Paul Greengard, Ph.D. - US grants

It is proposed to study the roles of cyclic AMP-dependent, cyclic GMP-dependent, and calcium-dependent protein kineases and their substrates in normal and abnormal brain function and in the mechanism of action of antipsychotic drugs. The specific program to be undertaken involves studies in several areas: (1) to search possible regulation by neurotransmitters and psychoactive drugs of the sate of phosphorylation of protein kinase substrates in slices prepared from various brain regions; (2) to prepare pure cyclic AMP-, cyclic GMP-, and calcium-dependent protein kinases, and their endogenous substrates; (3) to prepare antibodies to the protein kinases and their substrates, to develop radioimmunoassay procedures for the protein kinases and their substrates using these antibodies, and to determine the amount of the protein kinases and their substrates present (a) in various regions of the brain, (b) at various times during development of the brain, (c) after chronic treatment with various psychoactive drugs, and (d) in brain samples from normal and mentally-ill human subjects, using these radioimmunoassay procedures; (4) to identify, purify and characterize the phosphoprotein phosphatases for the endogenous protein kinase substrates in brain; and (5) to develop procedures for measuring the amount of mRNA for the endogenous substrates and to study the effects of various physiological and pharmacological manipulations on the amount and rate of transcription of the mRNA for these substrates.

phosphoproteins; phosphorylation; basal ganglia;

It is proposed to study the role of cyclic AMP, calcium and protein phosphorylation in the peripheral nervous tissue. One approach is to determine the effect of neuronal activity on the state of phosphorylation Protein I in the superior cervical ganglia. A second approach is to determine the site of such phosphorylation in the Protein I molecule. A third approach is to determine the effect of impulse conduction on the state of phosphorylation of Protein I in the vas deferens. A fourth approach is to determine the site of such phosphorylation in the Protein I molecule. A fifth approach is to carry out cytochemical and radioimmunoassay studies, at the light and electromicroscope levels, on the distribution in the peripheral nervous system of Protein X, a protein found in mammalian brain.

Protein III is a synaptic vesicle-associated neuronal phosphoprotein which has been shown to be phosphorylated in intact nerve cell preparations by electrical stimulation in the presence of calcium, by depolarization in the presence of calcium, and by exogenous 8-bromo-cAMP. These and other data have led to the hypothesis that protein III pays some role in synaptic vesicle function. Protein III has recently been studied in the brain of normal and alcoholic humans. Protein III from the brains of normal humans and of alcoholics who died intoxicated (blood alcohol levels = more than 50mg/100 ml) appeared to migrate normally (i.e., protein IIIa was Mr=74,000 and protein IIIb was Mr=55,000) in SDS-polyacrylamide gels. However, in the brains of alcoholics who died sober (no detectable blood alcohol) variant forms of protein III (protein IIIa, Mr=76,000 and 78,000; protein IIIb, Mr=57,000 and 59,000) were seen in all brains examined (n=12). These data raise the possibiity that these variant forms of protein III may reflect sme underlying proces of significant import in alcoholism. The primary purpose of this proposal is to investigate protein III in terms of its role in normal neuronal function as well as its possible relationship to the neuropathology associated with alcoholism. The first step in this proposal involves purification and characterization of protein III in bovine brain and in the brains of normal and alcoholic humans. Molecular biological studies of the protein will then be conducted in order to determine the amino acid sequence of protein III in bovine brain and in the brains of normal and alcoholic humans. These studies will also be designed to anzlyse the synthesis and expression of protein III in bovine and human brain. Protein III will also be studied in human cerebrospinal fluid.

It is proposed to study the role of protein phosphorylation in neurotransmitter release from presynaptic terminals in mammalian brain and the effects of psychoactive drugs on this system. This proposal will focus on two neuronal phosphoproteins: synapsin I, a synaptic vesicle-associated protein that is phosphorylated at specific sites by cAMP- and Ca/calmodulin-dependent protein kinases, and "87k" protein, a cytosolic protein that is specificallly phosphorylated in nerve terminals by protein kinase C. The pharmacological regulation of the phosphorylation of synapsin I and 87k will be studied with standard endogenous ATP labelng and "back-phosphorylation" techniques in brain slice and brain synaptosome preparations using agents that affect presynatpic receptors. We will sequence the regions on synapsin I and 87k containing the phosphoacceptor sites. We will then synthesize peptides of these sites and raise antibodies specific for either the phospho or de-phospho forms of these peptides. We will study the effect of these phosphosite peptides and antibodies (a) on the interaction of synapsin I with synaptic vesicles and (b) on neurotransmitter release from synaptosomes in "microinjection" experiments. We will isolate and characterize the synaptic vesicle component to which synapsin I binds. A "microinjection" technique that we have just developed will be used to introduce into brain synaptosomes molecules that modulate the phosphorylation of either synapsin I or 87k, and appropriate phosphosite peptides and antibodies. Neurotransmitter release will be studied in these same preparations under those conditions found to regulate the phosphorylation of these two phosphosubstrates. We will study the effects of several classes of psychoactive drugs on the phosphorylation of synapsin I and 87k and on neurotransmitter release. Also, brains and CSF from schizophrenic individuals and individuals with affective disorders will be examined for any changes in synapsin I or other phosphoproteins.

The research supported by our current grant has investigated the role of protein phosphorylation in the regulation of neurotransmitter release. Specifically, the data obtained from a variety of experimental approaches have provided support for a model by which synapsin I, a synaptic vesicle- associated phosphoprotein, regulates neurotransmitter release by affecting the movement of synaptic vesicles from a "reserve" pool to a "readily-releasable" pool. We have further demonstrated that the phosphorylation state of synapsin I regulates this biological function of the protein. We have established that synapsin I consists of two polypeptides, synapsin Ia and synapsin lb, which we have cloned and sequenced. The sequencing data indicate that synapsins Ia and lb are highly homologous to another pair of synaptic vesicle-associated phosphoproteins, synapsins IIa and lIb, which we have also cloned and sequenced. The current grant also supported the study of another phosphoprotein, MARCKS (myristoylated, alanine-rich, C-kinase substrate), previously called 87 k protein. This renewal application proposes to study the mechanisms by which the family of synapsin proteins (Ia, lb, IIa, and IIb) regulates neurotransmitter release. To date, most of our experiments have studied the effects of synapsins Ia and lb when present together. We now propose to study the potential functional differences among the four isoforms of synapsin. Using a variety of model neuronal systems, we will introduce each of the four synapsin isoforms alone and in various combinations. We will introduce the synapsins in both phosphorylated and dephosphorylated states, in order to determine the functional effects of phosphorylation on specific sites of the synapsin molecules. We will utilize mutated forms of synapsin to determine functional effects following deletions of phosphorylation sites and other functional domains. The principal measures of function will be neurotransmitter release, the development of facilitation, and the maintenance of long-term potentiation. We will study the molecular mechanisms underlying the function of the synapsins by using systems composed of purified components which have been reconstituted in vitro to examine the interactions between synapsin molecules, synaptic vesicles, and F-actin. Finally, we will study the differential distribution of the synapsins and the regulation of synapsin gene expression because our data indicate (a) that the four synapsin isoforms are differentially expressed in subpopulations of neurons, (b) that the expression of the synapsins can be regulated by transcriptional control via extracellular messengers (e.g., hormones), and (c) that the expression of the synapsins induces formation of presynaptic terminals.

It is proposed to continue a multidisciplinary investigation of basal-ganglia specific neuronal phosphoproteins. The program project for which renewed support is being requested consists of five parts. The first project (Principal Investigator, Dr. Angus C. Nairn) would continue the purification and characterization of phosphoprotein substrates (for cAMP dependent protein kinase) identified during the previous funding period, and develop immunological procedures to measure the state of phosphorylation of these proteins. It also proposes to carry out a detailed characterization of protein phosphatase- 1, since an understanding of the role of this phosphatase is essential to clarifying the role of DARPP-32 in the basal ganglia. The second project (Principal Investigator, Dr. Eric L. Gustafson) would continue studies involving the use of antibodies against phosphoproteins to carry out detailed immunocytochemical studies, at both the light and electron microscopic levels, of the regional, and subcellular distribution of these phosphoproteins. It proposes to study the neuroanatomical framework underlying neurotransmitter-phosphoprotein interactions in the basal ganglia. It would also study the regulation of specific phosphoprotein mRNAs in the basal ganglia, and examine these mRNAs during development. The third project (Principal Investigator, Dr. Paul Greengard) would involve a comprehensive analysis of the pharmacological and physiological regulation of the levels and phosphorylation states of the basal ganglia-enriched phosphoproteins in intact animals and brain slices. It also proposes to introduce electrophysiological techniques to study the physiological roles of protein phosphorylation in the basal ganglia by directly injecting purified phosphoproteins in their phospho and dephospho forms, protein kinases and protein phosphatases into neuronal cells and studying their effects on neuronal activity. The fourth project (Principal Investigator, Dr. Michelle E. Ehrlich) would use recombinant DNA technology to study the expression of the basal ganglia-enriched phosphoproteins, to characterize the human cDNAs for these proteins for use in human molecular genetics studies, and to establish transgenic mice to better understand the function of these proteins in vivo. The fifth project (Principal Investigator, Dr. Kang Tsou) would provide a core facility. Primary tissue cultures of neuronal and glial cells will be grown and maintained for the study of the factors that regulate the expression and phosphorylation state of basal ganglia-specific phosphoproteins. The core facility would also isolate and maintain continuous supplies of monoclonal antibodies against the basal ganglia specific phosphoproteins for the various components of the program project. It is believed that this multidisciplinary approach will provide vital information about the molecular nature of signal transduction in the nervous system as well as provide possible important clues toward the understanding of the etiology of a variety of neuropsychiatric disorders including schizophrenia.

Signal transduction via protein phosphorylation is a major mechanism whereby extracellular stimuli regulate intracellular functions. Protein phosphorylation is of particular importance in the brain: neurons are the body's richest cellular source of protein kinases (protein phosphorylating enzymes) and their substrate proteins. Changes in the phosphorylation state of these substrate proteins typically result in a profound effect on the biology's of those substrate proteins. Alzheimer's disease is the major cause of primary brain failure and is characterized by neurochemical deficits and the accumulation of intraneuronal and extracellular pathological structures. In the Alzheimer brain, there are likely to be important interactions between the structural-neurochemical pathology and the systems of signal transduction via protein phosphorylation. The overall goal of this application is to define and characterize those interactions, with an emphasis on identifying pathways with potential for therapeutic intervention. Specifically, we propose a multidisciplinary approach, from several perspectives. We propose to study the effects of protein phosphorylation on the expression, catabolism, and intracellular trafficking of the Alzheimer amyloid precursor protein (APP), the primary biochemical component of parenchymal and perivascular amyloid deposits. In order to provide independent and parallel evidence for the significance of effects of phosphorylation on APP metabolism, a highly related protein from a divergent species (the APP-like protein from Drosophila) will also be studied. A comprehensive study of the role of protein phosphorylation in APP expression will also be undertaken, with particular emphasis on the APP promoter-binding homeoproteins, several of which are known to be phosphoproteins. A broad and systematic regional survey of protein phosphorylation systems will be performed in rapidly processed human brain, including normal young, normal aged, and Alzheimer tissue. Enzymological, immunochemical, immunocytochemical and molecular biological approaches will be used. A multidisciplinary effort will also be made to determine whether APP, like many other transmembrane proteins, can function to transduce signals across the plasmalemma. Chimeric molecules, joining APP domains with counterparts from characterized transducing proteins, will be expressed in cells, forming the basis for assays of signalling capability. In summary, the experiments proposed in this application are designed to identify interactions of Alzheimer-related neuropathology with the vital neuronal system of signal transduction via protein phosphorylation. We place particular emphasis on identifying potential target pathways by which protein phosphorylation would either regulate the production of Alzheimer- type pathology, or alternatively, pathways of protein phosphorylation which might be aberrant as a result of the disease and whose therapeutic correction might ameliorate the cognitive failure of Alzheimer's disease.

