GSK3β Ser9 Phosphorylation Research Articles

The intermittent administration of ethanol during the juvenile period produces changes in the expression of hippocampal genes and proteins and deterioration of spatial memory

BackgroundBinge drinking is a pattern of alcohol intake characterized by excessive and intermittent alcohol consumption over a very short period of time that is more used during adolescence. We aim to compare the lasting effects of a chronic-moderate vs. this intermittent-excessive way of alcohol intake during adolescence in spatial memory and in the expression of glutamatergic receptors and GSK3β activity. MethodsAdolescent male Wistar rats were given ethanol/saline i.p. injections in four different groups: High-I (4 g/kg of a 25% (vol/vol) every 3 days), Low-I (1 g/kg of a 5% (vol/vol) every 3 days), M (0.3 g/kg of a 2.5% (vol/vol) daily) and Control (C, sterile isotonic saline daily). Rats received ethanol for up to five 3-day cycles. Spatial memory was measured by spontaneous alternation in the Y-Maze. Gene and protein expression of hippocampal proteins were also analysed. ResultsBoth high- and low-intermittent ethanol administration produced spatial memory impairment and changes in glutamatergic receptors gene expression were observed regardless of the pattern of exposure. High doses of intermittent alcohol administration produced an increase of phosphorylation of GSK3β Ser9. Moreover, moderate alcohol administration produced a down-regulation of the AMPAR 2/3 ratio despite lack of spatial memory deficits. ConclusionsExcessive and intermittent ethanol exposure during adolescence impaired the spatial memory processes during adulthood regardless of the amount of alcohol administered. Moreover, chronic-moderate and intermittent pattern induced changes in the expression of glutamatergic receptors. In addition, high-intermittent ethanol exposure during adolescence inactivated GSK3β by increasing its phosphorylation in Ser9.

Hydrophilic Glycoproteins of an Edible Green Alga Capsosiphon fulvescens Prevent Aging-Induced Spatial Memory Impairment by Suppressing GSK-3β-Mediated ER Stress in Dorsal Hippocampus.

Endoplasmic reticulum (ER) stress is involved in various neurodegenerative disorders. We previously found that Capsosiphon fulvescens (C. fulvescens) crude proteins enhance spatial memory by increasing the expression of brain-derived neurotrophic factor (BDNF) in rat dorsal hippocampus. The present study investigated whether the chronic oral administration of hydrophilic C. fulvescens glycoproteins (Cf-hGP) reduces aging-induced cognitive dysfunction by regulating ER stress in the dorsal hippocampus. The oral administration of Cf-hGP (15 mg/kg/day) for four weeks attenuated the aging-induced increase in ER stress response protein glucose-regulated protein 78 (GRP78) in the synaptosome of the dorsal hippocampus; this was attenuated by the function-blocking anti-BDNF antibody (1 μg/μL) and a matrix metallopeptidase 9 inhibitor 1 (5 μM). Aging-induced GRP78 expression was associated with glycogen synthase kinase-3 beta (GSK-3β) (Tyr216)-mediated c-Jun N-terminal kinase phosphorylation, which was downregulated upon Cf-hGP administration. The Cf-hGP-induced increase in GSK-3β (Ser9) phosphorylation was downregulated by inhibiting tyrosine receptor kinase B and extracellular signal-regulated kinase (ERK)1/2 with cyclotraxin-B (200 nM) and SL327 (10 μM), respectively. Cf-hGP administration or the inhibition of ER stress with salubrinal (1 mg/kg, i.p.) significantly decreased aging-induced spatial memory impairment. These findings suggest that the activation of the synaptosomal BDNF-ERK1/2 signaling in the dorsal hippocampus by Cf-hGP attenuates age-dependent ER stress-induced cognitive dysfunction.

HPCAL1 promotes glioblastoma proliferation via activation of Wnt/β-catenin signalling pathway.

