic CRF! or Y2 signaling and decreases in anxiolytic Yi activity, each of which contributes to passive anxiety behaviors during protracted ethanol abstinence. SPECIFIC AIM 1 will determine whether a chronic history of self-administered binge drinking leads to increased anxiety-like behavior upon acute or protracted ethanol withdrawal. SPECIFIC AIM 2 will determine whether rats rendered ethanol dependent via chronic passive ethanol vapor exposure or via chronic voluntary binge drinking show similar abnormalities in expression of CRF, NPY or their cognate receptors in components of the central extended amygdala or lateral septum, brain regions that subserve anxiety-like behavior and ethanol reinforcement. Using LC-MS, immunoassay, and quantitative and functional autoradiographic techniques, studies seek to identify which neurochemical changes coincide with the resurgence of anxiety-like behavior observed from early (2 weeks) to late (6-12 weeks) protracted withdrawal. Finally, SPECIFIC AIM 3 microinjects selective CRF and NPY receptor ligands into discrete brain regions to determine the functional relevance of neurochemical changes seen in AIM 2 for the increased anxiety of protracted withdrawal.

Previous work in our laboratory as part of the present program project grant has contributed significantly to our understanding of the role of central nervous system corticotropin-releasing factor (CRF) and urocortin (Ucn) peptides and their receptors in behavioral responses to stressors and energy regulation. In the previous funding period, differential effects of CRF/Ucn systems and CRF1 and CRF2 receptors both in the extended amygdala and the periphery were shown for stress-like behavior and modulation of appetite. The purpose of the experiments outlined in the present proposal are to build on these results and test the overall hypothesis that CRF/Ucn-related peptides have functional roles in the central nervous system and periphery to modulate behavioral and physiological responses to stressors in the domain of excessive energy intake. A subhypothesis is that CRF/Ucn activation, via CRF1 receptors in the extended amygdala, mediates the withdrawal-like aversive responses that contribute to binge-like eating. A second subhypothesis is that excessive, binge-like intake drives dysregulation in peripheral CRF/Ucn systems that contribute to prediabetic syndromes and obesity risk. A third subhypothesis is that rats selectively bred for vulnerability to diet-induced obesity show dysregulation of their CRF/Ucn systems that resemble those elicited by environmental models of binge eating. To test these hypotheses, in Specific Aim 1, the functional significance of alterations in the extended amygdala CRF/Ucn system for the stress-like behavior of rats withdrawn from diet-induced bingeing will be explored. In Specific Aim 2, the functional significance of peripheral CRF/Ucn systems in the glucose regulation, whole-body metabolism and obesity risk of rats exposed to diet-induced bingeing will be explored. In Specific Aim 3, functional alterations in central or peripheral CRF/Ucn systems produced by binge eating will be explored in rats with differential genetic vulnerability to obesity. Results of the present series of studies will provide key information regarding the roles of CRF/Ucn-related peptides in stress-like responses associated with dysregulation of appetite and energy balance. As a result, the results may provide insight into the role of CRF/Ucn systems in several stress-related pathologies of energy homeostasis, including obesity, binge-eating disorder, and diabetes.

