Craig C. Garner - US grants

Alzheimer's Disease (AD) is an age-related neurological disorder that affects almost exclusively associational areas of the human brain resulting in senile dementia. AD has been characterized neuropathologically by the presence of neurofibrillary tangles, neuritic plaques, and neuronal cell loss in specific cortical and subcortical brain regions. While these are the most characteristic features of this disease, evidence from clinico- pathological studies indicate a rather poor correlation between the occurrence of these pathologies and the severity of dementia. At present a decrease in synaptic density provides the best correlation to cognitive dysfunction in AD. The mechanisms mediating synaptic loss in AD are unknown. It is unclear, for example whether this is due to changes in the postsynaptic neuron and its ability to perceive changes in synaptic activity or whether a combination of pre- and post-synaptic events are required. A better understanding of the molecular mechanisms that regulate the stability of synapses or their dynamics behavior would contribute greatly to our understanding of the neuropathology of AD and consequently to the understanding of the behavioral abnormalities associated with this illness. This proposal focuses on investigating specific hypotheses concerning the possible functional roles three novel synaptic junctional proteins, SAP90, SAP97 and SAP102, may play in modulating the assembly state of synapses in AD. The primary goal of this proposal is to utilize biochemical, molecular, cell biological and immunohistochemical techniques to examine what roles SAP90, SAP97 and SAP102 perform in synaptic junctions. Our comprehensive working hypothesis is that the synaptic associated guanylate kinases (GKs) are involved in modulating the release of neurotransmitter (Nt) via second messenger systems, which may also regulate the efficiency and stability of synaptic junctions. Dysfunctional modifications of these GKs may contribute to the etiology of AD and other neurodegenerative diseases by altering synaptic stability. The first goal of this proposal is to examine whether SAP90, SAP97 and SAP102 are required for t he formation and maintenance of synaptic junctions. The second goal is to determine whether members of this family encode authentic guanylate kinases. The third goal is to test whether SAPs at synaptic junctions possess a GK activity that is modulated by synaptic activity. The final goal is to determine whether SAPs are differentially expressed in aging and in Alzheimer's brain.

DESCRIPTION (provided by applicant): Synapses of the mammalian CNS are highly specialized cellular junctions designed for rapid and regulated signaling between nerve cells and their targets. Morphologically, synapses are asymmetric structures composed of a presynaptic bouton filled with synaptic vesicles (SVs), a synaptic cleft and a postsynaptic specialization. Proper synaptic function requires the precise functional integration of numerous, appropriate localized components. However, little is known about the molecular and cellular mechanisms underlying the assembly of CNS synapses. Recent advances in determining the molecular composition of CNS synapses has allowed neuroscientists to address a number of fundamental questions regarding the assembly and function of CNS synapses. In our recent studies, we have begun to analyze how presynaptic active zones (AZs) are formed. These studies have lead to the discovery of two AZ cytoskeletal proteins, Piccolo and Bassoon, which appear to be central organizers of these sites of synaptic vesicle exocytosis and neurotransmitter release. More recently we have examined when, how and in what form these AZ proteins are transported and recruited into nascent synapses. These studies have answered several basic neurobiological questions regarding the timing of synaptogenesis and whether AZ formation precedes the formation of the postsynapse. In particular, we have found that glutamatergic synapses form in less than two hours and that AZs form prior to the postsynapse. Mechanistically, we could show that the rapid formation of AZs (less than 20 mins) appears to be accomplished by the fusion of a small number of AZ precursor vesicles carrying numerous components of the AZ that are essential for SV exocytosis. Experiments outlined in this proposal will test the hypothesis that these putative AZ precursor vesicles are not only essential for the assembly of functional AZs, but also for the formation of a functional postysynapse. This will be accomplished by determining the molecular composition of these precursor vesicles, whether their fusion at nascent synaptic sites is required for AZ formation and whether they facilitate the release of factors that promote the clustering of postsynaptic glutamate receptors and structural proteins. These studies are critical to the understanding of how synapses are formed during normal development, as well as in developmental disorders and degenerative diseases.

