A NOVEL INDUCIBLE TYROSINE-KINASE RECEPTOR TO REGULATE SIGNAL TRANSDUCTION AND NEURITE OUTGROWTH

Nervous system growth factor gene delivery can promote axonal growth and prevent cell death in animal models of CNS trauma and neurodegenerative diseases. The ability to regulate growth factor expression or signaling pathways downstream from growth factor receptors remains a desirable goal for in vivo gene transfer. To achieve precise pharmacological modulation of neurotrophin activity, we have generated a chimeric trkA receptor (ItrkA) by fusing the entire intracellular domain of the trkA high-affinity NGF receptor to two intracellular, modified FK506 binding domains for the synthetic small molecule dimerization ligand, AP20187. Rat PC12 cells were transduced with lentiviral vectors containing ItrkA and GFP (via an internal ribosome entry site). Treatment of ItrkA-expressing PC12 cells with AP20187 induced neurite outgrowth and differentiation in a time- and dose-dependent fashion, with a half-maximal response at a concentration of 1 nM AP20187. 70% of cells responded to AP20187 by day 3. Western blots demonstrated that AP20187 treatment resulted in phosphorylation of Erk1/2 and Akt in ItrkA-transduced PC12 cells, but not in non-transduced, naive cells. Phosphorylation levels were comparable to levels obtained with 50ng/ml NGF. In addition, ItrkA lentiviral transduction of primary E15 dorsal root ganglion neurons significantly increased neurite growth 3–4-fold in the presence of AP20187 compared to control GFP transduced and naive neurons. These results demonstrate that small ligand-induced dimerization of the intracellular domain of trkA can efficiently simulate the biological activity of NGF and provide a means to regulate intracellular neurotrophin receptor signaling.