Thomas C. Sudhof - US grants

Many intracellular processes have been shown to be regulated by Ca2+, presumably via its interaction with specific Ca2+- dependent regulatory proteins. We have recently described the purification of members of a novel family of Ca2+-binding proteins called calelectrins and have shown that one of these proteins, the 32.5 kDa calelectrin, is highly expressed primarily in ductal epithelial cells of various organs and the cardiac intercalated disk. In contrast, the 67 kDa calelectrin, another member of this protein family, is ubiquitously expressed. Several lines of evidence suggest that the calelectrin-like Ca2+-binding proteins may be involved in signal-dependent phospholipid metabolism, such as arachidonic acid or phosphoinositol release, and/or in the regulation of membrane traffic. Here, we propose to purify biochemical amounts of several members of the calelectrin Ca2+-binding protein family, to clone their cDNAs and to deduce their amino acid sequences. The purified proteins will be used to obtain affinity-purified antisera and monoclonal antibodies for immunocytochemistry and inhibition experiments, and to test various biochemical hypotheses concerning the functions of the calelectrins in arachidonic acid and phosphatidylinositol metabolism and membrane traffic. These experiments will yield insight into the functional and evolutionary relationships and regulatory roles of these proteins. This information in turn will help guide our future studies which will investigate the role of calelectrins in disease states such as cystic fibrosis and certain cardiomyopathies.

Many intracellular processes have been shown to be regulated by Ca2+, presumably via its interaction with specific Ca2+- dependent regulatory proteins. We have recently described the purification of members of a novel family of Ca2+-binding proteins called calelectrins and have shown that one of these proteins, the 32.5 kDa calelectrin, is highly expressed primarily in ductal epithelial cells of various organs and the cardiac intercalated disk. In contrast, the 67 kDa calelectrin, another member of this protein family, is ubiquitously expressed. Several lines of evidence suggest that the calelectrin-like Ca2+-binding proteins may be involved in signal-dependent phospholipid metabolism, such as arachidonic acid or phosphoinositol release, and/or in the regulation of membrane traffic. Here, we propose to purify biochemical amounts of several members of the calelectrin Ca2+-binding protein family, to clone their cDNAs and to deduce their amino acid sequences. The purified proteins will be used to obtain affinity-purified antisera and monoclonal antibodies for immunocytochemistry and inhibition experiments, and to test various biochemical hypotheses concerning the functions of the calelectrins in arachidonic acid and phosphatidylinositol metabolism and membrane traffic. These experiments will yield insight into the functional and evolutionary relationships and regulatory roles of these proteins. This information in turn will help guide our future studies which will investigate the role of calelectrins in disease states such as cystic fibrosis and certain cardiomyopathies.

Inositol 1,4,5-trisphosphate (InsP3) mediates calcium release from intracellular stores, thereby regulating cell activity. Neuronal InsP3-levels are strongly depressed by chronic lithium treatment, an effective therapy in manic-depressive psychosis, suggesting a relationship between the intracellular actions of InsP3 and the symptoms of manic-depressive psychosis. InsP3 acts by binding to specific receptors that are coupled to calcium. channels. In the present proposal, the functional domains and regulation of the InsP3-receptor will be studied. The long-term objective will be the elucidation of the molecular mechanisms by which InsP3-receptors release calcium from intracellular stores. The specific aims of the experiments described are: 1. To characterize the structures of the functional and regulatory domains of the InsP3-receptor. In preliminary studies an InsP3-receptor has been purified, molecularly cloned, and functionally expressed in COS cells. Its structure-function relationships will be investigated by expressing and characterizing mutant forms of the receptor and by biochemical assays utilizing site-specific antibodies. 2. To characterize the intrinsic calcium channel of the receptor. Novel expression approaches will be employed to localize the transmembrane regions that form the calcium channel, and mutagenesis experiments will delineate amino acid residues important in channel function. 3. To clone and characterize novel InsP3-receptors by homology with the currently available receptor. The experiments will employ partial clones obtained in preliminary experiments as probes to isolate full-length cDNA's. Novel receptors will be characterized biochemically by sequence-specific antibodies and by expression in heterologous cells. Together, these experiments will provide a molecular description of the functions of the InsP3-receptor and of the pathway(s) by which InsP3 modulates calcium levels in cells.

In brain, synaptic connectivity determines which neurons communicate with each other, thereby establishing the wiring diagram of the brain. Changes in synaptic connectivity are probably important in the pathogenesis of a number of mental and neurological diseases, such as AIzheimer's disease and epilepsy. However, the molecular basis for the specificity of neuronal connections is poorly understood. Neurexins are polymorphic, neuron- specific cell surface receptors that may function in specifying cell-cell interactions between neurons. This application describes a genetic approach to elucidate the functions of neurexins in the brain and to test their potential involvement in disease. Three specific aims will be pursued: First, genes encoding murine and human neurexins will be cloned and their exon-intro structure and chromosomal localizations will be determined. This will form the basis for the homologous recombination studies described below and allow the search for candidate diseases in which neurexins may be mutated. Second, the cloned murine genes will be used to delete or selectively mutate endogenous neurexin genes in embryonic stem cells, and mice carrying mutations in different neurexin genes will be generated. Mutant mice with defined deletions or mutations of neurexins will serve as models for functional and pathophysiological studies. Third, mice heterozygous and homozygous for neurexin gene mutations will be analyzed using biochemical, morphological, and physiological techniques to determine the in vivo functions of neurexins and their potential as disease models. Together, these experiments will give insight into the biological functions of neurexins and their potential involvement in mental and neurological diseases.

