Patent ID: 7169568
Filing Date: 2007-01-30
Classification: C12Q,G01N

Abstract:
1. A method for screening and identifying molecules that mediate neuronal cell survival in the absence of neurotrophic factors and transactivate a neurotrophic receptor, comprising assay A or assay A in combination with either or both of assay B and assay C, wherein: assay A comprises: treating neuronal cells, PC12 cells, or N2a neuroblastoma cells with a candidate transactivator molecule; reacting the neurotrophic receptor, obtained from a cell lysate of the treated cells, with an anti-phosphotyrosine antibody specific for the phosphorylated form of the neurotrophic receptor; and detecting binding of the anti-phosphotyrosine antibody to the phosphorylated form of the neurotrophic receptor to identify a transactivator molecule of the neurotrophic receptor that mediates neuronal cell survival in the absence of neurotrophins by transactivating the neurotrophic receptor; assay B comprises: treating neuronal cells, PC12 cells, or N2a neuroblastoma cells with a candidate transactivator molecule; reacting either phosphotidylinositol 3′-kinase (PI3-K), obtained from a cell lysate of the treated cells, with an anti-phospho-PI3-K antibody specific for the phosphorylated form of PI3-K or Akt, obtained from a cell lysate of the treated cells, with an anti-phospho-Akt antibody specific for the phosphorylated form of Akt; and detecting binding of the anti-phospho-PI3-K antibody to the phosphorylated form of PI3-K or binding of the anti-phospho-Akt antibody to the phosphorylated form of Akt to identify a transactivator molecule of the neurotrophic receptor and its downstream Akt target; and assay C comprises: culturing neuronal cells, PC12 cells, or N2a neuroblastoma cells in the presence of neurotrophic factors; treating and culturing the cells with a candidate transactivator molecule in the absence of neurotrophic factors; and determining the level of cell survival to identify a transactivator molecule of the neurotrophic receptor, wherein an increase in cell survival, or a decrease in cell death, over a control in which the cells are cultured in the absence of neurotrophic factors and the candidate transactivator molecule, identifies a transactivator molecule.