Glioblastoma (GBM) is the most prevalent primary malignancy of the central nervous system with obvious aggressiveness, and is associated with poor clinical outcome. Studies have indicated that calcium ion (Ca2+) can positively regulate the initiation of malignancy with regard to GBM by modulating quiescence, proliferation, migration and maintenance. Hippocalcin like‐1 protein (HPCAL1) serves as a sensor of Ca2+. However, the understanding of HPCAL1 activity in GBM is limited. The present study revealed that the gene HPCAL1 was up‐regulated by Ca2+ in the tissues and cells of GBM. Ectopic expression of HPCAL1 promoted proliferation of cells. Exhaustion of HPCAL1 inhibited cell growth not only in vivo, but also in vitro. In addition, HPCAL1 enhanced the Wnt pathway by stimulating β‐catenin accumulation and nuclear translocation in GBM cells, while β‐catenin silencing significantly inhibited the proliferation and growth of the GBM cells. Our results showed that Ser9 phosphorylation of GSK3β was significantly decreased after HPCAL1 knockdown in GBM cells, and knockdown of the gene GSK3β in GBM cells enhanced cell proliferation and promoted transcription of the genes CCND1 and c‐Myc. Furthermore, the phosphorylation of ERK was decreased in the cells with HPCAL1 knockdown, while it was promoted via overexpression of HPCAL1. The suppression or depletion of the gene ERK decreased proliferation triggered by overexpression of HPCAL1 and impaired transcription of the genes c‐Myc and CCND1. These studies elucidate the tumour‐promoting activity of HPCAL1. They also offer an innovative therapeutic strategy focusing on the HPCAL1‐Wnt/β‐catenin axis to regulate proliferation and development of GBM.

Glucocorticoid induces osteonecrosis of the femoral head in rats through GSK3β-mediated osteoblast apoptosis

ObjectiveOne of the important causes of glucocorticoids (GCs)-induced osteonecrosis of the femoral head (ONFH) is osteoblast apoptosis. Glycogen synthase kinase 3β (GSK3β) has been reported to be related to dexamethasone (Dex)-induced osteoblast apoptosis. This study aimed to determine whether GSK3β plays role in GC-induced ONFH and investigate the underlying mechanism. Methods18 male Sprague-Dawley rats were divided into 3 groups. Rats from ONFH group underwent lipopolysaccharide and methylprednisolone injection. Lithium chloride (LiCl, a GSK3β inhibitor) group were fed with LiCl solution. The control group were untreated. Osteonecrosis, apoptosis and bone loss were evaluated by HE staining, TUNEL staining and micro-CT respectively. Protein expressions were examined by western blotting. In addition, primary osteoblast cells were transfected by GSK3β-siRNA and related signaling pathway and proteins were examined. ResultsONFH group showed a relative high percentage of empty lacunae and apoptotic cells, whilst LiCl treatment markedly decreased the percentage. LiCl treatment decreased GC-induced bone loss. Immunoblot analysis for GSK3β showed decreased level of Ser9-phosphorylated GSK3β in ONFH group compared with control group. Knockdown of GSK3β by siRNA in primary osteoblast cells attenuated DEX-induced apoptosis and loss of mitochondrial transmembrane potential (Δψm). GSK3β knockdown also reversed the release of cytochrome C (Cyt C) from mitochondria to the cytosol. GSK3β decreased apoptosis-related protein expression both in vitro and in vivo. ConclusionOur findings suggest that GC induces ONFH in rats through GSK3β-mediated osteoblast apoptosis, with involvement of mitochondrial apoptotic pathway.

Decrease of GSK3β Ser-9 Phosphorylation Induced Osteoblast Apoptosis in Rat Osteoarthritis Model.

Nowadays, the cumulative intake of glucocorticoids has become the most common pathogenic factor for non-traumatic osteonecrosis of the femoral head (ONFH). Apoptosis of osteoblasts is considered as the main reason of ONFH at the molecular level. Glycogen synthase kinase 3β (GSK3β) is an important regulator of cellular differentiation and apoptosis pathway, which can modulate the balance between osteoblasts and osteoclasts. Several studies have reported about its function in osteoporosis, but little is known about it in osteonecrosis. In our study, lipopolysaccharide and methylprednisolone were utilized to establish a rat ONFH model. The phosphorylation of GSK3β Ser-9 was decreased in the model. Western blotting examination of β-catenin, Bcl-2, Bax and caspase-3 revealed that the osteoblasts were apoptotic. In dexamethasone (Dex)-incubated primary osteoblasts, the expression profile of GSK3β phosphorylation and apoptotic factors were consistent with those in the rat ONFH model. To further investigate the regulation of osteonecrosis caused by GSK3β, the expression and function of GSK3β were inhibited in Dex-incubated primary osteoblasts. The knockdown of GSK3β by siRNA decreased the expression of Bax and cleaved caspase-3, but increased Bcl-2 and β-catenin. On the other hand, selective inhibition of GSK3β function by LiCl counteracted the activation of caspase-3 induced by Dex. Our work is the first study about the GSK3β phosphorylation in ONFH, and provides evidence for further therapeutic methods.

