Inhibition Of GSK-3beta Research Articles

Lithium treatment reduced microglia activation and inflammation after irradiation to the immature brain (P6256)

Abstract To evaluate the effects of lithium on microglia activation and inflammation after irradiation to the immature brain, male rat pups were injected 2 mmol/kg lithium chloride i.p. on postnatal day 7 (P7), additional lithium injections, 1 mmol/kg, were administered at 24 h intervals. Pups were subjected to whole brain 6Gy irradiation on P11. The pups were sacrificed at 6h and 24h after IR. Microglia scattered in the brain can be detected by counting their numbers, their size, engulfment of cell debris or by the production of cytokines and chemokines. Microglia were stained using the marker Iba-1, revealing the presence of these cells throughout the brain under normal conditions, and apparently higher numbers and bigger sizes after IR, particularly in areas where cell death occurred. Quantification of Iba-1+ cells in the GCL of DG showed a significant increase after IR, but with a lower increase in the lithium-treated brains. The concentration of MCP-1, IL-1α, IL-1β, GRO/KC in the hippocampus were increased significantly at 6h after IR compared with non-irradiation control. Lithium treatment significantly inhibited the increase. There was no significantly difference with these cytokines/chemokines at 24h after IR between lithium and vehicle treated group. These data indicate that lithium can specifically reduce inflammation through GSK3-beta inhibition or modified microglia activation.

Group II Metabotropic Glutamate Receptor Agonist LY379268 Regulates AMPA Receptor Trafficking in Prefrontal Cortical Neurons

Group II metabotropic glutamate receptor (mGluR) agonists have emerged as potential treatment drugs for schizophrenia and other neurological disorders, whereas the mechanisms involved remain elusive. Here we examined the effects of LY379268 (LY37) on the expression and trafficking of the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits GluA1 and GluA2 in prefrontal neurons. We show that LY37 significantly increased the surface and total expression of both GluA1 and GluA2 subunits in cultured prefrontal neurons and in vivo. This effect was mimicked by the selective mGluR2 agonist LY395756 and was blocked by mGluR2/3 antagonist LY341495. Moreover, we found that both GluA1 and GluA2 subunits were colocalized with PSD95 but not synapsin I, suggesting a postsynaptic localization. Consistently, treatment with LY37 significantly increased the amplitude, but not frequency, of miniature excitatory postsynaptic currents. Further, actinomycin-D blocked LY37's effects, suggesting a transcriptional regulation. In addition, application of glycogen synthase kinase-3beta (GSK-3β) inhibitor completely blocked LY37's effect on GluA2 surface expression, whereas GSK-3β inhibitor itself induced decreases in the surface and total protein levels of GluA1, but not GluA2 subunits. This suggests that GSK-3β differentially mediates GluA1 and GluA2 trafficking. Further, LY37 significantly increased the phosphorylation, but not total protein, of extracellular signal-regulated kinase 1/2 (ERK1/2). Neither ERK1/2 inhibitor PD98059 alone nor PD98059 combined with LY37 treatment induced changes in GluA1 or GluA2 surface expression or total protein levels. Our data thus suggest that mGluR2/3 agonist regulates postsynaptic AMPA receptors by affecting the synaptic trafficking of both GluA1 and GluA2 subunits and that the regulation is likely through ERK1/2 signaling in GluA1 and/or both ERK1/2 and GSK-3β signaling pathways in the GluA2 subunit.

Inhibition of WNT signalling interferes with HCV replication

Background and Aims: The tumor relevant wnt pathway activity is associated with higher HCV replication. HCV core and NS5 protein have recently been shown to induce wnt pathway activity, implicating a cause for progression to HCC. We now investigated if wnt pathway activity might affect HCV replication. Methods: For in vitro experiments the HCV replicon system expressing HCV genes NS3 to NS5B alone (Huh5–15) or in combination with firefly luciferase (LucUbiNeo-ET) was used. To promote wnt pathway activity the glycogen synthase kinase 3 beta (GSK3b) inhibitors Kenpaullone or SB216763 were used. Inhibition of the wnt pathway was achieved by use of either Quercetin or 4,5,6,7-tetrabromo-2-azabenzimidazole (TBB), an inhibitor of beta-catenin stabilizing kinase CK2, or by transient knockdown of beta-catenin by siRNA. Gene expression was measured by real time RT-PCR and Western Blot analysis. Wnt pathway activity was measured by a reporter assay for beta catenin related transcription (CRT assay). Results: Our results confirm that activity of the wnt pathway is increased in replicon cell lines compared to the replicon background cell line Huh7 and that the HCV protein NS5A is involved. Additionally we found that NS3B is able to increase Wnt pathway activity. Inhibition of the wnt signaling pathway by quercetin, TBB or siRNA directed against beta-catenin reduced HCV replication significantly without toxic effects on cultured cells. Moreover, activation of wnt pathway activity by inhibitors of GSK3b increased HCV replication. Conclusions: HCV replication induces wnt signaling which in turn seems to promote HCV replication. Therefore, inhibition of the wnt signaling pathway might not only represent a way to interfere with tumor growth but might also become a novel approach for HCV therapy.

