Paul A. Slesinger - US grants

The aberrant activity of K+ channels and subsequent changes in membrane excitability have been implicated in neurological diseases. G protein-gated inwardly rectifying K+ channels (GIRK) in particular are linked to seizures and neurodegeneration in mice. The long term objective of this research grant is to elucidate the molecular mechanisms underlying G protein activation of GIRK channels. GIRK channels, mostly likely in groups of four subunits (tetramers), are opened during stimulation of G protein-coupled neurotransmitter receptors. While there is general agreement that G protein-derived Gbetagamma subunits activate GIRK channels, and that both the N- and C- termini of GIRK channels bind Gbetagamma, little is known about the physical interaction between Gbetagamma subunits and GIRK channels that governs channel activity. A combination of electrophysiological, molecular genetic, and biochemical techniques will be employed to: (1) Define the functional interactions among the Gbetagamma binding domains in the context of a native GIRK channel. The optimal arrangement of N- and C- termini from GIRK channels will be determined biochemically. The critical number of GIRK subunits and the position of GIRK subunits which donate obligate N- and C-termini for Gbetagamma activation will be determined in genetically engineered GIRK multimers expressed in Xenopus oocytes. (2) Identify the consensus sequences in GIRK channels that are essential for Gbetagamma activation. Gbetagamma activation in genetically altered GIRK channels will be assessed in Xenopus oocytes by monitoring changes in channel activity in the presence of exogenous Gbetagamma subunits. Gbetagamma binding will be measured in affinity-tagged fusion GIRK proteins containing mutant sequences. Gbetagamma channel activity will be tested in the presence of peptide fragments corresponding to regions found to bind Gbetagamma. (3) Identify interacting pairs of amino acids on Gbetagamma subunits and GIRK channels. Potential pairs of interacting amino acids in Gbetagamma and GIRK channels will be identified through biochemical crosslinking techniques. Functional crosslinking of Gbetagamma to GIRK during receptor activation will also be explored. Delineating the signal transduction mechanisms involved in the G-protein activation of these channels may bear directly on design of drug therapies for diseases due to aberrant membrane excitability.

DESCRIPTION (provided by applicant): Acetylcholine, a neurotransmitter implicated in memory and Alzheimer's disease, binds to and activates ligand-gated ion channels, G protein Gq-coupled receptors, and G protein Gi-coupled receptors. Hence, a single neurotransmitter can access a multitude of signaling pathways in neurons. Because activation of each of these pathways could result in a different biological response, neurons must functionally segregate these different signaling pathways. The molecular mechanism by which neurons isolate signaling pathways remains an enigma and is one of the most important questions facing Neuroscientists today. The study of G protein-gated inwardly rectifying K+ channels (GIRK) is an excellent system for identifying the molecular determinants of receptor specificity. GIRK channels are opened by the G protein G beta gamma subunits but show little discrimination among the different combinations of G beta and Ggamma, subunits in vitro. Because specific combinations of G beta gamma subunits do not selectively open GIRK channels, any G protein-coupled neurotransmitter receptor could theoretically open GIRK channels. However, neurons ensure that acetylcholine will open GIRK channels following stimulation of G protein Gi-coupled but not Gq-coupled cholinergic receptors. This type of receptor specificity is common among many different types of neurotransmitter signaling pathways. In this proposal, the hypothesis that a protein-protein interaction between the GIRK channel and a subset of G proteins determines the specificity of receptor coupling will be examined. The hypothesis will be tested using biochemistry for examining direct protein-protein binding interactions, electrophysiology for assessing the physiology of GIRK channels, laser confocal microscopy for visualizing the localization of GIRK channels and neurotransmitter receptors, and finally fluorescence spectroscopy for measuring protein-protein interactions in living cells. In addition, we will be using cultures of hippocampal neurons to identify the domains in GIRK channels that are important for targeting to the dendritic shafts. Results from this grant will significantly advance our understanding of the principles governing coupling of G protein-coupled receptors to GIRK channels, and may contribute to specific pharmaceutical strategies for treating humans with diseases that are caused by abnormal neuronal membrane excitability.

