Pietro De Camilli - US grants

The long term goal of the project is to broaden our understanding of neurological diseases due to autoimmune mechanisms and to find ways to treat such diseases. The objective of this grant is to investigate whether autoimmunity may be involved in the pathogenesis of stiff-man syndrome. Stiff-man syndrome is a rare, highly incapacitating, human diseases of the central nervous system characterized by progressive rigidity of the body musculature associated with painful spasms. The rigidity is thought to result from an impairment of suprasegmental and spinal inhibitory systems that operate through GABA. The pathogenesis of stiff-man syndrome is unknown. Recent studies have demonstrated the presence of signs of central nervous system autoimmunity and, more specifically, of autoantibodies directed against GABA-ergic nerve terminals, in a high percentage of patients affected by stiff-man syndrome. They have also suggested that glutamic acid-decarboxylase (GAD), the enzyme involved in the synthesis of GABA, may be a primary autoantigen in the disease. These results have raised the possibility that autoimmunity may be the cause of the neurological symptoms. A series of studies aimed at further testing this hypothesis are planned. It is proposed to further characterize the specificity of the autoantibodies that are found in the serum and cerebrospinal fluid of stiff-man syndrome patients and to determine whether GAD is always the primary antigen involved; to characterize other possible autoantigens by biochemical and recombinant DNA techniques; to determine if, and how, these autoantigens are directly involved in the pathogenesis of the disease; to reproduce the diseases in laboratory animals by injecting the relevant antigens. These studies may lead to new effective ways to treat stiff-man syndrome. In addition, if the working hypothesis of the project turns out to be correct, stiff-man syndrome may represent an important model disease to further elucidate the pathogenetic mechanisms involved in central nervous system autoimmunity and in autoimmunity in general.

The long term goal of my project is to broaden our understanding of how neurons communicate with each other and with other cells via chemical signals. Over the last several years it has become clear the exchange of information between neurons involves the cooperation of fast and slow; point-to-point and diffuse messages. One of the bases for this complexity appears to be the property of neurons to secrete a cocktail of neurotransmitters, via at least two types of secretory organelles, small synaptic vesicles [SSVs] (which are thought to contain classical neurotransmitters only) and large dense-core vesicles [LDCVs] (which contain peptides and may also contain amines). LDCVs may be seen as the equivalent organelles in neurons of secretory granules (SGs) in endocrine cells. Typical SSVs are present only in neurons, but recent evidence suggests that closely related vesicles (synaptic-like microvesicles [SLMVs]) are present also in endocrine cells. The biogenesis of SSVs and SLMVs is controversial and some authors have proposed that they are generated at the level of the plasmalemma as a result of endocytosis of LDCV and SG membranes. The working hypothesis of this research proposal is that SSVs represent the neuronal adaptation of SLMVs, that the two organelle share important functional similarities and that they have a biogenesis distant from that of LDCVs and SGs respectively. Studies aimed at testing this hypothesis are proposed. We will isolate SLMVs from anterior pituitary tissue and from naive PC12 cells and compare their biochemical characteristics with those of SSVs purified from brain. In PC12 cells we will analyze the intracellular traffic of SLMVs and characterize, with biochemical and morphological procedures, the transition from a SLMVs phenotype to a SSVs phenotype in parallel with neuronal differentiation. We will test the possibility that also SLMVs might have a secretory function. To study the biogenesis of SLMVs and SSVs we will carry out metabolic labeling experiments in PC12 cells and immunocytochemical experiments of organelles in transit to the nerve terminal in peripheral nerves. Finally, we will try to establish the relationship of SSVs and SLMVs to exocytic and endocytic pathways present in all cells by studying the expression, via cDNA transfection, of SSV proteins in fibroblastic CHO cells. I expect that the improved understanding of the mechanisms of chemical signalling between nerve cells will provide new clues towards the understanding of the molecular basis of neurological and psychiatric diseases and towards the development of novel pharmacological tools to treat such diseases.

