Monitoring Signaling by the p75 super(NTR) Receptor Utilizing a Caspase-3 Activation Assay Amenable to Small-Molecule Screening

p75 super(NTR) is a neurotrophin receptor that can mediate either survival or death of neurons depending on the cell context. Modulation of p75 super(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 super(NTR), no p75 super(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 super(NTR) modulators and studying p75 super(NTR) function. Stimulation of p75 super(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 super(NTR)-dependent caspase-3 activation. We validated the method by demonstrating that a signaling-incompetent p75 super(NTR) mutant could not substitute for wild-type p75 super(NTR) in mediating caspase-3 activation. A focused library screen identified new inhibitors of p75 super(NTR) signaling. This method will be useful for identifying small-molecule modulators of p75 super(NTR) as well as further characterizing downstream signaling events.