Alzheimer disease (AD) is a characterized by the deposition of cerebrovascular and cerebral parenchymal beta/A4-amyloid and progressive deficiency of effective cholinergic neurotransmission. These two features are the focus of the proposed Drug Discovery Group. Investigators in Project 1 will focus on amyloid precursor protein (APP) processing in brain and cerebral vessels, using synthetic APP peptides and recombinant APP holoprotein and fragments as APPases will be purified and characterized. In addition to these enzymological studies, APP processing will be studied in cultured endothelial cells, smooth muscle cells and platelets. Investigators in Project 2 will also use an APP processing assay in intact cultured cells, rationally choosing compounds based on the cell biology of APP and screening the compounds for efficacy in regulating APP processing. Where lead compounds exist, derivatives will be examined in order to determine structure-activity relationships of the various classes of compounds. Various host cell systems and an in vivo assay in the brain of the freely moving rodent will be used as screens. Complementing the cell biology and biochemical approaches of Projects 1 and 2, investigators in Project 3 will seek an in vivo model of cerebral amyloidogenesis, overexpressing in transgenic mice mutant APPs which are likely to favor amyloidogenesis. Progressive deficiency in cholinergic neurotransmission is another prominent feature of AD. Fine molecular and physiological characterization of the cholinergic system forms another focus of Project 3 and is the primary focus of Project 4. Newly developed antibodies that differentiate brain muscarinic receptor subtypes will be used to analyze levels and distributions of subtypes in the basal forebrain, hippocampus, and neocortex of aged humans. Also along this line, Project 4 will provide details regarding the possible therapeutic opportunity offered by manipulation of galanin, a neuropeptide and functional antagonist of cholinergic neurotransmission. The preservation in AD of galaninergic systems, coupled with loss of cholinergic neurons, suggests that galanin antagonists might be clinically useful. A Core Facility will serve to provide scientific support to the Projects of the Drug Discovery Group. Among the functions of the Core will be the provision of recombinant wildtype and mutant APP and APP fragments, synthetic peptides, protein purification and sequencing, and industrial liaison for compound derivatization and large-scale synthesis. Liaison for legal and patent matters will also be provided as well as the facilitation of timely communication among all members of the Group.

The broad, long-term objective of this proposal is to contribute to our understanding of the molecular basis of Alzheimer disease (AD). This objective will be advanced by characterizing APP in human platelets, by determining how APP is metabolized, and by relating these findings to the pathogenesis of AD. The four specific aims of this proposal are: 1) To study the species (isoforms and/or proteolytic fragments) of APP present in human platelets; 2) To investigate how APP is processed in human platelets, including determining which signal transduction mechanisms initiate processing, and which enzymes catalyze APP cleavage; 3) To establish human platelets as an in vitro model for APP processing with the intention of using it for testing agents that might modulate processing; 4) To study whether processing of APP in human platelets can generate APP metabolites that might be involved in AD-associated amyloidosis. The health-relatedness of this project is its aim of identifying the causes of AD and effective therapeutics for the AD- associated amyloidosis. The experimental design for these studies is to identify APP isoforms and metabolites in human platelets using antibodies with specificities for different regions of the APP molecule as well as antibodies against different isoforms of APP. Metabolism of APP will be studied in platelets with and without stimulation by physiological and pharmacological agents that induce degranulation. The subcellular distribution of APP and APP-metabolites will be localized using subcellular fractionation techniques. Phosphorylation and dephosphorylation as processes which might regulate APP metabolism will be studied. The enzyme(s) which are responsible for the proteolytic processing of APP will be purified and characterized. Metabolites of APP that possibly can be involved in Beta/A4-amyloidogenesis and pathogenesis of AD will be identified by purification and limited amino- terminal sequencing of APP metabolites from stimulated and non- stimulated platelets.

It is proposed to continue a multidisciplinary investigation of basal-ganglia specific neuronal phosphoproteins. The program project for which renewed support is being requested consists of five parts. The first project (Principal Investigator, Dr. Angus C. Nairn) would continue the purification and characterization of phosphoprotein substrates (for cAMP dependent protein kinase) identified during the previous funding period, and develop immunological procedures to measure the state of phosphorylation of these proteins. It also proposes to carry out a detailed characterization of protein phosphatase- 1, since an understanding of the role of this phosphatase is essential to clarifying the role of DARPP-32 in the basal ganglia. The second project (Principal Investigator, Dr. Eric L. Gustafson) would continue studies involving the use of antibodies against phosphoproteins to carry out detailed immunocytochemical studies, at both the light and electron microscopic levels, of the regional, and subcellular distribution of these phosphoproteins. It proposes to study the neuroanatomical framework underlying neurotransmitter-phosphoprotein interactions in the basal ganglia. It would also study the regulation of specific phosphoprotein mRNAs in the basal ganglia, and examine these mRNAs during development. The third project (Principal Investigator, Dr. Paul Greengard) would involve a comprehensive analysis of the pharmacological and physiological regulation of the levels and phosphorylation states of the basal ganglia-enriched phosphoproteins in intact animals and brain slices. It also proposes to introduce electrophysiological techniques to study the physiological roles of protein phosphorylation in the basal ganglia by directly injecting purified phosphoproteins in their phospho and dephospho forms, protein kinases and protein phosphatases into neuronal cells and studying their effects on neuronal activity. The fourth project (Principal Investigator, Dr. Michelle E. Ehrlich) would use recombinant DNA technology to study the expression of the basal ganglia-enriched phosphoproteins, to characterize the human cDNAs for these proteins for use in human molecular genetics studies, and to establish transgenic mice to better understand the function of these proteins in vivo. The fifth project (Principal Investigator, Dr. Kang Tsou) would provide a core facility. Primary tissue cultures of neuronal and glial cells will be grown and maintained for the study of the factors that regulate the expression and phosphorylation state of basal ganglia-specific phosphoproteins. The core facility would also isolate and maintain continuous supplies of monoclonal antibodies against the basal ganglia specific phosphoproteins for the various components of the program project. It is believed that this multidisciplinary approach will provide vital information about the molecular nature of signal transduction in the nervous system as well as provide possible important clues toward the understanding of the etiology of a variety of neuropsychiatric disorders including schizophrenia.

It is generally believed that abnormalities in either the processing of the amyloid protein precursor (APP) and/or the accumulation of the amyloid protein (A/beta) derived from APP play a central role in the etiology of Alzheimer's disease (AD). For this reason, understanding the function of APP and its catabolic fragments, as well as identifying the regulators of APP expression, APP processing, A/beta formation, and A/beta accumulation, are important steps towards understanding the causes of AD and defining therapeutic targets for this disease. This grant application is to support a multi-disciplinary study of APP function, expression, trafficking and processing. Dr. Nairn's project will include: identification and characterization of proteins that interact with the intracellular domain of APP; analysis of the effects of phosphorylation of APP on its interaction with these proteins; and, analysis of the effects of these APP binding proteins on APP processing. Dr. Gandy's project will include: study of the regulation of APP metabolism in in vitro cell-free protein processing and trafficking assays; study of the regulation of APP and alpha-factor processing in wild-type and modified Saccharomyces cerevesiae; and, study of the regulation of the level and turnover of various APP holoforms and metabolites within subcellular fractions and isolated organelles. Dr. Greengard's project will include: identification of isoforms of protein kinases and protein phosphatases involved in the regulation of APP processing; characterization of the role protein phosphatase plays in regulating APP expression and processing in vivo; and, identification and characterization of proteins that interact with the extracellular domain of APP. Core will include: the production and supply of key reagents, including knockout mice, enzymes and antibodies for the other sections of the Program Project.

The research supported by our current grant has investigated the role of protein phosphorylation in the regulation of neurotransmitter release. Specifically, the data obtained from a variety of experimental approaches have provided support for a model by which synapsin I, a synaptic vesicle- associated phosphoprotein, regulates neurotransmitter release by affecting the movement of synaptic vesicles from a "reserve" pool to a "readily-releasable" pool. We have further demonstrated that the phosphorylation state of synapsin I regulates this biological function of the protein. We have established that synapsin I consists of two polypeptides, synapsin Ia and synapsin lb, which we have cloned and sequenced. The sequencing data indicate that synapsins Ia and lb are highly homologous to another pair of synaptic vesicle-associated phosphoproteins, synapsins IIa and lIb, which we have also cloned and sequenced. The current grant also supported the study of another phosphoprotein, MARCKS (myristoylated, alanine-rich, C-kinase substrate), previously called 87 k protein. This renewal application proposes to study the mechanisms by which the family of synapsin proteins (Ia, lb, IIa, and IIb) regulates neurotransmitter release. To date, most of our experiments have studied the effects of synapsins Ia and lb when present together. We now propose to study the potential functional differences among the four isoforms of synapsin. Using a variety of model neuronal systems, we will introduce each of the four synapsin isoforms alone and in various combinations. We will introduce the synapsins in both phosphorylated and dephosphorylated states, in order to determine the functional effects of phosphorylation on specific sites of the synapsin molecules. We will utilize mutated forms of synapsin to determine functional effects following deletions of phosphorylation sites and other functional domains. The principal measures of function will be neurotransmitter release, the development of facilitation, and the maintenance of long-term potentiation. We will study the molecular mechanisms underlying the function of the synapsins by using systems composed of purified components which have been reconstituted in vitro to examine the interactions between synapsin molecules, synaptic vesicles, and F-actin. Finally, we will study the differential distribution of the synapsins and the regulation of synapsin gene expression because our data indicate (a) that the four synapsin isoforms are differentially expressed in subpopulations of neurons, (b) that the expression of the synapsins can be regulated by transcriptional control via extracellular messengers (e.g., hormones), and (c) that the expression of the synapsins induces formation of presynaptic terminals.

The broad, long-term objective of this proposal is to contribute to our understanding of the molecular basis of Alzheimer disease (AD). This objective will be advanced by characterizing APP in human platelets, by determining how APP is metabolized, and by relating these findings to the pathogenesis of AD. The four specific aims of this proposal are: 1) To study the species (isoforms and/or proteolytic fragments) of APP present in human platelets; 2) To investigate how APP is processed in human platelets, including determining which signal transduction mechanisms initiate processing, and which enzymes catalyze APP cleavage; 3) To establish human platelets as an in vitro model for APP processing with the intention of using it for testing agents that might modulate processing; 4) To study whether processing of APP in human platelets can generate APP metabolites that might be involved in AD-associated amyloidosis. The health-relatedness of this project is its aim of identifying the causes of AD and effective therapeutics for the AD- associated amyloidosis. The experimental design for these studies is to identify APP isoforms and metabolites in human platelets using antibodies with specificities for different regions of the APP molecule as well as antibodies against different isoforms of APP. Metabolism of APP will be studied in platelets with and without stimulation by physiological and pharmacological agents that induce degranulation. The subcellular distribution of APP and APP-metabolites will be localized using subcellular fractionation techniques. Phosphorylation and dephosphorylation as processes which might regulate APP metabolism will be studied. The enzyme(s) which are responsible for the proteolytic processing of APP will be purified and characterized. Metabolites of APP that possibly can be involved in Beta/A4-amyloidogenesis and pathogenesis of AD will be identified by purification and limited amino- terminal sequencing of APP metabolites from stimulated and non- stimulated platelets.

