Changes in heat shock protein 27 phosphorylation and immunocontent in response to preconditioning to oxygen and glucose deprivation in organotypic hippocampal cultures.

Organotypic hippocampal cultures have been recently used to study in vitro ischaemic neuronal death. Sub-lethal periods of ischaemia in vivo confer resistance to lethal insults and many studies have demonstrated the involvement of heat shock proteins in this phenomenon. We used organotypic hippocampal cultures to investigate the involvement of heat shock protein (HSP) 27 in preconditioning to oxygen and glucose deprivation. Neuronal damage was assessed using propidium iodide fluorescence; HSP27 phosphorylation and immunocontent were obtained using (32)Pi labelling followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis and immunoblotting. We observed that immunocontent of HSP27 was increased after lethal or sub-lethal treatment, indicating it is a response to metabolic stress. Treatments with 5 or 10 min of oxygen and glucose deprivation (OGD) or 1- microM N-methyl-D-aspartate (NMDA) induced tolerance to 40 min of OGD associated with an increase in HSP27 immunocontent and phosphorylation. These data suggest that, in vitro, phosphorylated HSP27 might be involved in preconditioning, probably acting as a modulator of actin filaments or by the blockage of neurodegenerative processes.

Effects of global cerebral ischemia and preconditioning on heat shock protein 27 immunocontent and phosphorylation in rat hippocampus.

Global cerebral ischemia, with or without preconditioning, leads to an increase in heat shock protein 27 (HSP27) immunocontent and alterations in HSP27 phosphorylation in CA1 and dentate gyrus areas of the hippocampus. We studied different times of reperfusion (1, 4, 7, 14, 21 and 30 days) using 2 min, 10 min or 2+10 min of ischemia. The results showed an increase in HSP27 immunocontent of about 300% after 10 min of ischemia in CA1 and dentate gyrus. CA1, a hippocampal vulnerable area, showed an increase in HSP27 phosphorylation, parallel with immunocontent. In dentate gyrus, a resistant area, the increase in HSP phosphorylation was lower than immunocontent. After preconditioned ischemia (2+10 min), when CA1 neurons are protected to a lethal, 10 min insult, we observed an increase in HSP immunocontent and a decrease in phosphorylation in both regions of the hippocampus, suggesting that, when there is no neuronal death, HSP27 in a vulnerable area responds similarly to the resistant area.When dephosphorylated, HSP27 acts as a chaperone, protecting other proteins from denaturation. As it is markedly expressed in astrocytes, we suggest that HSP27 could be protecting hippocampal astrocytes, which could then be helping neurons to resist to the insult, maintaining tissue normal homeostasis.