Promoting CNS regeneration by targeting cell signalling

Injured nerve processes, called axons, cannot usually regrow in the central nervous system (CNS). We are trying to find ways to encourage repair in individuals that suffer brain or spinal cord injury by studying signalling molecules in nerve cells that are important for axon growth.

Axon regeneration is actively prevented by a variety of proteins made by surrounding cells. An axon responds to these proteins using receptors on its surface that set off a chain of signals within. Axon behaviour can be radically changed using drugs to alter the activities of these signalling molecules.

We want to improve our understanding of the functions of one very important signalling molecule, called cyclic AMP, which can enhance axon regeneration when activated. We can study its activity and the functions of proteins that it activates in living rat nerve cells in culture using the most modern cell imaging techniques. We can therefore monitor the events that accompany axon contact with tissues, cells and proteins that they encounter in vivo, as well as the effects of using drugs to alter cyclic AMP activity. We aim to provide information that will lead to new approaches to promoting CNS repair in humans.

Injured axons in the adult mammalian central nervous system (CNS) fail to regenerate, due to a robust inhibitory extracellular environment and developmental changes in the regulation of intrinsic axonal signalling mechanisms. Appropriate manipulation of some signalling molecules can overcome growth cone collapse and repulsion in vitro, and promote some axon regeneration in vivo. However, little is known of the consequences of manipulating key signalling molecules within the growth cone or in the surrounding cells in vivo, because neither the activities of these molecules nor the cellular responses to their manipulation have been adequately quantified. Thus the potential of such a strategy to promote nerve repair remains to be properly investigated. A major focus of this proposal is to image cAMP activity in live growth cones by fluorescence resonance energy transfer (FRET), using tissue culture models to test the hypothesis that manipulating growth cone signalling mechanisms could overcome axon growth inhibition. Growth cone behaviour will be studied on contact with CNS glial cells or appropriate molecular boundaries. Specifically, a number of signalling molecules will be modified, including cyclic nucleotides, Ca2+ and members of the Rho family of small GTPases that mediate growth cone extension and collapse. The proposal will also focus on the roles of the cAMP targets PKA and Epac, a guanine exchange factor that is activated by cAMP independently of protein kinase A. Data will be quantitated by immunoreactivity, Western blotting, calcium imaging, FRET and timelapse microscopy. These studies will considerably enhance our knowledge of the signalling mechanisms that underlie a growth cone?s contact with its molecular and cellular environment, and inhibitory barriers specifically. They will also reveal how growth cone signalling changes during development, and what signals might be best targeted to overcome the inhibition of axon regeneration, providing new strategies for promoting CNS repair in vivo.