Overexpression of Stat3 promotes neuronal cell death through activation of microglia

Background and aims: Previously, we reported that Stat3 was activated following focal cerebral ischemia in mouse [1]. However, little is known about the function of Stat3 in the ischemic brain. Here we investigated the effect of Stat3 overexpression and co-culture with microglia on neurons. Methods & Results: Recombinant adenovirus expressing Stat3 wild type (St3.Wt), Stat3 dominant negative type (St3.F) and LacZ were constructed as described previously [2]. In vitro : Rat cultured neurons were exposed to adenovirus vector expressing St3.Wt(AdSt3.Wt), St3.F(AdSt3.F) or LacZ(AdLacZ) for 2h and incubated with fresh medium up to 72h. Then, neurons were co-cultured with rat cultured microglia in two different ways. 1) direct co-culture: microglia were added directly to 24 well plates containing neurons. 2) insert co-culture: microglia were seeded into cell culture inserts that were placed on the top of the 24 well plates containing neurons. At 48 hr after co-culture, cells were homogenized in lysis buffer and immunoblotted with antibody against Stat3, p-Stat3(Tyr-705) and MAP2. In vivo: A fine glass micropipette was introduced into the left caudoputamen in the rats. Twenty microliters of adenovirus vectors, AdSt3.Wt, AdSt3.F or AdLacZ were administered over a 20 min period. Two days later, the left middle cerebral artery (MCA) of the infected animal was occluded. One day after MCA occlusion, infarct volume was evaluated using TTC staining. Results: As shown in Figs A and B, overexpression of St3.Wt induced p-Stat3 and cell death in neurons after direct co-culture with microglia but not after insert co-culture with microglia. These results suggested that direct contact of microglia with neurons expressing St3.Wt induced stat3 activation and cell death in neurons. Moreover, in vivo experiment revealed that the infarct volume of the AdSt3.Wt-treated group was significant larger than that of AdLacZ-treated group while the infarct volume of the AdSt3.F-treated group was significant smaller than that of AdLacZ-treated group suggesting deteriorative effects of Stat3 on the ischemic brain. Conclusion: Our data revealed that overexpression of Stat3 promoted neuronal cell death through activation of microglia. These results suggest that activation of Stat3 might play a crucial role in the inflammatory reaction after cerebral ischemia.