Lonnie Wollmuth - US grants

AMPA receptors; NMDA receptors; glutamate receptor; synapses; calcium flux; ion channel blocker; sodium ion; mathematical model; protein structure function; biophysics; calcium channel blockers; calcium channel; magnesium ion; calcium ion; clone cells; voltage /patch clamp; microspectrophotometry; calcium indicator;

DESCRIPTION (provided by applicant): The long-term objective of my laboratory is to understand the molecular mechanisms underlying signaling in the brain, especially as it relates to higher brain function and disease states. My laboratory focuses mainly on synaptic physiology, specifically those synapses that use glutamate as their neurotransmitter. Glutamate receptors (GluRs) mediate basic information processing in the brain and contribute to the cellular and molecular mechanisms underlying learning and memory, the development and maintenance of cellular connections, and pain transduction and perception. When dysfunctional, GluRs have also been implicated in numerous psychiatric disorders such as schizophrenia as well as in acute and chronic cell death including that associated with various neurodegenerative diseases such as Alzheimer's and Parkinson's disease. The goal of the present proposal is to define GluR structure-function, focusing mainly on the ion channel component of these ligand-gated ion channels. Such information is key to understanding the role of GluRs in brain function as well as the development of drugs that attenuate the cell death they mediate under pathological conditions. This issue will be studied in recombinant GluRs using a variety of techniques including site-directed mutagenesis, cysteine scanning, chimeras, protein chemistry, available crystal structures, fast agonist application, and whole-cell, outside-out patches, and single channel recordings. Specific Aim#1 will study the dynamics of the linker regions coupling the ligand-binding domain to M3, the major transmembrane gating domain. This region represents a critical juncture regulating receptor function and hence sites for modulating receptor dynamics under pathological states. Specific Aim#2 will study the functional and structural contribution of the M4 segment to channel function. All mammalian GluRs subunits have a transmembrane segment, M4, located C-terminal to the core of the ion channel (M1-M3). M4 is a necessary requirement for mammalian GluR function. Defining its contribution to this process will clarify fundamental mechanisms of gating in GluRs. Specific Aim#3 will further pursue issues related to channel gating specifically to desensitization, focusing on electrostatic interactions between the ion channel and the ligand-binding domain. This work will define fundamental principles of GluR structure-function and provide new tools and insights into means to attenuate the cell death GluR mediate under psychiatric states and pathological conditions. PROJECT NARRATIVE: Synapses mediate the transfer of information in the brain and are at the center of how we think, act and learn. Many drugs that have proven successful in the treatment of brain diseases such Alzheimer's and Parkinson's Diseases and mental illness act on synapses so they are a major focus of medical and pharmaceutical research. Our work studies one element -glutamate receptors- found at the most wide spread excitatory synapse in the mammalian brain, trying to define how this element carries out its function and how it might be modulated in disease states.

DESCRIPTION (provided by applicant): A long-term objective of my laboratory is to understand the molecular mechanisms underlying signaling in the brain, especially as it relates to higher brain functions and disease states. In vertebrates, processing of visual information-how details are extracted and recombined to form our perceptions-depends critically on the local circuitry in layers 2/3 of the visual cortex. Although a variety of details are required to define this circuitry, our work focuses on the dynamic factors regulating the excitatory (glutamatergic) pyramidal inputs to inhibitory interneurons, focusing primarily on postsynaptic mechanisms. In particular, we are interested in how protein-protein interactions can affect the gating properties of glutamate receptors. To do so, we will take advantage of heterologous expression systems to study mechanisms of interaction between glutamate receptors and intracellular proteins and how these interactions affect channel function and use paired recordings from synaptically connected (pyramidal-to-interneuron) cells in layers 2/3 to study how these protein-protein interactions directly affect synaptic amplitudes and their frequency dependence. Specifically, we will address (1) The mechanism of synapse-associated protein 97 (SAP97) interaction with AMPAR GluR-A channels, specifically as it relates to changing their biophysical properties. Preliminary data indicate that SAP97 alters the gating properties of GluR-A AMPARs, and experiments proposed here will define the molecular basis for these novel actions providing fundamental insights into protein-protein interactions and how these interactions affect the dynamic function of a key ligand-gated ion channel. (2) The functional significance of SAP97 interaction with GluR-A in layers 2/3. SAP97 is expressed in multipolar interneurons and our proposed experiments will specifically address the functional significance of GluR-A/SAP97 interaction to the dynamics of synaptic function. This work is basic research in the molecular basis of signaling in the brain. Defining basic principles of signaling provides a foundation for understanding the disruptions that can occur in disease states.