G-Protein-Coupled Receptor 5 (LGR5) Overexpression Activates β-Catenin Signaling in Breast Cancer Cells via Protein Kinase A.

BackgroundTargeting cancer stem cells (CSCs) in breast cancer (BrCa) may improve treatment outcome and patient prognosis. Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a well-recognized adult stem cell and CRC marker, and previous reports have suggested the cancer-promoting role of LGR5 in breast cancer, but the mechanism remains unclear.Material/MethodsPotential LGR5-associating genes were explored using STRING database, and LGR5 overexpression and knockdown was constructed in MCF-7 and MDA-MB-453 human BrCa cells, respectively. PKA catalytic subunit activation and PKA kinase activity in human BrCa cells was examined by Western blot and PKA kinase activity assay, respectively. Protein expression level or activation of β-catenin and GSK-3β in human BrCa cells was investigated by Western blot. Cell proliferation, colony formation, Transwell migration, cisplatin sensitivity, and in vivo tumor formation of human BrCa cells were examined.ResultsLGR5 overexpression increased PKA activation and its kinase activity in human BrCa cells, which was decreased by LGR5 knockdown. LGR5 expression level or PKA kinase activity were correlated with β-catenin Ser 552 phosphorylation but inversely correlated with GSK-3β Ser9 phosphorylation in human BrCa cells in vitro. LGR5/PKA increased cell proliferation, colony formation, Transwell migration, and cisplatin resistance in vitro, as well as tumor formation in vivo, of human BrCa cells.ConclusionsLGR5 activates the Wnt/β-catenin signaling pathway in human BrCa cells in vitro via PKA.

Amyloid Β Oligomers Induce Sex-Specific Pathophysiological mGluR5 Signaling in Alzheimer Mice

Sex is an important modifier of Alzheimer’s disease (AD) prevalence and progression. Aβ oligomers engage metabotropic glutamate receptor 5 (mGluR5) to mediate pathological signaling in AD. We find here, that mGluR5 signaling is intrinsically different in male versus female mouse neurons, as mGluR5 agonist and Aβ oligomer treatments inactivate a GSK3β/ZBTB16/ATG14-regulated autophagy pathway via Ser9 phosphorylation of GSK3β in a mGluR5-dependent mechanism in male, but not female, primary neuronal cultures. These observed sex-specific differences in mGluR5 signaling translate into in vivo differences in mGluR5-dependent pathological signaling in male and female AD mice. We demonstrate that treatment of male, but not female, APPswe/PS1ΔE9 mice with the mGluR5-selective negative allosteric modulator (NAM), CTEP, improved cognition, reduced Aβ oligomer-mediated pathology, attenuated inflammatory responses and enhanced autophagy. These differences in male versus female APPswe/PS1ΔE9 responses to CTEP correlate with sex-specific differences in cell surface mGluR5 trafficking. This study demonstrates clear sex-specific differences in mGluR5 cellular signaling and trafficking in a mouse model of AD and strongly indicates a need to redesign and reanalyze sex-specific AD treatment strategies.

Nerigoside suppresses colorectal cancer cell growth and metastatic potential through inhibition of ERK/GSK3β/β-catenin signaling pathway

BackgroundNerigoside (NG), a cardenolide isolated from a commonfolk medicine, Nerium oleander Linn. (Apocynaceae), has not been explored for its biological effects. To date, cardenolides have received considerable attention in pharmacology studies due to their direct effects of apoptosis-induction or growth-inhibitory against tumor in vitro and in vivo. Whether and how NG exerts anticancer effects against colorectal cancer remains to be elucidated. PurposeThe aim of this study was to investigate the anticancer effect of NG in human colorectal cancer cells. MethodsTo test anticancer effect, we compared potency of NG in two colorectal cancer cell lines, HT29 and SW620 by WST-1 and colony proliferation assays. And we investigated mechanism of anticancer activities by analyzing players in apoptotic and ERK/GSK3β/β-catenin signaling pathways in HT29 and SW620 cells treated with NG. ResultsIn this study, we showed that NG markedly suppressed the cell viability and colony formation of colorectal cancer cells HT29 and SW620, with no significant toxic effect on non-cancer cells NCM460. Annexin V-FITC/PI and CFSE labeling results revealed that NG suppressed cell proliferation in low concentration, along with reducing expression of PCNA, while NG induced apoptosis in high concentration,. Meanwhile, NG significantly arrested cell migration by reversal of EMT and cell cycle on G2/M. Then, we found that the ERK and GSK3β/β-catenin signaling pathway were noticeably blocked in CRC cells after treatment with NG. According to western blot, NG upregulated the expression of p-GSK3β/GSK3β and decreased especially the expression of β-catenin in nuclear. In addition, Wnt signaling and its target genes were suppressed in response to NG. Then, the Ser9 phosphorylation of GSK3β can be reduced / raised by GÖ 6983 / LiCl, respectively. Thus, we further confirmed that the GSK3β/β-catenin axis is involved in NG-prevented cell proliferation. ConclusionNG inhibited the growth of colorectal cancer cells by suppressing ERK/GSK3β/β-catenin signaling pathway. And the GSK3β/β-catenin axis is involved in preventing cell proliferation and migration by NG-treatment. These results suggest that NG may be used to treat colorectal cancer, with better outcome by combining with GSK3β inhibitor to block Wnt pathway.

