Mary B. Kennedy - US grants

We are interested in the molecular structure and function of synaptic connections between neurons in the central nervous system. Derangements in the regulation of these connections are an important part of the pathology of several neurological and mental diseases including epilepsy, Alzheimer's disease, Huntington's chorea, schizophrenia, and depression. Many neurotransmitters and neurohormones regulate synaptic function by altering intracellular levels of calcium ion. We are studying the mechanisms by which these fluctuations in calcium levels alter synaptic function. We will focus our studies primarily on the molecular characterization and cellular localization of a synaptic regulatory pathway that has as its central element and abundant, brain-specific calcium and calmodulin-dependent protein kinase. This enzyme is composed of two types of subunits that share certain chemical characteristics, but appear to be present in different ratios in different regions of the brain. We will compare the structure and function of the two subunits by biochemical and recombinant DNA methods. We will also compare the distributions of both subunits in different brain nuclei, quantitatively by radiommunoassay, as well as by immunohistochemistry. We will determine whether neurons containing the kinase are associated with particular transmitter systems or specific anatomical pathways. Biochemical experiments suggest that the kinase is a component of certain brain postsynaptic densities and may also be associated with synaptic vesicles and microtubules. We will determine the subcellular locations of both kinase subunits in different brain regions by biochemical and immunochemical methods and by electron microscopic immunohistochemistry. Focusing on the hippocampus, where kinase activity is most highly concentrated, we will examine the proteins that are phosphorylated by the kinase in brain slices and in homogenates. Our long term goal is two-fold. First, we want to understand the functions of this calcium-mediated regulatory pathway. Second, we want to correlate information about it with similar information about other calcium-regulated pathways (e.g. calcium-dependent adenylate cyclase, phosphodiesterase, phosphatases, etc.), in order to understand the concerted responses to changing calcium levels in CNS neurons.

We are studying the molecular structure and function of synaptic connections between neurons in the central nervous system. Derangements in the regulation of these connections are an important part of the pathology of several neurological and mental diseases including epilepsy, Alzheimer's disease, schizophrenia, and depression. Many neurotransmitters and neurohormones regulate synaptic function by altering intracellular levels of calcium ion. We are studying the mechanisms by which these fluctuations in calcium levels alter synaptic function. We will focus on the study of a synaptic regulatory pathway that has as its central element an abundant, brain-specific calcium and calmodulin-dependent protein kinase. This kinase is a large oligomer of two distinct but homologous catalytic subunits called alpha and beta. In the forebrain, including the hippocampus, cortex and striatum, the kinase is extremely abundant (1% of total protein) and is composed mainly of alpha subunits. It is a major component of synapses and is concentrated in a cytoskeletal structure called the postsynaptic density. When activated by a brief rise in calcium concentration, the kinase phosphorylates itself and then remains active to phosphorylate other proteins even after the calcium concentration falls. We will test the hypothesis that this is a mechanism by which long-lasting changes in synaptic function are generated following brief bursts of synaptic activity. We will determine the structure of the autophosphorylation sites by recombinant DNA and biochemical methods, then characterize the brain phosphatases responsible for dephosphorylation of each of these sites. We will raise antibodies that specifically recognize the autophosphorylated sites on the kinase and others that recognize the phosphorylated form of kinase substrates. We will use these to study, with high spatial and temporal resolution, the physiological circumstances under which the kinase is activated and specific substrates become phosphorylated. We will continue a study of the association of the kinase with the cytoskeleton by biochemical and recombinant DNA techniques. Our goal in the next few years is to clarify the possible regulatory functions of this calcium-dependent protein kinase system. Our long-term goal is to correlate information about this pathway with similar information about other calcium regulated pathways in order to understand the concerted responses to changing calcium levels in CNS neurons.

