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.     

Acetyl-L-carnitine ameliorates spatial memory deficits induced by inhibition of phosphoinositol-3 kinase and protein kinase C

Glycogen synthase kinase-3β (GSK-3β) plays a crucial role in memory deficits and tau hyperphosphorylation as seen in Alzheimer's disease, the most common dementia in the aged population. We reported that ventricular co-injection of wortmannin and GF-109203X (WT/GFX) can induce tau hyperphosophorylation and memory impairment of rats through activation of GSK-3 [Liu S. J., Zhang A. H., Li H. L., Wang Q., Deng H. M., Netzer W. J., Xu H. X. and Wang J. Z. (2003) J. Neurochem. 87, 1333]. In the present study, we found that feeding the rats with Acetyl-L-Carnitine (ALCAR, 50 mg/day·rat, per os) for 2 weeks rescued the WT/GFX-induced spatial memory retention impairment of the rats by antagonizing GSK-3β activation independent of Akt, PKCζ and Erk1/2. We also found that ALCAR arrested microtubule-associated protein tau hyperphosphorylation at multiple Alzheimer's disease sites in vivo and in vitro. Moreover, ALCAR enhanced the expression of several memory-associated proteins including c-Fos, synapsin I in rat hippocampus. These results suggest that ALCAR could ameliorate WT/GFX-induced spatial memory deficits through inhibition tau hyperphosphorylation and modulation of memory-associated proteins.     

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.     

Primed phosphorylation of tau at Thr231 by glycogen synthase kinase 3beta (GSK3beta) plays a critical role in regulating tau's ability to bind and stabilize microtubules

Site-specific phosphorylation of tau negatively regulates its ability to bind and stabilize microtubule structure. Although tau is a substrate of glycogen synthase kinase 3beta (GSK3beta), the exact sites on tau that are phosphorylated by this kinase in situ have not yet been established, and the effect of these phosphorylation events on tau-microtubule interactions have not been fully elucidated. GSK3beta phosphorylates both primed and unprimed sites on tau, but only primed phosphorylation events significantly decrease the ability of tau to bind microtubules. The focus of the present study is on determining the importance of the GSK3beta-mediated phosphorylation of a specific primed site, Thr231, in regulating tau's function. Pre-phosphorylation of Ser235 primes tau for phosphorylation by GSK3beta at Thr231. Phosphorylation by GSK3beta of wild-type tau or tau with Ser235 mutated to Ala decreases tau-microtubule interactions. However, when Thr231 alone or Thr231 and Ser235 in tau were mutated to Ala, phosphorylation by GSK3beta did not decrease the association of tau with the cytoskeleton. Further, T231A tau was still able to efficiently bind microtubules after phosphorylation by GSK3beta. Expression of each tau construct alone increased tubulin acetylation, a marker of microtubule stability. However, when cells were cotransfected with wild-type tau and GSK3beta, the level of tubulin acetylation was decreased to vector-transfected levels. In contrast, coexpression of GSK3beta with mutated tau (T231A/S235A) did not significantly decrease the levels of acetylated tubulin. These results strongly indicate that phosphorylation of Thr231 in tau by GSK3beta plays a critical role in regulating tau's ability to bind and stabilize microtubules.     

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.     

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.     

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.     

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.     

Val97Leu mutant presenilin-1 induces tau hyperphosphorylation and spatial memory deficit in mice and the underlying mechanisms

Although the pathological role of presenilin-1 mutation in early onset familial Alzheimer's disease has been widely studied, few focused on how the presenilin-1 mutations result in memory impairment and tau hyperphosphorylation. In the present study, we expressed human Val97Leu mutant presenilin-1, which is reported in Chinese pedigrees by our group, in transgenic mice and found that the mutant presenilin-1 induced spatial memory deficit and tau hyperphosphorylation at PHF-1, pS199/202, pT231 and pS396 epitopes, but not at pS214 and pS422 epitopes. Pearson analysis showed that the memory deficit was only significantly correlated with tau phosphorylation level at PHF-1, pS199/202, pT231 and pS396 epitopes. Additionally, the hyperphosphorylated tau and tangle-like argentophilic structures were detected at CA3 and CA4, but not CA1, region of hippocampus, and we also found tangle-like structure and wizened degenerative neurons in frontal cortex. We demonstrated the tau hyperphosphorylation at the same epitopes in N2a cells expressing the mutant presenilin-1, which is caused by inhibition of phosphoinositol-3 kinase/Akt and activation of glycogen synthase kinase-3 specifically. Our data demonstrated that human Val97Leu mutant presenilin-1 causes spatial memory deficit in mice and increases tau phosphorylation level in glycogen synthase kinase-3-dependent manner.     

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.     

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.     

