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Multi-drug resistance P-glycoprotein (P-gp/MDR1) is one of the most clinically relevant ABC transporters, highly enriched at the blood-brain barrier (BBB) with a broad substrate spectrum including therapeutic drugs and metabolic waste products. Altered P-gp transport function has been implicated in multi-drug resistance and in the pathogenesis and progression of neurological diseases. Recent studies have shown that P-gp expression is modulated by micro-RNAs in peripheral organs. Particularly, miR-27a-3p has been shown to play a critical role in the regulation of P-gp in multi-drug resistant cancer cells. In brain disorders, altered levels of miR-27a-3p were reported in several diseases associated with alterations in P-gp expression at the BBB. However, effect of altered miR-27a-3p expression on P-gp expression at the BBB remains to be determined. In this study, we investigated the role of miR-27a-3p in the regulation of P-gp expression and activity at the brain endothelium. Levels of miR-27a-3p were modulated by mimic and inhibitor transfection in an in-vitro model of human brain endothelial hCMEC/D3 cells. Effect of miR-27a-3p modulation on P-gp expression and activity was examined and the underlying regulatory mechanisms explored. Our results showed that transfection of hCMEC/D3 cells with miR-27a-3p mimic induces expression and activity of P-gp while miR-27a-3p inhibition exerted opposite effects. Mechanistic studies revealed that miR-27a-3p regulates P-gp by mediating Glycogen Synthase Kinase 3 Beta (GSK3s) inhibition and activating Wnt/s-catenin signaling. These findings shed light on miR-27a-3p/GSK3s/s-catenin as a novel axis that could be exploited to modulate P-gp efflux activity at the brain endothelium and help improving CNS diseases treatment or brain protection.

International audience; Merkel cell carcinoma is an aggressive skin cancer frequently caused by the Merkel cell polyomavirus (MCPyV). Since proliferation of MCPyV-positive MCC tumor cells strictly depends on expression of the virus-encoded T antigens (TA), these proteins theoretically represent ideal targets for different kinds of therapeutic approaches. Here we developed a cell-based assay to identify compounds which specifically inhibit growth of MCC cells by repressing TA expression. Applying this technique we screened a kinase inhibitor library and identified six compounds targeting glycogen synthase kinase 3 (GSK3) such as CHIR99021 as suppressors of TA transcription in MCC cells. Involvement of GSK3 alpha and -beta in the regulation of TA-expression was confirmed by combining GSK3A knockout with inducible GSK3B shRNA knockdown since double knockouts could not be generated. Finally, we demonstrate that CHIR99021 exhibits in vivo antitumor activity in an MCC xenograft mouse model suggesting GSK3 inhibitors as potential therapeutics for the treatment of MCC in the future.

The Akt/GSK3 signal pathway exerts an impact on the apoptosis of chondrocytes. We planned to elucidate the role and mechanism of Akt/GSK3 signal transduction in ovariectomized osteoporosis. 50 female mice in SPF-graded and healthy condition were processed to establish the ovariectomized osteoporosis model, while 12 of the healthy mice were set as blank controls. The successfully established ovariectomized osteoporosis models were divided into model group (12 mice) and Astragalus group (12 mice). The following measurements were conducted: the bone mineral density in the left humerus, serum estrogen level and Cx43 protein level in osteoblasts, proportion of apoptotic cells, as well as the protein and mRNA levels of Akt and GSK3β in murine bone tissues via Western blotting and RT-PCR detection, respectively. The bone mineral density of mice in Astragalus group was the highest (0.174±0.04) g/cm2. The positive rate of Cx43 protein expression, apoptosis rate, as well as the protein and mRNA levels of Akt in osteocytes were significantly decreased (P < 0.05), but still significantly higher than model group. Estrogen level in Astragalus group was (87.52 ±8.69) pmol/L. The positive rate of Cx43 expression, apoptosis rate, as well as the protein and mRNA levels of GSK3β in osteocytes were decreased in comparison to model group (P < 0.05). Astragalus polysaccharide injection could ameliorate the apoptosis of chondrocytes and downregulate Cx43 protein via modulating Akt/GSK3β signal transduction, thereby exerting a therapeutic effect on ovariectomized osteoporosis.

International audience; Myotonic dystrophy type 1 (DM1) is the most common monogenetic muscular disorder of adulthood. This multisystemic disease is caused by CTG repeat expansion in the 3-untranslated region of the DM1 protein kinase gene called DMPK. DMPK encodes a myosin kinase expressed in skeletal muscle cells and other cellular populations such as smooth muscle cells, neurons and fibroblasts. The resultant expanded (CUG)n RNA transcripts sequester RNA binding factors leading to ubiquitous and persistent splicing deregulation. The accumulation of mutant CUG repeats is linked to increased activity of glycogen synthase kinase 3 beta (GSK3␤), a highly conserved and ubiquitous serine/threonine kinase with functions in pathways regulating inflammation, metabolism, oncogenesis, neurogenesis and myogenesis. As GSK3␤-inhibition ameliorates defects in myogenesis, muscle strength and myotonia in a DM1 mouse model, this kinase represents a key player of DM1 pathobiochemistry and constitutes a promising therapeutic target. To better characterise DM1 patients, and monitor treatment responses, we aimed to define a set of robust disease and severity markers linked to GSK3␤by unbiased proteomic profiling utilizing fibroblasts derived from DM1 patients with low (80-150) and high (2600-3600) CTG-repeats. Apart from GSK3␤ increase, we identified dysregulation of nine proteins (CAPN1, CTNNB1, CTPS1, DNMT1, HDAC2, HNRNPH3, MAP2K2, NR3C1, VDAC2) modulated by GSK3␤. In silico-based expression studies confirmed expression in neuronal and skeletal muscle cells and revealed a relatively elevated abundance in fibroblasts. The potential impact of each marker in the myopathology of DM1 is discussed based on respective function to inform potential uses as severity markers or for monitoring GSK3␤ inhibitor treatment responses.