Regulation of FANCL by Glycogen Synthase Kinase-3beta Links the Fanconi anemia pathway to Self Renewal and Survival Signals

Abstract 1263The molecular basis for how a Fanconi anemia (FA) genetic background contributes to hematopoietic stem cell defects and hypoplastic organ development remains poorly understood. Protein modification by ubiquitination is a mechanism that diversifies the function and regulation of proteins. In light of this, we focus on the dysfunction of FANCL, the E3 ubiquitin ligase of the FA pathway, as a key molecular defect in Fanconi anemia.Here we report our studies investigating mechanisms of post-translational regulation of FANCL. We view these mechanisms as potential targets to augment the function of the FA core complex and correct hematopoietic stem cell defects. We provide evidence that FANCL is exquisitely regulated by ubiquitin-proteosome degradation. Ligase-inactive mutants (FANCL-C307A and -W341G) are less sensitive to this regulation, suggesting a role for auto-ubiquitination in directing lysine-48 polyubiquitination. This constitutive negative regulation of FANCL is partially reversed with an ATP-competitive glycogen synthase kinase-3beta (GSK-3beta) inhibitor. GSK-3beta is a serine/threonine kinase that phosphorylates proteins and marks them for ubiquitin-mediated proteolysis. Mitogenic and survival pathways, including Ras/MAPK and PI3K/Akt, negatively regulate GSK-3beta by serine-9 phosphorylation. We show that the regulation of FANCL by GSK-3beta is likely direct because FANCL and GSK-3beta co-immunoprecipitate in cell lysates and as GST-fusion proteins. To define the biochemical mechanisms of FANCL regulation, we generated N-terminal deletion mutants of FANCL and we show that the regulation of FANCL is dictated by a region at the N-terminus (aa1-78). Mutational analysis of FANCL (lysine to arginine) in this N-terminus region does not affect the overall protein level or ubiquitination of FANCL, suggesting that FANCL may be targeted for degradation by phosphorylation and/or in a complex with other proteins.The potential biological relevance of our findings, that FANCL is regulated by GSK-3beta is revealed in studies overexpressing constitutively active, myristoylated-Akt. This experimental condition increases FANCL protein levels and suggests a role for FANCL as a downstream effector of PI3K/Akt signaling. In turn, FANCL likely regulates non-canonical targets that alter the transcriptome profile favoring self-renewal and survival of hematopoietic stem cells. We recently published our studies identifying beta-catenin as one such downstream target (Blood 2012 Jul 12;120:323). Suppression of FANCL expression severely disrupts Wnt/beta-catenin signaling and expression of downstream Wnt-responsive targets MYC and CCND1. We also identified that GSK3B gene expression is approximately 5-fold higher in Fancc-deficient hematopoietic stem cells exposed to TNF-alpha compared to untreated cells or to wildtype cells with or without TNF-alpha. Our current studies show that inhibition of GSK-3beta preserves the number of murine Fancc-deficient hematopoietic stem cells exposed to TNF-alpha compared with no GSK-3beta inhibition.Taken together, we have accumulated evidence suggesting that GSK-3beta is a promising molecular target to improve the self-renewal and survival of FA hematopoietic stem cells. Disclosures:No relevant conflicts of interest to declare.