Our long-term goal is to address molecular determinants of brain disorders. Fast synaptic transmission in the brain is mediated by ion channels that are directly activated by a chemical neurotransmitter. NMDA and AMPA receptors are glutamate-gated ion channels that convert the presynaptic release of glutamate, the predominant excitatory neurotransmitter in the brain, into a postsynaptic signal. By defining the operation of NMDA and AMPA receptors, we will gain a better understanding of how they control brain function. We will also learn how to modulate their function with greater precision and specificity to help understand, and potentially treat, brain disroders such as schizophrenia, epilepsy, and the excitotoxicity associated with acute and chronic brain disorders. Our experiments will focus on a eukaryotic transmembrane segment, the M4 segment, which is positioned around the pore domain. Recent published and preliminary data from our lab has indicated that the M4 segments act in novel ways to regulate core synaptic functions of NMDA and AMPA receptors. Highlighting their significance is that inherited and de novo mutations in the M4 segments induce neurodevelopmental disorders and epileptic encephalopathies. Aim 1 will address the novel hypothesis that the unique kinetics of NMDA receptors at synapses are due to two kinetically distinct gates and that the M4 segments regulate these gates in a subunit-specific manner. We will address this hypothesis using cysteine cross-linking, rigorous single channel analysis, and molecular dynamic simulations. Aim 2 will address the hypothesis that the M4 segments in NMDA receptors are a major allosteric conduit coupling external domains to transmembrane and internal domains. Here, we will test this hypothesis by decoupling external domains from transmembrane and internal domains and assay this decoupling using electrophysiological and FRET based measurements. Aim 3 will address the hypothesis that the M4 segments in AMPA receptors carry out distinct functional roles including acting as a conduit for auxiliary proteins found at synapses. Here, we will compare functional properties between the M4 segments in NMDA and AMPA receptors using electrophysiological recordings and molecular dynamic simulations. Our experiments will delineate molecular features of NMDA and AMPA receptors that contribute to synaptic function. This information will aid in developing specific therapies to target these receptors in nervous system disorders.

The sensory receptor neurons of the visual and auditory systems transmit information about stimulus properties via synaptic outputs that are specialized for transmission of both fast, transient and slower, sustained signals. The hallmark of these specialized synapses is the presence at the active zone of a complex organelle, the synaptic ribbon, which enhances the size of the readily releasable pool of synaptic vesicles. Although vision and hearing are impaired when synaptic ribbons are disrupted, exactly how the ribbon supports neurotransmitter release has been unclear. Numerous synaptic vesicles are attached to the surface of the ribbon, and these vesicles are thought to support both the fast and sustained components of transmission during stimulation. Previously in this project, methods were developed to track single synaptic vesicles associated with the ribbon at super-resolution in living synapses during neurotransmitter release, and to detect when vesicles fuse to release their contents. The results showed that ribbons have the dual role of serving as a conduit for diffusion of tethered vesicles and as a scaffold that supports compound fusion of vesicles. In the proposed work, the novel direct-imaging approach will be used to quantify all aspects of the life cycle of synaptic vesicle at ribbon active zones, including capture by the ribbon, translocation, fusion, clearance, and recycling by endocytosis. Direct tests of the compound-fusion model will also be carried out using cryofixation electron microscopy of ribbon synapses rapidly frozen while in the process of release. An additional goal of the project is to determine the molecular mechanisms that govern vesicle trafficking at ribbon active zones, in order to provide a comprehensive understanding that integrates molecular and physiological views of synaptic function. The results of the project will lead to significant new information about fundamental cellular and molecular mechanisms that control the early steps in transmission of sensory information in both vision and hearing.

