Specific Glycogen Synthase Kinase-3 Inhibitor Research Articles

GSK3: A potential target and pending issues for treatment of Alzheimer's disease.

Glycogen synthase kinase-3 (GSK3), consisting of GSK3α and GSK3β subtypes, is a complex protein kinase that regulates numerous substrates. Research has observed increased GSK3 expression in the brains of Alzheimer's disease (AD) patients and models. AD is a neurodegenerative disorder with diverse pathogenesis and notable cognitive impairments, characterized by Aβ aggregation and excessive tau phosphorylation. This article provides an overview of GSK3's structure and regulation, extensively analyzing its relationship with AD factors. GSK3 overactivation disrupts neural growth, development, and function. It directly promotes tau phosphorylation, regulates amyloid precursor protein (APP) cleavage, leading to Aβ formation, and directly or indirectly triggers neuroinflammation and oxidative damage. We also summarize preclinical research highlighting the inhibition of GSK3 activity as a primary therapeutic approach for AD. Finally, pending issues like the lack of highly specific and affinity-driven GSK3 inhibitors, are raised and expected to be addressed in future research. In conclusion, GSK3 represents a target in AD treatment, filled with hope, challenges, opportunities, and obstacles.

Abstract P021: Genomic biomarkers for response to 9-ING-41, a small molecule selective glycogen synthase kinase-3 (GSK-3) inhibitor, in pancreas cancer: Preliminary results

Abstract Background: 9-ING-41, a small molecule specific GSK-3 inhibitor, demonstrated favorable efficacy and safety in a Phase I clinical trial evaluating 9-ING-41 monotherapy and 8 chemotherapy combinations in 236 patients (pts) that has since been expanded to a Phase II multicenter study (NCT03678883). Interim results documented encouraging clinical activity, especially in patients with advanced pancreatic ductal adenocarcinoma (PDAC) and durable responses in melanoma and ATLL. Potential genomic biomarkers for 9-ING-41 response have not yet been evaluated. Methods: Next-generation sequencing (NGS) of tumor samples was obtained, whenever possible, for those receiving 9-ING-41 alone or in combination with chemotherapy across all sites. Genomic sequencing reports were reviewed for somatic alterations deemed potentially actionable or biologically relevant. For those with PDAC, best response was determined using RECIST 1.1 for those who completed at least two cycles of treatment. Chi-square frequency statistics were used to show the observed versus expected rate of pathogenic variants between patients with disease control (complete response [CR], partial response [PR] or stable disease [SD]) and progressive disease (PD). Results: NGS results were available for 135 pts across 11 sites. Most common histologies were PDAC (n=32), colorectal (n=17), and melanoma (n=9). Among pts with PDAC, NGS results were available for 32 pts (23 tumor samples, 9 ctDNA). Four patients received 9-ING-41 monotherapy; 28 received 9-ING-41 combined with gemcitabine/nab-paclitaxel (n=18), gemcitabine (n=7), or irinotecan (n=3). The most frequently mutated genes among PDAC included KRAS (n=22 pts), TP53 (n=21), CDKN2A (n=12), SMAD4 (n=4), CDKN2B (n=4), MTAP (n=3), ATM (n=3), AKT2 (n=2), and ARID1A (n=2). Of these 32 pts, 25 pts were evaluable for response: 2 pts had CR, 2 PR, 9 SD, and 12 PD. Among the pts with CR, one had tumor without pathogenic variants, the other displayed TP53 and KRAS mutations. Pts with PR: one tumor had no pathogenic mutations, the other tumor harbored mutations in ARID1A, TP53, FGF14, and ROS1. Of the nine patients with SD, 8 had KRAS mutations, 5 had CDKN2A loss of function, and 4 inactivating mutations in SMAD4. Eight out of 12 pts with PD had KRAS mutations. There were no significant differences between pts who had disease control and those with PD in the frequency of KRAS (χ2= 0.0189, P=0.89), TP53 (χ 2= 0.0712, P=0.78), or SMAD4 (χ 2= 1.9631, P= 0.1611). Conclusions: 9-ING-41 has shown clinical benefit in patients with PDAC independent of tumor somatic mutational profile. Preliminary analysis does not reveal pathogenic mutations that are associated with clinical benefit. Additional biomarker studies are ongoing. Citation Format: Brittany A. Borden, Andre De Souza, Devalingam Mahalingam, Steven Powell, Pamela N. Munster, Kelsey Huntington, Andrew Mazar, Ludmila Calvacante, Francis J. Giles, Wafik S. El-Deiry, Howard P. Safran, Benedito A. Carneiro. Genomic biomarkers for response to 9-ING-41, a small molecule selective glycogen synthase kinase-3 (GSK-3) inhibitor, in pancreas cancer: Preliminary results [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P021.

