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Title: The role of cyclic nucleotide signalling pathways in axon growth and guidance
Author: Murray, Andrew J.
ISNI:       0000 0001 3435 2096
Awarding Body: University of Aberdeen
Current Institution: University of Aberdeen
Date of Award: 2007
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Finding an effective treatment to repair the damaged mammalian central nervous system (CNS) is one ofthe greatest challenges facing medical science. When damaged, CNS axons are prevented from regenerating by a combination ofenvironmental factors, such as those present on myelin and astrocytes. Neurons themselves though, also have a reduced capacity for regeneration when they mature. Embryonic neurons, for example, are able to regenerate, 'even when transplanted into the mature nervous system, that contains myelin and astrocytic inhibitors, but this ability is lost shortly after birth. One key difference between embryonic and adult neurons that contributes to the lack of regeneration in older cells, is levels ofthe intracellular second messenger, cyclic adenosine monophosphate (cAMP). cAMP levels decline in neurons as the nervous system matures and this correlates with the loss ofregenerative ability with age. Furthermore, artificial cAMP elevation promotes substantial axon regeneration in vivo, and is ofthe most promising strategies for repairing the damaged nervous systems This study has focussed on targeting cAMP and other signalling molecules to dissect the mechanisms ofaxon growth and guidance and find a more effective target for promoting axon regeneration. Firstly, we tested the strategy pfcAMP elevation in rat dorsal root ganglion (DRG) neurons with an in vitro model ofaxon regeneration. We found that cAMP elevation was effective in promoting regeneration only in early postnatal neurons, adult neuron regeneration was not increased with this treatment. However, targeting of another signalling molecule, cyclic guanosine monophosphate (cGMP) in combination with cAMP was effective at promoting adult axon regeneration. Furthermore,-this could be increased with the concurrent application ofa Nogo receptor blocking peptide, until regeneration comparable to that seen in embryonic neurons was achieved. We feel that this strategy represents an extremely promising approach for examining axon regeneration in vivo. Within cells cA1v1P has two direct targets, protein kinase A (pKA) and the exchange protein activated by cAMP (Epac). Previous studies have indicated that PKA controls cAMP-dependent ~on growth and regeneration. However, Epac has only recently been characterised and a possible role for Epac in axon growth and regeneration has not previously been examined. We have found for the first time that Epac plays a critical role in cAMP-dependent axon growth and regeneration. Selective activation ofEpac mimics cAMP's ability to promote neurite outgrowth and axon regeneration. Furthermore, we have shown that Epac is required for cAMP's ability to promote neurite outgrowth and mediate axon regeneration. This 'provides a target for inducing axon regeneration without altering the cAMP-PKA signalling pathway.. cAMP levels are known to be critical for growth cone responses to extracellular guidance cues. These cues are important during development where they guide an axon to its ultimate target, but some ofthese proteins continue to be expressed in the mature nervous system and contribute to an inhibitory environment after damage. In general high levels ofcAMP in growth cones are thought to mediate attraction to a group of guidance cues, whereas low ~AMP levels initiate repulsion. The exact mechanisms as to how cAMP controls both growth cone attraction and repulsion are not known, although previous studies have indicated that PKA controls both ofthese processes. In this study we have developed a growth cone turning assay that ~llows analysis ofgrowth con~ responses to extracellular gradients in rat DRG neurons. Using this assay along with cAMP analogues specific for Epac orPKA and small interfering RNA (siRNA) we examined growth cone turning to the well characterised guidance molecules, myelin associated glycoprotein (MAG) and netrin-I. We have found that cAMP controls growth cone repulsion and attraction by activating either ofits direct targets. cAMP acts upon Epac to initiate growth cone attraction to MAG or netrin-I, when Epac signalling is perturbed attraction to these molecules is switched to repulsion, and selective Epac activation mimics cAMP's ability to switch repulsion to attraction. Furthermore, growth cones are attracted to a gradient ofan Epac agonist in the same way as they are to cAMP analogues. cAMP acting on PKA controls growth cone repulsion, and repulsion from MAG and netrin-I is abolished when PKA signalling is perturbed, however growth cone attraction is not affected. Additionally, repulsion is observed when growth cones are presented with gradients ofcompounds that selectively activate PKA. In summary, in this work we have examined the signalling pathways that control axon growth and guidance with an aim to provide a future strategy for induCing CNS axon regeneration. For the first time we have shown that Epac plays an important role in cAMP-dependent axon growth, guidance and regeneration andalso that cAMP controls growth cone attraction and repulsion by activating its two direct intracellular targets. We feel that this represents a substantial development in the understanding ofaxon growth mechanisms and hope that it will lead to improved therapies for promoting repair in the damaged CNS.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available