Alex Kolodkin - US grants

DESCRIPTION (provided by applicant): The establishment of precise neuronal connectivity during development requires that neurons respond to a myriad of guidance cues, both attractive and repulsive, as they extend toward their targets and ultimately establish final connectivity patterns. Importantly, many of these guidance cues and their receptors are also expressed in the adult nervous system where they may play important roles in the assembly and regulation of neuronal circuits. In addition, the expression patterns of several of these cues and their receptors show changes in response to neuronal injury. In this proposal we will explore how semaphorins, a large protein family which includes many members capable of functioning as repulsive neuronal guidance cues, direct the formation of mammalian central and peripheral neural connections during development and in the adult. Secreted semaphorins are robust neuronal repellents in vitro and in vivo, and we have shown during the initial funding period of this grant that certain secreted semaphorins and their receptors the neuropilins are indeed required in vivo for the establishment of normal connectivity in a wide variety of neural systems. Recent genetic and biochemical analyses have also shown that members of the plexin family of transmembrane proteins serve as signaling subunits of secreted semaphorin receptors. Thus neuropilins are the ligand binding subunits and plexins are the signaling subunits of holoreceptors for the secreted semaphorins. Using several mouse genetic models generated as part of our studies, and employing certain new mouse mutants, we propose here to provide a comprehensive understanding of how neuropilins, plexins and their six secreted semaphorin ligands Sema3A, B, C, D, E, and F (all defined as Class 3 semaphorins) participate in the establishment of neuronal connectivity in both the CNS and PNS. In all, these studies will help us move beyond our current, incomplete, understanding of functional ligand-receptor relationships between secreted semaphorins and their receptors. They will provide insight into how secreted semaphorins, acting as repellents and possibly as attractants, work in concert to regulate neuronal migration, morphology, pathway formation, and target selection in the embryonic and postnatal nervous system. Finally, these studies will begin to address how class 3 semaphorins and their receptors function once neural circuits are established to modulate neuron morphology and synapse function.

DESCRIPTION (provided by applicant): The central goal of the experiments in this proposal is to understand how neurons find their targets during neural development. Our current understanding of how various guidance cues and their receptors influence the establishment and maintenance of neuronal trajectories provides a platform upon which a thorough understanding of the signaling cascades which govern neuronal guidance can be built. The semaphorin family of phylogenetically conserved proteins contains many well characterized repulsive guidance cues. However, some semaphorins can act as attractants, and certain individual semaphorins have the capacity to act as both a repellent and an attractant. Therefore, defining the semaphorin signaling cascade will contribute to our understanding of how repulsive guidance is signaled to neuronal processes and also how this signaling is modulated. In addition to aiding our understanding of how growth cones navigate through complex extracellular environments during neural development, elucidation of semaphorin signaling cascades has important clinical implications. Semaphorin signaling has been linked to inhibition of neuronal extension following injury, to the progression of certain cancers, and to immune system function. Therefore, work proposed here has potential implications that extend beyond understanding how neuronal connectivity is established and maintained. We propose here to investigate how key semaphorin signaling cascade components steer neuronal processes. We have previously characterized two phylogenetically conserved different protein families, the MICALs and nervy/MTG proteins, each of which includes cytosolic proteins that play critical, but distinct, roles in promoting and modulating semaphorin-mediated repulsion in vivo. This work provides new insights for our current understanding of semaphorin signaling which can be applied to both invertebrate and vertebrate guidance events. Therefore, using approaches in both Drosophila and in rodents, we will address how these and other semaphorin signaling cascade intermediates ultimately facilitate the establishment and maintenance of neuronal connectivity.

The aim of the Embryonic Stem (ES) Cell Engineering facility is to accelerate research programs that depend on the development of genetically altered mouse models (knock-outs, knock-ins, and inducible knock-outs) that require ES cell manipulation. Successful ES cell engineering is a major hurdle for the realization of research goals for many NINDS investigators at JHU SOM (see below), and is currently performed by individual laboratories with varying degrees of success. The ES Cell Engineering Core will implement cutting-edge approaches for ES cell manipulation to assure that investigators use the most efficient strategies to generate animal models for NINDS supported research. These strategies will include generation of ES cells from existing mouse mutant lines for subsequent gene targeting and chimeric embryo production, and multi-hit transgenic targeting protocols that use novel recombination approaches for controlled site-directed mutagenesis. By establishing ES cell engineering as a core resource we will standardize protocols from the most successful laboratories, markedly increase the efficiency of model generation, and speed the pace of NINDS-supported research at JHU SOM. There is currently no core facility at JHU SOM that can perform ES cell engineering. The ES Cell Engineering Core will work in synergy with the current Transgenic Animal facility at JHU SOM, which provides complementary services such as injection of ES cells into blastocysts and animal husbandry necessary to achieve high chimera mice. There will be no overlap in the services provided by the ES Cell Engineering Core and the Transgenic Animal facility.

