Starting from a known compound, identified as the first inhibitor of the kinesin MKLP-2 and named Paprotrain, we have investigated its reactivity to produce through photochemistry a potent nanomolar inhibitor of the kinase DYRK1A. Using similar and different chemical pathways, we have designed several families of compounds that have been screened on a panel of five protein kinases: CK1δ/ε, CDK5/p25, GSK3α/β, DYRK1A and CLK1, all involved in neurodegenerative disorders such as Alzheimer's disease. We have identified a first group of multi-targeted compounds, a second group of dual inhibitors of DYRK1A & CLK1 and a last group of selective inhibitors of CLK1. Then, our best submicromolar to nanomolar inhibitors were evaluated towards the closest members of the aforementioned kinases: DYRK1B and CLK4, as well as the subfamily CLK2-3. Several compounds appear to be particularly promising for the development of tools in the battle against Alzheimer's disease.

Extracellular amino acid (AA) withdrawal/restriction invokes an integrated stress response (ISR) that induces global suppression of protein synthesis whilst allowing transcription and translation of a select group of genes, whose protein products facilitate cellular adaptation to AA insufficiency. Transcriptional induction of the System A/SNAT2 AA transporter represents a classic adaptation response and crucially depends upon activation of the General Control Nonderepressible-2 kinase/Activating transcription factor 4 (GCN2/ATF4) pathway. However, the ISR may also include additional signalling inputs operating in conjunction or independently of GCN2/ATF4 to upregulate SNAT2. Herein, we show that whilst pharmacological inhibition of MEK-ERK, mTORC1 and p38 MAP kinase signalling has no detectable effect on System A upregulation, inhibitors targeting GSK3 (e.g. SB415286) caused significant repression of the SNAT2 adaptation response. Strikingly, the effects of SB415286 persist in cells in which GSK3α/β have been stably silenced indicating an off-target effect. We show that SB415286 can also inhibit cyclin-dependent kinases (CDK) and that roscovitine and flavopiridol (two pan CDK inhibitors) are effective repressors of the SNAT2 adaptive response. In particular, our work reveals that CDK7 activity is upregulated in AA-deprived cells in a GCN-2-dependent manner and that a potent and selective CDK7 inhibitor, THZ-1, not only attenuates the increase in ATF4 expression but blocks System A adaptation. Importantly, the inhibitory effects of THZ-1 on System A adaptation are mitigated in cells expressing a doxycycline-inducible drug-resistant form of CDK7. Our data identify CDK7 as a novel component of the ISR regulating System A adaptation in response to AA insufficiency.

Glycogen synthase kinase 3 (Gsk3) α and β are both constitutively active and inhibited upon stimulation by N-terminal serine phosphorylation. Although roles of active Gsk3 in liver ischemia reperfusion injury (IRI) have been well appreciated, whether Gsk3 N-terminal serine phosphorylation has any functional significance in the disease process remains unclear. In a murine liver partial warm ischemia model, we studied Gsk3 N-terminal serine mutant knock-in (KI) mice and showed that liver IRI was decreased in Gsk3αS21A but increased in Gsk3βS9A mutant KI mice. Bone marrow chimeric experiments revealed that the Gsk3α, but not β, mutation in liver parenchyma protected from IRI, and both mutations in bone marrow-derived cells exacerbated liver injuries. Mechanistically, mutant Gsk3α protected hepatocytes from inflammatory (TNF-α) cell death by the activation of HIV-1 TAT-interactive protein 60 (TIP60)-mediated autophagy pathway. The pharmacological inhibition of TIP60 or autophagy diminished the protection of the Gsk3α mutant hepatocytes from inflammatory cell death in vitro and the Gsk3α mutant KI mice from liver IRI in vivo. Thus, Gsk3 N-terminal serine phosphorylation inhibits liver innate immune activation but suppresses hepatocyte autophagy in response to inflammation. Gsk3 αS21, but not βS9, mutation is sufficient to sustain Gsk4 activities in hepatocytes and protect livers from IRI via TIP60 activation.

