Henry A. Lester, PhD - US grants

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The overall goal is to clone and to study the structure and expression of genes coding for ion channels expressed in heart, especially of the rat. These studies will illuminate the basic molecular mechanisms of the rhythmic electrical activity controlling contraction and relaxation of the heart. The cloned genes will provide a new set of heart specific probes for the study of gene expression in normal and diseased heart tissue. The ion channels of special interest are those coding for voltage sensitive sodium, potassium, and calcium channels. Heart specific chromosomal and cDNA genes coding for sodium channels will be isolated using rat brain dDNA clones already isolated and characterized in these laboratories as probes. cDNAs for voltage sensitive potassium and calcium channels will be cloned by a new method under development in these laboratories. This method is based on high resolution gel electrophoretic fractionation of heart or brain poly(A) mRNA. Fractions that contain an mRNA coding for an ion channel will be identified by microinjection into Xenopus oocytes and electrophysiological assays. Active fractions will be converted into cDNA libraries. Hybrid arrest of translation and an efficient sib selection process will be used to isolate the cDNA clone coding for a given channel.

We intend to continue studying the mechanisms that render the nicotinic synapse a minute but highly sophisticated electrochemical machine, specialized to function on a time scale of milliseconds and a distance scale of micrometers. Electrophysiological and photochemical methods will be applied to electroplaques from Electroporus and Torpedo and to muscle fibers from frogs and fish. We shall investigate the agonist-receptor interaction using photoisomerizable agonist molecules. The experiments will probe (a) the role of membrane surface charge in accelerating the agonist-receptor interaction, (b) the number of bound agonist molecules directly linked to channel activation, (c) gating charge movements associated with agonist-receptor interaction, and (d) the mechanism of desensitization. We shall investigate the interaction between antagonists and the receptor by employing photochemically induced increases and decreases of antagonist concentration. We shall study the molecular nature of the rate-limiting steps leading to channel activation. On a millisecond time scale, binding of fluorescent drugs (agonists, antagonists, and open-channel blockers) will be measured and compared with the simultaneously measured membrane conductance. We shall develop methods for internally perfusing Electrophorus electroplaques. We shall then examine the role of internal drugs and ions in channel gating, ion movement through the channel, and desensitization. We shall conduct quantitative electrophysiological investigations on Torpedo electroplaques, for comparison with published binding and flux data on receptor-rich membrane fragments and on reconstituted membranes.

The overall goal is to clone and to study the structure and expression of genes coding for ion channels expressed in heart, especially of the rat. These studies will illuminate the basic molecular mechanisms of the rhythmic electrical activity controlling contraction and relaxation of the heart. The cloned genes will provide a new set of heart specific probes for the study of gene expression in normal and diseased heart tissue. The ion channels of special interest are those coding for voltage sensitive sodium, potassium, and calcium channels. Heart specific chromosomal and cDNA genes coding for sodium channels will be isolated using rat brain dDNA clones already isolated and characterized in these laboratories as probes. cDNAs for voltage sensitive potassium and calcium channels will be cloned by a new method under development in these laboratories. This method is based on high resolution gel electrophoretic fractionation of heart or brain poly(A) mRNA. Fractions that contain an mRNA coding for an ion channel will be identified by microinjection into Xenopus oocytes and electrophysiological assays. Active fractions will be converted into cDNA libraries. Hybrid arrest of translation and an efficient sib selection process will be used to isolate the cDNA clone coding for a given channel.

This project concerns the function of neurotransmitter receptors that directly gate ion channels. Nucleic acid techniques will be used in conjunction with electrophysiological measurements (voltage clamp, patch clamp) to test hypotheses about the role of specific polypeptide domains and of individual amino acids in agonist binding and channel gating. The molecular biological manipulations will employ cDNA clones for the receptors; site-specific mutations of these clones will be constructed. Available enzymatic methods will be used to transcribe the normal and mutated clones. The mRNA will be introduced by microinjection or by membrane fusion into Xenopus oocytes or mammalian cells in culture. The target cells will express the receptors in the cell membrane; the functional ion channels will be studied electrophysiologically. The work will include both the nicotinic acetylcholine receptor of Torpedo electric organ (clones now available and under study) and the receptor from mouse muscle (clones expected soon). Analogous studies will be pursued on the GABA receptor-channel complex of rat and chick brain. Work will continue on assaying fractionated mRNA preparations that express GABA channels and on screening cDNA clones constructed from the active fractions. When sequenced clones are available, voltage-clamp and patch-clamp analysis of specifically constructed mutant channels will begin.

A new goal is to exploit the Xenopus oocyte/mRNA injection system to study the diversity of voltage-dependent Ca2+ channels expressed in rat tissue. RNA will be extracted from a few stable cell lines known to express diverse Ca channels, from a few rat central nervous system regions known to express diverse Ca channels, and from rat skeletal and cardiac muscle. The electrophysiological techniques will include the two-electrode voltage clamp, the about 400 MU2 "big patch" for high-resolution recordings of macroscopic currents, and the usual about 1 MU-M2 patch for single-channel recordings. The data will form a catalogue of channel properties including waveform, voltage dependence, conductance, ion selectivity, drug sensitivity, and modulation by kinases. High-resolution gel fractionation will be used to begin the molecular characterization of the mRNA encoding the channels. A second new goal is to develop the excised big and little patch techniques with Xenopus oocytes so that these techniques can be used, in conjunction with standard intracellular injection, to test for RNA-directed synthesis of channels that are directly gated by intracellular messengers. The technique will be developed by surveying the endogenous responses to known intracellular messengers, probably with endogenous Ca-activated C1- channels and with Ca-activated K+ channels induced by mRNA from various rat organs. A search will then be made for RNA-directed responses to newly described intracellular messengers such as cGMP, ATP, and GTP.

The 1990 Gordon Conference on Ion Channels will be held in Wolfeboro, New Hampshire, two years after the previous conference. The conference will be noteworthy in two respects. (1) It has been designated an official satellite symposium of the 1990 International Biophysics Congress and will be held the week after the Congress. Therefore, a significant international attendance is anticipated. Although this grant will not be used to pay the expenses of non-U.S. personnel, the large international attendance should significantly enhance the Conference's quality. (2) The name of the Conference has been shortened to eliminate the term "excitable cells", and the Conference will focus on channels in all types of animal cells. In particular, it is hoped that there will be a very useful exchange of information between workers who emphasize excitable cells and epithelial physiology.

DESCRIPTION (Based on the applicant's abstract): This project concerns the membrane proteins -- receptors and ion channels -- that underlie cellular excitability. In the next project period, VV will be exploited as a new system for the heterologous expression of these proteins in the membrane of mammalian and avian cells. 1. Vaccinia affords a new system for studying the 7-helix receptor-G protein interactions and the ion channel pathway, which employs direct coupling rather than soluble second messengers. Experiments will therefore work toward a complete reconstitution of this pathway. As a first step, the serotonin 5-HT-1A receptor will be expressed in primary cultures of cardiac atrial cells (chicks or guinea-pigs). It is expected that it will couple to the existing G protein(s), which in turn will activate K+ channels. Quantitative studies will follow. 2. It is of interest to understand the molecular basis for the diversity of voltage-dependent Ca++ channels from rat brain. Efforts will be continued to construct full-length clones for four classes of putative voltage-dependent Ca++ channels from rat brain. These clones will then be expressed in mammalian cell lines using VV and electrophysiological testing. Site-directed mutagenesis will follow. 3. Milligram quantities of channel proteins will be expressed and purified for structural studies.