GSK3\u03b2 inhibition attenuates LPS-induced IL-6 expression in porcine adipocytes

IL-6 is not only a proinflammatory cytokine associated with inflammatory responses but also a regulator on the energy and glucose metabolism in adipose tissue. Glycogen synthase kinase 3β (GSK3β) has fundamental roles in the regulation of pro- and anti-inflammatory cytokines production. However, the regulatory role for GSK3β in the pig inflammatory response in adipocytes remains unknown. We show here that SB216763 and LPS increased the phosphorylation of GSK3β (Ser9), and decreased the phosphorylation of GS (Ser641) in adipocytes. The activity of porcine GSK3β was inhibited by SB216763, an inhibitor of GSK3β, attenuated the production of IL-6 in LPS-stimulated adipocytes. Additionally, the essential core region of the pig IL-6 promoter located at −191 bp to −59 bp, and an NF-κBp65 element in this region was responsible for IL-6 promoter activity. The transcription activity of NF-κBp65 was activated by LPS stimulation, and the GSK3β inhibition repressed LPS-induced luciferase activity of the IL-6 promoter. Furthermore, LPS increased p65 binding to the NF-κB site, and GSK3β inhibition had no effect on the association of NF-κBp65 with IL-6 gene promoter after LPS treatment. These results demonstrate that GSK3β has important regulatory roles in the LPS-induced inflammatory response of IL-6 production in pig adipocytes.

The anti-tumor effect of regorafenib in lung squamous cell carcinoma in vitro

Lung squamous cell carcinoma (LSCC) is a common type of non-small-cell lung cancer (NSCLC) and lacks effective treatment. Regorafenib, an oral multikinase inhibitor, has demonstrated promising anti-tumor activity in various solid tumors. To study whether regorafenib inhibits LSCC cells, we investigate the compound in several LSCC cell lines and explore the possible mechanism. In this study, we confirmed that regorafenib had anti-proliferation effect on LSCC cell lines by inducing G0/G1 arrest. In addition, glycogen synthase kinase 3β (GSK3β) remained at the same level and Ser9 phosphorylation of GSK3β decreased with increasing incubation time and increasing regorafenib concentration in LSCC cells. GSK3β inhibition enhanced the anti-tumor activity of regorafenib. Thus, GSK3β activation restricted the anti-cancer effect of regorafenib on LSCC. In conclusion, regorafenib might be a promising drug for LSCC therapy. GSK3β might be a potential target to increase the anti-tumor effect of regorafenib in LSCC cells.

Antidepressant effects of creatine on amyloid β1–40-treated mice: The role of GSK-3β/Nrf2 pathway