PROJECT SUMMARY/ABSTRACT The goal of this project is to advance knowledge of mechanisms in synaptic transmission. More specifically, the project will investigate pathways of synaptic vesicle endocytosis and recycling following the collapse of a fused synaptic vesicle into the plasma membrane. While it is well established that clathrin and dynamin- dependent endocytosis plays a major role in this process, key mechanistic aspects of this process remain elusive. In addition, there is evidence for clathrin and dynamin-independent bulk endocytic pathways at the synapse, but such pathways remain poorly understood. The role of endosomal compartments in the vesicle cycle also remains controversial. This grant application plans first to characterize endocytic traffic that operates in parallel to the classic clathrin-dependent endocytic pathway and to test the hypothesis that bulk endocytosis leads to the generation of new synaptic vesicles independently of clathrin and of the GTPase dynamin. Second, the project will investigate the properties, potential heterogeneity and interrelationships of endocytic compartments of nerve terminals, determine the function of 3-phosphorylated phosphoinositides in their dynamics and test the hypothesis that most intermediates in synaptic vesicle recycling are not classical endosomes. Third, mechanistic aspects of the clathrin and dynamin-dependent endocytic reaction will be investigated. These studies will involve mouse genetics, studies in cultured neurons and in cell-free systems. This work is of specific relevance in neuroscience, but given the fundamental nature of endocytosis and post- endosomal sorting, it will have broad implications for a variety of fields of biology and medicine.

ABSTRACT 9730059 Michael Nathanson Yale University A Two-Photon Imaging System for Biological Research and Education This proposal will fund the purchase of a two-photon excitation imaging system for the Center for Cell Imaging at Yale University School of Medicine. Specifically, the Center's confocal microscope will be upgraded to have two- photon capability. Two photon laser excitation imaging has several properties that make it unique, even relative to point scanning confocal imaging. Two photon imaging permits more highly localized excitation of fluorophores, excitation of dyes that excite at lower wavelengths, much deeper sample penetration, less photobleaching, and less cytotoxicity than conventional confocal microscopy. These features improve spatial resolution, especially in thicker tissue preparations such as intact organs, and make it possible for the first time to photolyse caged compounds or photobleach dyes in highly localized, submicron-sized intracellular regions. No two-photon imaging system is currently available at the School of Medicine, although a number of investigators require this technology to advance their research efforts. The range of projects that will make immediate use of this technology include flash photolysis of caged second messengers to examine regulation of Cai2+ signals at the subcellular level, photolysis of caged oligonucleotides to perform in situ PCR at the single cell level, immunochemistry of intact embryos and larvae, examination of acid production in intact gastric glands, examination of intercellular Cai2+ waves in intact, perfused livers, and distribution and trafficking of GFP-labeled proteins in tissues (including brain slices) and in whole brains of transgenic animals. In addition, the Center for Cell Imaging is the only such imaging resource available to all investigators at the School of Medicine. Therefore, the acquisition of the two-photon imaging system by the Center will insure that any investigator at the school will have access to this technology now and in the future.

The toxins responsible for the clinical manifestations of botulism and tetanus are neurotoxins produced by strains of the bacterial genus Clostridium. These toxins block exocytosis of synaptic vesicles and thus inhibit neuronal signalling. Recent work has established that all clostridial neurotoxins function as metalloproteases. The toxins are composed of a heavy chain that targets and delivers them to neurons and a light chain that carries out their proteolytic activity. Individual toxins from each of several strains of Clostridium botulinum selectively proteolyze one of three synaptic proteins: synaptobrevin (VAMP), SNAP-25, or syntaxin. Synaptobrevin is a resident of synaptic vesicles, while SNAP- 25 and syntaxin are found on the synaptic plasma membrane. Several independent lines of evidence, including biochemical and genetic experiments, have recently converged to strongly implicate these three proteins as the core of a multiprotein complex which mediates exocytotic membrane fusion. Furthermore, the regulated assembly and disassembly of these proteins, in concert with cytoplasmic factors, are probably underlying vesicle docking and vesicle fusion. In this application, we plan to determine the molecular and physiological consequences of toxin poisoning in the nerve terminal with the goal of understanding how individual neurotoxins affect the protein-protein interactions which underlie neuronal exocytosis. This information should contribute to our understanding of the neuronal exocytotic, fission machine. We plan to use three approaches including in vitro studies with recombinant proteins, biochemical analysis of toxin-treated nerve terminals and experiments with cultured neurons. First, we will study the effects of toxin cleavage on protein-protein interactions between the components of the putative exocytotic fusion machine. These interactions include assembly and dissociation of the toxin substrates synaptobrevin, SNAP-25, and syntaxin, as well as their binding to regulatory proteins such as synaptophysin. The experiments will be carried out using purified proteins in solution as well as proteins reconstituted in proteoliposomes in order to imitate their native environment. Second, the interactions between the toxin substrate proteins will be studied in material isolated from nerve terminals which have been poisoned by the various toxins. Subfractionation of nerve terminals into defined membrane pools will allow us to determine if there is a specific point in the cycling of synaptic vesicles which is blocked by toxin action. Finally, we will use cultured neurons as a model system to expand on the information obtained in the in vitro experiments. These experiments will examine the effects of exogenous substrate proteins, protein fragments and toxin-resistant mutant proteins on synaptic transmission.We expect these studies to clarify how individual neurotoxins perturb neuronal exocytosis.