Glycogen synthase kinase-3beta regulates differentiation-induced apoptosis of human neural progenitor cells

Glycogen synthase kinase-3beta is a multifunctional key regulator enzyme in neural developmental processes and a main component of the canonical Wnt signaling pathway. It is already known that the Wnt-driven differentiation of neural progenitor cells is accompanied by an increase of apoptosis at which the pro-apoptotic function of GSK-3beta is still discussed. The aim of the present study was to investigate whether the phosphorylation level of GSK-3beta at serine 9 is the primary regulatory mechanism of differentiation-induced apoptosis.Differentiating human neural ReNcell VM progenitor cells were treated with the specific GSK-3beta inhibitor SB216763 (10μM) and analyzed in respect to the intrinsic apoptosis pathway regulation using microscopy and protein expression analysis.Differentiation of ReNcell VM cells was accompanied by cell morphological changes, cytoskeleton rearrangement and apoptosis increase. Treatment of differentiating cells with SB216763 induced a significant dephosphorylation of GSK-3beta at serine 9 accompanied by a significant decrease of apoptosis of about 0.7±0.03% and reduced activation of caspase-3 as well as BAX and PARP cleavage during the first 12h of differentiation compared to untreated, differentiating cells.Dephosphorylation of GSK-3beta at serine 9 appears not solely to be responsible for its pro-apoptotic function, because we observed a decrease of intrinsic apoptosis after treatment of the cells with the specific GSK-3beta inhibitor SB216763. We assume that GSK-3beta drives neural progenitor cell apoptosis by direct interaction with pro-apoptotic BAX or by indirect influence on the canonical Wnt/beta-catenin target gene transcription.

A Design Principle for a Posttranslational Biochemical Oscillator

Multisite phosphorylation plays an important role in biological oscillators such as the circadian clock. Its general role, however, has been elusive. In this theoretical study, we show that a simple substrate with two modification sites acted upon by two opposing enzymes (e.g., a kinase and a phosphatase) can show oscillations in its modification state. An unbiased computational analysis of this oscillator reveals two common characteristics: a unidirectional modification cycle and sequestering of an enzyme by a specific modification state. These two motifs cause a substrate to act as a coupled system in whicha unidirectional cycle generates single-molecule oscillators, whereas sequestration synchronizes the population by limiting the available enzyme under conditions in which substrate is in excess. We also demonstrate the conditions under which the oscillation period is temperature compensated, an important feature of the circadian clock. This theoretical model will provide a framework for analyzing and synthesizing posttranslational oscillators.

P2‐097: Inhibition of PLA2 inactivates GSK3B via Akt and CAMKII in primary cultures of hippocampal neurons

Glycogen synthase kinase 3-beta (GSK3B) is overactive in Alzheimer's disease (AD), favoring the hyperphosphorylation of microtubule-associated protein Tau and the formation of neurofibrillary tangles. For this reason, GSK3B inhibition may be considered as a candidate disease-modifying approach in AD. In addition to the direct enzymatic inhibition (eg. by lithium), GSK3B can be also indirectly inactivated by phosphorylation at the Ser9 residue. This is a downstream effect of several intracellular cascades, including Akt and CaMKII signaling. Phospholipase A 2 (PLA 2) are key enzymes in membrane homeostasis and cell signaling. Reduced PLA 2 activity has been demonstrated in cerebral and peripheral tissues of patients with AD, and recent studies suggested that PLA 2 is further implicated in GSK3B homeostasis. The objective of this study is to evaluate the effect of the inhibition of PLA 2 on the GSK3B activity and to determine whether this effect involves CAMKII and Akt. Primary cultures of cortical and hippocampal neurons started from embryonic rats (E18) were treated after 4 four days in culture with different concentrations (5-250 μM) of methyl-aracdonyl-fluorophosphonate (MAFP), which is an irreversible, dual inhibitor of calcium-dependent and -independent cytosolic PLA 2. Treatment effects were evaluated by Western-blot, addressing the expression of total- and phosphorylated (Ser9) GSK3B, phosphorylated Akt (Ser473), and total CAMKII at protein level. The inhibition of GSK3B is a downstream effect of cytosolic PLA 2 inhibition, which apparently involves the activation of Akt and CAMKII. This effect seems to be specific to hippocampal neurons.

