The overall goal of the studies in the present proposal is to characterize neuronal molecules involved in neurite outgrowth with particular emphasis on proteases released by growing neurites. Various histological and electrophysiological studies have shown that the formation of the nervous system consists of a highly precise series of events that creates a complex interconnected network of synapses. However, the biochemical and molecular mechanisms involved in neurite outgrowth and synaptogenesis and the genetic programs regulating these events are not understood. The focus of the current application is to test hypotheses concerning the role of 3 neuronal proteases in neurite outgrowth. Each protease appears to be involved in neurite outgrowth; yet their mechanisms of action appear to uniquely different. OUr working hypothesis are that: 1) tissue plasminogen activator is released and binds to Thy-1 on the neuronal surface, resulting in an increase in intracellular Ca2+ which then alters growth cone dynamics and/or other functions, 2) the neuronal metalloproteinase is released from growing neurites to degrade components of the ECM and to open """"""""channels"""""""" within the ECM, and 3 urokinase plasminogen activator is released and binds t receptors on the bottom surface of the growth cone and alters adhesion between the growth cone and substratum by producing a small number of cleavages in neuronal or matrix proteins.
The specific aims of the present application are to: 1. Analyze the biochemical interactions and functional consequences of binding tissue plasminogen activator (tPA) to sympathetic neurons and its neuronal binding site, Thy-1. 2. Develop immunological and molecular probes for urokinase plasminogen activator (uPA) and use these probes to help define the role of uPA in neurite outgrowth. 3. Characterize the neuronal Ca2+ dependent metalloproteinase(s) (CDMP) and determine the consequences of inhibiting its activity on neurite outgrowth in vivo. The information that will be generated by these experiments is clinically important for understanding processes such as regeneration following traumatic or pathological injuries and for aiding recovery from diseases in which neurons and/or synaptic connections are lost.
