Src and FAK are key early signalling intermediates required for neurite growth in NGF-responsive adult DRG neurons

Axonal regeneration is influenced by factors in the extracellular environment, including neurotrophins, such as NGF, and adhesion molecules, such as laminin. The provision of both NGF and a permissive substrate to cultured adult NGF-responsive DRG neurons results in enhanced levels of neurite growth not achievable by either factor alone. In this study, we have investigated the early signalling events that contribute to NGF and laminin-induced neurite growth. Adult NGF-responsive DRG neurons were plated on poly-d-lysine for 2 h then stimulated with NGF, laminin, or laminin plus NGF for 10 min, 1 h, or 6 h. Signalling pathways were subsequently analysed using Western blotting and pharmacological inhibition of specific signalling components. While activation of the various signalling intermediates (Src, FAK, Akt, MAPK) could be detected as early as 10 min-1 h after stimulation, significant neurite growth was observed mainly at the 6 h time point. The results of the time course experiments showed differential activation of the signalling intermediates. Src was activated by all treatments (NGF, laminin and the combination) at the earliest time point analysed, 10 min. NGF stimulation also resulted in detectable activation of FAK, Akt and MAPK by 10 min. However, laminin stimulation alone did not result in detectable activation of FAK, Akt or MAPK until the 1 h time point. Inhibition of either Src or FAK activity attenuated both laminin and/or NGF-induced PI 3-K/Akt and MEK/MAPK signalling pathways, as well as neurite growth. Downstream inhibition of Akt by Akt knockdown also blocked observed neurite growth, while inhibition of MEK/MAPK had no significant effect. Together, these results demonstrate that signalling underlying neurite growth can be detected within minutes of stimulation and provide a mechanism for the observed enhancement of neurite growth when both NGF and the permissive substrate, laminin, are provided.