The Effect of Manipulation of the Phosphoinositide 3-kinase Pathway on Axon Regeneration in vivo

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Evans, Rachel Shane  ORCID logo

Millions worldwide are visually impaired by optic nerve diseases, and glaucoma, the leading cause of irreversible blindness, is due to affect approximately 80 million people by 2020. The optic nerve is comprised of retinal ganglion cell (RGC) axons, and like all mammalian central nervous system axons, they fail to regenerate due to a combination of extrinsic and intrinsic factors. Current treatments have limited success preventing disease progression, and this failed regeneration presents a major barrier to restoring vision.

Previous research has demonstrated that manipulation of the phosphoinositide 3-kinase (PI3K) pathway by transgenic knockout of phosphatase and tensin homolog (PTEN) promotes survival and regeneration of murine RGC axons in an optic nerve crush (ONC) model. However, translation issues are yet to be addressed. PI3K converts phosphatidylinositol (3,4)-bis-phosphate (PIP2) lipids to phosphatidylinositol (3,4,5)-tris-phosphate (PIP3). PTEN acts as a pathway regulator, converting PIP3 back into PIP2. PIP3 is a second messenger molecule for several pathways, including the mammalian target of rapamycin (mTOR) pathway, which has been demonstrated to promote axon regeneration in RGCs, the spinal cord, and cortical neurons. The PI3K pathway is activated by growth factors, integrins and cytokine receptors. Integrins have also been shown to promote axon regeneration via the focal adhesion kinase pathway.

This thesis aimed to determine the effects on axon regeneration of manipulating the PI3K pathway in various ways. A transgenic approach was adopted to conditionally express the hyperactive p110α and p110δ PI3K isoforms using viral Cre recombination. Both survival and axon regeneration were significantly increased 4 weeks post-ONC injury in young and aged mice. No significant difference between the two isoforms was observed, except that p110α promoted RGC survival in aged mice whereas p110δ had no significant effect. This was developed further using viral vectors to upregulate PI3K and to knockdown PTEN for a more translational approach: AAV2.shPTEN.GFP and AAV2.PI3K(p110δ). PTEN knockdown promoted both RGC survival and axon regeneration, while PI3K upregulation promoted axon regeneration. No significant difference in axon regeneration was seen between the two strategies. Where regeneration was seen, activation of the mTOR pathway was demonstrated.

It is becoming increasingly clear that strategies need to be combined to achieve long-distance, robust regeneration. In this thesis, PI3K upregulation was combined with PTEN knockdown and then with integrin activation using AAV2.Integrin.V5 and AAV2.Kindlin.GFP viruses. While PI3K upregulation and PTEN knockdown promoted RGC survival and axon regeneration compared to control, this was not significantly more than PI3K upregulation alone. The experiments involving integrin activation were unsuccessful and the viruses need to be investigated further.

While the field has achieved small numbers of regenerating axons up to the optic chiasm, key challenges for future work are reaching central targets in the brain and assessing axon regeneration in an aged model to better reflect the clinical setting, where neurodegenerative diseases predominately affect the aging population.

In summary, the work in this thesis investigated the pro-regenerative effects of potential gene therapy targets, advancing our knowledge for developing future clinical strategies.

Martin, Keith R
axon regeneration, optic nerve crush, phosphoinositide 3-kinase pathway, retinal ganglion cell survival
Doctor of Philosophy (PhD)
Awarding Institution
University of Cambridge
I am grateful for the funding I received to complete this PhD. My MRC and Sackler PhD scholarship was funded by the UK Medical Research Council and the Sackler fund. In addition, the work in this thesis was also funded by several grants written by Dr Amanda Barber and Prof Keith R Martin: Wellcome Trust, Fight for Sight (The Eye Research Charity), Cambridge Eye Trust, International Glaucoma Association, and the National Eye Research Centre.