This Center application is being submitted in response to the National Institute of Mental Health's Program Announcement "Centers for Neuroscience Research". The Caltech Center for Neuroscience Research brings together eight research groups in cellular and molecular neuroscience. A large number of proteins that mediate specialized communication within and among neurons have recently been discovered. Key questions now include the following: What is the significance of the large variety of isozymes of closely related neurotransmitter and neurohormone receptors? How do the small variations in structure of these molecules influence signal transduction? How are the networks of intracellular regulatory pathways that process receptor signals organized within different kinds of neurons? How do variations in concentration and spatial arrangement of key regulatory molecules, such as G proteins, protein kinases and protein phosphatases, influence patterns of electrical activity and other major signaling properties of different neuronal types? How is transmitter release controlled at the molecular level in different classes of neurons? Are there variations in the release machinery that produce major differences in mechanisms of regulation of transmitter release? To what extent do intracellular signaling pathways important in neuronal function overlap with those important for development? And finally, what new signaling pathways remain to be discovered? The central hypotheses emphasize both unity and diversity. It is suggested (1) that most classes of signaling protein -- 7-helix receptors, G proteins, enzyme effectors, kinases, ligand-gated channels, voltage-gated channels, transporters, and others now being discovered -- participate in several distinct types of signaling pathways within neurons; and (2) that the complexity and diversity of molecular and cellular signaling pathways accounts for much of the functional diversity of CNS neurons. The range of favorable preparations constitutes a strength of the Center and includes: rat CNS neurons in slices, organotypic cultures, and dissociated cultures; rat olfactory epithelium; neuronal cell lines; insect central nervous systems; Xenopus oocytes expressing signaling proteins; Xenopus brains; non-neuronal cells modified to function as artificial neurons; and bacteria expressing G proteins. The technical approaches are highly appropriate and promise rapid progress: nucleic acid molecular biology; antisense suppression; heterologous expression in mammalian cells and oocytes; protein chemistry; electrophysiology; immunocytochemistry; optical recording and imaging; numerical simulation and analysis; and bacterial genetics. Research on the cellular and molecular basis of neuronal function is relevant to several neurological diseases, to mental disease, and to substance abuse.

9352275 Lester A new laboratory course in cellular and molecular neuroscience has been established that is specifically tailored to undergraduate instruction, and uses the latest techniques and concepts in the molecular biology of excitable cells. The course emphasizes (1) the molecular biological and fine- structure function of the "excitability proteins" of neuronal and other membranes, (2) the nature of the intracellular signaling pathways that are activated by cell-surface receptors, and (3) the nature of the intracellular signaling pathways that are activated by cell-surface receptors. Most experiments take place in 2 sessions. In the first session, students begin with a cDNA clone encoding an ion channel or receptor. The students use PCR to generate an optimal DNA, suitable for in vitro cRNA synthesis, and carry out the cRNA synthesis reaction. The students inject the cRNA into Xenopus oocytes. In the second session, the students perform electrophysiological and pharmacological measurements on the injected oocytes. The initial sessions concentrate on relatively simple manipulations such as intracellular recording; then advance to 2-electrode voltage clamp and, finally, to patch-clamp experiments. Provisions are made for maximal utilization of the requested equipment in undergraduate research projects during the terms when the course is not offered formally. ***

This project focuses on the human cystic fibrosis transmembrane conductance regulator (CFTR), with the overall goals of (l) providing a map of the conduction pathway and (2) determining the interaction between the R-domain and this pathway. The major experimental tool is electrophysiological measurements on wild-type and mutant human CFTR expressed in Xenopus oocytes. (l) The ion channel will be mapped as follows. Based upon the recent characterization of the voltage-dependent blocking action of two acidic inhibitors of the wild-type CFTR -- diphenylamine-2-carboxylate (DPC) and flufenamic acid (FFA) --site-directed mutant channels are being designed and constructed. The mutant channels will be tested with respect to blockade and single-channel conductance, with the goal of localizing the amino acid residues that line the pore. Preliminary studies have outlined interesting regions to investigate. (2) The project will also map the contacting surfaces between the regulatory (R) domain and the bulk of the channel using the same methods that have proven successful for Specific Aim (l). Site-directed mutations will be made within (a) portions of the R-domain that may serve as donor regions and (b) portions of the bulk of the channel that may serve as acceptor regions. These mutations will be studied with respect to kinetics of activation/deactivation, in order to understand the mechanism of gating within CFTR. In parallel experiments, data will be used from two additional sources: construction of chimeric channels from CFTR/P- glycoprotein sequences (since the P-glycoprotein does not include an R- domain) and from human/shark CFTR sequences (if gating kinetics in the shark channel differ substantially from those of the human). These studies will provide an increased understanding of the function of CFTR, and may lead to new therapeutic strategies.

This project will study the structure and function of monoamine transporters. Our approach builds upon cDNA cloning and heterologous expression in Xenopus oocytes. Functional measurements will include flux of radiolabeled substrates, binding of ligands, electrophysiology, and electrochemical detection of monoamines. 1. Cloning and characterization of additional monoamine transporters will continue. Several partial cDNA clones from Drosophila will be used to screen for full-length clones, these will be sequenced and characterized functionally. Present and future Drosophila clones will be used as probes to isolate possible homologous mammalian transporters. 2. Physiological studies will continue on cloned monoamine transporters in heterologous expression systems. Dopamine and norepinephrine transporters will be characterized with methods proven for 5-HT transporters. The discrepancy between integrated current flow and [3H]5-HT uptake for 5-HT transporters will be studied. The nature of the irreversible transition to high leakage current levels that occurs with a mammalian 5-HT transporter will be studied. 3. Structure-function analysis will be performed with monoamine transporters. Experiments will utilize chimeras and site-directed mutations. The focus will be on localizing those domains and residues responsible for pharmacological properties and for permeation properties. 4. Systems will be developed for high-level expression with the eventual goal of structural studies. Expression will be optimized using the available techniques and assays. Initial characterization will utilize freeze-fracture and atomic force microscopy. Monoamine transporters are the targets for (a) many drugs of abuse, (b) many antidepressants, and (c) a toxin that produces Parkinsonism.

This Center application is being submitted in response to the National Institute of Mental Health's Program Announcement "Centers for Neuroscience Research". The Caltech Center for Neuroscience Research brings together eight research groups in cellular and molecular neuroscience. A large number of proteins that mediate specialized communication within and among neurons have recently been discovered. Key questions now include the following: What is the significance of the large variety of isozymes of closely related neurotransmitter and neurohormone receptors? How do the small variations in structure of these molecules influence signal transduction? How are the networks of intracellular regulatory pathways that process receptor signals organized within different kinds of neurons? How do variations in concentration and spatial arrangement of key regulatory molecules, such as G proteins, protein kinases and protein phosphatases, influence patterns of electrical activity and other major signaling properties of different neuronal types? How is transmitter release controlled at the molecular level in different classes of neurons? Are there variations in the release machinery that produce major differences in mechanisms of regulation of transmitter release? To what extent do intracellular signaling pathways important in neuronal function overlap with those important for development? And finally, what new signaling pathways remain to be discovered? The central hypotheses emphasize both unity and diversity. It is suggested (1) that most classes of signaling protein -- 7-helix receptors, G proteins, enzyme effectors, kinases, ligand-gated channels, voltage-gated channels, transporters, and others now being discovered -- participate in several distinct types of signaling pathways within neurons; and (2) that the complexity and diversity of molecular and cellular signaling pathways accounts for much of the functional diversity of CNS neurons. The range of favorable preparations constitutes a strength of the Center and includes: rat CNS neurons in slices, organotypic cultures, and dissociated cultures; rat olfactory epithelium; neuronal cell lines; insect central nervous systems; Xenopus oocytes expressing signaling proteins; Xenopus brains; non-neuronal cells modified to function as artificial neurons; and bacteria expressing G proteins. The technical approaches are highly appropriate and promise rapid progress: nucleic acid molecular biology; antisense suppression; heterologous expression in mammalian cells and oocytes; protein chemistry; electrophysiology; immunocytochemistry; optical recording and imaging; numerical simulation and analysis; and bacterial genetics. Research on the cellular and molecular basis of neuronal function is relevant to several neurological diseases, to mental disease, and to substance abuse.

This project studies G protein-gated inward rectifier K+ channels (GIRKs), continuing electrophysiological studies and also emphasizing biochemical studies of interactions between GIRKs and other molecules of signal transduction. (1) We shall address the physiological significance of the GIRK-integrin interaction. We shall study possible colocalization of the two molecules with confocal immunocytochemistry, study the identity of the integrin(s), and test for interactions between other inward rectifier K channels (non G protein-gated) and integrins. We shall test the hypothesis that the interaction also affects signal transduction by integrins. (2) We shall address the hypothesis that cells lacking normal RGS protein function will display postsynaptic responses. We shall study the detains of RGS action at the single-channel level, compare effects of RGS proteins on the kinetics of modulation at either GIRK or Ca/2+ channels, and use BIAcore measurements to address the hypothesis of ternary complexes among G proteins, RGS proteins, and GIRK channels. (3) We shall investigate surface exposure and topology of residues in GIRK channels using incorporated biocytin, domain structure and domain organization using site-specific nitrobenzyl-induced photochemical proteolysis of the main chain (SNIPP), and kinetic aspects of signal transduction using flash decaging of phosphorylatable tyrosine residues. (4) We shall investigate the hypothesis that the pathophysiology of the weaver mutation arises because of regenerative Na+ fluxes. We shall simultaneously conduct Ca2+ and Na+ imaging and electrophysiological investigations on weaver granule cells in culture following sudden activation of the GIRK channels. We shall investigate the hypothesis that some cells are spared because weaver channels appear in the cell membrane but are inactivated or inhibited. We shall attempt to understand the basis for activation by intracellular Na+. G protein-gated K+ channels (GIRKs) control the strength and frequency of the heartbeat, some responses to drugs of therapy and abuse, and some aspects of insulin secretion.

