Gianluca Gallo - US grants

DESCRIPTION (provided by applicant): Injury to the nervous system decreases the quality of life of the injured individual. Therefore, it is a priority to develop therapeutic approaches aimed at the amelioration and prevention of damage to the nervous system. The rational development of therapies requires a concrete knowledge of the response of neurons to injury. During injury, nerve fibers are often damaged and respond by retracting. The retraction of nerve fibers results in the loss of connectivity between neuronal populations that manifests as functional disabilities (e.g., loss of motor control and sensation). The retraction of nerve fibers in injured tissue is detrimental to nervous system function and occurs initially in response to direct physical damage, and subsequently in response to myelin-derived inhibitory signals that develop in response to injury. Therefore, it is important to understand the cellular mechanisms involved in nerve fiber retraction in order to develop rational therapeutic approaches. Three aims will be pursued to test the hypothesis that nerve fiber retraction in response to physical injury and myelin-derived inhibitory signals share a common mechanism requiring actomyosin-dependent contractility. Aim 1. Elucidate the role of the cytoskeleton in injury-induced nerve fiber retraction. The cytoskeleton of nerve fibers that have been severed will be studied to determine how it reorganizes in response to injury. The role of actin filaments and microtubules in injury-induced nerve fiber retraction will also be determined. Aim 2. Determine the role of actomyosin contractility in the retraction of severed nerve fibers. A number of techniques will be used to interfere with actomyosin function and study how these treatments affect the retraction of nerve fibers. The role of enzyme systems known to regulate actomyosin function will also be investigated. Aim 3. Determine the role of actomyosin contractility in the response of nerve fibers to myelin-derived inhibitory signals. Similar to aim 2, the effects of inhibiting actomyosin function on myelin-induced axon retraction will be investigated. These studies will use dorsal root ganglion neurons, a population of neurons that sends nerve fibers into the spinal cord. Damage to the nerve fibers of these neurons in the spinal cord can result in the loss of sensation and proprioception. The cellular basis of retraction in vitro will be studied using live video microscopy to directly determine the responses of nerve fibers. A number of methods will be used to inhibit the activity of the actomyosin system, and novel approaches to the inhibition of actomyosin activity will be developed. Collectively, these studies will determine whether the actomyosin system is a viable target for the development of therapies aimed at the inhibition of nerve fiber retraction in injured nervous tissue.

DESCRIPTION (provided by applicant): The generation of axon collateral branches is a fundamental aspect of the development of synaptic connectivity between neurons and their targets, and branching/sprouting is also involved in the recovery of the nervous system following injury. The protrusion of axonal filopodia is the first step in the initiation of a collateral branch. Filopodia that become invaded by axonal microtubules subsequently mature into collateral branches. In this proposal we seek to determine the mechanisms of the initiation of axonal filopodia and their maturation into nascent collateral branches. In the first period of support for this project, our studies have unveiled the earliest steps in the cytoskeletal dynamics underlying the formation of axonal filopodia. We demonstrated that axonal filopodia are formed from precursor structures that we term axonal F-actin patches. The competitive renewal application for this project is based on the novel observation of axonal microdomains of phosphoinositide 3-kinase (PI3K) activity that precede the formation of axonal filopodia, which is in turn dependent on PI3K activity. The studies we propose will advance the field by testing a specific hypothesis that merges the earliest signaling and cytoskeletal events underlying branch formation into a coherent model. Using in vitro and in vivo approaches we will test the hypothesis that localized axonal microdomains of PI3K activity orchestrate the formation of axonal F-actin patches through the recruitment and activity of the Rac1 GTPase, the F-actin nucleating system Arp2/3 and septin GTPases, resulting in the coordinated reorganization of the actin and microtubule cytoskeleton giving rise to collateral branches.

DESCRIPTION (provided by applicant): The goal of the proposed work is to determine the mechanisms of the early structural consequences of neural trauma and to develop strategies for acute intervention in traumatic brain injury (TBI) that address the primary mechanisms of axonal injury. The underlying hypothesis for this research is that initial mechanical trauma to the cell membranes leads to cytoskeletal disruptions and alterations of axonal transport and that acute intervention to restore membrane integrity and preserve axonal cytoskeleton and transport processes can dramatically reduce secondary degeneration and cell death. Specific Hypotheses to be tested are 1) Axonal cytoskeletal disruption and impaired axonal transport are causally related to membrane disruption, and acute repair of the axolemma by poloxamer P188 can prevent these effects; 2) JNK-3 activation is causally related to membrane damage, and axonal transport is, in part, impaired by the actions of JNK-3; and 3) Mild injury will be manifest in axonal pathology, the time course of which is modulated by injury severity. We have developed and in vitro model of focal axonal injury in primary chick forebrain (CFB) neurons that mimics many features observed in vivo. In particular, focal swelling, or axonal beads, appeared within one hour following the mechanical insult. Co-localized with the beads were focal disruptions of microtubules and the accumulation of membrane bound organelles indicating a disruption of axonal transport. We characterized the membrane damage as a result of the mechanical trauma and showed that treatment with Poloxamer 188 (P188), a water soluble, non-ionic surfactant restored membrane integrity and significantly inhibited axonal beading in CFB neurons. We also have an in vivo model that produces axonal injury in the deep white matter. In this model, we have demonstrated membrane damage, focal accumulation of amyloid precursor protein (APP), and focal activation of JNK-3. Significantly, injured JNK-3 deficient mice do not exhibit the severe cognitive deficits seen in age-matched WT littermates. The following Specific Aims were developed to test these hypotheses: Aim 1: To determine the causal relationship between mechanically-induced membrane damage and subsequent alterations of cytoskeletal structure and axonal transport and to test whether acute treatment with an agent that promotes membrane repair can preserve axonal structure and function and thereby prevent secondary degeneration. Aim 2: To determine the mechanism of focal activation of the MAP kinase, JNK-3, in injured axons and its role in axonal pathology and to test the effect of acute membrane repair on JNK activation. Aim 3: To determine the window of opportunity for therapeutic intervention for both membrane repair and inhibition of JNK-3. PUBLIC HEALTH RELEVANCE: Currently, acute care for trauma victims deals mainly with preserving or restoring basic life support systems, e.g., cardiac and respiratory function, and managing mass lesions in the brain to prevent death and/or further brain damage. This research, if successful, will provide the basis for a new approach to the treatment of traumatic brain injury in which early intervention to preserve the structural integrity of neurons will stave off the secondary degenerative processes that result in persistent neurological deficits. Successful completion of the proposed research will hopefully emphasize the importance of the early treatment of neuronal injury as an important therapeutic consideration in addition to the current focus on delayed treatments aimed at halting secondary degeneration.

