David C. Spray - US grants

This award supports the participation of Dr. David C. Spray and others of the Albert Einstein College of Medicine in a program of cooperative research with Dr. Antonio Campos de Carvalho of the Federal University of Rio de Janeiro in a project aimed at understanding the role of gap junctions in the heart ventricle in maintaining normal cardiac function. Gap junctions are a class of membrane channels that uniquely span the membranes of adjacent cells to form intercellular pathways for ions and uncharged molecules. The functional and biochemical properties of these junctions, and of the ions which they transmit, may be of profound importance in understanding how muscle impulses are transmitted within the heart. The U.S. principal investigator has participated in pioneering work on gap junctions between isolated pairs of ventricular myocytes, and has also studied the electrical behavior of adult rat ventricular myocytes and cell pairs. He has also studied the properties of junctional proteins in liver tissue, thereby becoming expert in techniques that he will extend, in this research, to the study of heart tissue. The Brazilian collaborator has considerable experience in the isolation of cardiac gap junctions, and also has experience in cardiac tissue electro-physiology. Complementary expertise will be utilized as U.S. investigators perform electrophysiological experiments in Brazil, on gap junctions isolated there; and in a subsequent visit by the Brazilian collaborators to the U.S., where they will participate in biochemical studies. By increasing our understanding of the operation and function of these important components of the heart, more will be learned about the maintenance of normal human cardiac function.

DESCRIPTION (provided by applicant): Electrical synapses between neurons are formed by gap junction channels, through which ions and metabolites pass directly from one cell to the next. Recent evidence indicates that other proteins are associated with connexins at gap junctions, and we have termed this macromolecular complex the Nexus. We hypothesize that the Nexus components may regulate both the properties of the gap junction channels and also may function in intracellular signal transduction. A new gap junction protein, connexin36, has been identified that is largely restricted in its expression to neurons, and we have shown that the channels formed by this connexin have properties that seem to uniquely suit it as a neuronal connexin. The goals of this grant application are to characterize the gating, permeability and conductance properties with regard to the protein domains involved, to identify other proteins in brain and neuron lysates that bind to connexin36, measure the strengths of interaction using surface plasmon resonance, and perform physiological experiments on neuron-like cells to determine the effects of Cx36 expression on neuronal differentiation and gene expression patterns. In addition, a major goal of this application is to obtain structural information using spectroscopic methods regarding domains of Cx36 that interact with each other and with other proteins. These studies use a multidisciplinary approach directed at exploring a new concept in the field and as such are expected to lead to novel understanding of regulation and roles of gap junctions in the nervous system.

DESCRIPTION (provided by the applicant): Long range intercellular communication among astrocytes is in large part mediated by gap junction channels through which ions and metabolites pass directly from one cell to the next. The pore of astrocyte gap junctions is formed primarily of the gap junction protein connexin43. Recent evidence indicates that other proteins are associated with Cx43 at gap junctions, and we have termed this macromolecular complex the Nexus. We hypothesize that the Nexus components may regulate both the properties of the gap junction channels and also may function in intracellular signal transduction. In order to test this hypothesis, we will determine the identities of the other proteins that bind to Cx43 in astrocytes and in transfected cells, implement high throughput and quantitative methods to identify novel Nexus proteins, measure the strengths of interaction, and perform physiological experiments with Cx43 mutants and with Cx43 binding partners to determine the functional consequences of such interactions. In addition, a major goal of this application is to obtain structural information regarding domains of Cx43 that interact with each other and with other proteins. These studies use a multidisciplinary approach directed at exploring a new concept in the field and as such are expected to lead to novel understanding of roles that gap junctions play in the nervous system and elsewhere.