Electron microscopy is a technique of fundamental importance in molecular and cell biology. Most of what we know about cell ultrastructure was learned from electron microscopy. Recently, several advancements have made possible he use of electron microscopy to obtain insight which go beyond descriptive information and the electron microscope has become a powerful tool to study molecular mechanisms. In large part, these developments reflect the advent of highly sensitive immunocythochemical techniques, of genetic methods to disrupt the function of individual proteins, and of cell free systems which make possible the analysis of isolated organelles or macromolecular complex. Furthermore, the advancements in cryoelectron microscopy are narrowing the gap between classical cell biology and structural biology. This application proposes the purchase of new transmission electron microscope (Philips CM120 BioTwin) and supporting equipment digital imaging. This instrumentation will be housed in the electron microscopy unit of the Center for Cell Imaging at the Yale School of Medicine. This is a shared facility, which is managed by the Department of Cell biology, but is subsidized by the School. It is used primarily by faculty from the Department of Cell Biology and other Departments whose research focuses on molecular and cell biology. The instrument will replace a Jeol 100cX which has been recently taken out of service The Yale Electron Microscopy Core Facility has traditionally been among the leaders in the application of electron microscopy to biology and medicine. While this continues to be the case, progress is now being hampered by microscope availability and by the outmoded instrumentation present in the facility. There have been major advances in electron microscope design and image digitalization and the facility is in urgent need of a state-of-the-art microscope. The productivity of Yale Cell Biology is crucially dependent on the availability of such an instrument.

Secretory granules (SGs) of endocrine cells are responsible for storage and regulated secretion of peptide hormones, including insulin. Pharmacological studies suggest that both tyrosine phosphorylation and dephosphorylation pathways modulate the trafficking of SGs, but the molecules involved in these pathways have not yet been identified. Islet Cell Autoantigen (ICA) 512 is a novel member of the receptor protein tyrosine phosphatase (RPTP) family that we have shown to be an intrinsic membrane protein of SGs. Interestingly, ICA512 contains an atypical protein tyrosine phosphatase (PTP) domain and does not display enzymatic activity. In view of its localization and homology with RPTPs, we hypothesize that ICA512 represents a link between regulated secretion and signal transduction pathways mediated via tyrosine phosphorylation. We plan to test this hypothesis by establishing the protein's function. To this aim we propose first to identify ICA512 interacting proteins using three complementary approaches: 1) the two- hybrid system in yeast, 2) co-purification by solid phase affinity chromatography and immunoprecipitation with anti-ICA512 antibodies, and 3) affinity purification on recombinant ICA512. ICA512 interactors will be cloned and characterized for their affinity to wild-type and ICA512 mutants. Knowledge of ICA512 interactors will provide clues about the cell pathways to which ICA512 insulinoma cell clones overexpressing wild-type or mutant ICA512 alleles in a tetracycline-inducible fashion. The role of ICA512 in vivo will be tested by conditionally over- expressing wild-type or a PTP active mutant of ICA512 in the pancreatic beta-cells of transgenic mice. This will be accomplished using the reverse tetracycline-inducible expression system driven by the rat insulin promoter II. As RPTPs are known to play an important developmental role, we will test the potential impact of ICA512 transgenes on islet organogenesis and beta-cell differentiation by inducing their overexpression at different stages of mouse development. The potential role of ICA512 in beta-cell physiology will be established by monitoring glucose homeostatis in transgenic mice and by studying insulin secretion from purified transgenic islets. These studies could establish a direct relationship between regulated secretion and signal transduction via tyrosine phosphorylation. In addition, since atypical PTP domains similar to that of ICA512 are present in an increasing number of orphan genes, our studies on ICA512 may provide insight into the general significance of such protein motifs.

