Aurelio Galli - US grants

[unreadable] DESCRIPTION (provided by applicant): The long-term objective of this project is to develop an understanding of the acute modulation of dopamine (DA) transporter (DAT) function by amphetamine (AMPH) and other DAT substrates. DA signaling at CNS synapses regulates a variety of cognitive, emotional and behavioral functions. Abnormalities in the dopaminergic system have been implicated in a number of psychiatric and neurological disorders, including drug addiction, schizophrenia and Parkinson's disease. AMPH induces reverse transport of DA, thereby increasing extracellular DA levels and leading to its psychostimulant effects. Exciting new experiments suggest that AMPH has unique interactions with the DAT. Thus, in contrast to DA, AMPH stimulates CaMKII activity, causes DAT associations with the SNARE protein syntaxin 1A, and induces DAT channel-like activity that results in rapid (msec) bursts of DA efflux. This project will combine molecular and biochemical approaches with biophysical and real-time imaging methodologies to characterize AMPH regulation of DAT function both in heterologous and neuronal preparations. The key issues to resolve include how AMPH differs from DA in its ability to induce DAT-mediated reverse transport of DA and DAT channel-like activity and whether DAT N-terminal phosphorylation and subsequent protein associations are essential for these actions of AMPH. Our experimental plan links the AMPH-induced functional regulation of DAT to stimulation of cytoplasmic signals that regulate the association of DAT and other proteins, thereby modulating DA efflux. The proposed studies address the following Specific Aims: 1) To identify differences in the abilities of AMPH and other DAT substrates to stimulate cytoplasmic signals leading to DA efflux. 2) To identify elements in the N-terminus that regulate the action of AMPH and to assess the impact of phosphorylation on the ability of AMPH to induce DA efflux. 3) To determine whether kinase activity and phosphorylation of DAT N-terminal serines regulate the interaction between DAT and syntaxin 1A, thereby modulating AMPH-induced DA efflux. The goal is to generate an experimentally-based model advancing our understanding of acute AMPH regulation of the DAT and to discover novel pathways and molecules that may contribute to disrupted dopaminergic signaling in disease states such as addiction. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand how changes in insulin signaling regulate the actions of amphetamine (AMPH). Dopamine (DA) transporters (DATs), which largely control DA clearance, are targets for psychostimulants such as AMPH and cocaine. By acting on the DAT, AMPH attenuates DA clearance efficiency and induces reverse transport of DA. As a consequence, AMPH increases synaptic DA levels and enhances dopaminergic transmission, with profound effects on behavior. Exciting new experiments suggest that insulin, through phosphatidylinositol 3 kinase (PI3K) signaling, regulates DA clearance by fine-tuning DAT plasma membrane expression. Consistent with these data, in experimentally-induced diabetic rats, where insulin signaling is impaired, DA clearance measured by in vivo chronoamperometry is reduced, as is the ability of AMPH to cause DA efflux. This project will combine biochemistry, biophysics, imaging and in vivo chronoamperometry to elucidate the relationships between changes in PI3K signaling in brain and changes in AMPH-induced increases in extracellular DA. Therefore, the key issues to resolve include how PI3K signaling regulates AMPH-induced DA efflux and to determine, in vivo, whether perturbations in PI3K signaling caused by changes in food intake and disease states such as diabetes regulate the ability of AMPH to increase extracellular DA levels. Interestingly, repeated systemic AMPH, which parallels social intake in humans, overrode the ability of hypoinsulinemia to reduce the effect of acute AMPH. This phenomenon appears to be mediated by D2 receptors. Therefore, we will also evaluate how signaling pathways activated by repeated AMPH exposures (e.g. D2 receptor through ERK1/2 activation) restore the acute actions of AMPH. The proposed studies address the following Specific Aims: 1) To define how PI3K signaling regulates AMPH-induced DA efflux. 2) To demonstrate, in vivo, that hypoinsulinemia or insulin resistance induced by changes in diet, reduce DA clearance and AMPH-induced DA efflux and, ex vivo that these modifications are regulated by DAT trafficking. 3) To determine whether in hypoinsulinemic and insulin resistant rats, repeated administration of AMPH restores AMPH-induced DA efflux by stimulating D2 receptors and consequently ERK1/2. These studies will illuminate pathways that may contribute to the development of psychostimulant abuse. PUBLIC HEALTH RELEVANCE: Stimulant abuse and potentially other dopamine-related pathologies such as schizophrenia and motor disorders (e.g. Parkinson's disease), are a tremendous public health burden. The dopamine transporter, which regulates extracellular brain dopamine, is the major molecular target of several psychoactive drugs, including amphetamine and cocaine. This proposal will analyze how perturbations of insulin signaling induced by diet and diseases states, such as diabetes, regulate dopamine clearance and the ability of amphetamine to increase extracellular brain dopamine, which can lead to addiction. Defining how insulin signaling affects dopamine neurotransmission may help to explain the mechanistic basis of how food intake regulates dopamine neurotransmission and drug abuse.

