This grant addresses an anatomical problem in signal transduction that is unique to nerve cells. How do target-derived growth factors regulate events in the nerve cell body following presentation to nerve endings that can be meters away from that ultimate target? Conventional growth factor signal transduction pathways would seem inadequate to the challenge without special adaptations. Retrograde transport, though well documented for neurotrophins, is not a proven component of the signal transduction process. The goal of this research is to learn how target-derived neurotrophins transmit information through nerve axons. A key to our study plan is a panel of activation state-specific antibodies we've raised to high affinity neurotrophin receptors (Trks). These antibodies resolve and report both catalytic and signaling functions of Trks in vivo. We will draw upon the sensitivity and specificity of these antibodies to study signal transduction by focal sources of BDNF in two complementary systems - sciatic nerve, and cerebellar granule neurons grown in a compartmented culture system (Campenot chambers). Sciatic nerve axons are long enough to clearly separate the target, axon and cell body. Granule cells, which can be readily purified in large numbers, express high levels of the BDNF receptor Trk B and so are ideal for certain mechanistic studies. We've done extensive preliminary work on both of these BDNF-responsive nerve cell systems. We have shown that a holus of BDNF at nerve endings leads to phosphorylation of Trk B within axons - that the phosphate is at a functional tyrosine (the SHC binding site) - that the change in Trk B phosphorylation state propagates through the axon towards the cell body- and that it travels at a rate of at least 60 mm/hr. This rate of signal propagation is roughly five times faster than conventional retrograde vesicular transport and it raises the possibility that other mechanisms are involved in target derived signal transduction. Our study plan builds upon these preliminary observations. We have three objectives. Objective one is to determine whether phospho Trk B executes either of its two known functions while in transit through the axon. Bearing in mind that activated Trk B is both a tyrosine kinase and a """"""""platform"""""""" for assembly of the signal generating particle, our specific aims are to learn; i. whether phospho Trk B in the axons is catalytically active and ii. whether it is associated with signal generating proteins SHC, PLC- gamma and PI3 kinase. Objective two is to determine how the Trk B phosphorylation state changes propagate so quickly through the axon.
Specific aims are to learn; i. whether we are observing the transport of phospho Trk B itself; ii. whether propagation of the phospho Trk B signal is linked to retrograde vesicular transport. Objective three is to learn how phospho Trk B transduces a signal at the neuronal cell body.
Specific aims are to learn whether; i. the Ras/Raf signal generating apparatus is activated; ii, MAP kinase is activated and relocated into the nucleus and iii. immediate early genes are induced in response to BDNF.
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