The understanding of the molecular basis for membrane traffic has made rapid progress in recent years. By convergence of work from yeast genetics, molecular neurobiology, and classical cell biology, major discoveries that have been made include the identification of proteins involved in membrane traffic and of their point of action; the demonstration of the roles of key proteins in fusion such as rab proteins, NSF, and synaptotagmins; and initial insight into fusion mechanisms. In addition, it is now becoming clear that several important diseases, such as Alzheimer's disease and Parkinson's disease, may involve mechanisms related to membrane traffic (e.g. APP processing, synuclein). The major problem in membrane traffic now is to gain a mechanistic understanding of processes such as docking, fusion, and budding, and to determine what role aberrations of these processes have in diseases. In this regard, understanding the physicochemical basis for membrane trafficking reactions such as fusion is probably the most important future challenge. This understanding will require a multidisciplinary approach uniting different methodologies. The purpose of th0e proposed meeting is to bring together key investigators studying molecular mechanisms in membrane traffic in diverse systems, including selected pathologies. We hope that by bringing people with different expertise and sometimes opposing viewpoints together, their discussions will help to further focus the field into a productive area. This will allow formulation of new concepts and approaches for studying mechanisms of membrane traffic, and will give students and postdoctoral fellows a chance to gain access to different opinions in this field.

DESCRIPTION (Verbatim from the Applicant's Abstract): Parkinson's disease is a severe neurodegenerative disorder affecting millions of people annually. Alpha, beta and gamma synucleins constitute a family of abundant presynaptic proteins that probably function in regulating neurotransmitter release. Interestingly, alpha synuclein forms pathological aggregates in Parkinson's disease, and mutations in alpha synuclein are associated with Parkinson's disease in humans. This suggests a role for alpha synuclein in the pathogenesis of Parkinson's disease. The overall goal of the current application is to achieve such an insight. It aims to elucidate the physiological functions of synucleins to determine the structural properties that underlie these functions, and to unravel the mechanisms that mediate the role of synucleins in Parkinson's and other neurodegenerative diseases. Five specific aims are proposed to pursue this goal. First we describe experiments to study the relative expression and localization of synucleins. In the second specific aim we suggest studies to examine the biological properties, binding to phospholipids and other targets, and phosphorylation of wild type and mutant synucleins. Synucleins are natively unfolded but fold upon binding to a target. Therefore we propose to study in the third specifc aim the three dimensional structure of synucleins after binding to a target using circular dichroism and nuclear magnetic resonance spectroscopy. The fourth specific aim will examine the essential functions of synucleins in knock out mice. In the fifth specific aim we propose to analyse the effect of overexpressing wild type and mutant synucleins in transgenic mice. These transgenic experiments will complement the knock out approach to testing function, and will determine if a transgenic model of Parkinson's disease can be generated. We anticipate that these experiments will establish a molecular understanding of synuclein functions in the brain and provide insight into the role of an alpha synuclein in Parkinson's disease.

DESCRIPTION (Verbatim from applicant's abstract): Synapses represent polarized,[unreadable] specialized intercellular junctions and constitute the major points of[unreadable] communication between neurons in the brain. At a synapse, the presynaptic[unreadable] neuron secretes neurotransmitters that are then recognized by the postsynaptic[unreadable] cell. Synaptic junctions are formed by interactions between pre- and[unreadable] postsynaptic membranes but little is known about the molecular basis for these[unreadable] interactions. Alpha- and beta-neurexins constitute a polymorphic family of[unreadable] neuron-specific, cell surface proteins that are expressed from three genes.[unreadable] Indirect evidence suggests that these proteins function as cell adhesion and[unreadable] signaling molecules in synaptic junctions. The evidence includes the[unreadable] observation of a large number of neurexin isoforms generated by alternative[unreadable] splicing (>1000 isoforms), the finding that an alternatively-spliced subset of[unreadable] beta-neurexins binds to a novel neuronal cell adhesion molecule called[unreadable] neuroligin, which is also localized to synapses, and the fact that[unreadable] intracellular complexes of synaptic proteins assemble on neurexins via[unreadable] PDZ-domain interactions. Furthermore, knockout mice revealed that the deletion[unreadable] of alpha-neurexins causes a selective deficit in symmetric synapses. The[unreadable] overall hypothesis that will be tested in the current grant application is that[unreadable] neurexins function as synaptic cell adhesion and recognition molecules and[unreadable] contribute to the formation and maintenance of synaptic junctions. Four[unreadable] specific aims are proposed to test this hypothesis. The first specific aim will[unreadable] examine the precise localization of neurexins. The second analyzes their[unreadable] functions genetically in knockout mice. The third specific aim will[unreadable] characterize the functions of neurexins as cell adhesion molecules and[unreadable] signaling receptors, and the fourth specific aim will study the intracellular[unreadable] interactions of neurexins with PDZ-domain proteins that link the neurexins to[unreadable] synaptic vesicle traffic and the actin cytoskeleton. Together, these[unreadable] experiments will provide insight into the function of this highly conserved[unreadable] neuron-specific family of proteins and extend our understanding of how synapses[unreadable] are formed and maintained.[unreadable]

