To regulate trkA signal transduction and to allow for stable gene expression in neurons, we generated a lentiviral vector expressing the intracellular domain of trkA including the juxtamembrane and activation loop domains (trkA-ICD), fused to two modified FK506 binding protein domains (FKBP36V) and an amino-terminal myristoylation signal (myr) for membrane localization. A carboxy-terminal hemagglutinin tag (HA) was included for immunocytochemical detection (), and an internal ribosome entry site followed by a GFP expression cassette monitored gene expression.
Schematic vector map and components of the inducible trkA receptor (ItrkA) system
To examine the ability of this inducible trkA (ItrkA) construct to activate trkA signaling cascades in response to a small molecule ligand for dimerization (AP20187), an ItrkA expressing cell line was generated by transducing PC12 cells with ItrkA lentivirus followed by fluorescence activated cell sorting for GFP expression. Treatment of transduced PC12 cells with AP20817 in the cell culture medium for 3 days resulted in robust extension of neurites whereas vehicle treated PC12 cells showed very little neurite extension. These findings indicate that morphological responses normally observed after NGF treatment can be obtained by AP20187-induced dimerization of ItrkA ().
PC12 cell neurite outgrowth in response to ItrkA dimerization by AP20187
A time course analysis of neurite outgrowth in the presence or absence of AP20187 indicated that as early as 1 day after addition of AP20187 to the cell culture medium, neurite growth responses could be observed, with increasing numbers of cells extending neurites after 2 and 3 days of AP20187 treatment (, repeated measures ANOVA p<0.0001 for time, and treatment versus time, posthoc analysis p<0.001 compared to untreated and vehicle treated cells at each time point). In contrast, untreated cells or cells treated with vehicle (0.1% ethanol) showed very few neurites and were not significantly different from each other at any time point.
Dose dependence and kinetics of neurite extension in ItrkA transduced PC12
In initial experiments, cells were treated with 1.25 μM AP20187. However, substantially lower levels of AP20187 might be capable of inducing ItrkA signaling, and ideally various levels of morphological responses should be obtainable depending on the amount of AP20187 present. Analysis of the dose response curve of ItrkA transfected PC12 cells exposed to various doses of AP20187 demonstrated that concentrations of AP20187 as low as 1 picomolar induced significant neurite outgrowth compared to vehicle treated cells (, ANOVA p<0.0001; posthoc analysis p<0.05 for concentrations of 1.25 pM). No significant further increases in neurite outgrowth were observed at concentrations above 125nM (p=0.62 comparing 125nM to 1.25 μM) and half-maximal responses within the AP20187 concentration range tested were obtained around1 nM. Thus, morphological responses are dependent on the concentration of AP20187 in the medium and can be accordingly modulated, similar to dose-response curves following NGF modulated neurite outgrowth.
NGF-induced cell signaling via trkA is mediated through two separate signaling cascades. Signals for differentiation are thought to depend on the activation of Erk1/2 and signals for cell survival utilize the PI3K/AKT cascade (Ashcroft et al. 1999
; Cowley et al. 1994
; Patapoutian and Reichardt 2001
). To determine whether ItrkA dimerization induces the same downstream signaling cascade as NGF binding to trkA, phosphorylation of AKT and ERK1/2 were investigated in naïve and ItrkA transduced PC12 cells in response to NGF and AP20187.
Stimulation of naïve and ItrkA-transduced PC12 cells with 50 ng/ml NGF resulted in similar increases in AKT and ERK1/2 phosphorylation in comparison to untreated control cells indicating that ItrkA overexpression did not interfere with normal NGF downstream signaling (). Treatment of ItrkA-transduced cells with AP20817 resulted in similar downstream signaling events as NGF binding to the full-length receptor. AP20817 increased phosphorylation of AKT and ERK1/2 compared to untreated cells or cells treated with vehicle (0.1% ethanol). In contrast, AP20187 treatment of naïve control cells did not stimulate AKT and ERK1/2 phosphorylation.
Western blot analysis of trkA downstream signaling in naïve and ItrkA-transduced PC12 cells
Taken together, ItrkA dimerization by AP20817 appears to result in the same downstream signaling events as NGF binding to full-length trkA. Baseline levels of phosphorylated ERK1/2 and AKT are similar in ItrkA-transduced and naïve cells indicating a tight control of ItrkA phosphorylation. Furthermore, ItrkA overexpression does not interfere with normal NGF signaling mechanisms in PC12 cells.
To determine whether ItrkA signaling can also induce classical responses to neurotrophin receptor activation in primary neurons, embryonic DRG neurons were isolated and transduced with ItrkA lentivirus or GFP virus as control. Examination of GFP fluorescence indicated that virtually all cells were transduced by lentivirus. Incubation of ItrkA transduced DRG neurons with AP20187 potently stimulated neurite growth compared to ItrkA transduced neurons incubated with vehicle. In contrast, neurite length of cells transduced with GFP control virus did not differ between AP20187 or vehicle treated cells and was not significantly different from non-transduced naïve neurons (). Quantification of mean neurite lengths indicated a highly significant 3–4 fold increase in neurite length in AP20187-stimulated ItrkA-transduced neurons compared to all other groups (ANOVA; p < 0.0001). Neurite length of vehicle-treated ItrkA transduced neurons was not significantly different from GFP-transduced neurons, and AP20187 treatment did not affect neurite length in GFP-transduced neurons ().
Inducible neurite outgrowth in E15 DRG neurons