The aim of the Monoclonal Antibody Core is to generate mouse and rat monoclonal antibodies against proteins studied by NINDS investigators at JHU SOM. Antibodies are central for the analysis of protein function, and ongoing studies by the Primary Center Investigators and other NINDS-funded investigators at the JHU SOM have been severely limited by the availability of reliable quantities of unique mono-specific antibodies. The monoclonal facility will develop new antibodies with enhanced selectivity, providing a much-needed resource to the Center's primary investigators and also other NINDS-funded scientists at JHU SOM. No such facility currently exists at JHU SOM.

The aim of the Multiphoton Imaging/EIectrophysiology Core is to provide instrumentation for analyzing protein localization, protein dynamics, and protein-protein interactions with high resolution. This facility will also allow users to perform time-lapse imaging of multiple fluorophores in living cells and tissues, and combine high resolution imaging of fluorescently tagged proteins or ion indicator dyes with electrophysiological monitoring of electrical activity. This Core is anchored by a Zeiss LSM 510 META NLO confocal microscope equipped with two visible lasers for performing traditional single-photon confocal imaging, and one near-infrared pulsed laser for two-photon imaging. In addition, the stage of this microscope has the capacity to either be enclosed to create an incubator for long-term time-lapse imaging, or to be surrounded by electrophysiology equipment to allow simultaneous whole-cell recording and imaging. The unique capabilities of this facility will serve as a resource for other NINDS funded investigators at JHU SOM and will establish new approaches for studying neurobiological processes that will benefit the greater JHU community. The equipment and capabilities of this core are not duplicated in any core facilities at JHU.

DESCRIPTION (provided by applicant): In this Institutional Center Core Grant to Support Neuroscience Research we propose to establish three Core facilities. These facilities will provide necessary resources and will perform required services that are impractical for individual laboratories to provide on their own. They consist of a Multiphoton Imaging/Electrophysiology Core, an Embryonic Stem Cell Engineering Core, and a Monoclonal Antibody Core. The experimental opportunities and technical services to be offered by these Cores complement and do not duplicate existing Core facilities available to NINDS-funded investigators at the Johns Hopkins University School of Medicine (JHU SOM). The use of these Core facilities will greatly benefit the NINDS funded research programs of the ten primary investigators who constitute the primary investigators of this Center, and they will also be an important resource for all other NINDS-funded investigators at JHU SOM. Nine of the Center's ten primary investigators are members of the Department of Neuroscience, and the remaining investigator is a member of the Department of Neurology. The research programs of the Center's primary investigators address unresolved issues in the areas of neural development, regulation of synaptic structure and function, ion channel physiology and neurotransmitter transporter function, and activity dependent regulation of gene expression. The Specific Aims of these primary investigator's NINDS research programs address critical clinical issues, including the developmental basis of neurological disorders, the promotion of neuronal regeneration folowing injury or degeneration, and the origin of neurodegenerative disorders such as Alzheimer's Dementia and ALS. The Center's Primary Investigators define a highly interactive group with a strong history of seamless collaborative research efforts. It is the primary goal of this Center to provide these investigators, and all other NINDS-funded investigators at JHU SOM with core facilities that are not currently available in order to augment their existing research programs.

DESCRIPTION (provided by applicant): The formation of neural circuits relies on axonal and dendritic growth, precise guidance events during development, recognition of appropriate target cells, and the subsequent formation and refinement of synaptic connections. Characterization of each of these stages is critical for understanding the assembly of neuron circuits that mediate all behavior. Deficits in these developmental processes underlie cognitive impairments associated with disease and neurologic disorders. Indeed, aberrant dendritic morphologies and synapses are associated with a range of neuropsychiatric disorders, underscoring the need for understanding the cellular and molecular basis of these events during circuit formation. The objective of this work is to understand how extracellular cues present within the postnatal brain control the morphological development of projection neurons whose cell bodies are located within layer V of the cortex. We will elucidate the functions and mechanisms of action of secreted members of the semaphorin protein family of guidance cues and their neuropilin and plexin receptors on the morphological development of these neurons. Our preliminary work shows that semaphorin 3F (Sema3F) and its neuropilin-2 (Npn-2) receptor function in vivo to govern cortical pyramidal neuron apical dendritic spine morphology and synaptogenesis, whereas semaphorin 3A (Sema3A) promotes the elaboration of basal dendritic arbors. Thus, structurally related cues instruct distinct steps in the development of layer V pyramidal neurons, the location, morphology, and number of synapses that form upon them, and hence the genesis of normal functioning cortical circuits. Mechanistically, we found that localization of the Npn-2 receptor is restricted to primary apical dendritic processes, while Npn-1 is located on both basal and apical dendrites. In addition, Npn-2 is enriched at sites of synapse formation-the PSD. These findings lead to the hypothesis that semaphorin receptor localization underlies Sema3A and Sema3F specificity of action. We propose here to investigate the regulation of neuropilin and plexin receptor distribution, secreted semaphorin receptor signaling mechanisms, and the source and mode of action of these semaphorin ligands during postnatal cortical neuron development. Since our findings will shed light on the mechanisms underlying spatially restricted regulation of dendritic morphology and synapses, the proposed Aims will begin to address how complex cortical connectivity patterns are generated and maintained. Finally, while the focus of the proposed work is on the morphologic and synaptic development of the primary projection neuron of the cortex, the layer V pyramidal neuron, the discoveries made here will have important implications for defining the molecular and cellular basis of neural circuit assembly throughout the brain. PUBLIC HEALTH RELEVANCE: The proposed studies will define the molecular mechanisms that organize precise and spatially restricted neuronal connectively patterns in the mammalian cortex. These discoveries will provide new insight into our current understanding of how neuronal morphology and connections are formed in the brain. Importantly, since aberrant neuronal morphology and synapses in the cortex are associated with a range of neuropsychiatric disorders, this work will inform diagnostic and therapeutic strategies for ameliorating these disorders.