Protein kinases are an important class of regulatory enzymes in all cells but are particularly important in the nervous system. When these enzymes are activated within cells in response to extracellular signals, they transfer phosphate to serine, threonine, or tyrosine residues on other proteins. This transfer may alter protein folding and function. In this way, protein kinases regulate cellular function. In the studies proposed here, a brain protein kinase that is activated by transient high calcium concentrations will be studied in the genetic organism, Drosophila melanogaster (the fruit fly). Mammalian genes for this enzyme have been sequenced. The genes encoding this important brain enzyme in the fly will be sequenced and mapped within the Drosophila genome. This approach will facilitate the generation of mutants in the kinase and will allow studies of the behavior of flies with mutant kinase genes. These studies will significantly advance our understanding of the basic molecular mechanisms that underlie information processing and storage in nervous systems.

DESCRIPTION (adapted from applicant's abstract) Derangements in synaptic transmission are an important part of the pathology of several neurological and mental diseases including epilepsy, schizophrenia, depression and perhaps Alzheimer's disease. Despite the medical significance of synaptic transmission and the important roles of synapses in information processing and storage in the brain, relatively little is known about the molecular mechanisms underlying regulation of synaptic transmission. The proposed research involves a study of the molecular structures of synapses in the central nervous system (CNS). It focuses on the identification and study of proteins associated with the postsynaptic density (PSD) a large fibrous specialization of the submembrane cytoskeleton that adheres to the postsynaptic membrane opposite presynaptic terminals. PSDs are especially prominent in glutamatergic terminals in the CNS where they are believed to function as anchoring sites for synaptic receptors and signal transduction molecules as well as for molecules mediating adhesion between the pre and postsynaptic membranes. The applicants have applied modern cell biological and microchemical methods to sequence and characterized several proteins associated with the PSD, including NR2B, a subunit of the NMDA receptors, PSD-95, an apparent clustering and adapter molecule, and densin-180, an apparent new adhesion molecule. Here they propose to extend their studies by completing the characterization of two additional PSD proteins that we have sequenced, by beginning to study the association among the PSD proteins that mediate assembly and function of the PSD, and by using recombinant DNA methodology to test hypothesis about the specific functions of PSD proteins in the synapse.

With a Faculty Award for Women Scientists and Engineers from the National Science Foundation, Dr. Mary Kennedy will continue her long-term study on elucidating the molecular structure of synaptic modification during learning and memory. Presently, Dr. Kennedy is studying the biochemical mechanisms that underlie synaptic plasticity, the basis for information storage in the brain. Dr. Kennedy's laboratory was the first to identify and purify type II CAM kinase, a brain calcium dependent protein enzyme. This enzyme is a specialized product of nerve cells in the forebrain that plays a role in the initiation of long-term potentiation, an important form of synaptic regulation that underlies the early stages of memory formation. The major goals of this research will be to identify functionally important synaptic proteins in CNS nerve cells that are regulated by CAM kinase, and to develop novel quantitative methods to measure the time course and cellular location of regulatory phosphorylation events in nerve cells.

The Gordon Research Conference on Neural Plasticity has been held in alternate years since 1977 in July at Brewster Academy, Wolfeboro, New Hampshire. We are requesting partial support for the Conference planned for July 19-23, 1993. The Gordon Research Conferences were established to stimulate scientific interchange in an informal setting. Uninhibited discussion is fostered by a rule prohibiting publication of the meetings and presentations, or indeed their citation. This format has proved particularly useful for the Conference on Neural Plasticity, a highly interdisciplinary meeting in which the subject of modifiability of the nervous system is examined at the molecular, cellular and systems levels. The participants in this meeting come from varied backgrounds (biochemical, pharmacological, anatomical, electrophysiological, behavioral, and computational) and find the opportunity for free exchange of ideas and information highly stimulating. For the 1993 Conference, one evening has been set aside for a keynote talk by Dr. Solomon Snyder, a leading scientist in the area of signal transduction in the brain. The remaining eight sessions will focus on specific issues of current interest. The formal speaking time is limited to allow for ample discussion. During the afternoon, no formal sessions are scheduled so that informal discussion can continue. As in past conferences, we will have poster sessions in the late afternoon as a further stimulus for mixing and discussion. Past participants have found that these informal interactions are one of the distinct advantages of the Gordon Conference format. The proposed program for 1993 includes sessions on: adaptive regulation of gene expression in the adult nervous system, molecular mechanisms of receptor regulation, regulation of synaptic transmission by nitric oxide, control of transmitter release at the molecular level, the molding of sensory systems by sensory inputs, conversion of short- term modulation into long-term structural changes, modulatory influences on simple neuronal networks, and the role of the amygdala in fear conditioning.

