Diane M. Snow - US grants

Proteoglycans (PGs) are a structurally diverse class of molecules that interact with many extracellular matrix (ECM) and cell surface components. PGs play a role in many cellular processes, including inhibition of retinal ganglion and dorsal root ganglion cell outgrowth. The mechanisms by which PGs inhibit neurite outgrowth are unknown. Therefore, the goal of this proposal is to investigate the hypothesis that PGs may inhibit growth cone migration by two fundamental mechanisms: 1) limiting access to growth promoting adhesive molecules, and 2) triggering the reorganization of the growth cone cytoskeleton through transient rises in [Ca2+]. First, we will use 35S-CSPG and 3H-laminin to determine the amounts of these molecules bound to the substrata in DRG cultures. We will then do behavioral assays to determine whether PGs limit cell surface access to the growth-promoting adhesive molecule laminin, and to determine whether such masking can lead to growth cone turning at a PG border. Second, we will determine if there are changes in the cytoskeletal proteins tubulin and actin, or in related proteins, as growth cones contact and are inhibited by PGs. We will use injection of fluorescent actin and tubulin, or immunocytochemistry of fixed cultures, and treatment with cytochalasin to inactivate growth cone filopodia. Third, contact with PGs induces a large rise in [Ca2+]i within growth cones. Therefore, we will determine the role of the transient elevation of [Ca2+]i in growth cones that contact CSPG, and test whether changes in [Ca2+]i can induce inhibition of growth cone migration. Fluorescence imaging of the calcium indicator fura-2/AM will be used in combination with pharmacological reagents to block the influx or release of Ca2+. Elevation of [Ca2+]i may affect the morphology and trajectory of the elongating growth cone, 1) through direct action on cytoskeletal components of the growth cone, or 2) through second messenger systems that regulate the dynamic motile apparatus of growth cones. These experiments are important because: 1) they will lead to an understanding of the mechanisms by which PGs regulate growth cone guidance, and 2) they may provide molecular methods by which to manipulate PGs in adult tissue to enable CNS regeneration, since PGs are expressed and are inhibitory to regenerating nerve cells, following CNS injury.

DESCRIPTION (provided by applicant): Cocaine use during pregnancy is associated with neurobehavioral abnormalities in offspring that include alterations in attentiveness. Understanding the mechanisms by which maternal cocaine abuse adversely affects the developing fetus is critical for the development of appropriate treatments and intervention strategies. The actions of cocaine stem from nonselective inhibition of catecholaminergic neurotransmitter reuptake systems. New evidence shows that norepinephrine (NE) cell bodies within the locus coeruleus (LC), but not dopamine cell bodies with the substantia nigra, are affected following prenatal cocaine exposure using a clinically relevant model. This, combined with previous data showing that cocaine affects LC neuron behavior, give strong reason to pursue this system rigorously to refine previously held hypotheses and to determine new mechanisms for the deleterious effects of prenatal cocaine at the cell biological level. This study focuses on the LC neurons and one target, the hippocampus (limbic system), in rats prenatally exposed to cocaine in vivo and in vitro. Our hypothesis is: maternal cocaine use during a critical period of pregnancy causes selective alterations in the central noradrenergic system evidenced, in part, by effects on neuronal development such as neurite initiation, directed process outgrowth, appropriate neuronal pathfinding, and connectivity. We will test this hypothesis by first, determining effects of maternal cocaine exposure on the noradrenergic cells of the fetal rat LC using a well-established, clinically relevant model for fetal cocaine exposure, followed by analysis during critical outgrowth periods of neurite initiation, the rate of neurite outgrowth, total neurite length, growth cone behaviors (e.g. pathfinding), and characteristics of target (hippocampus) selectivity and connectivity. Second, we will determine effects of physiologically relevant concentrations of cocaine in tissue culture on the noradrenergic cells of the fetal rat LC, by analyzing neurite initiation, the rate of neurite extension, total neurite length, neuronal growth cone behaviors (e.g. pathfinding), and characteristics of target (hippocampus) selectivity and connectivity. The long term goal of this study is to uncover mechanisms of the adverse effects of prenatal cocaine on developing nerve cells that lead to inappropriate connectivity and impaired function. This study represents a novel and innovative approach toward an understanding of the basic cell biology of noradrenergic neurons as they respond to cocaine during development, and may ultimately permit the development of effective pharmacotherapeutic interventions for the treatment of cocaine-exposed offspring.