DESCRIPTION (provided by applicant): Alzheimer's disease (AD), a major cause of morbidity and mortality, is caused at least in part by the toxicity of Abeta, a proteolytic product of a receptor-like protein called amyolid-beta precursor protein (APP). Although much progress was made in the understanding of how APP cleavage generates Abeta, the normal functions of APP, the relation of these functions to Abeta production and AD pathogenesis, and the mechanism by which Abeta damages neurons are incompletely understood. Recent studies suggest that AD is initially a disease of synapses, and that the characteristic cognitive impairment of early AD is due to a loss of synapse function. AD may affect synapses because synapses are exquisitely sensitive to injury, and/or because the function of APP acts directly or indirectly on synapses. The overall goal of the current application is to follow up on this hypothesis by utilizing the tools and reagents that our laboratory has developed to analyse synapse function. We aim to characterize the possible physiological role of APP at the synapse, and to examine how the synaptic etiology of AD is related to this physiological role. Since diverse functions have been associated with APP and a variety of mechanisms have been invoked in the pathogenesis of AD, this application proposes a wide spectrum of approaches ranging from cell biology and biochemical protein purification to genetic experiments in mice. These experiments will investigate the functions of APP, and relate these functions to the production and pathogenicity of Abeta. Specifically, we propose to identify and functionally characterize extracellular ligands for APP as a first approach to understanding the function of the conserved extracellular sequences of APP (specific aim 1), a goal that was already partially achieved in preliminary results with the isolation of specific APP ligands. We also propose to test the possible role of APP as a transcriptional regulator (specific aim 2), a role that we recently identified in transactivation experiments, and to analyze the functions of Mints, which bind to APP (specific aim 3). Furthermore, we propose to examine the relation of APP cleavage and Abeta production to intracellular membrane traffic and its regulation, and to test the physiological significance of APP cleavage (specific aim 4). Finally, we propose to clarify the question whether Abeta exerts a specifically synaptic toxicity, and to investigate the mechanism of its toxicity by using novel mouse models of AD that allow a more mechanistic approach than currently available models (specific aim 5). Together these studies will contribute to our understanding of APP function and Abeta toxicity in AD, and may suggest new avenues for interfering with the pathogenesis of AD.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] a-Latrotoxin is a presynaptic neurotoxin that stimulates massive synaptic vesicle exocytosis. a-latrotoxin acts by binding to high-affinity neuronal cell-surface receptors that are thought to represent specific markers of presynaptic neurotransmitter release sites. Our previous studies identified three a- latrotoxin receptors that belong to different signaling pathways: The CIRLs are G protein-coupled receptors, neurexins are cell adhesion-like proteins with a single transmembrane domain and short cytoplasmic tail, and PTP6 is a receptor-like protein tyrosine phosphatase. To understand how a-latrotoxin receptors couple synaptic adhesion to neuronal exocytosis, we propose to identify receptor-interacting proteins and to study the mechanisms of their interactions. Our experimental approach is based on the use of a unique tool, a-latrotoxin, that binds the receptors with high affinity, thus allowing the one-stage isolation of the receptors together with associated proteins. We propose two sets of experiments. The first one is to identify less abundant protein components present in the preparations of affinity-purified a-latrotoxin receptors, and to analyze the specificity of their interaction with the receptors. This will be achieved by modern proteomics techniques complemented by immunoprecipitation with receptor-specific and associated protein-specific antibodies. As a key negative control, receptor preparations obtained from brains of knockout mice lacking a-latrotoxin receptors will be used. The second set of experiments is to study the mechanism of the interaction of individual a-latrotoxin receptors with synaptotagmin and syntaxin, proteins, involved in exocytosis that bind to a-latrotoxin receptors. In particular, we will identify to which receptors these proteins bind, and test if this interaction is direct. We will also determine the interacting domains of the receptors and their associated proteins. Obtaining a better biochemical definition of the a-latrotoxin receptor complexes is important because it promises to provide insight into the signaling mechanisms involved in their function, and may provide insight into presynaptic cell adhesion. This research will be performed primarily in Russia at the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry in collaboration with Alexander Petrenko as an extension of NIH grant R37 MH5280. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Although autism spectrum disorders (ASDs) are highly heritable, ASDs are heterogeneous, and no single genetic cause contributes to ASDs in a large proportion of patients. Instead, heterogeneous genetic changes, including many single gene mutations and copy-number variations (CNVs) are found in ASDs. Thus, a key question is whether different genetic changes contribute to ASDs via multiple, independent, pathogenic pathways, or whether the various genetic changes in ASDs converge onto a single pathogenic pathway. Several independent mutations in genes encoding synaptic proteins, such as neurexin-1, neuroligins, and SHANK3, suggested that ASDs may generally involve an impairment of synaptic communication between neurons. However, most of the other genetic changes observed in ASDs have no known effect on synapses in fact, have no known effect on any brain function. Thus, the major goal of the present proposal is to conduct a large scale, systematic analysis of the synaptic effects of genetic changes in ASDs. The approach will be to over express (to mimic gene duplications) or knock down (to mimic gene inactivations) mRNAs corresponding to 81 ASD candidate genes, and to test the effect of these manipulations on synapses using standardized assays. Cell viability, neuronal development, synapse density and synapse function will be assessed in cultured mouse neurons using optical and electro-physiological assays that are well established in the PI's laboratories. Genes that were found to affect neuronal development, synapse formation, or synapse function in cultured neurons will be studied by the same manipulations in vivo after stereotaxic injection of lentiviruses into the mouse hippocampus, or after in utero electroporation. Changes in synapse function and plasticity will then be examined in acute slices from these mice using standard electrophysiological techniques well established in the PI's laboratories. All results from this project will be posted on a dedicated public website, and all reagents generated will be made readily available to the scientific community. The results of this project will provide a standardized reference point for the function of ASD candidate genes, and provide an initial test of the hypothesis that despite their clinical and genetic heterogenity, ASDs involve a common, if diverse, pathway acting on synaptic communication in the brain. PUBLIC HEALTH RELEVANCE: Autism spectrum disorders are known to be clinically and genetically heterogeneous, but it is unclear whether these two types of heterogeneity are related, and how specifically the various genetic changes affect brain function. This project will address these issues by studying the changes in neuron-to-neuron communication caused by the genes associated with autism.

