Glycogen synthase kinase-3 signaling in Alzheimer's disease

Alzheimer's disease (AD) is the most common form of neurodegenerative disorder with dementia, accounting for approximately 70% of the all cases. Currently, 5.8 million people in the U.S. are living with AD and by 2050 this number is expected to double resulting in a significant socio-economic burden. Despite intensive research, the exact mechanisms that trigger AD are still not known and at the present there is no cure for it. In recent years, many signaling pathways associated with AD neuropathology have been explored as possible candidate targets for the treatment of this condition including glycogen synthase kinase-3β (GSK3-β). GSK3-β is considered a key player in AD pathophysiology since dysregulation of this kinase influences all the major hallmarks of the disease including: tau phosphorylation, amyloid-β production, memory, neurogenesis and synaptic function. The present review summarizes the current understanding of the GSK3-β neurobiology with particular emphasis on its effects on specific signaling pathways associated with AD pathophysiology. Moreover, it discusses the feasibility of targeting GSK3-β for AD treatment and provides a summary of the current research effort to develop GSK3-β inhibitors in preclinical and clinical studies.     

Glycogen synthase kinase-3 is an early determinant in the differentiation of pathogenic Th17 cells

CD4(+) T cells are critical for host defense but are also major drivers of immune-mediated diseases. The classical view of Th1 and Th2 subtypes of CD4(+) T cells was recently revised by the identification of the Th17 lineage of CD4(+) T cells that produce IL-17, which have been found to be critical in the pathogenesis of autoimmune and other diseases. Mechanisms controlling the differentiation of Th17 cells have been well described, but few feasible targets for therapeutically reducing Th17 cells are known. The generation of Th17 cells requires IL-6 and activation of STAT3. During polarization of CD4(+) T cells to Th17 cells, we found that inhibition of glycogen synthase kinase-3 (GSK3) blocked IL-6 production, STAT3 activation, and polarization to Th17 cells. Polarization of CD4(+) T cells to Th17 cells increased by 10-fold the expression of GSK3β protein levels in Th17 cells, whereas GSK3β was unaltered in regulatory T cells. Diminishing GSK3 activity either pharmacologically or molecularly blocked Th17 cell production, and increasing GSK3 activity promoted polarization to Th17 cells. In vivo inhibition of GSK3 in mice depleted constitutive Th17 cells in intestinal mucosa, blocked Th17 cell generation in the lung after Francisella tularensis infection, and inhibited the increase in spinal cord Th17 cells and disease symptoms in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. These findings identify GSK3 as a critical mediator of Th17 cell production and indicate that GSK3 inhibitors provide a potential therapeutic intervention to control Th17-mediated diseases.     

Glycogen synthase kinase-3 is required for optimal de novo synthesis of inositol

Studies have shown that the inositol biosynthetic pathway and the enzyme glycogen synthase kinase-3 (GSK-3) are targets of the mood-stabilizing drugs lithium and valproate. However, a relationship between these targets has not been previously described. We hypothesized that GSK-3 may play a role in inositol synthesis, and that loss of GSK-3 may lead to inositol depletion, thus providing a mechanistic link between the two drug targets. Utilizing a yeast Saccharomyces cerevisiae gsk-3Delta quadruple-null mutant, in which all four genes encoding homologues of mammalian GSK-3 are disrupted, we tested the hypothesis that GSK-3 is required for de novo inositol biosynthesis. The gsk-3Delta mutant exhibited multiple features of inositol depletion, including defective growth in inositol-lacking medium, decreased intracellular inositol, increased INO1 and ITR1 expression, and decreased levels of phosphatidylinositol. Treatment of wild-type cells with a highly specific GSK-3 inhibitor led to a significant increase in INO1 expression. Supplementation with inositol alleviated the temperature sensitivity of gsk-3Delta. Activity of myo-inositol-3 phosphate synthase, the rate-limiting enzyme in inositol de novo biosynthesis, was decreased in gsk-3Delta. These results demonstrate for the first time that GSK-3 is required for optimal myo-inositol-3 phosphate synthase activity and de novo inositol biosynthesis, and that loss of GSK-3 activity causes inositol depletion.     

Glycogen synthase kinase-3beta is complexed with tau protein in brain microtubules.

