Richard L. Huganir - US grants

Protein phosphorylation is widely accepted as one of the principal mechanisms in the control of almost all cellular processes. Recent studies have provided evidence that protein phosphorylation plays a major role in the regulation of neuronal function. A newly discovered unique class of protein kinases which exclusively phosphorylates the tyrosine residues of their substrate proteins has been shown to be abundant in nervous tissue. The determination of the role of tyrosine-specific protein kinases in the regulation of neuronal function is the major goal of this research proposal. To accomplish this goal, the tyrosine phosphorylation of the nicotinic acetylcholine receptor will be studied and used as a model system for the regulation of neurotransmitter receptors and ion channels by tyrosine kinases. The nicotinic acetylcholine receptor is a neurotransmitter-regulated ion channel and is the most well-characterized neurotransmitter receptor and ion channel in biology today. The specific aim of this project is to identify the tyrosine kinase(s) that phosphorylate(s) the nicotinic receptor in vitro and in vivo and to determine the functional consequences of tyrosine phosphorylation of the receptor. To accomplish this goal the tyrosine kinase(s) from postsynaptic membranes enriched in the nicotinic receptor will be purified and biochemically characterized. The nicotinic receptor will be phosphorylated on tyrosine and the sites of phosphorylation determined by protein sequencing techniques. Receptor phosphorylated on tyrosine residues will be purified and reconstituted into phospholipid vesicles. The functional properties of the reconstituted phosphorylated receptor will then be analyzed. In addition, the tyrosine phosphorylation of the nicotinic acetylcholine receptor in intact muscle cell will be studied and the regulation of this phosphorylation investigated. The functional effects of the tyrosine phosphorylation in muscle will also be analyzed. The proposed research project will provide a better understanding of the role of a basic regulatory mechanism in the modulation of neuronal function. Such knowledge is critical for the understanding of both normal and abnormal neuronal function.

Protein phosphorylation is widely accepted as one of the principal mechanisms in the control of almost all cellular processes. Recent studies have provided evidence that protein phosphorylation plays a major role in the regulation of neuronal function. A newly discovered unique class of protein kinases which exclusively phosphorylates the tyrosine residues of their substrate proteins has been shown to be abundant in nervous tissue. The determination of the role of tyrosine-specific protein kinases in the regulation of neuronal function is the major goal of this research proposal. To accomplish this goal, the tyrosine phosphorylation of the nicotinic acetylcholine receptor will be studied and used as a model system for the regulation of neurotransmitter receptors and ion channels by tyrosine kinases. The nicotinic acetylcholine receptor is a neurotransmitter-regulated ion channel and is the most well-characterized neurotransmitter receptor and ion channel in biology today. The specific aim of this project is to identify the tyrosine kinase(s) that phosphorylate(s) the nicotinic receptor in vitro and in vivo and to determine the functional consequences of tyrosine phosphorylation of the receptor. To accomplish this goal the tyrosine kinase(s) from postsynaptic membranes enriched in the nicotinic receptor will be purified and biochemically characterized. The nicotinic receptor will be phosphorylated on tyrosine and the sites of phosphorylation determined by protein sequencing techniques. Receptor phosphorylated on tyrosine residues will be purified and reconstituted into phospholipid vesicles. The functional properties of the reconstituted phosphorylated receptor will then be analyzed. In addition, the tyrosine phosphorylation of the nicotinic acetylcholine receptor in intact muscle cell will be studied and the regulation of this phosphorylation investigated. The functional effects of the tyrosine phosphorylation in muscle will also be analyzed. The proposed research project will provide a better understanding of the role of a basic regulatory mechanism in the modulation of neuronal function. Such knowledge is critical for the understanding of both normal and abnormal neuronal function.

Protein phosphorylation is widely accepted as one of the principle mechanisms in the control of almost all cellular processes. Studies over the last two decades have provided evidence that protein phosphorylation plays a major role in the regulation of neuronal function. A newly discovered unique class of protein kinases which exclusively phosphorylates tyrosine residues of their substrate proteins has recently been shown to be abundant in neurons. However, the role of protein tyrosine kinases in the regulation of neuronal function is not known. The nicotinic acetylcholine receptor (AChR) is a neurotransmitter gated ion channel which is the most well-characterized neurotransmitter receptor and ion channel in biology. Recent studies have demonstrated that the AChR is phosphorylated on tyrosine residues. To study the role of tyrosine phosphorylation in the regulation of synaptic transmission, the tyrosine phosphorylation of the AChR will be studied and used as a model system for the regulation of neurotransmitter receptors and ion channels by protein tyrosine kinases. The specific aims of this research proposal are to identify the protein tyrosine kinase that phosporylates the AChR and the phosphotyrosine phosphatase that dephosphorylates the receptor and to characterize the functional consequences of tyrosine phosphorylation of the AChR. To accomplish these goals the protein tyrosine kinases and the phosphotyrosine phosphatases from postsynaptic membranes enriched in the AChR will be purified and biochemically characterized. The purified AChR will be phosphorylated on tyrosine residues and reconstituted into phospholipid vesicles and the single channel properties of the reconstituted phosphorylated AChR will be analyzed in detail. In addition the tyrosine phosphorylation of the AChR in muscle cell cultures as well as in intact muscle will be studied and the regulation of this phosphorylation by innervation and denervation will be investigated. The functional effects of tyrosine phosphorylation in muscle will also be analyzed. The proposed research will provide a better understanding of the role of a basic regulatory mechanism in the modulation of synaptic function. Such knowledge is essential for the understanding of both normal and abnormal neuronal function.