There are major medical reasons for understanding the nature of dopaminergic neurotransmission in the neostriatum. Parkinson's disease,, Huntington's chorea, schizophrenia and attention deficit hyperactivity disorder all reflect disorders of dopamine signaling pathways. This grant application proposes a multi-disciplinary study of signal Project (Biochemical Analysis of Basal Ganglia Phosphoproteins) will include: biochemical characterization of the role of phosphorylation of Th475 and DARPP-32; elucidation of the molecular basis of the interaction of neurabin. Project (Pharmacological Regulation of Basal Ganglia- enriched Phosphoproteins) will include: characterization of the phosphorylation of DARPP-32 at Th473 in intact cells; characterization of the roles of Th475 or Th434 in the regulation of glutamate and GABA receptor phosphorylation using genetically modified mice in which the Th434 and Th475 phosphorylation sites are substituted with alanine; characterization of the role of phosphorylation of spinophilin and neurabin in intact cells; characterization of gene knockout mice to evaluate the roles of PP1 isoforms, and neurabin in the phosphorylation of glutamate and GABA receptors. Project (Physiological and Cell Biological Studies of Phosphoproteins in the Basal Ganglia) will include: electrophysiological analysis of mice lacking either PP1 isoforms, spinophilin, neurabin, or in which the Thr34 and Thr75 phosphorylation sites in DARPP-32 will have been substituted with alanine; characterization of the regional and subcellular localization of spinophilin and neurabin; examination of the mechanism of targeting and the dynamic regulation of the spinophilin-PP1 and neurabin-PP1 complex in neurons. Project (Molecular Biology of Basal Ganglia and Phosphoproteins) will include: analysis of the behavioral and biochemical responses to dopamine in mice lacking striatal PP1 isoforms, mice lacking spinophilin and neurabin, and mice with mutated DARPP-32 genes, in which the Thr34 and Thr 75 phosphorylation sites are substituted with alanine; identification of proteins that interact with spinophilin and neurabin in yeast 2-hybrid systems. Project (Scientific Core) will produce key materials and perform routine required for the other projects.

The major objective of this application is to seek an understanding of the-intracellular signalling mechanisms by which dopamine exerts its effects in striatal neurons in the basal ganglia. This includes an understanding not only of the intracellular messengers that mediate the dopaminergic signal but also an understanding of how this signal is integrated together with that of a variety of other neurotransmitters impinging on striatal neurons. A major goal of the proposed studies is to generate, by novel technologies, genetically altered animals that will serve as models in which to study dopamine signalling pathways. These studies will allow the characterization of the involvement of individual components of the dopaminergic signalling pathways without the use of non-specific inhibitors. In addition, it also allows the analysis of the effect of these mutations in whole animals in which the complexity of interacting neurotransmitter pathways is preserved in the basal ganglia as well as other brain regions. Additional goals of the proposed studies are to use recently introduced molecular biological techniques to identify novel components of the dopamine signalling pathways. Specifically the yeast two-hybrid technique will be used to isolate proteins that interact with the phosphoproteins or with the enzymes that regulate their phosphorylation and dephosphorylation. These studies will be of particular importance in understanding such diseases as Parkinson's disease and schizophrenia which result in part from defects in dopamine- signalling pathways. In addition, the availability of mouse models in which specific components of dopamine-signalling pathways are deficient may be useful in the development of new therapeutic strategies for the treatment of such diseases.

amyloid proteins; monoclonal antibody; immunological substance; biomedical facility; hybridomas; hybrid antibody; cryopreservation; laboratory mouse; molecular cloning; protein purification; immunoprecipitation; western blottings; enzyme linked immunosorbent assay;

The synapsins are a family of neuron-specific, vesicle-associated phosphoprotein which are important for the regulation of neurotransmitter release from mature neurons, and may also play a critical role in synaptogenesis. The phosphorylation-dependent cross-linking by synapsin I of synaptic vesicles and the actin based cytoskeleton is believed to regulate the availability of vesicle for exocytosis. Proline-directed protein kinases represent a broad family of kinase, including cyclin-dependent protein kinases (cdks) and mitogen-activated protein kinases (MAP kinases). These kinases are known to play an essential role in cell division, growth and differentiation. Recently, we have demonstrated that synapsin I is a downstream effector of the MAP kinase pathway, and is also phosphorylated by cdc2 and cdk 5, a cdk that is enriched in adult, post-mitotic neurons. Synapsin II, the second member of the synapsin family, can also be phosphorylated in vitro by MAP kinase and cdc2 kinase. We wish to identify the cdk5-dependent site in synapsin I and each proline-directed site in synapsin II. Since the amounts of synapsin II that are available are very limited, even when the protein is produced by recombinant technology, we hope to take advantage of mass spectrometric analysis to identify these sites.

The Alzheimer's amyloid precursor protein (APP) can be processed via alternative pathways. The amyloidogenic pathway, which occurs normally in both neuronal and non-neuronal cells, leads to the production of A beta which is found deposited in plaques of AD (Alzheimer's disease) patients. Stimulation of the non-amyloidogenic pathway, in which APP is cleaved within A beta, can be brought about via activation of protein kinase C or inhibition of protein phosphatase 1. These observations may represent first leads towards therapeutic strategies for AD. The current application proposes to extend these findings by addressing the following questions: 1) which isoforms of the protein kinases and phosphatases are responsible for regulating APP processing and A beta formation; 2) what are the effects of modulating net phosphorylation on APP expression and processing in the living brain; and, 3) what are the biological consequences of stimulating APP secretion via increasing net phosphorylation. Understanding the normal and pathological metabolism of APP, as well as the biological effects of regulating APP processing, could advance rational therapeutic strategies for APP. The long-term objective, and health-relatedness, of the proposal is discovering new avenues for the rational design of therapies for AD directed at altering APP processing and/or A beta accumulation. The techniques to be used include molecular biological, cell biological and biochemical techniques.

DESCRIPTION (Applicatnt's Abstract) The addictive properties of drugs of abuse such as psychostimulants depend on their ability to augment dopamine neurotransmission in the basal ganglia. The major target for dopaminergic neurons in the basal ganglia are the medium-sized spiny neurons. Our laboratory has accumulated considerable experience in the study of the intracellular signal transduction pathways regulated by dopamine in medium spiny neurons. This Program Project Grant proposes to elucidate the role of signal transduction pathways in the actions of drugs of abuse, particularly psychostimulants. A group of experts in various disciplines of biomedical research will carry out these studies using distinct but complementary approaches. Project I, entitled "Drugs of abuse: Role of cdk5 in actions of psychostimulants" will characterize of the role of cdkS and phosphorylation ofDARPP-32 in the actions of cocaine. These studies will utilize gene knockout mice to identify the contributions of selected elements of the cdk5/DARPP-32 signaling cascade. Project II, entitled, "Drugs of abuse: casein kinases 1 and 2, and the DARPP-32/protein phosphatase-l signaling cascade," will characterize the role of casein kinases and DARPP-32 in the actions of drugs of abuse, including psychostimulants, opiates, cannabinoids and caffeine. These studies will utilize gene knockout mice to identify the contributions of selected elements of the casein kinase l/casein kinase 21 DARPP-32 signaling cascade. Project III, entitled "Role of DARPP-16 and DARPP-21 in mediating the actions of drugs of abuse," will study the dopamine targets, DARPP-16 and DARPP-21 in the actions psychostimulants. These studies will utilize gene knockout mice, and will also examine the phosphorylation of DARPP-16 and DARPP-2l. Project IV, entitled "Novel molecular targets for drugs of abuse," will characterize the role of the protein phosphatase-l targeting proteins, spinophilin and neurabin, in the actions of psychostimulants. These studies will utilize gene knockout mice to identify the contributions of the spinophilin/protein phosphatase-l and neurabin/protein phosphatase-l signaling cascades. Finally, these studies will be supported by a Scientific Core that will provide maintenance of all colonies of knockout mice, and will produce key materials and perform routine, yet critical, tasks that will be required to accomplish the studies described in the other Projects.

The acute and chronic actions of cocaine and amphetamine are thought to be mediated, in large measure, through augmentation of dopamine neurotransmission in forebrain terminal fields. Research in The laboratory of Molecular and Cellular Neuroscience has emphasized athe study of the molecular mechanisms of dopamine action and has identified a protein kinase/protein phosphatase cascade as one of the major intracellular pathways for dopamine neurotransmission. This project proposes to analyze the role of this cascade in the acute and chronic actions of cocaine and amphetamine. The proposed experiments utilize mouse strains lacking particular components of the postsynaptic dopamine signaling pathway; the genes coding for the phosphatase inhibitors, DARPP-32 and inhibitor-1. These mice represent the only animal models lacking a known intracellular target for dopamine action and provide a unique resource with which to study the role of altered dopaminergic signaling in the a c ute and chronic effects of drugs of abuse. The proposed studies will examine the role of DARPP-32 and inhibitor-1 in mediating or modulating (a) the effects of cocaine and amphetamine on the phosphorylation of known dopamine-regulated intracellular targets. (b) the psychomotor stimulant and reward properties of cocaine and amphetamine and (c) the neurotoxic actions of substituted amphetamines. The overall goal of this project is to gain further insight into the neurochemical mechanisms that play critical roles in human substance abuse.

proteins; nervous system; biomedical resource; Mammalia;

proteins; nervous system; enzymes; biomedical resource; Mammalia;

proteins; nervous system; biomedical resource; Mammalia;

A synaptic vesicle-associated form of CaM kinase II was identified as a major binding protein for the C-terminal region of synapsin I. The enzyme can regulate vesicle- and actin-binding activity of synapsin I by phosphorylation. The presence of a preassembled complex of CaM kinase II and synapsin I at the vesicle membrane may mediate sustained Ca2+-independent phosphorylation of synapsin I beyond the termination of the Ca2+ signal. The mechanism responsible for the targeting of CaM kinase II to the synaptic vesicle membrane is unknown. The vesicle-associated form might represent a novel gene product (e.g., the product of a previously unidentified gene, or a new isoform derived by alternative splicing of one of the four identified subunits) that contains a structural domain that is responsible for membrane targeting. Alternatively, it might be identical in primary structure to the soluble form, and is targeted to the membrane by a posttranslational lipid modification or via an interaction with a specific "receptor" protein on the synaptic vesicle membrane. To distinguish between these possibilities, purified soluble kinase and vesicle-associated kinase will be subjected to proteolytic/chemical fragmentation, followed by analysis using mass spectroscopy. Determining the exact mass of derived peptides of both proteins will allow us to detect changes in either the primary sequence or a posttranslational modification. In preliminary studies with Dr. Chait, several novel phosphorylation sites on CaM kinase 11 have also been revealed. Further studies will be performed to establish the identity of these sites.

