RNA Binding Protein Motif 3 Inhibits Oxygen-Glucose Deprivation/Reoxygenation-Induced Apoptosis Through Promoting Stress Granules Formation in PC12 Cells and Rat Primary Cortical Neurons

Abstract

As a sensitive cold-shock protein, RNA binding protein motif 3 (RBM3) exhibits a neuroprotective function in the condition of brain injury. However, how RBM3 is involved in acute ischemic stroke by affecting stress granules (SGs) remains unclear. Here, we established an oxygen-glucose deprivation/reperfusion (OGD/R) model in rat primary cortical neurons and PC12 cells to explore the potential mechanism between RBM3 and SG formation in acute ischemic/reperfusion (I/R) condition. The immunofluorescence results showed that the SG formation significantly decreased in rat primary cortical neurons and PC12 cells during the reperfusion period after 6 h of OGD stimulation. The western blot results, flow cytometry analysis, and cell viability assessment showed that the RBM3 expression and ratio of cell viability significantly decreased, while the rate of apoptosis increased in PC12 cells during the reperfusion period after 6 h of OGD stimulation. Co-immunoprecipitation (Co-IP) and immunofluorescence indicated that RBM3 and GTPase-activating protein-binding protein 1 (G3BP1) colocalized cytoplasm of PC12 cells after 6 h of OGD stimulation when the SGs formation reached the highest level. Besides, overexpression and knockdown of the RBM3 were achieved via plasmid transfection and CRISPR-Cas9 technology, respectively. The results of overexpression and knockdown of RBM3 gene illustrated the pivotal role of RBM3 in affecting SG formation and apoptosis level in OGD-treated PC12 cells. In conclusion, RBM3 could combine with G3BP1 resulted in increasing stress granules generation in rat primary cortical neurons and PC12 cells after 6 h of oxygen-glucose deprivation (OGD) injury, which ultimately reduced the apoptosis in OGD-induced cells. Our study may enable a new promising target for alleviating ischemia-reperfusion injury in cells.

Cooling and Sterile Inflammation in an Oxygen-Glucose-Deprivation/Reperfusion Injury Model in BV-2 Microglia

“…This difference in the dynamics of coldshock protein regulation may play an important role in the mechanism of neuroprotection induced by cooling. RBM3 has been shown to reduce neuronal apoptosis in ischemic brain injury [24,59,60]. Apoptosis is an active and, therefore, energy-dependent cell death mechanism that primarily contributes to reperfusion-induced injury.…”

“…Apoptosis is an active and, therefore, energy-dependent cell death mechanism that primarily contributes to reperfusion-induced injury. Si et al provide promising data that RBM3 is a key player in the formation of stress granules after OGD-induced injury, a cellular rescue mechanism prohibiting apoptosis [60,61]. Overexpression of RBM3 in PC12 cells resulted in attenuated apoptotic cell death and increased cell viability, whereas RBM3 knockdown had the opposite effect [60].…”

“…Si et al provide promising data that RBM3 is a key player in the formation of stress granules after OGD-induced injury, a cellular rescue mechanism prohibiting apoptosis [60,61]. Overexpression of RBM3 in PC12 cells resulted in attenuated apoptotic cell death and increased cell viability, whereas RBM3 knockdown had the opposite effect [60]. Another recent study investigating RBM3 knockout in mice after ischemic brain injury focusing on neural stem/progenitor cells (NSPC) shows that RBM3 plays an important role in neuronal regeneration after ischemic brain injury [59].…”

Stress Granules and Acute Ischemic Stroke: Beyond mRNA Translation

“…The RNA-binding protein motif 3 (RBM3) is a well-known cold shock protein with beneficial roles following a stroke in both animal models and humans [ 31 , 32 ]. Recently, the interaction between RBM3 and G3BP1 was suggested to mediate the survival of both the PC12 cell line and rat primary cortical neuronal cultures in an in vitro model of ischemia [ 33 ]. In particular, RBM3–G3BP1 union might increase the ratio of SGs by facilitating their nucleation and formation, leading to a reduction in the rate of cell death [ 33 ].…”

“…Recently, the interaction between RBM3 and G3BP1 was suggested to mediate the survival of both the PC12 cell line and rat primary cortical neuronal cultures in an in vitro model of ischemia [ 33 ]. In particular, RBM3–G3BP1 union might increase the ratio of SGs by facilitating their nucleation and formation, leading to a reduction in the rate of cell death [ 33 ]. Another RBP related to SGs, called argonaute RISC catalytic component 2 (AGO2), was found to colocalize with G3BP1 [ 34 ].…”