Alzheimer-like changes in protein kinase B and glycogen synthase kinase-3 in rat frontal cortex and hippocampus after damage to the insulin signalling pathway

The insulin-resistant brain state is related to late-onset sporadic Alzheimer's disease, and alterations in the insulin receptor (IR) and its downstream phosphatidylinositol-3 kinase signalling pathway have been found in human brain. These findings have not been confirmed in an experimental model related to sporadic Alzheimer's disease, for example rats showing a neuronal IR deficit subsequent to intracerebroventricular (i.c.v.) treatment with streptozotocin (STZ). In this study, western blot analysis performed 1 month after i.c.v. injection of STZ showed an increase of 63% in the level of phosphorylated glycogen synthase kinase-3alpha/beta (pGSK-3alpha/beta) protein in the rat hippocampus, whereas the levels of the unphosphorylated form (GSK-3alpha/beta) and protein kinase B (Akt/PKB) remained unchanged. Three months after STZ treatment, pGSK-3alpha/beta and Akt/PKB levels tended to decrease (by 8 and 9% respectively). The changes were region specific, as a different pattern was found in frontal cortex. Structural alterations were also found, characterized by beta-amyloid peptide-like aggregates in brain capillaries of rats treated with STZ. Similar neurochemical changes and cognitive deficits were recorded in rats treated with i.c.v. 5-thio-d-glucose, a blocker of glucose transporter (GLUT)2, a transporter that is probably involved in brain glucose sensing. The IR signalling cascade alteration and its consequences in rats treated with STZ are similar to those found in humans with sporadic Alzheimer's disease, and our results suggest a role for GLUT2 in Alzheimer's pathophysiology.     

A glycogen synthase kinase 3-like kinase MpGSK regulates cell differentiation in Marchantia polymorpha

Plants precisely coordinate the balance between cell proliferation and differentiation to ensure the continuous development. In Arabidopsis thaliana, members of glycogen synthase kinase 3 (GSK3) family, which are highly conserved serine/threonine protein kinases among eukaryotes, play important roles in regulating cell proliferation and differentiation during various developmental processes. However, functional roles of GSK3s in the plant lineages except angiosperms remain to be elucidated. Here, we utilized a model liverwort, Marchantia polymorpha, for studies of GSK3, because it has a single GSK3-like kinase, MpGSK. When M. polymorpha was treated with a chemical compound, bikinin, which is known as a specific inhibitor for GSK3-like kinases, growth and morphologies were altered with an expansion of the meristematic region. Similarly, Mpgsk loss-of-function mutants accumulated undifferentiated cell mass with no differentiated tissues. By contrast, overexpression of MpGSK reduced the size of the meristem region. These results suggest that MpGSK plays important roles as a regulator for the balance between cell differentiation and proliferation in M. polymorpha.     

Glycogen synthase kinase 3β in the nucleus accumbens core mediates cocaine‐induced behavioral sensitization

Glycogen synthase kinase 3β (GSK‐3β) is a ubiquitous serine/threonine protein kinase involved in a number of signaling pathways. Previous studies have demonstrated a role for GSK‐3β in the synaptic plasticity underlying dopamine‐associated behaviors and diseases. Drug sensitization is produced by repeated exposure to the drug and is thought to reflect neuroadaptations that contribute to addiction. However, the role of GSK‐3β in cocaine‐induced behavior sensitization has not been examined. The present study investigated the effects of chronic cocaine exposure on GSK‐3β activity in the nucleus accumbens (NAc) and determined whether changes in GSK‐3β activity in the NAc are associated with cocaine‐induced locomotor sensitization. We also explored whether blockade of GSK‐3β activity in the NAc inhibits the initiation and expression of cocaine‐induced locomotor sensitization in rats using systemic or brain region‐specific administration of the GSK‐3β inhibitors lithium chloride (LiCl) and SB216763. GSK‐3β activity in the NAc core, but not NAc shell, increased after chronic cocaine (10 mg/kg, i.p.) administration. The initiation and expression of cocaine‐induced locomotor sensitization was attenuated by systemic administration of LiCl (100 mg/kg, i.p.) or direct infusion of SB216763 (1 ng/side) into the NAc core, but not NAc shell. Collectively, these results indicate that GSK‐3β activity in the NAc core, but not NAc shell, mediates the initiation and expression of cocaine‐induced locomotor sensitization, suggesting that GSK‐3β may be a potential target for the treatment of cocaine addiction.     