DESCRIPTION: The synaptic cytoskeleton plays a critical role in the formation and maintenance of synapses in the central nervous system. Recent studies have identified a new protein motif called a PDZ domain which may be important in the proper targeting of proteins to cell-cell junctions. PDZ domains within cytoskeleton associated proteins mediate the interaction of the cytoskeleton with the C-termini of a variety of membrane proteins. The PDZ domains of the SAP/PSD family of proteins have recently been implicated in the synaptic targeting of NMDA receptors and K+-channels in neurons. The investigator has recently identified a novel PDZ domain containing protein, GRIP (Glutamate Receptor Interacting Protein), that specifically interacts with the C-termini of the GluR2 and GluR3 subunits of AMPA receptors. GRIP appears to link AMPA receptors to the synaptic cytoskeleton and may be critical for the clustering of AMPA receptors at excitatory synapses in the brain. In this research proposal it is planned to further characterize the structure and function of GRIP and related proteins and determine their role in the synaptic targeting of AMPA receptors. Specifically, the regional and subcellular distribution of GRIP will be examined using immunocytochemical and in-situ hybridization techniques. The structural domains of GRIP and GluR2 that are involved in their interaction will be determined by a combination of techniques, including the yeast two hybrid system, fusion protein binding studies and cell transfection techniques. In addition, other proteins (GRASPs -GRIP associated Proteins) that interact with GRIP and form a PDZ domain-based cytoskeleton at synapses will be identified. The functional effect of GRIP on the AMPA receptor ion channel will also be examined using patch clamp techniques. Dominant negative constructs of GRIP and GluR2 will be used to disrupt the interaction of AMPA receptors with GRIP in neurons in culture. Finally, gene targeting techniques will be used to knockout the GRIP gene to determine its role in the mechanisms underlying synaptic transmission in the brain. Such knowledge is essential for understanding the function of the brain

The synaptic cytoskeleton plays a critical role in the formation and maintenance of synapses in the central nervous system. Recent studies have identified a new protein motif called a PDZ domain which may be important in the proper targeting of proteins to cell-cell junctions. PDZ domains within cytoskeleton associated proteins mediate the interaction of the cytoskeleton with the C-termini of a variety of membrane proteins. The PDZ domains of the PSD/SAP family of proteins have been implicated in the synaptic targeting of NMDA receptors and K+-channels in neurons. We have recently identified novel PDZ domain-containing proteins, GRIP1 and 2 (Glutamate Receptor Interacting Protein), that specifically interact with the C-termini of AMPA receptors, the major excitatory neurotransmitter receptors in the brain. GRIP1 and 2 contain seven distinct PDZ domains and appear to serve as adapter proteins to link AMPA receptors to other neuronal proteins and may be critical for the regulation of AMPA receptor function. In this research proposal we plan to further characterize the structure and function of GRIP1 and 2 and related proteins, and determine their role in the synaptic targeting of AMPA receptors. Specifically, we will identify proteins that interact with GRIP1 and 2 (GRASPs-GRIP associated proteins) to form a PDZ domain- based complex at synapses. In addition, we will examine the functional effect-of GRIP1 and 2 on AMPA receptor ion channel function using patch clamp techniques. Dominant negative constructs of GRIP1 and 2, and GluR2 will be used to disrupt the interaction of AMPA receptors with GRIP1 and 2 in neurons to investigate the functional role of GRIPs in AMPA receptor targeting. Finally, gene targeting techniques will be used to knockout the GRIP1 and 2 genes to determine their role in the development, maintenance, and regulation of excitatory synapses. This research will elucidate basic molecular mechanisms underlying synaptic transmission in the brain. Such knowledge is essential for understanding the function of the brain in health and disease.

