Figure 8
Kainic acid (KA)-induced excitotoxicity causes truncation of GSK-3β and an increase in tau phosphorylation in mouse brain.
(A, B) Western blots show C-terminal truncation of GSK-3β (A) and hyperphosphorylation of tau (B) in brains of mice intraperitoneally injected with KA. Homogenates of forebrains of mice sacrificed at the indicated time points after single intraperitoneal KA injection were analyzed by Western blots developed with GSK-3β antibodies indicated at right side of each blot. (C, D) KA-induced excitotoxicity increased phosphorylation of tau at Ser 199 and Ser 396. Forebrain homogenates of KA-injected mice were analyzed by Western blots (B) developed with anti-pSer199, PHF-1 (pS396), anti-total-tau, anti-NeuN and anti-GAPDH. The levels of total tau (C) were normalized with NeuN or GAPDH and Phosphorylation of tau at Ser 199 and Ser 396 (D) were normalized with total tau level and plotted against the time after KA-injection. The data are presented as mean of two mice. (E, F) Inhibition of calpain prevents kainic acid (KA)-induced truncation and phosphorylation of GSK-3β in mouse brains. Calpain inhibitor calpeptin was intracerebroventricularly injected 3 hr before KA intraperitoneal injection. Homogenates of forebrains of mice sacrificed after 12 hr KA injection were analyzed by Western blots developed with GSK-3β and quantified densitometrically. The level of pSer9-GSK-3β (normalized with GSK-3β) (F) is shown as mean ± S.D. (n = 4–6). (G, H) Inhibition of calpain suppressed tau phosphorylation at Ser396 caused by KA even though inhibitory phosphorylation of GSK-3β at Ser 9 was decreased. Total tau and phosphorylated tau at Ser 396 in above samples were analyzed by Western blots (G) and quantified as described above. The level of phosphorylated tau at Ser 396 normalized with total tau (H) is shown as mean ± S.D. (n = 4–6); *, KA Via Con and p < 0. 05; #, KA+Calp via KA and p < 0. 05. *, indicates truncated GSK-3β.