Inhibition of GSK-3β ameliorates hepatic ischemia-reperfusion injury through GSK-3β/β-catenin signaling pathway in mice

Glycogen synthase kinase (GSK)-3beta/beta-catenin signaling regulates ischemia-reperfusion (I/R)-induced apoptosis and proliferation, and inhibition of GSK-3beta has beneficial effects on I/R injury in the heart and the central nervous system. However, the role of this signaling in hepatic I/R injury remains unclear. The present study aimed to investigate the effects and mechanism of GSK-3beta/beta-catenin signaling in hepatic I/R injury. Male C57BL/6 mice (weighing 22-25 g) were pretreated with either SB216763, an inhibitor of GSK-3beta, or vehicle. These mice were subjected to partial hepatic I/R. Blood was collected for test of alanine aminotransferase (ALT), and liver specimen for assays of phosphorylation at the Ser9 residue of GSK-3beta, GSK-3beta activity, axin 2 and the anti-apoptotic factors Bcl-2 and survivin, as well as the proliferative factors cyclin D1 and proliferating cell nuclear antigen, and apoptotic index (TUNEL). Real-time PCR, Western blotting and immunohistochemical staining were used. SB216763 increased phospho-GSK-3beta levels and suppressed GSK-3beta activity (1880+/-229 vs 3280+/-272 cpm, P<0.01). ALT peaked at 6 hours after reperfusion. Compared with control, SB216763 decreased ALT after 6 hours of reperfusion (4451+/-424 vs 7868+/-845 IU/L, P<0.01), and alleviated hepatocyte necrosis and vacuolization. GSK-3beta inhibition led to the accumulation of beta-catenin in the cytosol (0.40+/-0.05 vs 1.31+/-0.11, P<0.05) and nucleus (0.62+/-0.14 vs 1.73+/-0.12, P<0.05), beta-catenin further upregulated the expression of axin 2. Upregulation of GSK-3beta/beta-catenin signaling increased Bcl-2, survivin and cyclin D1. Serological and histological analyses showed that SB216763 alleviated hepatic I/R-induced injury by reducing apoptosis (1.4+/-0.2% vs 3.6+/-0.4%, P<0.05) and enhanced liver proliferation (56+/-8% vs 19+/-4%, P<0.05). Inhibition of GSK-3beta ameliorates hepatic I/R injury through the GSK-3beta/beta-catenin signaling pathway.

Tracing the Conversion Process from Primordial Germ Cells to Pluripotent Stem Cells in Mice1

To understand mechanisms underlying acquisition of pluripotency, it is critical to identify cells that can be converted to pluripotent stem cells. For this purpose, we focused on unipotent primordial germ cells (PGCs), which can be reprogrammed into pluripotent embryonic germ (EG) cells under defined conditions. Treatment of PGCs with combinations of signaling inhibitors, including inhibitors of MAP2K (MEK), GSK3B (GSK-3beta), and TGFB (TGFbeta) type 1 receptors, induced cells to enter a pluripotent state at a high frequency (12.1%) by Day 10 of culture. When we employed fluorescence-activated cell sorting to monitor conversion of candidate cells to a pluripotent state, we observed a cell cycle shift to S phase, indicating enrichment of pluripotent cells, during the early phase of EG formation. Transcriptome analysis revealed that PGCs retained expression of some pluripotent stem cell-associated genes, such as Pou5f1 and Sox2, during EG cell formation. On the other hand, PGCs lost their germ lineage characteristics and acquired expression of pluripotent stem cell markers, such as Klf4 and Eras. The overall gene expression profiles revealed by this system provide novel insight into how pluripotency is acquired in germ-committed cells.

Glycogen synthase kinase-3 beta inhibitor suppresses Porphyromonas gingivalis lipopolysaccharide-induced CD40 expression by inhibiting nuclear factor-kappa B activation in mouse osteoblasts

Bone-forming osteoblasts have been recently reported capable of expressing the critical co-stimulatory molecule CD40 upon exposure to bacterial infection, which supports the unappreciated role of osteoblasts in modulating bone inflammation. Recent studies highlight the anti-inflammatory potential of glycogen synthase kinase-3β (GSK-3β) inhibitors; however, their effect on osteoblasts remains largely unclear. In the present study, we showed that treatment with SB216763, a highly specific GSK-3β inhibitor, resulted in a dose-dependent decrease in the mRNA and protein expression of CD40, as well as production of pro-inflammatory cytokines IL-6, TNF-α and IL-1β, in the Porphyromonas gingivalis-lipopolysaccharide (LPS)-stimulated murine osteoblastic-like MC3T3-E1 cells. Furthermore, inhibition of GSK-3β remarkably represses the LPS-induced activation of the nuclear factor kappa B (NF-κB) signaling pathway by suppressing IκBα phosphorylation, NF-κBp65 nuclear translocation, and NF-κBp65 DNA binding activity. Closer investigation by immunoprecipitation assay revealed that β-catenin can physically interact with NF-κBp65. The negative regulation effect of GSK-3β inhibitor on CD40 expression is mediated through β-catenin, for siRNA of β-catenin attenuated the GSK-3β inhibitor-induced repression of NF-κB activation and, consequently, the expression of CD40 and production of pro-inflammatory cytokines in LPS-stimulated MC3T3-E1 cells. Thus our results elucidate the molecular mechanisms whereby GSK-3β inhibitor prevents the LPS-induced CD40 expression on osteoblasts and provide supportive evidence of the potential role of GSK-3β inhibitors in suppressing the immune function of osteoblasts in inflammatory bone diseases.