DESCRIPTION (provided by applicant): Data have accrued implicating interleukin-18 (IL-18), a cytokine discovered for its immune system-modulating properties, in the control of food intake and energy metabolism. We and others found that the IL-18 modulates energy homeostasis during adulthood in mice. Mice deficient in IL-18 (IL-18-/-) or its binding receptor subunit (IL-18Ra) develop maturity-onset obesity and insulin resistance. As young adults, before they become substantially overweight, IL-18-/- mice are fattier, show reduced energy expenditure, and overeat both low-fat and high-fat diets as compared to wildtype mice. Because heterozygote IL-18+/- mutant mice show an intermediate hyperphagic phenotype, the findings appear to be regulatory, rather than an artifact of non- specific developmental lesion effects. Converse to these loss-of-function studies, central administration of IL- 18 suppresses food intake and weight regain in hungry mice as potently as does the adipocyte hormone leptin and does so without producing the adverse malaise, fever or HPA-axis activation classically associated with proinflammatory cytokine-driven sickness anorexia. Human genetic studies have recently identified single nucleotide polymorphisms (SNPs) in the gene for IL-18 or its receptor associated with increased risk for obesity and metabolic syndrome disorders. IL-18 and its receptors are constitutively expressed in the hypothalamus, with levels increased by peripheral immune challenge. Therefore, the guiding hypothesis tested in the present application is that central IL-18 systems can control food intake and energy expenditure. Translationally, this function would make IL-18 a clinically relevant target for sickness syndrome/cachexia. Moreover, because IL-18 administration does not share sickness syndrome effects of the pro-inflammatory system, exogenous IL-18 may have translational relevance as an anti-obesity treatment. To test this hypothesis, the present interdisciplinary proposal combines complementary but distinct, expertise of two PI's to determine mechanistically how central IL-18-responsive systems modulate food intake, energy expenditure, and, ultimately, adiposity. Experiments in two Specific Aims combine global and Cre/lox-targeted genetic loss- of-function mouse models deficient in IL-18 or the IL-18R; classic molecular, pharmacological, and physiologic approaches; and innovative behavioral analyses. AIM 1 seeks to identify physiologic and receptor mechanisms for central IL-18 control of food intake and energy expenditure. The potency and IL-18R- dependence of central IL-18 reductions in food intake and increases in energy expenditure will be determined. Pair-feeding is used to determine the functional role of excess food intake in the obesity of IL-18 null mutants. Feeding microstructure analysis is used to explore whether IL-18 modulates meal size or post-meal satiety in controlling food intake. Finally, the influence of diet-induced obesity on molecula expression and functional sensitivity of the central IL-18 system is determined. AIM 2 seeks to identify central sites and molecular mechanisms of IL-18 action on energy homeostasis. Expt. 2.1 will yield key information regarding the cell types the express IL-18 and its cognate receptors within the hypothalamus under basal conditions and following peripheral immune challenge (lipopolysaccharide), histochemically differentiating neuronal from glial populations, modes of fast synaptic transmission, and neuroanatomical interactions with anorectic pathways implicated in cytokine anorexia. In Expts 2.2-2.3, Cre/lox targeting will be used to generate mice that lack IL- 18R subunits selectively in the paraventricular nucleus of the hypothalamus (PVN) and the ventromedial hypothalamic nucleus (VMH). Studies will involve phenotyping the offspring for energy balance parameters to allow comparison with results obtained in the global knockout and also to determine whether the site-specific deletion alters feeding or metabolic responses to central IL-18 administration. The studies provide a powerful test of the functional significance of IL-18/IL-18R systems in the PVN and VMH for the control of food intake and energy expenditure. Collectively, the results will provide seminal data regarding the sites and mechanisms of action by which IL-18 systems modulate food intake and energy expenditure. Such information will be key for beginning to understand the pathophysiologic and potential therapeutic relevance of central IL-18 signaling for disorders of energy homeostasis, such as obesity or cachexia.

Alcoholism is a chronic, relapsing disorder, characterized by withdrawal syndromes of negative emotional symptoms that putatively promote relapse via negative reinforcement mechanisms. The present application tests the overarching hypothesis that excitatory glutamatergic signaling in the amygdala is altered during ethanol withdrawal, partly due to actions of corticotropin-releasing factor systems and neuronal pentraxin 2, and thereby promote negative emotional symptoms and relapse risk during abstinence. To test this hypothesis, SPECIFIC AIM 1 will use site-specific administration of recently available, highly available classand subunit selective antagonists of ionotropic glutamate receptors to test their functional involvement in the increased anxiety-like behavior and ethanol self-administration of alcohol withdrawal. SPECIFIC AIM 2 will use glutamate system-restricted knockdown of CRF1 signaling to test the hypothesis that CRF1-driven increases in glutamatergic signaling originating from the BLA promotes anxiety-like behavior and ethanol self-administration during withdrawal from chronic intermittent ethanol exposure. Finally, SPECIFIC AIM 3 will use dominant negative Narp-mediated knockdown of Narp function and glutamate system-restricted overexpression of the Nptx2 gene to test the hypothesis that chronic intermittent ethanol-induced Narp, via increased trafficking of AMPAR to the post-synaptic density leads to increased anxiety-like behavior and ethanol self-administration behavior. The studies will involve close collaboration with the Roberto/Siggins, Parsons and Mandyam research components, benefit from the consultation of Dr. Catherine Rivier, and rely closely upon the Animal Models (Koob/George) and Viral Vector (Contet) Cores of the proposed TSRI-ARC.

