Promoting CNS regeneration by targeting cell signalling

Lead Research Organisation: University of Aberdeen
Department Name: Cal Unit (Medical Faculty)

Abstract

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.

Technical Summary

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.

Publications

10 25 50
 
Description The potential of combining Epac activation and novel silk-based biomaterial implants in promoting CNS axonal regeneration
Amount £90,993 (GBP)
Organisation International Spinal Research Trust (Spinal Research) 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2015 
End 09/2018
 
Description Uncovering new therapeutic targets for inhibiting vascular smooth muscle cell migration in cardiovascular disease
Amount £100,266 (GBP)
Organisation British Heart Foundation (BHF) 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2011 
End 04/2015
 
Title Advanced knowledge of cAMP signalling 
Description Discovery that cAMP mediates growth cone chemoattraction by signalling through Epac and chemorepulsion by signalling through PKA, answering a long-standing question of how cAMP can be involved in each of these distinct events. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Year Produced 2009 
Provided To Others? Yes  
Impact New understanding in cAMP signalling, and potentially a new target for axon regeneration studies - Epac. 
 
Title FRET analysis in primary mammalian neurons 
Description Fluorescence resonance energy transfer (FRET) in cultured primary rat (dorsal root ganglion) neurons. It involves transfection of a constructs that encodes a protein expressing two different fluorophores. When the fluorophores are in close proximity, activation of one with a specific wavelength of light leads to energy transfer to the other, which emits light at a separate wavelength that can be detected by computer software. The computer uses a formula to calculate the ratio of excitation and emission wavelengths to measure the amount of energy transfer. When the protein is activated a conformational change results in fluorophores moving closer together or further apart, causing a change in FRET that indicates changes in protein activity. 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact The method has largely been used in cell lines or non-neuronal cells. Transfection of primary neurons has historically been difficult due to the very low viability. We are one of the very few labs in the world who can successfully perform FRET in primary neurons. The method allows direct measurement of protein activity in live neurons. Thus predicted mechanisms can be tested and confirmed, or novel or unexpected signalling mechanisms may be uncovered. We have found that Epac and PKA distinctly mediate cyclic AMP-depednent neuronal signalling mechanisms that differentially influence axon growth, guidance and regenration. This will impact on the mode of cyclic AMP signalling mechanisms being re-assessed and could lead to novel targets for promoting CNS repair. Tha work is due to be published online in teh Journal of Neuroscience on December 2nd 2009. 
 
Title Growth cone turning assays on mammalian neurons 
Description The growth cone turning assay allows axon guidance mechanisms to be analysed when axons are presented a gradient of protein or chemical. This has provided great insights into axon guidance mechanisms used by developing neurons in lower vertebrates, particulalry frogs. We have developed a means to use this assay in mammalian neurons, and are possibly the only group in the world (or at least one of very few indeed) reporting on this method. 
Type Of Material Technology assay or reagent 
Year Produced 2007 
Provided To Others? Yes  
Impact It allows study of neuronal signalling mechanisms and axonal responses in higher animals, more related to humans. It also allows the combination of more modern applications, such as FRET and siRNA, to more fully understand mechanisms important in axon guidance. This will hasten our understanding of developmental processes that could address the question of why the mammalian CNS does not regenerate and what might be done to make it do so. The technique will also allow insights into cell signalling mechanisms that have been conserved or altered in evolution from, for example, fish and amphibians. 
 
Description Epac FRET 
Organisation Netherlands Cancer Institute (NKI)
Country Netherlands 
Sector Academic/University 
PI Contribution Carried out all primary research and prepared manuscript for publication
Collaborator Contribution Provided Epac FRET constructs by material transfer agreement, and approved manuscript for publication
Impact Also resulted in prominent publications (PMID 20007468 and PMID 20007468) as described in the PKA FRET collaboration.
Start Year 2007
 
Description PKA FRET 
Organisation University of Glasgow
Department Institute of Biomedical and Life Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We carried out all the primary research and prepared a manuscript reporting the results
Collaborator Contribution Provision of PKA FRET constructs, advice on their use, advice on MRC project grant application, and advice on manuscript preparation
Impact Publications (PMID 20007468 and PMID 20851749) reporting alternative downstream signalling via either PKA or Epac that determines repulsive or attractive growth cone turning respectively to axon guidance cues. We expect this to have huge impact on the axon guidance and regeneration fields, as it explains polarised growth cone responses reported over the last 20 years and more, and having received positive feedback from prominent researchers in the fields. Our publication (PMID 20007468) was highlighted in Nature Reviews Neuroscience in February 2010.
 
Description SRU-funded Lecturer in Neuroscience 
Organisation Scottish Rugby Union
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution Data from the MRC award "G0500801 Promoting CNS regeneration by targeting cell signalling" formed a major contribution in attracting financial support from the Scottish Rugby Union and the Aberdeen University Development Trust to fund a Neuroscience Lecturer position in the School of Medical Sciences, University of Aberdeen.
Collaborator Contribution Financial support to attract a researcher with in vivo experience with whom to forge potential future collaborations to take the in vitro work supported by the MRC to a new level.
Impact None
Start Year 2013
 
Description Deputy First Minister of Scotland visit 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Primary Audience Policymakers/politicians
Results and Impact Demonstrated our research laboratory and experimentation, describing results and aims, as part of an Institutional visit by the Deputy First Minister of Scotland.

Highlighted spinal injury issues to the leader of the Scottish Liberal Democrats and Deputy First Minister of the Scottish Parliament.
Year(s) Of Engagement Activity 2006
 
Description Poster presentation at Houses of Parliament 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Primary Audience Policymakers/politicians
Results and Impact A poster presentation and oral account of our research given at the House of Commons, Westminster, London. PhD student represented the University in informing MPs of our research activities at Aberdeen University.

MPs, including local MP Anne Begg, MP for Aberdeen South, kept informed of our research activities and results.
Year(s) Of Engagement Activity 2007
 
Description School visit 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Primary Audience Schools
Results and Impact Hosted Secondary school pupils (16-17 years old) for one week's work experience in academic research. The pupils took part in experimental procedures, learned about our research activities and wrote a report for their schools.

Pupils and their families learned about spinal injury issues that they were otherwise unaware of, and pupils declared interest in seeking University places in Biomedical Science degree schemes.
Year(s) Of Engagement Activity 2008,2009
 
Description University Open days 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? Yes
Primary Audience Schools
Results and Impact School pupils and parents invited to hear and ask about courses provided in biomedical sciences and future relevant careers. As part of this results of our research and their implications on human health and welfare are discussed.

Many school pupils are encouraged to pursue interests in biomedical science degree schemes with a view to pursuing biomedical careers.
Year(s) Of Engagement Activity 2006,2007,2008,2009