This application seeks renewal of the National Institute of Mental Health Silvio Conte Center for Neuroscience Research at Caltech. The Center brings together eight research groups and continues to emphasize cellular and molecular signaling as the basis for neuronal function in the central nervous system. In the next phase of operation, there will be an added an emphasis on molecules and pathways that were first identified in the context of developmental signaling. The central hypotheses emphasize both unity and diversity. It is suggested (1) that most classes of signaling protein--7-helix receptors, G proteins, enzyme effectors, kinases, ligand-gated channels, voltage-gated channels, transporters, receptor tyrosine kinases and phosphatases, integrin receptors, DNA- binding proteins, and others now being discovered--participate in several distinct types of signaling pathways within neurons; and (2) that the complexity and diversity of molecular and cellular signaling pathways accounts for much of the functional diversity of CNS neurons. The range of favorable preparations constitutes a strength of the Center and includes: rat CNS neurons in slices, organotypic cultures, and dissociated cultures; rat olfactory epithelium in primary culture; human olfactory neuroblastoma; mammalian cell lines; neural crest cells in primary culture; embryonic stem cells transfected with genes of interest; transgenic mice and tissues and cells from these mice; Xenopus oocytes expressing signaling proteins; Xenopus eyes and brains; and bacteria expressing G proteins. These questions are being investigated with a variety of appropriate technical approaches that promise rapid progress. The range includes: nucleic acid molecular biology; heterologous expression in mammalian cells via adenovirus; heterologous expression in oocytes; transgenic, knockout and knock- in mice; protein chemistry; electrophysiology; immunocytochemistry; optical recording of intracellular ions; confocal and 2-photon imaging; and bacterial genetics. Research on the cellular and molecular basis of neuronal function is relevant to mental diseases, to neurological diseases, and to substance abuse.

DESCRIPTION: (Applicant's Abstract) Functional aspects of cloned neurotransmitter transporters will be studied in heterologous expression systems, with emphasis on the rat serotonin transporter rSERT and the rat GABA transporter rGAT1. It is scientifically wise and efficient to intermix studies on these two homologous transporters (~ 40% primary sequence similarity). The first specific aims probe these themes of structure and function with established techniques: single-channel measurements, including simulations; proton permeation; functional characteristics of coexpressed mutants; substituted cysteine mutagenesis; and localization of the gates. The second set of specific aims probes these same themes, with innovative techniques that are obviously riskier but that may provide novel information: biocytin labeling with the nonsense suppression technique; photolytic cleavage, also with unnatural amino-acid residues; fluorescence assays of conformational changes during function. Novel proteins will be sought that interact with the transporters, using the yeast two-hybrid system. Predictions about the timing of substrate flux after a pulse of neurotransmitter will be tested using time-resolved amperometry. Fluoxetine typifies a class of antidepressants, introduced in the past decade, called serotonin-selective reuptake inhibitors (SSRI's). Many patients diagnosed with attention-deficit disorder (ADD) or attention deficit-hyperactivity disorder (ADHD) benefit from methylphenidate, amphetamines, or other agents thought to act on monoamine transporters either by blocking uptake or by enhancing release. Therefore neurotransmitter transporters deserve continued close study with modern molecular techniques.

This project continues to obtain high-resolution data about the functional roles played by ion channels and neurotransmitter receptors in neuronal signaling. We exploit two present ideas in the molecular genetics of ion channels: (1) "Hyperexcitable/Depolarizing" mutations produce the most dramatic phenotype for CNS ion channels; and (2) Mouse gene targeting is now a robust method for assessing the role of individual ion channel and receptor proteins. We shall design, construct and test a class of hyperexcitable/depolarizing mutants for channels and receptors expressed in the mouse CNS. The channels studied will be those for which hyperexcitable mutations are already known or expected. In the first year, we shall study inward rectifier K channels with the P- region GYG->SYG "weaver" mutation, and nicotinic AChR channels with mutations in the M2 region. In later years, we shall study cyclic nucleotide-gated channels with mutations in the cng binding regions, voltage-gated K channels with P-region mutation, P2X receptors, glutamate receptors, and other channel mutations that are discovered as a result of mouse or human genetic screens. These mutants will be tested and refined in systems of increasing complexity: Xenopus oocytes; mammalian cells in culture; and finally targeted replacement of the existing mouse gene-the major goal. Functional analysis of these mice will include electrophysiology, anatomy, and behavior.

This project focuses on the functional characteristics of molecules that render "fast" synapses, exquisite electrochemical machines, specialized to function on a time scale of milliseconds and a distance scale of micrometers. 1. Functional studies will continue on the nicotinic receptor superfamily. Tethered agonists and cation-it interactions will be studied. Fluorescent probes of receptor conformation will be developed and studied. 2. Structure/function relations will be studied in the P2X receptor superfamily. The mechanism for the large pore that appears after sustained exposure to ATP will be explored. The disulfide topology of the extracellular region will be explored. Mechanisms will be studied for the functional interaction between P2X and nicotinic receptors. 3. The experiments will develop ways to count local densities Of synaptic proteins: transporters, starting with the mouse GABA transporter mGAT1; and receptors, starting with the nAChR alpha4 subunit and P2X2 subunit Ion channels and transporters, including but not limited to those involved in synaptic transmission, are important in many aspects of pathophysiology and therapeutics.

DESCRIPTION (provided by applicant): This project aims to understand the density, intracellular processing, PDZ interactions, trafficking, and possible dimerization of the GABA transporter, mGAT1, by studying knock-in mice carrying fusions between mGAT1 and fluorescent proteins (XFP, where G = green; C, cyan; Y, yellow). The major tool is quantitative fluorescence microscopy. A mGAT1-XFP knock-in mouse strain will be developed that preserves a crucial PDZ interaction. mGAT1 will be counted in the synaptic structures of mGAT1-XFP knock-in mice. GAT1 interactions will be studied using hippocampal and cerebellar culture systems for the mGAT1-XFP mice. mGAT1-XFP mice will be mated with knockout mice for several synaptic proteins known to interact with GAT1, in order to study the effects on GAT1 distribution, trafficking, and sorting, using the culture system. The hypothesis that GAT1 dimerizes in vivo will be tested using fluorescent resonance energy transfer (FRET) between CFP-mGAT1 and YFP-mGAT1. The hypothesis will be tested that GAT1 is carried on a special vesicle, and the number of mGAT1 molecules in a vesicle will be counted using evanescent wave-total internal reflection fluorescence (TIR-FM) microscopy. Also the project aims to understand the functions of GAT1 by continuing to study the intron 14-neo-mGAT1 strain, which is essentially a GAT1 knockout. The major tools are whole-animal physiology and electrophysiology. The motor phenotype produced in WT mice by acute administration of GAT1 inhibitors will be studied, to ask whether acute blockade explains the motor and behavioral anomalies of the intron 14-neo-mGAT1 strain. GAT1 will be selectively reintroduced in various brain areas and cell types to study the motor defects, by mating with various strains that have restricted expression of cre recombinase. Electrophysiology of the knockout will be studied. Similar knowledge will also be gathered about another GABA transporter, GAT3, by replicating the research program for GAT3-XFP mice. A GAT3-XFP fusion protein will be selected that displays proper function, sorting, and targeting. Knock-in mice will be constructed bearing this protein. Density, intracellular processing, trafficking, and possible dimerization of GAT3 will be studied. Similar knowledge will be sought about the serotonin transporter, SERT, by replicating specific aim 1 for SERT-XFP knock-in mice. Neurotransmitter transporters are targets for many modern drugs of therapy and abuse.

