David Zenisek - US grants

DESCRIPTION (provided by applicant): In sensory neurons of the eye and inner ear the neurotransmitter, glutamate, is released at active zones in a graded and continuous manner. In these neurons specialized structures have evolved, known as synaptic ribbons. Synaptic ribbons are osmiophilic proteinaceous structures that tether synaptic vesicles near active zones. Because of their morphology, location within the cells and the cell types where they are found, these organelles are likely essential for the continuous release of glutamate; how ribbons aid in this task, however, remains unclear. The focus of this grant is to study the role of synaptic ribbons in sensory synaptic transmission, with the long term goal to resolve the temporal sequence of molecular and cellular events that are involved in the release of neurotransmitter from these important cells. To do this we use a combination of electrophysiology, fluorescence and biochemical tools to study the properties of synaptic transmission from retinal bipolar cells. Aim 1 is to investigation the properties of spontaneous and multi-vesicular release from the mouse rod bipolar cell synapse to determine the role of the ribbon in coordinating multi-vesicular release and to investigate the relationship between vesicles involve in evoked release and those involved in spontaneous release. In Aim 2, we propose to investigate modulation of synaptic transmission from ribbon synapses by calcium calmodulin kinase ii. In Aim 3, we investigate the role of the ribbon in priming vesicles for continuous neurotransmitter release. Understanding ribbon function may provide clues to help understand diseases that specifically affect vision and hearing, such as Usher syndrome. In addition, the fundamental understanding of presynaptic processes in these specialized neurons will have broader implications for neuronal communication in general and thus, may contribute to our understanding of various aspects of mental health and neurological disorders.

DESCRIPTION (provided by applicant): The goal of this project is to establish the zebrafish bipolar cells as a system for studying the molecules involved neurotransmitter release from ribbon-type retinal neurons. Zebrafish are an excellent vertebrate model for physiology and disease, conducive to both large-scale mutagenesis screens and the rapid and cheap generation of transgenic animals. Hence, several mutants have been generated that serve as human disease models and many more will be developed in the future. We intend to extend the utility of this preparation to the study of the physiology of presynaptic terminals from ribbon-type synapses after vision has been established. This proposal aims at developing tools and techniques for manipulation and investigation of presynaptic processes in these cells. Aim 1 establishes two zebrafish preparations for studying neurotransmitter release and vesicle recycling from retinal neurons. Aim 2 generates several transgenic lines for monitoring the properties of exocytosis and endocytosis in retinal neurons. Aim 3 attempts to develop new tools for silencing gene expression in adult zebrafish. Information in the nervous system is transmitted between nerve cells at the synapse, where an electrical impulse in the "presynaptic" nerve cell causes the release of neurotransmitter from small membrane bound structures, known as synaptic vesicles. Deficiencies in this process have been suggested to underlie and contribute to the pathologies of a number of neurological disorders, including Huntington's disease, Alzheimer's disease, bipolar disorder, Schizophrenia, Parkinson's Disease and mental retardation, yet it is unclear how presynaptic function is affected by these disorders. This proposal develops new tools to study how presynaptic nerve cells release neurotransmitter.

The purpose of the molecular biology core is i) to provide and maintain a wide range of equipment to be shared among vision researchers; 2) aid investigators by providing a service for those without the facilities for basic or advanced molecular biology techniques; 3) help new investigators and students acquire basic molecular biological techniques; 4) provide genotyping services for laboratories working with transgenic animals. The resources provided by this module will be applied toward maintenance of existing equipment for shared usage among vision researchers throughout the university, to employ two technical staff and to purchase a quantitative RT-PCR system to relieve pressure on existing machines due to heavy usage. One technician, Ms. Adrienne LaRue Marabelle, will have bench space in Brady Memorial Laboratory (BML) 224 and will handle the genotyping of transgenic animals and will oversee the use of the robotic workstation and microarray core facility. Ms. Marabelle has over 15 years of experience in molecular biology techniques and has been running the microarray facility in the Department of Ophthalmology for the past 10 years. A second technician, Mr. Steven Viviano, will have bench space in SHM-B1O3 and will work with and train post-doctoral fellows and graduate students in various molecular techniques including Western blotting, quantitative PCR, immunohistochemistry. He will also design and prepare plasmids for electroporation or viral transfection for vision research investigators that are not equipped to do so in their own laboratories. The work of Mr. Viviano will be carried out using equipment in the laboratory of Dr. Zenisek and core facilities within the Department of Physiology and will be closely supervised by Dr. Zenisek. Several vision laboratories have primary expertise in electrophysiological, anatomical and computational techniques and core support has been instrumental in enabling these vision researchers to increasingly employ molecular biology techniques in their research. This module has enjoyed considerable usage in the past and will continue to do so in the future. It is anticipated that Drs. Crair, McCormick, Tian and Zenisek will make use of the genotyping service, Drs. Hoh, Rizzolo and Zeiss will make use of the microarray facility, Drs. Coca- Prados, Crair, Hoh, Rizzolo, Tian and Zenisek will make use of core facilities in the Brady Memorial Laboratory building and Drs. Coca-Prados, Crair, Hoh, Rizzolo, Tian, Zeiss and Zenisek will make use of Mr. Viviano's services.