Alzheimer's disease (AD) is characterized by progressive synaptic dysfunction and neuronal lost in specific brain areas including hippocampus, resulting in memory/learning deficits and cognitive impairments. In addition, non-cognitive symptoms are reported in AD patients, such as anxiety, apathy and depressed mood. The current antidepressant drugs present reduced efficacy to improve depressive symptoms in AD patients. Here, we investigated the ability of creatine, a compound with neuroprotective and antidepressant properties, to counteract amyloid β1–40 peptide-induced depressive-like behavior in mice. Moreover, we addressed the participation of the intracellular signaling pathway mediated by glycogen synthase kinase-3β (GSK-3β)/nuclear factor erythroid-2-related factor 2 (Nrf2) in the creatine effects. Aß1–40 administration (400 pmol/mouse, i.c.v.) increased the immobility time in the tail suspension test and decreased the grooming time and increased latency to grooming in the splash test, indicative of depressive-like behavior. These impairments were attenuated by creatine (0.01 and 10 mg/kg, p.o.) and fluoxetine (10 mg/kg, p.o., positive control). No significant alterations on locomotor performance were observed in the open field. Aß1–40 administration did not alter hippocampal phospho-GSK-3β (Ser9)/total GSK-3β, total GSK-3β and heme oxygenase-1 (HO-1) immunocontents. However, Aß1–40-infused mice treated with creatine (0.01 mg/kg) presented increased phosphorylation of GSK-3β(Ser9) and HO-1 immunocontent in the hippocampus. Fluoxetine per se increased GSK-3β(Ser9) phosphorylation, but did not alter HO-1 levels. In addition, Aß1–40 administration increased hippocampal glutathione (GSH) levels as well as glutathione reductase (GR) and thioredoxin reductase (TrxR) activities, and these effects were abolished by creatine and fluoxetine. This study provides the first evidence of the antidepressive-like effects of creatine in Aß1–40-treated mice, which were accompanied by hippocampal inhibition of GSK-3β and modulation of antioxidant defenses. These findings indicate the potential of creatine for the treatment of depression associated with AD.

K313, a novel benzoxazole derivative, exhibits anti‐inflammatory properties via inhibiting GSK3β activity in LPS‐induced RAW264.7 macrophages

Benzoxazole and its derivatives have been widely studied in recent years due to their various biological properties. A previous study has demonstrated that K313 (1H-indole-2,3-dione 3-(1,3-benzoxazol-2-ylhydrazone)), a novel benzoxazole derivative, inhibits T cell proliferation to yield immunosuppressive effects. However, there are no related reports about its anti-inflammatory effects. In the present study, we investigated the anti-inflammatory properties and the underlying molecular mechanism of K313 in lipopolysaccharide (LPS)-induced RAW264.7 macrophages. K313 dose-dependently (5, 10, and 20 μM) inhibited LPS-stimulated nitric oxide (NO), interleukin (IL)-6, tumor necrosis factor (TNF)-α, and 3-nitrotyrosine (3-NT) production and significantly decreased the gene transcription levels of inducible nitric oxide (iNOS), IL-6, and TNF-α. In addition, the results showed that the inflammatory cytokines suppressed by K313 were not regulated by p65 NF-κB, ERK1/2, AKT, or p38 MAPK. Instead, K313 increased phosphorylation of glycogen synthase kinase-3 beta (GSK-3β) (Ser9) resulting in GSK-3β deactivation. Moreover, in LPS-stimulated RAW264.7 macrophages, K313 and lithium chloride (LiCl) had a synergistic effect on the anti-inflammatory response. These results indicated that K313 exhibited anti-inflammatory properties and revealed the potential mechanism. K313 can increase GSK-3β (Ser9) phosphorylation to decrease GSK-3β activation in LPS-induced RAW264.7 macrophages.

Glycogen synthase kinase 3β promotes liver innate immune activation by restraining AMP-activated protein kinase activation

Glycogen synthase kinase 3β (Gsk3β [Gsk3b]) is a ubiquitously expressed kinase with distinctive functions in different types of cells. Although its roles in regulating innate immune activation and ischaemia and reperfusion injuries (IRIs) have been well documented, the underlying mechanisms remain ambiguous, in part because of the lack of cell-specific tools in vivo. We created a myeloid-specific Gsk3b knockout (KO) strain to study the function of Gsk3β in macrophages in a murine liver partial warm ischaemia model. Compared with controls, myeloid Gsk3b KO mice were protected from IRI, with diminished proinflammatory but enhanced anti-inflammatory immune responses in livers. In bone marrow-derived macrophages, Gsk3β deficiency resulted in an early reduction of Tnf gene transcription but sustained increase of Il10 gene transcription on Toll-like receptor 4 stimulation in vitro. These effects were associated with enhanced AMP-activated protein kinase (AMPK) activation, which led to an accelerated and higher level of induction of the novel innate immune negative regulator small heterodimer partner (SHP [Nr0b2]). The regulatory function of Gsk3β on AMPK activation and SHP induction was confirmed in wild-type bone marrow-derived macrophages with a Gsk3 inhibitor. Furthermore, we found that this immune regulatory mechanism was independent of Gsk3β Ser9 phosphorylation and the phosphoinositide 3-kinase-Akt signalling pathway. In vivo, myeloid Gsk3β deficiency facilitated SHP upregulation by ischaemia-reperfusion in liver macrophages. Treatment of Gsk3b KO mice with either AMPK inhibitor or SHP small interfering RNA before the onset of liver ischaemia restored liver proinflammatory immune activation and IRI in these otherwise protected hosts. Additionally, pharmacological activation of AMPK protected wild-type mice from liver IRI, with reduced proinflammatory immune activation. Inhibition of the AMPK-SHP pathway by liver ischaemia was demonstrated in tumour resection patients. Gsk3β promotes innate proinflammatory immune activation by restraining AMPK activation. Glycogen synthase kinase 3β promotes macrophage inflammatory activation by inhibiting the immune regulatory signalling of AMP-activated protein kinase and the induction of small heterodimer partner. Therefore, therapeutic targeting of glycogen synthase kinase 3β enhances innate immune regulation and protects liver from ischaemia and reperfusion injury.