DESCRIPTION (provided by applicant): This project tests the hypothesis that knock-in mice carrying nicotinic receptor mutations linked to autosomal dominant nocturnal frontal-lobe epilepsy (nADNFLE) will display seizures resembling the human disease. Breeding will continue of a knock-in mouse strain harboring a nicotinic receptor alpha4 subunit with the M2 domain Serl0'Leu mutation, which corresponds to one of the five known nADNFLE alleles. This strain will be tested for spontaneous and nicotine-induced seizures using chronic video-EEG monitoring. Pharmacological tests will be performed to assess nicotinic specificity. Additional knock-in mouse strains will be developed that harbor nicotinic receptor beta2 subunits with the M2 domain Va122'Leu and Va122 Met mutations (other nADNFLE alleles) and will also be tested for seizures. If the hypothesis is confirmed, this project will provide an animal model appropriate for detailed pathophysiological studies as well as for testing of new therapeutic drugs, targeted at ADNFLE and at other types of frontal lobe epilepsy.

DESCRIPTION (provided by applicant): Mouse strains will be developed to test the concept of selective neuronal silencing using a combined genetic and pharmacological approach. The strategy employs the glutamate-gated chloride channel / ivermectin (GluCl / IVM). The mice will selectively express optimized GluCl channels, with behavioral testing as appropriate for each strain. Three "proof of concept" conventional mouse strains are envisioned that have straightforward behavioral assays. The first will employ the promoter for TrkA, a peripheral nervous system promoter. The assay is pain. The second, for retinal ganglion cells, will employ the Brn3b promoter. The assays are visual behavior and visual evoked potentials. The third, for the cerebellum, will employ the L7 promoter. The assay is ataxia. For each strain, experiments will detail the extent, IVM concentration dependence, onset, and recovery time course of silencing based on the behavioral assays. Pitfalls seem surmountable. The project seeks to develop a technique that permits one to interfere deliberately, delicately, specifically, transiently and reversibly with discrete subpopulations of genetically identified neurons in mice. If the "proof of concept" experiments succeed, an R01 grant will be sought to extend and exploit the system. There are important applications in many fields of neuroscience.

DESCRIPTION (provided by applicant): Chronic exposure to nicotine leads, in some individuals, to the set of behaviors that constitute nicotine dependence. Chronic nicotine exposure produces selective upregulation of certain high-sensitivity (HS) nicotinic acetylcholine receptors (nAChRs). This project works within an emerging hypothesis that selective upregulation of certain nAChRs is both necessary and sufficient for the initial stages of nicotine dependence- minutes, hours, days, and weeks. The power of the emerging hypothesis derives from the selectivity displayed by upregulation at every level thus far examined-brain region, cell type, subcellular region, major pharmacological subtype, and detailed subunit composition. These are "tiers of selectivity" in upregulation. As a result, upregulation selectively modifies neuronal excitability and neuronal interactions. These pathological patterns comprise, in part, the cellular and circuit basis of nicotine dependence. Aim 1 studies selective upregulation in the ventral tegmental area-nucleus accumbens (VTA-NAcc) pathway, which accounts for some aspects of both tolerance and sensitization to the rewarding aspects of nicotine. Previous data, especially on the substantia nigra-dorsal striatum pathway, show tiers of selectivity in upregulation. Now five linked sub- hypotheses will be tested about the cellular and subcellular details of alpha4beta2 nAChR upregulation in the VTA and NAcc. Aim 2 tests hypotheses about upregulation in medial entorhinal cortex and dorsal hippocampus, a partial model for cognitive sensitization during chronic exposure to nicotine. Sub-hypothesis 2.1 further characterizes the known upregulation of a4* nAChRs by chronic nicotine on the axons of the medial perforant path. This upregulation leads to increased glutamate release in the presence of nicotine. Sub- hypothesis 2.2 states that chronic nicotine upregulates a4* nAChRs in some hippocampal interneurons. Aim 2 also characterizes upregulation in dorsal hippocampus after only 24 hours of nicotine exposure. Aim 3 tests hypotheses about the amygdala, which is a partial locus for nicotine's calming responses to environmental stress. The project tests whether nicotine effects in amygdala arise partially via alpha4beta2 nAChRs on axon terminals of dopaminergic VTA projections. Experiments will focus on two amygdala cell types with dense dopaminergic innervation: intercalated neurons, and corticotrophin-releasing factor neurons in central amygdala, lateral region. The project then tests whether upregulation of these nAChRs amplifies the response to acute nicotine, providing a form of sensitization to the calming effects of nicotine during environmental stress. Aim 4 tests a hypothesis linking the alpha5 subunit to aspects of nicotine addiction. Specifically, the project tests whether lower levels of the alpha5 subunit produce more alpha4beta2 nAChRs, and therefore greater upregulation by chronic nicotine. The techniques, all established in the investigator's laboratory, will be applied as appropriate: a. Patch-clamp electrophysiology in brain slices;b, Electrochemical measurements of DA release;c, Quantitative fluorescence measurements in existing mouse strains with fully functional a4* receptors;d, Measurements in KO mice for the a5 subunits, and selectively in other strains;e, Multielectrode array-based single-unit extracellular recordings from intact mice. This project is a part of a broad context. Tobacco use presents challenges requiring data at many levels-from genome-wide association studies, through the intracellular molecular bases of upregulation, through more complex circuits, through animal and human behavior, through public policy. PUBLIC HEALTH RELEVANCE: Roughly 400,000 Americans and roughly 4,000,000 people worldwide die annually of the sequelae to nicotine dependence. This project attempts to explain nicotine dependence.

DESCRIPTION (provided by applicant): The Caltech-Boulder-Targacept National Cooperative Drug Discovery and Development Group (NCDDDG) continues to be motivated by the hypothesis that successful smoking cessation compounds can selectively target just one or a few nicotinic acetylcholine receptor (nAChR) combinations. Nicotine addiction begins with the activation of these receptors, which are ligand-gated channels. Progress in this NCDDG and around the world during the 2005-2010 Project Period provided additional knowledge about the range of homo- and hetero-pentameric subtypes that do exist in the brain; about their localization; and about their specific roles in the constellation of phenomena that comprise nicotine addiction (tolerance, dependence, sensitization, and goal-seeking behavior). Nonetheless, knowledge remains partial, especially about the long-term effects of both nicotine and smoking cessation drugs. The 2010-2015 Project Period provides an iterative series of experiments to test hypotheses in these areas. (1) The NCDDDG will use accumulating knowledge to optimize lead compounds for smoking cessation. We will devote the first 2 yr of the Project Period to three lead compounds that emphasize a6-oriented and/or a4-oriented activity: varenicline, TCD-1010, and one other compound to be chosen by the Executive Committee. The compounds are expected to exhibit varying ratios of a6* to a4* activity. All these compounds will be at least partial agonists. We expect that all compounds will also desensitize a4* nAChRs; a6* desensitization will be studied in detail in Project 3. (2) It is also likely that successful smoking cessation compounds will themselves produce long-term changes in these target nAChRs. Therefore Projects 2 and 3 will use mouse models to explore possible upregulation, an adaptive change in nAChRs caused by chronic exposure to lead compounds. (3) The iterative experiments will, in turn, lead to synthesis of new compounds at Targacept (Project 1), and further testing during the final three yr of the 2010-2015 Project Period. This scientific approach will enable the Group, as well as other groups throughout the world, to optimize and develop lead compounds for smoking cessation. (4) While pursuing these goals in parallel, we intend to continue publishing all of our studies according to the usual standards that have characterized research in academic neuroscience.

