An Inducible Tyrosine Kinase Receptor for Axonal Regeneration

Abstract

The prevention or reduction of neuronal degeneration remains a challenge in neurotrophins therapy. An inducible trkA (ItrkA) system has been shown to regulate embryonic dorsal root ganglion (DRG) neuronal survival and neurite outgrowth in vitro. A new ItrkA plasmid ItrkA-membrane (ItrkAmemb) with one adenine at 3’ terminal was established by correcting the sequence of the original plasmid ItrkA-cytosol (ItrkAcyto). Adult DRGs were dissected from adult Fischer 344 rats (8-14 weeks) for the treatment with AP20187 (membrane-permeable small-molecule ligand), vehicle or NGF (Nerve Growth Factor). Neurite outgrowth assessments were done by manually tracing the longest neurite of each neuron. Cell diameters were also measured and averaged for each well. Protein expression after ItrkAmemb transfection and trkA downstream signaling were investigated by Western-blotting. Neurite length of ItrkAmemb transfected DRGs was not influenced by AP20187 or NGF but cells displayed shorter neurites compared to GFP control groups. While ItrkAcyto transfected DRGs cultured with AP20187 had the longest neurite growth compared to ItrkAmemb transfected neurons and ItrkAcyto transfected cells treated with vehicle or NGF, no significant difference to GFP controls was detected. Quantification of the mean diameter of transfected DRGs demonstrated that ItrkAmemb electroporation significantly increased cell diameter, while the diameter of ItrkAcyto transfected neurons and GFP controls were almost the same as naïve neurons. In contrast to electroporated adult DRG neurons, ItrkAmemb virus transfection did not affect the diameter of infected adult DRG Neurons. No obvious difference was observed between the ItrkAmemb and GFP electroporated cells, and only cells transduced with ItrkAmemb treated with AP20187 seemed to show higher phosphorylation both of Akt and Erk1/2. The effect of adult DRG neurons after ItrkA transfection differs, which depends on the change of cell soma size and/or neurite growth, gene delivery technique, expression level and the localization of ItrkA.