β-catenin stimulates Tcf7l1 degradation through recruitment of casein kinase 2 in mouse embryonic stem cells

Activation of the Wnt/β-catenin signaling pathway by the inhibition of glycogen synthase kinase-3 (GSK-3) will induce Tcf7l1 protein degradation to effectively promote embryonic stem cell (ESC) self-renewal. However, the exact mechanism remains unclear. Here, we found that inhibition of casein kinase 2 (Csnk2) by TBB or DMAT was sufficient to block the reduction of the Tcf7l1 protein induced by CHIR99021, a specific inhibitor of GSK-3. Similarly, downregulation of Csnk2 increased the Tcf7l1 level. In contrast, overexpression of Csnk2 significantly decreased Tcf7l1 protein stability in mouse ESCs. Notably, Csnk2α1 controls Tcf7l1 turnover to a greater degree than the other two isoforms of Csnk2, Csnk2α2 and Csnk2β, as Csnk2α1-overexpressing mouse ESCs exhibited the lowest level of Tcf7l1. Csnk2α1 interacted with and phosphorylated Tcf7l1. In addition, the association of Csnk2α1 and Tcf7l1 was enhanced by CHIR99021. Our study demonstrated, for the first time, that Csnk2 is involved in Tcf7l1 turnover mediated by the Wnt/β-catenin signaling pathway. These results expand our understanding of the function and circuit of Wnt/β-catenin signaling pathway in ESCs.

Bi-directional genetic modulation of GSK-3\u03b2 exacerbates hippocampal neuropathology in experimental status epilepticus

Glycogen synthase kinase-3 (GSK-3) is ubiquitously expressed throughout the brain and involved in vital molecular pathways such as cell survival and synaptic reorganization and has emerged as a potential drug target for brain diseases. A causal role for GSK-3, in particular the brain-enriched GSK-3β isoform, has been demonstrated in neurodegenerative diseases such as Alzheimer’s and Huntington’s, and in psychiatric diseases. Recent studies have also linked GSK-3 dysregulation to neuropathological outcomes in epilepsy. To date, however, there has been no genetic evidence for the involvement of GSK-3 in seizure-induced pathology. Status epilepticus (prolonged, damaging seizure) was induced via a microinjection of kainic acid into the amygdala of mice. Studies were conducted using two transgenic mouse lines: a neuron-specific GSK-3β overexpression and a neuron-specific dominant-negative GSK-3β (GSK-3β-DN) expression in order to determine the effects of increased or decreased GSK-3β activity, respectively, on seizures and attendant pathological changes in the hippocampus. GSK-3 inhibitors were also employed to support the genetic approach. Status epilepticus resulted in a spatiotemporal regulation of GSK-3 expression and activity in the hippocampus, with decreased GSK-3 activity evident in non-damaged hippocampal areas. Consistent with this, overexpression of GSK-3β exacerbated status epilepticus-induced neurodegeneration in mice. Surprisingly, decreasing GSK-3 activity, either via overexpression of GSK-3β-DN or through the use of specific GSK-3 inhibitors, also exacerbated hippocampal damage and increased seizure severity during status epilepticus. In conclusion, our results demonstrate that the brain has limited tolerance for modulation of GSK-3 activity in the setting of epileptic brain injury. These findings caution against targeting GSK-3 as a treatment strategy for epilepsy or other neurologic disorders where neuronal hyperexcitability is an underlying pathomechanism.