[unreadable] DESCRIPTION (provided by applicant): The long-term objective of this project is to elucidate mechanisms supporting acute modulation of the norepinephrine (NE) transporter (NET) as established by coordinated trafficking and intrinsic activity modulation. NE signaling at PNS and CNS noradrenergic synapses regulates a variety of physiological functions including attention, motivation, vasoconstriction and heart rate. The NET dictates the magnitude and duration of NE signaling. For decades, the main focus on NET has been as a target for antidepressant and psychostimulant (e.g. amphetamine (AMPH), cocaine, and methylphenidate (Ritalin)) action. Our recent findings indicate that the stability of NET protein-protein associations, including interactions with syntaxin (SYN) 1A and protein phosphatase 2A (PP2A), dictate NET cell surface distribution and NET intrinsic activities, including transport rates, NET-gated currents and efflux potential. This renewal application unites two laboratories at the forefront of neurotransmitter transporter research, linking methods in biochemistry and molecular biology with real-time imaging and biophysical approaches, and includes studies on neuronal cell lines expressing NET and NET mutants as well as an evaluation of native noradrenergic neurons. We focus our efforts on three distinct, but coordinated, aspects of modulated NET behavior: the exocytosis of NET proteins, the endocytosis of NET proteins and the modulation of NET associated ion/substrate flux properties. Our specific aims are to: 1) Determine whether activity-dependent changes in NET surface density reflect increased insertion of cytoplasmic transporters or reduced transporter endocytosis (or both) and define the sites supporting SYN 1A/PP2Ac associations and their functional consequences with respect to NET localization and surface insertion; 2) Elucidate structural and signaling determinants of NET endocytic trafficking as triggered by GPCRs and AMPH; and 3) Determine relationships between NET/associated proteins/Ca2+ and AMPH-triggered NE efflux. Our overall goal then is to develop a more sophisticated understanding of the nature of acute NET regulation and to determine how the formation/dissolution of NET protein complexes promotes the coordination of NE clearance with NE release and clarifies intrinsic mechanisms by which psychostimulants impact NET mediated ion and NE flux.