Lithium: A switch from LTD- to LTP-like plasticity in human cortex

Lithium, a simple cation, is the mainstay treatment of bipolar disorder. Deficient synaptic plasticity is considered one important mechanism of this disease. Lithium inhibits glycogen synthase kinase-3beta (GSK-3β), which is involved in the regulation of synaptic plasticity. In animal preparations, inhibition of GSK-3β by lithium up-regulated long-term potentiation (LTP) of excitatory synapses but down-regulated long-term depression (LTD). The effects of lithium on plasticity in the human brain are unexplored. We tested the effects of a single oral dose of 900 mg of lithium on LTP-/LTD-like plasticity in human motor cortex induced by established paired associative transcranial magnetic stimulation (PASLTP, PASLTD) protocols. We studied 10 healthy adults in a placebo-controlled double-blind randomized crossover design. PAS-induced plasticity was indexed by change in motor evoked potential amplitude recorded in a hand muscle. In the placebo session, subjects were stratified, according to the known variability of the PASLTP response, into PASLTP ‘LTP responders’ and PASLTP ‘LTD responders’ (n = 5 each). Lithium did not affect the PASLTP-induced LTP-like plasticity in the ‘LTP responders’, but switched the PASLTP-induced LTD-like plasticity in the ‘LTD responders’ to LTP-like plasticity. In contrast, lithium had no effect on the PASLTD-induced LTD-like plasticity in the ‘LTD responders’. We provide first-time evidence that lithium significantly modulates brain stimulation induced plasticity in human cortex. The switch from LTD- to LTP-like plasticity is best explained by the inhibitory action of lithium on GSK-3β. This conclusion is necessarily circumstantial because GSK-3β activity was not directly measured. We discuss that other important plasticity-related modes actions of lithium cannot explain our findings.

Kisspeptin-10 inhibits cell migration in vitro via a receptor-GSK3 beta-FAK feedback loop in HTR8SVneo cells

Kisspeptin inhibits cancer cell metastasis and placental trophoblast cell migration. Kisspeptin gene expression in the placenta and circulating kisspeptin levels change during normal pregnancy and they are altered in preeclampsia. We therefore assessed the effect of kisspeptin-10 on the in vitro migration of a human placental cell line derived from first trimester extravillious trophoblasts (HTR8SVneo). HTR8SVneo cells specifically bound 125I-Kisspeptin-10 but kisspeptin-10 did not induce inositol phosphate production. Cell migration was inhibited by kisspeptin-10 with a maximal inhibition at 100nM. The signaling pathways involved in inhibition of cell migration were examined. Treatment with kisspeptin-10 elicited phosphorylation of GSK3 beta at Ser9 (which inhibits activity), with a 3-fold increase at 5min. Transient phosphorylation of ERK1/2 and p38MAPK peaked at 10min. Phosphorylation of focal adhesion kinase (FAK) at Tyr925 increased 3-fold at 10min. Inhibition of GSK3 beta correlated with release of beta-catenin into the cytoplasm. These signaling events were differentially blocked by inhibitors of Gq/11, Src, EGFR, PI(3)K, PKC and MEK. The data suggest that kisspeptin/GPR54 EGF-receptor transactivation leads to phosphorylation of ERK1/2, causing activation of p90rsk which in turn inhibits GSK3 beta via Ser9 phosphorylation. Inactivation of GSK3 beta results in release of beta-catenin into the cytoplasm, affecting cell–cell adhesion and Tyr925 phosphorylation of FAK, which increases phosphorylation of ERK1/2 via RAS/Raf-1 creating a feedback loop to enhance the effects on migration. These findings indicate that kisspeptin-10 inhibits the migration of human placental trophoblast-derived HTR8SVneo cells by stimulating complex ERK1/2–p90rsk–GSK3 beta–FAK feedback interactions.