A grant has been awarded to Yale University School of Medicine to acquire an electron tomography microscope for quantitative high-resolution imaging of cells and nanomaterials in three dimensions (3D). With its impressive high resolution (~5 nanometers) the new microscope will make it possible to visualize thick biological sections and other objects volumetrically, and will be used by more than 17 laboratories from 11 Yale departments. Specific focus areas will include high-resolution imaging of neurons and synapses, vesicular traffic, cytoskeleton, and nanomaterials, as well as the development of new methods of image analysis.

The grant will have a broad impact in the Yale scientific community and beyond. It will help train students and postdoctoral fellows in powerful new imaging techniques, forge new collaborations across discipline boundaries, bring scientists from diverse backgrounds together, create new partnerships with the private sector, develop methodology to analyze and mine tomography datasets, and provide a strong program for education and outreach to the local community (via links to local high schools and Yale ?STARS?, SURF, Biostep programs). In all of these ways, the award will support the scientific, educational, and diversity aims of Yale University and the National Science Foundation.

DESCRIPTION (provided by applicant): A decrease in the export of nascent proteins from the endoplasmic reticulum with aging may shorten cell life by two mechanisms: 1) initiation of ER stress responses, leading to enhanced apoptosis, and 2) secretion and trafficking of proteins, such as cytokines and their receptors that are required for cell growth and differentiation. A hallmark of the aging processing is the accumulation of inactive proteins that have cleared by cellular degradation systems. This pool is formed by both proteins that have been misfolded and those that have been inactivated and reached the end of their biologic half-lives. Three general mechanisms could account for the accumulation of such proteins: 1) increased misfolding, 2) enhanced protein denaturation and 3) decreased function in degradation pathways. We propose that dysfunction of protein export from the endoplasmic reticulum during aging leads to protein accumulation in the ER, protein misfolding, stimulation of ER stress responses, and enhanced apoptotic cell death. This proposal will examine the role of COPII coat proteins in the age-dependent accumulation of inactive cellular proteins. Molecular export from the ER is thought to be mediated by COPII vesicles. It has been shown that an essential COPII coat component, Sec31 in yeast, is phosphorylated and its phosphorylation has been implicated in COPII vesicle budding. However, the molecular mechanisms, including the phosphorylation sites and the kinases for this phosphorylation, are unknown. Hypothesizing that the phosphorylation-dephosphorylation cycle of the coat components of COPII vesicles may regulate molecular export from the ER, we recently discovered the phosphorylation of mammalian Sec31. In this proposal, we will determine: 1) whether the cycle of phosphorylation-dephosphorylation of mammalian Sec31 regulates protein export from the ER, 2) whether trafficking of the klotho protein, as well as other cargoes, are affected by dephosphorylation of Sec31, and 3) whether there are age-associated changes in the phosphorylation of Sec31, which may correlate with potential age-related impairment of the early secretory pathway. The success of this study will lead to a further understanding of the molecular mechanisms of aging, which may contribute to cures for age-related diseases.