Alterations in prolyl oligopeptidase (PREP) activity have been connected, for example, with bipolar and major depressive disorder, and several studies have reported that lack or inhibition of PREP blocks the effects of lithium on inositol 1,4,5-triphosphate (IP3) levels. However, the impact of PREP modulation on other intracellular targets of lithium, such as glycogen synthase kinase 3 beta (GSK3b) or protein kinase B (Akt), has not been studied. We recently found that PREP regulates protein phosphatase 2A (PP2A), and because GSK3b and Akt are PP2A substrates, we studied if PREP-related lithium insensitivity is dependent on PP2A. To assess this, HEK-293 and SH-SY5Y cells with PREP deletion or PREP inhibition (KYP-2047) were exposed to lithium, and thereafter, the phosphorylation levels of GSK3b and Akt were measured by Western blot. As expected, PREP deletion and inhibition blocked the lithium-induced phosphorylation on GSK3b and Akt in both cell lines. When lithium exposure was combined with okadaic acid, a PP2A inhibitor, KYP-2047 did not have effect on lithium-induced GSK3b and Akt phosphorylation. Therefore, we conclude that PREP deletion or inhibition blocks the intracellular effects of lithium on GSK3b and Akt via PP2A activation.

Neurodegenerative disorders are illnesses that are responsible for neuronal cell death and resulting in lifelong cognitive problems. Due to their unclear mechanism, there are no effective drugs available for the treatment. For a long time, herbal drugs have been used as a role model in the field of the drug discovery process. Withania somnifera (Ashwagandha) in the Indian medicinal system (Ayurveda) is used for several neuronal disorders like insomnia and memory loss for decades. This study aims to identify active components of W. somnifera (WS) as potential inhibitors for the treatment of neurodegenerative diseases (ND). To fulfill this objective, Network pharmacology approach, gene ontology, pharmacokinetics analysis, molecular docking, and molecular dynamics simulation (MDS) studies were performed. A total of 77 active components in WS, 175 predicted neurodegenerative targets of WS, and 8085 ND-related targets were identified from different databases. The network analysis showed that the top ten targets APP, EGFR, MAPK1, ESR1, HSPA4, PRKCD, MAPK3, ABL1, JUN, and GSK3B were found as significant target related to ND. On the basis of gene ontology and topology analysis results, APP was found as a significant target related to Alzheimer's disease pathways. Molecular docking results found that Anahygrine, Cuscohygrine, Isopelletierine, and Nicotine showed the best binding affinities -5.55, -4.73, -4.04, and -4.11 Kcal/mol. Further, MDS results suggested that Isopelletierine and Nicotine could be used as potential inhibitors against APP protein and could be useful for the treatment of Alzheimer's disease.Communicated by Ramaswamy H. Sarma.

In rats, lisdexamfetamine (LDX) induces manic-like behaviors such as hyperlocomotion and increases in appetitive 50-kHz ultrasonic vocalizations (USV), which are prevented by antimanic drugs, such as lithium. Inhibition of glycogen synthase kinase 3 beta (GSK3β) and antioxidant activity have been associated with antimanic effects. Thus, the aim of the present study was to evaluate the possible antimanic-like effects of andrographolide (ANDRO), a GSK3β inhibitor, on LDX-induced hyperlocomotion and 50-kHz USV increases. In addition, the effect of ANDRO was studied on LDX-induced oxidative stress. Lithium was used as positive control. Adult Wistar rats were treated with vehicle, lithium (100 mg/kg i.p., daily) or ANDRO (2 mg/kg i.p., 3 times a week) for 21 days. On the test day, either 10 mg/kg LDX or saline was administered i.p. and USV and locomotor activity were recorded. LDX administration increased the number of 50-kHz calls, as well as locomotor activity. Repeated treatment with lithium or ANDRO prevented these effects of LDX on 50-kHz USV and locomotor activity. LDX increased lipid peroxidation (LPO) levels in rat striatum and both lithium and ANDRO prevented this effect. LPO levels in rat striatum were positively correlated with increases in 50-kHz USV emission as well as hyperlocomotion. In conclusion, the present results indicate that ANDRO has antimanic-like effects, which may be mediated by its antioxidant properties.