Lithium Treatment of APPSwDI/NOS2−/− Mice Leads to Reduced Hyperphosphorylated Tau, Increased Amyloid Deposition and Altered Inflammatory Phenotype

Lithium is an anti-psychotic that has been shown to prevent the hyperphosphorylation of tau protein through the inhibition of glycogen-synthase kinase 3-beta (GSK3β). We recently developed a mouse model that progresses from amyloid pathology to tau pathology and neurodegeneration due to the genetic deletion of NOS2 in an APP transgenic mouse; the APPSwDI/NOS2−/− mouse. Because this mouse develops tau pathology, amyloid pathology and neuronal loss we were interested in the effect anti-tau therapy would have on amyloid pathology, learning and memory. We administered lithium in the diets of APPSwDI/NOS2−/− mice for a period of eight months, followed by water maze testing at 12 months of age, immediately prior to sacrifice. We found that lithium significantly lowered hyperphosphorylated tau levels as measured by Western blot and immunocytochemistry. However, we found no apparent neuroprotection, no effect on spatial memory deficits and an increase in histological amyloid deposition. Aβ levels measured biochemically were unaltered. We also found that lithium significantly altered the neuroinflammatory phenotype of the brain, resulting in enhanced alternative inflammatory response while concurrently lowering the classical inflammatory response. Our data suggest that lithium may be beneficial for the treatment of tauopathies but may not be beneficial for the treatment of Alzheimer's disease.

Valproic acid mediates the synaptic excitatory/inhibitory balance through astrocytes — A preliminary study

Valproic acid (VPA) is one of the most widely used anticonvulsant and mood-stabilizing agents for the treatment of epilepsy and bipolar disorder. However, the underlying therapeutic mechanisms of the treatment of each disease remain unclear. Recently, the anti-epileptic effect of VPA has been found to lead to modulation of the synaptic excitatory/inhibitory balance. In addition, the therapeutic action of VPA has been linked to its effect on astrocytes by regulating gene expression at the molecular level, perhaps through an epigenetic mechanism as a histone deacetylase (HDAC) inhibitor. To provide insight into the mechanisms underlying the actions of VPA, this study investigated whether the synaptic excitatory/inhibitory (E/I) balance could be mediated by VPA through astrocytes. First, using the primary rat neuronal, astroglial, and neuro-glial mixed culture systems, we demonstrated that VPA treatment could regulate the mRNA levels of two post-synaptic cell adhesion molecules(neuroligin-1 and neuregulin-1) and two extracellular matrices (neuronal pentraxin-1and thrombospondin-3) in primary rat astrocyte cultures in a time- and concentration-dependent manner. Moreover, the up-regulation effect of VPA was noted in astrocytes, but not in neurons. In addition, these regulatory effects could be mimicked by sodium butyrate, a HDAC inhibitor, but not by lithium or two other glycogen synthase kinase-3 beta inhibitors. With the known role of these four proteins in regulating the synaptic E/I balance, we further demonstrated that VPA increased excitatory post-synaptic protein (postsynaptic density 95) and inhibitory post-synaptic protein (Gephyrin) in cortical neuro-glial mixed cultures. Our results suggested that VPA might affect the synaptic excitatory/inhibitory balance through its effect on astrocytes. This work provides the basis for future evaluation of the role of astroglial cell adhesion molecules and the extracellular matrix on the control of excitatory and inhibitory synapse formation.

Characterization of motor neuron loss in animal models featuring prolonged disease duration