Famotidine inhibits glycogen synthase kinase-3β: An investigation by docking simulation and experimental validation

Famotidine was investigated as an inhibitor of glycogen synthase kinase-3β (GSK-3β) in an attempt to explain the molecular mechanism of its hypoglycemic side effects. The investigation included simulated docking experiments, in vitro enzyme inhibition assay, glycogen sparing studies using animal models and single dose oral glucose tolerance test (OGTT). Docking studies showed how famotidine is optimally fit within the binding pocket of GSK-3β via numerous attractive interactions with some specific amino acids. Experimentally, famotidine could inhibit GSK-3β (IC₅₀ = 1.44 μM) and increased significantly liver glycogen spares in fasting animal models. Moreover, a single oral dose of famotidine was shown to decrease the glycemic response curve after 75 g OGTT     

Cocaine regulates protein kinase B and glycogen synthase kinase-3 activity in selective regions of rat brain

Protein kinase B (also known as Akt) signaling regulates dopamine-mediated locomotor behaviors. Here the ability of cocaine to regulate Akt and glycogen synthase kinase 3 (GSK3) was studied. Rats were injected with cocaine or saline in a binge-pattern, which consisted of three daily injections of 15 mg/kg cocaine or 1 mL/kg saline spaced 1 h apart for 1, 3, or 14 days. Amygdala, nucleus accumbens, caudate putamen, and hippocampus tissues were dissected 30 min following the last injection and analyzed for phosphorylated and total Akt and GSK3(alpha and beta) protein levels using western blot analysis. Phosphorylation of Akt on the threonine-308 (Thr308) residue was significantly reduced in the nucleus accumbens and increased in the amygdala after 1 day of cocaine treatment; however, these effects were not accompanied by a significant decrease in GSK3 phosphorylation. Phosphorylation of Akt and GSK3 was significantly reduced after 14 days of cocaine administration, an effect that was only observed in the amygdala. Cocaine did not alter Akt or GSK3 phosphorylation in the caudate putamen or hippocampus. The findings in nucleus accumbens may reflect dopaminergic motor-stimulant activity caused by acute cocaine, whereas the effects in amygdala may be associated with changes in emotional state that occur after acute and chronic cocaine exposure.     

Prolonged Alzheimer-like tau hyperphosphorylation induced by simultaneous inhibition of phosphoinositol-3 kinase and protein kinase C in N2a cells

Co-injection of wortmannin (inhibitor of phosphatidylinositol-3 kinase, PI3K) and GF109203X(inhibitor of protein kinase C, PKC) into the rat brain was found to induce spatial memory deficiency and enhance tau hyperphosphorylation in the hippocampus of rat brain. To establish a cell model with durative Alzheimer-like tau hyperphosphorylation in this study, we treated N2a neuroblastoma cells with wortmannin and GF109203X separately and simultaneously, and measured the glycogen synthase kinase 3 (GSK-3)activity by y-32p-labeling and the level of tau phosphorylation by Western blotting. It was found that the application of wortmannin alone only transitorily increased the activity of GSK-3 (about 1 h) and the level of tau hyperphosphorylation at Ser^396/Ser^404 and Ser^199/Ser^202 sites (no longer than 3 h); however, a prolonged and intense activation of GSK-3 (over 12 h) and enhanced tau hyperphosphorylation (about 24 h) were observed when these two selective kinase inhibitors were applied together. We conclude that the simultaneous inhibition of PI3K and PKC can induce GSK-3 overactivation, and further strengthen and prolong the Alzheimerlike tau hyperphosphorylation in N2a cells, suggesting the establishment of a cell model with early pathological events of Alzheimer‘s disease.     

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.     

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.     

A novel glycogen synthase kinase-3 inhibitor 2-methyl-5-(3-{4-[(S )-methylsulfinyl]phenyl}-1-benzofuran-5-yl)-1,3,4-oxadiazole decreases tau phosphorylation and ameliorates cognitive deficits in a transgenic model of Alzheimer's disease

Alzheimer's disease (AD) is a neurodegenerative disorder leading to a progressive loss of cognitive function and is pathologically characterized by senile plaques and neurofibrillary tangles. Glycogen synthase kinase-3 (GSK-3) is involved in AD pathogenesis. GSK-3 is reported not only to phosphorylate tau, a major component of neurofibrillary tangles, but also to regulate the production of amyloid β, which is deposited in senile plaques. Therefore, pharmacological inhibition of GSK-3 is considered an attractive therapeutic approach. In this study, we report the pharmacological effects of a novel GSK-3 inhibitor, 2-methyl-5-(3-{4-[(S)-methylsulfinyl]phenyl}-1-benzofuran-5-yl)-1,3,4-oxadiazole (MMBO), which displays high selectivity for GSK-3 and brain penetration following oral administration. MMBO inhibited tau phosphorylation in primary neural cell culture and also in normal mouse brain. When administered to a transgenic mouse model of AD, MMBO significantly decreased hippocampal tau phosphorylation at GSK-3 sites. Additionally, chronic MMBO administration suppressed tau pathology as assessed by AT8-immunoreactivity without affecting amyloid β pathology. Finally, in behavioral assessments, MMBO significantly improved memory and cognitive deficits in the Y-maze and in novel object recognition tests in the transgenic AD mouse model. These results indicate that pharmacological GSK-3 inhibition ameliorates behavioral dysfunction with suppression of tau phosphorylation in an AD mouse model, and that MMBO might be beneficial for AD treatment.