Edwin R. Chapman, Ph.D. - US grants

Neurons transmit information by releasing neurotransmitters into the synaptic cleft. Release is triggered by increases in intracellular Ca2+ concentration and is mediated by the fusion of transmitter-filled synaptic vesicles with the presynaptic plasma membrane. While these aspects of synaptic transmission are well established, the molecular mechanism that couples Ca2+ to exocytosis is not known. The synaptic vesicle protein synaptotagmin binds Ca2+ and is essential for rapid and efficient Ca2+-triggered exocytosis. However, little is known concerning the molecular mechanism(s) by which synaptotagmin operates in the release process. The long term goal of our research is to elucidate the biochemical function of synaptotagmin in excitation- secretion coupling. To address this question, three Specific Aims are proposed: first, we will continue to delineate the synaptotagmin signaling pathway by identifying and characterizing synaptotagmin.target protein complexes. These studies include extensions of previously identified synaptotagmin.effector interactions, the characterization of preliminary effectors, and the identification of novel effectors. Second, we will determine whether individual synaptotagmin.effector interactions participate in Ca2+-triggered exocytosis. These studies will involve a detailed analysis of the structural determinants that mediate synaptotagmin.effector assembly. Binding domain data will be used to design peptides that potently and specifically inhibit discrete interactions in vitro. The function of individual interactions will then be determined by assessing the effects of the peptides on Ca2+- triggered exocytosis from semi-intact secretory cells. Third, we will determine the association kinetics of individual Ca2+- dependent synaptotagmin.effector interactions. From this analysis we can determine the temporal order of these interactions and discern which interactions are rapid enough to trigger release. This real time analysis will make us of synaptotagmin inserted into artificial liposomes and represents an initial step in the reconstitution of the molecular machinery that underlies Ca2+-triggered membrane fusion. A better understanding of the mechanisms of neuronal exocytosis will provide a framework for studying how this process is modulated and thus contributes to synaptic plasticity in both normal and pathophysiological states. Finally, defining this mechanism should ultimately provide targets for treatment of diseases in which synaptic transmission is impaired.

DESCRIPTION (provided by applicant): Communication between neurons relies on the release of neurotransmitters from pre-synaptic nerve terminals. Release is triggered by increases in intracellular [Ca2+] and is mediated by the fusion of transmitter-filled synaptic vesicles with the plasma membrane. The molecular mechanism that couples Ca2+ to exocytosis is not known. Synaptotagmin is a Ca2+ binding protein that has been proposed to function as a Ca2+ sensor that triggers release. We propose to examine the function of this protein, primarily on its two putative Ca2+-sensing domains, C2A and C2B. The structure and Ca2+-binding properties of the C2A-domain have been previously studied in detail. However, little is known concerning the function of the C2B-domain. We hypothesize that C2B is a Ca2+-sensing module that plays a critical role in Ca2+-triggered exocytosis. Support for this hypothesis is provided by preliminary data indicating that C2B must bind Ca2+, change conformation and oligomerize in order for docked synaptic vesicles to fuse in response to stimulation in vivo. To determine how C2B functions in exocytosis, three Specific Aims are proposed. (1) A series of biochemical studies will examine the kinetics of Ca2+-C2B interactions, the Ca2+ requirements for synaptotagmin oligomerization, and the role of C2B in the facilitation of SNARE complex assembly. (2) Time resolved amperometry, as well as genetic manipulations of Drosophila, will be used to address the role of C2B in fusion pore dynamics and in excitation-secretion coupling. A critical preliminary finding is that synaptotagmin regulates the opening and dilation kinetics of fusion pores, placing synaptotagmin at the final stages of the fusion reaction that is mediated by SNAREs. This finding supports the hypothesis that synaptotagmins and SNAREs are constituents of Ca2+-regulated exocytotic fusion pores. (3) Biochemical studies will be carried out to determine the subunit stoichiometry of this complex, its morphology, and the interfaces that mediate its assembly. The ability of the release machinery to respond to Ca2+ is subject to modulation and is likely to comprise an important locus for synaptic plasticity. Thus, a better understanding of the release process will provide insights into novel modes of synaptic plasticity and should ultimately provide targets for the treatment of diseases in which synaptic transmission is impaired.

