Haig Keshishian - US grants

DESCRIPTION (provided by applicant): This proposal examines the role of neural activity in regulating two forms of retrograde signaling during the development and maturation of synapses. The proposal will investigate this form of signaling as a model for understanding how early synaptic critical periods operate. The proposal has two specific aims. Aim 1 examines how neural activity modulates a retrograde chemorepulsive signal from muscle (Sema-2a) that acts on the motoneuron during the early refinement of synaptic connections at the neuromuscular junction (NMJ). The responsiveness of the motoneuron to the retrograde Sema-2a is modulated by Ca2+, potentially through the activation of CaMKII. Using genetic and molecular approaches we will define the downstream effectors that mediate the activity-dependent repulsion. In addition we will elucidate how patterned rhythmic electrical activity regulates the withdrawal of processes from off-target muscles. Aim 2 addresses a second form of retrograde control by a muscle-derived TGF-beta ligand, Gbb, that acts through a canonical BMP signaling pathway. We have found two distinct roles for the retrograde control: an early critical period that defines the future growth of the NMJ, and is required for both the mature size of the NMJ, and for activity-dependent expansion. There is also an ongoing requirement for retrograde signaling to regulate synaptic function. We will examine the molecular mechanisms that govern these signals, and use new genetic methods to screen for the downstream effectors, using both inducible gene expression and RNAi knockdown. These tools will allow us to narrow the candidate genes to those involved in the critical period, to better resolve the underlying molecular mechanisms. We will use molecular tools we have developed, that include reengineered ion channels that either suppress or enhance membrane excitability, expressed at specific times in development on either side of the synapse. In addition, we have developed an inducible bipartite gene expression system to perform experiments testing temporal requirements for induced genes at specific synapses. The proposal is structured as a series of well- defined hypotheses that will help resolve the molecular and cellular mechanisms that govern synaptogenesis in a model genetic system. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page PUBLIC HEALTH RELEVANCE: In this project we examine how muscle-derived molecular signals regulate the growth and maturation of the motoneuron. The cellular events we are studying are fundamental to the normal establishment of connections by nerve cells, and are relevant to events occurring both during normal development and in clinical conditions. As there are numerous neurological disorders that involve defects in synaptic growth, maintenance, and/or plasticity, these studies will contribute to a rational analysis of these disorders of the nervous system. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page

The goal of this project is to understand how developing nerve cells make connections with muscles, and to determine how those connections alter their properties as a function of activity. There is evidence that developing nerve connections are refined during development by activity mediated mechanisms. The influence of activity on changes in nerve ending size and projection patterns will be examined by use of mutations that disrupt the ability of neurons and muscles to generate normal electrical activity. These changes will also be investigated by the use of toxins that specifically interfere with nerve and muscle electrical activity. To observe the development of the nerve muscle connections, and their responses to experimental manipulations, time-lapse video movies will be made of the living nerve endings maintained in semi- intact cultures. These experiments will elucidate several fundamental features of normal nervous system development.

During development, neurons (nerve cells) develop elaborate processes that may extend over long distances to reach highly specific local targets. The mechanisms that determine such accurate pathfinding and connectivity are not fully known. The fruitfly, Drosophila, has provided valuable data on such development, and the ease of producing genetic crosses and mutations provides approaches to the molecular mechanisms involved. Ground-based studies show in great detail how particular motor neurons develop specific connections with specific muscles. With this very detailed background from identified single cells, this project uses the space flight of Neurolab as a unique opportunity to test, by lacking normal gravity, how gravity may be a subtle cue important in developing such neuronal outgrowths and connections normally. Drosophila has the advantages for space flight of having very low mass and needing minimal care, so this study can involve very large numbers of individuals to assay developmental variability. Results of this study will be important for developmental neuroscience, and also will raise issues important for developmental biology in general. Contribution to this project represents the NSF Biological Sciences Directorate's participation in the NASA/Interagency Neurolab activity on the space shuttle orbiter, which in turn is part of Decade of the Brain activities.