DESCRIPTION (provided by applicant): NMDA receptors play critical roles in the regulation of synaptic plasticity, neuronal development and several neurological and psychiatric diseases. Recent studies have shown that NMDA receptors bind to the PSD95/SAP9O adaptor protein to form a large macromolecular signaling complex. The PSD95/SAP9O protein family appears to play a critical role in the synaptic targeting of NMDA receptors and in the coupling of NMDA receptors to downstream signal transduction pathways. In this research proposal we plan to study the structure, function and regulation of the NMDA receptor macromolecular signaling complex and the role of this complex in synaptic transmission and plasticity. Recent studies in our lab have shown that phosphorylation of the NMDA receptor by a novel protein kinase disrupts receptor binding to the PSD95/SAP9O protein and thus may play a critical role in the regulation of NMDA receptor signaling. In the proposed research we plan to characterize the kinase responsible for this phosphorylation and examine the effect of this phosphorylation on NMDA receptor synaptic targeting and downstream signaling. Our laboratory has also recently shown that PSD95/SAP9O binds to a novel synapse specific rasGAP, SynGAP, which may regulate ras signaling at excitatory synapses. In this proposal we plan to analyze the role of SynGAP in the regulation of synaptic ras signaling and synaptic transmission and plasticity. The function of SynGAP will be studied in neurons transfected with wild-type and mutant forms of SynGAP and in recently obtained SynGAP knock-out mice. Finally, our laboratory has also recently found that the Rsk2 protein kinase binds to the synaptic scaffolding protein SHANK. SHANK is a component of the NMDA receptor complex that also interacts with several other synaptic proteins including GKAP, Homer and metabotropic glutamate receptors. Interestingly Rsk2 phosphorylates SHANK and may regulate the functional properties of the NMDA receptor complex. In this proposal we will further characterize the phosphorylation of SHANK by Rsk and analyze the functional effects of this phosphorylation on the regulation of synaptic function and plasticity. These studies investigate three different levels of the NMDA receptor protein complex and will help elucidate the function of this large macromolecular complex in excitatory synaptic function and its potential role in neurological and psychiatric diseases.

The molecular mechanisms involved in synapse formation in the central nervous system are unclear. Although it is known that contact of the presynaptic axon with the postsynaptic dendrite induces pre-and post-synaptic differentiation of the synapse, the molecules involved in this process have not been identified. Studies of the neuromuscular junction have shown that presynaptic release of the proteoglycan agrin induces postsynaptic differentiation and clustering of the postsynaptic nicotinic acetylcholine receptor. To investigate the factors involved in the postsynaptic differentiation of excitatory synapses in the CNS we have recently reconstituted synapse formation between axons and non-neuronal cells expressing glutamate receptors and other synaptic components. Incubation of neurons with non-neuronal cells under these conditions promotes differentiation of the presynaptic nerve terminal and clustering of AMPA receptors at the point of contact between the axon and the transfected non-neuronal cell. Electrophysiological recordings of the non-neuronal cells remarkably show synaptic excitatory postsynaptic currents (EPSCs) and mEPSCs. Using this system we will characterize the requirements for postsynaptic clustering of AMPA receptors. Initially we will characterize the structural domains of the AMPA receptor subunits required for this clustering. Deletions of the intracellular C-terminal region of the subunits will be performed to investigate whether intracellular protein interactions are required. Deletions of extracellular domains of the subunits will be performed to see if direct interaction of the AMPA receptor subunits with proteins secreted from the axon is required for receptor clustering. Information from these studies will be used for isolation of the molecules that promote AMPA receptor clustering. These studies will help identify molecules involved in synapse formation and modulation during development and in the adult. Since many neurological and psychiatric diseases result from defects in synaptic transmission, understanding the basic mechanisms regulating synaptic function is critical for the development of therapeutic treatments for these disorders.

DESCRIPTION (provided by applicant): The 100 billion neurons in the human brain have an average of 10,000 synapses. By establishing a dynamic network of synaptic connections, the brain is able to attain the level of functional complexity that underlies human behavior. The efficiency of signal transmission at synapses is constantly being adapted in response to experience as encoded by neural activity. This synaptic plasticity is critical for the fine- tuning of brain development as well as higher brain function such as learning and memory. The plasticity of synapses is modulated and maintained by processes that are sensitive to neuronal activity and cell-cell contact. Trans-synaptic protein interactions induce differentiation of the synapse and regulate the morphology and function of synapses. Release of neurotransmitter regulates the activity of the neuron and activates a variety of second messenger pathways including calcium-signaling systems, which have a central role in regulating both rapid synaptic plasticity and long-term changes in synaptic connections through the activation of gene transcription. These activity-regulated genes then modulate the function of the neuron and can directly affect synapse function. The proposed Conte Center will investigate the inter- and intracellular signaling to and from the synapse that induce synapse formation and differentiation and regulate synaptic efficacy. These signal transduction pathways are initiated at sites of neuronal cell contact by extracellular signals and are then relayed to the nucleus and finally cycle back to the synapse to regulate synaptic function. The proposed Center brings together six leading laboratories in the study of synaptic function to take multiple interdisciplinary collaborative approaches to investigate the molecular mechanisms involved in regulating synaptic transmission and plasticity. Richard Huganir will be identifying molecules involved in the formation, differentiation and regulation of excitatory synapses in the brain. Paul Worley and Sol Snyder will be analyzing how macromolecular signaling complexes at excitatory synapses control neuronal calcium signaling and synaptic function. David Linden and David Ginty will be analyzing how calcium regulates neuronal transcription factors and gene expression. Dwight Bergles will be studying the interaction of glutamate transporters and metabotropic glutamate receptors and the role of this interaction in regulating synaptic function. All of these projects center on the synapse and address how extracellular and intracellular signals converge on the synapse to sculpt its morphology and function. Many neurological and psychiatric diseases result from defects in the development and/or function of synapses. Thus, understanding the mechanisms regulating the formation and modulation of synaptic transmission in the brain is critical for the development of treatments for these diseases.