Synapses of the mammalian CNS are highly specialized cellular junctions designed for rapid and regulated signaling between nerve cells and their targets. Abnormal synaptogenesis and synaptic reorganization in the developing CNS has been strongly correlated with developmental disorders such as fragile X, epilepsy, schizophrenia and mental retardation. Our ability to understand how different genetic and environmental insults cause cognitive dysfunction and mental retardation requires a better understanding of the cellular mechanisms that lead to the proper assembly and function of CNS synapses. This requires a molecular description of the constituents of synaptic junctions and the mechanisms used by neurons to correctly sort traffic and localized each component. Our studies of CNS synapses have led to the identification and characterization of numerous synaptic junctional proteins. One of the most recently identified, Bassoon, is a novel component of the presynaptic cytoskeletal matrix assembled at the active zone. Based on its structure on its structure and distribution, we hypothesize that it is involved in the assembly and function of CNS synapses. With regard to mental retardation and cognitive dysfunction, our analysis of the Bassoon gene and its transcripts have revealed the presence of a CAG expansion similar to these found Huntingtin, Ataxins and the Fragile X mental retardation Moreover, Basson expression is selectively enhance in a neurodegenerative disorder, multiple system atrophy. To gain insights into the role played by Bassoon in the presynaptic cytoskeletal matrix, we proposed to examine the mechanisms directing that transport and assembly of Bassoon at CNS synapses In addition we propose to assess the function of Bassoon in presynaptic nerve terminals by analyzing loss of function mutations in the mouse Bassoon gene Bsn on the structure, assembly, and function of CNS synapses in the developing mouse brain.

DESCRIPTION (Adapted from applicant's abstract): Synaptogenesis in the developing rat central nervous system occurs primarily during the first three weeks of postnatal life. At present, we only have a rudimentary understanding of (i) the molecular constituents of synaptic junctions, (ii) in what form synaptic proteins are correctly sorted and trafficked to newly forming synapses, and (iii) how junctional proteins are able to define the various pre and postsynaptic subdomains. For example, the presynaptic terminal is a highly specialized cellular compartment designed to rapidly and efficiently release neurotransmitter from synaptic vesicles. Much progress has been made in understanding the molecular machinery involved in the Ca+2 regulated exocytosis of SVs at the presynaptic plasma membrane, as well as the endocytotic events that lead to the recycling of SV membranes. Knowledge is limited, however, concerning how the docking, fusion and recycling of SVs is restricted to the active zone. EM studies of nerve terminals have shown that the cytoplasm surrounding the readily releasable pool of SVs is composed of an electron dense network of fine filaments referred to as the junctional cytoskeletal matrix. In many respects, this presynaptic cytoskeletal matrix is the structural equivalent to that found at the PSD and is thought to hold the PSD and active zone in register. It may also help define the active zone by clustering components of the exo and endocytotic machinery. At present, little is known about the exact nature of the proteins that define this region, named the presynsptic junction (PSJ). Preliminary analysis of structural components of synaptic junctions has led to the identification of two novel structurally related presynaptic cytoskeletal matrix proteins of the PSJ, Piccolo and Bassoon. Biochemical, cellular and functional studies strongly indicate that they are structural elements of the PSJ and have led to the hypotheses that they play a fundamental role in defining the active zone. Experiments outlined in this application will test this hypothesis for Piccolo. Moreover, the investigator will test the hypotheses that the central region of Piccolo is involved in anchoring it in the PSJ and that components of the PSJ are sorted and trafficked to nerve terminals as a pre-PSJ particle. These studies will provide an entry point to understanding the molecules and mechanisms underlying PSJ assembly.

DESCRIPTION (provided by applicant): The assembly and plasticity of CNS synapses is intimately linked to the dynamic recruitment and turnover of its individual components. Of particular importance is the insertion and removal of synaptic -a-amino-5- hydroxy-3methyl-4-isoxacole propionate (AMPA) receptors. This has been shown to be critically important for the establishment of recurrent signaling at excitatory synapses as well as in setting the dynamic range of synapses as occurs during the establishment of certain forms of long term depression (LTD) or potentiation (LTP). Adapter proteins such SAP97, Grip and Pick1, known to interact with the cytoplasmic domains of AMPA receptors are thought to be important for receptor trafficking as well as synaptic localization and function. Of these SAP97 is emerging as one of the most important for trafficking and synaptic localization of AMPA receptors containing GluR1 subunits. In particular, this multidomain scaffold protein has been found to associate with GluR1 subunits in the endoplasmic reticulum (ER), Golgi and synapses. Excitingly, in our recent preliminary data, we have found that synaptic isoforms of SAP97 functionally affect the synaptic recruitment of AMPA receptors contain GluR1 subunits. As such, we hypothesize that SAP97 isoforms play a direct and integral role in the trafficking and dynamic insertion of synaptic GluR1 subunits of the AMPA receptor. In this application, we propose to test these and other hypotheses regarding the role that specific SAP97 isoforms play in the trafficking of GluR1 receptors as well as the assembly of multi-component protein complexes around GluRI. These issues are critical for understanding the cellular and molecular mechanisms underlying the dynamic properties of synaptic AMPA receptors and are relevant to issues of learning and memory and how drugs of abuse dramatically influence the behavior of these receptors and the activity of neural circuits.