In Alzheimer's disease, microtubule-associated protein tau is hyperphosphorylated by an unknown mechanism and is aggregated into paired helical filaments. Hyperphosphorylation causes loss of tau function, microtubule instability, and neurodegeneration. Glycogen synthase kinase-3beta (GSK3beta) has been implicated in the phosphorylation of tau in normal and Alzheimer's disease brain. The molecular mechanism of GSK3beta-tau interaction has not been clarified. In this study, we find that when microtubules are disassembled, microtubule-associated GSK3beta dissociates from microtubules. From a gel filtration column, the dissociated GSK3beta elutes as an approximately 400-kDa complex. When fractions containing the approximately 400-kDa complex are chromatographed through an anti-GSK3beta immunoaffinity column, tau co-elutes with GSK3beta. From fractions containing the approximately 400-kDa complex, both tau and GSK3beta co-immunoprecipitate with each other. GSK3beta binds to nonphosphorylated tau, and the GSK3beta-binding region is located within the N-terminal projection domain of tau. In vitro, GSK3beta associates with microtubules only in the presence of tau. From brain extract, approximately 6-fold more GSK3beta co-immunoprecipitates with tau than GSK3alpha. These data indicate that, in brain, GSK3beta is bound to tau within a approximately 400-kDa microtubule-associated complex, and GSK3beta associates with microtubules via tau.     

Glycogen synthase kinase-3beta is tyrosine-phosphorylated by MEK1 in human skin fibroblasts

Glycogen synthase kinase-3beta (GSK-3beta) can be associated with several proteins in cell. We analyzed the immunoprecipitates by an anti-GSK-3beta antibody from cell lysate of human fibroblasts and found that this protein was co-precipitated with mitogen-activated protein kinase kinase (MEK1/2). U0126, a MEK1/2 inhibitor, inhibited tyrosine phosphorylation of GSK-3beta, suggesting that MEK1/2 was involved in the phosphorylation of Tyr(216) in GSK-3beta. In vitro kinase assay was carried out using a recombinant human active MEK1 and we found that GSK-3beta was phosphorylated on Tyr(216) by this kinase in a dose- and time-dependent manner. Further, the pretreatment of fibroblasts with U0126 inhibited serum-induced nuclear translocation of GSK-3beta. These results suggested that MEK1/2 induces tyrosine phosphorylation of GSK-3beta and this cellular event might induce nuclear translocation of GSK-3beta. This is the first report to suggest that MEK1/2 phosphorylates not only ERK1/2 but also GSK-3beta.     

CD74 is a regulator of hematopoietic stem cell maintenance

Hematopoietic stem and progenitor cells (HSPCs) are a small population of undifferentiated cells that have the capacity for self-renewal and differentiate into all blood cell lineages. These cells are the most useful cells for clinical transplantations and for regenerative medicine. So far, it has not been possible to expand adult hematopoietic stem cells (HSCs) without losing their self-renewal properties. CD74 is a cell surface receptor for the cytokine macrophage migration inhibitory factor (MIF), and its mRNA is known to be expressed in HSCs. Here, we demonstrate that mice lacking CD74 exhibit an accumulation of HSCs in the bone marrow (BM) due to their increased potential to repopulate and compete for BM niches. Our results suggest that CD74 regulates the maintenance of the HSCs and CD18 expression. Its absence leads to induced survival of these cells and accumulation of quiescent and proliferating cells. Furthermore, in in vitro experiments, blocking of CD74 elevated the numbers of HSPCs. Thus, we suggest that blocking CD74 could lead to improved clinical insight into BM transplant protocols, enabling improved engraftment.

Hematopoietic stem and progenitor cells (HSPCs) can self-renew and differentiate into all blood cell lineages, making them useful for clinical transplantations and regenerative medicine. This study shows that blocking the MIF receptor CD74 increases the accumulation of HSPCs and could improve the efficacy of bone marrow transplantation protocols.     

Glycogen synthase kinase-3beta induces neuronal cell death via direct phosphorylation of mixed lineage kinase 3

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase member that activates the c-Jun N-terminal kinase (JNK) pathway. Aberrant activation of MLK3 has been implicated in neurodegenerative diseases. Similarly, glycogen synthase kinase (GSK)-3beta has also been shown to activate JNK and contribute to neuronal apoptosis. Here, we show a functional interaction between MLK3 and GSK-3beta during nerve growth factor (NGF) withdrawal-induced cell death in PC-12 cells. The protein kinase activities of GSK-3beta, MLK3, and JNK were increased upon NGF withdrawal, which paralleled increased cell death in NGF-deprived PC-12 cells. NGF withdrawal-induced cell death and MLK3 activation were blocked by a GSK-3beta-selective inhibitor, kenpaullone. However, the MLK family inhibitor, CEP-11004, although preventing PC-12 cell death, failed to inhibit GSK-3beta activation, indicating that induction of GSK-3beta lies upstream of MLK3. In GSK-3beta-deficient murine embryonic fibroblasts, ultraviolet light was unable to activate MLK3 kinase activity, a defect that was restored upon ectopic expression of GSK-3beta. The activation of MLK3 by GSK-3beta occurred via phosphorylation of MLK3 on two amino acid residues, Ser(789) and Ser(793), that are located within the C-terminal regulatory domain of MLK3. Furthermore, the cell death induced by GSK-3beta was mediated by MLK3 in a manner dependent on its phosphorylation of the specific residues within the C-terminal domain by GSK-3beta. Taken together, our data provide a direct link between GSK-3beta and MLK3 activation in a neuronal cell death pathway and identify MLK3 as a direct downstream target of GSK-3beta. Inhibition of GSK-3 is thus a potential therapeutic strategy for neurodegenerative diseases caused by trophic factor deprivation.     