We propose to study the acute effects of the increasingly popular designer drug gamma-hydroxy butyric acid (GHB) on neurons in the ventral tegmental area (mesolimbic dopamine system). The actions of GHB are mediated through G protein-coupled receptors (GPCRs)of the GABAB type, activating downstream effectors, such as G protein-gated inwardly rectifying potassium (Kir3 or GIRK) channels. We hypothesize that specific combinations of GIRK channels play a primary role in mediating acute rewarding effects of GABAg receptor agonists on mesolimbic dopamine system. We will (1) elucidate the role of regulator of G-protein signaling (RGS) proteins in modulating the coupling efficiency of GABAB receptors, (2) asses the role of GIRK channel subunits (GIRK1, GIRK2, and GIRK3) on setting the coupling efficiency of GABAB receptors in dopamine and GABA neurons of the VTA, and (3) determine single-channel properties and rules of assembly of heteromeric GIRK channels in the VTA. We will use a two-pronged approach [unreadable] a systems level approach involving patch-clamp recordings and two-photon imaging in acute brain slices from wild-type, GIRK- deficient, and transgenic mice;and a cellular approach, involving patch-clamp recordings and advanced imaging techniques from GIRK channels expressed in heterologous cells and in cultured neurons of the VTA. Together, these studies will reveal the cellular and molecular events underlying GIRK channel assembly and coupling to GABAs receptors in the brain, as well as elucidate the role of GIRK channels in determining the efficacy of GHB and related drugs in the brain. Public health relevance. Drug abuse is a USand world-wide problem affecting millions of people. Drugs of abuse impart strong sensations of reward, which may lead to repetitive drug administration, dependence and addiction. Addiction is characterized by relapse in response to uncontrollable cravings. The initial rewarding effects as well as the cravings are associated with the activation of the mesolimbic dopamine system. The abuse of GHB has increased dramatically over the last few years. By unraveling the role of GIRK channels in mediating GHB actions, we may reveal targets for new drugs that either directly activate these channels or alter the coupling efficiency to GPCRs. Such an approach may establish GIRK channels as formidable drug targets for treating addiction.

DESCRIPTION (provided by applicant): Drug addiction is a major health problem in the U.S. Although there have been significant advances in our understanding the neurochemical changes in addiction, there is a need to develop new approaches and to identify novel targets for elucidating the molecular underpinnings of drug addiction. In this R21 proposal, this fundamental need will be addressed by integrating a mouse behavioral model for psychostimulant addiction with electrophysiological, molecular and cellular techniques to investigate the role of two novel proteins implicated in psychostimulant addiction, G protein-gated potassium (GIRK) channels and sorting nexin 27 (SNX27). GIRK channels have been implicated previously in drug efficacy in the mesolimbic dopamine pathway. SNX27 has been shown to associate directly with GIRK channels, leading to down-regulation of channels on the plasma membrane. Whether inhibition mediated by GABAB receptor activation of GIRK channels is reduced in dopamine neurons of the ventral tegmental area (VTA) from mice sensitized to methamphetamine remains an important unanswered question. Furthermore, it is unknown if a stimulant-induced increase in SNX27, which could be involved in the down-regulation of GIRK channels, occurs in the VTA of mice sensitized to methamphetamine. To address these, (1) GIRK channel currents in the dopamine neurons of the VTA will be measured in control and methamphetamine-sensitized mice, and (2) immunohistochemical studies will determine whether changes in protein levels occur. The potential interaction of SNX27 with GIRK channels in a model of psychostimulant addiction has the potential to enhance health-related research on drug addiction. Current drug treatments for addiction fail to address the underlying causes of addiction and current pharmacological treatments are lacking. Identifying key proteins and changes in neural circuitry in addiction could lead to the development of novel pharmacological therapies for treating addiction. The proposed work in this proposal will provide a direct molecular pharmaceutical target to treat the underlying cause, rather than the symptoms of addiction, directly benefiting human health. PUBLIC HEALTH RELEVANCE: Neuronal connections in the drug reward center of the brain change with chronic use of drugs, leading to addiction and dependence. The goal of this grant is to learn more about which proteins are changed by chronic use of methamphetamines and to also explore whether potassium channel signaling is altered. Results from these experiments may help identify novel therapeutic strategies for treating drug addiction.