The strength of transmission at individual synapses in the central nervous system is often highly plastic and appears to be tightly regulated. This regulation plays a critical role in processing and storage of information. Furthermore, derangements in the regulation of synaptic strength seem likely to contribute to many neurological and psychological disorders including epilepsy, depression, schizophrenia, and Alzheimer's disease. The long-term goal of this study is to understand the molecular mechanisms that control synaptic transmission in the central nervous system and to learn how these mechanisms are distributed among different types of synapses. The proposed experiments will examine local regulation of autophosphorylation of a prominent brain Ca2+/calmodulin-dependent protein kinase in hippocampal neurons and phosphorylation of its substrate proteins, including the presynaptic vesicle protein, synapsin I, and proteins of the postsynaptic density. An immunocytochemical procedure will be developed for visualizing and quantifying phosphorylation of functionally significant sites on these proteins in situ after pharmacological and/or physiological manipulation of organotypic cultures or acute hippocampal slices. The method will make use of an existing monoclonal antibody that recognizes the CaM kinase only when it is autophosphorylated at a specific functional site and a complementary antisera that recognizes it only when it is not autophosphorylated at that site. Changes in autophosphorylation of the kinase in subcellular compartments will be recorded by this method at various times after inducing post-tetanic potentiation or long-term potentiation. We will raise similar antibodies against sites on synapsin I that are phosphorylated by CaM kinase II. We will use the antibodies to visualize changes in phosphorylation of synapsin I during induced changes in synaptic efficacy. Proteins in postsynaptic densities that are phosphorylated in situ by the CaM kinase, the A-kinase, or the C-kinase will be identified by biochemical labeling methods. These proteins will be purified from the postsynaptic density fraction, and the sites on the proteins that are phosphorylated in situ will be sequenced in preparation for immunocytochemical studies of their phosphorylation during induced changes in synaptic efficacy. The proposed immunocytochemical method will provide data about the kinetics of individual regulatory phosphorylation reactions and the sequence of those reactions at defined locations within neurons. Such data will permit testing of predictions made by explicit models of the organization of regulatory pathways in different types of synapses.

Derangements in synaptic transmission are an important part of the pathology of several neurological and mental diseases including epilepsy, schizophrenia, depression, and perhaps Alzheimer's disease. Despite the medical significance of synaptic transmission and the important roles of synapses in information processing and storage in the brain, relatively little is known about the molecular composition of the key synaptic organelles involved in transmission or about the mechanisms by which the functions of these organelles are regulated. The proposed research involves a study of the regulation by protein phosphorylation of identified proteins at synapses in the central nervous system. The goal of this project is to measure the time course of regulatory phosphorylation events in different subcellular domains of hippocampal neurons after a variety of physiological and pharmacological treatments. Our studies will focus on phosphorylation events at the postsynaptic membrane of glutamatergic synapses following tetanic stimulation that can induce long-term potentiation, and following perfusion with modulatory agents that can alter induction of long-term potentiation. By comparing the time course of phosphorylation of different sites, we will attempt to "chart" the sequence of regulatory phosphorylation events that is triggered by various stimuli. To make the proposed measurements, we will use a laser-scanning confocal immuno- fluorescence microscopy technique that we developed and have nearly perfected in the first three years of this grant. We will use phospho-site specific antibodies raised against proteins associated with the postsynaptic membrane of glutamatergic synapses, including CaM kinase II, the 2B and 2A subunits of NR2B, AMPA receptors, and densin-180. The antibodies will be engineered to recognize the proteins either when they are phosphorylated or when they are not phosphorylated at specific sites identified as targets for phosphorylation in vivo. Double-immunofluorescence labeling with these antibodies allows us to visualize the ratio of phosphorylated and nonphosphorylated protein at various cellular and subcellular sites within tissue slices. We will extend the immunofluorescence labeling method to the level of single neurons, by marking neurons that have been induced to undergo LTP by pairing of depolarization and tetanic stimulation under whole cell clamp, then examining the relative levels of phosphorylation along the dendrites and at synapses of the marked neuron. These data will help us to develop and test theories about the complex regulatory events governing control of synaptic strength in the hippocampus.