Selective small-molecule inhibitors of glycogen synthase kinase-3 activity protect primary neurones from death

The phosphatidylinositol 3-kinase (PI 3-kinase)/protein kinase B (PKB; also known as Akt) signalling pathway is recognized as playing a central role in the survival of diverse cell types. Glycogen synthase kinase-3 (GSK-3) is a ubiquitously expressed serine/threonine protein kinase that is one of several known substrates of PKB. PKB phosphorylates GSK-3 in response to insulin and growth factors, which inhibits GSK-3 activity and leads to the modulation of multiple GSK-3 regulated cellular processes. We show that the novel potent and selective small-molecule inhibitors of GSK-3; SB-415286 and SB-216763, protect both central and peripheral nervous system neurones in culture from death induced by reduced PI 3-kinase pathway activity. The inhibition of neuronal death mediated by these compounds correlated with inhibition of GSK-3 activity and modulation of GSK-3 substrates tau and beta-catenin. Thus, in addition to the previously assigned roles of GSK-3, our data provide clear pharmacological and biochemical evidence that selective inhibition of the endogenous pool of GSK-3 activity in primary neurones is sufficient to prevent death, implicating GSK-3 as a physiologically relevant principal regulatory target of the PI 3-kinase/PKB neuronal survival pathway.     

Overactivation of glycogen synthase kinase-3 by inhibition of phosphoinositol-3 kinase and protein kinase C leads to hyperphosphorylation of tau and impairment of spatial memory

Neurofibrillary tangles (NFTs) consisting of the hyperphosphorylated microtubule-associated protein tau are a defining pathological characteristic of Alzheimer's disease (AD). Hyperphosphorylation of tau is hypothesized to impair the microtubule stabilizing function of tau, leading to the formation of paired helical filaments and neuronal death. Glycogen synthase kinase-3 (GSK-3) has been shown to be one of several kinases that mediate tau hyperphosphorylation in vitro. However, molecular mechanisms underlying overactivation of GSK-3 and its potential linkage to AD-like pathologies in vivo remain unclear. Here, we demonstrate that injection of wortmannin (a specific inhibitor of phosphoinositol-3 kinase) or GF-109203X (a specific inhibitor of protein kinase C) into the left ventricle of rat brains leads to overactivation of GSK-3, hyperphosphorylation of tau at Ser 396/404/199/202 and, most significantly, impaired spatial memory. The effects of wortmannin and GF-109203X are additive. Significantly, specific inhibition of GSK-3 activity by LiCl prevents hyperphosphorylation of tau, and spatial memory impairment resulting from PI3K and PKC inhibition. These results indicate that in vivo inhibition of phosphoinositol-3 kinase and protein kinase C results in overactivation of GSK-3 and tau hyperphosphorylation and support a direct role of GSK-3 in the formation of AD-like cognitive deficits.     

Purification and partial characterization of glycogen synthase kinase-3 from rabbit liver

Summary Glycogen synthase kinase-3 (GSK-3) was purified from rabbit liver to homogeneity by ultracentrifugation, ion-exchange chromatography on DEAE-cellulose, Cellulose phosphate, CM-Sephadex and Fast Protein Liquid Chromatography (FPLC) on Mono-S column. The enzyme was purified approximately 20,000 fold with an approximate 2% recovery. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis. GSK-3 is a monomeric enzyme with a molecular weight of 50,000–52,000 as derived from SDS-polyacrylamide gel electrophoresis and gel filtration. The purified enzyme was indeed a GSK-3 since it phosphorylated three sites, i.e., 3a, 3b, and 3c on liver glycogen synthase. GSK-3 incorporated up to 2.6 mol Pi/mol glycogen synthase subunit with a concomitant inactivation of glycogen synthase activity.     