The cerebral amyloid-β accumulation that begins in middle age is considered the critical triggering event in the pathogenesis of late-onset Alzheimer's disease (LOAD). However, the molecular mechanism remains elusive. The Shugoshin 1 (Sgo1^-/+ ) mouse model, a model for mitotic cohesinopathy-genomic instability that is observed in human AD at a higher rate, showed spontaneous accumulation of amyloid-β in the brain at old age. With the model, novel insights into the molecular mechanism of LOAD development are anticipated. In this study, the initial appearance of cerebral amyloid-β accumulation was determined as 15-18 months of age (late middle age) in the Sgo1^-/+ model. The amyloid-β accumulation was associated with unexpected GSK3α/β inactivation, Wnt signaling activation, and ARC/Arg3.1 accumulation, suggesting involvement of both the GSK3-Arc/Arg3.1 axis and the GSK3-Wnt axis. As observed in human AD brains, neuroinflammation with IFN-γ expression occurred with amyloid-β accumulation and was pronounced in the aged (24-month-old) Sgo1^-/+ model mice. AD-relevant protein panels (oxidative stress defense, mitochondrial energy metabolism, and β-oxidation and peroxisome) analysis indicated (a) early increases in Pdk1 and Phb in middle-aged Sgo1^-/+ brains, and (b) misregulations in 32 proteins among 130 proteins tested in old age. Thus, initial amyloid-β accumulation in the Sgo1^-/+ model is suggested to be triggered by GSK3 inactivation and the resulting Wnt activation and ARC/Arg3.1 accumulation. The model displayed characteristics and affected pathways similar to those of human LOAD including neuroinflammation, demonstrating its potential as a study tool for the LOAD development mechanism and for preclinical AD drug research and development.

Keywords: Shugoshin1; amyloid-β; cohesinopathy; genomic instability; late-onset Alzheimer's disease; mitosis; mouse model; neuroinflammation.

The effective treatment of bipolar disorder (BD) represents a significant unmet medical need. Although lithium remains a mainstay of treatment for BD, limited knowledge regarding how it modulates affective behavior has proven an obstacle to discovering more effective mood stabilizers with fewer adverse side effects. One potential mechanism of action of lithium is through inhibition of the serine/threonine protein kinase GSK3β, however, relevant substrates whose change in phosphorylation may mediate downstream changes in neuroplasticity remain poorly understood. Here, we used human induced pluripotent stem cell (hiPSC)-derived neuronal cells and stable isotope labeling by amino acids in cell culture (SILAC) along with quantitative mass spectrometry to identify global changes in the phosphoproteome upon inhibition of GSK3α/β with the highly selective, ATP-competitive inhibitor CHIR-99021. Comparison of phosphorylation changes to those induced by therapeutically relevant doses of lithium treatment led to the identification of collapsin response mediator protein 2 (CRMP2) as being highly sensitive to both treatments as well as an extended panel of structurally distinct GSK3α/β inhibitors. On this basis, a high-content image-based assay in hiPSC-derived neurons was developed to screen diverse compounds, including FDA-approved drugs, for their ability to mimic lithium's suppression of CRMP2 phosphorylation without directly inhibiting GSK3β kinase activity. Systemic administration of a subset of these CRMP2-phosphorylation suppressors were found to mimic lithium's attenuation of amphetamine-induced hyperlocomotion in mice. Taken together, these studies not only provide insights into the neural substrates regulated by lithium, but also provide novel human neuronal assays for supporting the development of mechanism-based therapeutics for BD and related neuropsychiatric disorders.

Frozen-thawed boar sperm have less motility and fertility capacity in comparison to fresh sperm. Glycogen Synthase Kinase 3 (GSK3) contributes to sperm motility in fresh semen. In addition, GSK3 inhibition in boar spermatozoa in fresh semen improves motility variables. The role of GSK3 on boar cryopreserved sperm, however, is still unknown. The hypothesis in the present study was that GSK3 pathway inhibition by alsterpaullone (AST) could result in enhancement of the quality of sperm afer cryopreservation. Two different strategies were evaluated: i) AST supplementation to the freezing medium (AST + Cryo); ii) AST supplementation after sperm thawing (AST + Thaw). Sperm motility was evaluated using the CASA system and different sperm quality variables were evaluated using flow cytometry, as well as amount of GSK3 phosphorylation of thawed spermatozoa after 30 and 90 min incubation at 38.5 °C. Results indicate that AST supplementation had detrimental effects on sperm viability (live spermatozoa) and mitochondrial membrane potential when it was added after thawing (P < 0.05) The AST supplementation after thawing, however, had a protective effect on plasma membrane lipid disorganization (P < 0.05). The percentage of motile spermatozoa was not modified by AST supplementation. Nonetheless, after 30 min post-thawing, STR and LIN variables (related to straightness of the movement) as well as the percentage of rapid lineal spermatozoa were increased with both AST supplementation protocols. The GSK3α phosphorylation was not modified through the incubation time in boar thawed sperm. In summary, results do not support the idea of adding AST to the cryopreservation/thawing medium to improve boar sperm quality after cryopreservation.