DESCRIPTION (provided by applicant): The recent identification of transcription factors (TFs) that can induce conversion of fibroblasts into pluripotent stem (iPS) cells makes it potentially possible to generate patient-specific neurons from fibroblasts. However, the neurons thus produced are difficult to obtain. The present project builds on preliminary results in which we demonstrate direct conversion of adult fibroblasts cells into neurons, referred to as 'induced neuronal cells'(iN cells), without an iPS intermediate. The resulting iN cells have the functional properties of neurons, including the ability to form functional synapses as assayed by electrophysiology. Thus, the iN cell technology provides a novel, more facile approach to generating and studying human neurons, and opens up a new avenue to investigating human disease processes. However, at this point the iN cell technology has only been developed for mouse fibroblasts, fundamental questions regarding the conversion process and the molecular identity of iN cells were not determined, the generation of iN cells from human fibroblasts has not yet been established, and most importantly, the feasibility of the iN cell technology to study diseases affecting neuronal function has not been demonstrated. In this project, we propose to address these important challenges in an interdisciplinary approach capitalizing from the combined expertises of the Wernig and S|dhof laboratories. We propose experiments that will systematically investigate the cellular and molecular identity of iN cells, and develop protocols to induce specific neuronal subpopulations from fibroblasts. These protocols will then be employed to model genetic diseases in mouse iN cells. Furthermore, we will extend our findings to human fibroblasts, with the long term goal to establish a cell model for neuropsychiatric diseases. Our goals will be pursued by a combination of tissue culture experiments with cells cultured from mice and humans, cell biology, molecular biology, and electrophysiology. We believe our proposed experiments have the potential to fundamentally change existing paradigms of cellular differentiation and epigenetic gene regulation, and could provide a novel platform to study human neurons from patients suffering from a variety of brain diseases. PUBLIC HEALTH RELEVANCE: This application will develop methods to generate neurons directly from non-neuronal cells, allowing the production of neurons from skin fibroblasts of human patients. These methods will then be used to test the effects of mutations associated with neuropsychiatric disorders on neuronal biology, with the long-term goal of establishing a better understanding of the pathomechanism of these diseases in human neurons.

DESCRIPTION (provided by applicant): This application proposes development of an integrated array of assays to quantitatively measure synaptic function in cultured neurons and acute brain slices, with the potential for scaling up these assays for high- throughput screens. As suggested by RFA-MH-11-40 Scalable Assays for Unbiased Analysis of Neurobiological Function, this grant does not address a specific biological question, but describes new tools for large-scale analysis of neuronal function. Specifically, our applications proposes in eight specific aims a series of related but independent new assay systems, including new methods of achieving controlled expression of neuronal genes in mice, and new techniques for measuring properties of synaptic function in neurons, ranging from pre- and postsynaptic calcium-signaling over analysis of glutamate receptor trafficking to imaging of neuronal excitation or silencing and various forms of neuronal stress. With these assays, our overall goal is to develop tools to meet the increasingly obvious need for better approaches to study neuro-psychiatric disorders such as autism and schizophrenia. A growing human genetics literature describes many candidate pathogenic genes for these disorders, with a synaptic function likely for some of the implicated genes such as neurexins, suggesting that synapses could represent a pathogenetic hotspot for at least a subset of cases in these diseases. Analyzing candidate disease genes, however, has proven difficult with current approaches that require long-term studies of single genes in time-consuming and expensive experiments. Thus, new approaches that can be scaled up and quantitated without enormous investments in time and effort are needed. The tools we describe here are meant to address this need, at least in part, and are based on a series of technical innovations. The tools can be applied to cultured neurons, acute slices, or in vivo experiments in mice, and primarily use optical detection methods as readout to allow scalability. All of the tools developed under the auspices of this application will be freely and immediately distributed to the community, with the hope that they will become standardized approaches for large-scale interrogation of synaptic function in projects performed throughout the country. This application attempts to address an urgent need for scalable assay systems for analysis of neuronal function, as enunciated by the RFA-MH-11-40. The proposed new assay systems focus on synaptic transmission because neuropharmacology and human genetics identified synaptic transmission as a possible site of impairment in many important brain diseases, including autism and schizophrenia.