A multi-disciplinary approach is proposed in this Program Project to identify and characterize the basis by which the synapsin family of phosphoproteins promote synaptogenesis and stabilize synapses. A greater understanding of the molecular mechanisms which underlie the trophic actions of the synapsins may lead to novel therapeutic targets for the treatment of Alzheimer's disease. To define the trophic function of the synapsins in synapse formation and maintenance, studies will be performed at several levels of organization complexity, encompassing in vitro biochemical studies with purified molecules, cell biological systems, and studies in intact animals Project I will focus on elucidating the mechanisms by which the synapsins regulate the temporal expression and spatial organization of cytoskeletal and synaptic proteins during synaptogenesis and synapse stabilization. Immunocytochemical and biochemical analyses will be used to assess the effects of targeted deletion of the synapsins, and the expression of mutant synapsins, in primary neuronal cultures. Project II will characterize the mechanisms by which the synapsins act as downstream effectors in the signal transduction cascade that is activated by the neurotrophins. The phosphorylation of the synapsins by MAP kinase, the physiological regulation of this kinase by the neurotrophins, and the functional consequences of this phosphorylation will be studied in vitro and in vivo. Project III will examine the effects of altered synapsin expression on neural degeneration and reactive synaptogenesis in animal models. We will compare wild-type mice with aged mice and with which have been experimentally modified by targeted genetic alterations and by physical/chemical insults. A Core facility will provide a range of technical support services to the other components of the Program Project. The Core will be responsible for the preparation and maintenance of key reagent, and will maintain the colonies of genetically altered mice.

The synapsins are a family of four neuronal specific phosphoproteins, the expression of which has been associated with dynamic reorganization of the neuronal cytoskeleton and with trophic actions manifested in axon formation, synapse formation, and synapse maintenance. Synapsins are known to bind and regulate the stability of actin filaments, microtubules, and acidic phospholipids, and synapsin expression alters the expression of several other synaptic proteins. It is unclear, however, which of the synapsin bind activities might be important for the trophic actions of the synapsins, and relatively little is known about the differential contribution of the four synapsin proteins. The long-term objective of this section of the Program Project application will be to learn more about the role of synapsins in axon formation, synapse formation, and synapse maintenance. Specifically, we will focus on the relationship between the cytoskeletal and the genomic effects of synapsin expression, and the relative contributions of the synapsin isoforms to these trophic effects. Specific Aim 1 will focus on characterizing cytoskeletal dynamics in relation to the temporal expression, spatial distribution, and phosphorylation state of the synapsin isoforms. Cultures of primary hippocampal neurons from wild type and synapsin knock out mice will be used to determine how the normal steps in cytoskeletal development and synaptogenesis are affected by deletion of synapsin I, synapsin II, or synapsin I and II. Specific Aim 2 will focus on determining which of the synapsin binding activities are important during neuronal differentiation. Synapsins which contain deletions in one or more of the bind domains will be reintroduced into cultures from synapsin knock out mice and the ability of the mutant synapsins to rescue the developmental defects will be assessed. Specific Aim 3 will focus on determining the mechanism whereby synapsin levels regulate the expression levels of other synapsins and of synaptic proteins. Finally, Specific Aim 4 will focus on determining whether the ability of the synapsins to regulate expression of other proteins is dependent on their ability to induce cytoskeletal reorganization.

Ca2+/calmodulin-dependence protein kinase II phosphorylates synapsin I. The phosphorylated synapsin I dissociates from synaptic vesicle and causes the release of neurotransmitter. Two forms of Ca2+/calmodulin-dependent protein kinase II have been identified the soluble form and the synaptic vesicle-associated form. The vesicle-associated form of the kinase functions both as a binding protein for synapsin I and promotes its dissociation from the vesicles. The relationship between these two forms of the kinases is not clear. In the present study, we are attempting to use mass spectrometric techniques to structurally characterize these two forms of kinases by measuring their masses and obtaining their peptide maps.

Inhibitor-1 (l-1) and DARPP-32, which share sequence homology in the amino-terminal region, are endogenous regulators of protein phosphatase-1 (PP-1). l-1 is expressed in a wide variety of tissues, while DARPP-32 is highly enriched in caudate nucleus and striatum in mammalian brain. When a homologous site in each protein is phosphorylated by cAMP-dependent protein kinase, l-1 and DARPP-32 are converted to potent inhibitors of PP-1. Additional phosphorylation sites in DARPP-32 can regulate the functional state of DARPP-32. Phosphorylation at Ser-102 by casein kinase II regulates the ability of DARPP-32 to serve as a substrate for cAMP-dependent protein kinase. Phosphorylation of Ser-137 by casein kinase I inhibits the dephosphorylation of Thr-34 by the calcium/calmodulin-dependent protein phosphatase, calcineurin. Thus, a complex regulatory pathway for the control of PP-1 activity converges on a single molecule, DARPP-32, by way of multi-site phosphorylation by distinct cla sses of protein kinase. As described above for synapsin II, we have recently found that DARPP-32 and l-1 can serve as in vitro substrates for MAP kinase, cdc2 kinase and cdk5. We wish to identify these novel phosphorylation sites, determine the functional effects of these specific phosphorylations on phosphatase inhibition, and then prepare phospho-specific antibodies to these sites in order to study the physiological regulation of the state of phosphorylation.

Ca2+/calmodulin-dependence protein kinase II phosphorylates synapsin I. The phosphorylated synapsin I dissociates from synaptic vesicle and causes the release of neurotransmitter. Two forms of Ca2+/calmodulin-dependent protein kinase II have been identified the soluble form and the synaptic vesicle-associated form. The vesicle-associated form of the kinase functions both as a binding protein for synapsin I and promotes its dissociation from the vesicles. The relationship between these two forms of the kinases is not clear. In the present study, we are attempting to use mass spectrometric techniques to structurally characterize these two forms of kinases by measuring their masses and obtaining their peptide maps.

Inhibitor-1 (1-1) and DARPP-32, which share sequence homology in the amino-terminal region, are endogenous regulators of protein phosphatase-1 (PP-1). 1-1 is expressed in a wide variety of tissues, while DARPP-32 is highly enriched in caudate nucleus and striatum in mammalian brain. When a homologous site in each protein is phosphorylated by Camp-dependent protein kinase, 1-1 and DARPP-32 are converted to potent inhibitors of PP-1. Additional phosphorylation sites in DARPP-32 can regulate the functional state of DARPP-32. Phosphorylation at ser-102 by casein kinase II regulates the ability of DARPP-32 to serve as a substrate for cAMP-dependent protein kinase. Phosphorylation of Ser-137 by casein kinease I inhibits the dephosphorylation of Thr-34 by the calcium/calmodulin-dependent protein phosphatase, calcineurin. Thus, a complex regulatory pathway for the control of PP-1 activity converges on a single molecule, DARPP-32, by way of multi-site phosphorylation by disti nct classes of protein kinease. As described above for synapsin II, we have recently found that DARPP-32 and 1-1 can serve as in vitro substrates for MAP kinease, cdc2 kinase and cdk5. We wish to identify these novel phosphorylation sites, determine the functional effects of these specific phosphorylations on phosphatase inhibition, and then prepare phospho-specific antibodies to these sites in order to study the physiological regulation of the state of phosphorylation.

A synaptic vesicle-associated form of CaM kinase II was identified as a major binding protein for the C-terminal region of synapsin I. The enzyme can regulate vesicle- and actin-binding activity of synapsin I by phosphorylation. The presence of a preassembled complex of CaM kinase II and synapsin I at the vesicle membrane may mediate sustained Ca2+-independent phosphorylation of synapsin I beyond the termination of the Ca2+ signal. The mechanism responsible for the targeting of CaM kinase II to the synaptic vesicle membrane is unknown. The vesicle-associated form might represent a novel gene product (e.g., the product of a previously unidentified gene, or a new isoform derived by alternative splicing of one of the four identified subunits) that contains a structural domain that is responsible for membrane targeting. Alternatively, it might be identical in primary structure to the soluble form, and is targeted to the membrane by a posttranslational lipid modification or via an interaction with a specific "receptor" protein on the synaptic vesicle membrane. To distinguish between these possibilities, purified soluble kinase and vesicle-associated kinase will be subjected to proteolytic/chemical fragmentation, followed by analysis using mass spectroscopy. Determining the exact mass of the primary sequence or a posttranslational modification. In preliminary studies with Dr. Chait, several novel phosphorylation sites on CaM kinase 11 have also been revealed. Further studies will be performed to establish the identity of these sites.

DESCRIPTION (provided by applicant): This research program has focused on characterizing the synapsin family of phosphoproteins and its regulation of neurotransmitter release. By a variety of in vitro and in vivo techniques, we have demonstrated that synapsins are tightly associated with the cytoplasmic face of synaptic vesicles and can bind simultaneously to both synaptic vesicles and actin. Phosphorylation of synapsin I by calcium/calmodulin-dependent protein kinase II (CaMKII) or MAP kinase decreases its affinity for synaptic vesicles and actin, thus allowing vesicles to move within the vesicle cluster from a reserve pool to a readily releasable pool, where they can participate in exocytosis. In addition, we have recently discovered that synapsins directly modulate the dynamics of the readily releasable pool and thus the kinetics of exocytosis. This revised application for renewal proposes to extend our studies on the mechanisms by which synapsins regulate synaptic transmission. To understand the function(s) of synapsins at the molecular level, we propose the following three Specific Aims: I. To determine the impact of synapsin mutations on neurotransmitter release, synaptic vesicle trafficking and the trafficking to and within nerve terminals. We will make use of GFP-labeled synapsins, site-directed mutagenesis, transient cell transfection techniques and live cell imaging combined with physiological approaches for these purposes. II. To study the regulation of Src-family tyrosine kinases by synapsins in nerve terminals. We have previously demonstrated that synapsins are excellent ligands for the Src homology-3 (SH3) domain of c-Src. and that this interaction leads to activation of endogenous vesicle-associated tyrosine kinase, resulting in phosphorylation of endogenous vesicle substrates including synapsins. III. To identify novel binding partners for specific regions of the synapsins and to study their functional significance. Synapsins have been shown to interact with the SH3 domains of c-Src and Grb2. Preliminary results indicate that synapsin-SH3 domain interactions also include many proteins involved in signal transduction and regulation of membrane trafficking at the nerve terminal. We will use in vitro methods to investigate the interactions of synapsins mediated by SH3 domains of proteins relevant to nerve terminal function. We will also investigate those interactions mediated by the highly conserved domains A, C, and E of synapsins. The effects of acute disruption of the identified synapsin interactions on presynaptic morphology and neurotransmitter release will be determined in the in vivo models of the lamprey reticulospinal synapse and the squid giant synapse.

Investigations into the molecular targets of neuroleptic drugs and their associated intracellular signaling pathways have yielded a wealth of information regardingthe cellular perturbations associatedwith schi/ophrenia. The combined studies outlined in Projects 1-5 will extend upon this existing knowledge with rigorouscell biological, molecular, biochemical,behavioral, and electrophysiological studies of neuronal cells upon neuroleptic drug treatment These studies have the potential to provide greater insights into the etiology of schi/ophrenia as well as lo make possible novel and/or more efficient neuroleptic drug treatment. The Transgcnic Animal Core will be a center devoted to facilitating those experiments proposed in Projects 1-5 thai require the use of genetically modified animals.The experimentsoutlined in these Projects require the creation and maintenance of large numbersof genetically modified animals; it will be the responsibility of tie Core to produce these transgenic, knockout, and knockin animalsin an efficient, timely, and cost-effective manner. The existence of a centralized facility staffed with experienced personnel will ensure that this is feasible. In order to produce lurge numbers of transgenic animalsefficiently, Specific Aim I of the Core will be the production of BACs carrying transgenes. The Core will then use these modified BACs in Specific Aim II, the production of transgenic and knockout mouse lines and the testing of these lines. Upon production of transgenic and conditional knockout animals, the Core will test and characterize these animals to validatethem for use in the proposed studies, l-'inally, Specific Aim III of the Core will be the breeding and maintenance of these and other mouse lines (hat will be used throughout the studies in Projects 1-5.