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to develop therapeutic strategies for the treatment of two human diseases, Oculo-Cerebro-Renal syndrome of Lowe (Lowe syndrome) and Dent disease, which result from mutations in the gene encoding the inositol 5-phosphatase OCRL. Lowe syndrome is a severe X-linked disorder characterized by reabsorption defects in the kidney proximal tubule (renal Fanconi syndrome), mental retardation and congenital cataracts. Dent disease is another X-linked disorder in which the clinical manifestations are limited to kidney defects that are similar to those observed in Lowe syndrome. While it is known that the main function of OCRL is to dephosphorylate PI(4,5)P2 and PI(3,4,5)P3 at the 5 position of the inositol ring, the mechanisms through which a defect in this protein causes disease is unclear. The objective of this project is to elucidate these mechanisms in the kidney, as it is the organ consistently affected by mutations in the OCRL gene. Recent studies at the cellular level have suggested that the main site of action of OCRL is the early endocytic pathway, where several major OCRL interactors are concentrated, such as clathrin, the clathrin adaptor AP-2, Rab5 and the endocytic adaptor APPL1. A main working hypothesis is that abnormal levels of PI(4,5)P2, and possibly PI(3,4,5)P3, resulting from impaired OCRL function result in abnormal traffic and sorting of apical plasma membrane proteins in kidney proximal tubule cells. In this proposal we plan to further characterize the molecular properties and interactions of OCRL and of its homologue INPP5B, to elucidate the role of OCRL in endocytic traffic and endosomes dynamics in kidney proximal tubule cells and in model cell lines, to determine the impact of mutations in OCRL/INPP5B on kidney function in mice and to identify proteins whose function enhances or suppresses defects resulting from lack of OCRL. The identification of such modifier genes may provide clues relevant to understanding the impact of different OCRL mutations in Lowe syndrome and Dent disease and toward developing therapies for these conditions. Given the key role of PI metabolism and of the endosomal system in cell physiology and pathology, the results of these studies will be additionally relevant to the elucidation and treatment of a variety of diseases of the kidney as well as other organs. PUBLIC HEALTH RELEVANCE: OculoCerebroRenal Syndrome of Lowe (Lowe Syndrome) is a severe disorder characterized by kidney dysfunction, mental retardation and congenital cataracts, which results from mutations in the gene encoding a lipid metabolizing enzyme called OCRL. Mutations in OCRL can also cause Dent disease, whose clinical manifestations are limited to kidney defects that are similar to those observed in Lowe syndrome. The goal of this proposal is to understand how disruption of OCRL function leads to kidney disease with the hope that this information may help to develop therapeutic strategies for these conditions.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. It is well known that phophoinositides (PIs) play a pivotal role in almost all aspects of cell physiology both as precursors of intracellular second messengers and as regulators of membrane-cytosol interfaces. Phosphoinositide metabolism is tightly regulated by a large number of phosphoinositide metabolizing enzymes, and mutations of several such enzymes are responsible for human disease. Mutations in OCRL (also referred to as INPP5E) cause OculoCerebroRenal Syndrome of Lowe, an X-linked disorder characterized by congenital cataracts, mental retardation, neonatal hypotonia, and renal Fanconi syndrome. Despite many studies of the biochemical properties and cellular functions of OCRL, structural information on this multi-domain enzyme, which is essential for our understanding of its biological functions, is still limited. We successfully crystallized the C-terminal part of OCRL, which contains a novel domain named the ASH domain and a RhoGAP like domain. With the help by the NE-CAT team, we collected an anomalous diffraction data set and solved the structure of the C-terminal part of OCRL. Our crystallographic studies of the COOH-terminal region of OCRL reveal the first structure of a member of the newly defined ASH domain family. Our structure reveals with an unusual clathrin box protruding from a large loop inside the RhoGAP-like domain. Moreover, our structural studies elucidate a potential link between OCRL mutations and the phenotypic manifestations of Lowe syndrome.

The goal of the Cell Biology Core is to make available to members of the Yale DRC the instrumentation, technical personnel, and expertise for the analysis of cell function in areas of research related to diabetes. The Core focuses primarily on imaging techniques (both light and electron microscopy), and also offers quantitative infra-red imaging of gels and multiwall plates. Emphasis is given to immunocytochemical methods, and to the dynamic light microscopy imaging of living cells containing fluorescent markers, using standard epifluorescence, total internal reflection, and confocal (including spinning disk) techniques. In addition, the core offers leading edge techniques such as electron microscopy tomography and three different types of super-resolution microscopy (STED, FPALM, and SIM). DRC investigators will be trained in various imaging techniques as required for their work. It is anticipated that the services provided by the Core will permit the elucidation of wide-ranging aspects of cell function that are critical to understanding diabetes pathophysiology.