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

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.     

Inflorescence-specific expression of AtK-1, a novel Arabidopsis thaliana homologue of shaggy/glycogen synthase kinase-3

We report here the isolation of the Arabidopsis thaliana gene AtK-1. The predicted protein sequence of AtK-1 show 70% identity to the Arabidopsis ASK and alfalfa MsK kinases that are homologs of the Drosophila shaggy and rat GSK-3 serine/threonine protein kinases playing an important role in signal transduction processes in animals. Northern analysis of different organs revealed exclusive expression in inflorescences suggesting an involvement of the AtK-1 kinase in reproduction-specific processes.     

CREB DNA binding activity is inhibited by glycogen synthase kinase-3 beta and facilitated by lithium

The regulatory influences of glycogen synthase kinase-3 beta (GSK3 beta) and lithium on the activity of cyclic AMP response element binding protein (CREB) were examined in human neuroblastoma SH-SY5Y cells. Activation of Akt (protein kinase B) with serum-increased phospho-serine-9-GSK3 beta (the inactive form of the enzyme), inhibited GSK3 beta activity, and increased CREB DNA binding activity. Inhibition of GSK3 beta by another paradigm, treatment with the selective inhibitor lithium, also increased CREB DNA binding activity. The inhibitory regulation of CREB DNA binding activity by GSK3 beta also was evident in differentiated SH-SY5Y cells, indicating that this regulatory interaction is maintained in non-proliferating cells. These results demonstrate that inhibition of GSK3 beta by serine-9 phosphorylation or directly by lithium increases CREB activation. Conversely, overexpression of active GSK3 beta to 3.5-fold the normal levels completely blocked increases in CREB DNA binding activity induced by epidermal growth factor, insulin-like growth factor-1, forskolin, and cyclic AMP. The inhibitory effects due to overexpressed GSK3 beta were reversed by treatment with lithium and with another GSK 3beta inhibitor, sodium valproate. Overall, these results demonstrate that GSK3 beta inhibits, and lithium enhances, CREB activation.     

Genetic correlational analysis of glycogen synthase kinase-3 beta and prepulse inhibition in inbred mice

In humans, GSK-3β activity is diminished in schizophrenic patients as is prepulse inhibition of the startle response (PPI). We performed a genetic correlational analysis between published PPI values and frontal cortex GSK-3 activity analyzed in our laboratory in 10 inbred mouse strains. This methodology could indicate relevant parameters for study in an animal model. Indeed, we obtained significant correlations between the enzyme's activity and PPI measured by two different methods. This may indicate that investigation of the genetics of GSK-3β regulation holds promise for understanding some of the biochemical underpinnings of schizophrenia.     

Regulation of phosphorylation of tau by cyclin-dependent kinase 5 and glycogen synthase kinase-3 at substrate level

Microtubule associated protein tau, which is expressed in six alternatively spliced molecular isoforms in human brain, is abnormally hyperphosphorylated in Alzheimer disease and related tauopathies. Here, we show (i) that GSK-3alpha and neither GSK-3beta nor cdk5 can phosphorylate tau at Ser262 and phosphorylation at Ser235 by cdk5 primes phosphorylation at Thr231 by GSK-3alpha/beta; (ii) that tau isoforms with two N-terminal inserts (tau4L, tau3L) are phosphorylated by cdk5 plus GSK-3 at Thr231 markedly more than isoforms lacking these inserts (tau4, tau3); and (iii) that Thr231 is phosphorylated approximately 50% more in free tau than in microtubule-bound tau, and the phosphorylation at this site results in the dissociation of tau from microtubules. These findings suggest that the phosphorylation of tau at Thr231 and Ser262 by cdk5 plus GSK-3, which inhibits its normal biological activity, is regulated both by its amino terminal inserts and its physical state.     

