PP2, a potent inhibitor of Src family kinases, protects against hippocampal CA1 pyramidal cell death after transient global brain ischemia

It has been indicated that Src family protein tyrosine kinases (SrcPTKs) potentiate N-methyl-d-aspartate (NMDA) receptor function by phosphorylating NR2A subunits and that postsynaptic density protein 95 (PSD-95) facilitates this regulation. In this paper, we define the role of SrcPTKs in delayed neuronal damage following transient brain ischemia and explore the underlying mechanisms involved in this event. Transient global brain ischemia was induced by the four-vessel occlusion method. A specific Src family kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine) and a PP2 negative control PP3 (4-amino-7-phenylpyrazolo[3,4-d]pyramidine) were infused into rat cerebroventricule 30 min before occlusion. Hematoxylin and eosine staining showed that the number of surviving pyramidal neurons in rat hippocampal CA1 subfield increased markedly in PP2-treated rats comparing to PP3-treated groups after 5 days of reperfusion following ischemia. Additionally, immunoprecipitation and immunoblot analysis revealed that preadministration of PP2, but not PP3, attenuated not only the increased tyrosine phosphorylation of NR2A but also the enhanced interactions among Src, NR2A and PSD-95 induced by ischemia/reperfusion. In conclusion, SrcPTKs promote binding of the kinases and their substrate NR2A attributed to the scaffolding effect of PSD-95 during transient brain ischemia and reperfusion, which are responsible for the elevation of NR2A tyrosine phosphorylation and consequent delayed neuronal cell death.