Abstract Many people with obesity or binge eating show addiction-like behavioral changes that can be modeled in rodents with intermittent, extended access to palatable food. New neurobiological understanding of and therapeutic targets for compulsive eating are needed. PPARs are lipid-sensing transcription factors encoded by 3 genes (PPAR?, PPAR?, PPAR?/?) that were identified for their roles in peripheral regulation of fuel homeostasis. PPAR? and PPAR? also have received intense attention for their anti-addiction-like actions. Yet, the role of brain PPAR? receptors in the control of compulsive eating, which has overlapping striatal substrates with alcohol and substance use disorders, is entirely unknown. Here, we test the overarching hypothesis that brain peroxisome proliferator-activated receptors-delta subtype (PPAR?) inhibit addiction-like, aspects of eating. With a novel blood-brain barrier-penetrant selective PPAR? agonist (KD3010) and cre/lox tools to manipulate PPAR? function now available, the proposed studies address these gaps in the field and may yield new translational approaches and insight into the biology of brain PPAR? and their role in the control of compulsive eating and potentially other addiction phenotypes. Using an innovative, drug abuse-like mouse model, based on intermittent, extended access to highly palatable food, Aim 1 tests the roles of central vs. peripheral PPAR? receptors in compulsive-like food intake. Guided by preliminary data, Aim 2 test the functional role of PPAR? receptors in dopamine Drd2/Adora2a- vs Drd1- expressing medium spiny neurons. The resulting data and novel genetic and translationally-relevant pharmacological tools for this understudied PPAR? isotype will lay the groundwork for cell type- and anatomically-specific mechanistic studies and may lead to interventions for people affected by compulsive eating and potentially other forms of addiction.

ABSTRACT Alcohol use disorder (AUD) is a chronic, relapsing disorder that afflicts 29% of Americans in their lifetime1,2, is disabling2 and increases mortality3. New drug targets and neurobiological insight for AUD are needed. Compulsive drinking putatively involves a transition to dorsal (vs. ventral) striatal control over drinking and a relative underactivity of indirect pathway MSNs (iMSNs) that enable adaptive behavioral selection in contrast to overactive direct pathway MSNs (dMSNs) that drive drinking behaviors. Compulsive drinking also involves a shift to dorsal (caudate-putamen) from ventromedial (nucleus accumbens) striatal control of ethanol-related behavior. Guided by novel preliminary data, this multidisciplinary project tests the overarching hypothesis that decreasing dorsal striatal phosphodiesterase 10A (PDE10A) type 2 activity in indirect medium spiny neurons (MSN) reduces compulsive drinking. In 4 Specific Aims, we seek to fill molecular, circuitry, pharmacological, behavioral and human genetic gaps in our understanding of the role of PDE10A isoforms in activation of distinct striatal MSN pathways and compulsive drinking. Aim 1 seeks to identify translatable PDE10A inhibitors that reduce compulsive-like ethanol self-administration, with consideration of enzyme off-rate, lipophilic efficiency and neuroactivational effects on distinct MSN circuits. Aim 2 will intersect Adora2a-Cre rats with expression of a floxed, validiated PDE10A shRNA to knockdown dorsal iMSN PDE10A in order to determine this the role of caudate-putamen PDE10A in iMSNs in escalated and aversion-resistant self- administration. Aim 3 seeks to determine the causal role of the striatal-restricted, membrane-associated PDE10A2 isoform in compulsive-like ethanol intake. Finally, Aim 4 seeks PDE10A gene variants that associate with problematic alcohol use as well as their functional, expression, and psychiatric genetic correlates. The collective work of our assembled, multidisciplinary collaborative team will shed light on the neurobiological and genetic role of PDE10A isoforms in distinct striatal circuits and compulsive drinking behaviors as well as the potential impact of novel translatable PDE10A inhibitors to treat AUD.

ABSTRACT Loss of control (LOC) eating, the sense of being unable to control one?s intake, is a hallmark of binge eating and a defining, causal antecedent of binge-type eating disorders1,2. LOC eating also predicts anxiety disorders and depression and, as a core symptom of food addiction is seen in millions of overweight individuals8,12,17-21. Clinically, the severity of LOC eating predicts more eating disorder pathology and emotion dysregulation; impulsivity and impaired inhibitory control; and less weight loss post-bariatric surgery22-30. LOC predicts these measures independent from and more effectively than does the amount eaten28,31-41 and foretells weight gain and metabolic syndrome risk1,2,29,45-51. Recent psychometric finding also suggest that behavioral signs of LOC correlate more with eating disorder pathology and binge eating than do cognitive or affective LOC criteria43. We thus developed a novel model of LOC eating in which rats with intermittent, extended access to palatable food show hallmarks of behavioral LOC in human scales, including increased food-directed effort and intake despite negative consequences, In the present application we will develop the model further to obtain additional, translationally-relevant measures of behavioral LOC (Aim 1). We also will use a sensitive, specific AAV E-SARE to enable neuronal activity-dependent targeted recombination in active populations (TRAP) (Aim 2) in order to dissect a recently identified anterior insula neuronal ensemble implicated in LOC-like eating. The studies will inform the role of intermittent, extended access in the etiology and modeling of behavioral LOC for translational studies. They also will identify the LOC-associated ensemble?s location, molecular markers and causal role. Finally, by leveraging ensemble translatome results with UK Biobank gene association data, the project may identify novel therapeutic targets for LOC eating.