[unreadable] DESCRIPTION (provided by applicant): This project studies the structure and function of molecules in the Cys-loop receptor superfamily: the muscle nicotinic acetylcholine receptor (nAChR), the neuronal alpha4beta2 nAChR, and the serotonin 5-HT3 receptor. Hypothesis 1 states that three events occur in the following sequence at the agonist binding site, (a) The charged amine / ammonium group of the agonist is attracted to the site by a monopole-monopole interaction with fixed negative .charges on side chains, (b) For agonists with an amino group (not a quaternary ammonium group), this interaction is stabilized by an H-bond to the backbone carbonyl of the 149-150 peptide bond, (c) The earliest conformational change places the agonist in a cation-pi interaction at tryptophan alpha149. Hypothesis 2 states that ye M2-M3 linker undergoes a change in backbone conformation during gating. Hypothesis 3 states that during channel activation, the upper M2 helix of all five subunits re-orients with respect to neighboring helices. Hypothesis 4 states that the dynamic, history- dependent functional interaction between alpha4beta2 and P2X2 receptors occurs via the beta2-M3-M4 loop and the P2X2 C-terminal tail. Hypotheses 1 and 2 will be tested with macroscopic and single-channel electrophysiological assessments of receptors bearing unnatural amino-acid side chains and unnatural backbone linkages. Hypotheses 1, 3, and 4 will be tested in measurements based on direct fluorescence of tethered probes, fluorescence resonance energy transfer (FRET), and lanthanide-based resonance energy transfer (LRET). The resulting knowledge about acetylcholine receptors and 5-HT3 receptors may provide both pathophysiological insights and better drug therapies for health challenges including smoking cessation, Parkinson's disease, Alzheimer's disease, pain, Crohn's disease, sudden infant death syndrome, attention deficit disorder, autosomal dominant nocturnal frontal lobe epilepsy, and schizophrenia. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): This project seeks to develop a novel gene transfer-based approach as an alternative to deep brain stimulation (DBS) in the treatment of advanced Parkinson's disease (PD). Viral gene transfer will be used to manipulate neuronal activity in basal ganglia structures. The underlying mechanism of DBS has not been clarified; the current hypotheses suggest either activation or inhibition of the brain regions targeted by high-frequency stimulation. The project employs an emerging technique to inhibit electrical activity in mammalian neurons by expressing a modified glutamate-gated chloride channel (GluCl) from C. elegans. Channel activation by ivermectin, an FDA-approved, a widely used anthelmintic, elicits a chloride conductance, clamping these neurons to the Nernst potential for chloride, thereby inhibiting action potentials ('silencing'). For this study the GluCl channel will be modified to convert it into a Na and K-selective, excitatory channel ("GluNa"). Adeno-associated and lentivirus vectors will be developed that encode both GluCl and GluNa. Thus, tools will be developed to stimulate or to inhibit neuronal activity. Injection of virus encoding either GluCl or GluNa channel into the subthalamic nucleus or substantia nigra pars reticulata will (1) provide a minimally invasive, adjustable approach to accomplish pharmacological DBS based on an FDA-approved drug, and (2) will generate further insights into the underlying mechanism of DBS. Aim 1 will provide a cation-selective channel. Aim 2 and 3 will test applicability of GluCl and GluNa (respectively) in the approach to pharmacological DBS. Both aims will give insights into the mechanism of DBS. to public health If successful, the project will establish a new treatment option for Parkinson's disease and could also be used in other applications where deep brain stimulation is now being tested. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This project will generate and validate fourteen strains of mice with fully functional fluorescent mouse nicotinic receptor (nAChR) subunits. These strains will be constructed using the proven and reasonably efficient method of exon replacement via homologous recombination in embryonic stem cells, leading to "knock-in mice". In each case, one mGFP and one mCherry allele will be generated, so that strains can be crossed to study receptor assembly with Foerster resonance energy transfer. Fluorescent proteins (FPs) have been successfully integrated into the M3-M4 intracellular loop of most of these subunits (the final two will be verified before funding starts), and these fluorescent subunits are fully functional and correctly targeted in mammalian cell lines. The Caltech group will immediately construct targeting vectors for the alpha3, alpha4, alpha5, alpha6, beta2, beta3, and beta4 FP-labeled subunits. The project will generate embryonic stem cells harboring these subunits, then generate knock-in mice. Further experiments will validate that these strains display nAChRs whose key parameters of expression and function are within a factor of two of the wild type level. Thus, at Boulder autoradiographic and synaptosome-based experiments will be performed on the initial lines to verify faithful expression and function;at Caltech, cellular-level fluorescence quantification will also verify faithful expression and gene dose dependence. Simultaneously, the project will begin to generate congenic strains on the C57Bl/6 background. These mice will be valuable for future studies, for two key reasons which bear on multiple biomedical disciplines. First, evidence is emerging that changes in the level and composition of the nAChRs themselves underlie some components of the responses to chronic exposure to nicotine during nicotine addiction. Changes in the level of the receptors themselves are also thought to underlie two inadvertent therapeutic effects of tobacco use: the inverse correlation between a person's history of tobacco use and his risk of Parkinson's disease (PD), and the seizure-suppressing effects of nicotine use in autosomal dominant nocturnal frontal lobe epilepsy. Second, SNPs found in two clusters of nAChR subunit genes, and in two other nAChR subunit genes, are associated with nicotine dependence, number of cigarettes smoked per day, "pleasurable buzz" elicited by smoking, age of tobacco and alcohol initiation, early subjective response to tobacco, "dizziness" after the first few cigarettes, and lung cancer. Other studies have linked one or both clusters with alcohol and cocaine dependence. Smoking is also clearly associated with chronic obstructive pulmonary disease. Time lines and decision points for this research are established, as are procedures for distribution to the research community. It is likely that congenic strains will be available less than a year after the project terminates. PUBLIC HEALTH RELEVANCE: The mice generated in this project will be valuable for future studies, for reasons which bear on several diseases. First, evidence is emerging that changes in the level and composition of the nicotine receptors themselves underlie some components of the responses to chronic exposure to nicotine during nicotine addiction. Changes in the level of the receptors themselves are also thought to underlie two inadvertent therapeutic effects of tobacco use: the inverse correlation between a person's history of smoking and his risk of Parkinson's disease, and the seizure-suppressing effects of nicotine use in autosomal dominant nocturnal frontal lobe epilepsy. Second, variations found in two clusters of receptor subunit genes, and in two other receptor subunit genes, are associated with nicotine dependence;number of cigarettes smoked per day;"pleasurable buzz" elicited by smoking;age of tobacco and alcohol initiation;early subjective response to tobacco;and lung cancer. Other studies have linked one or both clusters with alcohol and cocaine dependence. Smoking is also clearly associated with chronic obstructive pulmonary disease.

DESCRIPTION (provided by applicant): The overall goal of these experiments is to reveal a mechanistic basis for the inverse correlation between smoking and Parkinson's disease. The project tests whether chronic exposure to nicotine up-regulates functional nicotinic acetylcholine receptors (nAChRs) in the basal ganglia, changing cell and circuit function. The cell-specific alpha4 up- regulation (CSAUR) hypothesis states that the key up-regulated receptors are characterized by a high sensitivity (HS) to nicotine and are therefore mostly of the alpha4beta2* and possibly of the alpha6* subtypes; that cell-specific increases in alpha4* nAChR(s) occur during chronic exposure to nicotine; and that this cell-specific upregulation provides the basis for circuit-based changes in neuronal activity. Aim 1 uses mouse brain slices containing basal ganglia to detect the distribution of alpha4* receptors, then to determine CSAUR. The studies will employ, where appropriate, quantitative electrophysiological assessment of nAChR function, mouse strains with modified nAChRs, cell labeling, and immunohistochemical techniques. Substantia nigra pars compacta (SNc) will be studied to confirm the absence of up-regulation in these neurons. The hypothesis will be tested that chronic nicotine renders SNc neurons less sensitive to burst firing mediated by GABAA receptor blockade or by NMDA receptor activation. Subthalamic nucleus (STN) will be studied to confirm preliminary data suggesting that it expresses a* receptors. If CSAUR occurs in STN, burst firing will be studied. Medium spiny neurons (MSNs) in dorsal thalamus will be studied to determine whether chronic nicotine changes alpha4* function at the DA terminals. If so, electrochemical detection of DA release will be used. GABAergic interneurons (INs) will be tested for alpha4* receptors, and then for alpha4* up-regulation. If CSAUR is detected, the alpha4*-expressing INs will be identified by anatomy; and the particular subclass of inhibited MSNs will be identified. In Aim 2, single DA and GABAergic cells will be monitored in basal ganglia of intact animals, to assess effects of chronic nicotine that are revealed when neuronal circuits are intact . In Aim3, fluorescence will be measured in knock-in mice that express fully functional fluorescent alpha4* receptors. Up-regulation of this fluorescence will be measured in specific cell types. High-resolution subcellular studies with 2-photon microscopy will determine the sub-cellular specificity of alpha4* expression and up-regulation. Understanding the cellular and circuit changes induced by chronic nicotine in basal ganglia could lead to new therapies for Parkinson's disease.