Proposal IBN 0091184
PI: Keshishian, Haig

Abstract:
One of the ways a nervous system undergoes change is to modify the connections between nerve cells. This often requires the action of specialized secreted enzymes, which degrade or otherwise modify the local cellular environment, thus permitting modifications in the connections between neurons and their targets. An important class of these secreted enzymes is the "serine proteinases". These proteinases are widely conserved in evolution, and have been shown to be important in both the establishment and modification of neural connections during development, as well as in the later modifications associated with learning and memory. Serine proteinases are in turn regulated by a special class of proteins known as "serpins" (serine proteinase inhibitors). The serpins and serine proteinases, therefore, function as an interdependent system of secreted proteins of key importance in regulating nervous system change. In order to understand these proteins better Dr. Keshishian will examine their biological functions in a model genetic system, Drosophila. In Drosophila Dr. Keshishian has already identified the key "neuroserpin" of the nervous system, and has noted that it is expressed at connections between motoneurons and muscles. The major goal is to examine its functions at the developing neuromuscular connections, and to test the effects of both its loss and overexpression. The Drosophila neuroserpin is closely related to its mammalian counterpart, and insights gained in Drosophila about its function and regulation will be of value in understanding how the serpins influence nervous system development in general.

? DESCRIPTION (provided by applicant): This proposal is for continued NIH support for graduate student training within the Interdepartmental Neuroscience Program (INP) of Yale University. The INP is Yale's university-wide interdepartmental doctoral program, currently in its 27th year. Currently five students each in years 1 and 2 are supported by the INP Jointly Sponsored NIH Predoctoral Training Program. Tuition and stipend support is requested for support for an additional sixth student for each year. The faculty of the INP's T32 Jointly Sponsored NIH Predoctoral Training Program consists of 82 neuroscientists from departments of the Faculty of Arts and Sciences (FAS) and the Yale Medical School (YMS). For the 2014-15 academic year there are 55 predoctoral graduate students, of which 37 are from the US or are permanent residents. The INP has two Co-Directors, Haig Keshishian and Charles Greer, and is supervised by an executive committee representing a cross-section of the neurosciences at Yale. Both individuals also serve as program Co-Director for the INP's T32 Training Program. The INP receives strong university support, including a salaried administrator, office space, 8 full fellowships with tuition, and stipend supplementation. The doctoral program undergoes provostial-level academic reviews. Students are admitted through a neuroscience admissions committee that is part of the Biological and Biomedical Sciences (BBS) program of Yale. Upon affiliating with the INP the students remain within the interdepartmental program through their graduation. On average over the past funding cycle, 137 US/permanent resident students have applied annually, with 17% offered admission, for an entering class averaging 8 students (2014 class: 8 US students). The INP is actively involved in educating students from underrepresented ethnic and/or racial groups. Since 2010 11% of the US/permanent resident neuroscience students in the program were from these groups. Students are supervised by the Co-Directors, an executive committee, and the program is reviewed by an outside advisory committee. All INP students take four core graduate classes in neuroscience and bioethics, three advanced course electives, and two 1st year research rotations. They attend invited seminars, research in progress talks, an annual retreat and attend the Society for Neuroscience meeting at the program's expense. In the 2nd year the students select a doctoral adviser from the pool of participating faculty. They also take the doctoral qualifier examination, which has tutorial, written, and oral components. The students advance to candidacy for the PhD upon defending a prospectus in the 3rd year. All students are provided travel funds to attend and present their work at national meetings. A PhD in Neuroscience is awarded to graduates by the INP.

Abstract: In the developing nervous system many key proteins are modified or activated by proteolytic enzymes or proteases. These enzymes act both within the neuron as well as in the extracellular environment near growing processes and synapses. By cleaving key regulatory proteins the nervous system proteases influence the development of connections between nerve cells and their targets, and control the processing of secreted hormones and signaling molecules that are necessary for synapse function. The "serine proteases" are an important class of proteolytic enzymes in neurons. The serine proteases are widely conserved in evolution, and are important for both development as well as for changes associated with learning and memory. Understanding how these enzymes are controlled is the central goal of this project. Serine proteases are often regulated by a special class of proteins known as "serpins" (serine protease inhibitors). The serpins and serine proteases function as an interdependent system. In order to understand these proteins better Dr. Keshishian will examine their biological functions in a model genetic organism, Drosophila. In Drosophila Dr. Keshishian has already characterized the key serpins of the nervous system (Spn4), and has noted that Spn4 is expressed at connections between motoneurons and muscles. He has also discovered a second role for Spn4 in the enzymatic processing of precursor forms of secreted peptides and hormones. One variant of Spn4 binds to and regulates peptide processing serine proteases in vitro. When it is experimentally expressed in vivo the protein disrupts endogenous peptide hormone maturation, resulting in developmental abnormalities. A major goal will be to examine serpin functions at both developing neuromuscular connections, and in the processing of peptide hormones, and to test the effects of both its loss and overexpression. Studying Drosophila Spn4 will be of value in understanding how these regulatory molecules influence nervous system development and function in general.