Wnt and GSK3 Signaling Pathways in Bipolar Disorder: Clinical and Therapeutic Implications.

The neurobiology of bipolar disorder, a chronic and systemic ailment is not completely understood. The bipolar phenotype manifests in myriad ways, and psychopharmacological agents like lithium have long term beneficial effects. The enzyme glycogen synthase kinase 3 (GSK3) has come into focus, as lithium and several other mood stabilizing medications inhibit its activity. This kinase and its key upstream modulator, Wnt are dysregulated in mood disorders and there is a growing impetus to delineate the chief substrates involved in the development of these illnesses. In May 2016, a comprehensive literature search was undertaken which revealed that there is over activity of GSK3 in bipolar disorder with deleterious downstream effects like proinflammatory status, increased oxidative stress, and circadian dysregulation leading to declining neurotrophic support and enhanced apoptosis of neural elements. By developing specific GSK3 inhibitors the progressive worsening in bipolar disorder can be forestalled with improved prospects for the sufferers.

Inhibition of Glycogen Synthase Kinase 3 Accelerated Liver Regeneration after Acetaminophen-Induced Hepatotoxicity in Mice

Overdose of acetaminophen (APAP) is the leading cause of acute liver failure (ALF) in the United States. Timely initiation of compensatory liver regeneration after APAP hepatotoxicity is critical for final recovery, but the mechanisms of liver regeneration after APAP-induced ALF have not been extensively explored yet. Previous studies from our laboratory have demonstrated that activation of β-catenin signaling after APAP overdose is associated with timely liver regeneration. Herein, weinvestigated the role of glycogen synthase kinase 3 (GSK3) in liver regeneration after APAP hepatotoxicity using a pharmacological inhibition strategy in mice. Treatment with specific GSK3 inhibitor (L803-mts), starting from 4 hours after 600 mg/kg dose of APAP, resulted in early initiation of liver regeneration in a dose-dependent manner, without modifying the peak regenerative response. Acceleration of liver regeneration was not secondary to alteration of APAP-induced hepatotoxicity, which remained unchanged after GSK3 inhibition. Early cell cycle initiation in hepatocytes after GSK3 inhibition was because of rapid induction of cyclin D1 and phosphorylation of retinoblastoma protein. This was associated with increased activation of β-catenin signaling after GSK3 inhibition. Taken together, our study has revealed a novel role of GSK3 in liver regeneration after APAP overdose and identified GSK3 as a potential therapeutic target to improve liver regeneration after APAP-induced ALF.

Up-regulation of insulin-like growth factor 2 by ketamine requires glycogen synthase kinase-3 inhibition

An antidepressant dose of the rapidly-acting ketamine inhibits glycogen synthase kinase-3 (GSK3) in mouse hippocampus, and this inhibition is required for the antidepressant effect of ketamine in learned helplessness depression-like behavior. Here we report that treatment with an antidepressant dose of ketamine (10mg/kg) increased expression of insulin-like growth factor 2 (IGF2) in mouse hippocampus, an effect that required ketamine-induced inhibition of GSK3. Ketamine also inhibited hippocampal GSK3 and increased expression of hippocampal IGF2 in mice when administered after the induction of learned helplessness. Treatment with the specific GSK3 inhibitor L803-mts was sufficient to up-regulate hippocampal IGF2 expression. Administration of IGF2 siRNA reduced ketamine's antidepressant effect in the learned helplessness paradigm. Mice subjected to the learned helplessness paradigm were separated into two groups, those that were resilient (non-depressed) and those that were susceptible (depressed). Non-depressed resilient mice displayed higher expression of IGF2 than susceptible mice. These results indicate that IGF2 contributes to ketamine's antidepressant effect and that IGF2 may confer resilience to depression-like behavior.