? DESCRIPTION (provided by applicant): Photoreceptors and bipolar cells of the retina and hair cells of the auditory and vestibular systems signal sensory stimuli as graded changes in neurotransmitter release. To do so, these cells have evolved synaptic ribbons, proteinaceous structures that tether large numbers of synaptic vesicles near release sites. The molecular machinery underlying synaptic ribbon function is poorly understood. A handful of proteins have been localized to the synaptic ribbon and the importance of these individual molecules as well as how they contribute to the unique functions of the synaptic ribbon remains elusive. Of these proteins, the most abundant is Ribeye, a protein unique to the synaptic ribbon and thought to constitute most of the synaptic ribbon and hypothesized to form the core of the synaptic ribbon. Ribeye arises from an alternative start site of the transcriptional corepressor CtBP2. The precise role of Ribeye remains unknown and the long-term goal of this proposal is to determine the functional role of Ribeye in the synaptic ribbon. To study Ribeye function, we will employ a combination of molecular biology, genetic and electrophysiology primarily using zebrafish as a primary model system. In Aim 1 we generated and characterize zebrafish with targeted mutations in both ribeye genes. In Aim 2, we the effect of one or both Ribeye gene products on the structure of photoreceptor and hair cell ribbons. In Aim 3, we will look at the effect of Ribey removal on the distribution and localization of other synaptic ribbon proteins. In Aim 4, we will investigate the effects of Ribeye loss on exocytosis and calcium current in neuromast hair cells. Understanding synaptic ribbon function at the molecular level will ultimately aid in understanding how visual and auditory information is processed and communicated. In addition, it may provide clues to help understand diseases that specifically affect vision and hearing. In addition, the fundamental understanding of presynaptic processes in these specialized neurons will have broader implications for neuronal communication in general and thus, may contribute to our understanding of various aspects of mental health and neurological disorders.

Genotyping and Virus Production Core Module Abstract The Genotyping and Virus Production Core Module will provide the means to incorporate modern molecular and genetic techniques into the research programs of the entire vision research community at Yale. First, the Module will supply genotyping and virus preparation assistance and services for laboratories working with transgenic animals and/or viral techniques. Second, it provides and maintains a range of molecular biology equipment to be shared among vision researchers and aids investigators by providing a service for those without the facilities for basic or advanced molecular biology techniques. Third, it trains new investigators and students in basic molecular biology techniques, including the use of viral vectors. Viral vectors are powerful tools for gene delivery in terminally differentiated cells. For example, adeno-associated viruses (AAVs) have enjoyed increasingly wide application in vision research for their many favorable features that include the lack of pathogenicity, low immunogenicity, and the ability to maintain long-term transgene expression in neurons. The Genotyping and Virus Production core module will be sufficient and well equipped to provide both virus vector design and virus production to meet a variety of research needs among vision researchers at Yale. Although many of the common viruses can be purchased from viral core facilities outside of Yale, having an in- house capability for virus design and production will significantly enhance vision research by saving time and cost and facilitating more efficient and innovative virus design with the convenience of in-person consultation for specific research needs.