Neuroleptics that antagonize G-protein coupled, dopaminergic and serotonergic receptors to alleviate the symptoms of schizophrenia. Prolonged treatment with neuroleptics 'remodels'circuits of the prefrontal cortex (PFC) and striatum, ameliorating the symptoms of the disease. It is our central hypothesis is that the induction of remodeling depends upon the ability of neuroleptics to antagonize G-protein coupled, D2, D1, and 5-HT2 receptors, triggering cell-type specific adaptations in striatal and cortical neurons. Neuronal dendrites are likely to be critical targets oi this remodeling, being directly implicated in the schizophrenic neuropathology. D2, D1 and 5-HT.. receptors richly invest dendritic regions of key PFC and striatal neurons and have been implicated in modulating dendritic electrogenesis, synaptic integration and plasticity. Dysregulation of dendritic function by D2, D1 and 5-HTj receptors provides a potential explanation for the apparent involvement of glutamatergic signaling in schi/ophrcnia. Yet, very little is known about how neuroleptics and neuroleptic-sensitive receptors influence dendritic electrogenesis, synaptic integration and plasticity in functionally relevant subpopulations of PFC and striatal neurons. The central goal of the project is to help fill this gap in our understanding. There are two major obstacles blocking achievement of this goal. One obstacle is that the neurons within both the PFC and striatum are heterogeneous in their expression of neuroleptic-sensitive GPCRs. The recent development of BAC transgenic mouse lines in which neurons expressing D2 and D1 receptors are fluoresccntly tagged has effectively removes this obstacle. Another major obstacle is the inaccessibility of dendritic regions to physiological study. The recent development of two photon laser scanning microscopy (2PLSM), when used in conjunction with patch clamp techniques, diminishes this obstacle, opening these critical dendritic regions to investigation. This proposal takes full advantage of these new developments to complement our skills in single cell molecular profiling, electrophysiological analysis of ion channel modulation and computational neuroscience to pursue three specific aims: Specific aim 1 is to characterize, in identified PFC and striatal neurons, short- and long-term neuroleptic-induced adaptations in i:he expression and modulation of voltage dependent ion channels thought to control dendritic electrogenesis. Specific Aim 2 is to characterize short- and long-term neuroleptic-induced adaptations in D2, D1, and 5-HT2 receptor modulation of dendritic electrogenesis and glutamatergic synaptic integration in identified PFC'and striatal neurons. Specific Aim 3 is to characterize in identified PFC and striatal neurons short- and long-term neuroleptic-induced adaptations in dopaminergic modulation of glutamatergic synaptic plasticity. For each of these aims, pursuit of the adaptations will be tightly linked to the other studies proposed in this Conte Center, providing a physiological complement to the molecular, biochemical and behavioral approaches they employ.

[unreadable] DESCRIPTION (provided by applicant): Project 1 will explore how typical and atypical neuroleptic drugs affect protein phosphorylation pathways in striatum and prefrontal cortex. It is anticipated that typical and atypical neuroleptics share some, but not all, intracellular signaling targets. The similarities could reflect the common ability of these compounds to counteract positive symptoms, whereas the differences could reflect their differential actions on negative symptoms and extrapyramidal side effects. Aim I will establish a comprehensive survey of protein phosphorylation pathways regulated by neuroleptics in cortex and striatum. Previous work has shown that DARPP-32 is implicated in the actions of neuroleptics. Up until the present time, it has not been possible to distinguish between the biochemical regulation of DARPP-32 in specific neuronal cell types of striatum. These neurons are morphologically indistinguishable and are intermixed anatomically. The goal of Aim II is to obtain a more detailed understanding of the specific cell population where neuroleptics act. Novel BAC transgenic mouse technology will be used to overexpress epitope-tagged DARPP-32, RCS and mGluR5 in striatonigral, striatopallidal or cortical neurons. Pull down experiments followed by immunoblotting or mass spectrometry from these animals will allow cell-specific analyses of phosphorylation events. DARPP-32 KO mice show altered responses to neuroleptics and psychotomimetics. The goal of Aim III is to define neuronal circuitries in which DARPP-32 mediates actions of neuroleptics. The Cre/loxP technology will be used to generate mice lacking DARPP-32 in striatonigral, striatopallidal or cortical neurons. The mouse lines will be studied in terms of their responsivity to neuroleptics in biochemical, behavioural and electrophysiological assays, finally, in Aim IV, studies on the cellular biology of novel proteins interacting with metabotropic glutamate, serotonin and muscarinic receptors will be conducted. Thus, the proposed studies should provide a detailed knowledge on how neuroleptics affect protein phosphorylation pathways in defined neuronal circuitries. [unreadable] [unreadable]

Project 1 will explore how typical and atypical neuroleptic drugs affect protein phosphorylaton pathways in striaium and prefrontal cortex. It is anticipated that typical and atypical neuroleptics share some, but not all, intracellular signalling targets. The similarities could reflect the common ability of these compounds to counteract positive symptoms, whereas the differences could reflect their differential actions on negative symptoms and extrapyramidal side-effects. Aim I will establish a comprehensive survey of protein phospho-rylation pathways regulated by neuroleptics in cortex and striatum. Previous work has shown that DARPP-32 is implicated in the actions of neuroleptics. Up until the present time, it has not been possible to distinguish between the biochemical regulation of DARPP-32 in specific neuronal cell types of striatum. These neurons are morphologically indistinguishable and are intermixed anatomically. The goal of Aim II is to obtain a more detailed understanding of the specific cell population where neuroleptics act. Novel BAG transgenic mouse technology will be used to overexpress epitope-iagged DARPP-32, RCS and mGluRS in striatonigral, striato-pallidal or cortical neurons. Pulldown experiments followed by immunoblotting or mass spectrometry from these animals will allow cell-specific analyses of ph.osphorylal-.on events. DARPP-32 KO mice show altered responses to neuroleptics and psychotomimetics. The goal of Aim III is to define neuronal circuitries in which DARPP-32 mediates actions of neuroleptics. The Cre/loxP technology will be used to generate mice lacking DARPP-32 in striatonigral, striatopallidal or cortical neurons. The mouse lines will be studied in terms of their responsivity to neuroleptics in biochemical, behavioural and electrophysiological assays, finally, in Aim IV, studies on the cellular biology of novel proteins interacting with metabotrpoic glutamate, serotonin and muscarinic receptors will be conducted. Thus, the proposed studies should provide a detailed knowledge on how neuroleptics affect protein phosphorylation pathways in defined neuronal circuitries.

The proposed Conte Center will seek to explore the central hypothesis that neurolcptic drugs act by affecting monoamine and glutamate signal transduction pathways. Validation of this hypothesis should provide very valuable insights into the etiology of schizophrenia and be highly relevant to the development of improved therapies for schizophrenia and related disorders. The proposed Conte Center will be comprised of 5 projects at 4 different universities. The five Pis involved have an established history of effective collaboration and will use their complementary expertise and resources to take a multi-disciplinary approach in the proposed research. The Center Director will be Paul Greengard (Project 1, Rockefeller Univ.);project leaders are Nathaniel Heintz (Project 2, Rockefeller Univ.), Angus Nairn (Project 3 leader, Yale Univ.);Eric Nestler (Project 4 leader, UT Southwestern);James Surmeier (Project 5 leader, Northwestern Univ.). In Project 3, Dr. Nairn will address the major hypothesis of the Conte Center by taking primarily biochemical and molecular approaches to characterize key signaling proteins believed to be involved in the actions of monoamines and glutamale. These studies will in part focus on studies of proteins and enzymes already implicated, from long lasting collaborative studies by Drs. Nairn and Greengard, in the actions of dopamine, serotonin and glutamate in the neostriatum. Specific Aims include: I. Characterization of the mechanism of altered dendritic structure caused by neuroleptic drugs these studies will analyze the role of the post-synaptic scaffold protein, spinophilin, in the action of neuroleptic drugs as well as characterize the role of other proteins in the long-term effects of these drugs; II. Characterization of the role of RCS (Regulator of Calniodulin Signaling) in the actions of neuroleptic drugs - these studies will analyze the role in neuroleptic action of the modulalory protein, RCS, known to be involved in mediating the actions of D2 dopamine receptors; III. Characterization of the role in neuroleptic action of novel signaling proteins interacting with GPCRsthese studies will analyze the structural basis for the interaction of a variety of GPCRs with novel interacting proteins. For each of the aims, the proposed studies will be closely linked with complementary cellular, electrophysiological and behavioral studies being performed in other Projects and Cores of the Conte Center.

Despite many decades of study, schizophrenia remains a poorly understood neurologicaldisorder.However, clinical and experimental studies have implicated dopaminergic, serotoriergic, and glutamatergicneurotransmission in the pathophysiology of this debilitatingpsychiatric disorder. A better understandingof the intracellular signal Iransduction mechanisms linked to these neurotransmitters may more effective treatments for this disease. The responsibilities of the Molecular and Biochemical Core will be to provide a foundation for many of the studies of neuroleptic drug action proposed in Projects 1-5. The Core will perform tasks ranging from sophisticatedscreens that will identify novel protein-protein interactions to basic molecular biology and routine biochemistry that will aid in the investigation these studies. The Core will also provide a technical support facility for the performanceof routine tasks that will be required to accomplish the studies described in Projects 1-5 in an efficient and cosl-effective manner. Thesi tasks will be to maintain stocks of purified protein kinases, protein phosphatases, affinity-purified antibodies, and substrate proteins. Standard purification protocols will be used to obtain these enzymes from native sources, and recombinant technologies will also be employed to produce specific proteins in quantities sufficient to carry out detailed enzymological and structural studies. The Core will also supervise the productionand testing of new polydonal anti-peptide antibodies and phosphorylation state-specific antibodies.