Apolipoprotein E and beta-amyloid (1-42) regulation of glycogen synthase kinase-3beta

Glycogen synthase kinase-3beta (GSK-3beta) is implicated in regulating apoptosis and tau protein hyperphosphorylation in Alzheimer's disease (AD). We investigated the effects of two key AD molecules, namely apoE (E3 and E4 isoforms) and beta-amyloid (Abeta) 1-42 on GSK-3beta and its major upstream regulators, intracellular calcium and protein kinases C and B (PKC and PKB) in human SH-SY5Y neuroblastoma cells. ApoE3 induced a mild, transient, Ca2+-independent and early activation of GSK-3beta. ApoE4 effects were biphasic, with an early strong GSK-3beta activation that was partially dependent on extracellular Ca2+, followed by a GSK-3beta inactivation. ApoE4 also activated PKC-alpha and PKB possibly giving the subsequent GSK-3beta inhibition. Abeta(1-42) effects were also biphasic with a strong activation dependent partially on extracellular Ca2+ followed by an inactivation. Abeta(1-42) induced an early and potent activation of PKC-alpha and a late decrease of PKB activity. ApoE4 and Abeta(1-42) were more toxic than apoE3 as shown by MTT reduction assays and generation of activated caspase-3. ApoE4 and Abeta(1-42)-induced early activation of GSK-3beta could lead to apoptosis and tau hyperphosphorylation. A late inhibition of GSK-3beta through activation of upstream kinases likely compensates the effects of apoE4 and Abeta(1-42) on GSK-3beta, the unbalanced regulation of which may contribute to AD pathology.     

Glycogen synthase kinase-3 regulation of chromatin segregation and cytokinesis in mouse preimplantation embryos

Glycogen synthase kinase-3 (GSK-3) is a highly conserved serine/threonine protein kinase implicated in diverse cellular processes. Activity of GSK-3 is essential for meiotic chromatin segregation in oocytes, yet expression and/or function of GSK-3 have not been reported in mammalian preimplantation embryos. Objectives of this study were to characterize GSK-3 protein expression/phosphorylation in mouse preimplantation embryos, to assess the effect of GSK-3 activity inhibition on early mitotic events, and to differentiate nuclear and cytoplasmic anomalies in GSK-3 inhibited embryos. Both GSK-3 isoforms were expressed during embryo development, with a differential expression of alpha versus beta. Phosphorylation of GSK-3alpha/beta at residues Y279/Y216 indicated constitutive activation throughout preimplantation development. Phosphorylation at N-terminal residues S21/S9 indicated inhibition of GSK-3alpha/beta activity that was differentially regulated during early development; both alpha and beta isoforms were phosphorylated during early divisions, whereas at the blastocyst stage, only beta was phosphorylated. Cytoplasmic microinjection of zygotes with anti-GSK-3alpha/beta antibody significantly compromised embryonic development past the two-cell stage compared to controls. Reversibility of developmental block was tested via pharmacological inhibitors of GSK-3, lithium chloride (LiCl) and alsterpaullone. Similar to immunoneutralization, significantly fewer zygotes cultured with either LiCl or alsterpaullone developed past the two-cell stage compared to controls and this mitotic block was not reversible. Inhibition of GSK-3 activity significantly compromised timing of pronuclear membrane breakdown and mitosis initiation, nuclear development, and cytokinesis. Inhibition of GSK-3 also resulted in abnormal chromatin segregation, evidenced by incomplete karyokinesis and micronuclei formation. These results suggest that GSK-3 activity is critical for early preimplantation embryonic development.     