Nonalcoholic fatty liver disease (NAFLD) is associated with dietary folate deficiency and mutations in genes required for one‑carbon metabolism. However, the mechanism through which this occurs is unclear. To improve our understanding of this link, we investigated liver morphology, metabolism and fuel storage in adult mice with a hypomorphic mutation in the gene methionine synthase reductase (Mtrr^gt ). MTRR enzyme is a key regulator of the methionine and folate cycles. The Mtrr^gt mutation in mice was previously shown to disrupt one‑carbon metabolism and cause a wide-spectrum of developmental phenotypes and late adult-onset macrocytic anaemia. Here, we showed that livers of Mtrr^gt/gt female mice were enlarged compared to control C57Bl/6J livers. Histological analysis of these livers revealed eosinophilic hepatocytes with decreased glycogen content, which was associated with down-regulation of genes involved in glycogen synthesis (e.g., Ugp2 and Gsk3a genes). While female Mtrr^gt/gt livers showed evidence of reduced β-oxidation of fatty acids, there were no other associated changes in the lipidome in female or male Mtrr^gt/gt livers compared with controls. Defects in glycogen storage and lipid metabolism often associate with disruption of mitochondrial electron transfer system activity. However, defects in mitochondrial function were not detected in Mtrr^gt/gt livers as determined by high-resolution respirometry analysis. Overall, we demonstrated that adult Mtrr^gt/gt female mice showed abnormal liver morphology that differed from the NAFLD phenotype and that was accompanied by subtle changes in their hepatic metabolism and fuel storage.

The angiotensin II type I receptor (AT1R) is involved in the regulation of cardiovascular function. Excessive activation of AT1R by angiotensin II (Ang II) leads to cardiovascular disease and may be involved in the development of insulin resistance and diabetes. Functionally selective Ang II analogues, such as the [Sar1, Ile4, Ile8]-angiotensin II (SII Ang II) analogue, that only activate a subset of signalling networks have been demonstrated to have beneficial effects on cardiovascular function in certain settings, including lowering blood pressure and increasing cardiac performance. Here, we studied the effect of SII Ang II on insulin receptor (IR) signalling and glucose metabolism in primary rat hepatocytes. We show that long-term pre-treatment of hepatocytes with SII Ang II increased insulin-stimulated glycogen synthesis, while Ang II and the AT1R antagonist losartan had no effect. Insulin-stimulated suppression of hepatic glucose output was not affected by Ang II or SII Ang II. It is well known that insulin regulates glycogen synthesis and glucose output through Akt-mediated phosphorylation of glycogen synthase kinase α/β (GSK3α/β) and forkhead box protein O1 (FOXO1), respectively. In line with this, we show that SII Ang II potentiated insulin-stimulated phosphorylation of Akt and GSK3α/β, but not FOXO1. Furthermore, we demonstrate that the effect of SII Ang II on insulin-stimulated signalling and glycogen synthesis was dependent on Src and Gαq, as inhibitors of these proteins abolished the potentiating effect of SII Ang II. Thus, our results demonstrate that SII Ang II may have a positive effect on IR signalling and glucose metabolism in hepatocytes.