GSK-3 inhibitor inhibits cell proliferation and induces apoptosis in human osteosarcoma cells.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that functions in numerous signaling pathways initiated by diverse stimuli. The functions of GSK-3 in cancer differ depending on cell type. In the present study, we examined the effects of a specific GSK-3 inhibitor on the regulation of osteosarcoma cell proliferation and apoptosis. Immunohistochemical analysis and real-time reverse transcription-polymerase chain reaction (RT-PCR) were performed to determine the expression pattern of GSK-3 in human osteosarcoma cells. We used the MTS assay, western blotting, measurement of single-stranded DNA and morphological analyses to study the effects of a GSK-3 inhibitor, SB216763 on osteosarcoma cell proliferation and survival. We detected an increase in mRNA expression of GSK-3 and aberrant nuclear accumulation of GSK-3 in the osteosarcoma cells. Pharmacological inhibition of GSK-3 led to a decrease in proliferation and survival of osteosarcoma cells. Inhibition of GSK-3 resulted in a decreased expression of Bcl-2 and a subsequent increase in osteosarcoma cell apoptosis via the mitochondrial pathway. The present study demonstrated that GSK-3 activity is critical for tumorigenicity and cell survival in osteosarcoma cells. Our findings suggest that GSK-3 is a potential therapeutic target for the treatment of human osteosarcoma.

Glycogen synthase kinase-3 (GSK3) controls deoxyglucose-induced mitochondrial biogenesis in human neuroblastoma SH-SY5Y cells

Mitochondrial biogenesis, a mitochondrial growth and division process, is crucial for adaptation to metabolic stress. The present study demonstrated that treatment with a specific inhibitor of GSK3, SB216763, attenuated induction of mitochondrial biogenesis by a glycolysis inhibitor, 2-deoxyglucose (2-DG), without affecting this biogenesis at basal condition. Additionally, overexpression of WT-GSK3β promoted whereas GSK3β-KD attenuated 2-DG-induced mitochondrial protein expression. The mitochondrial biogenesis attenuation by GSK3 inhibitor was not due to inhibition of protein degradation. Furthermore, GSK3 inhibition further reduced transcription of mitochondrial (COXII), but not nuclear (VDAC) gene by 2-DG suggesting its participation in 2-DG-induced mitochondrial transcription. Together, our results show that GSK3 regulates mitochondrial biogenesis induced by glycolysis inhibition.

Glycogen Synthase Kinase-3 Inhibitors Reverse Deficits in Long-term Potentiation and Cognition in Fragile X Mice

Identifying feasible therapeutic interventions is crucial for ameliorating the intellectual disability and other afflictions of fragile X syndrome (FXS), the most common inherited cause of intellectual disability and autism. Hippocampal glycogen synthase kinase-3 (GSK3) is hyperactive in the mouse model of FXS (FX mice), and hyperactive GSK3 promotes locomotor hyperactivity and audiogenic seizure susceptibility in FX mice, raising the possibility that specific GSK3 inhibitors may improve cognitive processes. We tested if specific GSK3 inhibitors improve deficits in N-methyl-D-aspartate receptor-dependent long-term potentiation at medial perforant path synapses onto dentate granule cells and dentate gyrus-dependent cognitive behavioral tasks. GSK3 inhibitors completely rescued deficits in long-term potentiation at medial perforant path-dentate granule cells synapses in FX mice. Furthermore, synaptosomes from the dentate gyrus of FX mice displayed decreased inhibitory serine-phosphorylation of GSK3β compared with wild-type littermates. The potential therapeutic utility of GSK3 inhibitors was further tested on dentate gyrus-dependent cognitive behaviors. In vivo administration of GSK3 inhibitors completely reversed impairments in several cognitive tasks in FX mice, including novel object detection, coordinate and categorical spatial processing, and temporal ordering for visual objects. These findings establish that synaptic plasticity and cognitive deficits in FX mice can be improved by intervention with inhibitors of GSK3, which may prove therapeutically beneficial in FXS.