8-Azabicyclo(3.2.1)octane-2-carboxylic acid, 3-(benzoyloxy)-8-methyl-, methyl ester, (1R-(exo,exo))-; Brain; CRISP; Calcium-Dependent Activator Protein; Calcium-Dependent Regulator; Calmodulin; Cell Communication and Signaling; Cell Signaling; Cocaine; Computer Retrieval of Information on Scientific Projects Database; Encephalon; Encephalons; Funding; Grant; Institution; Intracellular Communication and Signaling; Investigators; Mammals, Mice; Methods; Methods and Techniques; Methods, Other; Mice; Murine; Mus; NIH; National Institutes of Health; National Institutes of Health (U.S.); Nervous System, Brain; Phosphodiesterase Activating Factor; Phosphodiesterase Protein Activator; Phosphorylation; Phosphorylation Site; Protein Phosphorylation; Proteins; Research; Research Personnel; Research Resources; Researchers; Resources; Signal Transduction; Signal Transduction Systems; Signaling; Site; Source; Techniques; United States National Institutes of Health; biological signal transduction; gene product; psychostimulant

Drug addiction is defined as a compulsive pattern of drug-seeking and drug-taking behavior that takes places at the expense of most other activities. To address the question why addicts find it so difficult to stop using drugs, much research has been aimed at characterization ol brain systems that mediate the rewarding effects of addictive drugs. The brain reward circuits include dopaminergic innervations from ventral tegmental area and substantia nigra to the nucleus accumbens (ventral striatum) and caudate putamen (dorsal stnatum) as well as glutamate inputs from the prefrontal cortex, amygdala and hippocampus. The dendritic spines of medium spiny neurons in striatum are the cellular location for the integration of dopamine and glutamate transmission both of which are important for development and expression of the adaptive effects of psychostimulants. Because of the long lasting aspects of drug addiction, reorganization of synaptic connections and their maintenance have been suggested as a cellular mechanism of learning and enduring memory associated with addictive behaviors. However, the function and molecular mechanism of psychostimulant-induced dendritic spine proliferation are not fully understood. We propose to investigate cocaine-induced spine proliferation and its physiological significance in two major neuronal subpopulations in striatum: striatonigral and striatopallidal neurons (Aim I). We hypothesize that reorganization of actin filaments may underlie the mechanisms involved in spine proliferation induced by psychostimulants. Regulators of the actin cytoskeleton may play an essential role in drug-induced spine formation. We therefore propose to study five regulators of actin dynamics as targets of psychostimulants in spine proliferation: Cdk5 (Aim II),WAVE1 (Aim III)as a new substrate of Cdk5, spmophilin (Aim IV), neurabin (Aim IV) and Lfc (Aim IV) as a new molecule interacting with spinpphilin/neurabin. hi order to address the role of the five key regulators in psychostimulants-induced spine proliferation, we will analyze the biochemical, cell morphological, electrophysiological and behavioral responses to administration of cocaine in neuronal type-specific or conventional knockout mice and wild type mice. Furthermore, to identify novel targets involved in the actions of psychostimulants on spine formation, we propose to perform quantitative proteomic analysis of purified post-synaptic density after chronic treatment with cocaine (Aim I). Taken together, these studies will lead to elucidation of molecular mechanism(s) leading to spine formation after repeated exposure to addictive drugs and the role of spine formation in the actions of psychostimulants.

This is the second competing renewal of our Program Project Grant, "Drugs of Abuse: Role of Protein phosphorylation" (DA 10044), which was initially funded in 1996 and again in 2001. The grant has been highly successful based on research productivity and initiation of new research projects. The overall objective of the Program Project Grant remains the same as the original, namely, to elucidate the molecular basis of the actions of drugs of abuse, particularly psychostimulants, in the dorsal striatum and nucleus accumbens. The addictive properties of drugs of abuse such as psychostimulants depend on their ability to augment dopamine neurotransmission in the basal ganglia. The Program Project Grant is organized in four Projects, a Scientific Core and an Administrative Core. The Administrative Core staff will support the integration of the researchers and institutions involved in the Program Project Grant. The main goal of the Scientific Core is to provide a research and technical support facility for all of the Program projects. Responsibilities of the Scientific Core will include the creation, characterization, and breeding of genetically altered animals;the design and execution of yeast two-hybrid studies;the production and maintenance of key reagents stocks and new reagents;and the performance of certain routine tasks required to accomplish the studies described in Projects I-IV. Project I, "Psychostimulants and dendritic spines," will focus on the role five regulators of actin dynamics play as targets of psychostimulants in the dendritic spines. Project II, "The Role of DARPP-32, CK1, and CK2 in Mediating the Molecular and Behavioral Effects of Drugs of Abuse," will extend previous studies of four key phosphorylation sites of DARPP-32 upon in vivo administration of drugs of abuse and chronic exposure to drugs of abuse. Project III, "Striatal Phosphoproteins and the Actions of Psychostimulants," which will be a subcontract carried out at Yale University School of Medicine, study the role(s) of RCS and ARPP-16 in mediating or modulating the actions of psychostimulants as well as study the roles of the three isoforms of PP1 (PPla, P and y) in the actions of psychostimulants. Project IV, "The Role of GPCR-interacting Proteins in the Actions of Pscyhostimulants," will utilize yeast two-hybrid screening to further investigate the multiple intracellular signaling pathways of GPCR and its protein-interacting subunits in mediating actions of cocaine, D-amphetamine, ecstasy and caffeine.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. we are using hypothesis-driven mass spectrometry to identify novel phosphorylation sites on the metabotropic glutamate receptor, mGluR5. Mutants of the specific sites of phopshorylation will be constructed to obtain information about the roles of these sites on the function of the receptor.

The main goal of the Administrative Core of this Proposal is to facilitate and support the unification and interaction of the scientists and administrative personnel working on the five Projects and three Cores within the four institutions taking part in this Conte Center Proposal. Specific Aims. Aim I. Coordination and communication between the Cores and Projects in the Proposal - Facilitate communication between the five Projects and three Cores - Schedule all formal meetings - Facilitate the transfer of information for data sharing and manuscript generation - Supervise and assist in the design, implementation and maintenance of the Center website - Career development coordination center for students and postdoctoral trainees Aim II. Maintenance of the Physical Working Space, Supplies and Equipment for the Cores and Administrative Center of the Proposal - Maintenance of the physical laboratory and administrative space - Oversight of the laboratory safety guidelines - Purchase of and maintenance of supply stocks Aim III. Data and Progress Record Maintenance and Clerical Support for the Proposal - Clerical support and record maintenance - Maintenance of project-generated resources files - Publication generation and archive for the Proposal

Alzheimer's disease (AD) is characterized by the excessive generation and accumulation of p-amyloid peptides (Ap). y-secretase, the enzyme responsible for immediate release of Ap, is one of the most important drug targets in AD therapies. Most current y-secretase inhibitors lack discrimination between y-cleavage of APP and other substrates including Notch, and show severe toxicity after chronic administration in animals. We have shown that y-secretase generation of Ap in an N2a cell-free system is ATP dependent. In addition, Gleevec, an Abl kinase inhibitor, which acts by competing at the ATP-binding site of this tyrosine kinase, potently reduces Ap production. Gleevec also reduces Ap production in rat primary neuronal cultures and in vivo in guinea pig brains. However, Gleevec does not inhibit y-secretase-catalyzed cleavage of Notch-1. Our recent results suggest that ATP analogues and Gleevec enhance the binding of holo-APP and APP-CTF to presenilin-1. Furthermore, Gleevec retards egress of APP- but not Notch-containing vesicles from the ER in a PS1-dependent manner. More importantly, we have identified a novel Gleevec binding protein (GBP). We hypothesize that Gleevec achieves its actions on trafficking and cleavage of APP through this novel GBP, and propose specific experiments to test this hypothesis. One goal is to use Gleevec and other ATP analogues as tools to investigate the molecular steps involved in Ap formation. This should prove of enormous value in Alzheimer's disease research. To achieve this goal, in our revised application, we will characterize the effects of ATP analogues and Gleevec on kinetics, substrate binding, and conformational changes of y-secretase (Aim I);we will characterize the selective effects of Gleevec on trafficking of APP vs Notch (Aim II);we will examine the action of a newly discovered Gleevec binding protein (GBP) on the modulation of y-secretase activity and APP trafficking (Aim III);we will analyze the effects of Gleevec on ADrelated pathology, electrophysiology and behavior, using AD transgenic mice (Aim IV);we will characterize the mechanism by which inhibitor 2, another Abl kinase inhibitor, which also acts by competing at the ATPbinding site of this tyrosine kinase, modulates y-secretase activity (Aim V). Taken together, these studies should elucidate the underlying mechanism by which Gleevec regulates y-secretase activity. Our study should accelerate the development of novel therapeutic strategies against Alzheimer's disease.

Synaptic pathology has increasingly become recognized as a principle feature of Alzheimer's disease (AD) and occurs prior to the traditional hallmarks of AD, namely amyloid plaques and neurofibrillary tangles (NFT). Notably, our research group was the first to observe synapse loss in human AD brain. Loss of dendritic spines has been observed in young mouse models of AD before the buildup of plaques and NFT, and also correlates much better with cognitive loss. At a molecular level, several studies have demonstrated that Ap peptides, particularly Ap oligomers, potently inhibit hippocampal long-term potentiation (LTP). Our recent published results indicate that Ap causes endocytosis of NMDA and AMPA glutamate receptors, processes that are likely linked to perturbation of synaptic plasticity and remodeling of dendritic spines. In unpublished work, we have found that the kinase, Cdk5 (previously implicated in AD), regulates dendritic spine morphology via phosphorylation of WAVE1 (a regulator of actin polymerization), and that Ap may regulate WAVE1 phosphorylation and level of expression. Our observations strongly suggest that Ap-mediated glutamate receptor endocytosis and morphological changes or loss of dendritic spines in cortical and hippocampal neurons may contribute to the earliest stages of memory impairment. Moreover, our unpublished results suggest that synaptic dysfunction resulting from increased Ap levels is reversible, thus offering the possibility of alleviating cognitive defects in AD and related diseases. In the proposed studies, we will further investigate the effects of Ap on synaptic structure, transmission and plasticity. We propose to characterize the effect of Ap monomers, oligomers and fibrils on dendritic spines (AimI) as well as glutamate receptor endocytosis (Aim II). We will investigate the role of Cdk5, WAVE1 (as a new substrate of Cdk5) and CK1 (as a collaboration with Project 2) in Ap-induced spine loss (Aim I). In addition we propose to investigate the role of STEP, dephosphorylation of NR2B and GluR1, and Cdk5-WAVE1 in Ap-induced glutamate receptor endocytosis (Aim II). Furthermore, we will investigate whether cholinesterase inhibitors affect glutamate receptor localization (Aim II).We also propose to characterize the effects of Ap on synaptic plasticity and on learning and memory. We will examine the effect of Ap lowering agents on glutamate receptor endocytosis in vivo(Aim II). Finally, we will use Ts65Dn (Down syndrome mouse model) as well as AD mouse models (as a collaboration with Project 2) to investigate Ap-induced synaptic plasticity and behavioral abnormality (Aim III). Taken together, these studies will elucidate the molecular mechanism(s) leading to synaptic pathology in AD and provide potential targets for AD therapies.