Serotonin (5-HT) transporter (SERT) plays a crucial role in serotonergic transmission in the central nervous system, and any aberration causes serious mental illnesses. Nevertheless, the cellular mechanisms that regulate SERT function and trafficking are not entirely understood. Growing evidence suggests that several protein kinases act as modulators. Here we delineate the molecular mechanisms by which glycogen synthase kinase-3ß (GSK3ß) regulates SERT. When mouse striatal synaptosomes were treated with the GSK3α/ß inhibitor CHIR99021, we observed a significant increase in SERT function, V_max , surface expression with a reduction in 5-HT K_m and SERT phosphorylation. To further study how the SERT molecule is affected by GSK3α/ß, we used HEK-293 cells as a heterologous expression system. As in striatal synaptosomes, CHIR99021 treatment of cells expressing wild-type hSERT (hSERT-WT) resulted in a time and dose-dependent elevation of hSERT function with a concomitant increase in the V_max and surface transporters due to reduced internalization and enhanced membrane insertion; silencing GSK3α/ß in these cells with siRNA also similarly affected hSERT. Converting putative GSK3α/ß phosphorylation site serine at position 48 to alanine in hSERT (hSERT-S48A) completely abrogated the effects of both the inhibitor CHIR99021 and GSK3α/ß siRNA. Substantiating these findings, overexpression of constitutively active GSK3ß with hSERT-WT, but not with hSERT-S48A, reduced SERT function, V_max , surface density, and enhanced transporter phosphorylation. Both hSERT-WT and hSERT-S48A were inhibited similarly by PKC activation or by inhibition of Akt, CaMKII, p38 MAPK or Src kinase. These findings provide new evidence that GSK3ß supports basal SERT function and trafficking via serine-48 phosphorylation.

Keywords: glycogen synthase kinase-3; phosphorylation; serotonergic transmission; serotonin transporter; trafficking; uptake.

Hyperactivation and acrosome reaction of sperm are pre-requisite steps for fertilization. However, the hyperactivation and acrosome reaction are critically controlled through the phosphorylation of specific proteins. Glycogen synthase kinase-3 (GSK3), a serine/threonine kinase with two different isoforms (α and β), is involved in biochemical signaling pathways. This study was aimed to investigate whether the GSK3α/β is present in goat sperm and its regulatory role in sperm motility and acrosome reaction. GSK3α/β was detected with immunofluorescence and Western blotting. Sperm motility, membrane integrity, acrosome reaction, mitochondrial membrane potential, phospho-Ser21-GSK3α and phospho-Ser9-GSK3β were analyzed. The ATP production and activities of lactate dehydrogenase (LDH), malate dehydrogenase (MDH), and succinate dehydrogenase (SDH) were measured. It was observed that the GSK3α/β was expressed in goat sperm, especially in the peri-acrosomal, mid-piece and principal piece of the tail. The abundance of GSK3α/β in sperm was increased during transit along the epididymis. Addition of either 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or CHIR99021 significantly increased the sperm motility patterns and GSK3α/β phosphorylation. Interestingly, the adenosine triphosphate (ATP) production, activities of LDH, MDH and SDH were observed to be increased in the CHIR99021 treatment. The results suggested that GSK3α/β regulates sperm motility and acrosome reaction via phospho-ser21-GSK3α and phospho-ser9-GSK3β that involved in the regulation of sperm energy metabolism.

Dysregulation of the PI3K/Akt/mTOR signaling cascade has been associated with the pathology of neurodegenerative disorders, specifically Alzheimer's disease (AD). Both in-vivo models and post-mortem brain samples of individuals with AD have commonly shown hyperactivation of the pathway. In the present study, we examine how neuron subset-specific deletion of Pten (NS-Pten) in mice, which presents with hyperactive mammalian target of rapamycin (mTOR) activity, affects the hippocampal protein levels of key neuropathological hallmarks of AD. We found NS-Pten knockout (KO) mice to have elevated levels of amyloid-β, α-synuclein, neurofilament-L, and pGSK3α in the hippocampal synaptosome compared with NS-Pten wild type mice. In contrast, there was a decreased expression of amyloid precursor protein, tau, GSK3α, and GSK3β in NS-Pten KO hippocampi. Overall, there were significant alterations in levels of proteins associated with AD pathology in NS-Pten KO mice. This study provides novel insight into how altered mTOR signaling is linked to AD pathology, without the use of an in-vivo AD model that already displays neuropathological hallmarks of the disease.