DESCRIPTION (provided by applicant): Derangements in synaptic transmission are part of the pathology of several neurological and mental diseases including epilepsy, schizophrenia, depression, and Alzheimer's disease. We are studying the molecular mechanisms underlying regulation of synaptic transmission. Here we propose to study regulatory pathways at glutamatergic synapses governed by Ca2+/calmodulin-dependent protein kinase II (CaMKII). CaMKII is concentrated in the postsynaptic density where it can be activated by Ca 2+ influx through NMDA receptors. The proposal focuses on two principal postsynaptic substrates of CaMKII, SynGAP, a Ras GTPase-activating protein that is concentrated in the postsynaptic density, and densin, a proposed docking site for CaMKII. In the First Aim, we will test the hypothesis that synGAP participates in regulation of the cytoskeleton at synapses in brain slices. We have previously shown that synGAP helps to regulate the spine cytoskeleton during synapse formation in cultured neurons, and that the location and activation of the kalirin/PAK kinase pathway is altered in hippocampal slices heterozygous for synGAP. We will use slices from wild type and synGAP deficient mutants to map the role of synGAP in this and related pathways following synaptic stimulation. In the Second Aim, we will use electrophysiological studies in hippocampal slices from wild type and conditional synGAP deficient mice to investigate whether the effects of synGAP deficiency on LTP are a result of developmental abnormalities or of acute loss of synGAP. We will also test the hypothesis that synGAP participates in regulation of the modulation of dendritic excitability by MAP kinase. In the Third Aim, we will examine recruitment of CaMKII to the PSD in neuronal cultures prepared from knock-in mice that are missing the carboxyl terminal tails of the NR2A and NR2B subunits of the NMDA receptor. We will also examine recruitment of CaMKII in these cultures after introducing recombinant forms of the intracellular tails of densin, or reducing the expression of densin by introduction of siRNA. In the Fourth Aim, we propose to determine the physiological importance of densin, and test the hypothesis that densin is a docking site for CaMKII in the PSD by constructing knockout and conditional knockout mutants of densin.

The goal of this project is to implement simulations of activation of Ca 2+/calmodulin-dependent vrotein kinase II (CaMKII) within the models of calcium dynamics in glutamatergic spines created in Project 1. We will test the accuracy of our understanding of spine biochemistry by designing experiments to verify or falsify predictions made from the simulations of activation of CaMKII. The project includes four specific aims, some of which will be pursued in parallel. First, we will design an accurate kinetic model of activation of the CaMKII holoenzyme at levels of calcium and at ratios of calmodulin to CaMKII that are likely to occur in spines. We will develop theoretical kinetic models, determine boundaries on appropriate kinetic constants, and fit the kinetic models to experiments with purified CaMKII. Second, we will introduce representations of holoenzymes of CaMKII, programmed with appropriate activation kinetics, into the models of calcium dynamics in spines created in Project i and carry out simulations of activation of CaMKII. We will compare simulations in which kinetic parameters are varied within known or likely physiological boundaries. We will compare our results to those obtained with more traditional finite element methods. Third, We will test the importance, in our models, of calmodulin location and concentration, phosphatase location and concentration, and also of other parameters that control calcium availability. Fourth, in collaboration with Karel Svoboda (project 4), we will design experiments to test whether simulations of CaMKII activation match experimental observations, and accurately predict behavior of CaMKII in spines under a variety of physiological conditions. Our experimental systems will include rat hippocampal slices, and cultured hippocampal neurons. The second and third aims will require extensive collaboration with the Core and with Project 1. We will work closely with the Sejnowski group to implement additions to the MCell software that are necessary for representing CaMKII. The models developed in Project 1, with input from Projects 2 and 4, will form the basis for models constructed in this Project.

