Authors: B. NIEUWENHUIS, R.EVANS, C.S. PEARSON, A.C. BARBER, J. CAVE, P.D. SMITH, J. FUCHS, B.J. EICKHOLT, H.M. GELLER, K.R. MARTIN, R. EVA, J.W. FAWCETT
Injury to the central nervous system (CNS) has severe consequences because adult CNS axons do not regenerate. PtdIns-3,4,5-P3 (PIP3) signaling is essential for axon growth during development of the nervous system. Silencing PIP3 phosphatase PTEN leads to increased regeneration in the corticospinal tract and optic nerve after injury, showing that PIP3 is crucial for axonal regeneration. However as neurons mature, phosphoinositide 3-kinases (PI3Ks) activating receptors are excluded from axons. We hypothesize that a developmental decline in axonal PIP3 signaling contributes to restricted axonal regeneration in the CNS. The objective of this study is to explore whether overexpression of PI3Ks, which generates PIP3, could promote axonal regeneration of CNS neurons.
We first confirmed that there is a decline of PIP3 levels in cultured cortical neurons in line with maturation by using immunocytochemistry. Overexpression of activated PI3K increased axonal growth by 50% in developing neurons (4 days in vitro, DIV). Expression of activated PI3K in matured cortical neurons (14 DIV) resulted in an enlarged the soma size (by 100%) and more complex dendritic morphology in vitro. Expression of constitutively activated PI3K, but not wildtype PI3K, resulted in a six-fold increase of the PIP3 signaling pathway in these neurons – visualized by staining for the phosphorylated ribosomal protein S6. To investigate whether PI3Ks can enhance the regeneration capacity of CNS neurons, we overexpressed these in cortical neurons and applied in vitro laser axotomy. We confirmed that 14 DIV neurons have a restricted axon regeneration capacity after in vitro laser axotomy (only 15% of the axotomised neurons regenerate). Importantly, expression of PI3Ks increases the success rate of axonal regeneration (approximately 60% of the axotomised neurons regenerate). We recently started to explore whether PI3K can promote optic nerve regeneration in vivo. Using a transgenic mouse line with cre-inducible expression of constitutively activated PI3K, we found that the AAV2-cre condition had significantly increased axon regeneration beyond the optic nerve crush site compared to controls.
In summary, cultured cortical neurons have a decline of PIP3 signaling and this could contribute to their decreasing regeneration capacity during maturation. Overexpression of activated PI3Ks promotes developmental axon growth, and axonal regeneration of CNS neurons in vitro and in vivo. The mechanisms of PI3K-induced axon regeneration have not been investigated in great detail. We are currently investigating whether PI3K acts by stimulating axonal transport of regeneration-associated proteins.
*B. NIEUWENHUIS1,2, R. EVANS1, C. S. PEARSON3, A. C. BARBER1, J. CAVE1, P. D. SMITH4, J. FUCHS5, B. J. EICKHOLT5, H. M. GELLER3, K. R. MARTIN1, R. EVA1, J. W. FAWCETT1,6;
1Univ. of Cambridge, Cambridge, United Kingdom; 2Netherlands Inst. for Neurosci., Amsterdam, Netherlands; 3NIH, Bethesda, MD; 4Carleton Univ., Ottawa, ON, Canada; 5Charite Univ. Med., Berlin, Germany; 6Inst. of Exptl. Med., Prague, Czech Republic. Elevated phosphoinositide 3-kinase activity promotes axon regeneration of central nervous system neurons. Program No. 115.06. 2018 Neuroscience Meeting Planner. San Diego, CA: Society for Neuroscience, 2018. Online.
International Spinal Research Trust (NRB110) ERANET NEURON grant AxonRepair (013-16-002) Christopher and Dana Reeve Foundation (JFC-2013(3), JFC-2013(4)) Medical Research Council (G1000864 018556) Cambridge Eye Trust (RG80564) Fight for Sight (RG74504)