DESCRIPTION (provided by applicant): Modern dietary practices are out of control: despite knowing better, we consume too many calories, too much fat, and too much sugar. Herein we propose an idea that may explain our lack of success in combating obesity and that promises to transform our approach to this problem. This hypothesis arises from the recognized importance of midbrain dopamine signaling in complex aspects of food intake AND the seminal observation that insulin directly regulates dopamine signaling and reward. We propose that intact insulin signaling in midbrain areas such as striatum supports dopaminergic signaling and normal reward for food, which is adaptive when calories are scarce. In our modern, energy-dense food environment, reward drives poor dietary decisions. Reward-driven over-consumption of obesogenic foods quickly leads to neuronal insulin resistance and impaired dopamine signaling in striatum. In this stage the hypodopaminergic reward deficiency syndrome is established, in which decreased dopamine tone results in increased intake of obesogenic foods to achieve a normal level of reward in the setting of decreased dopamine tone. Our overarching hypothesis is that reward for food triggers midbrain insulin resistance, which sustains increased food intake, maladaptive feeding and behaviors, and as a consequence, obesity. Identification of the molecular mechanisms by which insulin fine-tunes control of feeding in the hypothalamus and reward centers in midbrain and identification of the mechanisms by which dysregulation of this system develops in obesity will yield tremendous insight. To achieve this goal, we will use a rodent model of diet-induced obesity in which dramatic changes in feeding behaviors occur. In this model a) for the first time we will quantify detailed pathological alterations in midbrain and hypothalamic insulin action and midbrain DA signaling over time, b) we will define the molecular mechanisms involved in these alterations in midbrain and hypothalamus using an array of cutting-edge tools, and c) as the model is refined and regulatory nodes identified, rescue the pathological alterations, proving the therapeutic potential of this work, and defining specific brain regions involved in obesity pathogenesis. We will initiate these studies in vivo, and will then model in vivo findings in ex vivo preparations, thereby distilling individual aspects of feeding regulation, a complex process involving cognition and reward. Finally, genetic tools in mouse models will illuminate the roles of insulin and dopamine signaling in the development of obesity in specific neuronal populations (e.g. dopamine neurons). Investigating this link between insulin and dopaminergic behavior will lay the foundation for understanding possible shared mechanisms of obesity and dopamine-related co- morbidities; cognitive dysfunction, bipolar disorder, schizophrenia, and attention-deficit disorder.

DESCRIPTION (provided by applicant): Bariatric surgery is currently the only effective treatment for severe obesity, and the only effective cure for type II diabetes. Research on the mechanism of action of the different bariatric surgical procedures in humans and model systems including pigs, dogs, rats, and mice supports the hypothesis that the beneficial effects result from more than the restrictive or malabsorptive effects of the procedures on food intake. Indeed, data argue that neuroendocrine changes in gut-brain signaling resulting from the Roux-en-Y and gastric sleeve procedures alter satiety, hunger, food preferences, and glucose homeostasis prior to the achievement of significant weight loss. Understanding the cellular and molecular basis of these changes induced by bariatric surgery might lead to the development of pharmaceutical interventions, or improved surgical procedures for the treatment of obesity and diabetes. While several animal models can be used for research on the physiology of bariatric surgery, the mouse provides the best model for studies of cellular and molecular mechanisms because transgenesis can be used to alter individual genes, and to label specific cell types. We show results here demonstrating successful creation of murine bariatric surgery models at Vanderbilt, and the use of the models to identify the first gene that plays an essential role in th efficacy of RYGB for long term maintenance of significant weight loss. The unique hypothesis to be tested is that the efficacy of bariatric surgery results not solely from a collection of changesto Gl signaling, but rather that essential changes in both Gl signaling AND in the plasticity and responsiveness of CNS homeostatic and hedonic circuits act synergistically to restore glucose homeostasis, and create a new weight set point. In this interdisciplinary team grant application, we bring together leading experts in human and murine bariatric surgery, murine pathology, Gl anatomy and function, obesity and diabetes, and quantitative human genetics to jointly study surgical preparations from humans and mice in order to identify the genes and cell types mediating the efficacy of bariatric surgery.

