Glycogen Synthase Kinase-3: A Promising Therapeutic Target for Fragile X Syndrome

Recent advances in understanding the pathophysiological mechanisms contributing to fragile X syndrome (FXS) have increased optimism that drug interventions can provide significant therapeutic benefits. FXS results from inadequate expression of functional fragile X mental retardation protein (FMRP). FMRP may have several functions, but it is most well-established as an RNA binding protein that regulates translation, and it is thought that by this mechanism FMRP is capable of affecting numerous cellular processes by selectively regulating protein levels. The multiple cellular functions regulated by FMRP suggest that multiple interventions may be required for reversing the effects of deficient FMRP. Evidence that inhibitors of glycogen synthase kinase-3 (GSK3) may contribute to the therapeutic treatment of FXS is reviewed here. Lithium, a GSK3 inhibitor, improved function in the Drosophila model of FXS. In mice lacking FMRP expression (FX mice), GSK3 is hyperactive in several brain regions. Significant improvements in several FX-related phenotypes have been obtained in FX mice following the administration of lithium, and in some case other GSK3 inhibitors. These responses include normalization of heightened audiogenic seizure susceptibility and of hyperactive locomotor behavior, enhancement of passive avoidance learning retention and of sociability behaviors, and corrections of macroorchidism, neuronal spine density, and neural plasticity measured electrophysiologically as long term depression. A pilot clinical trial of lithium in patients with FXS also found improvements in several measures of behavior. Taken together, these findings indicate that lithium and other inhibitors of GSK3 are promising candidate therapeutic agents for treating FXS

GSK3β N-terminus binding to p53 promotes its acetylation

The prevalence in human cancers of mutations in p53 exemplifies its crucial role as a tumor suppressor transcription factor. Previous studies have shown that the constitutively active serine/threonine kinase glycogen synthase kinase-3β (GSK3β) associates with the C-terminal basic domain of p53 and regulates its actions. In this study we identified the GSK3β N-terminal amino acids 78–92 as necessary for its association with p53. Inhibitors of GSK3 impaired the acetylation of p53 at Lys373 and Lys382 near the GSK3β binding region in p53, indicating that GSK3β facilitates p53 acetylation. We also found that acetylation of p53 reduced its association with GSK3β, as well as with GSK3α. These results indicate that the N-terminal region of GSK3β binds p53, this association promotes the acetylation of p53, and subsequently acetylated p53 dissociates from GSK3

Regulation of Cell Survival Mechanisms in Alzheimer's Disease by Glycogen Synthase Kinase-3

A pivotal role has emerged for glycogen synthase kinase-3 (GSK3) as an important contributor to Alzheimer's disease pathology. Evidence for the involvement of GSK3 in Alzheimer's disease pathology and neuronal loss comes from studies of GSK3 overexpression, GSK3 localization studies, multiple relationships between GSK3 and amyloid β-peptide (Aβ), interactions between GSK3 and the microtubule-associated tau protein, and GSK3-mediated apoptotic cell death. Apoptotic signaling proceeds by either an intrinsic pathway or an extrinsic pathway. GSK3 is well established to promote intrinsic apoptotic signaling induced by many insults, several of which may contribute to neuronal loss in Alzheimer's disease. Particularly important is evidence that GSK3 promotes intrinsic apoptotic signaling induced by Aβ. GSK3 appears to promote intrinsic apoptotic signaling by modulating proteins in the apoptosis signaling pathway and by modulating transcription factors that regulate the expression of proteins involved in apoptosis. Thus, GSK3 appears to contribute to several neuropathological mechanisms in Alzheimer's disease, including apoptosis-mediated neuronal loss

Lithium facilitates apoptotic signaling induced by activation of the Fas death domain-containing receptor