DESCRIPTION (provided by applicant): Many major neurodevelopmental disorders, including autism, epilepsy and schizophrenia, are believed to the caused by aberrant synapse formation during brain development, resulting in an excess or deficit of certain classes of synapses. Our long term goal is to understand the molecular mechanisms of synapse formation in the central nervous system (CNS), with the aim of developing therapeutics for these devastating diseases. The process of synapse formation has been best characterized in the peripheral nervous system, where the complete loss of neuromuscular junctions has been reported for multiple single gene knockout mice. In the central nervous system, despite the presence of many proteins that show strong synaptogenic activity in vitro, genetic deletion of several of these proteins result in only subtle changes in synapse density limited to small populations of neurons. These results suggest that the synaptogenic machinery in the CNS is heavily redundant;a situation that makes it inefficient to apply traditional genetic approaches to study the problem. We believe that an unbiased chemical screen for determinants of synapse formation, with its potential to block or enhance key pathways and entire classes of genes, may present a more efficient approach to studying synaptogenic mechanisms in the CNS. In addition, the study may also generate small molecule probes that will be useful in perturbing synapse formation and excitatory-inhibitory balance in vivo. An excess or deficit of specific synapses has been hypothesized to underlie many neurodevelopmental disorders, but to date, these hypotheses have been difficult to prove due to the lack of tools to perturb the underlying network connectivity. We believe our proposal will remedy this situation, and at the same time generate a high impact dataset which will shed light on the mechanisms of synapse formation in the CNS. PUBLIC HEALTH RELEVANCE: Defects in brain development leading to an excess or deficit of synapses may result in neurodevelopmental disorders such as autism, schizophrenia, epilepsy and mental retardation. Currently, no drug exists to directly target the development of synapses in the brain. We propose to screen a large chemical library for compounds that will modulate synapse development in the intact brain, and thereby shed light on the molecular mechanisms of the process as well as provide candidate compounds for both investigative and therapeutic purposes.

C1C. MONOCLONAL ANTIBODY CORE C1C1. Establishment of the Monoclonal Antibody Core: The Monoclonal Antibody Core was established with support from this grant and became operational in June of 2006. The Core currently occupies ~200 sq. ft. space on the 9th floor of the Wood Basic Science Building, and contains laboratory bench space, a clean bench for dissection, a Bio Safety tissue culture hood, two tissue culture incubators, two dissecting microscopes, an ELISA plate reader and a liquid nitrogen freezer (Fig. 8). Dr. Huganir serves as the Director for this facility, and Ms. Min Dai, a full time Research technician with extensive experience |n monoclonal antibody production, is the manager of the facility. Both the space and major equipment for this Core were provided by the Department of Neuroscience (see Appendix, Section 5, for letter of support from Dr. Huganir, Chairman, Department of Neuroscience).

