Monitoring signaling by the p75(NTR) 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.
Department of Biology, Elan Pharmaceuticals, South San Francisco, California, USA. michael.bradshaw@principiabio.com
J Michael
JM
Bradshaw
Lan
L
Nguyen
William
W
Wallace
Calvin
C
Li
John-Michael
JM
Sauer
Frédérique
F
Bard
Michael P
MP
Bova
D000818:Animals; D053148:Caspase 3 / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004789:Enzyme Activation / Q000187:drug effects*; D005455:Fluorescent Antibody Technique; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D020932:Nerve Growth Factor / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000502:physiology*; D051381:Rats; D020917:Receptor, trkA / Q000187:drug effects; D017475:Receptors, Nerve Growth Factor / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects*; D054852:Small Molecule Libraries; D014162:Transfection
C484065:NGFR protein, human; D009419:Nerve Tissue Proteins; D017475:Receptors, Nerve Growth Factor; D054852:Small Molecule Libraries; D020932:Nerve Growth Factor; D020917:Receptor, trkA; D053148:Caspase 3