Modulation of GSK3β autoinhibition by Thr‐7 and Thr‐8

Glycogen synthase kinase 3β (GSK-3β) is a pivotal signaling node that regulates a myriad of cellular functions and is deregulated in many pathological conditions, making it an attractive therapeutic target. Inhibitory Ser-9 phosphorylation of GSK3β by AKT is an important mechanism for negative regulation of GSK3β activity upon insulin stimulation. Here, we report that Thr-7 and Thr-8 residues located in the AKT/PKB substrate consensus sequence on GSK3β are essential for insulin-stimulated Ser-9 phosphorylation invivo and for GSK3β inactivation. Intestinal cell kinase (ICK) phosphorylates GSK3β Thr-7 invitro and invivo. Thr-8 phosphorylation partially inhibits GSK3β, but Thr-7 phosphorylation promotes GSK3β activity and blocks phospho-Ser-9-dependent GSK3β autoinhibition. Our findings uncover novel mechanistic and signaling inputs involved in the autoinhibition of GSK3β.

Delayed neurochemical effects of prenatal exposure to MeHg in the cerebellum of developing rats

Human fetuses and neonates are particularly vulnerable to methylmercury (MeHg)-induced brain damage and are sensitive even to low exposure levels. Previous work of our group evidence that prenatal exposure to MeHg causes cognitive and behavioral alterations and disrupt hippocampus signaling. The current study aimed to investigate the effect of gestational exposure of rats to MeHg at low doses (1 or 2 mg/kg) on parameters of redox imbalance and key signaling pathways in the cerebellum of their offspring. Pregnant females received MeHg (treated group) or 0.9% saline water (control group) by gavage in alternated days from gestational day 5 (GD5) until parturition and analyzes were proceed in the cerebellum of 30-day-old pups. We found increased lipid peroxidation and protein carbonylation levels as well as decreased SH content in pups prenatally exposed to 2 mg/kg MeHg. In addition, misregulated SOD/catalase activities supported imbalanced redox equilibrium. We found decreased GSK3β(Ser9) phosphorylation, suggesting activation of this enzyme and dephosphorylation/inhibition of ERK1/2 and JNK pathways. Increased PKAα catalytic subunit could be upstream of hyperphosphorylated c-Raf(Ser259) and downregulated MAPK pathway. In addition, we found raised levels of the Ca2+-dependent protein phosphatase 2 B (PP2B). We also found preserved immunohistochemical staining for both glial fibrillary acidic protein (GFAP) and NeuN in MeHg-exposed pups. Western blot analysis showed unaltered levels of BAX/BCL-XL, BAD/BCL-2 and active caspase 3. Together, these findings support absence of reactive astrocytes, neuronal damage and apoptotic cell death in the cerebellum of MeHg treated pups. The present study provides evidence that prenatal exposure to MeHg leads to later redox imbalance and disrupted signaling mechanisms in the cerebellum of 30-day-old pups potentially predisposing them to long-lasting neurological impairments in CNS.

