Intracellular signaling underlying neurite growth in adult sensory neurons

Jones, David M. (2003) Intracellular signaling underlying neurite growth in adult sensory neurons. Masters thesis, Memorial University of Newfoundland.

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Abstract

Regeneration in the peripheral nervous system (PNS) is relatively successful compared to that in the central nervous system. A major reason for this may be the permissive growth environment. In the PNS, a variety of factors contribute to promote neurite regeneration. These can be divided into two general categories i) trophic factors released by their targets, or supporting cells like Schwann cells and ii) components of the extracellular matrix (ECM). Generally, these factors can influence neurite growth by binding cell surface receptors and subsequent activation of intracellular signaling cascades. -- I have employed a tissue culture system of adult rat dorsal root ganglion (DRG) neurons to study the contribution of trophic factors (specifically NGF and IGF-1) and the ECM protein, laminin, in promoting neurite growth. Using computer-assisted analysis of neurite growth and analysis of protein expression and post-translational modifications with Western blotting, I have attempted to correlate the biological response (eg. neurite growth) with the biochemical response (signaling intermediates and activation). Through the use of pharmacological intervention, I have further tried to characterize the requirement for specific signaling cascades in the growth response. -- In Ch. Ill, further evaluation of this growth synergism with neuron tracing revealed that having both NGF and IGF (N+I) in the environment of regenerating neurites does not increase the number of neurons with neurites (neuritogenesis) but does enhance neuronal elongation and neurite branching above that seen with either growth factor alone. This synergistic effect correlates with the activation of the PI3- K>Akt>GSK-3 pathway more so than with the ras>Raf>MEK>ERK pathway. Pharmacological inhibitors of PI3-K (LY294002), GSK (LiCl), and ERK activity (U0126) confirmed the importance of the PI3-K>Akt>GSK-3 pathway. These experiments suggested that NGF and IGF signaling converge on the PI3-K>Akt>GSK-3 pathway to exert their growth synergism. To complement these experiments, I examined the biochemistry of two possible downstream effector proteins of both these signaling pathways; the microtubule associated protein tau, and the transcription factor cyclic adenosine monophosphate response element binding protein (CREB). Phosphorylation of tau appears to correlate with growth. -- In Ch. IV, preliminary data on the contribution that laminin has on NGF- dependent neurite growth was collected. The contribution of ECM components to neurite growth is well recognized. These experiments demonstrated that laminin can enhance NGF-dependent neuritogenesis. Like growth factors, the growth contribution of laminin is, at least, in part mediated by activating specific signal transduction cascades. The pi integrin subunit, paxillin, and FAK were phosphorylated when grown on laminin as compared to a polylysine substrate. However these integrin signaling proteins were not further phosphorylated by NGF when cultures were grown on laminin. The enhanced NGF-dependent neuritogenesis by laminin is however associated with decreased and increased activation of GSK-3 and ERK, respectively. This correlation suggests a convergence of both integrin and NGF signaling on the PI3-K>Akt>GSK-3 pathway. -- Taken together these results provide further evidence to the notion that many different factors are required for maximal neurite regeneration. In addition they suggest that signaling convergence upon the PI3-K>Akt>GSK-3 pathway and ras>Raf>MEK>ERK pathway underlies environmental factor growth synergism.

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