Method for screening molecules that exert a neurotrophic effect through activation of neurotrophin receptors

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.

2. The method of claim 1 , wherein the neurotrophic receptor is a Trk receptor.

3. The method of claim 2 , wherein the Trk receptor is TrkA receptor.

4. The method of claim 3 , wherein the cells are PC12 cells.

5. The method of claim 1 , wherein the candidate transactivator molecule is a ligand of a G protein coupled receptor (GPCR).

6. The method of claim 1 , wherein the neurotrophic receptor is a Ret receptor.

7. The method of claim 6 , wherein the cells are N2a neuroblastoma cells.

8. The method of claim 1 , wherein, in the reacting and detecting steps of assay B, Akt is reacted with anti-phospho-Akt antibody and binding of anti-phospho-Akt antibody to the phosphorylated form of Akt is detected.

9. The method of claim 8 , wherein assay B further comprises:

reacting Akt, obtained from a cell lysate of the treated cells, with an anti-Akt antibody; and

detecting binding of the anti-Akt antibody to Akt.

10. The method of claim 1 , wherein in the reacting and detecting steps of assay B, PI3-K is reacted with anti-phospho-PI3-K antibody and binding of anti-phospho-PI3-K antibody to the phosphorylated form of PI3-K is detected.

11. The method of claim 10 , wherein assay B further comprises:

reacting PI3-K, obtained from a cell lysate of the treated cells, with an anti-PI3-K antibody; and

detecting binding of the anti-PI3-K antibody to PI3-K.