BACKGROUND: Lithium, a mood stabilizer widely used to treat bipolar disorder, also is a neuroprotectant, providing neurons protection from apoptosis induced by a broad spectrum of toxic conditions. A portion of this neuroprotection is due to lithium's inhibition of glycogen synthase kinase-3. The present investigation examined if the neuroprotection provided by lithium included apoptosis induced by stimulation of the death domain-containing receptor Fas. RESULTS: Instead of providing protection, treatment with 20 mM lithium significantly increased apoptotic signaling induced by activation of Fas, and this occurred in both Jurkat cells and differentiated immortalized hippocampal neurons. Other inhibitors of glycogen synthase kinase-3, including 20 μM indirubin-3'-monoxime, 5 μM kenpaullone, and 5 μM rottlerin, also facilitated Fas-induced apoptotic signaling, indicating that the facilitation of apoptosis by lithium was due to inhibition of glycogen synthase kinase-3. CONCLUSIONS: These results demonstrate that lithium is not always a neuroprotectant, and it has the opposite effect of facilitating apoptosis mediated by stimulation of death domain-containing receptors

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 GSK3β and GSK3α, respectively. This study found that accurate measurements of phospho-Ser-GSK3 in brain are confounded by a rapid post-mortem dephosphorylation, with ~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 GSK3β and GSK3α 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

Lithium Regulates Glycogen Synthase Kinase-3β in Human Peripheral Blood Mononuclear Cells: Implication in the Treatment of Bipolar Disorder

Background: Bipolar disorder has been linked to alterations in the multifunctional enzyme glycogen synthase kinase-3β (GSK3β). The mood stabilizer lithium inhibits GSK3β in vitro and in mouse brain, and this is currently the strongest known potential therapeutic target of lithium. We tested whether lithium modified GSK3β in vivo or in vitro in peripheral blood mononuclear cells (PBMCs) from healthy control and bipolar disorder subjects. Methods: The PBMCs were obtained from 23 healthy control subjects, 9 bipolar subjects currently treated with lithium, and 13 lithium-free bipolar subjects. Immunoblot analyses were used to measure the inhibited, serine9-phosphorylated GSK3β. Results: The level of phospho-Ser9-GSK3β in PBMCs was regulated by agents that modified kinases and phosphatases acting on GSK3β and was increased by in vitro lithium treatment. More important, phospho-Ser9-GSK3β levels were eightfold higher in PBMCs from lithium-treated bipolar than healthy control subjects. Conclusions: Signaling pathways regulating serine9-phosphorylation of GSK3β can be studied in human PBMCs. Both in vitro and in vivo therapeutic lithium treatment is associated with a large increase in phospho-Ser9-GSK3β in PBMCs. Therefore, the inhibitory serine9-phosphorylation of GSK3β in human PBMCs may provide a biochemical marker to evaluate the association between GSK3β inhibition and therapeutic responses to lithium treatment

Free and bound choline blood levels after phosphatidylcholine

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/110000/1/cptclpt198264.pd

GSK3 Influences Social Preference and Anxiety-Related Behaviors during Social Interaction in a Mouse Model of Fragile X Syndrome and Autism

BACKGROUND: Nearly 1% of children in the United States exhibit autism spectrum disorders, but causes and treatments remain to be identified. Mice with deletion of the fragile X mental retardation 1 (Fmr1) gene are used to model autism because loss of Fmr1 gene function causes Fragile X Syndrome (FXS) and many people with FXS exhibit autistic-like behaviors. Glycogen synthase kinase-3 (GSK3) is hyperactive in brains of Fmr1 knockout mice, and inhibition of GSK3 by lithium administration ameliorates some behavioral impairment in these mice. We extended our studies of this association by testing whether GSK3 contributes to socialization behaviors. This used two mouse models with disrupted regulation of GSK3, Fmr1 knockout mice and GSK3 knockin mice, in which inhibitory serines of the two isoforms of GSK3, GSK3alpha and GSK3beta, are mutated to alanines, leaving GSK3 fully active. METHODOLOGY/PRINCIPAL FINDINGS: To assess sociability, test mice were introduced to a restrained stimulus mouse (S1) for 10 min, followed by introduction of a second restrained stimulus mouse (S2) for 10 min, which assesses social preference. Fmr1 knockout and GSK3 knockin mice displayed no deficit in sociability with the S1 mouse, but unlike wild-type mice neither demonstrated social preference for the novel S2 mouse. Fmr1 knockout mice displayed more anxiety-related behaviors during social interaction (grooming, rearing, and digging) than wild-type mice, which was ameliorated by inhibition of GSK3 with chronic lithium treatment. CONCLUSIONS/SIGNIFICANCE: These results indicate that impaired inhibitory regulation of GSK3 in Fmr1 knockout mice may contribute to some socialization deficits and that lithium treatment can ameliorate certain socialization impairments. As discussed in the present work, these results suggest a role for GSK3 in social behaviors and implicate inhibition of GSK3 as a potential therapeutic