DESCRIPTION (provided by applicant): Alcohol (ethanol) consumption alters neural activity in the brain by modulating different types of ion channels. An emerging concept in the field is that some of the physiological effects of ethanol are mediated by direct modulation of ion channels in the brain. One of the targets in the brain for ethanol is the G protein-gated inwardly rectifying potassium (GIRK) channel, which is activated by ethanol. Mice lacking GIRK2 channels exhibit diminished ethanol-induced tolerance to pain and self-administer more ethanol than wild-type mice. Moreover, a quantitative trait loci with a large effect on predisposition to sedative withdrawal, such as from ethanol, was narrowed to a region on chromosome 1 in mice that contains Girk3 gene. GIRK3 knockout mice exhibit less severe sedative-hypnotic withdrawal. Though GIRK2 and GIRK2/3 channels are implicated in ethanol-related behaviors, the molecular mechanism underlying this response is not well understood. Recently, we showed with high-resolution structural studies that alcohols bind directly to hydrophobic pockets of inwardly rectifying potassium channels. Mutations in the alcohol-binding pocket of GIRK2 channels significantly alter ethanol activation. We hypothesize that ethanol binds to hydrophobic pockets in GIRK2/3 channels and facilitate a conformational change that is relayed to the channel's gate and opens the channel. In this research proposal, we plan to use an innovative approach of high-resolution crystallographic studies, structure-based mutagenesis and advanced electrophysiological recordings to investigate this hypothesis. Specifically, we will conduct a structure-function analysis of the ethanol-binding pocket in GIRK2/3 channels (1), solve high-resolution structures of GIRK channels complexed with ethanol to reveal conformational changes in the channel protein that occur with ethanol-dependent gating (2), and elaborate mechanistic models for ethanol-dependent activation of GIRK channels, utilizing single-channel recordings and chemical modification of cysteine-substituted channels (3). Completion of these proposed experiments will reveal the structural basis of ethanol modulation of GIRK channels, which will provide insights into the mechanism of ethanol-modulation of other types of ion channels. Understanding the molecular mechanism underlying ethanol modulation of ion channels could lead to development of novel pharmaceutical agents for treating alcohol-dependence, directly benefiting human health. PUBLIC HEALTH RELEVANCE: Ethanol, a major drug of addiction and abuse in the U.S., directly modulates brain ion channels, which control the excitability of brain neurons. The goal of this grant is to investigate the molecular and structural mechanisms underlying ethanol-dependent activation of neuronal potassium channels. Results from these studies could lead to the development of novel pharmaceutical agents that specifically modulate potassium channels or antagonize actions of ethanol.

DESCRIPTION (provided by applicant): Addiction represents a complex genetic psychiatric disorder that likely involves a number of different genes. Addiction to cocaine and other psychostimulants, which are among the most addictive of substances, is also the most heritable. Recently, the ability to reprogram fully differentiated tissues, such as skin, into human induced pluripotent stem cells (hiPSCs) has made it possible to study a multigenic psychiatric disease, such as drug addiction. Cell-based human models for addiction will be created by directly reprogramming skin fibroblasts from methamphetamine-addicted patients and controls into hiPSCs (RFA-DA-11-012). Differentiating these hiPSCs into dopaminergic (DA) neurons will enable the identification of specific genetic and functional changes associated with hiPSC neurons treated with psychostimulants. There are three primary Aims for this R21 proposal. In Aim 1, fibroblasts from 'control'humans will be reprogrammed into hiPSCs and then differentiated into DA neurons. These DA neurons will be transcriptionally profiled in untreated, methamphetamine-treated and methamphetamine-withdrawal conditions to identify the gene pathways affected by methamphetamine treatment and withdrawal. In Aim 2, an electrophysiological characterization of DA neurons differentiated from control hiPSC lines will be completed, followed by studies examining the effect of exposure to methamphetamine treatment and withdrawal. In Aim 3, hiPSCs will be developed from humans addicted to psychostimulants and then examined for differences in properties of these patient-derived neurons and their response to methamphetamine treatment and withdrawal. The hypothesis that hiPSC DA neurons from patients addicted to methamphetamine respond uniquely to methamphetamine treatment in a manner distinct from controls will be tested. Preliminary data are provided that show reprogramming of adult cells into hiPSCs, differentiation of hiPSCs into DA neurons, and basic electrophysiological properties. Studies of patient-derived hiPSC neurons have the potential to significantly advance our understanding of a complex multigenic disorder like drug addiction. The proposed studies have the potential to reveal unique functional differences in DA neurons derived from addicted and control patients, as well as their response to methamphetamine. Results from the proposed studies will likely lead to both the development of improved animal models of addiction and provide an innovative approach for examining the effect of new drug therapies for the treatment of addiction. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to create human cell-based models for addiction by reprogramming skin samples from methamphetamine-addicted patients'human induced pluripotent stem (hiPS) cells. By differentiating these disease-specific hiPSCs into dopaminergic neurons, we will identify specific gene expression and electrophysiological changes associated with methamphetamine treatment and withdrawal in vitro. Ultimately, we hope that one day it will be possible to treat the disrupted molecular pathways in neurons that lead to addiction.

