Methods for identifying inhibitors of neuronal degeneration
The present invention concerns methods and means for identifying inhibitors of neuronal degeneration, and their use in the treatment of neurodegenerative disorders. In particular the invention concerns methods and means for identifying inhibitors of neuronal degeneration or cell death by taking advantage of the involvement of presenilin (PS) and Par-4 in NF-κB activation.
1. A method for identifying inhibitors of neuronal degeneration comprising:
(A) (1) cotransfecting eukaryotic host cells expressing a presenilin protein (PS), with a polynucleotide encoding a prostate apoptosis response-4 (Par-4) polypeptide, and an NF-κB dependent reporter construct, (2) exposing the cotransfected cells to a candidate molecule, (3) monitoring the ability of said candidate molecule to induce NF-κB activation, (4) comparing the level of NF-κB activation in the cells exposed to the candidate molecule to the level of NF-κB activation in at least one comparable control sample and (5) identifying an inhibitor of neuronal degeneration when the level of NF-κB activation in the exposed cells is significantly greater than the level of NF-κB activation in the comparable control sample; or
(B) (1) transfecting eukaryotic host cells endogenously expressing prostate apoptosis response-4 (Par-4) polypeptide and a presenilin (PS) protein with nucleic acid encoding an NF-κB dependent reporter construct, (2) exposing the transfected cells to a candidate molecule, (3) monitoring the ability of said candidate molecule to induce NE-κB activation, (4) comparing the level of NF-κB activation in the cells exposed to the candidate molecule to the level of NF-κB activation in at least one comparable control sample and (5) identifying an inhibitor of neuronal degeneration when the level of NF-κB activation in the exposed cells is significantly greater than the level of NF-κB activation in the comparable control sample.
2. The method of claim 1 wherein said eukaryotic host cells of part (A) are mammalian cells endogenously expressing PS.
3. The method of claim 1 wherein said eukaryotic host cells of part (A) are mammalian cells transfected with nucleic acid encoding PS.
4. The method of claim 1 wherein said PS is PS1.
5. The method of claim 4 wherein said PS1 is human.
6. The method of claim 1 wherein said PS is FAD PS.
7. The method of claim 6 wherein said FAD PS is FAD PS1.
8. The method of claim 7 wherein said FAD PS1 is human.
9. The method of claim 1 wherein said eukaryotic host cells of part (A) are neuronal cells.
10. The method of claim 9 wherein said neuronal cells are cerebellar granule cells.
11. The method of claim 9 wherein said neuronal cells are organotypic brain cells obtained from transgenic mice genetically engineered to express human PS1.
12. The method of claim 11 wherein said human PS1 is FAD PS1.
13. The method of claim 1 wherein said NF-κB dependent reporter construct comprises a luciferase reporter gene.
14. The method of claim 13 wherein said NF-κB dependent reporter construct comprises NF-κB-binding consensus sites linked to a luciferase reporter gene.
15. The method of claim 1 wherein the ability of said candidate molecule to induce NF-κB activation is monitored in comparison with a known inducer of NF-κB activation.
16. The method of claim 15 wherein said known inducer of NF-κB activation is TNF-α.
17. The method of claim 1 wherein said eukaryotic host cells of part (B) are HeLa cells.