Glycogen Synthase Kinase 3 Inhibition Promotes Lysosomal Biogenesis and Autophagic Degradation of the Amyloid-β Precursor Protein

Alzheimer's disease (AD) has been associated with altered activity of glycogen synthase kinase 3 (GSK3) isozymes, which are proposed to contribute to both neurofibrillary tangles and amyloid plaque formation. However, the molecular basis by which GSK3 affects the formation of Aβ remains unknown. Our aim was to identify the underlying mechanisms of GSK3-dependent effects on the processing of amyloid precursor protein (APP). For this purpose, N2a cells stably expressing APP carrying the Swedish mutation were treated with specific GSK3 inhibitors or transfected with GSK3α/β short interfering RNA. We show that inhibition of GSK3 leads to decreased expression of APP by enhancing its degradation via an increase in the number of lysosomes. This induction of the lysosomal/autophagy pathway was associated with nuclear translocation of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis. Our data indicate that GSK3 inhibition reduces Aβ through an increase of the degradation of APP and its carboxy-terminal fragment (CTF) by activation of the lysosomal/autophagy pathway. These results suggest that an increased propensity toward autophagic/lysosomal alterations in AD patients could have consequences for neuronal function.

Glycogen synthase kinase-3 (GSK-3) inhibition induces apoptosis in leukemic cells through mitochondria-dependent pathway

The roles of glycogen synthase kinase-3 (GSK-3) in cell survival and apoptosis are controversial. We examined the effect of a specific GSK-3 inhibitor (SB-415286) on the regulation of leukemic cells proliferation and apoptosis. SB-415286 (40μM) induced cell growth inhibition, β-catenin stabilization, cell cycle arrest in G2/M phase, cyclin B1 downregulation, and apoptosis in leukemic cell lines KG1a, K562, and CMK. Blocking the death receptor pathway by using a specific inhibitor of caspase-8, did not inhibit SB-415286-induced apoptosis. This indicates that activation of caspase-8 is part of the intrinsic apoptotic pathway and occurs downstream of mitochondria membrane potential depolarization mediated by other caspases. Furthermore, we found that depolarization of mitochondria membrane caused by GSK-3 inhibition is regulated by dephosphorylation of anti-apoptotic protein Bcl-2 and downregulation of Bcl-xL. Thus, inhibition of GSK-3-induced apoptosis of leukemic cells could be an attractive target for treatment of leukemia.

Abstract 2874: Treatment with the autophagy inhibitor 3-Methyladenine sensitizes pancreatic cancer cells to GSK3 inhibition-induced apoptosis