DESCRIPTION (provided by applicant): Neurons transmit information via Ca2+-trigged exocytosis of synaptic vesicles. The synaptic vesicle protein synaptotagmin I has been proposed to serve as a major Ca 2+sensor that regulates release. Its cytoplasmic domain contains two Ca 2+sensing modules, C2A and C2B. Recent studies indicate that perturbation of the Ca2+-sensing ability of either the C2A or C2B domain inhibits the evoked fusion of docked synaptic vesicles. Furthermore, changes in the expression levels of different synaptotagmin isoforms alter the time-to-opening, as well as the dilation kinetics, of fusion pores. Thus, synaptotagmins are likely to interact with, and regulate, the membrane fusion machinery in vivo. However, the mechanism by which synaptotagmin regulates exocytosis remains to be established. A number of studies point to three potential effectors for the action of Ca2+ synaptotagmin: t-SNAREs (SNAP-25 and syntaxin), membranes, and other copies of synaptotagmin. A goal of this proposal is to determine whether these interactions function as coupling steps in exocytosis. The strategies of aims 1 and 2 are to selectively alter the kinetics and affinities of synaptotagmin-t-SNARE and synaptotagmin-membrane interactions, respectively. To test the hypothesis that these interactions trigger exocytosis, loss-of-function mutant copies of synaptotagmin will be expressed on the large dense core vesicles of PC12 cells where their effects on secretion will be monitored using amperometry. In aim 3 we will reconstitute full-length synaptotagmin into proteoliposomes in order to determine whether the kinetics of Ca2+-triggered oligomerization, within the plane of the bilayer, are rapid enough to couple Ca 2+to fusion. Once active protein is reconstituted, we will test the hypothesis that Ca2+-synaptotagmin acts to accelerate SNARE catalyzed membrane fusion by co-reconstituting synaptotagmin with purified SNAREs. This will provide a defined in vitro system to analyze the activities of selective loss-of-function mutants. A better understanding of exocytosis will provide a framework to determine how fusion is modulated (both pre- and post-synaptically) and thus contributes to synaptic plasticity in both normal and pathophysiological states. Finally, defining this mechanism should ultimately provide targets for treatment of diseases in which synaptic transmission is impaired.

DESCRIPTION (provided by applicant): Botulism was first described almost 200 years ago. This disease is caused by the botulinum neurotoxins (BoNT), which are seven related toxins (A-G) produced by toxigenic strains of Clostridium botulinum. These toxins are the most poisonous substances known. They act by entering neurons and cleaving proteins that mediate the exocytosis of neurotransmitters, resulting in paralysis and death. BoNTs are thought to bind to the surface of neurons via a double-receptor mechanism in which the receptor is a complex composed of gangliosides and protein(s). Identification of the toxin receptors, and the pathways that mediate entry, might provide a means to block the action of these toxins. Recent evidence indicates that members of the synaptotagmin family serve as the proteins components of the BoNT/B receptor. Synaptotagmin I and II exhibit distinct abilities to bind and mediate entry of BoNT/B. The first Aim of this proposal explores these differences with the goal of relating the structure of synaptotagmin with its ability to function as a toxin receptor. The precise mechanism by which the other BoNTs gain entry into cells is not known. Therefore, in the second Aim we will identify the pathways through which other BoNTs enter target cells. Our preliminary data indicate that different toxins enter cells via distinct pathways. In the third Aim we will further explore the means of BoNT host recognition and entry by identifying the receptor(s) that mediate internalization of BoNT/E. We have focused on this serotype because we have demonstrated feasibility using a BoNT/E affinity matrix; the long term goal is to identify receptors for additional BoNTs. Finally, in the fourth Aim we will develop FRET-based sensors that can be used to monitor BoNT activity in vitro and in living cells in real time. These sensors will make it possible to carryout high throughput screening to identify small molecules that can antagonize the action of the toxins. BoNTs are currently being used clinically to treat a variety of muscle dystonias and are also produced on large scales as potential biological weapons. The studies proposed here will provide new insights into the molecular mechanism of action of the BoNTs, and may provide a novel means to prevent poisoning by these substances.

DESCRIPTION (provided by applicant): This proposal is aimed at understanding fundamental aspects of membrane fusion in neurons. Membrane fusion is mediated by SNARE proteins (v-SNAREs on the vesicle membrane, t-SNAREs on the target membrane) and is tightly controlled by regulatory factors. We have largely focused on the Ca2+triggered exocytosis of synaptic vesicles, which is controlled by the Ca2+binding protein synaptotagmin (syt) 1. Syt I operates through direct physical interactions with t-SNAREs and membranes. This proposal continues our studies of syt I but also extends our work to address the functions of the other fifteen isoforms of this protein. Aim 1a employs a defined reconstituted system to test the hypothesis that the syt gene family has diverged in ways that help confer specificity to intracellular membrane fusion reactions. We hypothesize that specificity is achieved, in part, through selective pairing between different isoforms of syt and t-SNAREs in various sub-cellular compartments. We will determine the syt-SNARE pairing-code and compare this with the distribution of these proteins in neurons. In Aim 1 b we continue to pursue our goal of reconstituting rapid membrane fusion that recapitulates the rapid kinetics of synaptic vesicle exocytosis in neurons. This is a key step toward defining the precise molecular mechanism that underlies exocytosis. In Aim 2 we will bridge the gap between the minimal fusion assay used in Aim 1 and studies of synaptic transmission (e.g. Aim 3 below), by analyzing directly the fusion activity of synaptic vesicles isolated from knock-out/knock-in mice. This approach compliments electrophysiological analysis because it reports the intrinsic fusion properties of vesicles lacking specific proteins. These experiments will also make it possible to address the function of synaptic vesicle proteins that we have been unable to reconstitute in an active form in Aim 1. In Aim 3, we will test the hypothesis that different isoforms of syt have diverged to impart synapses with distinct kinetic components of release. We predict that syt isoforms with fast kinetics mediate synchronous transmission, while syt isoforms with slower kinetics mediate asynchronous transmission. These studies will provide new insights into information flow in neuronal circuits. Together, the experiments proposed here will provide critical information regarding our understanding of membrane fusion at synapses. This will aid efforts to alter communication between neurons in disease states where synaptic transmission is impaired.