Project Summary Information in the nervous system is relayed mostly at synapses, where neurotransmitter is released with great temporal precision from a presynaptic terminal on to a post-synaptic cell via the fusion of membrane bound synaptic vesicles (SVs) with the cell membrane, in a process called exocytosis. The components of these SVs are subsequently retrieved via endocytosis and recycled for reuse. This grant aims to understand the interplay between SV recycling and membrane tension gradients and associated membrane flows. In neurons and neuroendocrine cells, both exocytosis and endocytosis are influenced by osmotic swelling or shrinking, suggesting they are influenced by membrane tension, ?. Conversely, membrane addition to the presynaptic terminal via exocytosis is expected to lower ?, while endocytosis should restore it. In addition, membrane tension has been suggested to be one of the possible signals for coupling exocytosis to endocytosis. However, despite these key roles, there are no measurements of membrane tension in synaptic terminals and how tension changes are related to exo-endocytosis is not known, mainly due to technical difficulties. The best method to probe ? is to pull a thin membrane tether from the cell surface using optical tweezers, manipulating a 1-3 ?m diameter bead as a handle. The bead's displacement from the trap center provides the tether force, which reflects ?. However, most terminals are small and are tightly coupled to post-synaptic structures, making tether pulling impractical. We overcome this challenge using goldfish bipolar cells which possess giant terminals, in a setup that combines optical tweezers with electrophysiology (to control stimulation and/or measure capacitance changes) and with high-resolution fluorescence microscopy (to label and identify sub- cellular structures and calcium imaging). We aim 1) to characterize the tether force response to electrical and mechanical perturbations that occur at a presynaptic terminal during activity. After stimulation, membrane added at an exocytic site needs to flow (and the associated tension perturbation propagate) over the terminal surface, then through the tether to produce a change in the measured tether force. We will characterize membrane flows in double-tether experiments and calibrate the tether response to step- changes in tether length. We will confirm that ? changes we observed in preliminary experiments (a drop ~1 s after stimulation, followed by recovery in ~10 s) are due to exo-endocytosis, and characterize rapid voltage- induced tether force changes. These will enable a quantitative understanding of measured ? changes associated with stimulation. Next, we will 2) characterize how membrane tension is regulated at a presynaptic nerve terminal. Combining pharmacological interventions with live imaging and ? measurements, we will test the hypothesis that F-actin is a major regulator of ? at the nerve terminal. We will manipulate ? and calcium independently to dissect calcium and ? requirements for SV turnover. These measurements will help generate a model of feedback between membrane trafficking and ? at the nerve terminal.

Light responses of rod and cone photoreceptors are encoded by the release of glutamate-filled vesicles at photoreceptor synapses. Synaptic transmission at the first synapse in the retina thus fundamentally shapes visual perception and damage to photoreceptor synapses by protein mutation or diseases such as macular degeneration and ischemia causes vision loss. To understand the consequences of damage to these synapses and how to restore vision by therapeutic means requires a thorough understanding of their normal operation. Release from photoreceptors involves a plate-like protein structure known as the synaptic ribbon. Unlike most central nervous system (CNS) synapses that release only one or two synaptic vesicles at a time, ribbon synapses in photoreceptors and other sensory neurons are specialized for continuous release. In addition to the ribbon itself, the specialized capabilities of ribbon synapses are also determined by the use of certain proteins that differ from those at more conventional synapses. Rod and cone photoreceptors differ further from both conventional and other ribbon synapses in their use of an exocytotic Ca2+ sensor with unusual Ca2+ dependence. At most synapses, synaptic vesicle release rate rises with the 5th power of [Ca2+]i but release from photoreceptors has a weaker 1-3rd order Ca2+-dependence. The identity of the atypical Ca2+ sensor that regulates vesicle release from photoreceptors is a major unresolved question about the mechanisms of release at the first synapse in the retina. Isoforms of the protein synaptotagmin (Syt) serve as the exocytotic Ca2+ sensors in most neurons. Our first aim is to identify the Ca2+ sensor controlling release from photoreceptors by testing mice in which specific Syt proteins have been selectively deleted from rods or cones. Our second aim is to confirm that the exocytotic Ca2+ sensors in mouse rod and cone synapses retain the unusually low Ca2+ cooperativity seen in lower vertebrates. In Aim 3, we propose to characterize how the Ca2+-dependence of release rate is shaped by different combinations of Syt, Complexin, and SNARE proteins that reproduce components of the rapid release machinery at different conventional and ribbon synapses, using unique in vitro approaches that can probe single fusion pores with sub-ms time resolution. Together, these experiments will reveal the mechanisms responsible for the atypical Ca2+-dependence of neurotransmission at the critical first synapse in vision and allow us to understand how the expression of particular proteins shapes the properties of release to meet specific signaling needs at different CNS synapses.