Motor neuron loss is a feature of amyotrophic lateral sclerosis (ALS), spinal muscle atrophy (SMA) and it is recently described in other neurodegenerative disorders. In humans these disorders vary from dramatically short up to long disease duration. A short disease duration in humans ranges from some months up to 1 or 2 years. In contrast animal models commonly used to evaluate motor neuron disorders are extremely short lasting, where motor impairment lasts about a few weeks. This is the case of the G93A mouse model which is expected to reproduce ALS in humans. Despite commonalities, this condensed time interval is likely to produce different pathological correlates. Therefore, it is not surprising that experimental morphology and therapeutics fail to detect some hallmarks of human disorders in these animal models. This temporal discrepancy is often missed out and genetic background as well as evolution-based structural differences in motor systems are considered as critical determinants instead. The present communication wish to introduce the benefits of a long-lasting animal model of motor neuron disorders. In detail, we had been able to characterize the motor failure, the biochemical abnormalities and the morphological alterations which develop only at prolonged time interval (18 months) using a single knock out double transgenic mouse model of human SMAIII. In these mice we had been able to dissect for the first time morphological alterations typical of human disorders such as motor neuron heterotopy as well as motor neuron loss. This is correlated with a profound loss of the survival motor neuron (SMN) protein. There was a remarkable somatotopic correlation between the specific motor deficit (proximal vs distal limb muscles) and the severity of motor neuron loss in the corresponding pool (medial vs lateral) of motor neurons in the spinal cord. This ideal experimental context provides the chance to evaluate the therapeutic effects of specific drugs administered in the long run ruling out bias due to experimental variability when the drugs are administered only for a few weeks. In this way it is also easy to discern symptomatic vs disease modifying effects. In line with this, we compared the effects of GSK3 beta inhibitors and/or autophagy activators and we found a remarkable induction in the SMN protein which was in line with protection from motor neuron loss and a steady effect counteracting the long term progressive motor deterioration.

Evidence for Irreversible Inhibition of Glycogen Synthase Kinase-3β by Tideglusib

Tideglusib is a GSK-3 inhibitor currently in phase II clinical trials for the treatment of Alzheimer disease and progressive supranuclear palsy. Sustained oral administration of the compound to a variety of animal models decreases Tau hyperphosphorylation, lowers brain amyloid plaque load, improves learning and memory, and prevents neuronal loss. We report here that tideglusib inhibits GSK-3β irreversibly, as demonstrated by the lack of recovery in enzyme function after the unbound drug has been removed from the reaction medium and the fact that its dissociation rate constant is non-significantly different from zero. Such irreversibility may explain the non-competitive inhibition pattern with respect to ATP shown by tideglusib and perhaps other structurally related compounds. The replacement of Cys-199 by an Ala residue in the enzyme seems to increase the dissociation rate, although the drug retains its inhibitory activity with decreased potency and long residence time. In addition, tideglusib failed to inhibit a series of kinases that contain a Cys homologous to Cys-199 in their active site, suggesting that its inhibition of GSK-3β obeys to a specific mechanism and is not a consequence of nonspecific reactivity. Results obtained with [(35)S]tideglusib do not support unequivocally the existence of a covalent bond between the drug and GSK-3β. The irreversibility of the inhibition and the very low protein turnover rate observed for the enzyme are particularly relevant from a pharmacological perspective and could have significant implications on its therapeutic potential.

Abstract 9595: Generation of Transgene-Free Induced Pluripotent Stem Cells from Fibroblasts by Small Molecule Chemicals

Backgrounds: Reprogramming of somatic cells using viral- and non-viral vectors encoding defined transcription factors to generate induced pluripotent stem (iPS) cells has successfully been achieved over the past few years. However, the current established methods to create iPS cells require viral vectors or plasmids to deliver transgenes into the cells. Methods and Results: To obviate the putative problems of genetic contamination in the cells for future clinical application, it is highly demanded to develop new methods to create “safer” iPS cells without any genetic modifications. Here, we for the first time demonstrate successful generation of iPS cell line from mouse fibroblasts with only combinations of small-molecule chemicals such as GSK-3 beta inhibitor, histone deacetylase inhibitor, and G9a histone methyltransferase inhibitor, which act as epigenetic modulators and self-renewal activators. These chemical induced pluripotent stem (ChemiPS) cells have almost the same properties of embryonic stem cells in the expression patterns of pluripotency-related markers, epigenetic status in promoter regions of Oct4 and Nanog, spontaneous differentiation into three germ layers by in vitro embryoid body formation, and formation of teratoma in vivo after injection into mice. Conclusion: This study provides the first evidence that sole chemical combinations can reprogram somatic cells, resulting in transgene-free iPS cells. This method will advance the use of iPS cells in clinical application and disease investigation.