Purification of proteins for the biophysical analysis of protein interactions occurring in human cells can benefit from methods that facilitate the capture of small amounts of natively processed protein obtained using transient mammalian expression systems. We have used a novel calcium-dependent fragment complementation-based affinity method to effectively purify full length glycogen synthase kinase 3 (GSK3) α and β isoforms to study their interaction with amyloid β peptide (Aβ42). Using these proteins, purified from 1 mg of total cell lysate, we measured an apparent K_D of ≤100 pM between GSK3α/β and immobilized Aβ42 with surface plasmon resonance technology. This approach can be used to retrieve useful quantities of protein for biophysical experiments with small scale mammalian cell culture.

Many substances in cigarette smoke can induce changes in DNA methylation. Our previous studies have confirmed paternal nicotine exposure causes hyperactivity in the offspring via mmu-miR-15b. The main aim of the present study is to explore the molecular mechanism underlying the cross-generation effects of paternal nicotine exposure more comprehensively. The male C57BL/6 mice were exposed to 2 mg/kg/d nicotine for 5 weeks, and then mated with wild-type females. The offspring male mice were subjected to behavioral tests at 8 weeks after birth. The results suggested that, paternal nicotine exposure led to hyperactivity in the offspring. An analysis of the changes in DNA methylation revealed that nicotine exposure induced a rise in the total DNA methylation level of Dat in murine spermatozoa, and the hyper-methylation could imprint in the brains of the offspring mice. Then these epigenetic modifications reduced the expression of DAT in the brain of the offspring, resulting in a rise in the level of extracellular dopamine. The activation of D2 receptors caused the dephosphorylation of AKT, which led to increased activation of GSK3α/β, and ultimately caused hyperactivity in the offspring mice. Further, in wild-type mice, injection of DAT inhibitors simulated this hyperactive phenotype, while the injection of D2s inhibitors reversed the hyperactivity of the offspring caused by paternal nicotine exposure. In conclusion, all results indicated that paternal nicotine exposure could induce hyperactivity in the offspring via the hyper-methylation of Dat. Consequently, Dat may be one of the genes that mediate the cross-generation effects of nicotine besides mmu-mmiR-15b.

The biological activity of the enzyme glycogen synthase kinase-3 (GSK3) is fulfilled by two paralogs named GSK3α and GSK3β, which possess both redundancy and specific functions. The upregulated activity of these proteins is linked to the development of disorders such as neurodegenerative disorders (ND) and cancer. Although various chemical inhibitors of these enzymes restore the brain functions in models of ND such as Alzheimer's disease (AD), and reduce the proliferation and survival of cancer cells, the particular contribution of each paralog to these effects remains unclear as these molecules downregulate the activity of both paralogs with a similar efficacy. Moreover, given that GSK3 paralogs phosphorylate more than 100 substrates, the simultaneous inhibition of both enzymes has detrimental effects during long-term inhibition. Although the GSK3β kinase function has usually been taken as the global GSK3 activity, in the last few years, a growing interest in the study of GSK3α has emerged because several studies have recognized it as the main GSK3 paralog involved in a variety of diseases. This review summarizes the current biological evidence on the role of GSK3α in AD and various types of cancer. We also provide a discussion on some strategies that may lead to the design of the paralog-specific inhibition of GSK3α.

Keywords: Alzheimer’s disease; GSK3; GSK3 structure; GSK3α; GSK3β; cancer.

Some patients with chronic hepatitis C also demonstrate liver steatosis, but the mechanism remains elusive. To analyze the hepatic expression of phosphorylated kinase Akt at Thr 308 and phosphorylated GSK-3 (Glycogen synthase kinase-3) isoforms, GSK3α at Ser 21 and GSK3β at Ser 9, in chronic hepatitis C patients with normal body weight, glucose, and lipid profiles depending on homeostasis model assessment of insulin resistance (HOMA-IR) levels and histological parameters. The study group consisted of 31 patients with chronic hepatitis C. The hepatic expression of kinase Akt (Thr³⁰⁸), GSK3β (Ser⁹), and GSK3α (Ser²¹) was measured using Western blot assay. Liver steatosis was observed in 41.93% of patients with HCV infection, in those with increased HOMA-IR index (p = 0.02). However, the hepatic expression of Akt (Thr³⁰⁸), GSK3β (Ser⁹), and GSK3α (Ser²¹) was not related to progression of liver steatosis, inflammation, and fibrosis. There was no significant difference in the hepatic expression of kinase Akt (Thr³⁰⁸), GSK3β (Ser⁹), and GSK3α (Ser²¹) in relation to HOMA-IR. Liver steatosis was found to be positively associated with HOMA-IR levels in patients with chronic hepatitis C without metabolic disorders. However, the hepatic expression of Akt (Thr308), GSK3β (Ser9), and GSK3α (Ser21) did not correspond to progression of liver disease.