Core A: Administrative Core An adminstrative core is esential for the effective management of a complex multi-disciplinary program project. This core will oversee all aspects of the daily operations of the Program Project. This will be performed by the principle investigator, Dr. Craig C. Garner, in consultation with members of the Executive Committee and the Internal Advisory Board. He will be responsible for budgetary planning, policy and guidelines, and in general making sure the program runs smoothly. He will also be responsible for the organizing the monthly Executive Committee tele-conference and inter-lab meetings. He will also oversee the organization of our triannual Stanford-UT Southwestern meetings. He will also work closely with the directors of Animal Core B and the different project directors to ensure there is a fair allocation of animal resources and animal genotyping. The Core in general will oversee budgetary planning and the preparations of annual reports and the non-competive annual renewal of the program project. Finally, it is the responsibility of the Principle Investigator ot assure that the PPG scientist work as an integrated team, and that through his scientific and organizational leadership, that the goal of the PPG are achieved.

DESCRIPTION (provided by applicant): Long-lasting neural circuit modifications are thought to underlie all forms of adaptive and pathological experience-dependent plasticity. Thus there has been great interest in elucidating the mechanisms and functions of various forms of synaptic plasticity. While historically NMDA receptor-dependent long-term potentiation (LTP) has been the prototypic and most extensively studied form of long-lasting synaptic plasticity, it is clear that several key circuits in the mammalian brain express an NMDA receptor-independent form of LTP that is triggered by increases in cAMP and mediated by a long-lasting enhancement of neurotransmitter release. The central goal of this program project is to elucidate the molecular mechanisms and functions of this presynaptic form of LTP. This will be accomplished by analyzing the functional properties of the presynaptic active zone protein RIM to both presynaptic forms of plasticity as well as its contribution to learning and memory in the cerebellum. We have assembled four projects to accomplish these goals. In project #1, we propose a biochemical and genetic analysis of RIM to elucidate the contributions that individual RIM isoforms and domains make to RIM function. In project #2, we will utilize electrophysiological approaches to assess the physiological functions of RIM isoforms and discrete RIM domains to different forms of presynaptic plasticity. In project #3, we propose a set of cellular and dynamic imaging studies to examine how RIM proteins regulate the dynamics of key active zone proteins in response to synaptic activity. Finally, in project #4, we proposed to integrate these RIM structure function studies to assess the role that presynaptic forms of plasticity contribute to VOR plasticity in the mouse cerebellum. These studies will advance our understanding of not only how RIM proteins regulate neurotransmitter release, but also how presynaptic forms of long-lasting plasticity contribute to both neural circuit behavior and experience dependent plasticity.

Project # 3: Molecular and Cellular Mechanisms of Presynaptic activity Long-lasting neural circuit modifications are thought to underlie all forms of experience-dependent plasticity, including learning and memory. Thus, there has been great interest in elucidating the mechanisms and functions of various forms of synaptic plasticity. While excellent progress has been made in elucidating the mechanisms of postsynaptic plasticity little is known about how presynaptic forms of plasticity are encoded. Recent genetic and physiological studies on the presynaptic active zone protein RIM have demonstrate that this molecule plays essential roles in the regulated release of neurotransmitter. Furthermore, RIM has been implicated in regulating a special type of cAMP dependent long-lasting increase in neurotransmitter release. However, mechanistically it is unclear how RIM accomplishes these diverse functions. Structural studies support a model wherein RIM promotes presynaptic plasticity through its ability to interact with a number of key active zone proteins including Munc13, a critical SV priming factor. In this project, we propose to explore whether RIM isoforms regulate neurotransmitter release probability by dynamically controlling the levels of Mund3 tethered to the active zone cytoskeletal matrix. This will be accomplished by addressing the following issues. In aim #1, we will define the cellular and molecular correlates of presynaptic plasticity in dissociated neuronal cultures. In aim # 2, we will dissect how RIM1a and its specific domains contribute to setting SV release probability. Finally in aim #3, we will assess the functional significance of RIM2 isoforms to presynaptic function? Furthermore, close collaborations with project #1 and #2 will explore the structural and physiological importance of discrete domains in RIMs and their interacting proteins for synaptic plasticity. Similar collaborations with project #4 will investigate whether RIM regulates the dynamic states of synaptic proteins at subclasses of synapses in the cerebellar system. These studies will advance our understanding of the mechanisms encoding synaptic plasticity.

