During peripheral nervous system development and regeneration, the axon dictates whether a Schwann cell differentiates into a myelin forming cell. In the event of injury, axonal breakdown signals Schwann cells to proliferate, repress myelin gene expression, and express cell adhesion molecules and neurotrophic factors. Our long term goal is to understand how extracellular signals orchestrate changes in the Schwann cell phenotype. In any cell, extracellular signals are known to activate a series of transcription factors which then regulate the expression of genes that ultimately define the phenotype. In preliminary studies we have identified four transcriptional-control genes that are expressed in Schwann cells at the onset of a change in phenotype. The immediate goals are to understand the role of these four factors in regulating Schwann cell properties. These genes were chosen on the basis of our preliminary data, which indicated that C-JUN and HLH462 (specific aim 1 and 2, respectively) play a role in the process of dedifferentiation, whereas AP- 2 and C/EBP (specific aim 3 and 4, respectively) are involved in differentiation to the myelin-forming phenotype. To ascertain a functional role for each of these genes, Schwann cells will be transfected with cDNA in the sense and antisense orientation, and expression will be regulated by either a constitutive, viral promoter or a synthetic, inducible promoter. Stable cell lines will be generated and studied with respect to their mitotic rate and expression of phenotypic markers. [3H] Thymidine incorporation and doubling times will be used as a index of proliferation. Indirect immunofluorescence staining, and Western and Northern blot analysis will be used to determine if expression of a gene leads to the display of phenotypic traits associated with either differentiation or dedifferentiation. Supporting evidence for a functional role of a gene will be provided if expression of the antisense prevents the response to mitogens or differentiating signals (agents that increase cAMP or neurites extended by dorsal root ganglia). In addition, the role of C-JUN and AP-2 in regulating transcription of the myelin P-O gene will be examined. Methods used for these studies include transient transfection, protein-DNA binding, in vitro transcription, and in vitro mutagenesis. Although several other genes will interplay in controlling the Schwann cell phenotype, we are attempting to dissect out the contribution of four individual genes. A greater understanding of how the Schwann cell phenotype is regulated may ultimately allow experimental manipulation, which could be of help in several medical areas. Schwann cell transplants are currently being studied for possible therapeutic use in spinal cord regeneration, in cografts with adrenal chromaffin cell for Parkinson's disease, and as a source of neurotrophic factors in neurodegenerative disease. Furthermore, detailed knowledge of the process of differentiation can ultimately prove useful in designing therapies for dysmyelinating and demyelinating diseases.
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