DESCRIPTION (provided by applicant): The transition from acute to chronic pain is likely to result from a complex combination of mechanisms. It is important to develop a useful preclinical animal model that can replicate the complexity of the human condition. Previous studies have shown that psychosocial and socio-environmental factors are involved in the development of chronic postsurgical pain. In our preliminary studies, we found that forced swim stress significantly enhances plantar incision-induced ¿-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor phosphorylation and greatly prolongs plantar incision-induced pain, but forced swim stress alone does not produce pain behaviors; we also found that targeted mutation of AMPA receptor GluA1 phosphorylation site Ser831 significantly inhibits stress-induced prolongation of incisional pain. Thus, stress may induce pain transition by regulating AMPA receptor phosphorylation. Recently, we further found that forced swim stress significantly increases GluA1 membrane surface expression and GluA2 internalization and thereby enhances synaptic AMPA receptor switch from Ca2+-impermeable (GluA2-containing) to Ca2+-permeable (GluA2-lacking) in the spinal dorsal horn neurons. This switch will increase Ca2+ influx and further activate Ca2+-dependent protein kinases, thereby promoting AMPA receptor phosphorylation and other phosphorylation-triggered activities. This positive feedback loop may contribute to the molecular mechanisms that underlie stress- induced pain transition. Therefore, we hypothesize that regulation of AMPA receptor phosphorylation and phosphorylation-triggered synaptic AMPA receptor switch from Ca2+-impermeable to Ca2+-permeable contribute to a key mechanism by which stress induces the transition from acute to chronic pain. To address this central hypothesis, pain scientists from Dr. Tao's laboratory and non-pain neuroscientists with expertise in neuroplasticity from Dr. Huganir's laboratory will work together. We will combine plantar incision with different levels of forced swim stress to develop a new animal model to study pain transition (specific aim 1), we will investigate stress-produced regulation of AMPA receptor activities (phosphorylation, trafficking, synaptic targeting, and subunit composition change) in our pain transition model (specific aim 2), and we will investigate the role of phosphorylation-triggered switch of AMPA receptors from Ca2+-impermeable to Ca2+- permeable in stress-induced pain transition (specific aim 3). The overall goal of this proposal is to develop a new animal model to study pain transition and provide critical evidence to characterize the pain transition model. The proposed studies will demonstrate the role of stress-produced AMPA receptor regulation in the transition from acute to chronic pain and shed new light on the pathogenesis of chronic postsurgical pain. PUBLIC HEALTH RELEVANCE: This project is focused on the development of a new animal model to study pain transition. Our hypothesis is that regulation of AMPA receptor phosphorylation and phosphorylation-triggered synaptic AMPA receptor switch from Ca2+-impermeable to Ca2+-permeable contribute to a key mechanism by which stress induces the transition from acute to chronic pain. The proposed studies will demonstrate the role of stress-produced AMPA receptor regulation in stress-induced pain transition and shed new light on the pathogenesis of chronic postsurgical pain.

DESCRIPTION (provided by applicant): Each of the billions of neurons in the human brain can have up to 10,000 synapses. By establishing a dynamic network of synaptic connections, the brain is able to attain the level of functional complexity that underlies human behavior. The efficiency of signal transmission at synapses is constantly being adapted in response to experience. This synaptic plasticity is critical for the fine-tuning of brain development as well a higher brain function such as learning and memory. The plasticity of synapses is modulated and maintained by processes that are sensitive to neuronal activity and astrocyte function. It is now clear that many neurological and psychiatric diseases result from defects in synaptic transmission and plasticity. Thus, understanding the mechanisms regulating synaptic transmission in the brain is critical for the development of therapeutic treatments for these diseases. The Conte Center will take several approaches to investigate the molecular and cellular mechanisms involved in the regulation of plasticity at the excitatory synapses. Richard Huganir will be examining the dynamics of receptor trafficking during synaptic plasticity in vivo i real time using two-photon microscopy. Sol Snyder will be analyzing how gaseous transmitters like NO and H2S modify AMPA receptor function and synaptic plasticity. David Ginty and Alex Kolodkin will be examining how the Sema3F-Npn-2/PlexinA3 signaling pathway regulates synaptic structure and function and AMPA receptor trafficking. Paul Worley will be analyzing how the Oral and STIM1 proteins control intracellular calcium stores and regulate synaptic plasticity in neurons and astrocytes. Dwight Bergles will be studying calcium signaling in astrocytes and how astrocytic signaling can regulate synaptic plasticity. David Linden will be examining the modulation of calcium transients in mossy fiber nerve terminals in the cerebellum and the effects of behavioral experience on presynaptic function using in vivo imaging techniques. All of these projects center on the synapse and address how presynaptic, postsynaptic and astrocytic mechanisms converge on the synapse to sculpt its morphology and function. Understanding these basic mechanisms of synaptic transmission and plasticity will provide insight into normal and abnormal brain function.

