Signalling pathways involved in the inhibition and promotion of axonal regeneration

Axonal regeneration following nerve injury requires the complex orchestration of various molecular events. Neurite outgrowth can be initiated by a variety of cues from the extracellular environment, including neurotrophins (e.g. nerve growth factor; NGF) and the extracellular matrix (ECM). Biological responses to neurotrophins are mediated by two distinct receptors: Trks, which initiate distinct signalling for the promotion of growth and survival, and p75NTR. Signalling pathways initiated by p75NTR exhibit considerable complexity and can elicit a variety of paradoxical physiological responses depending upon cellular context. Biological responses to ECM components, including laminin (LN), are mediated by a group of receptors known as integrins, which facilitate signal transmission to regulate cellular behaviour. Signalling pathways initiated by NGF and LN, have been previously reported to synergize resulting in optimized axonal regeneration of sensory neurons. The present series of studies sought to explore the molecular mechanisms underlying the enhanced growth initiated by stimulation with neurotrophins and LN, with a particular focus on the roles of the NGF receptors; specifically, TrkA phosphorylation-induced signalling cascades, and events associated with p75NTR ligand-dependent and independent signalling. -- My initial studies utilized a series of PC 12 cell derivatives expressing TrkA phosphorylation mutants, to investigate the potential role of TrkA in the regulation of p75NTR expression. I determined that TrkA played a role in the regulation of constitutive p75NTR expression, and further, controlled the upregulation of p75NTR in response to neurotrophin stimulation. In a subsequent study, I demonstrated that this occurred via a Phospholipase C γ-Protein Kinase C δ-dependent mechanism, and confirmed the existence of this regulatory pathway in cerebellar granule neurons (CGN). I further investigated the contribution of the ECM to regenerative growth, in both its capacity to signal synergistically with the TrkA receptor for the enhancement of early signalling intermediates, and its ability to elicit growth in a neurotrophin-independent scenario. Strikingly, integrin activation in the absence of neurotrophins was responsible for the promotion of neurite outgrowth via a rapid and potent Egr-1-dependent increase in the expression of the phosphatase PTEN, which relocalized to the nucleus where it dephosphorylated transcription factor Spl, thereby decreasing its ability to bind to the p75NTR promoter, resulting in the subsequent downregulation of p75NTR and depression of Rho activity. This novel ECM-induced signalling paradigm was also determined to occur in CGNs, and following the development of a unique motility assay, I demonstrated that interference with this cascade impaired motility, suggesting that this signalling cascade may contribute to the developmental migration of CGNs. -- p75NTR is a unique and flexible pleiotropic receptor which may promote or inhibit cell growth depending upon the presence or absence of neurotrophins. Taken together, the results of these studies detail the mechanisms involved in the upregulation of p75NTR expression in the presence of neurotrophins, but additionally present a novel signalling paradigm initiated by the ECM for the downregulation of the p75NTR in the absence of neurotrophin stimulation. Interestingly, both scenarios result in the promotion of neurite outgrowth and cellular motility as a result of the flexible signalling interactions of p75NTR, particularly those involving Rho, which can directly influence cytoskeletal dynamics