DESCRIPTION (provided by applicant): The generation of neural connectivity critically depends upon temporally and spatially coordinated regulation of neuronal process guidance. The central goal of this proposal is to understand the functions and mechanisms employed by specific neuronal semaphorin guidance cues to regulate axonal and dendritic targeting. A range of cues and their receptors play key roles in attracting and repelling neuronal processes as they extend toward their final targets, where guidance cues also play important roles in target recognition and synaptogenesis. During the previous funding period, we identified unique roles for semaphorin cues and their receptors in guiding neuronal processes in both Drosophila and in the mouse, and we also investigated signaling components that serve to facilitate these guidance events. Our cross-phylogenetic approach provides unique insight into select guidance cue-mediated neuronal targeting, and it uses strong complementary experimental strategies. In Drosophila, we have the ability to employ powerful molecular and genetic approaches to address complex aspects of guidance cue function and receptor signaling in vivo. In the mouse, we have the opportunity to employ one of the best characterized laminar structures in the nervous system, the vertebrate retina, to investigate the molecular mechanisms underlying the assembly of complex neuronal connectivity, utilizing powerful genetic and anatomical strategies. Our studies during the previous funding period raise several issues we propose investigating in this renewal application of our long-standing work on semaphorin- mediated neuronal guidance. Our results show that closely related Drosophila secreted semaphorins function through the same receptor to mediate short-range attraction or longer-range repulsion, and we have in place both in vivo and in vitro experimental paridigms that will allow us to begin to dissect critical ligand and receptor signaling interactions that lead to divergent guidance cue responses (Aim I). Our work on mouse transmembrane semaphorin regulation of retinal lamination raises intriguing issues regarding novel mechanisms by which distinct classes of retinal ganglion cells (RGCs) and amacrine cells establish their exquisite connectivity in the IPL, and also how select RGC axonal projections employ transmembrane semaphorins and their plexin receptors to regulate targeting to appropriate retinorecipient CNS targets (Aim II). These studies will contribute to our understanding of circuit assembly and function.

SUMMARY: Administrative Core The Administrative Core of this proposed continuation of the ?JHU Center for Neuroscience Research? NINDS P30 Center is critical for implementation of the primary goal of this Center: to provide the Primary Center Investigators, and other JHU neuroscientists that are engaged in research consistent with the mission of NINDS, with cutting-edge Core services, enhancing their research capabilities to enable fundamental scientific advances in our understanding of the nervous system that have the potential to address critical issues in the treatment of neurological disease. This Core, therefore, includes an administrative structure that allows for fair and equitable use of the two Center Scientific Cores: the Multiphoton Imaging (MPI) Core and the Murine Mutagenesis Core (MMC). It also addresses Scientific Core project prioritization to ensure fair access to the Cores by JHU neuroscience investigators, including: the NINDS-funded Primary Center Investigators, other NINDS-funded JHU investigators, and other neuroscientists at JHU who would benefit from access to the Center. Further, the Administrative Core is proposed to have in place mechanisms that provide a facile means to request Core access, and also strict reporting procedures that provide a record of Core use by all investigators. This includes an assessment of the success of projects performed by the Cores, quantification of services provided by the Cores, and feedback on the quality of Core service. The Administrative Core will oversee all financial issues relating to day-to-day activities of the Cores, and it will provide long-term oversight in order to assure adequate resources are available for all Core services. Finally, the Administrative Core will provide a forum for continual assessment of the quality of Core services and for incorporating into Core services and activities new technologies and, where appropriate, re-organization for Core services to best serve the needs of the users. All of these functions of the Administrative Core will serve to further the NINDS scientific mission by ensuring that this Center for Neuroscience Research functions effectively, efficiently and creatively to assist neuroscientists at JHU SOM in their research efforts.