AMPA receptors (AMPAR) mediate the majority of fast excitatory synaptic transmission in the central nervous system. Trafficking of AMPARs in and out of synapses is a highly dynamic process and regulation of this trafficking plays a critical role in synaptic plasticity and learning and memory. However, the direct observation of dynamic AMPAR trafficking in live animals during synaptic plasticity has not been accomplished. To examine plasticity in vivo, we will investigate AMPAR dynamics in live animals undergoing various physiologically relevant sensory experiences using two-photon microscopy. We have been able to express pHluorin tagged AMPARs in layer ll/lll pyramidal neurons in the mouse barrel cortex using in utero electroporation. Following electroporation we make a cranial window over the barrel cortex region, map out barrel columns in the cortex using intrinsic optical imaging and then image pHluorin tagged AMPARs with two-photon microscopy. In preliminary studies we have investigated AMPAR dynamics under acute whisker deflection and chronic whisker trimming and regrowth conditions. We have found that both acute whisker deflection and whisker deprivation and regrowth lead to specific changes in AMPAR synaptic levels. To further understand how AMPARs behave during learning tasks, we will observe AMPAR dynamics in the mouse visual cortex during stimulus-specific response potentiation, a well-studied learning paradigm in the visual system and in other cortical regions. To study the molecular mechanisms underlying the induction and long-term maintenance of plasticity we will investigate the AMPAR subunit dependence and the structural regions of each subunit required for the regulation of AMPAR synaptic trafficking in vivo. In addition, we will examine AMPAR trafficking in vivo in our collection of transgenic and knock out mouse lines in which various key synaptic proteins, such as PSD95, SAP97, GRIP1/2, PICK1 and PKC zeta have been removed or altered. The findings from these experiments will help us identify the essential regulators of AMPAR trafficking in vivo under conditions that elicit synaptic plasticity and learning and elucidate the molecular mechanisms underlying synaptic plasticity in the brain in health and disease.

An Administrative Core will provide support for all Conte Center activities. The Administrative Core will be directed by Richard Huganir and the daily activities will be performed by two administrators. The Administrators will arrange consultant activities, manage budgets, order supplies, monitor inventory, and provide secretarial support for the PI. In addition, the Core Administrator will also manage the Conte Center web site. The Administrative Core will be located in the Administrative Office Suite for the Department of Neuroscience, Room 1001 ofthe Woods Basic Science Building. This room is located near the office ofthe PI and within one or two floors of each of the Conte Center Labs.

PROJECT 3 PROJECT SUMMARY/ABSTRACT Project 3 of the Johns Hopkins Alzheimer s Disease Research Center (JHADRC) is focused on the regulation of ?-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid receptors (AMPARs) by amyloid ? (A?) in order to identify mechanisms by which A? alters synaptic transmission and plasticity during the early stages of Alzheimer s disease (AD) prior to cell death. AMPARs mediate the majority of fast excitatory neurotransmission in the central nervous system and are dynamically regulated by modifications such as phosphorylation and by interactions with other proteins. This regulation of AMPARs is critical for modulating synaptic transmission and plasticity. We propose that A? induces synaptic deficits by interfering with the normal regulation of AMPARs, resulting in down-regulation of AMPARs at excitatory synapses. Understanding this process is critical both for understanding early disease pathology and for generating effective therapies. Our project consists of the following three specific aims: (1) To test the hypothesis that elevated A? alters AMPAR phosphorylation to reduce surface AMPAR expression by altering interactions with proteins involved in receptor trafficking. This aim will allow us to identify cellular targets downstream of A? that may provide a mechanistic focus to directly slow or halt early progression of A?-mediated cognitive decline. (2) To use novel site-specific AMPAR phosphorylation mutant mice to determine the physiological and A?-mediated pathological role of phosphorylation in AMPAR trafficking, basal synaptic transmission, and synaptic plasticity. This aim will provide novel insight into the role of specific phosphorylation sites in different aspects of A?-induced synapse dysfunction. (3) To test the hypothesis that physiological and behavioral deficits induced by A? in vivo can be rescued with compensatory alterations in AMPAR modulation. In this aim we will take advantage of a transgenic mouse model of AD (APPswe/PS1dE9 mice) that more closely mimics the exposure of neurons to chronic, endogenously-produced A?, as occurs in AD patients. These mice will be used to determine if disruption of AMPAR phosphorylation or specific protein interactions can ameliorate deficits in synaptic plasticity and memory induced by chronic A? elevation. Through the studies in this proposal, we will identify mechanisms underlying A?-induced decreases in synaptic AMPAR expression and will determine if inhibition of these molecular changes is sufficient to rescue A?-induced deficits in synaptic transmission, synaptic plasticity, and memory.