During autophagy, the coordinated actions of autophagosomes and lysosomes result in the controlled removal of damaged intracellular organelles and superfluous substrates. The evolutionary conservation of this process and its requirement for maintaining cellular homeostasis emphasizes the need to better dissect the pathways governing its molecular regulation. In our previously performed high-content screen, we assessed the effect of 1530 RNA-binding proteins on autophagy. Among the top regulators, we identified the eukaryotic translation initiation factor 4A-3 (eIF4A3). Here we show that depletion of eIF4A3 leads to a potent increase in autophagosome and lysosome biogenesis and an enhanced autophagic flux. This is mediated by the key autophagy transcription factor, TFEB, which becomes dephosphorylated and translocates from the cytoplasm to the nucleus where it elicits an integrated transcriptional response. We further identified an exon-skipping event in the transcript encoding for the direct TFEB kinase, GSK3B, which leads to a reduction in GSK3B expression and activity. Through analysis of TCGA data, we found a significant upregulation of eIF4A3 expression across several cancer types and confirmed the potential relevance of this newly identified signaling axis in human tumors. Hence, our data suggest a previously unrecognized role for eIF4A3 as a gatekeeper of autophagy through the control of TFEB activation, revealing a new mechanism for autophagy regulation.

ETS1 − an evolutionarily conserved transcription factor involved in the regulation of a number of cellular processes − is overexpressed in several malignancies, including ovarian cancer. Most studies on ETS1 expression have been focused on the transcriptional and RNA levels, with post-translational control mechanisms remaining relatively unexplored in the pathogenesis of malignancies. Here, we show that ETS1 forms a complex with glycogen synthase kinase-3β (GSK3β). Specifically, GSK3β-mediated phosphorylation of ETS1 at threonine 265 and serine 269 promoted protein stability, induced the transcriptional activation of matrix metalloproteinase (MMP)-9, and increased cell migration. In vivo experiments revealed that a GSK3β inhibitor was able to suppress both endogenous ETS1 expression and induction of MMP-9 expression. Upon generation of a specific antibody against phosphorylated ETS1, we demonstrated that phospho-ETS1 immunohistochemical expression in ovarian cancer specimens was correlated with that of MMP-9. Notably, the cumulative overall survival of patients with low phospho-ETS1 histoscores was significantly longer than that of those showing higher scores. We conclude that the GSK3β/ETS1/MMP-9 axis may regulate the biological aggressiveness of ovarian cancer and can serve as a prognostic factor in patients with this malignancy.

Serum and glucocorticoid-regulated kinase 1 (SGK1) is one of the most frequently mutated genes in diffuse large B-cell lymphoma (DLBCL). However, little is known about its function or the consequence of its mutation. The frequent finding of truncating mutations has led to the widespread assumption that these represent loss-of-function variants and, accordingly, that SGK1 must act as a tumor suppressor. In this study, instead, the most common SGK1 mutations led to production of aberrantly spliced messenger RNA neoisoforms in which translation is initiated from downstream methionines. The resulting N-terminal truncated protein isoforms showed increased expression related to the exclusion of an N-terminal degradation domain. However, they retained a functional kinase domain, the overexpression of which rendered cells resistant to AKT inhibition, in part because of increased phosphorylation of GSK3B. These findings challenge the prevailing assumption that SGK1 is a tumor-suppressor gene in DLBCL and provide the impetus to explore further the pharmacological inhibition of SGK1 as a therapeutic strategy for DLBCL.

Background As the symbolic pathological changes of Alzheimer’s disease (AD), hyperphosphorylated tau and amyloid plaque play important roles in the progression of the disease. In AD patients, the neural activity in default mode network is abnormal at different stages of the disease, and showed a hypoconnective status. Inhibition of phosphatidylinositol-3-kinase (PI3K) activates glycogen synthase kinase 3 beta (GSK-3β) and induces tau phosphorylation. Objective We speculated that inhibiting cerebral PI3K altered the glucose metabolism in DMN. We aimed to explore the impacts of PI3K inhibition on tau phosphorylation, cerebral glucose metabolism, and synaptic plasticity. Methods We injected wortmannin, an inhibitor of PI3K, lateral ventricularly in rats to mimic the pathology of AD. Immunohistochemistry was carried out to analyze the expression of phosphorylated tau. Region-specific glucose metabolism in the brain was analyzed using 18F-FDG PET imaging. In vivo long-term potentiation (LTP) in the hippocampus was detected to assess the synaptic plasticity. Results The results show that the phosphorylated tau at T231 increased and the hippocampal LTP was suppressed 24 h after wortmannin administration. In the DMN, glucose uptake was significantly high, indicating a neural activity disturbance. Conclusion We conclude that targeting PI3K-GSK-3β pathway to mimic AD tau pathology interrupted the glucose metabolism of DMN brain regions.

Although initially discovered and extensively studied for its role in inflammation, Annexin A1 (ANXA1) has been reported to be closely related to cancer in recent years, and its role in cancer is specific to tumor types and tissues. In the present study, we identified ANXA1 as an interaction partner of glycogen synthase kinase 3 beta (GSK3β), a multi-functional serine/threonine kinase tightly associated with cell fate determination and cancer, and assessed the functional significance of GSK3β-ANXA1 interaction in the metastasis of non-small cell lung cancer (NSCLC). We confirmed the interaction between GSK3β and ANXA1 in vitro and in H1299 and A549 cells by Glutathione-S-transferase (GST) pull-down assay and co-immunoprecipitation. We found that ANXA1 negatively regulated the phosphorylation of GSK3β and inhibited the epithelial-mesenchymal transformation (EMT) process and migration and invasion of NSCLC cells. By functional rescue assay, we confirmed that ANXA1 inhibited EMT through the regulation of GSK3β activity and thereby inhibited the migration and invasion of NSCLC cells. Our study sheds light on the function of ANXA1 and GSK3β and provides new elements for the understanding of NSCLC pathogenesis.