In mouse and bovine sperm, GSK3 activity is inversely proportional to motility. Targeted disruption of the GSK3A gene in testis results in normal spermatogenesis, but mature sperm present a reduced motility, rendering male mice infertile. On the other hand, GSK3B testis-specific KO is fertile. Yet in human sperm, an isoform-specific correlation between GSK3A and sperm motility was never established. In order to analyze GSK3 function in human sperm motility, normospermic and asthenozoospermic samples from adult males were used to correlate GSK3 expression and activity levels with human sperm motility profiles. Moreover, testicular and sperm GSK3 interactomes were identified using a yeast two-hybrid screen and coimmunoprecipitation, respectively. An extensive in-silico analysis of the GSK3 interactome was performed. The results proved that inhibited GSK3A (serine phosphorylated) presents a significant strong positive correlation (r = 0.822, P = 0.023) with the percentage of progressive human sperm, whereas inhibited GSK3B is not significantly correlated with sperm motility (r = 0.577, P = 0.175). The importance of GSK3 in human sperm motility was further reinforced by in-silico analysis of the GSK3 interactome, which revealed a high level of involvement of GSK3 interactors in sperm motility-related functions. The limitation of techniques used for GSK3 interactome identification can be a drawback, since none completely mimics the physiological environment. Our findings prove that human sperm motility relies on isoform-specific functions of GSK3A within this cell. Given the reported relevance of GSK3 protein-protein interactions in sperm motility, we hypothesized that they stand as potential targets for male contraceptive strategies based on sperm motility modulation.

In an effort to develop ATP-competitive VEGFR-2 selective inhibitors, a novel series of tricyclic pyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4-amine derivatives were designed and synthesized. These compounds were characterized by IR, (1)H NMR, (13)C NMR, elemental and mass spectral analyses. Docking studies have given a partial insight into the molecular determinants of the activity of this novel series in VEGFR-2 kinase active site. Moreover, these compounds were assessed at 10μM for their selective inhibitory activities over a panel of 6 human kinases, namely VEGFR-1/Flt-1, VEGFR-2/KDR, EGFR, CDK5/p25, GSK3α and GSK3β. Compound N-(4,6-dimethylthieno[2,3-b]pyridine)-7,9-dimethylpyrido[3',2':4,5]thieno[3,2-d]pyrimidin-4-amine (9d) exhibited the most potent and selective inhibitory activity against VEGFR-2/KDR over the six human kinases, with an IC50 value 2.6μM. The identification of this hit candidate could aid the design of new tricyclic-based VEGFR-2 kinase modulators.

UNASSIGNED

Head and neck squamous cell cancer (HNSCC) is one of the most common tumors worldwide and there is an enormous need for innovative therapy approaches. Several recent studies suggest tumor entity specific roles of glycogen synthase kinase 3 (GSK3) in different human cancers, acting as tumor suppressor or as tumor promoter. Here we describe the role of GSK3 with respect to different parameters within HNSCC progression.

UNASSIGNED

Base line expression and activity profiles of p-GSK3α/β (Ser21/9) and p-GSK3α/β (Tyr279/216) were analyzed by immunohistochemistry and western blotting. Four different permanent HNSCC cell lines were exposed to the potent GSK3α/β inhibitor SB 216763. Cell viability was controlled via the MTT test. Cell migration was quantified with the Real Time Cell Analyzer (RCTA) xCELLigence. Regulation of the epithelial-mesenchymal transition (EMT) was measured with the Human Epithelial to Mesenchymal Transition (EMT) RT2 Profiler™ PCR Array and scratch assays. Taqman probes were used to detect the specific gene expression profiles of inflammatory cytokines Interleukin IL1β, IL6, IL8, IL10, TNFα and IFNβ.