DESCRIPTION (provided by applicant): In the adult mammalian brain and spinal cord, neuronal injury results in failed regeneration, in part due to the upregulation of chondroitin sulfate proteoglycans (CSPGs). The majority of CSPGs originate from reactive astrocytes of the glial scar surrounding the lesion. The glial scar is beneficial for the recovering nervous system and should not be "excised", but axonal growth could be promoted if the expression of specific inhibitory portions of CSPGs could be targeted selectively. Our previous data indicate that neuronal inhibition is due to specific CSPG microheterogeneities and to the specific configurations of CSPGs predicted by their structure and by the molecules to which they bind. Our goal is to identify the most significant of the CSPG motifs with respect to neurite inhibition and regeneration, and specifically, to manipulate these moieties to promote regeneration. To this end, we and our collaborators have engineered 1) an array of CSPG isoforms and mutants we call "Designer PCs", 2) a variety of unique bioassays to express CSPGs, including a novel model of the glial scar in vitro, and 3) imaging methods to measure subtle features of nerve responses to CSPGs. The hypothesis of this proposal is: Identification and manipulation of specific inhibitory CSPG motifs using designer PGs and novel models of the glial scar will promote plasticity and regeneration in vitro and in vivo. We propose three Specific Aims that represent independent but interrelated studies to test this hypothesis. One, we will determine the expression and relative abundance of specific, inhibitory CSPG types and posttranslational modifications or core protein domains by reactive astrocytes in vitro. Two, we will determine the inhibitory potential for specific CSPG posttranslational modifications or core protein domains by establishing an inhibitory quotient to evaluate the responses of adult neurons in vitro. Three, we will manipulate specific inhibitory moieties of CSPGs to promote neuronal regeneration of adult neurons in vivo. The long term goal of this study is to identify the mechanism(s) of CSPG-induced inhibition following brain and spinal cord injury. The significance of the studies lies in the tremendous potential for translational application through the manipulation of specific CSPG motifs in injured patients, providing a therapeutic avenue to stimulate neuronal plasticity, facilitate reconnectivity of injured neurons, and accomplish restoration of function.

Project Summary Neurotrauma is the largest cause of death and disability for persons under the age of 45 in the world and the societal cost of the resulting disability exceeds $60 billion per year in the United States alone. The National Neurotrauma Society (NNS) annual symposium, now in its 34th year, is the primary forum for the exchange of information in the fields of traumatic brain injury (TBI) and spinal cord injury (SCI), and a means of integrating new information regarding the pathophysiology and treatment of TBI and SCI. This application seeks funding to support 20 pre- and postdoctoral trainees to attend the Symposium, as well as partial support for travel of 102 invited speakers (which will be distributed equitably by consensus of the Program Planning Committee.) The meeting will be held in Lexington, KY at the downtown Lexington Convention Center and Hyatt. The 4-day meeting will focus on important and timely topics in the field of neurotrauma, including basic, translational, and clinical investigations. Given that Lexington, KY is the horse capital of the world, and given the focus of the meeting, the conference is entitled: ?The Triple Crown: Advances in Basic Sciences, Pre-clinical Modeling, and Clinical Approaches.? For the 6th year in a row, the 2016 meeting will be co-hosted by the NNS and the AANS/CNS Joint Section on Neurotrauma and Critical Care (AANAS/CNS JSNCC). Members from both societies are included on the Program Planning Committee to ensure a sufficient mix of topics, targeting both TBI and SCI, and both basic science and clinical research. This arrangement successfully closes the gap by bringing together clinicians and basic scientists to network, share concerns, and create solutions. An exciting collection of state-of-the-art sessions on the consequences of damage to the nervous system and treatment strategies for protection and repair are planned ? some that get attention annually and others that are novel and innovative. Plenary session topics include: discussions by world experts on concussion and on clinical trial design, the acute cellular pathophysiology of neurotrauma, engineering approaches for functional restoration after SCI, advances, challenges and opportunities in CNS regeneration, a primer on imaging from animals to humans, big data approaches to neurotrauma, and emerging therapeutics for neurotrauma. Symposia or break-out sessions will cover a wide variety of other important topics that will be of great interest to scientists and clinicians. Further, specialty events include: a trainee Data Blitz, a discussion on careers in science, a Patient's Perspective segment, networking and team-building exercises for trainees, and an interactive project on the lifecycle of the society to promote attendee interaction and involvement.