Hyperforin attenuates aluminum-induced Aβ production and Tau phosphorylation via regulating Akt/GSK-3β signaling pathway in PC12 cells

Aluminum (Al) is a neurotoxicant and cause β-amyloid (Aβ) peptides aggregation and tau hyperphosphorylation. Hyperforin (HF) is one of the major active constituents of the extracts of St. John’s Wort (Hypericum perforatum), can treat Alzheimer’s disease (AD) and other diseases involving peptide accumulation and cognition impairment. To determine the effects of HF on Al-induced Aβ formation and tau hyperphosphorylation, PC12 cells were cultured and treated with Al-malt (500μM) and/or HF (1μM). The results showed that HF treatment significantly attenuated Al-malt-induced Aβ1-42 production by reducing the expressions of APP, BACE1 and PS1, while increasing the expressions of sAPPα, ADAM9/10/17, and tau phosphorylation in PC12 cells. In addition, HF treatment also increased phosphorylation of AKT (Ser473) and inhibited GSK-3β activity by increasing phosphorylation of GSK-3β (Ser9). These results indicated that HF may exert the protection via regulating the AKT/GSK-3β signaling to reduce Aβ production and tau phosphorylation in PC12 cells. Furthermore, these results could lead a possible therapeutics for the management of Al neurotoxicity.

Resveratrol Ameliorates Tau Hyperphosphorylation at Ser396 Site and Oxidative Damage in Rat Hippocampal Slices Exposed to Vanadate: Implication of ERK1/2 and GSK-3β Signaling Cascades.

The objective of this study was to investigate the effect of resveratrol (a natural polyphenolic phytostilbene) on tau hyperphosphorylation and oxidative damage induced by sodium orthovanadate (Na3VO4), the prevalent species of vanadium (vanadate), in rat hippocampal slices. Our results showed that resveratrol significantly inhibited Na3VO4-induced hyperphosphorylation of tau at the Ser396 (p-S396-tau) site, which is upregulated in the hippocampus of Alzheimer's disease (AD) brains and principally linked to AD-associated cognitive dysfunction. Subsequent mechanistic studies revealed that reduction of ERK1/2 activation was involved in the inhibitory effect of resveratrol by inhibiting the ERK1/2 pathway with SL327 mimicking the aforementioned effect of resveratrol. Moreover, resveratrol potently induced GSK-3β Ser9 phosphorylation and reduced Na3VO4-induced p-S396-tau levels, which were markedly replicated by pharmacologic inhibition of GSK-3β with LiCl. These results indicate that resveratrol could suppress Na3VO4-induced p-S396-tau levels via downregulating ERK1/2 and GSK-3β signaling cascades in rat hippocampal slices. In addition, resveratrol diminished the increased extracellular reactive oxygen species generation and hippocampal toxicity upon long-term exposure to Na3VO4 or FeCl2. Our findings strongly support the notion that resveratrol may serve as a potential nutraceutical agent for AD.

Telmisartan mitigates hyperglycemia-induced vascular inflammation by increasing GSK3β-Ser9 phosphorylation in endothelial cells and mouse aortas

Telmisartan, an angiotensin II type 1 receptor blocker (ARB), attenuates hyperglycemia-aggravated vascular inflammation by decreasing IκB kinase β (IKKβ) expression in endothelial cells. Because glycogen synthase 3β (GSK3β) is involved in inflammatory process by regulating nuclear factor-κB (NF-κB) activity, we investigated whether GSK3β mediates telmisartan-ameliorated vascular inflammation in hyperglycemia-treated endothelial cells and high-fat diet (HFD)-fed mice. Telmisartan remarkably induced GSK3β-Ser9 phosphorylation in hyperglycemia-treated endothelial cells that accompanied a decrease in hyperglycemia-induced NF-κB p65-Ser536 phosphorylation, vascular cell adhesion molecule-1 (VCAM-1) expression, and THP-1 monocyte adhesion. Ectopic expression of GSK3β-S9A, a constitutively active mutant of GSK3β, significantly restored complete telmisartan-inhibited NF-κB p65-Ser536 phosphorylation, VCAM-1 expression, and THP-1 monocyte adhesion. In addition, it reversed telmisartan-repressed IKKβ expression. Among the ARB, including losartan and fimasartan, only telmisartan increased GSK3β-Ser9 phosphorylation, and telmisartan-induced GSK3β-Ser9 phosphorylation remained unchanged by pretreatment with GW9662, a specific and irreversible peroxisome proliferator-activated receptor γ (PPARγ) antagonist. Finally, in the aortas of HFD-fed mice, telmisartan treatment significantly attenuated HFD-induced upregulation of NF-κB p65-Ser536 phosphorylation, VCAM-1 expression, and IKKβ expression and downregulation of GSK3β-Ser9 phosphorylation. Taken together, our findings demonstrated that telmisartan ameliorates hyperglycemia-exacerbated vascular inflammation, at least in part, by inducing GSK3β-Ser9 phosphorylation, which consequently inhibits IKKβ expression, NF-κB p65-Ser536 phosphorylation, and VCAM-1 expression in a PPARγ-independent manner.

