Rosenberg, L.H.; (2010) Ras/Raf/ERK signalling and Schwann cell dedifferentiation. Doctoral thesis , UCL (University College London).

Abstract

Schwann cells are a highly specialized cell type whose function is to ensheathe and myelinate axons of the peripheral nervous system (PNS). Remarkably, Schwann cells retain the capacity to regenerate throughout the lifespan of an animal, responding to peripheral nervous system damage by dedifferentiating and reverting to a proliferating, progenitor like state. Following successful axon outgrowth, dedifferentiated Schwann cells re-differentiate, thereby reconstituting a repaired and functional nerve. Previously, we have shown that the Ras/Raf/ERK pathway is sufficient to drive Schwann cell dedifferentiation and that damage to peripheral nerves results in strong and sustained activation of ERK in Schwann cells. This data suggests an important physiological role of Ras/Raf/ERK signalling in controlling Schwann cell plasticity. The molecular mechanism by which this occurs is unknown. The transcription factor Krox-20 is required for both the initiation and the maintenance of the myelinated state. Loss of Krox-20 is sufficient to drive mature, differentiated Schwann cells towards the less differentiated, progenitor like state and decreased levels of Krox-20 are associated with damaged peripheral nerves. In this thesis I show that activation of Raf/ERK signalling in differentiated Schwann cells induces the rapid downregulation of Krox-20 and myelin associated mRNA expression. At the protein level however, Krox-20 persists past the point of myelin gene down-regulation both in vitro following activation of Raf, and in vivo in response to peripheral nerve damage. Moreover, constitutive expression of Krox-20 does not abolish the ability of Raf/ERK signalling to downregulate myelinassociated genes. Schwann cell dedifferentiation therefore occurs independently of Krox-20 protein loss. Further work shows that Raf/ERK signalling acts to block Krox- 20 dependent activation of myelin-associated genes and that this occurs via negative regulation of myelin gene promoters. To address the mechanism by which Raf/ERK signalling blocks Krox-20 dependent activity, I have studied the role of a number of candidate transcription factors, including the immature-Schwann cell associated factors cJun and Sox-2. Interestingly, activation of Raf is sufficient to drive the expression of these factors in differentiated Schwann cells. Nevertheless, further studies showed that neither cJun nor Sox-2 are required for Raf driven dedifferentiation. To further study the mechanism I have used microarray analysis to identify candidates whose expression changed following Raf activation. I have identified and examined the role of a number of transcription factors in this process.

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