The Clostridium botulinum neurotoxins (BoNTs) are the most toxic proteins known to humans and are classified as Category A Select Agents. They are considered as potential serious threat agents in bioterrorism. The Clostridium botulinum and Neurotoxin Core Facility at the University of Wisconsin is directed by Eric A. Johnson, whose laboratory has 24 years of experience with C. botulinum and BoNT, and is the senior reference source for BoNTs, C. botulinum strains, assay systems, animal models, and other relevant materials. The EAJ laboratory has considerable resources and expertise in various aspects C. botulinum and BoNTs. The primary functions of the Core are preparing BoNTs and associated materials, and assisting investigators within the GLRCE in their research projects. The specific aims are: Acquisition, characterization, and maintenance of strains (currently approx. 650) of Clostridium botulinum and nontoxigenic derivative strains. Production, purification and characterization of all seven serotypes and subtypes of botulinum neurotoxins (BoNTs) and their natural protein complexes. Production, purification, and characterization of recombinant forms of BoNTs, their domains and toxin complexes. Performance of animal studies, including mouse LDSOs, mouse challenges, immunization studies, and BoNT neutralization analyses. Implementation of non-animal assays, particularly neuronal cell assays, for detection and study of BoNTs and inhibitors. Maintain Select Agent Laboratory environment for research with C. botulinum and BoNTs. The Botulism Core will continue collaborations and facilitate the research efforts of other laboratories within the GLRCE.

DESCRIPTION (provided by applicant): Synaptotagmin 1, a protein found on synaptic vesicles, serves as a Ca2+ sensor in neurotransmitter release. This protein has 16 homologues, and while these other proteins are widely thought to serve related functions essentially nothing is known about where they are or what they do. Preliminary data presented in this application shows that several synaptotagmin isoforms are abundant in the posterior and/or anterior pituitary, glands that release peptide hormones that control essentially all vital aspects of mammalian physiology. Prior to these new preliminary results, the pituitary had been established as a preparation amenable to quantitative biophysical study of how peptide-containing vesicles fuse with the plasma membrane to release their content into the circulation. Thus, the finding of novel synaptotagmin isoforms in the pituitary offers a unique opportunity to test their function in detail and elucidate their precise roles in excitation-secretion coupling. This project will study the pituitary synaptotagmin isoforms with complementary approaches, investigating their binding properties in vitro, and their localization and physiological function in the pituitary. The functional studies employ a strategy of using transgenic mice for gene ablation of specific synaptotagmin isoforms, and gene targeting to identify specific cell populations that express those isoforms for electrical recording. By performing capacitance recording in identified cells and nerve terminals we will determine how syt isoforms regulate exocytosis in terms of Ca2+ sensitivity and fusion pores. The experiments proposed here will test the functions of synaptotagmins 4, 7, 9, 10, 11, and 12 in the release of hormones that control growth, metabolism, stress, fluid balance, and reproduction. PUBLIC HEALTH RELEVANCE: These experiments will shed light on the function of a class of proteins with broad roles in neurological, mental, and endocrine function and thus contribute to improving treatment of neurological disorders and mental illness. By illuminating the mechanisms of control of the pituitary hormones oxytocin, vasopressin, growth hormone, adrenocorticotropin, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, and prolactin, these studies will improve our understanding of growth defects, abnormal stress responses, metabolic disorders, and reproductive health.