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (06) and specific Challenge Topic, 06-MH-103 New technologies for neuroscience research. This project develops tools to address two hypotheses: first that specific types of neurons in localized brain regions or subregions can be altered so that they are susceptible to reversible inactivation or activation in response to systemically delivered low-toxicity chemicals or drugs;and second, that neuronal development, differentiation, and migration can be studied via manipulating ion fluxes, including calcium fluxes, which then lead to diverse signal transduction events. Reagents will be generated that can achieve adjustable, reversible, cell-specific, electrophysiologically measurable, and non-antigenic manipulation of neuronal excitability, and of Ca-activated signal transduction within neurons. Development will continue on a small repertoire of ligand-activated channels, avermectin receptors (AVMRs), that can be expressed in target neurons. These channels will be developed by systematically mutating subunits of existing pentameric Cys-loop receptors: the C. elegans GluCl alpha and beta subunits, and the human glycine receptor. The channels will be insensitive to endogenous ligands (such as neurotransmitters), but will be activated dose-dependently by ivermectin and its analogs, widely used drugs that can be given orally or by injection into the periphery. The chloride-permeable AVMR-Cl will be used as a starting point for constructing AVMR-Na and AVMR-Ca, sodium- and calcium-permeable versions of these channels. The "therapeutic or research index" of the AVMR system will be improved by finding an AVM analog with fewer CNS side effects, by increasing the AVM sensitivity of the existing optimized GluCl subunits, or by increasing the channel open time or conductance of the existing GluCl subunits, while maintaining reversibility. The project will also avoid immune reactions to AVMR proteins by generating a human glycine receptor-based version. The AVMRs will be useful for research in many vertebrate species: there will be considerable impact for research on circuit analysis;controlling differentiation, neurogenesis, and migration;models for excitotoxicity;and glial activation. Therapeutic impacts include "pharmacological deep-brain stimulation";neuroprotection;and other uses that extend beyond CNS neuroscience. PUBLIC HEALTH RELEVANCE: This project continues to use receptor ion channels to gain new insight into how neurons are connected within circuits and how such circuits control behavior. We will engineer new receptor channels that respond only to drugs, avermectins, that can be delivered in an animal's diet. Once these receptors are developed, it will be possible to study how activating or inhibiting selected neurons influences behavior. Ultimately, such procedures, involving both gene therapy and an FDA-approved set of drugs, could also help normalize neurons that are either too active, or not active enough, in psychiatric diseases.

nACfiR upregulation by ciironic exposure to nicotine and ottier nicotinic drugs The Overall Progress Report and Overall Description sections provide the logic and background for the study of receptor upregulation by candidate drugs. Upregulation in 2 pathways related to nicotine addiction Cellular and circuit-level research on nAChRs is governed by the fact that only a handful of papers present believable eEPSCs, sEPSCs, or mEPSCs associated with nAChR activation by ACh in the brain. Investigators believe that most HS nicotinic receptors are on presynaptic terminals, where they control transmitter release (and therefore pose challenges for detailed study) (MacDermott et al., 1999). Many studies also reveal the presence of somatodendritic HS nAChRs (mostly a4B2* and a6*). Little or no evidence shows that these somatodendritic receptors respond to circulating or released ACh. But there is much evidence that these nAChRs do respond to nicotine with both activation and, in some cases, desensitization, primarily because nicotine is not hydrolyzed by acetylcholinesterase.

(Parent Abstract): The Caltech-Boulder-Targacept National Cooperative Drug Discovery and Development Group (NCDDDG) continues to be motivated by the hypothesis that successful smoking cessation compounds can selectively target just one or a few nicotinic acetylcholine receptor (nAChR) combinations. Nicotine addiction begins with the activation of these receptors, which are ligand-gated channels. Progress in this NCDDG and around the world during the 2005-2010 Project Period provided additional knowledge about the range of homo- and hetero-pentameric subtypes that do exist in the brain; about their localization; and about their specific roles in the constellation of phenomena that comprise nicotine addiction (tolerance, dependence, sensitization, and goal-seeking behavior). Nonetheless, knowledge remains partial, especially about the long-term effects of both nicotine and smoking cessation drugs. The 2010-2015 Project Period provides an iterative series of experiments to test hypotheses in these areas. (1) The NCDDDG will use accumulating knowledge to optimize lead compounds for smoking cessation. We will devote the first 2 yr of the Project Period to three lead compounds that emphasize a6-oriented and/or a4-oriented activity: varenicline, TCD-1010, and one other compound to be chosen by the Executive Committee. The compounds are expected to exhibit varying ratios of a6* to a4* activity. All these compounds will be at least partial agonists. We expect that all compounds will also desensitize a4* nAChRs; a6* desensitization will be studied in detail in Project 3. (2) It is also likely that successful smoking cessation compounds will themselves produce long-term changes in these target nAChRs. Therefore Projects 2 and 3 will use mouse models to explore possible upregulation, an adaptive change in nAChRs caused by chronic exposure to lead compounds. (3) The iterative experiments will, in turn, lead to synthesis of new compounds at Targacept (Project 1), and further testing during the final three yr of the 2010-2015 Project Period. This scientific approach will enable the Group, as well as other groups throughout the world, to optimize and develop lead compounds for smoking cessation. (4) While pursuing these goals in parallel, we intend to continue publishing all of our studies according to the usual standards that have characterized research in academic neuroscience.

DESCRIPTION (provided by applicant): Lynx in organization and dynamics of nicotinic acetylcholine receptor complexes Channelopathies are diseases of ion channel dysfunction, arising either in channel proteins themselves or in associated proteins. They can arise either genetically or via autoimmune reaction. Mis-regulation of nicotinic acetylcholine receptors (nAChRs) is linked to neural disorders, including schizophrenia, some epilepsies, nicotine addiction, myasthenia gravis, Alzheimer's disease, and Parkinson's disease. Common findings are that the number and localization of nAChRs are altered during in the disease state, and that restoration of their correct number and distribution can ameliorate disease phenotypes. Localization in synaptic vs extrasynaptic areas is also a crucial aspect of nAChR function. Lynx proteins are widely expressed regulators of nicotinic receptor function. Removal of the lynx1 gene results in nAChR hypersensitivity, enhanced learning, and extended critical periods for binocular selectivity, but also susceptibility to neurodegeneration. Genetic, biochemical, and physiological studies indicate direct or indirect interactions between 17 and 1422 nAChRs. Lynx proteins have (a) GPI anchors and (b) structural and functional similarities to the soluble nAChR ligands, 1-bungarotoxin and related toxins from snakes and snails. GPI-anchored membrane proteins can influence the distribution and mobility of surface receptors, through their preferential association with well-ordered, cholesterol-rich domains and nanodomains that accumulate signaling molecules and cytoskeletal components at the plasma membrane. These molecular events can produce an altered microenvironment, influencing receptor distribution and lifetime at the cell surface. Therefore, this project investigates the effect of lynx-nAChR interactions on the mechanisms of nAChR trafficking to the plasma membrane, the distribution of nAChRs on the plasma membrane, the mechanisms of internalization of surface nAChRs, and the underlying interactions with cytoskeletal and trafficking machinery.

DESCRIPTION (provided by applicant): Chronic exposure to nicotine is a crosscutting phenomenon, leading to nicotine dependence as well as to inadvertent therapeutic effects such as protection against Parkinson's disease. The project tests the novel suggestion that effects of chronic nicotine exposure depend on intracellular nicotine-not on conventional signal transduction via receptors at the plasma membrane. It is known that nicotine passively enters cells and recent studies suggest that nicotine also enters organelles such as endoplasmic reticulum (ER). In ER, nicotine may pharmacologically chaperone nascent nicotinic acetylcholine receptors (nAChRs), matchmaking subunits as pentameric receptors assemble. Other studies suggest additional potential sequelae of intracellular nicotine-nAChR interactions: decreasing unfolded protein responses, escorting other proteins from ER, or abducting proteins to abnormal pathways. The proposed mechanism is inside-out, because it begins in the ER rather than on the plasma membrane. Nicotine's sustained inside-out effects proceed at concentrations much lower than its transient activation of plasma membrane nAChR channels. The project will invent new techniques to measure and control the initial steps in inside-out nicotinic pharmacology. We will develop NanoSIMS for measuring drug binding, and we will develop compartmentalized nicotinic ligands. We will also employ other state-of-the-art techniques: reconstitution of COPII vesicle budding, and FRET. Sub-Approach A invents tools measuring the compartmentalization of nicotine action. Sub-Approach B invents tools for confining pharmacology to the ER, both for nicotine and for the clinically important alpha4beta2 nAChR-selective ligand, varenicline. Sub-Approach C tests for interactions between nAChRs and candidate genes discovered by previous experiments. Inside-out pharmacology is a transformative concept that may reveal new therapeutic targets for addiction and neurodegeneration.

