title: Screening of immunophilin ligands by quantitative analysis of neurofilament expression and neurite outgrowth in cultured neurons and cells.
abstract: Immunophilins are protein receptors for the immunosuppressant drugs FK506, cyclosporin A (CsA), and rapamycin. Two categories of immunophilins are the FK506-binding proteins (FKBPs), which bind to FK506, rapamycin, and CCI-779 and the cyclophilins, which bind to CsA. Reports have shown that immunophilins are expressed in the brain and spinal cord, are 10-100-fold higher in CNS tissue than immune tissue, and their expression is increased following nerve injury, suggesting that their chemical ligands may have therapeutic utility in the treatment of neurodegenerative diseases. In this study, we report the development and utility of a rapid neurofilament (NF) enzyme-linked immunosorbent assay (ELISA) to quantify neuronal survival and the Cellomics ArrayScan platform to quantify neurite outgrowth following treatment with immunophilin ligands. Cultured neurons or F-11 cells were treated with various immunophilin ligands for 72 or 96h and their promotion of neuronal survival and neurite outgrowth were determined. The results showed that all immunophilin ligands, in a concentration-dependent manner, significantly increased neuronal survival and neurite outgrowth, when compared to control cultures. Taken together, these results demonstrate the potential utility of the neurofilament ELISA and Cellomics ArrayScan platform to efficiently quantify neurotrophic effects of immunophilin ligands on cultured neurons and cell lines.
authors.affiliation: Neuroscience Discovery Research, Wyeth Research, Princeton, NJ 08543, United States.
authors.forename: Danni
authors.initials: D
authors.lastname: Liu
authors.forename: H Beal
authors.initials: HB
authors.lastname: McIlvain
authors.forename: Myles
authors.initials: M
authors.lastname: Fennell
authors.forename: John
authors.initials: J
authors.lastname: Dunlop
authors.forename: Andrew
authors.initials: A
authors.lastname: Wood
authors.forename: Margaret M
authors.initials: MM
authors.lastname: Zaleska
authors.forename: Edmund I
authors.initials: EI
authors.lastname: Graziani
authors.forename: Kevin
authors.initials: K
authors.lastname: Pong
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000295:instrumentation / Q000379:methods*; D002452:Cell Count / Q000295:instrumentation / Q000379:methods; D018929:Cell Culture Techniques / Q000379:methods; D049108:Cell Enlargement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D020104:Immunophilins / Q000187:drug effects* / Q000378:metabolism; D007166:Immunosuppressive Agents / Q000494:pharmacology; D008024:Ligands; D016501:Neurites / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D016900:Neurofilament Proteins / Q000032:analysis* / Q000187:drug effects*; D055095:Optics and Photonics / Q000295:instrumentation; D011494:Protein Kinases / Q000187:drug effects / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D058570:TOR Serine-Threonine Kinases
chemical_list: D007166:Immunosuppressive Agents; D008024:Ligands; D016900:Neurofilament Proteins; D011494:Protein Kinases; D058570:TOR Serine-Threonine Kinases; D020104:Immunophilins