p75NTR is a neurotrophin receptor that can mediate either survival or death of neurons depending on the cell context. Modulation of p75NTR 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 p75NTR, no p75NTR 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 p75NTR modulators and studying p75NTR function. Stimulation of p75NTR-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 p75NTR-dependent caspase-3 activation. We validated the method by demonstrating that a signaling-incompetent p75NTR mutant could not substitute for wild-type p75NTR in mediating caspase-3 activation. A focused library screen identified new inhibitors of p75NTR signaling. This method will be useful for identifying small-molecule modulators of p75NTR as well as further characterizing downstream signaling events.
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