Alzheimer’s disease (AD) belongs to the most common forms of dementia that causes a progressive loss of brain cells and leads to memory impairment and decline of other thinking skills. There is yet no effective treatment for AD; hence, the search for new drugs that could improve memory and other cognitive functions is one of the hot research topics worldwide. Scientific efforts are also directed toward combating behavioral and psychological symptoms of dementia, which are an integral part of the disease. Several studies have indicated that glycogen synthase kinase 3 beta (GSK3β) plays a crucial role in the pathogenesis of AD. Moreover, GSK3β inhibition provided beneficial effects on memory improvement in multiple animal models of AD. The present review aimed to update the most recent reports on the discovery of novel multifunctional ligands with GSK3β inhibitory activity as potential drugs for the symptomatic and disease-modifying therapy of AD. Compounds with GSK3β inhibitory activity seem to be an effective pharmacological approach for treating the causes and symptoms of AD as they reduced neuroinflammation and pathological hallmarks in animal models of AD and provided relief from cognitive and neuropsychiatric symptoms. These compounds have the potential to be used as drugs for the treatment of AD, but their precise pharmacological, pharmacokinetic, toxicological and clinical profiles need to be defined.

Abnormal ion channel currents caused by myocardial electrical remodeling is one of the main causes of malignant arrhythmias. Glycogen synthase kinase 3β (GSK-3β) is the main therapeutic target following ischemia as it regulates nerve cell channels. However, few studies have investigated its role in myocardial electrical remodeling. The present study aimed to investigate the role of GSK-3β in a rat myocardial infarction (MI)–induced electrical remodeling and potential effects on cardiac ionic channels including KCNJ2/Kir2.1/IK1. Ligation of the left anterior descending artery in rats was performed to establish a MI model. The rats were randomly divided into three groups, the sham, MI, and MI + SB group. The animals in the latter group were administered SB216763 (GSK-3β inhibitor) at a dose of 0.6 mg·kg−1·day−1. The ventricular function was assessed by echocardiography, electrocardiography, and histological analysis 7 days post-surgery. Serum was collected to measure lactate dehydrogenase and cardiac troponin I levels, and the mRNA and protein levels of the KCNJ2/Kir2.1/IK1 channel in the heart tissues were assessed. H9c2 cells were cultured to examine the effects of SB216763 on the protein expression of Kir2.1 channel under hypoxic conditions. The results revealed that SB216763 ameliorated acute cardiac injury and improved myocardial dysfunction. Moreover, SB216763 increased the mRNA and protein expression of Kir2.1 during MI. Furthermore, SB216763 treatment abrogated the decreased expression of Kir2.1 in H9c2 cells under hypoxic conditions. GSK-3β inhibition upregulates Kir2.1 expression in a rat model of MI.

Glycogen synthase kinase-3β (GSK-3β), a serine/threonine kinase, has been implicated in the pathogenesis of many cancers, with involvement in cell-cycle regulation, apoptosis, and immune response. Small-molecule GSK-3β inhibitors are currently undergoing clinical investigation. Tumor sequencing has revealed genomic alterations in GSK-3β, yet an assessment of the genomic landscape in malignancies is lacking. This study assessed >100,000 tumors from two databases to analyze GSK-3β alterations. GSK-3β expression and immune cell infiltrate data were analyzed across cancer types, and programmed death-ligand 1 (PD-L1) expression was compared between GSK-3β–mutated and wild-type tumors. GSK-3β was mutated at a rate of 1%. The majority of mutated residues were in the kinase domain, with frequent mutations occurring in a GSK-3β substrate binding pocket. Uterine endometrioid carcinoma was the most commonly mutated (4%) tumor, and copy-number variations were most commonly observed in squamous histologies. Significant differences across cancer types for GSK-3β–mutated tumors were observed for B cells (P = 0.018), monocytes (P = 0.002), dendritic cells (P = 0.005), neutrophils (P = 0.0003), and endothelial cells (P = 0.014). GSK-3β mRNA expression was highest in melanoma. The frequency of PD-L1 expression was higher among GSK-3β–mutated tumors compared with wild type in colorectal cancer (P = 0.03), endometrial cancer (P = 0.05), melanoma (P = 0.02), ovarian carcinoma (P = 0.0001), and uterine sarcoma (P = 0.002). Overall, GSK-3β molecular alterations were detected in approximately 1% of solid tumors, tumors with GSK-3β mutations displayed a microenvironment with increased infiltration of B cells, and GSK-3β mutations were associated with increased PD-L1 expression in selected histologies. These results advance the understanding of GSK-3β complex signaling network interfacing with key pathways involved in carcinogenesis and immune response.

