William N. Green - US grants

9319656 Green Central to the mechanisms that underlie central nervous system function are the different neurotransmitter receptors responsible for the rapid signaling between neurons and between nerve and muscle. These different neurotransmitter receptors are membrane proteins composed of several subunits, which are structurally similar and form a single large molecular family. While much is known about how these receptors function, little is known about how the subunits fold into the correct conformation and assemble together to form the receptors. To address these questions, the research will focus on the best characterized member of this receptor family, the nicotinic acetylcholine receptor. A series of experiments will identify specific chemical processes that are part of subunit assembly and identify specific regions on the subunits responsible for interactions between subunits. ***

Central to synaptic transmission are the family of ionotropic neurotransmitter receptors, which are responsible for the rapid responses to neurotransmitters in nerve and muscle. In this proposal, a class of receptors that are members of this family, the neuronal nicotinic acetylcholine receptors (AChRS), will be studied. As the site where nicotine binds in the brain, these receptors are responsible for nicotine addiction and may also play a role in neurodegenerative diseases such as Alzheimers disease. Neuronal AChRs are composed of a number of subtypes as classified by their diverse pharmacology and distribution. Further evidence of the diversity has been the cloning of a large number neuronal AChR subunit isoforms. While neuronal AChRs are found throughout the central and peripheral nervous system, neither the subunit composition nor the function of single nAChR subtype is known. The neuronal alpha-bungarotoxin binding receptor (BuTxR), a neuronal AChR subtype, has been selected for study because recent studies suggest that these receptors are a Ca2+ influx pathway, which at presynaptic sites stimulates neurotransmitter release and underlie nicotine addiction. The first objective is to examine whether BuTxRs are homomers composed only of alpha7 subunits or heteromers composed of subunits in addition to alpha. In addition, the number of BuTx sites per BuTxR will be determined. The next objective is to begin to define the function of BuTxRs. Towards this objective, we will test how permeable BuTxRs are to Ca+2 and if Ca2+ entering through BuTxRs contributes to secretion in PC12 cells. We will also address why so few BuTxRs functional in our undifferentiated PC12 cells and test the hypothesis that the number of functional BuTxRs are regulated. The last part of the grant is concerned with questions about BuTxR expression. Heterologous expression of alpha7 homomers in mammalian cells is poor to nonexistent. In contrast, alpha7/5HT3 chimeric subunits readily express as homomers in mammalian cells. Using alpha7/5HT3 chimeric subunits, we will determine alpha7 subunit regions that prevent homomer formation. In PC12 cells,different subunit processing at folding events will be characterized and their effect on BuTxR expression tested. Finally, we will further characterize intracellular BuTxR subunit pools, which appear to be precursors of the cell-surface BuTxRs.

molecular biology; biomedical facility; nucleic acid sequence; plasmids; transfection; site directed mutagenesis; molecular cloning; Xenopus oocyte;

DESCRIPTION(Adapted from applicant's abstract): Tobacco smoking is a major health problem leading each year to $50 billion dollars in health costs and 400,000 deaths in the United States. Significant evidence indicates that nicotine is the component in tobacco leading to abuse and addiction. Nicotine binds to nicotinic receptors found in the brain. To characterize the molecular biology of nicotine addiction, our research will focus on nicotinic receptors in the brain and how nicotine-induced changes in nicotinic receptors correlates with aspects of addiction. Like other drugs of abuse, many of the addictive effects of nicotine appear to result from the stimulation dopaminergic, mesolimbic neurons, in particular dopaminergic neurons from the ventral tegmental area (VTA). The short-term effects of nicotine are caused by the activation of nicotinic receptors on these neurons. Long-term exposure to nicotine enhances or "sensitizes" the response of these neurons to nicotine, which correlates with the enhancement or "upregulation" of high-affinity binding to nicotinic receptors. The goals of this application are: 1) to identify receptor subtypes involved in nicotine upregulation, 2) to identify the mechanisms that cause nicotine upregulation of nicotinic receptors and 3) to test whether nicotinic receptor upregulation by nicotine is involved in the sensitization of the response to nicotine. The hypotheses to be tested are that: 1) at nicotine concentrations achieved during smoking, the upregulation of nicotinic receptors is primarily an upregulation of alpha4beta2-containing receptors; 2) nicotinic receptor upregulation in vivo is caused by a receptor state change that causes the receptors to enter a hypersensitive state; 3) sensitization of the nicotine response of VTA neurons is caused, at least in part, by receptor upregulation. Several possible nicotinic subunit combinations will be expressed to test whether upregulation occurs for each combination and the extent to which upregulation is a change in receptor number or entry into a hypersensitive state. After, the experiment will be extended to cells expressing "native" nicotinic receptors of unknown subunit composition, including PC12 rat pheochromocytoma cells, mouse N1E-115 neuroblastoma cells, and VTA neurons.