DESCRIPTION (provided by applicant): The recent identification of transcription factors (TFs) that can induce conversion of fibroblasts into pluripotent stem (iPS) cells makes it potentially possible to generate patient-specific neurons from fibroblasts. However, the neurons thus produced are difficult to obtain. The present project builds on preliminary results in which we demonstrate direct conversion of adult fibroblasts cells into neurons, referred to as 'induced neuronal cells' (iN cells), without an iPS intermediate. The resulting iN cells have the functional properties of neurons, including the ability to form functional synapses as assayed by electrophysiology. Thus, the iN cell technology provides a novel, more facile approach to generating and studying human neurons, and opens up a new avenue to investigating human disease processes. However, at this point the iN cell technology has only been developed for mouse fibroblasts, fundamental questions regarding the conversion process and the molecular identity of iN cells were not determined, the generation of iN cells from human fibroblasts has not yet been established, and most importantly, the feasibility of the iN cell technology to study diseases affecting neuronal function has not been demonstrated. In this project, we propose to address these important challenges in an interdisciplinary approach capitalizing from the combined expertises of the Wernig and S|dhof laboratories. We propose experiments that will systematically investigate the cellular and molecular identity of iN cells, and develop protocols to induce specific neuronal subpopulations from fibroblasts. These protocols will then be employed to model genetic diseases in mouse iN cells. Furthermore, we will extend our findings to human fibroblasts, with the long term goal to establish a cell model for neuropsychiatric diseases. Our goals will be pursued by a combination of tissue culture experiments with cells cultured from mice and humans, cell biology, molecular biology, and electrophysiology. We believe our proposed experiments have the potential to fundamentally change existing paradigms of cellular differentiation and epigenetic gene regulation, and could provide a novel platform to study human neurons from patients suffering from a variety of brain diseases.

In this Project 2, we propose to study mouse neurons in order to better define in a model system the neuronal and synaptic phenotypes induced by high-risk schizophrenia (SCZ) mutations, to test whether the mouse phenotypes concur with those observed in human neurons (cross-platform validation), and to explore the relation of such phenotypes to SCZ-associated symptoms in human patients, experiments that can only be performed in mice but not in humans. Thus, this project is an essential component for the pursuit of the overarching goals of this application, which are to determine whether different high-risk mutations for SCZ produce a synaptic phenotype, where such phenotypes exhibit commonalities, and whether future screens for therapeutics to ameliorate such phenotypes can be developed. Inherent in these goals is the need not only to validate the reproducibility of phenotypes across platforms, but also to relate such phenotypes to symptoms observed in SCZ. Project 2 will focus in mouse neurons on the same mutations that constitute the focus of the studies on human neurons in the other projects, namely the Nrxn1 heterozygous and homozygous deletions, the minimal critical 22q11.2 deletion, and the 16p11.2 duplications and deletions. The three specific aims of this project will be carried out in a collaborative fashion coordinated by Stanford University (Tom S?dhof and Marius Wernig), with contributions by Rutgers University (Zhiping Pang), the U. of Cincinnatti (Bruce Aronow), and Eli Lilly (John Isaac). These three specific aims are: (1) To determine the neuronal and synaptic phenotypes of primary medial prefrontal cortex neurons that carry Nrxn1 gene deletions, the minimal critical 22q11.2 deletion, or the 16p11.2 duplication or deletion, (2) to test whether iN cells produced from mouse embryonic fibroblasts (MEFs) and iPS cells derived from mutant mice replicate the phenotype of the respective mutations in primary neurons, and (3) to determine the effects of the Nrxn1 gene deletions, the minimal critical 22q11.2 deletion, and the 16p11.2 duplication on synaptic properties of neurons in situ in brain slices of the medial prefrontal cortex. Together, the three specific aims will utilize an interdisciplinary approach to study SCZ pathophysiology in mouse models of the disorder, and provide vertically and horizontally integrated cross- validations of the effects of SCZ-associated high-risk mutations on neuronal function in mice. They will enable not only validation of the results obtained with human iN cells in Projects 1 and 3, but also facilitate a translation of such results into a conceptual framework for understanding SCZ-associated behavioral changes.

DESCRIPTION (provided by applicant): The goal of the NCRCRG program is to create multidisciplinary research groups, in partnership with academia and industry, to use patient-derived reprogrammed cells to develop validated platforms for identifying novel targets and developing new therapeutics ... to reduce the burden of mental illness. Here we propose to use induced neuronal (iN) cells, derived from induced pluripotent stem (iPS) cells, to model defects in synaptic function due to three engineered or naturally-occurring mutations known to substantially increase the risk of schizophrenia (SCZ): NRXN1 exonic deletions, 22q11.2 deletions, and 16p11.2 duplications. The study includes three projects. Project 1 will analyze the functional characteristics of human iN cells. For each mutation, one component will study cells with engineered mutations vs. non-mutated cells from the same control individual, while a second component will study iN cells derived from 5 SCZ patients with the mutation of interest vs. 5 control individuals. Project 2 will analyze the same mutations in mouse models, providing both a cross-species validation of human findings, and an novel attempt to determine whether the synaptic phenotypes of these mutations are the same in iN cells, cultured primary neurons, and brain slices (medical pre-frontal cortex). Project 3 will develop and optimize stem cell methods that are required for this project (large-scale implementation of iN cell protocols; new targeted mutation strategies for large CNVs; derivation of pure inhibitory iN cells; development of a mouse-free iN cell protocol), and which will also be needed to develop future high-throughput screening assays based on the pathophysiological models developed in this study. These experiments will provide new insights into the characteristics of neurons derived by reprogramming method, into synaptic phenotypes produced by each of these mutations, and ultimately into the pathophysiology susceptibility to the risk of psychotic disorders including SCZ. We will determine whether these mutations produce distinct or at least partially overlapping synaptic phenotypes. Either observation has profound implications for future SCZ research. To accomplish this work, we have assembled an outstanding scientific team from Stanford University (Drs. Sudhof in molecular neuroscience, Wernig in stem cell biology and Levinson in genetics of schizophrenia); Rutgers University (Dr. Pang in neuroscience and stem cell biology); Cincinnati Children's Hospital Medical Center (Dr. Aronow in bioinformatics and high-content imaging); Eli Lilly and Company (Drs. Isaac and Ursu in electrophysiology, Merchant in drug development, Dage in high-content imaging and assay development, Collier in genomics and systems biology, and Eastwood in biostatistics); and Cellular Dynamics, Inc. (Dr. Swanson, representing a leading biotechnology company in stem cell biology). These projects represent a multidisciplinary effort of academic and industrial institutions to gain insight into te pathophysiology of psychotic disorders by studying three mutations that are associated with SCZ.