DESCRIPTION (provided by applicant): Addictive drugs usurp the brain's intrinsic mechanism for reward, leading to compulsive and destructive behaviors. While there have been significant advances in understanding the neurochemical and physiological changes in addiction, clinically effective treatments have eluded researchers. Remarkably, there are no FDA approved drugs for treating psychostimulant abuse. In the ventral tegmental area (VTA), the center of the brain's reward center, GABAergic inhibition controls the excitability of neurons. A primary component of the GABAergic inhibitory pathway is formed by GABAbeta receptors that couple to G protein-gated potassium (GIRK) channels. Studies show that an acute injection of psychostimulant in mice produces a robust depression of the GABAbeta receptor GIRK inhibitory pathway in VTA GABA neurons but not in DA neurons. This drug-evoked depression of GABAbetaR signaling in GABA neurons removes an intrinsic brake on GABA neuron firing that would result in enhanced GABA-mediated inhibition of DA neurons and potentially reducing reward perception. This acute effect of psychostimulants provides a unique opportunity learn how to control the VTA output through alterations in the levels of endogenously expressed GABAbeta-GIRK proteins in VTA GABA neurons. However, detailed pre-clinical studies are needed to better understand how adaptations in GABAbetaR-GIRK signaling in VTA GABA neurons affect the output of the VTA and motivated behavior. The main objectives of this project are to determine the role of phosphorylation of the receptor in this change of GABAbeta-GIRK signaling and assess whether down-regulation of GABAbeta receptor alters the addictive properties of psychostimulants. Specifically, we will (i) test the hypothesis that dephosphorylation of the receptor underlies the ability of psychostimulants to down regulate GABAbeta receptor activity in VTA GABA neurons, (ii) elucidate the structural and molecular interactions governing protein phosphatase 2A (PP2A) association with the GABAbeta receptor, and (iii) determine impact of GABAbeta-GIRK signaling in GABA neurons on VTA function and motivated behavior. The research team will use a comprehensive approach that combines molecular genetics, structural biology, biochemistry, physiology and behavior. The hypothesis that psychostimulants regulate synaptic inhibition by modulating GABAbeta receptor phosphorylation, a process that is dependent upon a fraction of PP2A directly associating with the receptor, is an innovative and untested model. The concept that the C terminal domain of the GABAbeta receptor serves as the epicenter for coordinating regulatory proteins for receptor function could provide the framework for understanding the molecular regulation of other GPCRs in the brain. Baclofen (Lioresal) is an agonist of the GABAbeta receptor and is being evaluated for treating alcoholism, addiction and autism. Selectively targeting the GABAergic inhibitory system as described here could lead to improved clinical use of baclofen and perhaps to new drugs for treating addiction and other neurological disorders.