DESCRIPTION (provided by applicant): Support is requested for the 56th Annual Symposium of the Society for General Physiologists to be held at the Marine Biological Laboratory in Woods Hole, MA on September 5-7, 2002. The 2002 Symposium, "The Trafficking of Transporters," will present and discuss recent progress in the area of the biogenesis, targeting and degradation of transport proteins, e.g., ion channels, receptors and transporters. The conference will focus on both normal cell biological events and pathological conditions that can lead to "folding" diseases such as Cystic Fibrosis and a number of neurodegenerative diseases. This symposium is unique in bringing together leading cell biologists and neurobiologists from diverse areas of expertise to focus on the subject of the cell biology of transport proteins. At the symposium, members of the Society of General Physiologists, who primarily study the structure and function of transport proteins, will interact with cell biologists and neurobiologists, who study transport proteins from a different perspective. Thus, we hope to attract a broad audience of cell biologists and cellular/molecular neurobiologists as well as physiologists.

DESCRIPTION (provided by applicant): Central to the mechanisms that underlie central nervous system function and disease are the different neurotransmitter receptors responsible for the rapid signaling between neurons and between nerve and muscle. Like other oligomeric membrane proteins, these neurotransmitter receptor subunits are synthesized and inserted into the membrane of the endoplasmic reticulum (ER) after which the receptor subunits undergo a series of folding and processing steps. The folding and processing steps that occur in the ER transform the individual subunits into mature neurotransmitter receptors before the receptors reach the cell surface. A critical part of the folding and processing that occurs in the ER are the 'quality control' mechanisms that identify, retain and degrade misfolded or misassembled receptor subunits. While much is known about how these receptors function, relatively little is known about how the receptor subunits fold and assemble into functional receptors. The long-term objective of the proposed research is to understand the mechanisms governing the quality control mechanisms in the ER that ensure that only properly assembled and functional receptors are expressed on the cell surface. To achieve this objective, studies will focus on the best-characterized member of this receptor family, the 'muscle-type nicotinic acetyicholine receptor (AChR). The first set of experiments will characterize the interactions between AChR subunits and a specific set of ER resident proteins called chaperone proteins. The second set of experiments will identify the mechanisms involved in the degradation of AChR subunits and determine how subunit degradation affects AChR assembly. The final set of experiments will test whether pathology caused by certain AChR subunit mutations identified in congenital myasthenic syndromes results from defects in AChR assembly or quality control. As the site where nicotine binds in the brain, neuronal AChRs are responsible for nicotine addiction and may play a role in Alzheimer's disease. In addition, the 'muscle-type' AChR is responsible for myasthenia gravis, the autoimmune disease, and many of the congenital myasthenic syndromes both of which cause deterioration of neuromuscular junctions. The level of AChR expression appears to be altered in all three disorders. Since our goal is to elucidate the molecular basis for AChR expression, this proposal should provide insights into certain pathological features of these disorders.