Abstract Pancreatic cancer (PC) is the fourth leading cause of cancer-related death worldwide and has the highest fatality rate of all cancers. The chemoresistance of PC cells contributes to the poor prognosis of PC patients which raises the need for the identification and development of new effective therapy. Studies have revealed that the glycogen synthase kinase 3 (GSK3) is overexpressed in human pancreatic adenocarcinoma as compared to normal pancreas and its inhibition leads to the growth arrest of established tumor xenografts thus suggesting a tumor promoting role for GSK3 in PC cells. Considering that GSK3 inhibitors are currently under clinical trial for treatment of different disorders such as neurodegenerative diseases and diabetes, inhibition of GSK3 activity might represent an attractive therapeutic option for PC patients. However, mechanisms under the control of GSK3 activity in PC cells need to be clarified. We have previously demonstrated that prolonged inhibition of GSK3 activity induces JNK-dependent cell death in human PC cells. Recently, it was suggested in prostate cancer cells that GSK3 promotes both apoptosis and autophagy, the latter playing a protective role against cell death. Interestingly, inhibition of autophagy was shown to sensitize diverse cancer cells to a wide array of stress conditions. The AIM of the study was to evaluate whether GSK3 controls autophagy in PC cells and determine the role of autophagy with regard to PC cell survival. METHODS. Experiments were performed using the human PC cell lines PANC1 and MIAPaCa2. GSK3 activity was inhibited by treatment with a specific GSK3 inhibitor SB216763 (20μM). Apoptosis was measured by assessment of PARP cleavage and caspase 3 and 7 activities. Autophagy was evaluated by the detection of the membrane-bound LC3B-II isoform. The autophagy inhibitor 3-methyladenine 3-MA (10mM) and the JNK inhibitor SP600125 (25μM) were also used. RESULTS. 1) Prolonged inhibition of GSK3 activity (48-72h) in PC cells induced both an apoptotic and autophagic responses. Treatment with 3-MA 2) elicited an apoptotic response and 3) strongly promoted the SB216763-induced apoptosis. 4) Mechanistically, inhibition of GSK3 activity led to a rapid and persistent activation of the JNK-cJun pathway. 5) Interestingly, blockade of the JNK pathway prevented the SB216763-induced apoptosis but not the SB216763-induced autophagy. CONCLUSION. Our results provide evidences that GSK3 regulates both apoptosis and autophagy in PC cells. On one hand, inhibition of GSK3 activity triggers apoptosis through JNK-dependent mechanisms. On the other hand, inhibition of GSK3, independently of the JNK pathway, promotes autophagy, a process that appears to protect PC cells against cell death. Inhibition of autophagy might represent an attractive avenue to promote cytotoxic activity of potential therapeutic agents such as GSK3 inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2874. doi:10.1158/1538-7445.AM2011-2874

Effects of streptozotocin-induced diabetes on tau phosphorylation in the rat brain

Brain protein kinase B (Akt) and glycogen synthase kinase-3 (GSK-3) activities are adaptable to changes of peripheral blood glucose level in vivo. GSK-3 phosphorylates microtube-associated protein tau at multiple sites, which can be antagonized by protein phosphatase-2A (PP-2A). The imbalance among these enzymes might have potential connections with diabetes mellitus (DM) and Alzheimer's disease (AD). In this study hyperglycemia rat DM model was achieved by streptozotocin (STZ) treatment. The phosphorylation of tau in the rat hippocampus was detected with specific antibodies. Insulin and Li2CO3 administration were also employed to find out the regulatory efforts of the kinases. We observed that rat hippocampus tau was hyperphosphorylated at Ser396/Ser404 (PHF-1 sites) in STZ-induced DM model, accompanied by lowered phosphorylation levels of Akt, GSK-3 and PP-2A. Lithium, a specific GSK-3 inhibitor, nearly reversed all phosphorylation of tau at above sites in 30days. Insulin administration restored the blood glucose level in DM rats but suppressed PP-2A activity, resulting in the PHF-1 sites of tau not being dephosphorylated. These findings strongly suggest that STZ-induced hyperglycemia may cause disorder of Akt/GSK-3/PP-2A regulations in rat brain and further lead to abnormal phosphorylation of hippocampus tau.

Tissue-Specific Analysis of Glycogen Synthase Kinase-3α (GSK-3α) in Glucose Metabolism: Effect of Strain Variation