DESCRIPTION (provided by applicant): Behavioral and cognitive disorders caused by genetic lesions, environmental insults or age related dementia are serious societal problems, contributing to a loss of quality of life for affected individuals and their families. The incidence of intellectual disability in the United States is 1-2%, costing tens of billions of dollars/year in health care and lost productivity. A key to defining therapeutic strategies that can ameliorate these disorders is a fundamental understanding of the cellular and molecular mechanisms regulating how neuronal circuits encode, process, and retain information. Synapses are the basic components of information storage and plasticity in our brains. Over the last decade, we have used molecular, cellular and reverse genetic approaches to identify and characterize proteins involved in the assembly, function and plasticity of vertebrate synapses. Increasingly, our data has shown that many of these proteins are transcribed from large multi-gene families spanning hundreds of kb of genomic DNA and comprised of multiple alternatively spliced exons. This complexity, as well as the cost and time associated with the generation of conditional knockout or knockin mice, has severely hampered progress in the field. What is needed is a simple, cost-effective strategy, akin to transgenics, for creating mice deficient in individual or combinations of proteins. Recent advances in transgenic technology using lentiviruses combined with interference RNAs are well poised to meet this challenge. Over the last five years, we have developed isoform specific short-hairpin RNAs (shRNAs) against numerous synaptic proteins as well as a collection of lentiviral vectors expressing XFP-tagged reporter proteins for the cell-autonomous and synapse specific analysis of pre and postsynaptic function. In the present application, we propose to integrate these technologies and the CRE/lox system to create an innovative set of lentiviruses capable of conditionally inactivating and expressing multiple neuronal proteins. Given their importance in the assembly and plasticity of synapses, we propose to use shRNAs against the structurally related presynaptic active zone proteins Piccolo and Bassoon to evaluate this new strategy. These conditional knockdown mice for Piccolo and/or Bassoon will be invaluable for assessing the shared and unique functions of these proteins during neuronal differentiation, axonal pathfinding, synapse formation, and in mechanisms of presynaptic plasticity at vertebrate synapses. PUBLIC HEALTH RELEVANCE: This grant application describes experiments designed to create a set of conditional transgenic knockdown mice deficient in the expression of the structurally related presynaptic active zone proteins Piccolo and Bassoon. In Aim 1, we propose to design, build and test the next generation of lentiviral vectors for the cost effective creation of conditional knockdown mice. Specifically, we will create a virus expressing three mini-genes: one an shRNA for Piccolo knockdown, the second a YFP-tagged Synapsin1a for labeling presynaptic boutons, and the third a red fluorescent protein variant (mCherry) for selecting mice with the integrated transgenes. The expression of each will be placed under the control of the CRE/lox system. The functionality of these vectors will be tested in HEK293 cells and cultured hippocampal neurons. In Aim 2, we will create and characterize a transgenic mouse using the lentiviral vector created in Aim1. In Aim 3, we will expand this technology to create a lentiviral vector capable of knocking down two or more synaptic proteins by expressing shRNAs for each protein under separate polymerase III promoters. This technology will be invaluable for studies of individual or families of proteins thought to perform similar functions. Moreover, it is ideally suited for a molecular replacement strategy that simultaneously eliminates the expression of one synaptic protein while replacing it with a mutated or altered version. Such a strategy could become crucial for the generation of mouse models of specific psychiatric, neurodegeneartive or neurodevelopment disorders such as depression, schizophrenia, or Autism Spectrum Disorders, or Alzheimer's disease.