ABSTRACT Visual deficits stemming from retinal dysfunction are associated with Alzheimer?s Disease (AD). As a result, assessment of retinal dysfunction may be a specific and non-invasive diagnostic tool of AD at the earliest stages of pathology (Mahajan & Votruba, 2017). Two hallmarks of AD are accumulation of senile plaques, composed mainly of amyloid-b (Ab), and neurofibrillary tangles (NFT), composed of hyperphosphorylated tau protein. In the retina of AD patients, the presence of senile plaques and neurofibrillary tangles is limited, but there is extensive hyperphosphorylated tau throughout the inner and outer plexiform layers (den Haan et al., 2018). We speculate that this hyperphosphorylated tau, alters glucose metabolism and oxidative stress, leading to disruption of signaling in the retina and disease progression. Indeed, metabolic diseases, diabetes and glaucoma that is not linked with intraocular pressure, are associated with AD (Mancino et al., 2018). Hence, altered metabolism may be a critical component of retinal dysfunction in AD. Still, the mechanisms underlying aging and AD related retinal problems are poorly defined, which limits development of therapies . Mitochondrial dysfunction is a critical component of aging and age-related neurodegeneration including AD. In the Parent Grant (see Specific Aims on subsequent page), we are studying the structure and dynamics of the synaptic ribbon in retinal cells, mainly bipolar cells and photoreceptors using ?flash- freeze? cryofixation electron (slam-freeze cryo-EM) microscopy, confocal microscopy with electrophysiology, immunohistochemistry, and measuring oculomotor responses. This work is mainly being done in zebrafish. In the present Supplement, we want to extend our experiments to study how glucose metabolism and oxidative stress ? elements associated with AD ? impact synaptic transmission in the retina focusing on mitochondria including mitophagy and structure and dynamics of the synaptic ribbon in retinal cells, mainly bipolar cells and photoreceptors. We will essentially overlay the question of glucose metabolism and oxidative stress on our on-going experiments. Critically, we will bring our highly refined and quantitative approach to address the role of metabolism in retinal function. In Objective 1, we will address how variations in glucose metabolism and oxidative stress impact the distribution and structure of mitochondria and ribbon synapses using slam-freeze cryo-EM microscopy (Mses. Henry-Vanisko & Akmentin & Dr. Joselevitch). This technique is central to the parent grant and will allow us to exam the impact of glucose metabolism and oxidative in quantitative detail. In Objective 2, we will address how NMDA receptors contribute to cell viability under conditions of altered glucose metabolism and oxidative stress in the retina. NMDA receptors play critical roles in cell health in the retina and are strongly linked to the neurodegeneration in AD (Wang & Reddy, 2017), and a putative treatment (neurosteroids) for glaucoma and AD act through NMDA receptors (Ishikawa et al., 2018). For these experiments, we will take advantage of NMDA receptor knock-outs we have generated to study NMDA receptor subunits in the retina and the visual system as a whole (Mr. Zoodsma & Drs. Sirotkin & Joselevitch). Finally, in Objective 3, we will obtain fish lines that express various markers for mitochondria (e.g., express TMRE which changes its fluorescence spectrum based on oxidation state)(Mandal et al., 2018)(Drs. Sirotkin & Joselevitch). In the long term, these tools will allow us to ultimately expand our studies to whole animals under chronic conditions. Our major goal with the Supplement would be to establish the feasibility of our overall approach to address glucose metabolism, oxidative stress, mitochondrial dynamics, mitophagy, and ribbon synapse structure/dynamics in the retina. If we are successful, we would develop a new research program, in collaboration with Dr. Howard Sirotkin, to continue to study glucose metabolism and oxidative stress in the retina. We would envision submitting a new RO1, possibly independent of the Parent Grant, either to NEI or NIA. Specific Aims of Parent Grant The major focus of the Parent Grant is to study the molecular structure and dynamics of synaptic ribbons in bipolar cells and photoreceptors. Technically, we study these issues mainly using "flash-freeze? cryofixation electron (cryo-EM) microscopy, confocal microscopy combined with electrophysiology to image single vesicles, immunohistochemistry to study overall retina, and oculomotor responses to assay how our manipulations affect visual acuity (note we have also set-up a visual acuity assay using prey-capture (Gahtan et al., 2005), which while not of the parent grant, we have been using for NMDA receptor knock-out fish). We will use these same techniques in the present Supplement. Specific Aim 1. Determine the dynamics of synaptic vesicle cycling at active zones of ribbon synapses. The goal of this aim is to answer fundamental questions about synaptic vesicle trafficking at the active zone, by exploiting our newly developed methods for tracking single vesicles in voltage- clamped synapses. The aim consists of several components. 1) Results obtained in the previous grant period are consistent with the hypothesis that synaptic vesicles distal to the plasma membrane undergo compound fusion on synaptic ribbons. We will test this hypothesis using "flash-freeze" (Watanabe et al., 2013a; Watanabe et al., 2013b) cryofixation electron microscopy to identify the ultrastructural correlates of compound fusion at ribbon synapses. 2) Clearance of fused vesicles from the active zone is an important aspect of the vesicle cycle. Here, we will use a combination of pHluorin signals, FM dye uptake, and electron microscopy to establish how long fused vesicles reside at the active zone, and where and when they are retrieved by endocytosis. 3) Synaptic vesicles also move on ribbons by diffusion. Single-vesicle tracking methods will be used to determine the detailed properties of this process, including the step size and dwell time at a specific position on the ribbon, as well as the calcium-dependence of vesicle-ribbon interactions. These experimentally determined parameters will then be used to generate a quantitative model of neurotransmitter release at ribbon synapses that incorporates tethered diffusion, vesicle dissociation from the ribbon, and compound fusion. 4) Photoreceptor ribbons are much larger than bipolar-cell ribbons, extending a micron or more into the terminal from the membrane. The methods we developed to track single synaptic vesicles and monitor their fusion will be used to test the hypothesis that the relative contributions of compound fusion and vesicle diffusion to release are different at large photoreceptor ribbons than at the small ribbons of bipolar cells. Specific Aim 2. Establish the molecular basis of ribbon synapse function. Increasing information is available about the molecular composition of ribbon active zones, but much less is known about the function of these molecules. Our ability to track single vesicles at ribbons and monitor their fusion with high temporal and spatial precision provides a sensitive assay for ribbon function and therefore offers a unique opportunity to establish molecular mechanisms. In Aim 2, we will combine molecular manipulations with multiple functional assays to establish how synaptic proteins regulate vesicle release at ribbon-type active zones and contribute to visual function. A number of proteins that colocalize in the CAZ of conventional synapses are instead spatially segregated to either the ribbon or the CAZ at ribbon synapses. The central hypothesis to be tested in this Aim is that the ribbon-associated proteins mediate capture,diffusion, and compound fusion of synaptic vesicles during sustained neurotransmitter release, whereas CAZ-associated proteins mediate the fast, transient component of release proximal to the membrane. We will use a variety of strategies to test this hypothesis by modifying specific proteins in order to localize them, track their movements at living synapses, and alter their function. The sensitive physiological assays at our disposal ensure that the effects of these molecular manipulations can be assigned precisely to specific aspects of ribbon synapse function. To accommodate the reduced budget, we will reduce the number of presynaptic proteins propose to be investigated from 11 (original) to 5.