Project Summary Cell signaling pathways in the brain are an essential part of a complex system regulating the activity and coordination of neuronal circuits. During learning and memory synaptic plasticity processes regulate the strength of synaptic connections and modify neuronal circuits. Intracellular signaling pathways play critical roles in regulating synaptic strength and are an important part of the molecular mechanisms underlying learning and memory. Kinase signaling pathways play a central role integrating neuromodulatory and excitatory and inhibitory synaptic inputs to control how neurons and neuronal circuits adapt during behavior. However, direct interrogation of signaling pathways in live awake animals has been challenging due to lack of appropriate tools. Monitoring of multiple signaling pathways in such a setting has not been achieved. The goal of this research proposal is to develop novel tools to simultaneously monitor the activity of several signaling pathways and to use in vivo imaging techniques to visualize cell-specific and circuit-specific activity of these dynamic signaling pathways in live animals during physiologically relevant sensory experience and learning. In initial studies the use of genetically encoded biosensors will be used to monitor signaling pathways in vivo using two-photon microscopy to image an existing biosensor in the somatosensory cortex of mice. New single- color fluorescent protein based biosensors with a greater dynamic range than existing FRET-based sensors will be developed for imaging signaling pathways (PKA, CaMKII, ERK, and mTOR complex 1) involved in synaptic plasticity. The ultimate goal of this research proposal is to introduce these new biosensors into the mouse brain and to monitor both rapid dynamics of signaling pathways on the order of seconds to minutes and long-term stability of signaling pathways on the order of weeks to months using two-photon microscopy in awake behaving animals. This proposed project would be the first investigation of cell-specific and circuit- specific neuronal signaling beyond calcium and voltage changes in live animals. These studies will allow us to uncover mechanisms underlying the regulation of signaling pathways and will shed light on the complexity of signaling pathways during synaptic plasticity in vivo.

Project Summary Recent genome-wide association studies of Intellectual disability (ID), Autism spectrum disorder (ASD) and Schizophrenia (SCZ) have improved our understanding of the molecular and cellular basis of human cognitive diseases. Functional categorization of these genes has revealed a significant enrichment of mutations affecting glutamatergic synapse structure and function. One protein that has been shown to regulate glutamatergic synapses is SynGAP, a RasGAP that is a critical negative regulator of spine morphogenesis and synaptic plasticity via Ras-ERK and protein synthesis-dependent signaling pathways. De Novo deleterious SYNGAP mutations are estimated to account for approximately 1% of ID cases and are highly comorbid with ASD. SYNGAP variants have also been found to be a significant risk factor in other neuropsychiatric disorders including SCZ and bipolar disorder (BP). We recently identified SynGAP as one of the most potent regulators of synaptic size and/or number using a high-throughput screen of 200 SCZ-associated risk genes. In addition, we found that ID/ASD-associated SynGAP mutations also affect synaptic structure and function. These data support the notion that human SynGAP mutations might alter synaptic transmission and plasticity. To determine how disease-associated SynGAP mutations impact synaptic pathophysiology and behavior, we will first characterize the effect of SYNGAP disease risk variants on synapse structure and function using a combination of approaches including real time imaging, biochemical and electrophysiological techniques. Next, we will use CRISPR/Cas9 genome editing to generate mouse models carrying SynGAP mutations that precisely mimic human disease risk variants of SynGAP. With these mice we will determine whether they have differential plasticity, circuit and behavioral phenotypes. Finally, we will perform mechanism based drug screens to target disrupted SynGAP-regulated signal transduction pathways to discover small molecule(s) that can ameliorate synaptic and behavioral deficits. This proposed project would be the first systematic investigation of disease-associated SynGAP mutations on synaptic pathophysiology and animal behavior. These studies will allow us to gain insight into mechanisms underlying SynGAP-associated diseases and pave the way for novel therapeutic strategies.

Abstract Autism spectrum disorders are clinically and genetically heterogeneous. Identification of convergent molecular pathways and neural circuits underlying autism endophenotypes are crucial to discovery of novel drug targets for development of effective therapies. Glutamate mediates the majority of excitatory neurotransmission in the CNS. Glutamate receptor interacting proteins 1/2 (GRIP1/2) are neuron-enriched scaffolding proteins with 7 PDZ domains. PDZ domains 4-6 of GRIP1/2 bind the c-terminal domain of AMPA receptor 2/3 (GluA2/3). Loss of Grip1/2 expression in mice results in delayed recycling of GluA2 in neurons and increased sociability and social interactions. Studies of AMPA-signaling proteins identified an enhanced GluA2-S880 phosphorylation in prefrontal cortex in the mutant mice. In a screen of glutamate signaling genes in patients with autism, we found gain-of-function mutations in GRIP1-PDZ4-6 that contribute to reduced social interactions in autism patients. To study mechanisms of GluA2 trafficking in modulating social behaviors, we generated knock-in mice carrying a human autism-associated mutation I586L. Grip1-I586L mice show increased binding with GluA2 in brain lysates and exhibit a reduced sociability in the modified three-chamber sociability tests. We hypothesize that Grip1-I586L alter GluA2 recycling and surface expression resulting in increased AMPA synaptic strength and enhanced local connectivity in prefrontal cortex. We will study molecular mechanisms responsible for GluA2 trafficking defects in Grip1-KO and Grip1-I586L mice. We will investigate neural mechanisms of disturbance of AMPA signaling in prefrontal cortex causing social behavioral deficits in autism using electrophysiology and optogenetic methods. The results shall provide valuable insights into neural mechanisms of AMPA signaling defects in social behavioral deficits in autism.

