High-Content Genome-Wide RNAi Screen Reveals CCR3 as a Key Mediator of Neuronal Cell Death

siRNA screening for genes required for DNA damage–induced neural cell death. A, Schematic diagram of lentiviral siRNA library screening. B, Fragments of siRNA inserts were amplified from lentivirus-infected, MNNG-treated surviving neural stem cells and reference control cells (lentivirus infection but no MNNG treatment) by two rounds PCR. C, Scatterplot analysis of siRNA inserts enriched in neural stem cells treated by MNNG.

Validation of the siRNA library screening. A, Seventeen genes were randomly selected from the 80 identified in the screen, and expression was knocked down in primary cultured cortical neurons (7 days in vitro) by lentiviral siRNAs for the 17 genes. B, Representative images of neurons treated with lethal OGD (90 min) and stained with Hoechst 33342 and propidium iodide (PI). Dead neurons were scored as red cells with condensed or fragmented nuclei. Non-virus-infected neurons were used as viral control, and neurons infected with lentiviral siRNA for DsRed were used as siRNA control. Scale bar =100 µm. C, Quantification of cell death in B. Experiments were performed three times, and data represent the mean ± SEM. Statistical significance from DsRed siRNA control indicated at *p < 0.05; differences between multiple groups were evaluated by one-way ANOVA followed by the Tukey–Kramer post hoc test.

GSEA of identified genes required for neural cell death. The expression data are ranked in the order of differential expression. A, Forty-seven core enrichment factors from the siRNA library screen for DNA damage–induced cell death genes classified by GSEA analysis. B, Five individual plots (p-value <0.05) of the running sum for the gene sets in the ranked gene list. C, Subcellular localization was categorized using the top 80 genes from the siRNA library screen. Genes with no annotations were excluded from the analysis.

Network analysis of identified genes required for DNA damage–induced neural cell death. Screen results for 80 genes from siRNA library screen and 20 evidence-based predicted genes were mapped onto the network, and highly interconnected clusters containing factors identified from the siRNA library screens were isolated, representing the programmed cell death clusters important for DNA damage–induced neural cell death. A network was initially constructed by Cytoscape v3.3.0 software with GeneMANIA plugin using limited database search for predicted and literature (evidence)-based coexpressed genes. The network was then visually rearranged with combination of GO term results [cellular components (CC) molecular functions (MF), and pathways] from the Database for Annotation, Visualization, and Integrated Discovery and GSEA.

CCR3 deletion or inhibition protects neurons against OGD-induced excitotoxicity. A, Primary cultured cortical neurons were isolated from CCR3 KO and WT embryos at embryonic day 15. Neurons (14 days in vitro) were pretreated with or without CCR3 inhibitor SB328437 at the indicated dose before being subjected to OGD treatment for 90 min. Twenty-four hours after OGD, neurons were stained with Hoechst 33342 and propidium iodide (PI). Dead neurons were scored as red cells with condensed or fragmented nuclei. Scale bar =100 µm. B, Quantification of cell death in different treatments. Experiments were performed at least three times. Data represent the mean ± SEM. * p<0.05, one-way ANOVA with Tukey–Kramer test.