The role of glycogen synthase kinase-3 beta in the pathogenesis of liver ischemia reperfusion injury

To investigate the role of the key intracellular signaling molecule glycogen synthase kinase-3 beta in the mechanism of liver ischemia reperfusion (IR). C57BL/6 mice were subjected to 90 min warm liver cephalad lobe ischemia, followed by various length of reperfusion. Experiment groups included sham control group, liver IRI model group and glycogen synthase kinase-3 beta inhibitor-treated group (SB216763 in DMSO, 25 g/kg, i.p, 2 hour prior to the onset of liver ischemia). The expression of glycogen synthase kinase-3 beta protein was analysed by Western blotting. The serum ALT levels were determined to reflect the function of liver. The affected liver lobes were harvested for histology analysis. The inflammatory gene expression was detected by Quantitative PCR. By western blot analysis, we found that ischemia itself activated glycogen synthase kinase-3 beta by a significant decrease of its phosphorylation. Glycogen synthase kinase-3 beta inhibitor SB216763-pretreatment ameliorated the liver damages significantly as compared to the controls (sALT: 2046+/-513 U/L vs 5809+/-1689 U/L, P = 0.0153), and suppressed the gene expressions of IL-12, TNFa, IL-1b and IL-6. This study demonstrated that the ischemia process modulated liver innate immune activation via a GSK-3-dependent mechanism which favored the development of a pro-inflammation response and lead to liver tissue damages. GSK-3b may be a new therapeutic target to ameliorate liver IRI in transplant patients.

Inhibition of Tat-mediated HIV-1 replication and neurotoxicity by novel GSK3-beta inhibitors

The HIV-1 protein Tat is a critical regulator of viral transcription and has also been implicated as a mediator of HIV-1 induced neurotoxicity. Here using a high throughput screening assay, we identified the GSK-3 inhibitor 6BIO, as a Tat-dependent HIV-1 transcriptional inhibitor. Its ability to inhibit HIV-1 transcription was confirmed in TZM-bl cells, with an IC50 of 40nM. Through screening 6BIO derivatives, we identified 6BIOder, which has a lower IC50 of 4nM in primary macrophages and 0.5nM in astrocytes infected with HIV-1. 6BIOder displayed an IC50 value of 0.03nM through in vitro GSK-3β kinase inhibition assays. Finally, we demonstrated 6BIO and 6BIOder have neuroprotective effects on Tat induced cell death in rat mixed hippocampal cultures. Therefore 6BIO and its derivatives are unique compounds which, due to their complex mechanisms of action, are able to inhibit HIV-1 transcription as well as to protect against Tat induced neurotoxicity.

Abstract 1985: The activation of GSK3 by HGF-MET axis is responsible for differentiation of rhabdomyosarcoma cells

Abstract Introduction: Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma among children. At the late stages (metastatic) the outcome for the patients is very poor. The molecular basis for loss of cell differentiation in the process of carcinogenesis is not well understood. Differentiation status of rhabdomyosarcoma cells strongly influences its progression and metastatic ability and therefore the overall patient survival. At the onset of myogenic differentiation MET receptor is rapidly downregulated and on the other hand the activation of MET receptor in rhabdomyosarcoma has been shown to influence proliferation, survival and migration. Aim: Dissecting the role of HGF-MET receptor axis and GSK3 activation in the process of RMS differentiation. Materials and methods: Cell lines used in experiments: RH30 (ARMS), SMS-CTR (ERMS). Real time RT-PCR and western blotting were used to evaluate gene expression and the activation of various intracellular signaling pathways, respectively. The evaluation of protein activation in RMS cell lines was performed after stimulation with HGF. Results: We found that, during differentiation process the expression of MyoD, characteristic for poorly differentiated rhabdomyosarcoma cells decreased and the expression of myogenin, protein characteristic for differentiated/ matured muscle cells increased. That was paralleled by decreased expression of MET receptor. In RMS cell lines with stable downregulation of MET receptor the expression of differentiation related genes was similar to differentiated RMS cells. When we study the activation of GSK3 we observed the phosphorylation of GSK3 beta on serine 9 after HGF stimulation. The intracellular pathway responsible for GSK3 beta phosphorylation turn out to be PI3K-AKT dependent. We observed also the accumulation of beta-catenin in the nucleus of RMS cells stimulated with HGF. This effect was father augmented when HGF was used together with BIO, small molecular inhibitor of GSK3 beta. Conclusion and future directions: In this study we have shown that the expression and signaling of MET receptor is responsible for differentiation of RMS cells. Moreover we postulate that MET receptor exerts its influence on RMS cells differentiation through GSK3 beta activation. These findings might have the significant clinical implication for the treatment of RMS cells because they suggest that induction of differentiation of RMS cells by blocking MET receptor could inhibit growth of RMS tumors. Finally, we think that also GSK3 beta could be used as the new therapeutic target to induce differentiation of RMS. 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 1985. doi:10.1158/1538-7445.AM2011-1985