[unreadable] DESCRIPTION (Provided by the Applicant): A major goal of neuroscience is to characterize neuronal connectivity in the CNS in order to understand the mechanisms that govern the development and function of neural circuits. This goal requires new tools for the direct labeling of neural circuits, using molecular genetic approaches for trans-synaptic transfer. A further goal is to generate molecular constructs that control gene expression within all the cells of specific neural circuits. This project will advance us toward these goals. There are three specific objectives: First, to generate novel transgenic lines where transsynaptic labeling constructs are expressed in vivo in specific targeted neurons. The constructs are based on both plant lectins (WGA and BL) and the tetanus toxin C chain (TTC), molecules that cross synapses. The carrier proteins will convey various cargos, such as GFP and RFP, across synapses in either a forward or reverse direction. Second, we will identify the essential domains of these carrier proteins through structure-function analysis, generating novel fusion proteins with improved carrier properties. Finally, we will develop a method for controlling gene expression within specific neural circuits, which we term "Inducible Transsynaptic Expression with Amplification" (ITEM). This entails generating molecules for transsynaptic transcriptional activation, based on fusions between known transsynaptic carriers and the GAL4::VP16 transcriptional activator. With ITEM, gene expression in a specific neural circuit is achieved when the transcriptional activator is transferred sequentially from cell to cell. By placing the transsynaptic transcriptional activator under UAS control, the molecule activates its own expression in recipient cells, resulting in amplification of the signal as it passes from cell to cell. The ITEM method will be a powerful tool for mapping neural connectivity, as well as for experimentally controlling the development or function of neural circuits. Although the studies will be pioneered in Drosophila, successful transsynaptic reporter gene activation would provide a proof of principle that could then be adapted for higher systems. Relevance to public health: In this project we will create new tools for looking at how nerve cells form connections with one another. These will help us understand how different patterns of connections give rise to different behaviors, and determine how nerve cells modify their connections in response to experience or to changes in brain function caused by injury or disease. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The goal of this project is to analyze the dynamic changes that occur at a cellular and molecular level during synaptic development and plasticity. These events will be examined in a well characterized genetic model system, the Drosophila neuromuscular junction (NMJ). We will generate a toolset of photoactivatable green fluorescent protein (PA-GFP) fusions made to a panel of pre- and postsynaptic proteins. Multiple transgenic lines will be made, including both promoter fusions and UAS effector lines. The molecular probes include PA- GFP fusions of the vesicular SNARE protein synaptotagmin, the PSD95/MAGUK adaptor protein disks large (Dlg) and the glutamate receptor subunit dGluRIIA. In addition, we will make fusion constructs of molecular components of the TGF-beta signaling cascade that is believed to be involved in the retrograde control of synaptic development. These include the Smad protein Mad, and the type II BMP receptor wishful thinking (wit). Extensive genetic and physiological validation experiments will be performed to demonstrate that the PA- GFP fusion reporters faithfully localize to the correct synaptic sites, are functional and can rescue mutant phenotypes, and do not confer dominant phenotypes when expressed in vivo. In the second specific aim, we propose to use the photoactivatable probes to investigate several outstanding questions in synaptic development and plasticity. The experiments include an analysis of activity-dependent growth of the synapse, the trafficking of retrograde growth factors involved in NMJ growth, the translocation of vesicular components between release sites, and the tagging of synapses undergoing plasticity. The proposed experiments were selected both for the importance of the question and for their potential for follow-on studies. The lines expressing the molecular probes will be of widespread use to researchers studying synaptic development and plasticity, and will be freely shared. The problems we will investigate are of universal interest to researchers studying synaptic development and plasticity, and the results will be of broad relevance. PUBLIC HEALTH RELEVANCE: In this project we will generate new molecular tools for examining how synapses modify their structures and deploy molecular components as a function of neural activity. We will make available to basic researchers a variety of valuable tools for imaging neuronal connections as they develop and as they are modified as a function of prior experience. These tools will allow researchers to study how specific proteins are deployed and regulated. As there are numerous neurological disorders that involve defects in synaptic growth, maintenance, and/or plasticity, these studies will contribute to a rational analysis of these disorders of the nervous system.