[unreadable] DESCRIPTION (provided by applicant): This is the second competing renewal of our Program Project Grant (PPG), entitled, "Signal Transduction and Alzheimer's Disease". This PPG was developed from a program established in the Laboratory of Molecular and Cellular Neuroscience in 1990 for the purpose of investigating the biochemistry, cell biology, molecular biology, and pharmacology of regulation, by protein phosphorylation, of the Alzheimer amyloid precursor protein (APP). Studies of the cellular and molecular mechanisms underlying the sequential cleavage of the APP to [unreadable]-amyloid (A[unreadable]) by [unreadable]-and ?-secretases have afforded great insight into the etiology of Alzheimer's Disease (AD).Understanding the mechanisms that regulate A[unreadable] generation may enable the development of pharmacologically active compounds that target A[unreadable] formation. A group of experts in various disciplines of biomedical research will carry out these studies using distinct but complementary approaches. Project 1, entitled "Effects of A[unreadable] on Synaptic Structure, Transmission and Plasticity", will investigate the effects of A[unreadable] on dendritic spine morphology, glutamate receptor trafficking, and long term potentiation in cellular and mouse models of AD. Project 2, entitled "Mechanisms of ?-Secretase Regulation" will characterize the underlying mechanism(s) by which ATP, and ATP analogs, including Gleevec, regulate [unreadable]APP processing and trafficking. Project 3, entitled "Role of CK1 in Alzheimer Disease Etiology" will evaluate the role of the protein kinase, CK1, in [unreadable]APP processing, and identify the targets for CK1 involved in this process. These studies will be supported by a Scientific Core (Core B) that will produce key materials and perform routine, yet critical, tasks that will be required to accomplish the studies described in the other Projects. An Administrative Core (Core A) will coordinate various aspects of the PPG, integrating day-to-day activities of the investigators and consultants involved in the various projects. These studies will lead to greater knowledge of mechanisms involved in the production of A[unreadable] in the brains of AD patients and will hopefully identify novel proteins that can be targeted by therapeutic agents. [unreadable] [unreadable] REVIEW OF INDIVDUAL COMPONENTS [unreadable] [unreadable] CORE A - ADMINISTRATIVE CORE; Dr. Paul Greengard (CL) [unreadable] [unreadable] DESCRIPTION (provided by applicant): The objective of the NIA Program Project funding mechanism is to provide for greater opportunities and capabilities through the unification of a shared commitment to a central investigative theme. The main goal of the Administrative Core is to facilitate and support the unification and interaction of the scientists and administrative personnel working on the Proposal at The Rockefeller University, Weill College of Cornell University and Yale University School of Medicine. [unreadable] [unreadable] Specific Aims: [unreadable] [unreadable] Aim I. Coordination of and communication among between the Cores and Projects in the Proposal [unreadable] [unreadable] - Facilitate communication between the three Projects and two Cores, the scientific and administrative staffs at Rockefeller, Cornell and Yale and consultants from other institutions [unreadable] - Schedule and coordinate all formal meetings [unreadable] - Facilitate the transfer of information for data sharing and manuscript generation [unreadable] - Career development coordination center for students and postdoctoral trainees [unreadable] [unreadable] Aim II. Maintenance of the Physical Working Space, Supplies and Equipment for the Proposal [unreadable] [unreadable] - Maintenance of the physical laboratory and administrative space [unreadable] - Oversight of the laboratory safety guidelines [unreadable] - Purchase and maintenance of supply stocks [unreadable] [unreadable] Aim III. Data and Progress Record Maintenance and Clerical Support for the Proposal [unreadable] [unreadable] - Clerical support and record maintenance [unreadable] - Maintenance of project-generated resources files [unreadable] - Publication generation and archive [unreadable] - Supply and equipment record maintenance [unreadable] - Budget tracking and allocation for the Cores and Projects [unreadable] - Data sharing of biological material resources and tools generated by the NIA Program Project Grant [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Our previous competing application of the Program Project Grant (PPG), entitled, "Signal Transduction and Alzheimer's Disease", was reviewed in October 2006 and the grant awarded for 5 years from 02/07 to 01/12. This supplemental application grew out of new studies that were related to part of Project 1 of that application (entitled "Effects of Ap on Synaptic Structure, Transmission and Plasticity1). Project 1 has focused on the very early stages of synapse loss associated with the actions of the (-amyloid peptide (A(), a causative factor in Alzheimer's disease (AD). In particular, our studies in Project 1 suggest that A( may negatively influence synaptic structure and function by virtue of its effects on the actin cytoskeleton. In new studies that represent a significant extension of Project 1, we have found that regulation of the actin cytoskeleton by A( appears to be linked to synaptic pathology including spine loss, and deficits in plasticity, through modulation of mitochondrial function. In this Supplement, we propose to examine the molecular details of the regulation of mitochondrial function by A(. In Specific Aim 1, we will investigate the dynamics of the actin cytoskeleton, and its role in mitochondrial motility, fission and translocation to dendritic spines, both under control conditions and in response to the actions of A(. In Specific Aim 2, we will investigate the role of WAVE1 and its phosphorylation in A(- and neuronal activity-regulated mitochondrial fission and translocation to spines. We will also investigate the role of cAMP and NO/cGMP signaling pathways in mitochondrial fission, motility and translocation and how this is influenced by A(. In Specific Aim 3, we will characterize the mechanisms involved in mitochondrial localization of WAVE1. We will investigate the role of proteins in the WAVE1 complex as well as of other proteins that interact with mitochondria in mediating the interaction between WAVE1 and mitochondria. The new studies carried out in this Supplement will complement continuing work being carried out in Project 1 and the other two projects. Together, this work will elucidate fundamental molecular mechanisms involved in the effects of A( on synaptic pathology and will provide potential new targets for AD therapies.

It is our hypothesis that typical and atypical antipsychotic drugs exert their actions via distinct molecular changes in cell populations that impinge upon and participate in striatal signaling. By identifying what these changes are, we will identify common signaling pathways that are linked to the therapeutic effects of antipsychotic drugs. Project 1 of this Conte center application will focus on striatal cell-specific changes in mRNA translation induced by antipsychotic drug administration. For this purpose we will use our newly developed Translating Ribosome Affinity Purification (TRAP) methodology. In Aim 1 we will use TRAP to identify mRNA translational alterations in the major populations of striatal dopaminoceptive and dopaminergic cells: striatonigral medium spiny neurons (MSNs); striatopallidal MSNs; cholinergic intemeurons; and dopaminergic neurons of the substantia nigra pars compacta and ventral tegmental area. Our preliminary TRAP studies have revealed that sphingosine 1-phosphate (SIP), a ligand for the striatopallidal MSN-enriched S1P receptor Gpr6, can alter both calcium levels and DARPP-32 phosphorylation levels in MSNs. Thus, S1P and Gpr6 may be capable of modulating striatopallidal cell physiology and their response to antipsychotic drugs. In Aim 2, we will characterize the role of 81P and Gpr6 signaling in the response of striatal MSNs to antipsychotic drug treatment using a combination of genetic, pharmacological, and biochemical approaches.

DESCRIPTION (provided by applicant): Schizophrenia is one of the most debilitating psychiatric disorders, affecting approximately 1% of the population worldwide. A number of antipsychotic drugs are available but these are often ineffective and do not treat all the symptoms of the disease. New therapeutics are needed but their discovery has been hampered by a limited understanding of the etiology of this complex neurological disorder, and a lack of clear understanding of the precise molecular mechanisms of action of available antipsychotic drugs. One of the major limitations in identifying the molecular mechanisms of antipsychotic drug action has been the heterogeneous and intermixed cellular nature of the central nervous system. The major goal of the proposed studies in the Conte Center is therefore to achieve a complete understanding of the cellular and molecular actions of antipsychotic drugs through innovative approaches that use novel rodent animal models to allow analysis of individual types of neurons within cortico-striatal circuits. The Center Director and leader of Project 1 is Paul Greengard (Rockefeller University). The other Project leaders are: Nathaniel Heintz (Project 2, Rockefeller University);Angus Nairn (Project 3, Yale University);Eric Nestler (Project 4, Mount Sinai Medical School);and James Surmeier (Project 5, Northwestern University. There will also be an Animal Core, a Molecular &Biochemical Reagents Core, and an Administrative Core. The five Pis involved have an established history of effective collaboration and will use their complementary expertise and resources to take a multi-disciplinary approach in the proposed research. Through the use of biochemical, cell biological, molecular, electrophysiological, structural and behavioral assays, the proposed Conte Center will achieve a fuller understanding of the cellular and molecular actions of antipsychotic drugs in the cortico-striatal circuits. PUBLIC HEALTH RELEVANCE: Relevance to public health: Schizophrenia is a debilitating psychiatric disorder, and new therapies are needed. This Conte Center will characterize the effects of antipsychotic drugs on the properties of specific neuronal subtypes involved in schizophrenia, allowing for a complete understanding of the normal and maladaptive actions of these drugs, and leading to better therapies with higher efficacy and fewer side-effects.

DESCRIPTION (provided by applicant): This is the third competing renewal of our Program Project Grant, Drugs of Abuse: Role of Protein Phosphorylation (DA010044), which was initially funded in 1996 and most recently in 2006. The grant has been highly successful based on research productivity and initiation of new research projects. The overall objective of the Program Project Grant remains the same as the original, namely, to elucidate the molecular basis of the actions of drugs of abuse, particularly psychostimulants, in the dorsal striatum and nucleus accumbens. The Program Project Grant is organized into three Projects, a Scientific Core and an Administrative Core. The Administrative Core will support the integration of the researchers and institutions involved in the Program Project Grant. The main goal of the Scientific Core is to provide research and technical support for all of the Projects. Responsibilities of the Scientific Core will include the creation, characterization, and breeding of genetically altered animals; the design and execution of yeast two-hybrid studies; the production and maintenance of key reagents stocks and new reagents; and the performance of certain routine tasks required to accomplish the studies described in Projects 1-3. Project 1, Cell type- and region-specific studies of psychostimulants and dendritic spines will focus on the role of Cdk5 and WAVE1, regulators of actin dynamics in the dendritic spines. Project 2, The role of the mGluR5/CK1-CK2/DARPP-32 pathway in mediating the effects of psychostimulants, will extend previous studies of novel signaling pathways that regulate DARPP-32 function. Project 3, Striatal Phosphoproteins and the Actions of Psychostimulants, which will be a subcontract carried out at Yale University School of Medicine, will study the role of Rap GTPase, and its modulators Rap1GAP and Epac2 in the actions of psychostimulants, as well as study the role of novel isoforms of PP2A in the actions of psychostimulants. Results from the three Projects will complement each other. In addition, there will be a significant level of collaboration between the three Projects, as well as close interaction of the three Projects with the Scientific Core.

PROJECT SUMMARY (See instructions): RESEARCH SUPPORT SUMMARY The sharing of strengths encouraged by the NIDA Program Project funding mechanism provides for greater opportunities and capabilities through the unification of a shared commitment to a central hypothetical question than would exist for any of the scientists involved individually. The main goal of the Administrative Core of this Proposal is to facilitate and support the unification and interaction of the scientists and administrative personnel working on the three Projects and two Cores of the Program Project Grant at The Rockefeller University and Yale University on this Proposal. The Administrative Core (AC) team have successfully supported the work of the researchers on this Program Project Grant for the last 14 years. Their combined expertise along with the flexibility to adapt to the changing needs of the researchers has led to the development of a proven set of methods for carrying out Specific Aims to aid the studies proposed. Through Aim I. the AC staff will facilitate coordination and communication between the Cores and Projects in the Proposal and data sharing. The AC staff will also oversee maintenance of the physical working space, supplies and equipment for the Cores and Projects 1 and 2 (Aim 11). The staff will also maintain the data and reports of the progress generated under this proposal and provide clerical support (Aim III). The AC staff will continue to utilize their expertise to integrate the various components and individuals of this Program Project Grant to ensure achievement of the goals of this application.

