Hsp20 Protects Neuroblastoma Cells from Ischemia/Reperfusion Injury by Inhibition of Apoptosis via a Mechanism that Involves the Mitochondrial Pathways

Hsp20 is a chaperone protein that is highly and constitutively expressed in the brain, cardiac tissue and many other organs. Recently, it is well established that Hsp20 can enhance cardiac function and render cardioprotection. However, the potential benefits of Hsp20 and its phosphorylation form action on ischemic stroke and the underlying mechanism(s) are largely unknown. To investigate whether Hsp20 exerts protective effects on in vitro ischemia/ reperfusion (I/R) injury, mouse neuroblastoma cells were subjected to oxygen-glucose deprivation (OGD) and reoxygenation. Expressions of Hsp20 were strongly downregulated in mouse N2A cells at the 0-hour and 6-hour recovery time points following 4 hours of OGD, and returned to basal level 12 and 24 hours after OGD treatment, both at mRNA and protein levels. The ratio of phosphorylated to total Hsp20 protein was not significantly affected at the 0-hour and 6- hour recovery time points following 4 hours of OGD. However, markedly higher serine phosphorylation of Hsp20 was observed 12 and 24 hours after OGD treatment. Furthermore, overexpression of Hsp20 reduced OGD-induced apoptosis by reducing the release of cytochrome c from mitochondria to cytosol. However, blockade of Hsp20 phosphorylation at Ser16 abrogated this anti-apoptotic effect. In conclusion, our data demonstrated that increased Hsp20 expression in mouse N2A neuroblastoma cells protected against I/R injury, resulting in reduced apoptosis with the decrease of the release of cytochrome c from mitochondria to cytosol. Phosphorylation of Ser16 played an important role in the neuroprotective effect of Hsp20. Thus, Hsp20 may constitute a new therapeutic target for cerebral ischemic diseases. Keywords: Apoptosis, cytochrome c, Hsp20, ischemia/reperfusion (I/R), mitochondria, oxygen-glucose deprivation (OGD), N2A cells, Phosphorylation, Ser16, Stroke, Alzheimer's disease, ATCC, DMEM, Quantitative Real-Time PCR, Western Blot Analysis, ECL, X-ray film, Immunoprecipitation, Plasmid, Cell Apoptosis Assays, Bonferroni's post-hoc test, pEGFP-Hsp20, apoptotic cells, pEGFP-N1, sHsp, aB-crystallin, cardiomyocytes, MicroRNAs, NF-κB, cerebral ischemia, Ca2+, neuroprotection