Glycogen synthase kinase-3beta regulates presenilin 1 C-terminal fragment levels

The majority of familial Alzheimer's disease cases have been attributed to mutations in the presenilin 1 (PS1) gene. PS1 is synthesized as an inactive holoprotein that undergoes endoproteolytic processing to generate a functional N- and C-terminal heterodimer (NTF and CTF, respectively). We identified a single residue in PS1, Ser(397), which regulates the CTF levels in a population of dimer that has a rapid turnover. This residue is part of a highly conserved glycogen synthase kinase-3beta (GSK-3beta) consensus phosphorylation site within the loop domain of PS1. Site-directed mutagenesis at the Ser(397) position increased levels of PS1 CTF but not NTF or holoprotein. Similar increases in only CTF levels were seen when cells expressing wild type PS1 were treated with lithium chloride, an inhibitor of GSK-3beta. Both wild type and PS1 S397A CTF displayed a biphasic turnover, reflecting rapidly degraded and stable populations. Rapid turnover was delayed for mutant PS1 S397A, causing increased CTF. These data demonstrate that PS1 NTF.CTF endoproteolytic fragments are generated in excess, that phosphorylation at Ser(397) by GSK-3beta regulates the discard of excess CTF, and that the disposal of surplus NTF is mediated by an independent mechanism. Overall, the results indicate that production of active NTF.CTF dimer is more complex than limited endoproteolysis of PS1 holoprotein and instead involves additional regulatory events.     

Glycogen synthase kinase-3 is the predominant insulin-regulated eukaryotic initiation factor 2B kinase in skeletal muscle

Eukaryotic initiation factor eIF2B is a guanine nucleotide exchange protein involved in regulation of translation initiation. Phosphorylation of the epsilon-subunit is thought to be important in insulin-mediated changes in eIF2B activity. However, elucidation of insulin's action has proven elusive, primarily because eIF2B epsilon is a substrate in vitro for at least three different protein kinases. In the present study, we observed changes in eIF2B epsilon kinase activity only in those muscles previously shown to exhibit alterations in protein synthesis in response to insulin. Specifically, eIF2B epsilon kinase activity was increased in psoas muscle from diabetic rats compared to controls. Treating diabetic rats with insulin rapidly reduced eIF2B epsilon kinase activity below control values. Changes were not observed in heart. To identify the kinase(s) in psoas responsible for phosphorylating eIF2B epsilon, the wildtype and two variant forms of the epsilon-subunit were expressed in and purified from Sf9 insect cells, and were used as substrates in protein kinase assays. The first variant contained a point mutation in the eIF2B epsilon cDNA that converted the glycogen synthase kinase-3 (GSK-3) phosphorylation site, Ser535, to a nonphosphorylatable Ala residue. In the second variant, the putative GSK-3 'priming' site, Ser539, was converted to Asp. Based on the pattern of phosphorylation of the wildtype and two variant forms of eIF2B epsilon using casein kinase (CK)-I, CK-II, or GSK-3 as well as that observed with skeletal muscle extracts, we conclude that the predominant eIF2B epsilon kinase in psoas muscle is GSK-3. Thus, insulin-mediated changes in eIF2B activity are likely to involve GSK-3.     

Glycogen synthase kinase-3 enhances nuclear export of a Dictyostelium STAT protein

Extracellular cAMP stimulates the rapid tyrosine phosphorylation and nuclear translocation of the DICTYOSTELIUM: STAT protein Dd-STATa. Here we show that it also induces serine phosphorylation by GskA, a homologue of glycogen synthase kinase-3 (GSK-3). Tyrosine phosphorylation occurs within 10 s of stimulation, whereas serine phosphorylation takes 5 min, matching the kinetics observed for the cAMP regulation of GskA. Phosphorylation by GskA enhances nuclear export of Dd-STATa. The phosphorylated region, however, is not itself a nuclear export signal and we identify a region elsewhere in the protein that mediates nuclear export. These results suggest a biphasic regulation of Dd-STATa, in which extracellular cAMP initially directs nuclear import and then, via GskA, promotes its subsequent export. It also raises the possibility of an analogous regulation of STAT nuclear export in higher eukaryotes.