[unreadable] DESCRIPTION (provided by applicant): The purpose of this application is to obtain a photon-counting camera and accessories to allow high temporal and spatial resolution of luminescence within cultured cells and in whole animals; accessories include both multiwell plate and single tube luminometers required for optimization of expression and activation of the photoproteins in vitro and an imaging station in which whole animal imaging will be performed. There is no such high sensitivity camera or whole animal imager currently at Einstein College of Medicine, and there is currently neither type of luminometer in the nine-floor Kennedy Center housing the Department of Neuroscience, where the proposed studies will be performed. The studies that are proposed are all natural extensions of NIH-funded projects of nine investigators that will use the relatively new technique of spatially resolved luminescence recording that has become more accessible due to new generations of vector constructs in which the photoproteins have been optimized with regard to intensity and wavelengths of light output and destabilized with regard to gene expression reporting. The studies of users outlined in this proposal are diverse, including bioluminescent resonance energy transfer to determine dimerization and binding partners for connexin molecules, luminescent reporter molecules to localize ATP release from within astrocytes and astrocytoma cells, luminescence-tagged bone marrow cells in order to characterize their tissue distribution following transplantation into mice, and gene promoters fused to luminescent reporter molecules to identify time course of gene activation in cytokine-stimulated astrocytes and to develop a moderately high throughput assay by which to identify connexin-specific gap junction channel blockers. The proposal also includes the plan for a bimonthly user group meeting, through which new users will be attracted to the instrument and new analytical tools and protocols will be discussed, as well as problems that may arise. It also includes plans for training as well as inclusion of relevant methods in a graduate student course. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Long range intercellular communication among astrocytes is in large part mediated by gap junction channels, through which ions and metabolites pass directly from one cell to the next. The pore of astrocyte gap junctions is formed primarily of the gap junction protein connexin43 (Cx43). Recent evidence indicates that other proteins are associated with Cx43 at gap junctions, forming a macromolecular complex that we have termed the Nexus. We hypothesize that the Nexus components may regulate both the properties of the gap junction channels and also may function in intracellular signal transduction and trafficking of Cx43 within the astrocyte, all of which are critical for maintaining the network of coupled astrocytes throughout the brain. During the last period of support, we found that both intra- and intermolecular interactions occur with Cx43, have quantified the affinities of several interactions under different pH and phosphorylation conditions and used NMR to solve structures of relevant Cx43 cytoplasmic domains and to determine how structures change upon binding. We have also identified new binding partners for Cx43 and have begun to examine how interaction with binding partners affects function of Cx43 gap junction channels and the intracellular trafficking of this protein to and from the membrane. The Nexus complex in astrocytes is thus dynamic, changing binding partner affinities due to local conditions and local concentrations of ligands. We now propose to extend these studies to determine how interactions of cytoskeletal proteins with Cx43 is linked to both rapid and gradual remodeling of the astrocyte network. The proposed studies use an interdisciplinary approach with several techniques that are new to the gap junction field to rigorously explore the novel concept that connexin-cytoskeletal interaction is a major determinant of gap junction function in astrocytes. As such, these studies are expected to lead to novel insight of roles that gap junctions play in the nervous system and elsewhere. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Osteocytes, the cells that reside within bone matrix, are the most abundant bone cells. They function as the mechanical sensors in bone, and are critical to activation and coordination of osteoclastic and osteoblastic activities by which bone adapts to mechanical usage, maintains its health and prevents fractures. The mechanisms underlying osteocyte mechanotransduction are not well understood, though changes osteocyte mechanosensitivity have been implicated in regulating the effect of both bone anabolic agents and sex hormones. We have developed engineering models which show that small whole bone strains can be amplified locally around osteocyte processes by focal attachments to the canalicular wall. Osteocyte cell bodies cannot see similar high strains as they are too compliant and lack the cellular attachments needed for local strain amplification. These mathematical models argue that the osteocyte cell process may be uniquely designed to function as a detector of small tissue strains. To test this hypothesis, we developed a broad-based multiple-PI program that combines expertise in ion channel physiology, in vivo osteocyte structure/biomechanics and bioengineering/modeling to understand how osteocytes perceive and transduce their local mechanical environment. This program will a) examine the functional polarity of osteocyte mechano- responsiveness using electrophysiological approaches on cultured osteocytes (Aim 1), b) identify the molecular components of mechanotransduction complexes in osteocytes (Aim 2), c) characterize the structure of the mechanotransduction complex in osteocytes in vivo (Aim 3) and d) build integrative mathematical models relating local hydrodynamic forces and membrane strains at osteocyte processes and cell bodies to cellular responses in vitro and in vivo (Aim 4). We have also developed a novel technology ("Stokesian" Fluid Stimulus probe) that allows us to hydrodynamically load osteocyte processes vs. cell bodies at extremely low forces (<10pN) typical of what bone cells actually experience in vivo. Expansion of this technology to interrogate mechano-responsiveness in a broad range of cell types is a developmental goal of this grant. Significance: Understanding how osteocytes "perceive" and transduce mechanical signals may provide key new insights into bone physiology leading to the identification of novel therapeutic targets against bone loss due to aging and disease. PUBLIC HEALTH RELEVANCE: Osteocytes are the cells in bone that sense mechanical loading and translate mechanical strain into biochemical signals that initiate modeling and remodeling through which bone adapts its structure to its mechanical loading environment. This ability is key to skeletal health;failure to adapt results in bone in fragility. Increases and decreases in osteocyte mechanosensitivity have been implicated in regulating the bone response to anabolic agents, and conversely the bone loss resulting from estrogen loss, respectively. Thus understanding how osteocytes "perceive" and transduce mechanical signals may provide key new insights into bone physiology leading to the identification of novel therapeutic targets against bone loss due to aging and disease.

