Anesthesia and post-mortem interval profoundly influence the regulatory serine phosphorylation of glycogen synthase kinase-3 in mouse brain

Abstract

Glycogen synthase kinase-3 (GSK3) is a crucial enzyme contributing to the regulation of neuronal structure, plasticity and survival, is implicated as a contributory factor in prevalent diseases such as Alzheimer's disease and mood disorders and is regulated by a wide range of signaling systems and pharmacological agents. Therefore, factors regulating GSK3 in vivo are currently of much interest. GSK3 is inhibited by phosphorylation of serine-9 or serine-21 in GSK3beta and GSK3alpha, respectively. This study found that accurate measurements of phospho-Ser-GSK3 in brain are confounded by a rapid post-mortem dephosphorylation, with approximately 90% dephosphorylation of both GSK3 isoforms occurring within 2 min post-mortem. Furthermore, three anesthetics, pentobarbital, halothane and chloral hydrate, each caused large in vivo increases in the serine phosphorylation of both GSK3beta and GSK3alpha in several regions of mouse brain. Thus, studies of the phosphorylation state of GSK3 in brain, and perhaps in other tissues, need to take into account post-mortem changes and the effects of anesthetics and there is a direct correlation between anesthesia and high levels of serine-phosphorylated GSK3.

Fig. 1

Phospho-Ser-GSK3 is rapidly dephosphorylated post-mortem and is increased by anesthesia. (a) Mouse brains were incubated at room temperature (22°C) for 0, 10, 20 or 30 min post-mortem before rapid dissection and homogenization. Samples of the cerebral cortex were immunoblotted for phospho-Ser9-GSK3β, phospho-Ser21-GSK3α, phospho-Tyr-GSK3α/β and total GSK3α/β (upper band, GSK3α; lower band, GSK3β). Anesthesia was induced in mice by administration of (b) pentobarbital (Pb; 100 mg/kg; 15 min), (c) halothane (HL; 5 min) or (d) chloral hydrate (CH; 600 mg/kg; 2 min). Anesthetic agents were tested individually in different experiments with paired control mice (Ctl) which received no anesthetic. Hippocampal and cerebral cortical samples were immunoblotted for phospho-Ser9-GSK3β, phospho-Ser21-GSK3α, total GSK3β and total GSK3α.

Fig. 2

Similar post-mortem loss of phospho-Ser-GSK3 occurs in brains from control and pentobarbital-treated mice. (a) Hippocampus and (b) cerebral cortex of control and pentobarbital-anesthetized (100 mg/kg; 15 min) mice were extracted following post-mortem delays of 0, 2, 5 or 10 min. Samples were immunoblotted for phospho-Ser9-GSK3β and the loss of phosphorylation was calculated from densitometric measurements and presented as a percentage of the 0 min value of control or pentobarbital-treated mice.

Fig. 3

Time dependence of pentobarbital-induced increases in phospho-Ser-GSK3. The levels of phospho-Ser9-GSK3β, phospho-Ser21-GSK3α, total GSK3β and total GSK3α were measured in the hippocampus, cerebral cortex, striatum and cerebellum after administration of pentobarbital (Pb; 100 mg/kg) during anesthesia at 1, 2 or 4 h and after recovery from anesthesia 6 h after pentobarbital administration.