Background Oral cancer represents the sixth most common cancer in the world and is associated with 40-50% survival at 5 years. Within oral malignancies, oral squamous cell carcinoma (OSCC) is commonly preceded by potentially malignant lesions, which, according to histopathological criteria, are referred to as oral dysplasia and their diagnosis are associated with higher rates of malignant transformation towards cancer. We recently reported that aberrant activation of the Wnt/β‑catenin pathway is due to overexpression of Wnt ligands in oral dysplasia. However, the expression of other regulators of this pathway, namely components of the β-catenin destruction complex has not been explored in oral dysplasia. Material and Methods Using immunohistochemical analyses, we evaluated nuclear expression of β‑catenin and its association with Wnt3a and Wnt5a. Likewise, components of the β-catenin destruction complex, including Adenomatous Polyposis Coli (APC), Axin and Glycogen Synthase Kinase 3 beta (GSK-3β) were also evaluated in oral dysplasia and OSCC biopsies. Results We found that moderate and severe dysplasia samples, which harbored increased expression of nuclear β‑catenin, depicted augmented cytoplasmic expression of GSK‑3β, Axin and APC, in comparison with OSCC samples. Also, GSK-3β was found nuclear in mild dysplasia and OSCC samples, when compared with other study samples. Conclusions Cytoplasmic levels of components of the β-catenin destruction complex are increased in oral dysplasia and might be responsible of augmented nuclear β‑catenin. Key words:Oral cancer, oral dysplasia, β-Catenin, Wnt ligands, destruction complex.

Background The aim of this study was to identify important miRNAs and their target genes involved in cell apoptosis in intervertebral disc degeneration (IDD) patients. Methods The dataset, GSE63492, was obtained from the gene expression omnibus platform. After preprocessing, the differentially expressed miRNAs (DEMs) and their target genes were identified using the Limma package and miRWalk2.0 database, respectively. The clusterProfiler package in R was used to perform functional enrichment analysis of these target genes. Subsequently, protein-protein interaction (PPI) network and subnet clusters of the coregulated genes were conducted using the STRING database and MCODE, respectively. Further, the co-regulatory network of the key miRNAs and PPI networks were visualized using Cytoscape. Finally, cell apoptosis-related pathways and the genes enriched in these pathways were identified. Results The genes targeted by the upregulated (hsa-miR-302c-5p, hsa-miR-631, hsa-let-7f-1-3p, hsa-miR-3675-3p, and hsa-miR-585-3p) and downregulated miRNAs (hsa-miR-185-5p, hsa-miR-486-5p, hsa-miR-4306, and hsa-miR-4674) were interrelated with cell apoptosis-related pathways. MAPK1 and MAPK3 were targeted by hsa-miR-185-5p, while GSK3B was targeted hsa-miR-4306, hsa-miR-486-5p, hsa-miR-185-5p, hsa-let-7f-1-3p, and hsa-miR-631. Besides, MAPK3 and VEGFA were regulated by hsa-miR-3675-3p and hsa-miR-631, respectively. Conclusions The expression of GSK3B may be coregulated by miR-4306, miR-185-5p, miR-486-5p, hsa-let-7f-1-3p, and miR-631 and may affect IDD development. Besides, miR-185-5p and miR-3675-3p may control nucleus pulposus (NP) cell apoptosis through the MAPK signaling pathway in IDD patients. VEGFA expression may be regulated by miR-631, and help maintain NP cell survival in IDD patients. Our findings may help guide further research into the role of miRNAs in IDD progression.

Myotonic dystrophy type 1 (DM1) is the most prevalent form of muscular dystrophy in adults and yet there are currently no treatment options. Although this disease causes multisystemic symptoms, it is mainly characterised by myopathy or diseased muscles, which includes muscle weakness, atrophy, and myotonia, severely affecting the lives of patients worldwide. On a molecular level, DM1 is caused by an expansion of CTG repeats in the 3' untranslated region (3'UTR) of the DM1 Protein Kinase (DMPK) gene which become pathogenic when transcribed into RNA forming ribonuclear foci comprised of auto complementary CUG hairpin structures that can bind proteins. This leads to the sequestration of the muscleblind-like (MBNL) family of proteins, depleting them, and the abnormal stabilisation of CUGBP Elav-like family member 1 (CELF1), enhancing it. Traditionally, DM1 research has focused on this RNA toxicity and how it alters MBNL and CELF1 functions as key splicing regulators. However, other proteins are affected by the toxic DMPK RNA and there is strong evidence that supports various signalling cascades playing an important role in DM1 pathogenesis. Specifically, the impairment of protein kinase B (AKT) signalling in DM1 increases autophagy, apoptosis, and ubiquitin-proteasome activity, which may also be affected in DM1 by AMP-activated protein kinase (AMPK) downregulation. AKT also regulates CELF1 directly, by affecting its subcellular localisation, and indirectly as it inhibits glycogen synthase kinase 3 beta (GSK3β), which stabilises the repressive form of CELF1 in DM1. Another kinase that contributes to CELF1 mis-regulation, in this case by hyperphosphorylation, is protein kinase C (PKC). Additionally, it has been demonstrated that fibroblast growth factor-inducible 14 (Fn14) is induced in DM1 and is associated with downstream signalling through the nuclear factor κB (NFκB) pathways, associating inflammation with this disease. Furthermore, MBNL1 and CELF1 play a role in cytoplasmic processes involved in DM1 myopathy, altering proteostasis and sarcomere structure. Finally, there are many other elements that could contribute to the muscular phenotype in DM1 such as alterations to satellite cells, non-coding RNA metabolism, calcium dysregulation, and repeat-associated non-ATG (RAN) translation. This review aims to organise the currently dispersed knowledge on the different pathways affected in DM1 and discusses the unexplored connections that could potentially help in providing new therapeutic targets in DM1 research.