Project Summary Neuropeptides are essential neuromodulators in the brain. They are released into the extrasynaptic space, where they diffuse over long distances and signal through G protein coupled neuropeptide receptors. Neuropeptides control cognition, sensorimotor processing, and energetics through changes in vascular tone and blood flow in the nervous system. Pharmacological and molecular genetic studies have implicated alterations in neuropeptide signaling as a contributor to brain dysfunctions, including migraines, addiction, motivation and stress. Although widely expressed in the brain, remarkably little is known about when and where neuropeptides are released. Monitoring the release of neuropeptides in real-time in awake animals performing complex behaviors would be transformative, enabling the elucidation of the function of neuropeptides in regulating neural circuits in the brain. In response to RFA-MH-16-775, we propose to develop and validate an innovative neurotechnique for optically measuring release of neuropeptides in a cell-specific and circuit-specific processes in the brain. The new technology is based on cell-based neurotransmitter fluorescent engineered reporters, referred to as CNiFERs, which were original developed for detecting the release of classical, small molecule neurotransmitters. A CNiFER is a clonal HEK293 cell that is engineered to express a specific G-protein coupled receptor and a genetically encoded fluorescence-based intracellular calcium sensor. CNiFERs are implanted in the brain, where they produce minimal inflammation and remain viable for days, and have been used successfully to measure volume transmission of dopamine, norepinephrine and acetylcholine in vivo during learning. Three neuropeptide CNiFERs will be developed and used for test-bed validation projects within our own laboratories: Orexin, which is important in sleep regulation as well as drug seeking and reinstatement, Somatostatin which has been implicated in depression, motivation and learning, and Vasoactive Intestinal Peptide, which as been implicated in neuroplasticity and learning. For collaborative projects, we will further construct four additional neuropeptide CNiFERs for detecting release of Dynorphin, Corticotropin-Releasing Factor, Neuropeptide Y and Substance P. Each neuropeptide CNiFER will be subjected to rigorous in vitro testing prior to their use to study the dynamics and consequences of release of neuropeptides in vivo.

PROJECT SUMMARY The inhibitory neurotransmitter GABA produces slow and prolonged inhibition in the brain through activation of metabotropic GABAB receptors (GABABR). Defects in GABABR signaling have been implicated in various neurological and mood disorders including spasticity, epilepsy, addiction and anxiety. GABABR is a member of the class C G protein-coupled receptor (GPCR) family, which typically functions as a dimer and possesses large extracellular domains. Fundamental questions remain concerning the molecular mechanisms underlying activation and modulation of these class C receptors. The GABABR is a heterodimer of GABAB1 and GABAB2 subunits and is modulated by the potassium channel tetramerization domain-containing (KCTD) protein auxiliary subunits. In this proposal, we will develop structural models for full-length GABABR, its auxiliary subunits and their complexes with G- protein. Building on our structural models for both the extracellular and intracellular components of human GABABR, we propose to determine the structural diversity of GABABR auxiliary subunits KCTDs, and elucidate the interactions between KCTDs and G proteins (Aim 1), describe the molecular association between GABABR and KCTD, and determine its impact on GABABR signaling (Aim 2), and solve the structures of full-length GABABR in multiple functional states, characterizing the allosteric interaction between the GABABR and G proteins (Aim 3). We will use an innovative strategy of combining structural and functional analyses, including cryo-electron microscopy (EM) and nanodiscs. Together, these studies will advance our understanding of the molecular basis of GABA action in the brain, leading to the development of novel therapeutics for treating neurological diseases.

PROJECT SUMMARY Schizophrenia, bipolar disorder and autism are common and debilitating neurodevelopmental disorders that together affect more than 5 million Americans. Despite more than fifty years of research, no cures exist and the standard of treatment remains unsatisfactory. Heterozygous mutations of neurexin-1 (NRXN1) have been repeatedly associated with schizophrenia (SZ) and autism spectrum disorder (ASD). The clinical presentations of NRXN1+/- mutations (including diagnosis, severity, prognosis and age-of-onset) in affected patients are diverse and the genetic mechanism affecting the penetrance of these mutations remains unknown. Moreover, mouse models do not permit researchers to study how and why some NRXN1+/- deletions have more deleterious effects in patients. Our objective is to resolve how NRXN1+/- deletions perturb the NRXN1 isoform repertoire and impact neuronal maturation and synaptic function. Our preliminary data defined the NRXN1 alternative splice repertoire in control fetal and adult cortical tissue, and compared this to human induced pluripotent stem cell (hiPSC)-derived neurons from NRXN1+/- cases and controls. Here, we propose to evaluate the effect of experimental manipulation of the NRXN1 isoform repertoire in both control and NRXN1+/- patient-derived excitatory and inhibitory neurons. Ultimately, we hope to directly correlate genomic and functional deficits across increasingly refined populations of NRXN1+/- patient-derived neurons.

