Timothy Gomez - US grants

calcium ion; spinal cord; neuronal guidance; growth cones; neurotransmitter receptor; neurogenesis; confocal scanning microscopy; fluorescent dye /probe; Xenopus;

DESCRIPTION (provided by applicant): The development of the nervous system requires the proper differentiation, migration and morphogenesis of neurons. The morphogenesis of individual neurons and the assembly of the trillions of neuronal connections that compose the human nervous system occurs through guided extension of axons and dendrites. The long-term objective of our research is to better understand the intracellular signaling cascades and effector mechanisms that are responsible for axon outgrowth and guidance in the developing brain. For this we must understand how nerve growth cones detect, integrate and respond to soluble, as well as cell- and substratum-associated guidance molecules in their environment. Mutations in genes involved in the detection and transduction of axon guidance information into directed neurite outgrowth are likely responsible for many deficits in cognitive function, including autisms, dyslexias, psychological disorders and mental retardations. Axon extension proceeds through a sequential process that involves leading edge membrane protrusion driven by actin polymerization, followed by adhesion and protrusion stabilization. New protrusions that do not adhere are retracted, as do existing protrusions that de-adhere. While extensive research has focused on the signals that control membrane protrusion and retraction, surprisingly little is known about the regulation of adhesion. Stabilization of growth cone protrusions to extracellular matrix (ECM) ligands occurs at specialized adhesion sites called point contacts. Point contacts are macromolecular complexes, containing both structure and signaling proteins, which link the cytoskeleton to the ECM through transmembrane integrin receptors. Our research is focused on understanding the molecular signaling events that control point contact assembly, maturation and disassembly and how axon guidance cues influence these processes to control axon pathfinding. Importantly, our evidence suggests that growth promoting axon guidance cues stimulate point contact assembly and turnover, while inhibitory cues slow the assembly of new point contacts and reduce turnover. We hypothesize that guidance of axons to their proper targets and stabilization of synaptic contacts requires modulation of integrin-dependent point contacts. We will test this hypothesis using a variety of approaches and model systems in three specific aims. In Aim 1, we will examine the role of Focal Adhesion Kinase (FAK) in the control of adhesion dynamics, veil protrusion and phosphotyrosine signaling at filopodial tips in response to axon guidance cues. In Aim 2, we will examine the role of p21-Activated Kinase (PAK) proteins in the regulation of integrin-dependent adhesion, cellular protrusion and axon outgrowth. Finally, in Aim 3, we will determine the role of adhesion site dynamics in axon guidance at several choice point in both Xenopus and zebrafish embryos. PUBLIC HEALTH RELEVANCE: The development of a functional nervous system requires accurate guidance of axons and dendrites to their target locations and establishment of synaptic connections. This proposal is focused on understanding how axon guidance cues regulate axon outgrowth by modulating integrin-dependent adhesions. As a number of cognitive disorders result from improper axon pathfinding, understanding the molecular basis for normal neural development is essential for designing therapeutic interventions.