DESCRIPTION (provided by applicant) Neurological and mental diseases result, in part, from derangements in regulation of synaptic transmission. In glutamatergic spines, calcium influx through NMDA receptors is a principal regulator of synaptic plasticity. Spines contain many signaling proteins that can be regulated by Ca2+. Different regulatory pathways are activated under different experimental conditions; and, thus, calcium influx can lead to increases or decreases, of varying durations, in synaptic strength. The objectives of the work proposed in this Program Project are to gain a quantitative understanding of Ca2+-regulated signal transduction triggered by Ca2+ in spines, and to apply computational methods to stimulate the dynamics of initial events during Ca2+ signaling in spines. The program includes four projects and a core that will provide new computer software. Project 1 will make use of the computer program Mcell to develop and test models of calcium dynamics in spines based on realistic synaptic geometries and measured spatial distributions and kinetic properties of relevant signaling molecules. The models will be constructed with the use of a streamlined program interface to be developed in the core, and will incorporate data generated in Projects 2 and 4. Project 2 will use quantitative immunocytochemistry at the light and electron microscope levels to study the organization of calcium sources and sinks in spines, as well as the distribution of the Ca2+ target, CaM kinase II. The data will be compared with measurements made in Project 4, and used to constrain simulations arising from Projects 1 and 3. Project 3 will develop and test accurate kinetic models of activation of CaMKII that will be incorporated into the models of Ca2+ dynamics in spines constructed in Project 1. Predictions of simulations of activation of CaMKII will be tested experimentally in conjunction with project 4. Project 4 will use 2-photon fluorescence microscopy to measure [Ca2+] signals and their regulation in individual spines. The data will be integrated with that from project 2, and used to construct and test models made in projects 1 and 3. The program addresses two goals of the Channels, Synapses, and Circuits program of NINDS: 1. To facilitate collaborations among researchers working at molecular and cellular levels to develop multidisciplinary approaches for analysis of channels and synapses and 2. To facilitate collaborations among neuroscientists, computer scientists, and physicists to develop computational tools for data analysis and modeling. The purpose of the models and simulations will be to quantify hypotheses about Ca+ function in spines in order to test them rigorously with experiments. We will attempt to predict the relative importance of measured variations in the structure and molecular composition of synapses for their signaling capabilities. The predictions will be tested by comparison to experiments. Thus, we view the models and the simulations we propose to generate as powerful quantitative tools with which to study the dynamics of synaptic signaling, and not as an end in themselves.

Abstract not provided.

The goal of this project is to implement simulations of activation of Ca 2+/calmodulin-dependent vrotein kinase II (CaMKII) within the models of calcium dynamics in glutamatergic spines created in Project 1. We will test the accuracy of our understanding of spine biochemistry by designing experiments to verify or falsify predictions made from the simulations of activation of CaMKII. The project includes four specific aims, some of which will be pursued in parallel. First, we will design an accurate kinetic model of activation of the CaMKII holoenzyme at levels of calcium and at ratios of calmodulin to CaMKII that are likely to occur in spines. We will develop theoretical kinetic models, determine boundaries on appropriate kinetic constants, and fit the kinetic models to experiments with purified CaMKII. Second, we will introduce representations of holoenzymes of CaMKII, programmed with appropriate activation kinetics, into the models of calcium dynamics in spines created in Project i and carry out simulations of activation of CaMKII. We will compare simulations in which kinetic parameters are varied within known or likely physiological boundaries. We will compare our results to those obtained with more traditional finite element methods. Third, We will test the importance, in our models, of calmodulin location and concentration, phosphatase location and concentration, and also of other parameters that control calcium availability. Fourth, in collaboration with Karel Svoboda (project 4), we will design experiments to test whether simulations of CaMKII activation match experimental observations, and accurately predict behavior of CaMKII in spines under a variety of physiological conditions. Our experimental systems will include rat hippocampal slices, and cultured hippocampal neurons. The second and third aims will require extensive collaboration with the Core and with Project 1. We will work closely with the Sejnowski group to implement additions to the MCell software that are necessary for representing CaMKII. The models developed in Project 1, with input from Projects 2 and 4, will form the basis for models constructed in this Project.

