Repair of the corticospinal tract

After spinal cord injury patients lose sensation and are paralysed because the nerve fibres connecting the brain with the body are cut in the spinal cord. To restore full function these damaged fibres must be made to regenerate. Effective regeneration in the spinal cord has not been possible, but recent advances in Cambridge have made it possible to restore sensory regeneration in the spinal cord. Regeneration of motor fibres is more difficult, because unlike sensory fibres, the motor fibres mature to exclude many of the molecules needed for regeneration.
The plan for this application is to bring together the method that made sensory regeneration possible with four candidate methods to enable growth-related molecules to be transported into the motor nerve fibres. The molecules that enable nerve fibre growth are integrins, which are transported into sensory fibres but not motor fibres. Research on transport mechanisms has developed four methods the enable integrins to be transported into mature nerve fibres and to restore their ability to regenerate in a tissue culture model.
The plan for the application is to develop viral vectors suitable for use in rats for each of these treatments. The treatments will then be tested in combination to find out the most powerful treatment for inducing regeneration. Some of the successful combinations will then be tested in quick proof of principle experiments in rats with partial spinal cord damage to identify the best method for restoring regeneration. Finally the best two treatments will be tested in a full experiment in which regeneration and behavioural recovery will be measured.

The overall aim of the project is to develop a method to reconstruct the corticospinal tract in the damaged spinal cord, The project follows on from recent work showing that long distance sensory axon regeneration can be stimulated by transducing sensory neurons with the tenascin-C-binding integrin alpha9beta1 together with integrin-activating kindlin-1. Tenascin-C is the main integrin ligand of the adult spinal cord, upregulated by injury. Kindlin-1 overcomes inactivation of integrins by CSPGs and NogoA.
Activated integrin treatment promotes long-distance regeneration of sensory axons, but a further intervention is needed for corticospinal regeneration. The issue is that transport in mature CNS neurons becomes polarised, sending some molecules to dendrites, some to axons. Many growth-related molecules are carried in Rab11 recycling endosomes, and these are excluded from axons as they mature. We have developed four effective and probably synergistic methods that can restore Rab11/integrin transport to mature cortical axons, and promote regeneration. These have been validated in an in vitro model in which cortical neurons mature, developed polarised transport excluding Rab11/integrin and lose the intrinsic ability to regenerate. The four interventions are: a) knockdown of the Arf6 GEF Efa6, b) dominant active transport adaptor protrudin, 3) constitutively active PI3K delta, 4) demolition of the axon initial segment.
The plan for the grant is:
1) Develop AAV vector tools for each of the for regeneration methods and validate them in vitro.
2) Test combinations of the treatments to find additive effects and effective combinations.
3) Four short proof-of-principle experiments to find the most effective combination of AAV vectors, including alpha9 integrin, kindlin-1 and two of the four candidate treatments injected into cortex to trace corticspinal regeneration.
4) A definitive regeneration experiment with behaviour to validate the most effective treatment.

Beneficiaries
1. The academic community. As described above, the work is of importance to research in several fields, and gives a practical output to areas of basic research. The knowledge gained on axon regeneration and PIP signalling will have widespread impact.
2. The pharmaceutical industry is starting to take an interest in regeneration again after previous setbacks. Fawcett is an advisor to Acorda Therapeutics who develop new treatments for spinal cord injury.
3. Patients. Patients with spinal injury.
4. Training. The project will involve PhD students in the participating groups, post-docs working on related projects and the personnel employed on this grant. The laboratories currently work closely together, and programme meetings are held in the UK, Holland and Germany approximately twice a year. This is a very strong training environment with an interdisciplinary background.