Psychostimulant-induced dendritic spine plasticity in brain reward circuits might be a cellular mechanism of learning and enduring memory associated with addictive behaviors. We hypothesize that two types of dendritic spine formation (silent spines and mature spines) may occur during chronic exposure, withdrawal and re-exposure to psychostimulants. Cell type- and region-specific studies of dendritic spines and of the role of Cdk5 and WAVE1 in the molecular mechanisms underlying the two steps of spine formation are designed with novel and innovative approaches to achieve the following aims. In Aim I, cell type- and region-specific analysis of 1) dendritic spine morphology of medium-sized spiny neurons (MSNs) and of 2) the levels of glutamate receptor expression in dendritic spines will characterize the structure and function of dendritic spines. We will use Bac-transgenic mice expressing a cell type-specific fluorescent marker protein to identify specific MSNs. We will generate three-dimensional images of dendritic spines from two types of MSNs in subregions of striatum (core and shell of nucleus accumbens and dorsal striatum) during chronic exposure, withdrawal and re-exposure to cocaine. Various morphological parameters of dendritic spines will be analyzed with automatic software. In Aim II and III, we will use D1- or D2-MSNs-specific Cdk5 or WAVE1 knockout mice. We will also restore the expression of Cdk5 or WAVE1 only in the specific types of MSNs in specific regions of striatum (nucleus accumbens or dorsal striatum) by injection of conditional AAV-floxed-STOP signal-floxed-Cdk5 or WAVE1 (wild-type or phosphorylation site mutants). With these knockout and add-back Cdk5 or WAVE1 mice, we will study the roles of Cdk5, WAVE1 and phosphorylation of WAVE1 in (a) spine morphology, (b) glutamate receptor trafficking, (c) electrophysiology and (d) behavior. In Aim III.C, we will characterize WAVE1 phosphorylation at tyrosine sites by TrkB, a receptor of mature BDNF, in the actions of psychostimulants. In addition, we will carry out collaborative studies with Project 2 in the analysis of dendritic spines in genetically modified animal models (e.g. CK1 overexpressing mice). We will also collaborate with Project 3 in studies of the role of PP2A in WAVE1 regulation and of the role of Rap1GAP in dendritic spine morphogenesis.

It is well established that DARPP-32 is a key integrator of striatal signaling in physiological conditions as well as in the context of psychostimulants. Beside dopamine, various neurotransmitters such as glutamate act on and modify DARPP-32 downstream signaling. Our studies have demonstrated the importance of multiple signaling pathways converging on DARPP-32 in the action of drugs of abuse, and recently we have developed state-of-the-art technologies proving that these signaling events are specifically regulated in different sub-populations of medium spiny neurons (MSNs) that differentially express Dl and D2 types of dopamine receptor. Our ongoing research will focus on studies of mGluRS, an important GPCR invovled in glutamate-dependent DARPP-32 regulation, and two kinases, CKI and CK2. CKI is a crucial player in the mGluR5/DARPP-32 pathway and CK2 is essential for regulation of DARPP-32 nuclear trafficking. We have generated novel mouse lines for each of the three proposed Aims that will allow us not only to study the impact of psychostimulants in vivo but also to evaluate the relative importance of D1- and D2-MSNs in these phenomena. To address these questions we propose three Aims in Project 2 of the Program Project Grant. In Aim I we will study the role of the newly discovered mGluRS regulator Norbin in the actions of psychostimulants. In Aim II we will study the role of CK1 in the mGluRS/DARPP-32 pathway in vivo. We will also further charcterize the CK16 over expressing mice that present some behavioral features that ressembles ADHD. We will further address differences observed between Dl and D2 receptor pathways. In Aim III we will study the role of CK2 in the actions of psychostimulants, in both Dl and D2-MSNs using specific KO strategies. Results from this Project will complement the other two Projects of this Program Project grant. In addition we will also carry out a number of collaborative studies with Projects 1 and 3, including studies involving spine morphology with Project 1 and phosphoproteomic studies and behavioral studies with Project 3.

Adverse effects; Affect; Aging; Alzheimer's Disease; Amyloid; analog; Attenuated; Behavioral Assay; Binding; Binding Proteins; Biochemical; Complex; CSNK1A1 gene; Data; Development; Electrophysiology (science); Enzymes; Funding; gamma secretase; Goals; Imatinib; In Vitro; in vitro Assay; in vivo; Kinetics; knock-down; Knock-out; Knockout Mice; Lipids; Mediating; Membrane Microdomains; Methods; mimetics; Molecular; Molecular Conformation; mouse model; Mus; Mutation; Names; neurotoxic; notch protein; novel; novel therapeutics; overexpression; Pathology; Peptide Hydrolases; Phosphorylation; Phosphotransferases; Post-Translational Protein Processing; Principal Investigator; Process; Production; protein transport; Proteins; Regulation; Reporting; Role; secretase; Site; Specificity; Substrate Interaction; Substrate Specificity; synergism; System; targeted treatment; Testing; Therapeutic; therapeutic target; Tissues; trafficking; Transgenic Mice; Ubiquitination; Vertebral column; Work;

No abstract provided

In early phases of Alzheimer?s disease (AD) only specific types of neurons are affected by pathological lesions. As the disease progresses, additional neurons types gradually start expressing signs of pathology and degeneration throughout the brain. A major obstacle to treating Alzheimer?s disease (AD) is our lack of understanding of the molecular mechanisms underlying this selective neuronal vulnerability. Neurons from the layer II of the entorhinal cortex (ECII) are the most vulnerable neurons of the brain, but there are currently no in vitro culture system for these neurons, preventing both an in-depth analysis of genes involved in neuronal vulnerability, and the search for drugs inhibiting early neurodegeneration affecting these neurons. In the parent RF1 grant, we proposed to identify transcription factors (TFs) involved in specifying ECII neuron identity, using both detailed molecular profiles for ECII and control neurons, which we generated at different points of their development, and cutting-edge functional genomics analyses. These TFs will then be used to drive the differentiation of induced pluripotent stem cells (iPSC) into ECII neurons. While iPSCs provide a versatile platform that can be differentiated into many different neuron types, concerns have been raised that neurons derived from iPSCs are biologically very young, as the iPSC generation protocol ?resets? the biological clock of a cell. AD-associated lesions are age-dependent, and we think that an in vitro paradigm adapted to this study of aging is crucial. In this administrative supplement, we thus want to adapt this iPSC protocol for the direct reprogramming of human fibroblasts, using the TFs identified in the parent grant. Directly reprogrammed fibroblasts are not reset biologically and present a biological age that correlates with the age of the fibroblast donor. After having optimized a protocol for direct reprogramming of fibroblasts into ECII neurons, we will provide a proof of concept that these ECII neurons are a good model for probing selective neuronal vulnerability by studying Tau pathology in neurons reprogrammed from fibroblasts of aging donors and of donors with AD mutations. Overall these ECII neurons directly reprogrammed from fibroblasts would be the first in vitro model for the study of the interplay between selective neuronal vulnerability in AD and aging.

Project Summary Alzheimer's disease (AD) is the most common cause of elderly dementia and currently there are no effective treatments. Selective neuronal vulnerability, appearance and accumulation of amyloid-? (A?) plaques, altered circuitry function, synaptic loss and degeneration are hallmarks of AD. Microglia and astrocytes, two major subtypes of glial cells, contribute to these neuropathological changes. However, the precise mechanisms controlling these processes remain elusive. One of the reasons may be that we still lack the information about the cell type-specific alterations and how they contribute to the onset and progression of AD. Here, we postulate that by revealing detailed changes in functional networks of genes in microglia and astrocytes, and how those processes intersect, we may be able to determine their contribution to neuropathological changes in AD. We will utilize Translational Ribosomal Affinity Purification combined with novel bioinformatics tools to construct functional network models of glial-specific functions and integrate these with AD quantitative genetic data. Using this framework, we will detect glial-specific genes most likely associated with AD pathology in an unbiased data-driven way and further investigate the functional network modules between astrocytes and microglia. Relevant candidate genes from this analysis will be examined in vitro for their role in AD progression, and emphasis will be given to cytokine-cytokine receptor pathways, especially interleukin 1 and colony stimulating factor 1 hubs. We will utilize primary cell cultures and co-cultures with astrocytes and microglia isolated from healthy and 5xFAD mice. Furthermore, we will investigate only genes that are relevant to the human pathology. To achieve this, we will test the appearance of AD hallmarks and/or the rescue phenotype in the microglia and astrocytes derived from the induced pluripotent stem cells (iPSC) isolated from AD patients and healthy controls. Finally, the most important gene candidates will be tested in vivo by gene expression manipulation in the appropriate mice lines. We will measure: i) A? accumulation, uptake, and degradation by microglia and astrocytes, ii) neuronal degeneration, iii) synaptic function, including synaptic markers and functional circuitry, iv) plaque formation, and v) cognitive function, assessed with a battery of behavioral tests. The proposed study will reveal the molecular profile of each cell type and examine their interaction. Generated datasets and bioinformatics tools will be shared with the public via web-based interface. These data will offer new insights into the appearance of the AD hallmarks and elucidate the basic mechanisms of the disease. Studying these changes, especially on the whole genome level, will engender tremendous insights into alterations in the individual genes, as well as pathways, and may offer new approaches to studying the cause of AD, as well as reveal novel therapy targets.

PROJECT SUMMARY Proteostatic regulatory mechanisms in the endoplasmic reticulum (ER) such as the ER-associated degradation (ERAD) and unfolded protein response (UPR) systems are commonly dysregulated in neurodegenerative proteinopathies such as Alzheimer?s disease (AD). We identify a new role for the ER-resident component membralin in ERAD function. Interestingly, membralin polymorphisms and splice variation have been implicated in AD, and our results indicate that membralin expression is reduced in AD brain. We also identify nicastrin as a membralin/ERAD substrate, and membralin downregulation results in elevated nicastrin levels, thereby enhancing ?-secretase activity, A? generation and cognitive impairment. Interestingly, we also identify ERAD components in complex with the ?-secretase activating protein (GSAP) by proteomic analysis and demonstrate that membralin and GSAP interact by coimmunoprecipitation. Although GSAP is thought to be pathogenic due to its role in enhancing A? generation, we unexpectedly find that GSAP KO mice show impaired fear memory and synaptic function. Moreover, synaptic response and long-term potentiation in with GSAP deletion is further impaired with A?, thereby implicating a protective ?-secretase-independent role for GSAP in synaptic function. Together, our results indicate that membralin/ERAD and GSAP physically and functionally interact, and that these two components likely support both ?-secretase dependent and independent modes of neuroprotection. Membralin splice variation has been previously characterized in AD, however, how this potentially impacts membralin/ERAD function is unclear. We will therefore characterize expression of long and short membralin isoforms in human AD brain and determine whether C-terminal truncation can impact membralin-associated ERAD function in the short isoform. Given that homozygous membralin deletion is lethal, while heterozygous membralin deletion yields no visible phenotype, it seems likely that membralin haploinsufficiency may induce compensatory changes in the ERAD complex, or associated UPR system to normalize proteostatic function. We will characterize changes in ERAD/UPR systems with membralin downregulation in the absence and presence of A? and determine whether enhancing these compensatory changes can enhance ERAD function. GSAP has been previously known to be processed into N- and C-terminal fragments (NTF/CTFs) through caspase-3 cleavage; we will determine whether full-length GSAP or its cleaved forms are involved in GSAP-dependent synaptic function by overexpression/complementation in GSAP KO mouse hippocampus. Since our previous results indicate that membralin depletion can induce caspase-3 activation, we will determine whether modulating membralin levels can affect GSAP-dependent synaptic function and cognitive behavior. Lastly, we will establish an AD/membralin haploinsufficiency model to determine whether enhancing membralin/ERAD, UPR, or GSAP pathways can ameliorate cognitive, synaptic and pathological AD defects.