BackgroundOver-activity and elevated expression of glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology of insulin resistance and Type 2 diabetes. Administration of specific GSK-3 inhibitors to diabetic or obese rodent models improves glycaemic control and insulin sensitivity. However, due to the indiscriminatory nature of these inhibitors, the relative contribution of the two isoforms of GSK-3 (GSK-3α and GSK-3β) is not known. Recently, we demonstrated that an out-bred strain of mice (ICR) lacking expression of GSK-3α in all tissues displayed improved insulin sensitivity and enhanced hepatic glucose metabolism. We also found that muscle (but not liver) inactivation of GSK-3β conferred insulin and glucose sensitization in an in-bred strain of mice (C57BL/6).Methodology/Principal FindingsHere, we have employed tissue-specific deletion of GSK-3α, to examine the relative contribution of two insulin-sensitive tissues, muscle and liver, towards the insulin sensitization phenotype originally observed in the global GSK-3α KO animals. We found that mice in which GSK-3α has been inactivated in either skeletal-muscle or liver displayed no differences in glucose tolerance or insulin sensitivity compared to wild type littermates. Given the strain differences in our original analyses, we examined the insulin and glucose sensitivity of global GSK-3α KO animals bred onto a C57BL/6 background. These animals also revealed no significant differences in glucose metabolism/insulin sensitivity compared to their wild type littermates. Furthermore, deletion of hepatic GSK-3α on the out-bred, ICR background failed to reproduce the insulin sensitivity manifested by the global deletion of this isoform.Conclusions/SignificanceFrom these data we conclude that the improved insulin sensitivity and hepatic glucose homeostasis phenotype observed upon global inactivation of GSK-3α is strain-specific. We surmise that the insulin-sensitization observed in the out-bred strain of mice lacking GSK-3α is mediated by indirect means that do not require intrinsic function of GSK-3α in skeletal muscle and liver tissues.

Glycogen synthase kinase-3α/β inhibition promotes in vivo amplification of endogenous mesenchymal progenitors with osteogenic and adipogenic potential and their differentiation to the osteogenic lineage

Small molecules are attractive therapeutics to amplify and direct differentiation of stem cells. They also can be used to understand the regulation of their fate by interfering with specific signaling pathways. Mesenchymal stem cells (MSCs) have the potential to proliferate and differentiate into several cell types, including osteoblasts. Activation of canonical Wnt signaling by inhibition of glycogen synthase kinase 3 (GSK-3) has been shown to enhance bone mass, possibly by involving a number of mechanisms ranging from amplification of the mesenchymal stem cell pool to the commitment and differentiation of osteoblasts. Here we have used a highly specific novel inhibitor of GSK-3, AR28, capable of inducing β-catenin nuclear translocation and enhanced bone mass after 14 days of treatment in BALB/c mice. We have shown a temporally regulated increase in the number of colony-forming units-osteoblast (CFU-O) and -adipocyte (CFU-A) but not colony-forming units-fibroblast (CFU-F) in mice treated for 3 days. However, the number of CFU-O and CFU-A returned to normal levels after 14 days of treatment, and the number of CFU-F was decreased significantly. In contrast, the number of osteoblasts increased significantly only after 14 days of treatment, and this was seen together with a significant decrease in bone marrow adiposity. These data suggest that the increased bone mass is the result of an early temporal wave of amplification of a subpopulation of MSCs with both osteogenic and adipogenic potential, which is driven to osteoblast differentiation at the expense of adipogenesis.

Potential Mechanisms Involved in the Prevention of Neurodegenerative Diseases by Lithium

Lithium is a monovalent cation that was introduced in 1949 by John Cade for the treatment of bipolar disorder. Clinical reports and subsequent studies confirmed this application and the beneficial effects of this compound. However, over the last 15 years, various authors have also demonstrated the neuroprotective effects of lithium against several neurotoxic paradigms. Thus, experimental studies in neuronal cell cultures and animal models of Alzheimer disease and others pathologies have provided strong evidence for the potential benefits of lithium. The main mechanism underlying its neuroprotective effects is thought to be inhibition of glycogen synthase kinase-3 (GSK-3), although other biochemical pathways in the brain could also be affected. In this review, the main mechanisms of lithium action are summarized, including the modulation of glutamate receptors, effects on arachidonic acid metabolism, its role with respect to AKT, and other potential mechanisms. In addition, its effects on neuroprotective proteins such as Bcl-2 and p53 are also discussed. Although the cellular and molecular biological effects of lithium may constitute an effective therapeutic strategy for Alzheimer disease, further clinical and experimental studies with this drug and specific GSK-3 inhibitors are necessary to confirm the use of lithium in therapeutic approaches to neurodegenerative diseases.

