Neuronal loss due to ischemic injury, trauma or disease can lead to devastating consequences for the individual. With the goal of trying limit neuronal loss a number of cell death pathways have been studied but there may be additional contributors to neuronal death that are yet unknown. To identify previously unknown cell death mediators we performed a high content genome-wide siRNA screen with a siRNA library in murine neural stem cells following exposure to N-Methyl-N-nitroso-N′- nitroguanidine (MNNG) which leads to DNA damage and cell death. 80 genes were identified as key mediators for cell death. Among these genes, 14 genes are known cell death mediators and 66 are genes that have not previously been linked to cell death pathways. Using an integrated approach with functional and bioinformatics analysis, we provide possible molecular networks, interconnected pathways and/or protein complexes that may participate in cell death. Of these 66 genes we selected CCR3 for further evaluation and find that CCR3 is a mediator of neuronal injury. CCR3 inhibition or deletion protects murine cortical cultures from oxygen-glucose deprivation induce cell death and CCR3 deletion in mice provides protection from ischemia in vivo. Taken together our findings suggest that CCR3 is a previously unknown mediator of cell death and future identification of the neural cell death network that CCR3 participates in will enhance our understanding of the molecular mechanisms of neural cell death.
Significance Statement: A high content siRNA genome-wide identified 80 mediators of cell death in neural stem cells in response to DNA damage injury. These cell death mediators possibly form a molecular network containing several partially overlapping and interconnected pathways and/or protein complexes. Through in vitro and in vivo experiments, we confirmed one of the identified molecules, CCR3 is a mediator of neuronal cell death. Further investigation of the mechanisms of CCR3 action in neuronal injury is warranted. Additionally, the other 65 genes may provide new opportunities to understand and prevent neuronal cell death.
Authors report no conflict of interest.
American Heart Associate (AHA) Postdoctoral Fellowship to Dr. J. Zhang (0725470U). AHA Grant-in-Aid to Dr. J. Zhang (11GRNT7810020). AHA Scientist Development Grant to Dr. J. Zhang (12SDG8940000). NIH RO1 grant to Dr. VL Dawson (RO1AG029368). Simons Foundation Autism Research Initiative (SFARI) to Dr. TM Dawson. National Natural Science Foundation of China to Dr. J. Zhang (81271415 and 31471016). PUMC Scholarship to Dr. J. Zhang (2012RC01).