Proceedings of The Physiological Society

King's College London (2011) Proc Physiol Soc 22, PC02

Poster Communications

NG2-glia use the retinoic acid signalling pathway to create a permissive environment for neurite outgrowth

E. J. Pawson1, R. Wigley1, S. McMahon1, J. Corcoran1

1. Biomedical and Health Sciences, Wolfson CARD, King's College London, London, United Kingdom.

Inhibitory chondroitin sulphate proteoglycans (CSPGs), such as NG2, are highly up-regulated following central nervous system (CNS) injury and removal of these CSPGs promotes functional recovery. The NG2 CSPG is also expressed by a population of glial cells known as NG2-glia that are present throughout the developing and adult CNS, and these NG2-glia have been shown to support regenerating axons. Interestingly, following injury NG2-glia also express retinaldehyde dehydrogenase (RALDH) 2, the key retinoic acid (RA) synthesising enzyme. Retinoic acid stimulates neurite outgrowth via activation of the retinoic acid receptor (RAR) β. We therefore hypothesised that activation of the RA signalling pathway in NG2-glia would create a permissive environment for axonal regeneration. Using a co-culture system comprising adult dissociated dorsal root ganglia (DRG) sensory neurons with NG2-glia, we have investigated the effects of NG2-glia and RA on neurite outgrowth. Briefly, optic nerves from postnatal day (P) 7 mice were used to culture NG2-glia which were then co-cultured with dissociated DRG neurons. After 24 hours co-cultures were fixed in 2% PFA and immunolabelled for NG2 to identify NG2-glia, β-III tubulin to identify neurons and RARβ. Cells were then imaged using a Zeis LSM 700 confocal microscope and the percentage of neurite bearing neurons was calculated. Our results show that NG2-glia, as a unique glial cell type, are permissive to neurite outgrowth from DRG, and specifically in presence of the RARβ agonist, CD2019 (400nM), axonal outgrowth from DRG neurons is significantly enhanced (p<0.05, Two way ANOVA with Bonferroni post-hoc test). Furthermore, DRG neurites are closely associated with NG2-glia. Digestion of the NG2 CSPG from NG2-glia, via incubation with the enzyme chondroitinase ABC (ChABC), also significantly enhances neurite outgrowth (p<0.001, Student’s T-test) and increases neurite branching from DRG neurons in DRG-NG2 co-cultures (p<0.01, Student’s T-test). This effect can be blocked and then rescued by the addition of the RALDH2 inhibitor disulphiram (10µM), followed by all-trans RA (100nM). Pharmacological intervention with the pan RAR antagonist (10µM) also demonstrates that NG2-glia constitutively secrete RA, and this significantly enhances neurite outgrowth (p<0.05, One way ANOVA followed by Bonferroni post hoc test). Together, these data demonstrate that despite the presence of the NG2 CSPG itself, NG2-glia offer a favourable environment for neurite outgrowth, which is mediated via the RA signalling pathway.

Where applicable, experiments conform with Society ethical requirements