Functional identification of neuroprotective molecules

Lethal ischemic injury sets in motion a series of events that kills tissue in and around ischemic core. However, ischemic preconditioning can trigger the activation of protective genes resulting in protection against subsequent exposure to lethal ischemia. Identification of these protective genes will be useful in protecting neuron against ischemic injury and may offer neuroprotection for other neurological diseases. We developed a functional cloning strategy to identify cytoprotective genes. A precondition library was expressed by retrovirus and screened in mouse fibroblast cells for survival following PARP-1 dependent ROS (reactive oxygen species)-induced cell death. Thirty-two genes were identified from this functional cloning strategy. Their protective effects were further confirmed by introducing the full-length genes into fibroblasts and primary cortical neurons. Most of the genes show protective effects against menadione toxicity to fibroblasts and against oxygen-glucose deprivation (OGD, 90 min) and NMDA (500 muM, 5 min) toxicity to primary cortical neurons. Analysis of this thirty-two genes set reveals a representation of proteins involved in diverse biologic activities. The highest representation of protein functions are in genes that regulate energy production, regulate nuclear activity, and regulate membrane trafficking or are localized to the synapse. Two genes of known function were further characterized, calmodulin or ADP-ribosylation factor-4-like gene (ARF4L). Knock down of calmodulin or ARF4L before OGD or NMDA preconditioning abolished protective effects of preconditioning, confirming their importance in the development of neuronal survival. 531-7 is a novel gene, and overexpression of 531-7 renders fibroblasts resistant to menadione toxicity. Primary cortical neurons transfected with 531-7 are also resistant to OGD and NMDA insults. 531-7 is mainly localized in brain, and OGD and NMDA preconditioning increase expression of 531-7. Subcellular localization of 531-7 protein shows that it is highly expressed in nucleus. Yeast two-hybrid screening of 531-7 and immunoprecipitation results revealed its possible interaction with GEFs (guanine nucleotide exchange factors). These results indicate that functional cloning is a powerful method for identifying cytoprotective genes and various genes may involve in preconditioning mediated cytoprotection. Further characterization of these and other protective signals could provide new treatment opportunities for neurological injury from ischemia or neurodegenerative disease.