Monitoring Signaling by the p75NTR Receptor Utilizing a Caspase-3 Activation Assay Amenable to Small-Molecule Screening.

p75^NTR is a neurotrophin receptor that can mediate either survival or death of neurons depending on the cell context. Modulation of p75^NTR is a promising strategy to promote neuronal survival for treatment of cognitive disorders such as Alzheimer's disease. Despite years of investigation into the signaling mechanisms of p75^NTR, no p75^NTR signaling assay has yet been developed that is compatible with efficient screening of small-molecule modulators. In this work, we developed a homogeneous cell-based assay for screening p75^NTR modulators and studying p75^NTR function. Stimulation of p75^NTR-transfected cells using either nerve growth factor (NGF) or Pro-NGF resulted in an enhanced caspase-3 activity as assessed by cleavage of a fluorescent caspase-3 substrate. Optimization of the assay with respect to time, cell density, NGF and Pro-NGF concentration, and other factors provided a twofold increase in the caspase-3 activity compared to background. Withdrawal of serum during the NGF or Pro-NGF treatment period was found to be essential for p75^NTR-dependent caspase-3 activation. We validated the method by demonstrating that a signaling-incompetent p75^NTR mutant could not substitute for wild-type p75^NTR in mediating caspase-3 activation. A focused library screen identified new inhibitors of p75^NTR signaling. This method will be useful for identifying small- molecule modulators of p75^NTR as well as further characterizing downstream signaling events. [ABSTRACT FROM AUTHOR]