DESCRIPTION (provided by applicant): Manufacturers add menthol as a flavoring in many tobacco products and in electronic cigarettes. Some smokers who use menthol find it more difficult to quit. Most suggestions about the basis for this phenomenon assume that menthol affects the pharmacokinetics of nicotine, but this explanation has several unsatisfactory aspects. The project explores a novel and transformative idea that menthol actually acts within cells, to modify the biosynthesis, stoichiometry, endoplasmic reticulum (ER) exit, trafficking, or membrane insertion of nicotinic acetylcholine receptors (nAChRs). Preliminary evidence is consistent with the idea that menthol, at submicromolar concentrations, is a chemical chaperone for both ?4?2 and ?6?2?3 nAChRs. The project expands the present measurements and also extends the studies to ?4?2 and ?6?2?3 nAChRs. An important underlying principle is the chaperoning generalization, which states that experiments in isolated cell lines are quite relevant for chaperoning processes in the neurons and in brains. Aim 1 distinguishes between acute (10 min) and chronic (24 hour) effects of menthol on the plasma membrane, including dose-response relations and waveform analysis for ACh and nicotine. A chaperoning effect is quite unlikely in 10 min but quite possible in 24 h. Aim 2 provides the mechanism of chronic effects of submicromolar menthol, by examining the entire pathway from synthesis of nAChRs in the ER, exit from the ER, cycling between Golgi and ER, nAChR movements within cells, to eventual plasma membrane (PM) composition of nAChRs. The experiments will proceed by analogy with the presumably more specific pharmacological chaperone effects of submicromolar nicotine. The effects of nicotine are under extensive study, and the methods adapt themselves well to examine the hypothesis that menthol is a chemical chaperone. Nearly all the tools and techniques now exist in our laboratory, requiring no extensive development. As in Aim 1, we will study any interactions between nicotine and menthol concentrations. Aim 2a Describes effects on functional nAChRs at the PM. The experiments utilize dose-response relations and for ACh itself and nicotine, waveform analysis, and superecliptic pHluorin measurements of PM and ER localization. Also, a novel set of measurements on fluorescent receptors in isolated plasma membrane will provide precise PM stoichiometry and subunit order within the assembled pentamer. Aim 2b then continues to define several additional possible effects of menthol on the cell biology of nAChRs, as the nAChRs are processed within cells. Experiment 2b1 measures menthol-produced changes to total / ER nAChR levels, using SEP-tagged nAChRs. We will also employ selective biotinylation (intact vs permeabilized cells) to determine the ratio of PM to total nAChRs. Experiment 2b2 monitors changes in intracellular stoichiometry, using F?rster resonance energy transfer (FRET). Experiment 2b3 studies changes in ER exit sites (ERES), a general assay for increased flux out of the ER. This experiment will be conducted both with and without expressed nAChRs, to test the generality of the proposed molecular chaperone effect. Experiment 2b4 monitors changes in COPI interactions, an assay for the importance of cycling between Golgi and ER. Experiment 2b5 monitors changes in the dynamics of intracellular nAChRs, using total internal reflection microscopy (TIRFM) video analysis. The data may provide novel insights about the actions of menthol on nAChRs, showing the details, limits, or alternatives to the chemical chaperone mechanism. The data will also shed light on menthol effects on the cell biology of other membrane proteins. With the data from this project in hand, a future R01 project can perform new sets of experiments on the effects of chronic menthol in cultured neurons, in brain slices, in neuronal circuits, in animal models, and in behavioral assays.

DESCRIPTION (provided by applicant): This project responds to several sections in Center for Tobacco Products Research Plan. Nicotine dependence (Tobacco Use Disorder, DSM-5, 305.1, and Tobacco Withdrawal, 292.0) is the tobacco-related disorder that underlies all other tobacco-related diseases. Establishing a biomarker for nicotine dependence in animals, and then exploiting this biomarker for information on menthol's actions, will contribute importantly to tobacco control research. A biomarker relevant to distinct components of human nicotine dependence must be localized at the level of brain region, of individual cell types, and of axonal vs. somatodendritic compartments. The biomarker must develop during maintained exposure to nicotine. Furthermore, this biomarker should immediately be exploited for studies on menthol. The biomarker chosen will consist of detecting the total level of beta2 nicotinic acetylcholine receptor (nAChR) subunits (intracellular plus plasma membrane). Mouse brain is an entirely appropriate model system. The approach develops, and then begins to exploit, a production-level assay for determining the upregulation of beta2* nAChRs in mouse brain. Maintained exposure to nicotine produces upregulation of beta2* nAChRs, and such upregulation is hypothesized to be both necessary and sufficient for some early stages of nicotine dependence. Aim 1 develops a rather efficient, quantitative, but anatomically low-resolution approach, refining an immunoblot technique (western blots) for quantifying the amount of beta2 subunits in various regions of mouse brain. The approach will lead to valuable data. Aim 2 develops a higher-resolution, semiquantitative approach that identifies the neuronal cell types in which upregulation occurs, as well as the subcellular regions (axonal vs. somatodendritic) in which upregulation occurs. Aim 2 employs a recently developed strain of beta2-GFP knock-in mice. Most of Aim 2 uses direct fluorescence for the GFP-labeled beta2 subunit; immunofluorescence is used to enhance sensitivity. Aim 2 is not on the production pathway for further work, but the data of Aim 2 are the minimum necessary for understanding and validating the beta2 biomarker. Aim 3 uses these tools to test an important hypothesis: that menthol upregulates nAChRs even when delivered intravenously. The underlying assumption is that menthol increases tobacco dependence via events at the level of neurons, in addition to possible increased absorption of nicotine through airway epithelium or possible decreased nicotine catabolism. The overall results of this project will be a production-level procedure that can be used to assess levels in the beta2 subunit biomarker as a function of key experimental variables that interest the Center for Tobacco Products. Furthermore Aim 3 will use the beta2 biomarker to begin answering the important question, where does menthol act? FDA requires this key information in order to evaluate / measure menthol. Future projects can test additional variables including flavorings, additional possible addictive components such as cotinine, and other compounds in tobacco smoke. The beta2 biomarker assay will also be appropriate for mice engineered to possess alterations in accessory proteins. Therefore this project responds to the RFA with high impact, high significance, and a highly appropriate approach.

Drug development for the central nervous system (CNS), especially for psychiatry, has slowed, partially because we do not know the mechanisms by which some drugs exert their therapeutic or harmful effects. The project provides data to test the hypothesis that several CNS drugs act, in addition to their acute effects, in a slower, ?inside-out? fashion. The drugs would start by binding to their classical molecular targets, but in organelles. By measuring neural drugs, and their target interactions, within organelles of living cells, this project helps to test inside-out pharmacology. The experiments invent, then exploit, genetically encoded fluorescent biosensors to measure drugs in organelles. The biosensors are bacterial and archaeal periplasmic binding proteins (PBPs), fused to circularly permuted green fluorescent protein (cp-GFP). Sub-Approach A is a solution-based screen of drugs x existing biosensors. The library of 92 compounds includes many orally available drugs approved for various indications, but emphasizing psychiatry. The collection of 60 purified biosensor proteins comprises five existing families, which now sense glutamate, dopamine, GABA, and serotonergic drugs. Sub-Approach B utilizes ?directed evolution? to improve the ?hits?, toward the goal of detecting the drugs at pharmacologically appropriate sub-micromolar concentrations. The major tools?site- saturation mutagenesis, atomic-scale structure, computational docking, and high-through fluorescence screening--are expected to converge on appropriate biosensors. Sub-Approach C expresses the refined biosensors in ER and performs live-cell, time-resolved imaging while the drugs are applied extracellularly. We begin with the simple questions, ?does the drug enter the ER, and how quickly?? We then analyze signals within organelles that also express the classical targets for the drugs. We expect a rich set of data on ?kinetic buffering? of diffusion by binding to the targets within ER, thus revealing drug-receptor interaction within organelles of live cells. The sub-approach then graduates to mouse preparations, using viral vectors, brain slices, and two-photon imaging in intact animals will be employed. Sub-Approaches D and E complement each other. D extends subcellular pharmacokinetics to acidic organelles, including secretory granules and neurotransmitter vesicles already suspected of accumulating drugs via ?acid trapping?. We'll retain the PBP portions of the biosensors, but employ additional cp-fluorescent proteins, known to function at low pH, and also modify linkers. The result will become a collection of fluorescent biosensor platforms, each specialized to perform best within, and targeted to, a class of organelles. Sub-approach E extends the drug biosensor strategy to new classes of PBPs, and to new classes of drugs. We will retain the cp-fluorescent protein part of the biosensors, but optimize the new PBPs and linkers. The transformative overall results will produce at least ten, and as many as 100, biosensors to detect drugs within organelles, and a clear roadmap for subcellular pharmacokinetics as a robust research tool. Data could suggest transformative therapeutic strategies for psychiatry, addiction, and neurodegeneration.