DESCRIPTION (provided by applicant): In this Project, we will characterize synaptic, cellular and biochemical phenotypes of high-risk Alzheimer Disease (AD) mutations in human induced neuronal (iN) cells derived from iPS cells. We believe that the new advances in pluripotent stem cell biology and epigenetic reprogramming will provide an important breakthrough as they allow the genetic modification and functional evaluation of human neurons. Therefore, it is now possible to functionally interrogate risk mutations and study their cell biological effects in huma neurons. In particular, recent advances of gene targeting tools in human induced pluripotent stem (iPS) cells and our recent development of rapid methods that generate fully functional induced neuronal (iN) cells from iPS cells provide ideal conditions to begin to apply this technology to disease modeling for brain diseases such as AD. We will introduce into control iPS cell line derived from a well-characterized healthy normal subject conditional mutations that confer high risk for AD. Mutations will be introduced using homologous recombination in a protocol that we have developed in preliminary studies, and the conditionally mutant iPS cells will then be converted into precisely matched wild-type and mutant iN cells. Mutant and control cells will be characterized for Ab and Tau biochemistry and importantly for detailed synaptic properties. We believe the focus on the precise synaptic characterization represents a key innovative factor of our proposal as synaptic dysfunction may be much more sensitive than other cell biological assays such as cell death. Finally, we have confirmed in our iN cell/ astrocyte co-culture system that ApoE is primarily produced by the glia and that ApoE is a critical mediator of the glia-induced synaptic maturation of primary neurons and human iN cells, with possibly different effects of ApoE3 and ApoE4. Building on these results, we propose to evaluate in this specific aim the precise effects of ApoE3 and ApoE4 on synaptic maturation in wild type and APP-mutant iN cells generated in Aim 1, with the goal of gaining insight into the role of ApoE4 in AD pathology. Applied together, these specific aims will allow us to perform a well-controlled assessment of the effect of AD-associated APP mutations on the properties of human neurons and their synapses.

Project 1 will characterize and define synaptic and cellular phenotypes of high-risk mutations associated with schizophrenia (SCZ) (NRXN1 exonic deletions, 22q11.2 deletions and 16p11.2 duplications), in human induced neuronal (iN) cells derived from iPS cells. We will identify the most robust phenotypic changes in mutated iN cells (engineered and naturally-occurring), and the electrophysiological, genomic and morphometric assays that detect those differences most cost-effectively. The most promising models and assays will be selected for future high-throughput screening, based on robustness (prioritizing those models and assays that reveal phenotypes that are observed in more than one mutation) and cross-validation across laboratories, within and across species, and across source tissues in mice (iN cells, primary cultured neurons and mPFC brain slices -- Project 2). The project includes 9 specific aims: (1) To generate iN cells with an engineered form of each high-risk mutation for functional evaluation of mutant and non-mutant cells. (2) To generate iN cells from iPS cells from patients carrying high-risk mutations and controls. (3) To characterize the synaptic phenotype(s) of these mutations in human iN cells using electrophysiology and functional imaging. (4) To identify novel morphological synaptic and cellular phenotypes of these mutations in iN cells using morphometric analysis of high-definition images. (5) To identify gene regulatory networks associated with high-risk mutations. (6) To test cross-lab reproducibility of all procedures and findings. (7) To integrate functional, molecular and morphological data from mouse and human, mutant and control iN cells. In this last Aim, which is ongoing throughout the study, taking into account all results from Aims 3-6 in human iN cells as well as from Project 2 (mouse models), the most robust and reproducible pathophysiological models will be identified; the extent to which synaptic and cellular phenotypes are overlapping across mutations vs. distinct will be evaluated; and recommendations will be made for the selection of one or more model systems and assays for future high- throughput screening of novel therapeutics. This work will proceed through the analysis of each of the three mutations of interest, studying iN cells first in the engineered form of the mutation compared with non-mutant cells from the same control line, and then in the naturally-occurring mutations observed in SCZ patients vs. control individuals. This work will develop one or more pathophysiological models of synaptic and cellular dysfunction in iN cells carrying specific mutations, for future use in high-throughput screening of novel therapeutics.