Project Summary/Abstract The overall goal of this proposal is to elucidate how to regrow and reconnect injured optic nerves and tracts to specific target neurons in the brain. Specifically, this proposal investigates mechanisms that promote the regeneration of connections made by direction selective retinal ganglion cells (DSGC) to their the accessory optic targets in the brainstem (collectively referred to as the ?Accessory Optic System,? or ?AOS?). The AOS serves a crucial role in vision by generating slip-compensating eye movements whenever the head or the eyes move at slow speeds. In the absence of proper AOS connectivity and function, images appear blurry and perceptual performance is severely degraded. From a practical standpoint, understanding how to regenerate the mammalian AOS, and defining the cellular and molecular underpinnings of that regeneration, represent an ideal model for parsing regeneration of other visual parallel pathways and also mammalian CNS circuits generally. The AOS is comprised of known retinal neurons and circuits, and the central targets and information carried in this pathway are rather well understood. Indeed, significant progress has been made by our and other groups in identifying genetic markers for the DSGCs that drive AOS function and also cellular and molecular pathways that wire them to their targets. Moreover, both of our laboratories have adopted and expanded state-of-the-art approaches to measure AOS function at the whole animal level with quantitative rigor. In parallel to our work, the field of CNS visual system regeneration has reached the crucial milestone of identifying molecular and activity-based manipulations that allow some retinal ganglion cell (RGC) axons to regenerate following axotomy. The next crucial milestone is to figure out how to ensure accurate reconnection of specific RGC types with their correct targets in the brain. Importantly, it remains unclear whether, after damage to the retina or optic nerve, RGCs and/or their targets re-express, or maintain expression of, the receptors or ligands that enabled them to correctly wire up with one another during development. It is also imperative to determine how the specificity of axon-target matching at the level of cell types and targets, impacts circuit function and behavior. Now that the molecular programs for these developmental steps have started to become clear, this essential issue relating to optic nerve regeneration can finally be approached with deep rigor, and we propose here do that in the context of the AOS. The four major aims of this proposal are to: 1) Test the hypothesis that AOS-projecting RGCs are among the cohort of RGC types capable of regenerating in response to increases in mTOR activation and/or RGC firing. 2) Test the hypothesis that damage to AOS-projecting RGCs and their axons triggers robust changes in axon guidance receptors and ligands in the relevant RGCs and targets. 3) . Test the hypothesis that re-introduction of specific guidance receptors and ligands to AOS-projecting RGCs can be used to steer their axons to desired brain areas. 4) Test the hypothesis that regeneration of a small fraction of total retinofugal connectivity is sufficient to replenish functional recovery of the optokinetic reflex and nystagmus necessary for image stabilization.

PROJECT SUMMARY The elaboration of neural circuits involves a complex series of events, including neuronal differentiation, settling of neurons in appropriate locations, neural process outgrowth and pathfinding, target selection, synaptogenesis and synapse refinement. Development of direction-selective (DS) circuits in the mammalian visual system relies on precise execution of each of these steps, however we are only beginning to understand how these connections are established. The central goal of this proposal is to understand the molecular mechanisms that allow components of DS circuits to mediate appropriate visual system responses to image motion. DS responses depend critically on distinct classes of bipolar cells, starburst amacrine cells (SACs), and direction-selective retinal ganglion cells (DSGCs). The development of these neurons, including their differentiation and the regulation of their morphology and synaptic contacts, is integral to the generation of functional DS circuitry. Here, we propose leveraging our recent gene profiling and additional Preliminary Findings to address key unresolved questions in DS circuit wiring. Subtypes of DSGCs are tuned to motion in distinct preferred directions, and this is due to differences in asymmetric wiring of SACs onto the dendrites of these different DSGC subtypes; however, the underlying basis of this asymmetric SAC-DSGC wiring is unknown. We have identified genes that are differentially expressed in subtypes of DSGCs that are components of the Accessory Optic System (AOS): On-DSGCs (oDSGCs) that differ only in their preferred directional preference?in this case for dorsal vs. ventral object motion. Analysis of these differentially expressed (DE) genes has the potential to reveal underlying molecular mechanisms governing the development of these oDSGCs and the synaptic wiring that determines their directional tuning, since the central difference between dorsal-oDSGCs and ventral-oDSGCs is the polarity of their preferred directional tuning. This proposal is focused on testing the hypothesis that differential gene expression in oDSGCs of the accessory optic system (AOS) tuned to detect either upward or downward motion instructs the development of functional DS circuits.