DESCRIPTION (provided by applicant): Psychostimulant abuse and addiction is a crushing public health problem. Our laboratories have begun to decipher the functional effects of glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) stimulation on dopamine uptake, clearance, and trafficking of presynaptic dopamine transporters. GLP-1 is an incretin hormone and neuropeptide that is released in response to food intake. GLP-1 acts through both peripheral and central mechanisms to regulate energy homeostasis and the hedonic components of food intake. We and others have hypothesized that peptides that modulate feeding behavior may also regulate brain circuitry responsible for drug reward. In fact, we recently discovered that systemic administration of the GLP-1 long-lasting analogue exendin-4, which is already used clinically in the treatment of type 2 diabetes, reduces the rewarding effects of cocaine in mice. Within the brain, GLP-1Rs are expressed within the hypothalamus, ventral tegmental area, and nucleus accumbens, but are especially enriched in the lateral septum (LS). The LS is (re)emerging as a crucial brain region involved in the hedonic properties of psychostimulants. In the current application, we will first define cellular heterogeneity in GLP-1 receptor expression patterns within the LS and test the hypothesis that GLP-1Rs modulate dopamine neurotransmission and signaling within the LS (Aim 1). These studies will use modern molecular neuroanatomical, biochemical and electrochemical methods. Next, we will test the hypothesis that local GLP-1 receptor signaling within the LS mediates the therapeutic effects of systemic exendin-4 on cocaine reward (Aim 2). We will also examine cocaine- and GLP-1 receptor agonist-induced changes in cellular activation. Our studies will use both pharmacological and genetic approaches, taking advantage of a recently created GLP-1R conditional knockout mouse. These multidisciplinary studies will provide essential foundational knowledge of the role of the GLP-1 receptor in psychostimulant abuse. The commercial availability of several FDA-approved GLP-1 agonists for the treatment of diabetes offers readily translational opportunities to improve human outcomes in psychostimulant abuse.

DESCRIPTION (provided by applicant): The dopamine (DA) transporter (DAT) controls DA homeostasis and neurotransmission by the active reuptake of synaptically released DA. The DAT is the major molecular target responsible for the rewarding properties and the abuse potential of amphetamine (AMPH) and cocaine. AMPH acts as a DAT substrate, promoting the reversal of DA transport, thereby resulting in DA efflux via DAT. This efflux leads to increased extracellular DA levels, an event of importance for the psychomotor stimulant properties of AMPHs. The N-terminus of the DAT is a structural domain that is critical for AMPH to cause DA efflux. We have shown that DAT N-terminus phosphorylation at the five most distal Ser is required for AMPH-induced DA efflux, but does not regulate DA uptake. Furthermore, our preliminary studies suggest that the DAT N-terminus interacts electrostatically with phosphatidylinositol-4,5-bisphosphate PIP2 (a key phospholipid enriched at the inner leaflet of the plasma membrane), and that this interaction impairs DA efflux, but not uptake. Our mechanistic hypothesis is that upon phosphorylation, the DAT N-terminus uncouples from PIP2 to disengage from the membrane. Both of these events are required to elicit the DA efflux produced by AMPH. We propose to test our hypothesis and its implications for the behavioral effects of AMPH in vivo, through the following specific aims: 1) To determine the nature of the interaction between DAT N-terminus and PIP2, and describe it in a structural context provided by computational modeling; 2) To determine the role of N-terminus phosphorylation in regulating how DAT and PIP2 interact, and its effect on DAT function; 3) To determine the role of DAT/PIP2 interactions in AMPH-induced behaviors in Drosophila melanogaster. In this animal model, we have established that locomotion is a DAT-dependent behavior and is stimulated by AMPH. Deletion of Drosophila DAT (dDAT) in DA neurons of flies inhibits AMPH-induced locomotion, an effect that is restored by the expression of the human DAT (hDAT) in these dDAT-deficient DA neurons. Using this strategy, we will translate in vivo our molecular findings from specific aims 1 and 2. This will allow us to understand how hDAT N-terminus phosphorylation and associations with PIP2 determine AMPH-induced behaviors. The long-term goals of this research are to understand how AMPH-induced DA efflux and its associated behaviors are dictated by the interactions of hDAT N-terminus with PIP2, and/or by phosphorylation of the N- terminus. By specifically impairing DA efflux, but not uptake, we will determine the contribution of DA efflux in AMPH behaviors. The intent is to uncover novel targets for the treatment of AMPH abuse. From a broad perspective of transporter biology, we will reveal how a DAT structural domain (the N-terminus) via its interactions with the plasma membrane dictates different aspects of transport function.