M Phase-Specific Phosphorylation of Histone H1.5 at Threonine 10 by GSK-3

H1 histones are progressively phosphorylated during the cell cycle. The number of phosphorylated sites is zero to three in late S phase and increases to five or six in late G2 phase and M phase. It is assumed that this phosphorylation modulates chromatin condensation and decondensation, but its specific role remains unclear. Recently, it was shown that the somatic H1 histone subtype H1.5 becomes pentaphosphorylated during mitosis, with phosphorylated threonine 10 being the last site to be phosphorylated. We have generated an antiserum specific for human H1.5 phosphorylated at threonine 10. Immunofluorescence labeling of HeLa cells with this antiserum revealed that the phosphorylation at this site appears in prometaphase and disappears in telophase, and that this hyperphosphorylated form of H1.5 is mainly chromatin-bound in metaphase when chromatin condensation is maximal. In search of the kinase responsible for the phosphorylation at this site, we found that threonine 10 of H1.5 can be phosphorylated by glycogen synthase kinase-3 in vitro, but not by cyclin-dependent kinase 1/cyclin B and cyclin-dependent kinase 5/p35, respectively. Furthermore, addition of specific glycogen synthase kinase-3 inhibitors led to a reduction in phosphorylation at this site both in vivo and in vitro.

GSK3 inhibitors show benefits in an Alzheimer's disease (AD) model of neurodegeneration but adverse effects in control animals

The dysregulation of glycogen synthase kinase-3 (GSK3) has been implicated in Alzheimer disease (AD) pathogenesis and in Aβ-induced neurotoxicity, leading us to investigate it as a therapeutic target in an intracerebroventricular Aβ infusion model. Infusion of a specific GSK3 inhibitor SB216763 (SB) reduced a downstream target, phospho-glycogen synthase 39%, and increased glycogen levels 44%, suggesting effective inhibition of enzyme activity. Compared to vehicle, Aβ increased GSK3 activity, and was associated with elevations in levels of ptau, caspase-3, the tau kinase phospho-c- jun N-terminal kinase (pJNK), neuronal DNA fragmentation, and gliosis. Co-infusion of SB corrected all responses to Aβ infusion except the induction of gliosis and behavioral deficits in the Morris water maze. Nevertheless, SB alone was associated with induction of neurodegenerative markers and behavioral deficits. These data support a role for GSK3 hyperactivation in AD pathogenesis, but emphasize the importance of developing inhibitors that do not suppress constitutive activity.

Lithium inhibits invasion of glioma cells; possible involvement of glycogen synthase kinase-3

Therapies targeting glioma cells that diffusely infiltrate normal brain are highly sought after. Our aim was to identify novel approaches to this problem using glioma spheroid migration assays. Lithium, a currently approved drug for the treatment of bipolar illnesses, has not been previously examined in the context of glioma migration. We found that lithium treatment potently blocked glioma cell migration in spheroid, wound-healing, and brain slice assays. The effects observed were dose dependent and reversible, and worked using every glioma cell line tested. In addition, there was little effect on cell viability at lithium concentrations that inhibit migration, showing that this is a specific effect. Lithium treatment was associated with a marked change in cell morphology, with cells retracting the long extensions at their leading edge. Examination of known targets of lithium showed that inositol monophosphatase inhibition had no effect on glioma migration, whereas inhibition of glycogen synthase kinase-3 (GSK-3) did. This suggested that the effects of lithium on glioma cell migration could possibly be mediated through GSK-3. Specific pharmacologic GSK-3 inhibitors and siRNA knockdown of GSK-3alpha or GSK-3beta isoforms both reduced cell motility. These data outline previously unidentified pathways and inhibitors that may be useful for the development of novel anti-invasive therapeutics for the treatment of brain tumors.