PROJECT SUMMARY Memories can last the entire lifetime of an organism. Dynamic communication among billions of neurons at synapses underlies information processing and enables the coding and storage of memory. Changes in synapse strength and structure through synaptic plasticity are widely speculated as the cellular basis of memory formation and storage. Studies have identified cellular signaling events and molecular rearrangements underlying the initiation of synaptic plasticity. However, considerably less is known regarding the molecular basis enabling synaptic strength and memories to persist for extended periods of time. While initial synaptic plasticity and long-term memory coding requires protein synthesis, following a period of consolidation, memory storage becomes independent of protein synthesis or neural activity, suggesting that the memory is stored in a remarkably stable molecular entity. During this time, however, most of the individual proteins that are known to make up the synapse will turnover, being degraded and replaced within hours to a few days. Therefore the question remains as to what physical substrates underlie the persistence of long-lasting memories. One possibility is that exceptionally long-lived proteins (LLPs) reside in synapses and act as molecular anchors to maintain the synaptic strength or a network property that defines a given memory. While previous studies have demonstrated the existence of LLPs in the central nervous system, particularly in the nuclei of non-dividing cells, no studies to date have addressed whether such proteins exist at synapses and contribute to the establishment and maintenance of long-term memories. To investigate this hypothesis we designed an unbiased, proteomics-based approach to identify LLPs resident in synapses and characterize their neuronal function. Stable isotope metabolic pulse-chase labeling will be used both in vivo and in vitro to measure the half-lives of the neuronal and synaptic proteomes. These experiments will further be combined with behavioral and pharmacological manipulations to examine how memory formation and neuronal activity influence protein turnover. Identified candidate proteins will be characterized using biochemical, cell-biological, electrophysiological, imaging and behavioral methodologies to determine how these LLPs contribute to synaptic/neuronal function and memory. Within the metabolically active environment of the cell it is known that proteins can undergo oxidative damage. Such damage to LLPs could be a source of vulnerability that may contribute to functional decline during aging. The experiments described in this proposal will significantly contribute to our understanding of LLP functions in the brain and their potential role in for memory formation, long-term storage and age-related cognitive decline.

Project Summary Developing new methodological and analytical tools to address currently insurmountable experimental questions is crucial to the future of neuroscience. While recent advances in two-photon microscopy and activity sensors have revolutionized our understanding of the cellular and circuit basis of behavior, many barriers still exist that preclude fully exploring the molecular basis of these processes in vivo. This is an important question, as modulating synaptic strength is thought to underlie higher brain functions such as learning and memory, whereas synaptic degradation is observed in many neurological pathologies. Despite the clear significance of synaptic communication, a large-scale analysis of how synapses across the brain are distributed and change during learning has not been performed, mainly due to technical difficulties arising from the immensely complex nature of synaptic networks. Here, we present a suite of novel methodologies that breaks through these barriers. Our novel approach leverages CRISPR-based labeling of endogenous synaptic proteins, in vivo two-photon microscopy to visualize fluorescently tagged synapses in behaving animals, and deep-learning-based automatic synapse detection. Using these minimally invasive methods, we will be able to longitudinally track how the strength of millions of individual synapses change during learning. By developing and enabling new strategies to automatically detect and track vast numbers of synapses across entire brain regions, this pioneering approach has the potential to provide us with an unprecedented view of synapses in behaving animals, enabling new discoveries regarding how dynamic regulation of synaptic strength encodes learning and memory.

The synapse is the fundamental unit of the nervous system, enabling communication between brain cells and providing a substrate for experience-dependent plasticity to drive adaptive behaviors. Altering the strength of synapses between specific cells or neuronal ensembles is thought to underlie higher brain functions such as learning and memory, whereas synaptic degradation is observed in many neurological pathologies, such as Alzheimer's disease (AD) and related dementias. Despite the clear significance of synaptic communication, the relationship between impaired synaptic function, progression of AD symptoms, and cognitive decline remains unclear. However, recent breakthroughs in molecular microscopy enable direct imaging of the progression of pathological synaptic deficits in mouse models of Alzheimer's disease. Our approach is to fluorescently tag synaptic proteins and AD markers to track them throughout disease progression using in vivo two-photon microscopy. By imaging large populations of synapses comprising entire cortical and hippocampal circuits, we strive to gain a detailed understanding of how molecular pathologies affect synaptic physiology and ultimately give rise to cognitive decline. This approach will yield a detailed time course of the progression of synaptic and cognitive Alzheimer's pathologies that may reveal effective treatment windows and novel avenues for therapeutic interventions for human disease.