DESCRIPTION (provided by applicant): Autism is a neurodevelopmental disorder characterized by abnormal social behavior, communication deficits, and repetitive or stereotyped behaviors. Cumulative prevalence literature suggests that approximately 1 in 1000 children are diagnosed with Autism, and as many as 1 in 150 are diagnosed with one of the Autism Spectrum Disorders (ASDs), including Asperger's Syndrome and PDD-NOS (pervasive developmental disorder not otherwise specified). Economic costs associated with ASDs are estimated at $35 billion/year, including special education services and treatments to reduce symptoms. These estimates do not even factor in the costs associated with lost productivity and specialized care for Autistic individuals once they reach adulthood. A key to developing therapeutic strategies to effectively treat ASDs is a fundamental understanding of the cellular and molecular mechanisms that underlie them. The goal of this grant application is to design an imaging-based screening assay in order to assess whether a group of genes associated with Autism Spectrum Disorders (ASDs) lie in a common signaling pathway. Our approach will not only define key molecular components of the signaling pathway(s) involved in ASDs, but also create a platform for screening small molecule libraries in order to identify potential pharmacotherapies for ASDs. Specifically, we will test the hypothesis that many of the genes mutated in ASD patients function to regulate the formation of complexes between two key synaptic proteins, the transsynaptic cell adhesion molecules Neurexin and Neuroligin, which in turn mediate the maturation, function, and plasticity of excitatory glutamatergic synapses. We will first establish an imaging-based assay to detect and quantify levels of transsynaptic Neurexin/Neuroligin (Nrx/Nlg) complexes. Here, we will combine the technologies of proximity labeling via BirA/AP biotinylation (developed by our collaborator Alice Ting at MIT) to label synaptic Nrx/Nlg complexes, bicistronic vectors to simultaneously introduce two pre- or postsynaptic proteins into the same neuron, and high-resolution quantitative imaging to monitor Nrx/Nlg complex formation. Next, we will evaluate whether at least a subset of ASD-associated genes regulate the formation of synaptic Nrx/Nlg complexes. Specifically, we will create short interfering (si) RNAs against known ASD-associated gene products, and perform a medium-throughput screen to assess whether downregulation of these molecules affects Nrx/Nlg complex formation. Once in place, this assay will be adaptable for automated, higher-throughput screens of siRNA and small molecule libraries, thus enabling the identification of other molecular components of the Nrx/Nlg-based signaling pathway, and of potential drug targets to normalize cognitive function in ASD patients carrying mutations in these genes. PUBLIC HEALTH RELEVANCE: The goal of this grant application is to design an imaging-based screening assay in order to assess whether subsets of genes associated with Autism Spectrum Disorders (ASDs) lie in a common signaling pathway. Specifically, we will test the hypothesis that a set of the genes mutated in ASD patients function to regulate the formation of transsynaptic complexes between two key synaptic proteins, the cell adhesion molecules Neurexin and Neuroligin, which in turn mediate the maturation, function, and plasticity of excitatory glutamatergic synapses. In Aim 1, we will establish an imaging-based assay to detect and quantify levels of transsynaptic Neurexin/Neuroligin (Nrx/Nlg) complexes in dissociated hippocampal cultures. Here, we will combine the technologies of proximity labeling via BirA/AP biotinylation (developed by our collaborator Alice Ting at MIT) to label synaptic Nrx/Nlg complexes, bicistronic vectors to simultaneously introduce two pre- or postsynaptic proteins into the same neuron, and high-resolution quantitative imaging to monitor Nrx/Nlg complex formation. In Aim 2, we will evaluate whether ASD-associated genes regulates the formation of synaptic Nrx/Nlg complexes. Specifically, we will create short interfering (si) RNAs for the known ASD-associated gene products, and perform a medium-throughput screen to assess whether downregulation of these molecules affects Nrx/Nlg complex formation. Once in place, this assay will be adaptable for automated, high-throughput screens of siRNA and small molecule libraries, thus enabling the identification of other molecular components of the Nrx/Nlg-based signaling pathway and of potential drug targets to normalize cognitive function in ASD patients carrying mutations in these genes.

DESCRIPTION (provided by applicant): Autism spectrum disorders (ASDs) have risen to approximately 1 in 88 in the Unites States over the past years, affecting an entire generation of children, families and communities. Currently, the diagnosis for most forms of ASD is based on a triad of behavioral symptoms, including social impairments, communication difficulties, and repetitive or stereotyped behaviors, with no quantitative measures for screening or assessment of potential drug therapies. Electrophysiological measurements of synapses and neuronal networks from these patients may hold the potential for diagnosing, characterizing and analyzing the effectiveness of potential treatment strategies. Here, we propose to apply a transformative technology for the long-term intracellular recording networks of neurons differentiated from patient-derived iPSC. To accomplish this goal, we have created a solid-state device comprised of 2D arrays of Stealth electrodes that sit passively within the membrane of neuronal cells and have the capacity to record synaptic, neuronal and network properties of multiple interconnected neurons simultaneously for days to weeks. Through the optimization of the fabrication of these Stealth probes and the transformation into a turn-key device, we will evaluate the feasibility of this platform as a diagnostic and research tool for ASD. We then propose to use this innovative scalable analytical platform to characterize the neuronal, synaptic and network signatures of neurons differentiated from iPS cells derived from patients with Phelan-McDermid Syndrome and then assess the effectiveness of emerging drug therapies to normalize aberrant signatures. If successful, our solid-state platform can be transformed into a high-throughput screening device that will allow investigators to recapitulate early developmental stages of ASD and evaluate the effects of ASD mutations and environmental insults on neuronal network and synaptic function and utilize this as a tool for drug screening, diagnosis and personalized treatment.