Promoting Central Nervous System Regeneration by Targeting Intracellular Pathways

Adult central nervous system neurons regenerate poorly after injury. One reason for this regenerative failure is that a developmental change occurs where essential growth-molecules become excluded from axons with maturation. In this thesis, two strategies were employed in order to improve axon regeneration: 1) restoring the axonal transport of growth-molecules in mature axons via manipulation of transport machinery; 2) reverting mature neurons to an earlier developmental state where growth-molecules are abundant in the axon. In order to restore axon transport of growth-molecules, protrudin - a membrane-associated protein involved in directional membrane trafficking, was studied. Phosphorylated protrudin preferentially binds Rab11, a small GTPase involved in axon transport of growth receptors. This association is required for neurite outgrowth and for anterograde movement of this complex. We found that endogenous protrudin is excluded from mature axons similar to other growth-related machinery and cargo. It was hypothesised that introducing a phosphomimetic form of protrudin to mature cortical neurons, could increase the phospho-protrudin/Rab11 interaction resulting in improved anterograde transport of growth-molecules and enhanced axon regeneration. We found that overexpression of two constitutively phosphorylated forms and also wild-type protrudin increased the proportion of regenerating axons after in vitro laser axotomy. Furthermore, overexpression of wild-type and phospho-protrudin in the retina resulted in enhanced axon regeneration in a mouse model of optic nerve crush 2 weeks after injury. Live-cell imaging experiments revealed that this increased regenerative ability is accompanied with increased transport of Rab11 endosomes as well as growth receptors into the axon. Importantly, by further studying protrudin’s mechanisms of action, we identified novel players in the process of axon regeneration, including an exciting new role for the endoplasmic reticulum. In summary, protrudin is a promising intervention which improves axon regeneration in vitro and in vivo and due to its participation in multiple molecular pathways, new targets for aiding and understanding axon regeneration could be uncovered. Another approach to aid axon regeneration is to rejuvenate mature neurons. We hypothesised that overexpressing different combinations of transcription factors that are developmentally down-regulated could bring neurons to an earlier developmental stage. Four maturity markers were identified – doublecortin as an early maturity marker and 68-kDa neurofilament, calcitonin, tubulin-4a as late maturity markers. Some transcription factors showed promising results in rejuvenating primary cortical neurons. Further studies are needed to identify correct combinations of transcription factors to achieve maximum effect and to examine the effects of this treatment on axon regeneration.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2019

Author dc:creator

• Petrova, Veselina

Advisor dc:contributor.advisor

• Fawcett, James

• axon regeneration
• protrudin
• axon transport
• transcription factors
• optic nerve crush
• glaucoma
• growth receptors
• integrins

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/5035f54f-5e78-4978-90f2-1a80baf19650/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

en

Author Identifier

0000-0002-5593-0198
0000-0002-7990-4568

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/294511