UNASSIGNED

Exposure of permanent HNSCC cell lines to the specific GSK3α/β inhibitor SB 216763 leads to significant growth inhibition, inhibition of migration and decreased levels of active GSK3α/β in a dose dependent manner.Exposure of HNSCC lines to SB 216763 also resulted in a markable shift of EMT markers and functional EMT dysregulation. Functionally GSK3 differentially mediates the expression of TLR4- and TLR3-induced inflammatory cytokines in HNSCC, whereas no effect of SB 216763 on the NFkB activity was noticed.

UNASSIGNED

GSK3α/β plays a crucial role in a variety of regulatory networks for HNSCC cancer progression as it drives proliferation or migration and thus GSK3 could serve as an interesting target for clinical drug development.

Background/aim: Demethoxycurcumin (DMC), one of the derivatives of curcumin, has been shown to induce apoptotic cell death in many human cancer cell lines. However, there is no available information on whether DMC inhibits metastatic activity in human glioblastoma cancer cells in vitro.

Materials and methods: DMC at 1.0-3.0 μM significantly decreased the proliferation of GBM 8401 cells; thus, we used 2.0 μM for further investigation regarding anti-metastatic activity in human glioblastoma GBM 8401 cells.

Results: The internalized amount of DMC has reached the highest level in GBM 8401 cells after 3 h treatment. Wound healing assay was used to determine cell mobility and results indicated that DMC suppressed cell movement of GBM 8401 cells. The transwell chamber assays were used for measuring cell migration and invasion and results indicated that DMC suppressed cell migration and invasion in GBM 8401 cells. Gelatin zymography assay was used to examine gelatinolytic activity (MMP-2) in conditioned media of GBM 8401 cells treated by DMC and results demonstrated that DMC significantly reduced MMP-2 activity. Western blotting showed that DMC reduced the levels of p-EGFR^(Tyr1068), GRB2, Sos1, p-Raf, MEK, p-ERK1/2, PI3K, p-Akt/PKBα^(Thr308), p-PDK1, NF-κB, TIMP-1, MMP-9, MMP-2, GSK3α/β, β-catenin, N-cadherin, and vimentin, but it elevated Ras and E-cadherin at 24 h treatment.

Conclusion: DMC inhibited cancer cell migration and invasion through inhibition of PI3K/Akt and NF-κB signaling pathways in GBM 8401 cells. We suggest that DMC may be used as a novel anti-metastasis agent for the treatment of human glioblastoma cancer in the future.

Keywords: Demethoxycurcumin (DMC); NF-κB; human glioblastoma multiforme (GBM 8401) cells; invasion; migration.

Objective: Focal cortical dysplasia (FCD) accounts for nearly half of all cases of medically refractory epilepsy in the pediatric and adult patient populations. This neurological disorder stems from localized malformations in cortical brain tissue due to impaired neuronal proliferation, differentiation, and migration patterns. Recent studies in animal models have highlighted the potential role of the Fragile X mental retardation protein (FMRP) levels in FCD. The purpose of this study was to investigate the status of FMRP activation in cortical brain tissues surgically resected from patients with FCD. In parallel, this study also investigated protein levels within the PI3K/AKT/mTOR and canonical Wnt signaling pathways.

Methods: Pathologic tissue from malformative lesions of FCD patients with medically refractory epilepsy was compared to relatively normal control non-epileptic tissue from patients with intracranial neoplasms. A series of western blotting assays were performed to assess key proteins in the PI3K/AKT/mTOR, canonical Wnt signaling pathways, and FMRP.

Results: There was suppression of S235/236-phosphorylated S6, GSK3α, and GSK3β protein levels in samples derived from FCD patients, compared to non-epileptic controls. FCD samples also had significantly greater levels of total and S499-phosphorylated FMRP.

Conclusion: These findings support our hypothesis that malformative lesions associated with FCD are characterized by high levels of FMRP activation along with dysregulation of both PI3K/AKT/mTOR and canonical Wnt signaling. These novel clinical findings extend previous work in animal models, further suggesting a potential unforeseen role of GSK3α and GSK3β in the pathophysiology of FCD and refractory epilepsy.