Alzheimer’s disease (AD) is the most common cause of dementia, affecting approximately 35 million people worldwide. The current treatment options for people with AD consist of drugs designed to slow the rate of decline in memory and cognition, but these treatments are not curative, and patients eventually suffer complete cognitive injury. With the substantial amounts of published data on targets for this disease, we proposed that machine learning software could be used to find novel small-molecule treatments that can supplement the AD drugs currently on the market. In order to do this, we used publicly available data in ChEMBL to build and validate Bayesian machine learning models for AD target proteins. The first AD target that we have addressed with this method is the serine–threonine kinase glycogen synthase kinase 3 beta (GSK3β), which is a proline-directed serine–threonine kinase that phosphorylates the microtubule-stabilizing protein tau. This phosphorylation prompts tau to dissociate from the microtubule and form insoluble oligomers called paired helical filaments, which are one of the components of the neurofibrillary tangles found in AD brains. Using our Bayesian machine learning model for GSK3β consisting of 2368 molecules, this model produced a five-fold cross validation ROC of 0.905. This model was also used for virtual screening of large libraries of FDA-approved drugs and clinical candidates. Subsequent testing of selected compounds revealed a selective small-molecule inhibitor, ruboxistaurin, with activity against GSK3β (avg IC50 = 97.3 nM) and GSK3α (IC50 = 695.9 nM). Several other structurally diverse inhibitors were also identified. We are now applying this machine learning approach to additional AD targets to identify approved drugs or clinical trial candidates that can be repurposed as AD therapeutics. This represents a viable approach to accelerate drug discovery and do so at a fraction of the cost of traditional high throughput screening.

Background Peri-implantitis is an inflammation that occurs around the implant, resulting in varying degrees of inflammatory damage to the soft and hard tissues. The characteristic criterion is the loss of the supporting bone in an inflammatory environment. However, the specific mechanisms and biomarkers involved in peri-implantitis remain to be further studied. Recently, competing endogenous RNAs (ceRNA) and immune microenvironment have been found to play a more important role in the inflammatory process. In our study, we analyzed the expression of immune related microRNAs (miRNAs), long noncoding RNAs (lncRNAs) and message RNAs (mRNAs) in peri-implantitis by analyzing GSE33774 and GSE57631. Methods In this study, we explored the expression profile data of immune-related lncRNAs, miRNAs and mRNAs, and constructed immune-related ceRNA network involved in the pathogenesis of peri-implantitis. In addition, the CIBERSORT was used to evaluate the content of immune cells in normal tissues and peri-implantitis to detect the immune microenvironment of peri-implantitis. Results In the analysis, 14 DElncRNAs, 16 DEmiRNAs, and 18 DEmRNAs were used to establish an immune related ceRNA network and the immune infiltration patterns associated with peri-implantitis was discovered. Through the mutual verification of the two datasets, we found that GSK3B and miR-1297 may have important significance in the immune microenvironment and pathogenesis of peri-implantitis and GSK3B was closely related to four types of immune cells, especially with the highest correlation with resting mast cells (P = 0.0003). Conclusions Through immune-related ceRNA network, immune-related genes (IRGs) and immune cell infiltration can further comprehensively understand the pathogenesis of peri-implantitis, which built up an immunogenomic landscape with clinical significance for peri-implantitis.

Background: Diabetes is a chronic metabolic disorder affecting the central nervous system. A growing body of evidence has depicted that high glucose level leads to the activation of the transient receptor potential melastatin 2 (TRPM2) channels. However, there are no studies targeting TRPM2 channels in diabetes-induced cognitive decline using a pharmacological approach. Objective: The present study intended to investigate the effects of 2-aminoethoxydiphenyl borate (2-APB), a TRPM2 inhibitor, in diabetes-induced cognitive impairment. Methods: Streptozotocin (STZ, 50 mg/kg, i.p.) was used to induce diabetes in rats. Animals were randomly divided into the treatment group, model group and age-matched control and pre se group. 2-APB treatment was given for three weeks to the animals. After 10 days of behavioural treatment, parameters were performed. Animals were sacrificed at 10th week of diabetic induction and the hippocampus and cortex were isolated. After that, protein and mRNA expression study was performed in the hippocampus. Acetylcholinesterase (AchE) activity was done in the cortex. Results: : Our study showed the 10th week diabetic animals developed cognitive impairment, which was evident from the behavioural parameters. Diabetic animals depicted an increase in the TRPM2 mRNA and protein expression in the hippocampus as well as increased AchE activity in the cortex. However, memory associated proteins were down-regulated, namely Ca2+/calmodulin-dependent protein kinase II (CaMKII-Thr286), glycogen synthase kinase 3 beta (GSK-3β-Ser9), cAMP response element-binding protein (CREB-Ser133), and postsynaptic density protein 95 (PSD-95). Gene expression of parvalbumin, calsequestrin and brain-derived neurotrophic factor (BDNF) were down-regulated while mRNA level of calcineurin A/ protein phosphatase 3 catalytic subunit alpha (PPP3CA) was upregulated in the hippocampus of diabetic animals. A three-week treatment with 2-APB significantly ameliorated the alteration in behavioural cognitive parameters in diabetic rats. Moreover, 2-APB also down-regulated the expression of TRPM2 mRNA and protein in the hippocampus as well as AchE activity in the cortex of diabetic animals as compared to diabetic animals. Moreover, the 2-APB treatment also upregulated the CaMKII (Thr-286), GSK-3β (Ser9), CREB (Ser133), and PSD-95 expression and mRNA levels of parvalbumin, calsequestrin, and BDNF while mRNA level of calcineurin A was down-regulated in the hippocampus of diabetic animals. Conclusion: : This study confirms the ameliorative effect of TRPM2 channel inhibitor in the diabetes- induced cognitive deficits. Inhibition of TRPM2 channels reduced the calcium associated downstream signaling and showed a neuroprotective effect of TRPM2 channels in diabetesinduced cognitive impairment.