Abstract The goal of this project is to describe the function of synaptic adhesion molecules of the Neuroligin family (Nlgns) in the mouse brain and in human neurons. Recent single cell expression studies have highlighted the obversation that Nlgns are expressed also in non-neuronal cells, in particular oligodendrocyte precursors cells (OPCs) and astrocytes who express Nlgns to even higher levels than neurons. Since little is known about the function of Nlgns in glia and their effect on neurons and neural circuits, we propose to specifically delete Nlgns in OPCs and astrocytes using our triple conditional Nlgn1-3 knock-out strain. Brains will be characterized morphologically, electrophysiologically on the cellular and circuit level, and mutant mice will be characterized by behavior. Next, we will perform an in-depth molecular characterization of the Neuroligin proteins by characterizing the molecular mechanisms underlying the surprising functional diversity of Nlgns. We will map their functional domains in mouse neurons by expressing various domain-mutant proteins in Nlgn1-4 quadruple knock-out cells. We will explore whether Nlgn sequence relates to functional specificity and investigate the notion of a synaptic Neurexin ?code? that may determine Nlgn specificity. To complement our mouse studies and explore human-specific Neuroligin function as well as human disease-associated mutations, we will capitalize on our previous human stem cell and reprogramming work in which we have developed human induced neuronal (iN) cells that exhibit all principal functional properties of primary mouse neurons including robust synapse formation. We propose to utilize this system to investigate the so far obscure function of NLGN4Y, a Y chromosomal gene closely related to NLGN4 on the X- chromosome and a member of the family not present in mouse. We will assess subcellular targeting by tagging the endogenous locus and assess the functional consequences of genetic deletion. Another frequently mutated Nlgn gene is NLGN3. Unlike NLGN4 it is better conserved in mice, but almost nothing is known about its function in human cells. In addition to generate loss-of-function alleles, we will study the functional consequences of distinct ASD-associated mutations introduced into the human NLGN3 gene. We will use a conditional mutagenesis approach as we have successfully done in the past, as it allows the generation of a perfect control conidition derived from the identical cell line as the experimental condition. Mutant human neurons and controls will be characterized biochemically, morphologically, by gene expression, and electrophysiologically. Finally, we propose to investigate the role of the proposed Nlgns-modulators MDGAs which are also found mutated in ASD and other neurodevelopmental disorders. We will assess their requirement for proper synapse formation and function by generating loss-of-function alleles in human neurons. We will further probe their function as Neuroligin modulators as competitive Nlgn binding molecules.

SUMMARY Alzheimer's Disease (AD) is thought to involve early synapse loss. Familial AD is most often caused by mutations in presenilins, which are the catalytic subunits of g-secretase. Inactivation of g-secretase in adult mice by conditional deletion of its presenilin or nicastrin subunits causes synaptic impairments followed by neurodegeneration, but the relation of the loss of g-secretase acitivity, the observed synaptic impairments, and the neurodegeneration is unclear, as is the connection between these processes and human AD. The present project will focus on clarifying the synaptic function of g-secretase in mature neurons and its relation to neurodegeneration, using mice as a model system. The project proposes four specific aims to address this overall goal. The first two specific aims will mechanistically characterize the synaptic impairments that are caused by g-secretase inactivation in young adult mice in vivo, and analyze their relation to the neurodegeneration that develops at a later stage after g-secretase inactivation. The third and fourth specific aim will then test the hypothesis that at least some of the synaptic functions of g-secretase that are impaired upon its inactivation may be mediated by g-secretase-dependent cleavage of presynaptic neurexins and postsynaptic neuroligins, which are trans-synaptic cell-adhesion molecules that bind to each other and act as master regulators of synaptic properties. Strikingly in this context, neurexins and neuroligins were shown previously to be substrates for g-secretase, were genetically linked to AD, and are arguably the most plausible mediators of g-secretase function at the synapse. To test the involvement of neurexins and neuroligins in the synaptic functions of g-secretase, the project will characterize the site and regulation of the g-secretase- dependent cleavage of neurexins and neuroligins, and probe the function of this cleavage using conditional knockout mice of these molecules. Moreover, the project will examine whether inactivation of neurexin- and/or neuroligin-cleavage promotes neurodegeneration. Viewed together, the experiments of this project will thus not only characterize the synaptic function of g-secretase and its relation to neurodegeneration, but also determine whether the synaptic function of g-secretase involves the cleavage of neurexins and/or neuroligins and whether such cleavage may play a contributory role in the pathogenesis of AD.

Project Summary/Abstract Parkinson's disease (PD) imposes a major burden on our aging population, and represents a neurodegenerative disorder that affects broad areas of the brain, but primarily manifests as a loss of dopaminergic neurons in the Substantia nigra. ?-Synuclein is thought to play a central role in PD because ?- synuclein mutations and polymorphisms are linked to PD, and because ?-synuclein containing inclusion bodies (Lewy bodies) are found neuropathologically in PD. Current data suggest that ?-synuclein promotes neurodegeneration in PD by misfolding into a toxic conformation, most likely as a microaggregate, that is deleterious to the neurons harboring the toxic ?-synuclein conformer. However, the mechanisms mediating the toxic effects of ?-synuclein remain incompletely understood. The present project proposes to investigate the mechanisms underlying ?-synuclein neurotoxicity in two specific aims, using cultured human neurons and mouse brains in situ as model systems, and focusing in particular on an understanding of the nature of ?- synuclein toxicity. The project will utilize an interdisciplinary approach with a particular emphasis on electrophysiological recordings to probe the relation of neuronal function, in particular as regards synaptic transmission, to ?-synuclein neurotoxicity. The results from these experiments will elucidate the extent and nature of ?-synuclein neurotoxicity in mouse and in human neurons.