DESCRIPTION (provided by applicant): Central to synaptic transmission are the family of ionotropic neurotransmitter receptors, which are responsible for the rapid responses to neurotransmitters in nerve and muscle. In this proposal, a class of receptors that are members of this family, the neuronal nicotinic receptors, will be studied. As the site where nicotine binds in the brain, these receptors are responsible for nicotine addiction and may also play a role in neurodegenerative diseases such as Alzheimer's disease. Neuronal AChRs are composed of a number of subtypes as classified by their diverse pharmacology and distribution. One such subtype, the neuronal a-bungarotoxin binding receptor (BgtR) will be studied in this grant. We have found that that BgtRs are composed only of nicotinic a7 subunits and functional expression of a7 BgtRs only occurs in cells of neuronal origin. The first objective is to identify and characterize the neuronal-specific processing required for BgtR expression. The identification of posttranslational events that regulate BgtR expression in a neuronal-specific manner should provide insights into how other receptors and channels are regulated. The other objective is to determine the number of Bgt binding sites on BgtRs and to begin to characterize the BgtR Ab peptide binding sites. Ab peptides, which are play a critical role in the onset of Alzheimer's disease, bind to BgtRs, altering BgtR function and trigger second messenger changes within neurons. The hypotheses to be tested are that: 1) a7 subunit palmitoylation is one of a series of processing events required for expression of functional BgtRs; 2) there are four ligand binding sites on each BgtR and the sites can be distinguished by different affinities for antagonists; and 3) Ab peptides bind to a site on the BgtR extracellular surface different from the ligand binding sites.

DESCRIPTION (provided by applicant): Evidence is mounting that the post-translational process, palmitoylation, has a major role in neuronal development and function, in particular, the formation and functioning of synapses. The standard methods for assaying protein palmitoylation are relatively insensitive, not quantitative, as well as laborious. Given these difficulties, it is apparent that many palmitoylated proteins remained unidentified. We have recently developed alternative assays of protein palmitoylation based on hydroxylamine cleavage of the thioester bond between the fatty acid and cysteine side chain followed by reaction of the newly generated free sulfhydryl with sulfhydryl-specific reagents, such as 3H-N-ethyl maleimide (NEM) and biotinylated reagents. These techniques are significantly more sensitive than metabolic labeling with 3H-palmitate and can be used quantitatively to measure levels of protein palmitoylation. They have the additional advantage of allowing protein palmitoylation to be assayed on preparations from brain and other nervous tissue, which is not possible using [3H]-palmitate labeling. The goal of this application is to adapt our new assays for palmitoylation to a proteomic scale in order to accomplish the following two specific aims. First, we propose to modify our techniques in order to identify the specific sites of palmitoylation on the different ionotropic glutamate receptors we are currently studying in our laboratory and all of which we have found to be palmitoylated. Second, we are developing protocols that allow a set of palmitoylated proteins to be specifically purified from highly complex protein extracts in order to quantify protein palmitoylation differences in neuronal preparations. To test these techniques, two different preparations will be analyzed. First, we will isolate palmitoylated proteins from rat cultured cortical neurons and assay for changes in their palmitoylation when long-term potentiation (LTP) has been chemically induced. In the second preparation, we will isolate palmitoylated proteins from rat nucleus accumbens punches and test for changes in their palmitoylation with amphetamine exposure in order to determine how behavioral sensitization alters palmitoylation. The proposed research is innovative because it will develop new proteomic-based technology to assay a protein post-translational modification that should provide new insights into basic synaptic biology and the neurotransmitter receptors, such as gluatamate receptors that are involved in drug abuse. PUBLIC HEALTH RELEVANCE: Protein palmitoylation is a reversible lipid modification on cysteine (Cys) residues and is involved in targeting proteins to membrane domains. The regulation of this modification has been shown to play a significant role in synapse formation and function. The large scale analysis of palmitoylated proteins will provide a comprehensive view of the scope of this modification by identifying many unknown protein targets, which will promote the development of subsequent hypothesis-driven research.

DESCRIPTION (provided by applicant): Tobacco smoking is a leading cause of preventable deaths in the United States and worldwide. Understanding why tobacco smoking is highly addictive and identifying the mechanisms underlying addiction would be of tremendous benefit in developing new therapies for smoking cessation needed for preventing addiction. Nicotine is the primary agent in tobacco leading to addiction. Addiction is initiated by nicotine binding to nicotinic acetylcholine receptors (nAChRs) in the brain. High-affinity nAChRs, mainly ?4ß2 nAChRs in the brain's reward areas, mediate the reinforcing effects of nicotine. The only long-lasting effect, i.e., greater than a few minutes, of nicotine directly on nAChRs is a phenomenon called nicotine-induced upregulation of nAChRs (upregulation). We have demonstrated that nAChR upregulation is much more complex than originally assumed consisting of multiple processes that occur at very different rates and are caused by different mechanisms. One goal of the proposed research is to characterize in more detail the two components of nAChR upregulation that we have recently discovered. In the first Aim, we will examine how nicotine exposure increases nAChR endocytosis and recycling of ?4ß2 nAChRs in neurons and heterologous cells. In the second Aim, we will test in more detail how nicotine exposure regulates the glycosylation of ?4ß2 nAChRs. Another goal of the proposed research is to examine how nAChR ligands and smoking cessation agents affect the different components of upregulation. These experiments will be performed in the third Aim.