Glycogen synthase kinase 3 in the world of cell migration

Glycogen synthase kinase 3 (GSK3) is one of the few master switch kinases that regulate many aspects of cell functions. Recent studies on cell polarization and migration have shown that GSK3 is also essential for proper regulation of these processes. GSK3 influences cell migration as one of the regulators of the spatiotemporally controlled dynamics of the actin cytoskeleton, microtubules, and cell‐to‐matrix adhesions. In this mini‐review, the effects of GSK3 on these three aspects of cell migration will be discussed.     

New insights into the autoinhibition mechanism of glycogen synthase kinase-3beta

It has been suggested that phosphorylation at serine 9 near the N-terminus of glycogen synthase kinase-3β (GSK-3β) mimics the prephosphorylation of its substrate and, therefore, the N-terminus functions as a pseudosubstrate. The molecular basis for the pseudosubstrate's binding to the catalytic core and autoinhibition has not been fully defined. Here, we combined biochemical and computational analyses to identify the potential residues within the N-terminus and the catalytic core engaged in autoinhibition of GSK-3β. Bioinformatic analysis found Arg4, Arg6, and Ser9 in the pseudosubstrate sequence to be extremely conserved through evolution. Mutations at Arg4 and Arg6 to alanine enhanced GSK-3β kinase activity and impaired its ability to autophosphorylate at Ser9. In addition, and unlike wild-type GSK-3β, these mutants were unable to undergo autoinhibition by phosphorylated Ser9. We further show that Gln89 and Asn95, located within the catalytic core, interact with the pseudosubstrate. Mutation at these sites prevented inhibition by phosphorylated Ser9. Furthermore, the respective mutants were not inhibited by a phosphorylated pseudosubstrate peptide inhibitor. Finally, computational docking of the pseudosubstrate into the catalytic active site of the kinase suggested specific interactions between Arg6 and Asn95 and of Arg4 to Asp181 (apart from the interaction of phosphorylated serine 9 with the “phosphate binding pocket”). Altogether, our study supports a model of GSK-3-pseudosubstrate autoregulation that involves phosphorylated Ser9, Arg4, and Arg6 within the N-terminus and identified the specific contact sites within the catalytic core.     

Functional significance of glycogen synthase kinase-3 regulation by serotonin

Serotonin modulates brain physiology and behavior and has major roles in brain diseases involving abnormal mood and cognition. Enhancing brain serotonin has been found to regulate glycogen synthase Kinase-3 (GSK3), but the signaling mechanism and functional significance of this regulation remain to be determined. In this study, we tested the signaling mechanism mediating 5-HT1A receptor-regulated GSK3 in the hippocampus. Using mutant GSK3 knock-in mice, we also tested the role of GSK3 in the behavioral effects of 5-HT1A receptors and the serotonin reuptake inhibitor fluoxetine. The results showed that activation of 5-HT1A receptors by 8-hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) increased phosphorylation of the N-terminal serine of both GSK3α and GSK3β in several areas of the hippocampus. The effect of 8-OH-DPAT was accompanied by an increase in the active phosphorylation of Akt, and was blocked by LY294002, an inhibitor of phosphoinositide 3-kinases (PI3K). Phosphorylation of GSK3β, but not GSK3α, was necessary for 5-HT1A receptors to suppress the hippocampus-associated contextual fear learning. Furthermore, acute fluoxetine treatment up-regulated both phospho-Ser21-GSK3α and phospho-Ser9-GSK3β in the hippocampus. Blocking phosphorylation of GSK3α and GSK3β diminished the anti-immobility effect of fluoxetine treatment in the forced swim test, wherein the effect of GSK3β was more prominent. These results together suggest that PI3K/Akt is a signaling mechanism mediating the GSK3-regulating effect of 5-HT1A receptors in the hippocampus, and regulation of GSK3 is an important intermediate signaling process in the behavioral functions of 5-HT1A receptors and fluoxetine.