Center PI: Malenka, Robert C. Principal Investigator (Project 2): Südhof, Thomas/Malenka, Robert Summary Although long-term synaptic plasticity has been studied for half a century, the fundamental mechanisms that mediate process such as NMDA-receptor-dependent LTP remain largely unknown, and the biological significance of LTP is incompletely understood. Here, we propose a novel avenue to understanding LTP by focusing on our recent unexpected observation that two different postsynaptic cell-adhesion molecules, LRRTMs and neuroligins which both bind to presynaptic neurexins, are essential for normal LTP. The present project is guided by the hypothesis that understanding trans-synaptic signaling mediated by neurexin-based cell adhesion may provide insight into the coordinated structural changes and vesicular trafficking events that occur postsynaptically during LTP. Four specific aims utilizing conditional knockout mice of LRRTMs and neuroligins are proposed to test this overall hypothesis. Specific Aim 1 will examine how LRRTMs and neuroligins contribute to LTP, Specific Aim 2 will map candidate molecular interactions of LRRTMs and neuroligins that underlie their function in LTP, Specific Aim 3 will test the role of these interactions in LTP using replacement of endogenous with mutant proteins in conditional knockout mice, and Specific Aim 4 will test the behavioral significance of the function of LRRTMs and neuroligins especially in learning and memory, with the aim to develop tests of the role of LTP in memory that involve highly selective changes in only LTP. Together, these experiments will advance our understanding of the relation between trans-synaptic cell adhesion mediated by neurexins and their ligands and long-term plasticity, thus contributing not only insight into how synapses are formed and function, but also into how LTP is induced and expressed. Relevance In studying LRRTMs and neuroligins, the present project will not only shed light on how these central organizers of synapses contribute to long-term plasticity and on the mechanisms of such plasticity, but will also provide a basic understanding of the potential role of these proteins in neuropsychiatric disorders such as autism and schizophrenia to which these proteins have been linked genetically. PHS 398/2590 (Rev. 11/07) Page 1 Summary

We found that the three transcription factors Ascl1, Myt1-like (Myt1l), and Brn2 can reprogram fibroblasts directly into functional neurons and are thus powerful neuronal lineage determination factors. Ascl1 and Brn2 are well studied genes. Myt1l on the other hand is a fairly uncharacterized zinc finger domain containting protein predicted to be a transcription factor. It has a remarkably unique expression pattern: it is expressed in virtually all neurons, but at the same time is also specific for neurons, to our knowledge the only transcription factor known to be specific and pan-neuronal at the same time. Independent of the reprogramming work, recent sequencing studies showed that MYT1L is frequently mutated in neuropsychiatric disease including autism and schizophrenia. Nevertheless, very little is known about this gene. Not even a mouse knock-out has been reported yet. We have therefore begun to investigate Myt1l's role in reprogramming and during normal development. Our first insights about its molecular function suggest that Myt1l is important for neuronal reprogramming and normal embryonic neurogenesis acting predominantly by transcriptional repression of non- neuronal lineage programs. The goal of this research project is to better understand the role of Myt1l in neurons after neurogenesis is completed. We propose to investigate its role on the molecular, cellular circuit, and behavioral level using the mouse as model system. We have intriguing preliminary data that about a third of high confidence autism- causing chromatin factors are also candidate binding partners of Myt1l. This suggests that all these mutations might converge on a hypothetical Myt1l-associated ?chromatin pathway? which is dysfunctional in at least subset of autism. This project will test this hypothesis and evaluate whether interference with the members of this chromatin ?pathway? might rectify molecular, cellular or behavioral phenotypes caused by Myt1l deletion. Since chromatin modifying enzymes are in principle pharmacologically tractable the hope would be that a functional intervention of such chromatin factors may be of therapeutic value for autistic children carrying MYT1L mutations. We will therefore test throughout all our three aims whether manipulation of these chromatin factors can rescue the molecular, cellular, or behavioral Myt1l phenotypes.

Neural circuits are constructed by synapses that connect neurons into vast networks. Although many neural circuits have been characterized, the molecular and cellular mechanisms that build their synaptic architecture remain largely unknown. During synapse formation that establishes the synaptic architecture of neural circuits, bi-directional signaling via trans-synaptic adhesion molecules is thought to control assembly of synapses. Strikingly, genetic changes in trans-synaptic adhesion molecules often predispose to neuropsychiatric disorders, suggesting that dysfunction of the synaptic architecture of neural circuits contributes to neuropsychiatric disorders, although the nature of these impairments is poorly understood. Our preliminary data show that in hippocampal neurons, formation of subsets of excitatory synapses requires latrophilins (Lphns), a family of three postsynaptic adhesion-GPCRs. Different Lphns mediate establishment of distinct synapses even in the same neuron, suggesting that they are involved not only in constructing synapses, but also in determining their specificity. How Lphns mediate synapse formation, and to what extent their synapse-formation function involves GPCR signaling or adhesive interactions, remains unknown. Moreover, SNPs in the human Lphn3 gene (ADGRL3) downregulate Lphn3 expression robustly. The present application proposes to examine the signaling mechanisms that mediate Lphn-dependent synapse formation, to explore how Lphns determine synapse specificity, and to investigate how changes in Lphn3 expression change synaptic function. Specifically, the proposed experiments will test the overall hypotheses that (1) Lphns control synapse formation and maintenance by a GPCR-mediated mechanism involving locally restricted signaling, that (2) different Lphn isoforms control formation of distinct synapses via sequence-specific differences in their protein interactions and GPCR function, and that (3) changes in Lphn3 expression impair formation of a specific subset of synapses. Three Specific Aims will test these hypotheses, thus targeting key questions that are most relevant for understanding how neural circuits are wired and how impairment of neural circuits alter cognition. Using both mouse and human neurons as a model system, the project will pursue broadly interdisciplinary approaches in both mice and human neurons that range from biophysical studies of ligand-receptor complexes to cell-biological investigations of intracellular signaling to behavioral studies probing for cognitive changes. Thereby, this application will provide insight into how Lphns drive synapse formation in mice, and how decreased expression of Lphn3 predisposes to synaptic changes in human neurons. Addressing these questions is of paramount interest in basic and translational neuroscience because neural circuits that process the brain?s information are constructed by synapse formation, and dysfunction or imbalance of synaptic communication in neural circuits likely underlies the pathogenesis of neuropsychiatric disorders.