DESCRIPTION (provided by applicant): The clostridial neurotoxins (CNTs) comprise a family of eight related toxins: tetanus (TeNT) and seven botulinum neurotoxins (BoNT/A-G), which cause the diseases tetanus and botulism, respectively. BoNT/A and BoNT/B are also used clinically to treat a wide range of serious medical conditions, including dystonia and pain; this represents a two billion dollar per year industry. The therapeutic action of the BoNT/A and B has traditionally been thought to involve the local - at the site of injection - inhibition of neurotransmitter release from neurons (by acting as proteases that selectively cleave SNARE proteins); in the case of dystonia, this presumably results in relaxation of skeletal muscles. However, a new hypothesis posits that in addition to having local effects at the site of injection (i.e. at the neuromuscular junction), BoNT/A can also undergo retrograde transport, away from the site of uptake (nerve terminals in the periphery), transcytosis from the axonal to the somatodendritic compartment, release from the latter compartment, and re-uptake into the nerve terminals of upstream, connected neurons in the central nervous system (CNS), where it exerts some of its medicinal effects. Whether any other BoNTs have distal effects is an issue that has not been explored. The goal of this proposal is to directly determine whether any of the BoNTs (A-G) do in fact undergo retrograde transport, transcytosis, release and re-uptake into upstream, connected neurons in a catalytically active form. In Aim 1 we will determine whether toxins that are added to the cis macrochannel of a compartmentalized microfluidic device (which contains only axons), are transported to, and act within, the trans macrochannel of the device (which contains axons, dendrites, and cell bodies). Strikingly, our preliminary data indicate that many of the BoNTs, including BoNT/A and B, do in fact undergo retrograde transport to the trans macrochannel in an active form. This work will include single particle tracking of toxins conjugated to quantum dots (Qdots) to directly visualize, and quantitatively analyze, transport. In Aim 2 we will address the question of whether the toxins undergo transcytosis, release, and re-uptake to act on neurons upstream of the 'primary' neurons that mediated the initial entry step. Release and re-uptake of the toxins would occur via vesicular carriers, and the fusion/recycling of these carriers can be blocked, in principle, using a CNT distinct from the toxi under study. For example, preliminary data indicate that prior cleavage of the SNARE synaptobrevin, in the trans macrochannel, with TeNT, prevents BoNT/A from cleaving its SNARE substrate, SNAP-25, within the trans macrochannel (after BoNT/A was initially taken up in the cis macrochannel). These data directly demonstrate that BoNT/A acts on neurons upstream of the 'primary' neuron that mediated initial entry. By conducting these experiments with all of the CNTs, we will determine which toxins have only local actions, and which toxins have previously undetected distal actions. This work will shed new light regarding the mechanism of action of these agents.

PROJECT SUMMARY/ABSTRACT During exocytosis, fusion pores form the first aqueous connection that allows escape of neurotransmitters and hormones from secretory vesicles. Although it is well established that SNARE complexes catalyze fusion, the structure and composition of fusion pores remain unknown. This is the central question in the field of membrane fusion, as the mechanism of fusion cannot be solved until the structure of the first key intermediate in this pathway, the fusion pore, has been elucidated. The main objective of this proposal is to gain new insights into fusion pore composition, structure, and dynamics, using both reconstitution and cell-based approaches. A major limitation in the biochemical study of fusion pores in cells concerns their low abundance and ephemeral nature. For example, in neuroendocrine cells, the duration of the initial open state of the fusion pore is of the order of msec; the pore then either closes (kiss-and-run exocytosis), or dilates to yield full fusion. To overcome this limitation, we have begun to study fusion pore structure, in vitro, by exploiting the rigid framework of nanodiscs. SNARE-bearing proteoliposomes dock and fuse with nanodiscs that harbor cognate SNAREs. Since nanodiscs are bounded by membrane scaffolding proteins, the pores cannot dilate, and hence can be studied biochemically. Using this system, we have begun to interrogate the properties of reconstituted fusion pores. Our preliminary data indicate that, contrary to the common view that fusion pores are purely lipidic, they are in fact hybrid structures, composed of both lipids and proteins. We will use a simulation approach to derive a new model for fusion pore structure, and conduct cryo-electron microscopy studies to visualize this structure. We will also use a variety of cargos of varying diameter, in conjunction with optical sensors that report their release during fusion, to determine the size of the pore, and to determine whether pore diameter is `plastic' and varies with the number of SNARE proteins. We will also probe for interactions between cargo and SNARE transmembrane domains by exploiting electrostatic interactions between them. The nanodisc system will be adapted to single molecule studies, to monitor pore opening and closing of individual pores in real-time, and to directly assess the impact of regulatory factors on pore stability. These in vitro experiments will be complimented by our ongoing direct measurements of fusion pores in chromaffin cells, using carbon fiber amperometry, by comparing the effects of SNARE mutations in these two systems. We will draw parallels between these systems so that we can arrive at unified, physiologically relevant models for pores. Finally, we will also design novel optical probes, based on pH sensitive dyes conjugated to quantum dots, to study fusion pores in cultured neurons. These latter studies will address the highly controversial topic of kiss-and-run exocytosis versus full fusion. Together, the work described here will provide unparalleled comparisons between in vitro and cell based observations, and will reveal new insights in the first crucial intermediate in the exocytotic pathway: the enigmatic fusion pore.