Cortical gamma oscillations are believed to be involved in mental processes which are disturbed in schizophrenia. For example, the magnitudes of sensory-evoked oscillations, as measured by auditory steady-state responses (ASSRs) at 40 Hz, are robustly diminished, whereas the baseline gamma power is enhanced in schizophrenia. Such dual gamma oscillation abnormalities are also present in a mouse model of N-methyl-D-aspartate receptor hypofunction (Ppp1r2cre/Grin1 knockout mice). However, it is unclear whether the abnormal gamma oscillations are associated with dysfunction in schizophrenia. We found that glycogen synthase kinase-3 (GSK3) is overactivated in corticolimbic parvalbumin-positive GABAergic interneurons in Grin1 mutant mice. Here we addressed whether GSK3β inhibition reverses both abnormal gamma oscillations and behavioral deficits with high correlation by pharmacological and genetic approach. We demonstrated that the paralog selective-GSK3β inhibitor, but not GSK3α inhibitor, normalizes the diminished ASSRs, excessive baseline gamma power, and deficits in spatial working memory and prepulse inhibition (PPI) of acoustic startle in Grin1 mutant mice. Cell-type specific GSK3B knockdown, but not GSK3A knockdown, also reversed abnormal gamma oscillations and behavioral deficits. Moreover, GSK3B knockdown, but not GSK3A knockdown, reverses the mutants’ in vivo spike synchrony deficits. Finally, ex vivo patch-clamp recording from pairs of neighboring cortical pyramidal neurons showed a reduction of synchronous spontaneous inhibitory-postsynaptic-current events in mutants, which was reversed by GSK3β inhibition genetically and pharmacologically. Together, GSK3β inhibition in corticolimbic interneurons ameliorates the deficits in spatial working memory and PPI, presumably by restoration of synchronous GABA release, synchronous spike firing, and evoked-gamma power increase with lowered baseline power.

Aging is a main risk factor for a number of debilitating diseases and contributes to an increase in mortality. Previous studies have shown that Ginkgo biloba extract (EGb) can prevent and treat aging-related diseases, but its pharmacological effects need to be further clarified. This study aimed to propose a network pharmacology-based method to identify the therapeutic pathways of EGb for aging. The active components of EGb and targets of sample chemicals were obtained from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) database. Information on aging-related genes was obtained from the Human Ageing Genomic Resources database and JenAge Ageing Factor Database. Subsequently, a network containing the interactions between the putative targets of EGb and known therapeutic targets of aging was established, which was used to investigate the pharmacological mechanisms of EGb for aging. A total of 24 active components, 154 targets of active components of EGb, and 308 targets of aging were obtained. Network construction and pathway enrichment were conducted after data integration. The study found that flavonoids (quercetin, luteolin, and kaempferol) and beta-sitosterol may be the main active components of EGb. The top eight candidate targets, namely, PTGS2, PPARG, DPP4, GSK3B, CCNA2, AR, MAPK14, and ESR1, were selected as the main therapeutic targets of EGb. Pathway enrichment results in various pathways were associated with inhibition of oxidative stress, inhibition of inflammation, amelioration of insulin resistance, and regulation of cellular biological processes. Molecular docking results showed that PPARG had better binding capacity with beta-sitosterol, and PTGS2 had better binding capacity with kaempferol and quercetin. The main components of EGb may act on multiple targets, such as PTGS2, PPARG, DPP4, and GSK3B, to regulate multiple pathways, and play an antiaging role by inhibiting oxidative stress, inhibiting inflammation, and ameliorating insulin resistance.

Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal cancers due to frequently late diagnosis and futile treatment. It is a crucial necessity to determine the mechanisms of PDAC. Y-box Binding Protein 1 (YBX1), a highly conserved transcription factor, has been previously reported to play a role in various hallmarks of cancer. We show here that YBX1 is significantly overexpressed in PDAC and correlates with poor prognosis and reduced survival. In PDAC cell lines, YBX1 regulated cell-cycle progression, proliferation, and the expression of glycogen synthase kinase 3 beta (GSK3B) and cell-cycle-related proteins cyclin D1 and E1. Dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays established that YBX1 binds to the promoter of GSK3B, suggesting that YBX1 promotes pancreatic cancer cell growth through induction of GSK3B expression. These findings offer important insights into the mechanisms underlying pathologic proliferation in PDAC., Gou and colleagues found that YBX1 levels were elevated in PDAC and correlated with poor prognosis, and YB-X1 silencing inhibited cell-cycle progression and reduced PDAC cell proliferation. Moreover, YBX1 activated transcription of GSK3B, an effector of PDAC proliferation.