? DESCRIPTION (provided by applicant): Gap junction-mediated intercellular ionic and metabolic coupling provides both homeostatic and signaling functions among astrocytes throughout the brain, and CNS pathology both results in and is caused by disruptions in astrocyte gap junction signaling. It is now well known that gap junction proteins (connexins) interact with other proteins, forming a molecular complex we term the Nexus. Recent data indicate that Nexus is a highly dynamic signaling unit, where direct linkages of connexins to adaptor and scaffolding molecules are bound to cytoskeletal, cytoplasmic and other membrane proteins, polarizing the astrocyte. The overall goal of this grant proposal is to understand how gap junctions in astrocytes control organization of this endfoot complex and thus control blood-brain-barrier integrity. To accomplish this goal, we propose to: Aim 1: Test the hypothesis that localization and mobility of gap junction proteins and their binding partners determines organization of astrocyte endfeet. Aim 2. Determine downstream effects of connexins on endothelial tight junction formation. Aim 3. Validate the hypothesis that localization and mobility of gap junction proteins and their binding partners determines organization of astrocyte endfeet using mice with modified connexin expression We believe that these interdisciplinary and innovative studies will provide the first molecular insights into the role of the astrocyte Nexus i establishment of cell polarity and thus identify important astrocyte functions that can be targeted to reverse disorders of astrocyte polarity.

ABSTRACT Bone adapts its structure to mechanical loading. This adaption is essential for growing the right skeleton and maintaining its integrity throughout life. Osteocytes are the cells responsible for sensing and coordinating response to mechanical load. Key recent discoveries reported by our group during the last several years established that osteocyte cell processes function as unique mechanosensory elements. Processes are >10-fold more sensitive to mechanical stimuli than osteocyte cell bodies. Moreover, this triggering of Ca2+ signaling from cell processes occurs through a unique complex of aVb3 integrins, membrane channels and receptors, that occur at attachment points to the canalicular walls, and which we call the ?Osteocyte mechanosome.? This proposal is based on the global hypothesis that a novel structure localized on osteocyte processes, the osteocyte mechanosome, detects and transduces mechanical signals. To date, we have identified four key osteocyte mechanosome components: ?V?3 integrin, pannexin1, P2X7 receptor (P2X7R) and the CaV3.2 T-type calcium channel. Our multidisciplinary team will test this hypothesis by multiple approaches in each of three aims. In Aim 1 we will combine biochemical techniques (co-immunoprecipitation, surface plasmon resonance) and imaging modalities (FRAP, FRET and STORM super-resolution microscopy) to define comprehensively the structural and dynamic properties of this heretofore unknown transduction complex, the osteocyte mechanosome in osteocytic cells in vitro. In Aim 2 we test how pharmacological and genetic alteration of individual mechanosome components alters upstream (Ca2+) and downstream (to bone) signaling in osteocytic cells in vitro. In Aim 3, we will combine our novel OtGP3 osteocyte Ca2+ reporter mice-in vivo loading/imaging system with pharmacological manipulations to confirm effects of key mechanosome components (as identified in Aims 1 and 2) on osteocyte Ca2+ response and on downstream signaling. We will also use this approach to answer the fundamental question of whether osteocyte Ca2+ responses to mechanical loading altered by loss of constitutive sex hormones (estrogen/androgen) or by anabolic PTH.

Gap junctions and Aquaporin4 (AQP4) water channels form a water distribution network that is crucial for brain water homeostasis and for generation of water movements hypothesized to drive the glymphatic circulation. Despite their importance, fundamental issues remain to be resolved regarding the nature of the channels formed by AQP4 and its newly discovered isoforms and the interdependence of gap junction and AQP4 distribution in astrocytes. We here propose to determine function and structure of novel AQP4 isoforms and interactions with gap junction plaques using transfected cells and transgenic mouse models that were newly generated by the MPIs. One particular focus will be on the AQP4 isoforms that aggregate into Orthologal Arrays of Particles (OAPs) in astrocyte endfeet and are believed to provide most water flux. Because AQP4 channels and gap junctions are primarily in the endfeet of astrocytes, they provide control of brain water homeostasis. We will test the hypothesis that changes in AQP4 OAPs correlate with changes in gap junction plaque structure/distribution and that the permeability of blood-brain-barrier (BBB) is modified by this interplay between the astrocyte AQP4 isoforms and gap junction plaques. We expect that these straightforward studies using methods routine in our laboratories on new mouse models will provide key fundamental information on the organization of AQP4 isoforms in astrocyte endfeet relative to gap junction plaques and on the functions of AQP4 isoforms and gap junctions in BBB regulation. This proposal represents a collaboration between groups of investigators who have published several studies together and is expected to provide the foundation for future studies aimed to understand the cellular mechanisms involved in the redistribution of water, ions and solutes mediated by the combined action of AQP4 and gap junctions. .