The project innovatively extends nicotine pharmacokinetics to the subcellular level. This is important because nicotine enters neurons, is then thought to enter the endoplasmic reticulum (ER), and then acts as a pharmacological chaperone for several types of nicotinic acetylcholine receptors (nAChRs). The downstream effects are ?inside-out? pharmacology?. Inside-out effects are thought to lead to some aspects of nicotine dependence, as well as neuroprotection by nicotine. However, three key questions remain. We have developed innovative tools for this study. iNicSnFR1 and iNicSnFR2 are genetically encoded, ?intensity- based nicotine-sensing fluorescent reporters?. In test tubes, at nicotine concentrations near those in the brain of a smoker, iNicSnFRs detect nicotine within < 1 s. Preliminary data in cells transfected with iNicSnFRs answer part of Question 1, affirmatively: nicotine enters the ER within a few s after nicotine is applied near an isolated cell, then leaves within a few s after nicotine is washed away. We use adeno-associated viral vectors that express iNicSnFRs. Question (1). There has been no direct proof for the central aspects of inside-out pharmacology: that nicotine enters the ER, that nicotine binds to nAChRs in the ER, and that this binding produces transitions to more tightly binding, chaperoned states of nAChRs. Aim 1 addresses Question 1, by continuing to develop and apply the iNicSnFR tools quantitatively, in mammalian clonal cell lines and in cultured neurons. We will verify the selective targeting of iNicSnFRs to either the ER or the plasma membrane (PM), using membrane-permeant vs impermeant compounds. We then employ iNicSnFR signals in the ER to measure kinetic buffering of nicotine, when it binds to heterologously expressed nAChRs in the ER. Multiple controls and comparisons are used to modify this binding by changing the subcellular localization of nAChRs, their binding affinity for nicotine, and the extent to which they undergo transitions to higher affinity. We also attempt to measure kinetic buffering by endogenously expressed nAChRs, in cultured dopaminergic neurons. We then image the localization of the iNicSnFR signals at greater resolution, to ask whether buffering occurs at sub-regions of ER. Question (2). The time course of nicotine entry and exit from various brain compartments after a pulse of nicotine (for instance, during smoking) is not known. Aim 2 addresses Question 2 by studying the iNicSnFRs expressed in mice. We study iNicSnFR in brain slices from these mice, to provide additional evidence of intended localization. We also study live mice. After a pulse of nicotine is introduced by peripheral injection, we measure the extent and time course of nicotine concentration in the ER. The experiment employs fiber photometry. We will compare these measurements with those for PM-localized iNicSnFR constructs, and with conventional measurements of nicotine in blood and CSF. Question (3). After nicotine accumulates in the acidic lumen of synaptic vesicles by the expected factor approaching ~ 100, is nicotine then released by presynaptic impulses? Aim 3 addresses Question 3. In the high- risk spirit of an R21, answering Question 3 is not on the project's critical path to success, but would enhance the tools' usefulness. Using the brain slice preparation, we ask whether iNicSnFR can detect when presynaptic stimulation releases nicotine from synaptic vesicles. Note that this accumulation and release is not thought to be restricted to cholinergic synaptic vesicles. Answering Questions 1, 2, and 3 prepares for additional research. After decades of uncertainty, we will finally have the answer to the question, which nicotine dose and time course should be used in testing the details of inside-out nicotine action? The measurements from mice can be extrapolated, using standard allometric assumptions, to the brains of smokers or vapers.

The project studies ketamine, its metabolites, and related drugs [Ketamine-Class Antidepressant Drugs (KCADs), our term]. Sub-anesthetic doses of ketamine produce antidepressant effects in just a few hours (2 h) via unknown mechanism(s). However, higher doses have adverse effects. Understanding the mechanism mediating KCAD antidepressant activity is an important step in the process of drug development. This research program has the goal to fundamentally change our understanding of how this rapid antidepressant mechanism works and holds promise for development of more robust and safer treatments. The molecular target(s) of KCAD action are not known. In the absence of such knowledge, one should investigate possible actions in the compartments where KCADs are most concentrated. We test the hypothesis that the effects of KCADs in the brain are mediated, in least in part, by the accumulation of KCADs in various subcellular compartments (organelles), including synaptic vesicles. Aim 1 develops a family of next-generation genetically encoded ?Intensity-based Ketamine-Sensing Fluorescent Reporters? (iKetSnFRs) for KCADs. These will dynamically image and quantify the presence of KCADs at sub-cellular levels. Aim 1 measures the time course of KCAD entry and exit from various organelles after the drugs appear or disappear near cells. Aim 2 tests the hypothesis that the antidepressant mechanism of KCADs involves accumulation in the lumen of acidic vesicles, especially in synaptic vesicles, followed by synaptic stimulation-induced release of KCAD from presynaptic terminals. Aim 3 detects KCAD-induced neurotransmitter release from presynaptic terminals, employing next generation genetically encoded biosensors for various neurotransmitters. The experiments also include electrophysiological studies of membrane and synaptic properties. The data from Aims 1, 2 and 3 will not in themselves develop a new ~2 h antidepressant drug. But the data from the proposed experiments can help to understand how KCADs exert their effects. The data will also guide the development of related molecules with fewer potential side effects as new fast-acting therapies for depression.

Biological membranes are permeable to exogenous opioid drugs--both plant-derived molecules such as morphine, and synthetic molecules of which hundreds exist. Now a genetically encoded fluorescent biosensor technique allows us to measure opioids within neutral organelles such as the endoplasmic reticulum (ER). We term these molecules the intensity-based opioid-sensitive fluorescent reporter, iOpioidSnFR, family (Figure 1). Ongoing experiments, before the project begins, will extend the iOpioidSnFR family to the major classes of µ-opioid agonists. Aim 1 Further extends the iOpioidSnFRs for measurements within acidic organelles such as endosomes and synaptic vesicles. Aim 1a utilizes the present circularly permutated green fluorescent protein (cpGFP) moiety. Aim 1b develops novel circularly permuted HaloTags, which are pH-insensitive. Aim 1c, Extends the existing measurements to measure the entry of opioids into organelles, and their exit from organelles. Quantification involves both dynamics and steady- state measurements. Aim 2 tests the hypothesis that some effects of opioid drugs result after synaptic vesicles accumulate opioids via acid trapping. The synaptic vesicles would then release the opioids upon presynaptic stimulation. This mechanism would extend the patho-pharmacology of exogenous opioids to their release from many types of presynaptic neurons?even those neurons that do not release endogenous opioid peptides. Aim 2a evolves iOpioidSnFR sensitivity further, to the required nanomolar levels. Aim 2b Identifies the most sensitive method for testing presynaptic release. Aim 3 tests the hypothesis that brain regions expressing µ-opioid receptors vary in the extent and timing of organellar opioids. Aim 3a generates adeno-associated viral vectors that encode ?floxed? iOpioidSnFRs. These will be expressed under the control of vesicular GABA transporter (vGAT) cre recombinase in suitable mouse lines. Aim 3b measures in brain slices from ventral tegmentum area (VTA) / substantia nigro pars reticulata (SnR), periaqueductal gray (PAG), and ventral pallidum (VP).The results will aid in the ongoing efforts to understand the cellular and molecular basis of tolerance to µ-opioid ligands.