Disentangling the signaling complexity of nerve growth factor receptors by CRISPR/Cas9

The binding of nerve growth factor (NGF) to the tropomyosin-related kinase A (TrkA) and p75^NTR receptors activates a large variety of pathways regulating critical processes as diverse as proliferation, differentiation, membrane potential, synaptic plasticity, and pain. To ascertain the details of TrkA-p75^NTR interaction and cooperation, a plethora of experiments, mostly based on receptor overexpression or downregulation, have been performed. Among the heterogeneous cellular systems used for studying NGF signaling, the PC12 pheochromocytoma-derived cell line is a widely used model. By means of CRISPR/Cas9 genome editing, we created PC12 cells lacking TrkA, p75^NTR , or both. We found that TrkA-null cells become unresponsive to NGF. Conversely, the absence of p75^NTR enhances the phosphorylation of TrkA and its effectors. Using a patch-clamp, we demonstrated that the individual activation of TrkA and p75^NTR by NGF results in antagonizing effects on the membrane potential. These newly developed PC12 cell lines can be used to investigate the specific roles of TrkA and p75^NTR in a genetically defined cellular model, thus providing a useful platform for future studies and further gene editing.