Microcystin-LR (MC-LR) is a widely produced monocyclic heptapeptides in eutrophication waterbodies. MC-LR can induce various toxic effects in different cells. Our previous studies have found that MC-LR exposure can disrupt insulin signaling pathway in human liver cells (HL 7702). Skeletal muscle is one of the major organs for glucose disposal and responsive to insulin. However, the effects of MC-LR on insulin signaling pathway in muscle cells have not been fully explored. By using C2C12 mice muscle cells, this study aims to investigate the toxic effects of MC-LR in muscle cells with a focus on its effects on insulin signaling pathways. It was found that MC-LR entered into cells and inhibited protein phosphatase 2A (PP2A) significantly. Furthermore, MC-LR increased phosphorylation of Ser302, Ser307, Ser612 of insulin receptor substrate 1, AKT-Ser473, GSK3α-Ser21, and S6K1-Thr389 by inhibiting the activity of PP2A. The results in this study demonstrate that exposure of MCLR can disrupt the insulin pathway in muscle cells.

Glycogen synthase kinase 3 (GSK3) was identified as an enzyme regulating sperm protein phosphatase. The GSK3α paralog, but not GSK3β, is essential for sperm function. Sperm lacking GSK3α display altered motility and are unable to undergo hyperactivation, which is essential for fertilization. Male mice lacking sperm-specific calcineurin (PP2B), a calcium regulated phosphatase, in testis and sperm, are also infertile. Loss of PP2B results in impaired epididymal sperm maturation and motility. The phenotypes of GSK3α and PP2B knockout mice are similar, prompting us to examine the interrelationship between these two enzymes in sperm. High calcium levels must exist to permit catalytically active calcineurin to function during epididymal sperm maturation. Total and free calcium levels are high in immotile compared to motile epididymal sperm. Inhibition of calcineurin by FK506 results in an increase in the net phosphorylation and a consequent decrease in catalytic activity of sperm GSK3. The inhibitor FK506 and an isoform-selective inhibitor of GSK3α, BRD0705, also inhibited fertilization of eggs in vitro. Interrelated functions of GSK3α and sperm PP2B are essential during epididymal sperm maturation and during fertilization. Our results should enable the development of male contraceptives targeting one or both enzymes.

Conjugated linoleic acids (CLA) have been extensively advertised as dietary supplements to reduce fat and increase muscle mass. However, the role of CLA in glycogen metabolism is still largely unknown. The aim of this study was to assess the effect of CLA on glycogen synthesis in vitro (CCL 136 cell line human) and CLA in vivo (C57BL/6J mice). The materials used were the CCL 136 muscle cell line and muscles of female C57BL/6J mice (n = 52), housed at animal laboratory facility and feed with "MURIGRAN", a standard feed prepared for rodents (Agropol, Poland). Chemically pure fatty acids were added to soybean oil. CLA isomers (c9,t11 CLA, t10,c12 CLA, and as a mixture (1:1)) were administered with feed. Supplementation in mice started at week 6 of age and lasted for 4 weeks. Methods used in the study were real time- PCR - quantification of gene expression, Western blot glycogen synthase kinase-3 (GSK3α 9) and glycogen synthase (GS) protein, glycogen staining by PAS. Quantitative determination of glycogen by spectrophotometry and intracellular reactive oxygen species was measured the intracellular oxidation of dichloro-dihydro-fluorescein diacetate (DCFH-DA). In vitro data showed that GS and GSK3 expression was lower in cells cultured with different CLAs and a mixture of CLAs. GS gene expression was significantly decreased in cells cultured with c9, t11 CLA (P < 0.04) and t10, c12 CLA (P < 0.05) as well as the mixture of both isomers. The GSK3α gene expression was reduced in cells cultured with a mixture of CLA (P < 0.02), whereas phosphorylation of GSK3α increased in cells cultured with c9, t11 CLA GSK3α (P < 0.05). In vivo data showed a reduction in the glycogen concentration among mice fed a diet containing t10, c 12 CLA and a mixture of CLA isomers. We conclude that both CLA isomers can affect the synthesis of glycogen in muscle cells through the regulation of GS and GSK3α gene expression.