During neural development motile growth cones at the leading edge of elongating axons and dendrites use environmental cues to navigate to their synaptic targets. While many diffusible, cell surface and extracellular matrix bound guidance cues and their receptors have been identified, relatively little is known of the downstream intracellular signal transduction cascades they activate. One intracellular signal that has particularly diverse effects on axonal and dendritic growth is cytosolic calcium ions (Ca2+). Transient elevations of intracellular Ca2+ in growth cones can promote, inhibit or orient motility depending on the size, duration and distribution of these signals, as well as on the downstream Ca2+-dependent targets available. Interestingly, the mode of Ca2+ entry has also been found to determine the specificity of some responses. The diverse effects of Ca2+ on neurite outgrowth are likely due to the many cytoskeletal regulatory targets expressed by growth cones. Importantly, recent evidence suggests that Ca2+ signaling may influence the activity of some of the Rho-family of small G proteins. Rho-family GTPases are potent and diverse regulators of the actin cytoskeleton in growth cones and could be key intermediaries between Ca2+ signals and growth cone motility. Dr. Gomez hypothesizes that Ca2+ influx through plasma membrane channels versus release from intracellular receptor stores regulate distinct subsets of Ca2+-sensors within local microdomains of growth cones. Further, the downstream signaling pathways activated by Ca2+ movement through different channels will have opposing effects on growth cone motility. The aim of this study is to begin by characterizing the effects of altering general and specific Ca2+ influx and release pathways on neurite outgrowth. Next, the effects of these Ca2+ changes on the activity of three different Rho GTPases will be measured and the necessity of GTPase function for motility determined. The long-term goal of Dr. Gomez's research is to understand how specific Ca2+ signals orchestrate the spatial and temporal activation of downstream targets that control growth cone pathfinding. Knowing the molecular mechanisms through which Ca2+ exerts such varied effects on growth cone motility will support treatment strategies for developmental disorders and neural injury.
Five graduate students from three different graduate programs and two undergraduates are current members of the Gomez Lab. The proposed project will directly involve two graduate students, but all students will receive some indirect support from this award. Students in the lab are trained as developmental neurobiologists using molecular and biophotonic based approaches. Student participation in research, as well as coursework, journal clubs, lab meetings, graduate program subgroup meetings, and international meetings reflect this focus. In addition to receiving training, graduate students are expected to teach junior graduate students, undergraduates or in some cases high school students. Several students are currently involved in University organized teaching programs. Dr. Gomez is also an active member of the Carnegie Initiative on the Doctorate (CID) planning committee. If awarded to the Neuroscience Training Program, CID will be a multi-year research program aimed at enriching and invigorating the education of doctoral students. Finally, as a member of the Biophotonics cluster hire, Dr. Gomez collaborates with labs in other departments such as Physiology and Electrical and Computer Engineering. These collaborations focus on technology transfer and as indicated by this agreement the Gomez lab provides imaging expertise as well as access to the lab's modern imaging facilities.

DESCRIPTION (provided by applicant): The development of the nervous system requires the proper differentiation, migration and morphogenesis of neurons. The morphological differentiation of individual neurons and assembly of the trillions of neuronal connections that compose the human nervous system occurs through guided extension of axons and dendrites. Molecular guidance cues in the environment of developing neurons guide neuronal growth cones at the tips of extending axons and dendrites. mTOR-mediated local synthesis of new proteins within growth cones as emerged as an important mechanism that controls axon guidance. Mutations in genes involved local protein synthesis are responsible for several human autism spectrum disorders, including Fragile X syndrome and Tuberous Sclerosis Complex (TSC). While modulation of mTOR-dependent protein synthesis is known to be required downstream of both attractive and repulsive axon guidance in several animal model systems, it is unknown if similar mechanisms function in developing human neurons. This proposal will first test whether human Retinal Ganglion Cells (RGCs) derived from human induced pluripotent stem cells (hiPSCs) use mTOR-mediated protein synthesis to respond to positive and negative axon guidance cues. In Aim 2 we will test the role of the TSC1/TSC2 complex, which is a key upstream negative regulator of mTOR. For this we will generate new lines of hiPSCs by reprograming fibroblast cells from patients with TSC. While this proposal will focus on RGCs, a wide variety of other cell and neuronal types can be studied using these new hiPSC lines. Therefore, these important new cell lines will be a valuable resource for many investigators and will be made available for distribution through WiCell. In Aim 3 we will test whether RGCs derived from TSC hiPSCs exhibit abnormal response to axon guidance cues tested in Aim 1.

DESCRIPTION (provided by applicant): The development of the nervous system requires the proper differentiation, migration, morphogenesis and maturation of neurons. The morphological differentiation of individual neurons and assembly of the trillions of neuronal connections that compose the human nervous system occurs through guided extension of axons and dendrites. The long-term objective of our research is to better understand the intracellular signaling cascades and effector mechanisms that are responsible for axon outgrowth and guidance in the developing brain. For this we must understand how nerve growth cones detect, integrate and respond to soluble, as well as cell- and substratum-associated guidance molecules in their environment. Mutations in genes involved in the detection and transduction of axon guidance information into directed neurite outgrowth are responsible for many neuro- developmental disorders, including autisms, dyslexias, psychological disorders and cognitive deficits. Therefore, our work aimed at better understanding the molecular basis of normal neural development, may help inform treatments for conditions leading to abnormal neural network assembly. While extensive studies have investigated the molecular mechanisms that regulate axon guidance over two- dimensional substrata in vitro or along axonal tracks in vivo, little is known about the signals that control axon guidance across three dimensional tissues. Our preliminary data suggest that along with planar filopodia and lamellipodia, growth cones generate orthogonal protrusions in vitro and in vivo that resemble podosomes or invadopodia. Podosomes and invadopodia, collectively referred to as invadosomes, are actin-based cellular protrusions associated with extracellular matrix (ECM) degradation and tissue invasion. We hypothesize that growth cone invadosomes function to actively detect ligands through receptor interactions that regulate actin polymerization and participate in ligand and receptor degradation to modulate ligand-mediated guidance. The three aims of this proposal will use a series of molecular gain of function and loss of function manipulations, together with super resolution three dimensional fluorescence imaging, to assess the signals that control invadosome formation and their roles in controlling neural network formation.

