Liu Danni, McIlvain H Beal, Fennell Myles, Dunlop John, Wood Andrew, Zaleska Margaret M, Graziani Edmund I, Pong Kevin
Neuroscience Discovery Research, Wyeth Research, Princeton, NJ 08543, United States.
J Neurosci Methods. 2007 Jul 30;163(2):310-20. doi: 10.1016/j.jneumeth.2007.03.018. Epub 2007 Apr 5.
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.
免疫亲和素是免疫抑制剂药物FK506、环孢素A（CsA）和雷帕霉素的蛋白质受体。免疫亲和素分为两类，即FK506结合蛋白（FKBPs），它可与FK506、雷帕霉素和CCI-779结合；还有亲环蛋白，可与CsA结合。报告显示，免疫亲和素在脑和脊髓中表达，在中枢神经系统组织中的含量比免疫组织高10到100倍，并且在神经损伤后其表达增加，这表明它们的化学配体可能在神经退行性疾病的治疗中具有治疗作用。在本研究中，我们报告了一种快速神经丝（NF）酶联免疫吸附测定（ELISA）用于量化神经元存活情况的开发和应用，以及Cellomics ArrayScan平台用于量化免疫亲和素配体处理后神经突生长情况的开发和应用。将培养的神经元或F-11细胞用各种免疫亲和素配体处理72或96小时，然后测定它们对神经元存活和神经突生长的促进作用。结果表明，与对照培养物相比，所有免疫亲和素配体均以浓度依赖性方式显著增加了神经元存活和神经突生长。综上所述，这些结果证明了神经丝ELISA和Cellomics ArrayScan平台在有效量化免疫亲和素配体对培养的神经元和细胞系的神经营养作用方面的潜在应用价值。