DESCRIPTION (provided by applicant): The dopamine (DA) transporter (DAT) controls DA homeostasis and neurotransmission by the active reuptake of synaptically released DA. The DAT is the major molecular target responsible for the rewarding properties and the abuse potential of amphetamine (AMPH) and cocaine. AMPH acts as a DAT substrate, promoting the reversal of DA transport, thereby resulting in DA efflux via DAT. This efflux leads to increased extracellular DA levels, an event of importance for the psychomotor stimulant properties of AMPHs. The N-terminus of the DAT is a structural domain that is critical for AMPH to cause DA efflux. We demonstrated that DAT N-terminus phosphorylation at the five most distal Ser is required for AMPH-induced DA efflux. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein syntaxin1 (Stx1) interacts with the DAT N-terminus and this interaction supports the ability of AMPH to cause DA efflux. Stx1 is phosphorylated at Ser14 by casein kinase 2 (CK2)1, an event we hypothesize is promoted by AMPH and regulates DAT-Stx1 association. Finally, we discovered that phosphatidylinositol-4,5-bisphosphate (PIP2) (a cofactor we show interacts with Stx1) directly interacts with the DAT N-terminus, and here we hypothesize coordinates Stx1 phosphorylation, leading to DA efflux. Our mechanistic hypothesis is that AMPH-induced Stx1 phosphorylation mediated by CK2 leads to DAT N-terminus phosphorylation. These events are coordinated by DAT-PIP2 interaction and are required to transmit the actions of AMPH. We propose to test our hypothesis through the following specific aims: 1) To define how DAT-Stx1 association is coordinated under basal and AMPH conditions; and 2) To determine the role of Stx1 phosphorylation in AMPH-induced DA efflux. To test our molecular discoveries in vivo, we have developed a behavioral model in Drosophila melanogaster. In this system, we have established that locomotion is a DAT-regulated behavior, and is stimulated by AMPH. Deletion of Drosophila DAT (dDAT) in DA neurons of flies inhibits AMPH-induced locomotion, an effect that is restored by the expression of the human DAT (hDAT) in these dDAT-deficient DA neurons. Using this strategy, we will translate in vivo our molecular observations. We will elucidate how CK2-mediated Stx1 phosphorylation and Stx1 interaction with the DAT N-terminus determine AMPH behaviors. Thus, our last specific aim is: 3) To determine the role of DAT-Stx1 interactions in AMPH-induced behaviors. The long-term goal of this research is to understand how AMPH-induced DA efflux and its associated behaviors are dictated and coordinated by Stx1 phosphorylation. Supported by our preliminary data, we hypothesize that inhibiting CK2 function impairs specifically DA efflux, but not uptake. Thus, we will learn how to selectively manipulate different aspects of the DAT transport cycle and determine the contribution of DA efflux in AMPH behaviors. This will uncover a new druggable target (CK2) for the treatment of AMPH abuse.

Project Summary/Abstract Substance abuse (e.g. cocaine) evolved to hijack a system already in place to respond to natural, adaptive rewards such as calorie-dense foods. It should therefore come as little surprise that robust crosstalk between food and abused substances may be observed in neuronal responses and adaptations, circuits, and even hormonal regulators. Cocaine abuse and addiction is a crushing public health problem that medical approaches have resoundingly failed to address ? new ideas and targets are sorely needed. Our group discovered that a novel gut-based weight-loss surgery (biliary diversion) alters neuronal responses to cocaine, including behavioral measures of cocaine reward. Biliary diversion is capable of chronically elevating circulating bile acids through ligation of the common bile duct and anastomosis of the gallbladder to the ileum (GB-IL). In the control surgery, the gallbladder is anastomosed to the duodenum (GB-D), restoring normal bile flow. Bile acids act as steroid hormones with targets in the brain, including the G protein-coupled bile acid receptor 1 (TGR5) which is expressed in the nucleus accumbens (NAc). Biliary diversion was recently developed at Vanderbilt University to treat high fat diet-induced obesity in mice. GB-IL mice exhibited weight loss and reduced high fat food consumption as compared to GB-D animals. Notably, the weight loss and decreased caloric intake occurred only in animals fed a high fat diet and not in animals fed a regular chow diet. Thus, we hypothesized that this reduction in the intake of rewarding, calorically-dense food could stem, at least in part, from altered reward for palatable food (hedonic eating). Reward is a process regulated by mesolimbic dopamine (DA). Dysregulated mesolimbic DA circuitry has been linked to high fat, high calorie food consumption and, importantly, to cocaine abuse. Thus, understanding how to prevent or correct this dysregulation, is pivotal for developing treatments for cocaine abuse. This proposal stems from the observation that GB-IL, which reduces high fat intake and increases circulating bile acids, also reduces the reinforcing properties of cocaine and impairs the ability of cocaine to enhance released DA. Our overarching hypothesis is that GB-IL and central bile acid signaling regulate cocaine behaviors by impairing cocaine-induced changes in accumbal DA neurotransmission. This hypothesis will be tested within the two Specific Aims below: Aim 1: To determine the neuroadaptations induced by GB-IL and GB-D (control surgery) to accumbal DA homeostasis and to cocaine-induced increase in extracellular DA. Aim 2: To determine the role of GB-IL and central bile acids signaling in modulating cocaine behaviors.