Project Summary The development of the nervous system requires the proper differentiation, migration, morphogenesis and maturation of neurons. The morphological differentiation of individual neurons and assembly of the trillions of neuronal connections that compose the human nervous system occurs through guided extension of axons and dendrites. The long-term objective of our research is to better understand the intracellular signaling cascades and effector mechanisms that are responsible for axon outgrowth and guidance in the developing brain. For this we must understand how nerve growth cones detect, integrate and respond to soluble, as well as cell- and substratum-associated guidance molecules in their environment. Mutations in genes involved in the detection and transduction of axon guidance information into directed neurite outgrowth are responsible for many neuro- developmental disorders, including autisms, dyslexias, psychological disorders and cognitive deficits. Therefore, our work aimed at better understanding the molecular basis of normal neural development, may help inform treatments for conditions leading to abnormal neural network assembly. While extensive studies have investigated the molecular mechanisms that regulate axon guidance over two- dimensional substrata in vitro or along axonal tracks in vivo, little is known about the signals that control axon guidance across three-dimensional tissues. Our preliminary and recently published data suggest that along with planar filopodia and lamellipodia, growth cones generate orthogonal protrusions in vitro and in vivo that resemble podosomes or invadopodia. Podosomes and invadopodia, collectively referred to as invadosomes, are actin-based cellular protrusions associated with extracellular matrix (ECM) degradation and tissue invasion. We hypothesize that growth cone invadosomes function to actively detect ligands through receptor interactions that regulate actin polymerization and participate in ligand and receptor degradation to modulate ligand-mediated guidance. The three aims of this proposal will use a series of molecular gain of function and loss of function manipulations, together with super resolution three-dimensional fluorescence imaging, to assess the signals that control invadosome formation and their roles in controlling neural network formation.

Growing evidence suggests that patients with Tuberous Sclerosis Complex (TSC), like other neuro-developmental disorders, have mis-wiring of neuronal connections that form during development. These defects in neuronal connectivity likely contribute to symptoms of TSC, such as cognitive deficits, autism and epilepsy. However, defective axon guidance by human neurons has only been suggested from brain imaging studies, as models to study the molecular basis for mis-guidance of developing human neurons have not been developed. To directly address these fundamental questions, we will study the development of human neurons that we differentiate from human induced pluripotent stem cells (hiPSCs) from TSC patient-derived cells and their genetically engineered counterparts. Using a series of cell behavior and molecular signaling assays, we will compare TSC2 mutant neurons with their gene-corrected, isogenic control neurons both in vitro and within 3D forebrain spheroids. We will examine changes in mTORC1 and mTORC2 signaling pathways in TSC2 mutant neurons to determine the relative contributions of each signaling pathway to neuronal development. While modulation of mTOR-dependent protein synthesis has been suggested to be required downstream of both attractive and repulsive axon guidance in several animal model systems, it is unknown if similar mechanisms function in developing human neurons. Our surprising preliminary data suggest that TSC2 functions independent of mTOR in growth cones to directly regulate the cytoskeleton to control axon guidance. In this proposal, we will determine how loss of TSC2 function alters the development of human forebrain neurons, with a current focus on axon extension and sensitivity to key axon guidance cues, two important cellular consequences of abnormal TSC2 function. We will determine the molecular mechanisms downstream of TSC2 and test functionally how these signaling pathway contribute to abnormal axon extension and guidance cue responses. Over the long term, we believe our research may help identify key druggable targets in patients with TSC.