Activation of G protein-gated inwardly rectifying potassium (GIRK) channels provides a key inhibitory signal in the brain that reduces neuronal firing. Many neurotransmitters in the reward pathway, such as dopamine and GABA, couple to GPCRs that signal through GIRK channels. Prior studies have established a role for GIRK channels in the response to psychostimulants. A complex network of proteins is hypothesized to support drug-dependent changes in trafficking and targeting of GIRK channels to postsynaptic inhibitory synapses, but the identity of these proteins remain largely unknown. To address this major gap in the field, an innovative technique of proximity-dependent biotin identification (referred to as iBio-ID) will be used to identify new proteins in the GIRK channel proteome (Aim 1). In this proposal, GIRK channels will be engineered to biotinylate proteins in vivo (within 50 nanometers of the channel), and then biotinylated proteins will be purified and identified using tandem mass spectrometry. Preliminary data show that GIRK channels fused to the biotinylating enzyme are functional, and can biotinylate proteins in vivo, leading to identification of putative GIRK channel regulators. Following quantitative analyses, candidate GIRK channel regulator proteins will be assessed for functional interaction with GIRK channels in neurons. Changes in the GIRK channel proteome with cocaine, using the locomotor sensitization as a model of addiction, will also be studied (Aim 2). These experiments will provide for the first time a comprehensive list of proteins in the GIRK channel proteome, and lead to the identity of new drug-dependent regulators of GIRK channels. Discovery of novel protein targets in the reward pathway could lead to the development of new treatments for addiction.

Neuropeptides are important signaling molecules that regulate brain states, modify neural activity and control vascular tone in the nervous system. Pharmacological and molecular genetic studies have implicated changes in neuropeptide signaling with brain dysfunctions, such as alcohol abuse, drug addiction, and stress. Released via dense-core vesicles into the extrasynaptic space, neuropeptides diffuse over long distances (i.e., volume transmission), and activate G protein coupled neuropeptide receptors. Although widely expressed in the brain, remarkably little is known about their actions on neural circuits. Lacking in the field is a technique to control the timing and spatial release of neuropeptides in real-time in awake animals. To address this unmet need, we will develop a new tool for photo-release of three neuropeptides (SST, VIP, Dyn) using an innovative nano- technology. We will create lipid nano-vesicles (50~100 nm) that encapsulate neuropeptides, and are coated with gold nanoparticles, making them exquisitely sensitive to disruption with two-photon (2p) or one-photon (1p) light. We will integrate the use of photosensitive neuropeptide nano-vesicle with cell-based neurotransmitter fluorescent engineered reporters (CNiFERs), a recently developed technique for optical detection of neuropeptides in vivo. Specifically, we propose to (1) develop photosensitive neuropeptide nano-vesicles (nVs) with efficient and multicolor two-photon release, (2) validate and investigate modulation of photo-released neuropeptide in brain slices, and (3) determine the properties of photo-released neuropeptides in vivo and the impact on neuronal excitability in the cerebral cortex. With the all-optical release and monitoring, we will investigate the diffusion rate and distance for neuropeptides in vivo. We provide preliminary data demonstrating feasibility of creating and studying photo-released peptides. Completion of the proposed research will establish a transformative technique to investigate neuropeptide volume transmission and its modulation of neural circuits. Success of this proposed research will advance studies on the transient and localized effects of neuropeptides that are currently not possible with today's technology, revealing new information on the function of neuropeptides in the brain.