Endoplasmic reticulum, Protrudin and Axon Regeneration

The aim of the application is to develop new methods to stimulate regeneration of nerve fibres in the injured spinal cord and optic nerve.
At present when the spinal cord or optic nerve are damaged the nerve fibres are unable to regenerate and little function can be restored.
There has been a steady increase in our understanding of why nerve fibre regeneration fails, and treatments have been developed that stimulate regeneration, although not sufficiently to make a successful treatment for patients. This application aims to develop new methods to increase the intrinsic regeneration potential of neurons that connect to the spinal cord and connect the eye to the brain, whilst increasing our understanding of the mechanisms that control regeneration in the adult central nervous system.

The current application focuses on the endoplasmic reticulum (ER). This is the largest organelle in cells, and has branches that travel down the length of nerve fibres. We believe that changes in the ER may be one of the main reasons that nerve fibres in the brain and spinal cord lose the ability to regenerate as they mature. This hypothesis came from our work with the molecule protrudin, which we have found to the be the strongest promoter of regeneration that we have seen. Protrudin is an adaptor molecule that brings together several molecules and structures that are needed for regeneration. Importantly, it attaches to the ER and drags it to the tip of nerve fibres. We find that ER diminishes in nerve fibres as they mature and lose the ability to regenerate, but protrudin restores the ER to nerve fibre tips and growth cones. If the ER-binding of protrudin is removed, it no longer promotes regeneration.

The grant focuses on the role of the ER in regeneration. The ER has many functions that should be essential for good regeneration, but it has not been studied in this context. The experiments will study the anatomy of the ER and its enzymes in nerve fibres that regenerate vigorously versus those that have matured and lost the ability to regenerate. There are many molecules that affect the distribution and anatomy of ER, and a selection of these will be used to increase and decrease ER in the different types of nerve fibre. Also some of the key functions of the ER will be manipulated. Together, these experiments should give conclusive proof of whether the ER is a key player in regeneration. The treatments that have been successful in promoting regeneration in tissue culture experiments will then be tested for their ability to promote regeneration in the optic nerve and spinal cord.

The application focuses on the role of endoplasmic reticulum (ER) in axon regeneration. This concept comes from previous work on protrudin. This scaffolding molecule brings together molecules and components that promote regeneration. Our recent work shows that it has a very strong effect on promoting regeneration of mature cortical axons in vitro and retinal axons in vivo. Protrudin has VAP-A and a hairpin domain that both fix it to the ER. Deletions of each of these domains renders protrudin ineffective at promoting regeneration. As cortical neurons mature in vitro and lose their ability to regenerate, ER in the distal axons declines. Axons that regenerate (immature CNS neurons, sensory neurons) have abundant ER in their axons. Protrudin restores ER in mature axons, and only ER-recruiting forms of protrudin promote regeneration.

It is surprising that ER has not been a focus of regeneration research. Its many functions in calcium regulation, phospholipid synthesis, direction of vesicle trafficking, endosome production etc. make it a strong and novel candidate. The application aims to determine the role of ER recruitment, caused by protrudin and several other ER-shaping molecules, in regeneration. First ER morphology in regenerating and non-regenerating axons will be studied. Next, several molecules that should increase or decrease axonal ER will be tested, and effects on regeneration measured in regenerative and non-regenerative neurons. Two key ER functions are phosphoinositol synthesis and formation of ER-plasma membrane contacts. The role of these in regeneration will be tested. Successful treatments will be tested in vivo for optic nerve and corticospinal tract regeneration. The results will inform current strategies in the lab to promote CNS axon regeneration.

The project will have impact with the following groups.
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 will have widespread impact.
2. The pharmaceutical industry is starting to take an interest in regeneration again after previous setbacks. Applicants advise AZ, ABBVIE, Acorda, Janssen.
3. Patients. Patients with spinal injury need a regeneration treatment. Axon regeneration is also a potential therapeutic strategy in optic nerve damage and glaucoma. Protrudin and associated findings will be part of a repair strategy.
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.