The long-term goal of this application is to gain information about mechanisms that control homeostasis of membrane lipids in cells of the nervous system. Such control is critical to ensure normal function and traffic of cellular membranes, and dysfunction of these mechanisms result in neurological and psychiatric diseases. The specific goal of this application is to advance knowledge of lipid transfer reactions that occur at contact sites between the endoplasmic reticulum (ER) and other membranes and that are mediated by membrane tethering proteins containing lipid transport modules of the TULIP domain superfamily. The occurrence of protein- dependent, but membrane traffic-independent, transport of lipids between the ER and other membranes has been known for decades. Recently, however, it has become clear that much of such transport occurs at membrane contact sites. Additionally, several new proteins that localize at membrane contact sites and contain lipid transport modules have been identified. These mechanisms so far have not been investigated in cells of the nervous system, although contacts between the ER and other organelles have been described in all neuronal compartments including synapses. Here I propose to investigate the properties, mechanisms of action and physiological functions of two proteins that function at contacts between the ER and other membranes. The first is TMEM24, an intrinsic ER protein by far preferentially expressed in neurons and neuroendocrine cells. TMEM24 contains a lipid transport module of the TULIP domain superfamily (an SMP domain) and functions as a regulated tether between the ER and the plasma membrane. We hypothesize that the lipid transport function of TMEM24 regulates signaling reactions at the plasma membrane. The second is Vps13A/chorein, a very large protein without transmembrane regions that we have found to be concentrated at ER-mitochondria contacts, where it tethers their membranes. Loss-of-function mutations in Vps13A result in chorea-acanthocytosis, a neurodegenerative condition resembling Huntington's disease with an associated defect of red blood cells. Mutations in a closely related protein, Vps13C, are responsible for a familial form of Parkinson's disease. Based on preliminary results we hypothesize that one function of Vps13A is to mediate lipid transport between the ER and mitochondria via SMP like domains. We will test these hypotheses with a variety of complementary experimental strategies ranging from in vitro reconstitution of lipid transport between artificial liposomes, to studies in cultured cells (including iPS cells) and mutant mice. Results of this research will provide insight into completely unknown aspects of neuronal and synaptic function and into pathogenetic mechanisms in neurodegenerative diseases.

An award is made to Yale University to implement a state-of-the-art Focused Ion Beam Scanning Electron Microscope (FIB-SEM) system. It provides a powerful new tool to study the 3D structure and function of cells and their organelles - especially those that require high-resolution, yet large volumetric views. It will also allow new correlative studies that bridge the gap between light microscopy (including super-resolution) and electron microscopy. The broader impacts are multifaceted. The instrument will be integrated into the Yale CCMI Electron Microscopy Core Facility and infrastructure created precisely for such multi-user equipment. The instrument will be used to train the next generation of scientists and bioengineers in quantitative imaging, fostering new interdisciplinary research between biological, engineering and physical sciences. Student diversity is a strong focus and will be actively promoted on multiple fronts through established Yale outreach programs. 3D EM datasets will be rendered in a stereoscopic virtual reality (VR) platform so that anyone can voyage into cells. Thus, the large high-resolution volumetric data will not only propel basic science, but also help educate and inspire a broad and diverse audience.

The new FIB-SEM system will enable new scientific research into how cell and tissue structure impacts function, with projects on how neurons communicate, how organelles such as primary cilia, Golgi complex, phagosomes, and the endoplasmic reticulum form and function. The main user group comprises leaders in a broad range of scientific disciplines stretching across the biological sciences, engineering, and computer sciences. The new system will act as a focal point, with faculty members, postdoctoral trainees, and students from different departments interacting and helping each other to reach common imaging and analysis goals. The instrument will be an extremely valuable tool not only for research on intracellular functions, but also for projects that address intercellular communications, such as those that occur in the nervous system and in the immune system.