PROJECT SUMMARY/ABSTRACT NMDA receptors (NMDARs) are glutamate-gated ion channels that mediate excitatory neurotransmission in the brain. Many higher order neural processes including synaptogenesis and the synaptic plasticity underlining learning and memory depend on NMDAR-mediated transmission. The NMDAR GluN2B subunit is critically involved in early brain development. Accordingly, missense and nonsense mutations in GRIN2B, the gene encoding GluN2B, are associated with autism spectrum disorder, intellectual disability, and schizophrenia among other neurodevelopmental disorders. The specific role of GluN2B in brain development and the causal link between GluN2B dysfunction and these diverse disorders is unknown. Our goal, taking advantage of the power of zebrafish, is to identify the role of GluN2B in brain development and neurodevelopmental disorders and to pioneer new therapies to treat such disorders. We must first establish the use of zebrafish as a model system to study GluN2B in neurodevelopmental disorders. To do so, we will test whether zebrafish GluN2B (zGluN2B) functions similarly to human GluN2B and if it displays a comparable pharmacology (Aim#1). In addition, we need to identify GluN2B-dependent zebrafish behaviors (Aim#2), which provide a pathway to study circuit development and a substrate for behaviorally-based drug screens. In Aim 1, we will use heterologous expression zebrafish and human NMDAR subunits in HEK293 cells and patch clamp electrophysiology to characterize functional and pharmacological properties. In Aim 2, we will examine how knockout of zebrafish GluN2B affects behaviors associated with human neurodevelopmental disorders. In the long-term, zebrafish have the potential to provide a platform to study the role of GluN2B in brain development and the effects of human GRIN2B missense mutations on neural circuit development and carry out behaviorally based high throughput small molecule drug screens to counter the effects of GluN2B dysfunction.

Wollmuth, Lonnie P ABSTRACT The trainee (Noele Certain) is a PhD student who is starting her 3rd year of graduate training in the Graduate Program in Molecular and Cellular Pharmacology at Stony Brook University. Noele?s long-term goal is to maintain an independent research lab in parallel with a teaching career ? she wants to be a strong role-model and mentor to other underrepresented minorities. I believe my laboratory will provide the necessary environment for Noele to achieve her career goals. Specifically, my laboratory and its surroundings will provide Noele with a strong intellectual and technical environment; she will study scientific issues of great clinical importance (structure/function and cell biology of AMPA receptors); she will obtain rigorous training ? an issue of fundamental importance for any scientist; she will be given guidance but also considerable freedom so that she can develop and mature as an independent scientist; she will be given numerous opportunities to present her data in public forums, both locally and nationally; and finally she will be given opportunities to teach undergraand mentor undergraduates under my guidance. With a rigorous but broad background in molecular and cellular pharmacology, Noele will be well prepared to pursue her goal of a research career and to research any topic that may be appropriate as she develops her own research lab. In addition, she will be well placed to act as a mentor for undergraduate and graduate students including underrepresented minorities. Page 1