Amphetamines (AMPHs) are psychostimulants commonly used for the treatment of neuropsychiatric disorders (e.g. attention deficit disorders). They are also abused, with devastating outcomes. The abuse potential of AMPHs has been associated with their ability to cause mobilization of cytoplasmic dopamine (DA), which leads to an increase in extracellular DA levels. This increase is mediated by the reversal of the DA transporter (DAT) function that causes non-vesicular DA release, herein defined as DA efflux. However, the molecular events underlying DA efflux and how these events translate to specific AMPH behaviors is not well understood and is the focus of this proposal. We have shown that the DAT N-terminus (NT) is a structural domain that upon phosphorylation supports AMPH-induced DA efflux, but does not regulate DA uptake. Also, our preliminary data indicate that this phosphorylation event regulates DA-associated behaviors. Previously, using a combination of biochemistry, electrophysiology, and atomistic molecular dynamics simulations, as well as behavioral assays, we have shown that the DAT NT contains structural elements (Lys) that interact with plasma membrane lipids, specifically, phosphatidylinositol (4,5)-bisphosphate (PIP2). Impairing the interaction of the DAT NT with PIP2, either pharmacologically or molecularly, inhibits both DA efflux and AMPH hyperlocomotion. This was the first demonstration that the interaction of a plasma membrane protein with PIP2 is essential for psychostimulant behaviors. It also raised the possibility, that this interaction is essential for AMPH to cause DAT NT phosphorylation. DA efflux also requires the NT to be present and highly dynamic, since either anchoring the DAT NT to the plasma membrane or deleting the NT impairs DA efflux, but not DA uptake. Our mechanistic hypothesis is that the interaction between the NT and PIP2 is pivotal for AMPH to cause NT phosphorylation. Upon phosphorylation, the DAT NT uncouples from PIP2 and disengages from the membrane, forming new interactions with a specific motif of intracellular loop 4 (IL4) as predicted by our preliminary data. These new interactions, facilitated by NT phosphorylation, are essential for AMPH actions. We propose to test this hypothesis through the following specific aims: 1) To determine the role of hDAT-plasma membrane interactions in regulating NT phosphorylation; 2) To determine how hDAT NT phosphorylation supports DA efflux and the involvement of IL4. Our molecular discoveries will be then translated in vivo using Drosophila melanogaster as an animal model in which we express the human DAT (hDAT) in DA neurons of flies lacking the Drosophila DAT (?humanized flies?). In this animal model, we developed the ability to study hDAT function in isolated brains, both biochemically and biophysically, and to determine whether molecular manipulations of hDAT impairing DA efflux, but not uptake, impair complex behaviors associated with AMPH, including reward/preference. Therefore, in specific aim 3) we will determine the requirement of hDAT IL4-PIP2 interactions for AMPH-induced behaviors and the role played by NT phosphorylation.