DESCRIPTION (provided by applicant): The immediate objective of this proposal is to build an accurate dynamic model of activation and autophosphorylation of the signaling protein Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII) during influx of Ca2+ into the postsynaptic spine through NMDA receptors. The work will proceed in three stages. First, investigators will validate a model of activation of individual monomeric catalytic subunits by Ca2+ and CaM and refine its kinetic parameters by comparing the model to experiments. The deterministic model is implemented in Mathematica;the output of the model will be tested against bench assays of the enzymatic activity of monomeric subunits of CaMKII under a wide range of concentrations of the subunits, CaM, and Ca2+. The concentrations will mimic both in vivo and in vitro conditions. In the second stage, investigators will construct a model of activation of the dodecameric holoenzyme of CaMKII, based on the model validated in the first stage. They will model cooperative activation of subunit dimers within the holoenzyme, and three different paths of autophosphorylation of its subunits. The models will be constructed in the program MCell, which supports spatially correct stochastic models of protein interactions and enzymatic activation, within biologically realistic geometries. This model will employ a new rule-based algorithm to specify the locations and behavior of subunits in holoenzymes. It will be constructed in a well-mixed volume to enable testing by comparison to bench experiments with holoenzymes under a wide range of concentrations of subunits, CaM, and Ca2+. The comparisons will be used to optimize four new parameters in the holoenzyme model, and to choose the most accurate model for progression of autophosphorylation within the holoenzyme. In the third stage, investigators will introduce optimized models of the CaMKII holoenzyme into a larger MCell model of Ca2+ influx into spines through NMDA receptors and its removal by pumps and exchangers. Simulations in MCell with this model will be used to test hypotheses about parameters governing activation of CaMKII in spines. The intellectual merit of the proposal lies in its utility in the study of mechanisms of learning in the central nervous system. The regulatory machinery in a spine controls synaptic strength by regulating activity-dependent changes such as LTP and LTD. We know much about the regulatory enzymes in a spine and we have hypotheses about enzymatic networks that regulate the cellular processes controlling synaptic plasticity, including insertion and removal of glutamate receptors and changes in the shape of the spine actin cytoskeleton. However, at the present stage of analysis, qualitative studies with mutant animals, or over-expression and knock-down of particular enzymes are the dominant paradigm in the field and they are not adequate to bring our knowledge to the next level, which is to establish the timing of the action of each of these players, and the precise conditions and position in the regulatory network at which each one becomes important. To reach that level of understanding, we need better quantitative models and methods. CaMKII is one of the the initial enzymes activated by Ca2+ coming through NMDA receptors during induction of LTP. A well-validated quantitative model of its activation in the powerful MCell program will provide a starting point and an example for the construction of dynamic models of successive steps in spine regulatory pathways. The broader impacts include the educational goal of fostering introduction of computational techniques into cellular neurobiological research. A female postdoctoral fellow will be trained in experimental techniques to test computational models, and in the use of MCell. Undergraduate students (including minority students) will be involved in the work through summer research programs at Caltech and Salk. All models will be made available to the community for download. The models of CaMKII holoenzymes will be a first example of simulation in MCell of interactions within a cytosolic multiprotein complex. The syntax for doing this will be published, and taught in the regular workshops on MCell sponsored by NSF. The proposal has medical significance. Deficiencies in spine signaling pathways that use CaMKII are associated with working memory deficits similar to those that underlie schizophrenia and related thought disorders. A quantitative understanding of the factors governing activation of CaMKII during synaptic activity, and its role in controlling synaptic plasticity will facilitate development of clinically useful pharmacological agents that target specific aspects of synaptic dysfunction with fewer undesirable side effects.