DESCRIPTION (provided by applicant): Tobacco smoking is a leading cause of preventable deaths in the United States and worldwide. Understanding why tobacco smoking is highly addictive and identifying the mechanisms underlying addiction would be of tremendous benefit in developing new therapies for smoking cessation needed for preventing addiction. Nicotine is the primary agent in tobacco leading to addiction. Addiction is initiated by nicotine binding to nicotinic acetylcholine receptors (nAChRs) in the brain. High-affinity nAChRs, mainly ?4ß2 nAChRs in the brain's reward areas, mediate the reinforcing effects of nicotine. The only long-lasting effect, i.e., greater than a few minutes, of nicotine directly on nAChRs is a phenomenon called nicotine-induced upregulation of nAChRs (upregulation). We have demonstrated that nAChR upregulation is much more complex than originally assumed consisting of multiple processes that occur at very different rates and are caused by different mechanisms. One goal of the proposed research is to characterize in more detail the two components of nAChR upregulation that we have recently discovered. In the first Aim, we will examine how nicotine exposure increases nAChR endocytosis and recycling of ?4ß2 nAChRs in neurons and heterologous cells. In the second Aim, we will test in more detail how nicotine exposure regulates the glycosylation of ?4ß2 nAChRs. Another goal of the proposed research is to examine how nAChR ligands and smoking cessation agents affect the different components of upregulation. These experiments will be performed in the third Aim.

Abstract Tobacco continues to be widely used world-wide, primarily via cigarette smoking, and is the leading cause of preventable deaths in the United States. Tobacco use is driven by nicotine addiction, which starts by nicotine binding to high-affinity nicotine binding sites in brain. 80-90% of the high-affinity sites are located on ?4?2-type nicotinic acetylcholine receptors (?4?2Rs). Prolonged nicotine exposure increases high-affinity ?4?2R binding sites in brain, a process termed ?upregulation?, linked to craving and withdrawal in nicotine addiction. This proposal is based on our recent discovery that ?4?2R ligands that are weak bases, such as the smoking cessation reagent varenicline (Chantix), can be selectively trapped in ?4?2R-containing acidic vesicles of cells and neurons. Slow release of trapped varenicline reduces the effects of nicotine upregulation. Selective trapping is further regulated by nicotine upregulation, which increases the numbers of ?4?2R-containing acidic vesicles. Nicotine, also a weak base, is not trapped because its ligand pKa and affinity for ?4?2Rs is lower than that of varenicline. These results provide a new paradigm for how varenicline causes smoking cessation. They also provide new information about the potential cellular distribution of??4?2R PET probes, all of which are weak bases. Like varenicline and nicotine, different ?4?2R PET probes have different ligand pKas and affinities for ?4?2Rs, which explains differences in kinetics, displaceable binding by varenicline and nicotine, non-displaceable binding and metabolism. While a number of studies have used PET probes specific for ?4?2R high-affinity binding sites in brain, these studies are complicated by the interpretations of the binding and binding kinetics especially when nicotine and/or varenicline are present. Using our concept about the trapping of ?4?2R weak base ligands in intracellular acidic vesicles, we will develop new cellular and whole models of PET probe kinetics that take into account ?4?2R ligand trapping in acidic vesicles. There is the potential of wider application of the PET methods that will be developed in this application, since for ?4?2R PET imaging is currently underway in a number of brain disorders. The goals of this proposal are to examine how our discovery of the trapping of weak base ?4?2R ligands in acid vesicles affects the imaging of ?4?2Rs using PET probes and to use PET probe imaging to examine how nicotine causes ?4?2R upregulation and how varenicline alters upregulation.