How scars shape the neural landscape: key molecular mediators of TGF-β1s anti-neuritogenic effects

Following injury to the peripheral and central nervous systems, tissue levels of transforming growth factor (TGF)-{beta}1 often increase, which is key for wound healing and scarring. However, active wound regions and scars appear to inhibit process outgrowth by regenerating neurons. We recently showed that corneal wound myofibroblasts block corneal nerve regeneration in vivo, and sensory neurite outgrowth in vitro in a manner that relies critically on TGF-{beta}1. In turn, delayed, abnormal re-innervation contributes to long-term sensory dysfunctions of the ocular surface. Here, we exposed morphologically and biochemically-differentiated sensory neurons from the ND7/23 cell line to TGF-{beta}1 to identify the intracellular signals regulating these anti-neuritogenic effects, contrasting them with those of Semaphorin(Sema)3A, a known inhibitor of neurite outgrowth. Neuronal morphology was quantified using phase-contrast imaging. Western blotting and specific inhibitors were then used to identify key molecular mediators. Differentiated ND7/23 cells expressed neuron-specific markers, including those involved in neurite extension and polarization. TGF-{beta}1 increased phosphorylation of collapsin response mediator protein-2 (CRMP2), a molecule that is key for neurite extension. We now show that both glycogen synthase kinase (GSK)-3{beta} and Smad3 modulate phosphorylation of CRMP2 after treatment with TGF-{beta}1. GSK-3{beta} appeared to exert a particularly strong effect, which could be explained by its ability to phosphorylate not only CRMP2, but also Smad3. In conclusion, TGF-{beta}1s inhibition of neurite outgrowth in sensory neurons appears to be regulated through a highly-conserved signaling pathway, which involves the GSK-3{beta}/CRMP-2 loop via both canonical and non-canonical mechanisms. It is hoped that by defining the signaling pathways that control neurite outgrowth in wound environments, it will become possible to identify optimal molecular targets to promote re-innervation following injury.