Hair cells of the auditory and vestibular systems signal sensory stimuli as graded changes in neurotransmitter release and employ unique anatomical and molecular components that differ from conventional synapses. Among the unique molecular features is the apparent lack of reliance on neuronal SNARE proteins and their various partners. Instead, hair cell exocytosis depends on a large protein called Otoferlin, by unknown mechanisms. In this proposal, we investigate the unique features of hair cell synaptic transmission using a combination of molecular biology, electrophysiological approaches in zebrafish, and novel in vitro membrane fusion assays. In Aim 1 we will determine whether Otoferlin can stimulate membrane fusion using well established cell-cell fusion assays with engineered cells and determine the requirements for SNAREs, lipids and calcium in otoferlin-dependent fusion. These experiments will also determine the functional domains required to mediate membrane fusion. Importantly, this strategy avoids difficulties with purification of Otoferlin that hindered advances in reconstituting Otoferlin-dependent fusion in the past. In Aim 2, we will measure the calcium-dependent membrane binding properties of Otoferlin and look for Otoferlin-interacting partners in native cells. In Aim 3, we use the zebrafish lateral line as a model system explore the role of SNAREs in hair cell exocytosis. In Aim 4, we test the effectiveness of truncation mutants to rescue synaptic function. Understanding hair cell synaptic function at the molecular level will ultimately aid in understanding how auditory information is processed and communicated. Moreover, mutations in Otoferlin lead to DFNB9 form of inherited deafness and thus study of its function has relevance for human disease. Otoferlin belongs to the ferlin class of proteins, which include myoferlin and dysferlin, which are also implicated in membrane fusion and human disease. It is reasonable to expect that what we learn in this project will be instructive for studies with other ferlins. Lastly, the fundamental understanding of presynaptic processes in these specialized cells will have broader implications for cellular communication in general and thus, may contribute to our understanding of various aspects of mental health and neurological disorders.

Project Summary Congenital heart disease (CHD) leads to severe morbidity and mortality to children in the US and worldwide. Despite this impact on child health, we simply do not understand the genetic causes of CHD. Recently, trio based whole exome sequencing has identified a class of voltage-gated potassium channels (multiple KCNH family members) as candidates for CHD and, specifically heterotaxy, a disorder of left-right (LR) patterning that has a severe effect on cardiac function. However, a molecular role connecting potassium channels to structural heart disease and heterotaxy is unprecedented. We propose, and our preliminary data support, that KCNH6 defines a new paradigm for cell signaling in early embryonic cells. Our data support an electrophysiological model where specific germ layers fates (paraxial mesoderm and ectoderm) are dependent on an ion channel network. Our overarching hypothesis is that K+ channels define electrical membrane potential and regulate voltage gated Ca2+ channels that establish an exit from pluripotency towards specific cell fates, gastrulation, and LR patterning providing a plausible mechanism for our patients with Htx and CHD. Our electrophysiological pathway then integrates with biochemical signaling pathways that define specific cell fates in the embryo. In this proposal revision, we will focus on KCNH6 to see if gene depletion leads to LR patterning defects in Xenopus. In addition, we will test where in the LR patterning cascade, KCNH6 plays a role. Then, using a series of judiciously chosen chemical and ionic perturbations, we will test if membrane potential is indeed essential for pluripotency, cell fate, and calcium regulation. Due to the novelty of this project, we will also perform unbiased genomics (RNAseq) for discovery of transcriptional targets of ? Vm. Finally, we will measure electrical properties electrophysiologically using both whole-cell voltage clamp and intracellular recordings and determine the various currents that define membrane potential in early germ cells. A major strength of our proposal is our expertise; we have forged a collaboration between Xenopus developmental biologists and electrophysiologists that will allow us to rigorously investigate membrane potential as an embryonic patterning mechanism.

In sensory cells of the eye and inner ear the neurotransmitter glutamate is released at active zones in a graded and continuous manner. These cells have evolved specialized structures known as synaptic ribbons. These proteinaceous structures tether synaptic vesicles near active zones. Based on their location, abundance of tethered vesicles, and properties of the sensory neurons in which they are found, these organelles have long been thought to be important for maintaining the continuous release of glutamate. Several other functions have also been ascribed to ribbons. However, recent evidence casts doubt on these ideas. The focus of this grant is to understand the role of synaptic ribbons in sensory synaptic transmission using animal models that lack Ribeye, a protein that is both the most abundant within the ribbon and not found anywhere else. Lack of Ribeye leads to loss of membrane associated synaptic ribbons, without loss of other presynaptic proteins. Specific Aim 1 investigates how Ribeye removal and loss of synaptic ribbons affects neurotransmitter release from photoreceptors. Specific Aim 2 looks at the properties of synaptic release in bipolar cells from the same animals. In Specific Aim 3, we will image single synaptic vesicles in cells lacking Ribeye to measure rates of vesicle movement and replenishment to test the role of the ribbon in these processes. Understanding ribbon function may provide clues to help understand diseases that specifically affect vision and hearing. In addition, the fundamental understanding of presynaptic processes in these specialized neurons will have broader implications for neuronal communication in general and thus, may contribute to our understanding of various aspects of mental health and neurological disorders.