RESUMEN
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.
Asunto(s)
Caspasa 3/efectos de los fármacos , Evaluación Preclínica de Medicamentos/métodos , Activación Enzimática/efectos de los fármacos , Proteínas del Tejido Nervioso/fisiología , Receptores de Factor de Crecimiento Nervioso/fisiología , Transducción de Señal/efectos de los fármacos , Animales , Supervivencia Celular/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Células HEK293 , Humanos , Microscopía Fluorescente , Factor de Crecimiento Nervioso/farmacología , Ratas , Receptor trkA/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas , TransfecciónRESUMEN
Alzheimer's disease (AD) is a devastating neurodegenerative disease affecting millions of people. The amyloid hypothesis suggests that the pathogenesis of AD is related to the accumulation of amyloid beta (Aß) in the brain. Herein, the authors quantify Aß-mediated changes in neuronal morphology in primary cultures using the Cellomics neuronal profiling version 3.5 (NPv3.5) BioApplication. We observed that Aß caused a 33% decrease in neurite length in primary human cortical cultures after 24 h of treatment compared with control-treated cultures. We also determined that quantifying changes of neuronal morphology was a more sensitive indicator of nonlethal cell injury than traditional cytotoxicity assays. Aß-mediated neuronal deficits observed in human cortical cultures were also observed in primary rat hippocampal cultures, where we demonstrated that the integrin-blocking antibody, 17E6, completely abrogated Aß-mediated cytotoxicity. Finally, we showed that Aß challenge to 21 days in vitro rat hippocampal cultures reduced synapsin staining to 14% of control-treated cultures. These results are consistent with the finding that loss of presynaptic integrity is one of the initial deficits observed in AD. The implementation of phenotypic screens to identify compounds that block Aß-mediated cytotoxicity in primary neuronal cultures may lead to the development of novel strategies to prevent AD.
Asunto(s)
Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/farmacología , Bioensayo/métodos , Hipocampo/efectos de los fármacos , Fragmentos de Péptidos/farmacología , Animales , Anticuerpos Monoclonales/metabolismo , Células Cultivadas , Evaluación Preclínica de Medicamentos/métodos , Hipocampo/patología , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Integrina alfaV/inmunología , Integrina alfaV/metabolismo , Citometría de Barrido por Láser/métodos , Neuronas/efectos de los fármacos , Neuronas/patología , Ratas , Programas InformáticosRESUMEN
The structure-activity relationship (SAR) of a novel, potent and metabolically stable series of sulfonamide-pyrazoles that attenuate ß-amyloid peptide synthesis via γ-secretase inhibition is detailed herein. Sulfonamide-pyrazoles that are efficacious in reducing the cortical Aßx-40 levels in FVB mice via a single PO dose, as well as sulfonamide-pyrazoles that exhibit selectivity for inhibition of APP versus Notch processing by γ-secretase, are highlighted.