Neuroprotection via RNA-binding protein RBM3 expression is regulated by hypothermia but not by hypoxia in human SK-N-SH neurons

OBJECTIVE Therapeutic hypothermia is an established treatment for perinatal asphyxia. Yet, many term infants continue to die or suffer from neurodevelopmental disability. Several experimental studies have demonstrated a beneficial effect of mild-to-moderate hypothermia after hypoxic injury, but the understanding of hypothermia-induced neuroprotection remains incomplete. In general, global protein synthesis is attenuated by hypothermia, but a small group of RNA-binding proteins including the RNA-binding motif 3 (RBM3) is upregulated in response to cooling. The aim of this study was to establish an in vitro model to investigate the effects of hypoxia and hypothermia on neuronal cell survival, as well as to examine the kinetics of concurrent cold-shock protein RBM3 gene expression. METHODS Experiments were performed by using human SK-N-SH neurons exposed to different oxygen concentrations (21%, 8%, or 0.2% O2) for 24 hours followed by moderate hypothermia (33.5°C) or normothermia for 24, 48, or 72 hours. Cell death was determined by quantification of lactate dehydrogenase and neuron-specific enolase releases into the cell cultured medium, and cell morphology was assessed by using immunofluorescence staining. The regulation of RBM3 gene expression was assessed by reverse transcriptase-quantitative polymerase chain reaction and Western blot analysis. RESULTS Exposure to hypoxia (0.2% O2) for 24 hours resulted in significantly increased cell death in SK-N-SH neurons, whereas exposure to 8% O2 had no significant impact on cell viability. Post-hypoxia treatment with moderate hypothermia for 48 or 72 hours rescued the neurons from hypoxia-induced cell death. Moreover, exposure to severe hypoxia led to observable cell swelling, which was also attenuated by moderate hypothermia. Finally, moderate hypothermia but not hypoxia led to the induction of RBM3 expression on both transcriptional and translational levels. CONCLUSION Moderate hypothermia protects neurons from hypoxia-induced cell death. The expression of the cold-shock protein RBM3 is induced by moderate hypothermia and could be one possible mediator of hypothermia-induced neuroprotection.