DESCRIPTION (provided by applicant): We will develop a new method to measure the time courses of activation of biochemical regulatory networks that control changes in synaptic strength which underlie processing and storage of information in neural networks. The proposed method will permit unprecedented time resolution and will enable measurement of the time courses of activation of at least 20, and eventually as many as 50 to 100 enzymes in brain tissue that has been rapidly frozen at intervals as small as one second following an electrical or pharmacological stimulus. The method will be immediately applicable to basic research on, and target development for, mental illnesses and Alzheimer's disease. Upon scale-up, it will be applicable to screening for drugs to treat these diseases. The method will involve substantial adaptation of two existing technologies: plunge-freezing and Selected/ Multiple Reaction Monitoring (S/MRM) by mass spectrometry. Once developed, both technologies can be scaled up for medium or high throughput screening. The project has three aims. First, we will develop a plunge freeze apparatus to rapidly freeze slices of hippocampal tissue at accurate time intervals following application of a stimulus to the perfused slice. We will accomplish this by making modifications and additions to a plunge-freeze apparatus now commercially available from Leica (Leica EM GP). We will devise an optimal design for a sample chamber to maintain the health of slices during perfusion, and to deliver electrical stimuli to the Schaffer collateral pathway, a major hippocampal axon tract, prior to rapidly freezing the slice by plunging it into a -1900 C liquid propane/ethane bath. We estimate that freezing time to the center of the slice upon plunge will be ~ 200 msecs or less. This freezing time is compatible with a resolution of one second for time intervals following application of a discrete stimulus. Second, we will develop methods to measure the activation of a panel of 20-25 protein kinases or their key substrate proteins located at positions in the regulatory networks that are believed to control synaptic plasticity in excitatory synapses in the hippocampus. Each enzyme or substrate that we will measure is regulated by addition of a phosphate group to key residues in the protein structure. Mass spectrometry will be used to measure changes in the levels of these phosphorylated sites in the frozen slice tissue. Third, once the assays are developed, we will carry out proof of principle experiments by combining the technologies developed in Aims 1 and 2 to acquire time courses of activation of each the enzymes in hippocampal slices after delivery of stimuli that alter synaptic plasticity.

SynGAP Haploinsufficiency is the cause of ~2-5% of sporadic Intellectual Disability (ID) accompanied by autism spectrum disorder (ASD) and/or epilepsy. SynGAP is specifically located postsynaptically and is located in the postsynaptic density (PSD). It has a GTPase activating (GAP) domain that accelerates inactivation of Ras and Rap. However, new evidence, including our own, suggests an additional activity that, when lost, may contribute significantly to the pathology. We have shown that SynGAP?1 binds to all three PDZ domains of the major PSD scaffold protein, PSD-95, and have measured its affinity for each PDZ domain. SynGAP?1 is abundant in the PSD; its ?1 isoform could occupy up to 15% of PSD95's PDZ domains in wild type animals and thus compete with binding of other PDZ-domain ligands. We have shown that in synGAP+/- mice (an animal model for synGAP haploinsufficiency), the amount of synGAP in the PSD is reduced and the amounts of other PDZ-domain binding proteins are increased, including TARP-?2,3,4, and 8, and LRRTM2, both of which are AMPA-type glutamate receptor (AMPAR) chaperone proteins. This change in composition would increase the excitatory/inhibitory balance of synapses onto neurons and contribute to abnormal brain function. Thus, we postulate that reduction of binding of synGAP to PDZ domains of PSD-95 is a major contributor to the ID and ASD observed in SynGAP Haploinsufficiency. We have found that phosphorylation by Ca2+/calmodulin- dependent protein kinase II (CaMKII) decreases the affinity of synGAP for PDZ domains. We postulate that phosphorylation of synGAP is important for reconfiguration of the PSD during early stages of induction of LTP. In Aim One, we will enable quantitative tests of the hypothesis that binding of synGAP to PDZ domains regulates the composition of the PSD by measuring the affinities between PDZ domains of PSD-95 and the carboxyl terminal tails of TARP-?2,3,4, and 8, LRRTM2, and NR2B. We will express soluble fusion proteins containing the cytosolic carboxyl termini of each protein. To determine affinities for each PDZ domain, we will use Biacore surface plasmon resonance detection of binding to recombinant PDZ domains by the ?affinity in solution? method that we perfected for use with synGAP. In Aim Two, we will construct computational models in MCell to simulate equilibrium binding of synGAP and each of these proteins to PSD-95. The models will make use of parameters measured in Aim One. We will construct models of in vitro experiments in order to test their concepts and parameters by comparing simulated results to experimental results. We will then construct spatially realistic models within reconstructed spine geometries to study how the spatial arrangement and high densities of proteins in the spine influence competition among the proteins for binding to PSD-95. In Aim Three, we will use cultured rodent neurons to test the hypothesis that phosphorylation of synGAP drives acute changes in the composition of PSDs in primary neuronal cultures before and after chemical induction of LTP.