A Lentivirus-Mediated Genetic Screen Identifies Dihydrofolate Reductase (DHFR) as a Modulator of β-Catenin/GSK3 Signaling

The multi-protein β-catenin destruction complex tightly regulates β-catenin protein levels by shuttling β-catenin to the proteasome. Glycogen synthase kinase 3β (GSK3β), a key serine/threonine kinase in the destruction complex, is responsible for several phosphorylation events that mark β-catenin for ubiquitination and subsequent degradation. Because modulation of both β-catenin and GSK3β activity may have important implications for treating disease, a complete understanding of the mechanisms that regulate the β-catenin/GSK3β interaction is warranted. We screened an arrayed lentivirus library expressing small hairpin RNAs (shRNAs) targeting 5,201 human druggable genes for silencing events that activate a β-catenin pathway reporter (BAR) in synergy with 6-bromoindirubin-3′oxime (BIO), a specific inhibitor of GSK3β. Top screen hits included shRNAs targeting dihydrofolate reductase (DHFR), the target of the anti-inflammatory compound methotrexate. Exposure of cells to BIO plus methotrexate resulted in potent synergistic activation of BAR activity, reduction of β-catenin phosphorylation at GSK3-specific sites, and accumulation of nuclear β-catenin. Furthermore, the observed synergy correlated with inhibitory phosphorylation of GSK3β and was neutralized upon inhibition of phosphatidyl inositol 3-kinase (PI3K). Linking these observations to inflammation, we also observed synergistic inhibition of lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines (TNFα, IL-6, and IL-12), and increased production of the anti-inflammatory cytokine IL-10 in peripheral blood mononuclear cells exposed to GSK3 inhibitors and methotrexate. Our data establish DHFR as a novel modulator of β-catenin and GSK3 signaling and raise several implications for clinical use of combined methotrexate and GSK3 inhibitors as treatment for inflammatory disease

Trypanosoma brucei Glycogen Synthase Kinase-3, A Target for Anti-Trypanosomal Drug Development: A Public-Private Partnership to Identify Novel Leads

Over 60 million people in sub-Saharan Africa are at risk of infection with the parasite Trypanosoma brucei which causes Human African Trypanosomiasis (HAT), also known as sleeping sickness. The disease results in systemic and neurological disability to its victims. At present, only four drugs are available for treatment of HAT. However, these drugs are expensive, limited in efficacy and are severely toxic, hence the need to develop new therapies. Previously, the short TbruGSK-3 short has been validated as a potential target for developing new drugs against HAT. Because this enzyme has also been pursued as a drug target for other diseases, several inhibitors are available for screening against the parasite enzyme. Here we present the results of screening over 16,000 inhibitors of human GSK-3β (HsGSK-3) from the Pfizer compound collection against TbruGSK-3 short. The resulting active compounds were tested for selectivity versus HsGSK-3β and a panel of human kinases, as well as their ability to inhibit proliferation of the parasite in vitro. We have identified attractive compounds that now form potential starting points for drug discovery against HAT. This is an example of how a tripartite partnership involving pharmaceutical industries, academic institutions and non-government organisations such as WHO TDR, can stimulate research for neglected diseases