DESCRIPTION (provided by applicant): The formation of axon collateral branches underlies the ability of neurons to make synaptic contacts with multiple target neurons and thus give rise to complex neuronal networks. In the context of nervous system injury, the formation of axon collateral branches can have either beneficial or undesired effects depending on the affected circuitry. Collateral branches are also affected by a variety of disease states. However, the cellular mechanisms of collateral branching are only minimally understood. The preliminary data presented in this proposal unveils for the first time that the branch-inducing signal nerve growth factor drives the intra-axonal synthesis of cytoskeletal proteins with roles in the formation of collateral branches. Importantly, the preliminary data also demonstrate that axonal protein synthesis is required for the induction of axon collateral branching by nerve growth factor. Although recent studies have revealed a large set of mRNAs targeted to axons, the functional roles of the axonal translation of axonal mRNAs remains minimally understood. The main aim of the proposal is to determine the roles of the axonal synthesis of individual cytoskeletal proteins in the formation of axon collateral branches. By bridging the expertise of the PI (Dr. Gallo; neuronal cytoskeletal cell biology) and the Co-I (Dr. Twiss; axonal protein synthesis), the project provides the unique opportunity to uncover a new aspect of the mechanism of axon branching and aims to directly link the axonal synthesis of specific cytoskeletal proteins to the regulation f the dynamics of the axonal cytoskeleton. Through the joint expertise of the PI and Co-I, the project takes a multi-pronged in vivo and in vitro approach to address the main Aims. Collateral branching is affected by nervous system injury and disease. However, the ability to manipulate branching in a therapeutic context is mostly lacking. The project has the potential to lead to strategies for the regulation of axon branching by targeting specific axonal mRNA species in the context of neuronal injury and disease. The reagents developed for use in the project (e.g., cell permeable tools to selectively inhibit the axonal translation of individual mRNA species) have the potential for translation to animal model system of collateral branching in future directions o the project. The work we propose will serve as the foundation for these long term goals by determining the relevant mRNA targets through the elucidation of their roles in branching.

PROJECT SUMMARY The branching of axons is a pivotal event in the development of neuronal circuitry and during the response of the nervous system to injury. We and others recently identified a crucial role for axonal mitochondria in axon branching. This proposal aims to advance the understanding of the role of mitochondria in axon branching and axonal physiology. The preliminary data unveil that nerve growth factor, a branch inducing signal, promotes the fission of axonal mitochondria which is in turn required for the ensuing branching. The PI3K signaling pathway is necessary and sufficient for the induction of fission by NGF. Aim 1 of this proposal aims to determine whether neurotrophins which induce branching in vivo also induce fission and the role of fission in in vivo branching, and further dissect the signaling mechanism used by NGF to drive the fission of axonal mitochondria. Aim 2 will address the role of the axonal cytoskeleton in the regulation of mitochondrial fission. Aim 3 will seek to determine how the fission of mitochondria contributes to axonal physiology and the mechanism of axon branching. Collectively, these aims have the potential to make impactful advances which will be of interest to a wide community of scientists.

The branching of axons is a fundamental aspect of neurodevelopment and the response of the nervous system to injury. This proposal seeks to understand the role of a protein called Sarm1 in developmental and injury induced axon branching. Sarm1 has been involved in axon degeneration following injury, but our preliminary data unveils a novel role for Sarm1 as a repressor of axon branching. The project has two Aims focused on elucidating how Sarm1 impacts the cytoskeleton underlying branching, and whether Sarm1 is involved in injury-induced axon branching. Completion of the project will advance our understanding of the mechanism of axon branching and identify a novel role for Sarm1 in the response of the nervous system to injury.

Axon extension, retraction and regeneration are energy dependent processes. ATP is the main form of cellular energy and it is generated through oxidative phosphorylation, in the mitochondrion, and glycolysis. This project aims to pioneer investigations into the role of glycolysis in neuronal morphogenesis with emphasis on axon development. The project also addresses the role of glycolysis in the response of axons to injury. In both contexts, the project also aims to determine if axonally targeted mRNAs encoding glycolytic enzymes undergo axonal translation in response to extracellular signals and injury.