DESCRIPTION (provided by applicant): Glia and endothelial cells play a central role in the homeostatic regulation of the extracellular environment of the nervous system. The ionic composition and overall extracellular volume is actively regulated by these cells. In many pathological conditions homeostatic balance is lost, leading to swelling of the extracellular space, neuronal dysfunction, and apoptosis. Despite the severe impact of glial dysfunction, much remains to the learned about the molecular mechanisms that govern this critical form of physiological regulation. The focus of the study is a signaling cascade mediated by the Drosophila ser/thr kinase Fray. Fray regulates the activity of its downstream effector, the cation-chloride cotransporter NCC69, in glial cells of the blood-nerve barrier. This cascade is involved in the regulation of extracellular volume in peripheral nerves: nerves defective in this signaling cascade have extensive swelling. Such swelling also occurs in hyperactie mutants that have increased neuronal electrical activity. We have found extensive molecular and functional homologies between the Drosophila proteins and their human counterparts, SPAK and NKCC1. There is good evidence that dysfunction of NKCC1 in mammals is associated with stroke, and leads to edema in the nervous system. In this project we will examine the molecular cascade in detail, with the goal of identifying novel components, and to better understand how neural activity affects glial function. In specific aim 1 we use a genetic approaches to characterize the molecular elements that are involved in activity-dependent swelling in peripheral nerves. In the second specific aim we will use a knock-down RNAi screen to identify a substantial number of previously unidentified molecular players that function at the blood-nerve barrier to regulate the extracellular environment of the nervous system. The use of a genetic model system with extensive mammalian homology is a powerful strategy to rapidly identify the key molecular components involved in glial homeostasis. With their identification, this R21 proposal will set the stage for follow-on studies to better resolve how glia regulate their extracellular environment.

? DESCRIPTION (provided by applicant): This proposal describes a novel genetic method for mapping and experimentally controlling the neurons that are functionally connected within neural circuits. The approach used involves a modified plant-derived lectin carrier that is released in an activity-dependent fashion from presynaptic neurons, and is endocytosed by post- neuronal partners. The carrier lectin molecule has been modified from its native form to improve its efficiency of transfer and trafficking within the neuron. The carrier lectin is fused t various molecular cargoes, including a variety of fluorescent proteins, as well as epitope tags. Significantly, we use the carrier to transfer GV16, for the activation of UAS effector transgenes. To our knowledge this is the first example of a bipartite gene expression system that can be targeted to the members of a neural circuit. This method, which we term ITEM (Inducible Transneuronal Expression with Amplification), includes potent gene amplification. Using ITEM we can activate specific transgenes within synaptic partners, opening a new technology where the neurons of specific neural circuits can have specific transgenes activated in a genetic fashion. The project includes a series of proposed improvements to the technology, and functional tests as to its effectiveness as a tool to control gene expression in neural circuits.

This proposal examines a novel mechanism for the activity-dependent regulation of synaptic targeting and error correction. At the developing Drosophila neuromuscular junction a low frequency (0.01-0.03Hz) Ca oscillation modulates or gates the cell?s response to retrograde transsynaptic chemotropic signals from target cells. The dependence on neural activity is presynaptic and heterosynaptic. This system provides a powerful avenue for studying an alternate mechanism for early synaptic refinement in the establishment of neural circuits, distinct from a Hebbian matching that involves spike timing and competition. A similar mechanism involving low frequency oscillations has been observed in the development of connectivity at the mammalian superior colliculus. In our system, the low frequency Ca oscillation regulates the response of the growth cone to a postsynaptically derived chemorepellant, Semaphorin 2a (Sema2a), acting through a Plexin B receptor in the neuron. Experimental manipulation of the frequency and duration of the Ca oscillator disrupts the synaptic wiring process. Our previous works shows that the responsiveness of the neuron to Sema-2a involves at least three Ca-dependent signaling systems: principally a Ca-activated adenylyl cyclase, as well as the Ca-activated serine/threonine kinase CaMKII, and the Ca-dependent PP2B phosphatase Calcineurin. This proposal will examine in detail the signaling events that are involved in early synaptic targeting and refinement, in the context of a time-dependent Ca-wave environment. This includes live imaging of growth cones and their contacts, molecular genetic manipulation of the second messenger systems, and genetic tests to examine the roles of candidate molecules, including kinases and phosphatases, in mediating this time-dependent system of second messenger activity. We use several molecular tools we have developed, that include reengineered ion channels that either suppress or enhance membrane excitability, and an inducible bipartite gene expression system to perform experiments testing the temporal requirements for induced genes at specific synapses.