Genetic Screen Identified Prmt5 as a Neuroprotection Target against Cerebral Ischemia [RNA-seq]

Epigenetic regulators present novel opportunities for both ischemic stroke research and therapeutic interventions. While previous work has implicated that they may provide neuroprotection by potentially influencing coordinated sets of genes and pathways, most of them remains largely uncharacterized in ischemic conditions. In this study, we used the oxygen-glucose deprivation (OGD) model in the immortalized mouse hippocampal neuronal cell line HT-22 and carried out an RNAi screen on epigenetic regulators. We identified Prmt5 as a novel negative regulator of neuronal cell survival after OGD, which presented a phenotype of translocation from the cytosol to the nucleus upon oxygen and energy depletion both in vitro and in vivo. Prmt5 bound to the chromatin and a large number of promoter regions to repress downstream gene expression. Silencing Prmt5 significantly dampened the OGD-induced changes for a large-scale of genes, and gene ontology analysis showed that Prmt5-target genes were highly enriched for Hedgehog signaling. Encouraged by the above observation, we treated mice with middle cerebral artery occlusion (MCAO) with the Prmt5 inhibitor EPZ015666 and found that Prmt5 inhibition sustain protection against neuronal death in vivo. Together, our findings revealed a novel epigenetic mechanism of Prmt5 in cerebral ischemia and uncovered a potential target for neuroprotection. HT-22 cells were kindly provided by Dr. Richard Dargusch (Salk). Cells were routinely cultured in Dulbecco’s Modified Eagle’s Medium (Gibco) supplemented with 10% fetal bovine serum (Gibco), and the cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2. The cultured neurons were used for experiments within 8-10 days. All experimental protocols and animal handling procedures were performed in accordance with the National Institutes of Health (NIH) guidelines for the use of experimental animals and were approved by the Animal Care and Use Committee of The Second Affiliated Hospital of Air Force Medical University. Oxygen-glucose deprivation (OGD) treatment was carried out as previously described. Briefly, cells were rinsed and cultured in the glucose-free Earle’s balanced salt solution (BSS) with the following composition (in mmol/L): 116 NaCl, 5.4 KCl, 0.8 MgSO4·7H2O, 1 NaH2PO4·2H2O, 26.2 NaHCO3, 0.01 glycine, 1.8 CaCl2·2H2O, and pH 7.4. The culture was then placed in an Hypoxia Incubator Chamber (StemCell Technology) and were equilibrated for 15 minutes with a continuous flux of 95% N2/5% CO2 gas. The chamber was then sealed and placed into a humidified incubator at 37°C for 8 hours (HT-22) or 60 minutes (primary cortical neuron). OGD was terminated by removing the cultures from the chamber, replacing BSS with normal culture medium, and returning cells to the normoxic conditions for 24 hours. Cells cultured under normal conditions during the experimental period were used as controls. For Prmt5 inhibitor treatment, EPZ015666 (Selleckchem Company) was prepared in dimethyl sulfoxide (DMSO) at a concentration of 100 mM and stored at -20 °C before use. EPZ (10μM final concentration) or DMSO was added to cells 20 minutes before the initiation of OGD and to the BSS during the OGD treatment. For siRNA transfection of the primary neurons, the culture medium was refreshed at day 7. Cells were transfected with siRNAs using Lipofectamine 3000 according to manufacturer’s instructions. The medium was replaced the next day, and OGD experiments were performed 48 h after transfection. For pLKO.1 shRNAs, complementary single-stranded oligos were annealed and cloned as suggested by the RNAi consortium. pGIPZ shRNAs were ordered from Dharmacon. All plasmids were confirmed by sequencing. The shRNA library used for the screen was as previous describe. shRNA lentivirus was prepared by transfecting 293T cells using TransIT-293 (Mirus) using a standard protocol from Addgene. Total RNA was isolated from cells using the GeneJet RNA purification kit (Thermo Scientific), and 0.5 μg total RNA was reverse transcribed to generate cDNA using the iScriptTM cDNA Synthesis Kit (Bio-Rad) according to manufacturer’s instructions. RT-qPCRs were performed using the SsoFastTM EvaGreen Supermix (Bio-Rad) on the Bio-Rad CFX-384 Real-Time PCR System. Actin was used for normalization. All experiments were performed at least three times, and representative results were shown in the figures. For RNA-seq, libraries were prepared from two biological replicates using the TruSeq RNA Sample Prep Kit and sequenced on the NextSeq (Illumina).