Nerve growth factor (NGF) is a target-derived polypeptide that acts through its receptor, trkA, to serve as a trophic factor for neurons in the peripheral nervous system (PNS) and central nervous system (CNS). The NGF signal must be transported retrogradely in axons from the target of neurons to their cell bodies. The molecular and cellular basis of retrograde signalling is unknown. Preliminary data suggests that activated trkA receptors in intracellular vesicles could be the signalling entity. We propose to test the hypothesis: that intracellular signalling of activated trkA in vesicles provides the means by which the NGF signal is carried from axon tips to the cell body.
Four Specific Aims are proposed. In the first, we will define membrane trafficking of NGF receptors in PC12 cells. Biotinylation will be used to define the rates for constitutive and ligand-induced internalization of trkA and p75. Using trkA mutants, we will characterize the relationship between signal transduction and endocytosis. We will isolate coated vesicles and determine whether trkA is internalized via a clathrin-coated pit mechanism. In the second Aim, we will characterize activated trkA in intracellular vesicles. We will define conditions for producing vesicles in which trkA tyrosine phosphorylation and trkA kinase activity are optimized. The signalling proteins associated with activated trkA in vesicles will also be defined.
In Aim 3 we will test the ability of activated trkA-containing vesicles to signal in vitro and in vivo. In vitro studies will examine phosphorylation of signalling intermediates (e.g. PLC-gamma, SHC) and activation of kinase activities (e.g. ERK1). In in vivo studies we will microinject naive PC12 cells and assess induction of immediate early gene expression, neurite outgrowth and cellular hypertrophy.
In Aim 4 we will use the Campenot compartmented culture system to examine retrograde signalling in sympathetic neurons. Using biochemical and morphological criteria, we will characterize retrograde transport of NGF, the NGF signal and activated trkA to establish whether or not they are identical in their kinetics and susceptibility to colcimid block. The proposed studies are expected to provide important new insights into how it is that neurons and their targets communicate. Through elucidation of signalling molecules and mechanisms, our studies may make possible novel opportunities for understanding and treating disorders of the nervous system, including Alzheimer's disease.
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