Fast glutamatergic synaptic transmission is based on a precise and complex molecular organization which requires the control of the number of AMPA-type glutamate receptors (AMPARs) at the postsynaptic sites of glutamatergic synapses on dendritic spines. The number of AMPARs varies as a function of pre- and postsynaptic activation history of the synapse. It is now well described that synapses can change their number of AMPARs and therefore, their response properties through biochemical mechanisms of synaptic plasticity. In this way, information is stored in the brain. The overall goal of this project is to use quantitative models and experiments to answer two fundamental questions about the role of an abundant postsynaptic protein, synGAP, in regulation of the numbers of AMPARs. Numerous experiments in intact neurons have revealed that the level of synGAP expressed at synapses is inversely correlated with the amount of AMPARs available at the synapses, and that synGAP helps to regulate changes in AMPAR numbers during synaptic plasticity. The enzymatic GAP domain of synGAP acts as a ratchet to adjust the rates of addition and removal of AMPARs from the surface of the dendrite. SynGAP also contains a sight that binds tightly to the major scaffold protein PSD-95 via its three protein-binding PDZ domains. Important to the mental health mission of the NIMH, SynGAP plays a critical role in learning and memory in the Brain and mutation of SynGAP is implicated in cognitive disabilities. The project is divided into two broad Aims. In Aim 1, we will answer the question: What are the mechanisms by which synGAP controls the amount of AMPA receptor in the postsynaptic density (PSD) - by control of surface amount and/or by control of availability of PDZ domain binding sites in the synapse? We will improve our existing computational model of the competition between synGAP and AMPARs for binding to PSD-95 by incorporating it into our model of AMPAR trafficking. We will use genetics and sophisticated molecular engineering to experimentally disentangle the two mechanisms. Effects on the nano-organization of AMPARs will be measured by super- resolution fluorescence microscopy and electrophysiology. Results of these experiments will be used to constrain our model of AMPAR trafficking. Aim 2, Through the synergy of experimental and computational approaches, we will address the questions: How does the formation of the condensate between synGAP and PSD-95, and the presence of additional PDZ domain-binding proteins (GluN2 receptor subunits, neuroligin, nNOS, CRIPT, etc.) influence the nano-organization of AMPAR-TARPs in the PSD in the basal state and during synaptic plasticity? RELEVANCE (See instructions): We propose a combination of computational and experimental work that will help clarify the role of synGAP in regulation of AMPARs in CNS synapses, including its role in mental illness. The work will impact a specific medical condition termed ?SynGAP haploinsufficiency? or ?MRD5?, in which SynGAP is mutated in ~1% of children with sporadic non-syndromic cognitive disability accompanied by autism and/or epilepsy. The medical impacts of this work are potentially quite significant as it could help to point toward specific molecular interventions with therapeutics that could improve the lives of patients with these afflictions.