Potentiation of TRAP-6-induced platelet dense granule release by blockade of P2Y12 signaling with MRS2395

The release of ADP from platelet dense granules and its binding to platelet P2Y12 receptors is key to amplifying the initial hemostatic response and propagating thrombus formation. P2Y12 has thus emerged as a therapeutic target to safely and effectively prevent secondary thrombotic events in patients with acute coronary syndrome or a history of myocardial infarction. Pharmacological inhibition of P2Y12 receptors represents a useful approach to better understand the signaling mediated by these receptors and to elucidate the role of these receptors in a multitude of platelet hemostatic and thrombotic responses. The present work examined and compared the effects of four different P2Y12 inhibitors (MRS2395, ticagrelor, PSB 0739, and AR-C 66096) on platelet function in a series of in vitro studies of platelet dense granule secretion and trafficking, calcium generation, and protein phosphorylation. Our results show that in platelets activated with the PAR-1 agonist TRAP-6 (thrombin receptor-activating peptide), inhibition of P2Y12 with the antagonist MRS2395, but not ticagrelor, PSB 0739 or AR-C 66096, potentiated human platelet dense granule trafficking to the plasma membrane and release into the extracellular space, cytosolic Ca2+ influx, and phosphorylation of GSK3β-Ser9 through a PKC-dependent pathway. These results suggest that inhibition of P2Y12 with MRS2395 may act in concert with PAR-1 signaling and result in the aberrant release of ADP by platelet dense granules, thus reducing or counteracting the anticipated anti-platelet efficacy of this inhibitor.

AKT signaling is essential for functional and structural integrity of the heart

The mammalian heart expresses predominantly two isoforms of protein kinase B (AKT), namely AKT1 and AKT2. Knockout mice for AKT1 and AKT2 respectively, have demonstrated that AKT1 primarily appears to regulate growth whereas AKT2 is rather involved in regulating metabolism. Here we have analyzed common functions of both AKT isoforms in the heart by generating Tamoxifen(OHTX)‐inducible, cardiac myocyte specific AKT1 and AKT2 single and double KO mice (iCMAKT1/2KO).Inactivation of AKT1 and AKT2 resulted in a maximal loss of both isoforms 5 days after start of OHTX‐treatment without a compensatory upregulation of AKT3. The basal level of Ser9 Phosphorylation of GSK3β was significantly reduced in hearts of double KO only, demonstrating that GSK3β is controlled by AKT1 and AKT2 isoforms in CM. Insulin did not stimulate GSK3β phosphorylation. Furthermore, insulin dependent phosphorylation of classical direct and indirect AKT downstream targets like mTor (Ser2448, Ser2841), p70S6 kinase (Thre 389) or S6 ribosomal protein, among others, was shown to be significantly downregulated in iCMAKT1/2KO heart.Functional analysis by echocardiography revealed a progressive decline of pump function characterized by a drop of ejection fraction from initially 62 to 37 % and 31 % on day 10 and day 21, respectively resulting in cardiac failure on day 24. In contrast, the single iCMAKT1 and iCMAKT2 KO mice showed no cardiac depression. Interestingly, wall thickness declined from day 14 to day 21. Concomitantly, cardiac mass declined to 70 %. Gene expression analysis using Agilent 60K microarrays were performed using hearts from single and double knock out mice. On day 10 after KO induction 1095 genes were differentially expressed (P<0.05) and on day 21 this number increased to 7707 (P<0.01, moderated t‐test, Benjamini‐Hochberg correction). On day 10 upon Txf induction Gdf15 (4.9 × up), a typical stress marker for diseased hearts but not ANP was induced in double knock out hearts. On day 21 this expression increased to Gdf15 (58 × up) and also ANP was induced (17.8 × up). None of these expression alterations coud be detected in Akt1 or Akt2 single knock out heart. Ingenuity Pathway analysis identified particularly metabolic pathways as well as EIF2 signalling to be substantially affected.ConclusionExpression of either AKT1 or AKT2 in cardiac myocytes is sufficient to preserve cardiac function whereas loss of both isoforms results in progessive cardiac atrophy and failure.Support or Funding InformationThis work was funded by DFG Grant IRTG1902.