The long-term objective of this research is to resolve an anatomical problem in signal transduction. How do myelinated axons signal to the nuclei of cells that enwrap them? The nuclei of oligodendrocytes (in the CNS) and Schwann cells (in the PNS) are segregated from the axon by multiple concentric layers of bimolecular lipid leaflet and myelin basic protein. Conventional cell signals and signal transduction pathways would seem inadequate to the challenge without special adaptations. Insights into these adaptations might translate into new therapies for dysmyelinating disease states (multiple sclerosis, Guillian-Barre), peripheral neuropathies due to cancer chemotherapy and nerve injury. ? ? A favored biological context to address signaling in myelinated axons is the Wallerian degeneration response of rodent sciatic nerve. In preliminary studies, we have shown that transmembrane receptor tyrosine kinase erbB2 is localized to paranodal loops and Schmidt-Lanterman incisures of myelinating Schwann cells at the point of contact with rat sciatic nerve axons. Transecting the sciatic nerve results in activation of erbB2 at these positions within 1 hr. Coincident with erbB2 activation, Akt is activated in the paranodal loops and """"""""immediate-early"""""""" genes are induced in the Schwann cell nuclei. The research described here draws upon these preliminary observations. We have three question-oriented specific aims: 1) Determine how erbB2 is initially targeted to the loop/incisure regions, 2) determine whether activated erbB2 has remote signaling functions in the Schwann cell nucleus and 3) determine the cellular fate of erbB2 following activation at the loop/incisure domains. ? ? Our study plan exploits a new system for preparation of artificial, myelinated sciatic nerves in vitro with the use of compartmentalized cell culture chambers (Campenot cultures). Damage to the axons of these synthetic nerves triggers a response that emulates Wallerian degeneration in axotomized rodents. However, the axons and Schwann cells in these compartmental cultures lend themselves to targeted genetic and pharmacological intervention. To resolve localization issues raised in aims 1 and 3, we will used a tetracycline-regulated retroviral vector to express epitope-tagged erbB2 in myelinating Schwann cells. To resolve signaling functions (aim 2), we will examine the """"""""gain-of-function"""""""" and """"""""loss-of-function"""""""" erbB2 signaling phenotypes in myelinating Schwann cells. The gain-of-function phenotype will be generated by transfecting myelinating Schwann cells with chimeric erbB2 receptor constructs that can be gratuitously activated by membrane-permeable, synthetic ligands. The loss-of-function phenotype will be produced by using new """"""""small interfering RNA"""""""" expression vectors to knockdown erbB2 protein in myelinating Schwann cells. ? ?
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