In indirect co-culture system, chondrocytes can induce differentiation of bone marrow mesenchymal stem cells (BMSCs) to chondrocytes without additional inducer. The participation of microRNAs (miRNAs) may take part in the chondrogenic differentiation. Present study aimed to investigate the effect and mechanism of chondrocytes-derived exosomal miRNA in BMSCs chondrogenic differentiation. Our data showed that miR-8485 was the exosomal miRNA derived from chondrocytes and transmitted to BMSCs. Functionally, miR-8485 silence in chondrocytes impaired exosome-induced chondrogenic differentiation of BMSCs. Mechanistically, exosomal miR-8485 targeted GSK3B to repress GSK-3β expression and targeted DACT1 to induce p-GSK-3β (Ser9), activating Wnt/β-catenin pathways. Our study firstly showed that chondrocytes-derived exosomal miR-8485 regulated the Wnt/β-catenin pathways to promote chondrogenic differentiation of BMSCs, providing innovative thoughts for cartilage reconstruction.

Lithium is the smallest monovalent cation with many different biological effects. Although lithium is present in the pharmacotherapy of psychiatric illnesses for decades, its precise mechanism of action is still not clarified. Today lithium represents first-line therapy for bipolar disorders (because it possesses both antimanic and antidepressant properties) and the adjunctive treatment for major depression (due to its antisuicidal effects). Beside, lithium showed some protective effects in neurological diseases including acute neural injury, chronic degenerative conditions, Alzheimer's disease as well as in treating leucopenia, hepatitis and some renal diseases. Recent evidence suggested that lithium also possesses some anticancer properties due to its inhibition of Glycogen Synthase Kinase 3 beta (GSK3β) which is included in the regulation of a lot of important cellular processes such as: glycogen metabolism, inflammation, immunomodulation, apoptosis, tissue injury, regeneration etc. : Although recent evidence suggested a potential utility of lithium in different conditions, its broader use in clinical practice still trails. The reason for this is a narrow therapeutic index of lithium, numerous toxic effects in various organ systems and some clinically relevant interactions with other drugs. Additionally, it is necessary to perform more preclinical as well as clinical studies in order to a precise therapeutic range of lithium, as well as its detailed mechanism of action. The aim of this review is to summarize the current knowledge concerning the pharmacological and toxicological effects of lithium.

Background: Little is known about the pathophysiological diversity of myocardial injury in type 2 diabetes mellitus (T2DM), but analyzing these differences is important for the accurate diagnosis and precise treatment of diabetic cardiomyopathy. This study aimed to elucidate the key cardiac pathophysiological differences in myocardial injury between obese and non-obese T2DM from mice to humans. Methods: Obese and non-obese T2DM mouse models were successfully constructed and observed until systolic dysfunction occurred. Changes in cardiac structure, function, energy metabolism and oxidative stress were assessed by biochemical and pathological tests, echocardiography, free fatty acids (FFAs) uptake fluorescence imaging, transmission electron microscopy, etc. Key molecule changes were screened and verified by RNA sequencing, quantitative real-time polymerase chain reaction and western blotting. Further, 28 human heart samples of healthy population and T2DM patients were collected to observe the cardiac remodeling, energy metabolism and oxidative stress adaptations as measured by pathological and immunohistochemistry tests. Results: Obese T2DM mice exhibited more severe cardiac structure remodeling and earlier systolic dysfunction than non-obese mice. Moreover, obese T2DM mice exhibited severe and persistent myocardial lipotoxicity, mainly manifested by increased FFAs uptake, accumulation of lipid droplets and glycogen, accompanied by continuous activation of the peroxisome proliferator activated receptor alpha (PPARα) pathway and phosphorylated glycogen synthase kinase 3 beta (p-GSK-3β), and sustained inhibition of glucose transport protein 4 (GLUT4) and adipose triglyceride lipase (ATGL), whereas non-obese mice showed no myocardial lipotoxicity characteristics at systolic dysfunction stage, accompanied by the restored PPARα pathway and GLUT4, sustained inhibition of p-GSK-3β and activation of ATGL. Additionally, both obese and non-obese T2DM mice showed significant accumulation of reactive oxygen species (ROS) when systolic dysfunction occurred, but the NF-E2-related factor 2 (Nrf2) pathway was significantly activated in obese mice, while was significantly inhibited in non-obese mice. Furthermore, the key differences found in animals were reliably verified in human samples. Conclusion: Myocardial injury in obese and non-obese T2DM may represent two different types of complications. Obese T2DM individuals, compared to non-obese individuals, are more prone to develop cardiac systolic dysfunction due to severe and persistent myocardial lipotoxicity. Additionally, anti-oxidative dysfunction may be a key factor leading to myocardial injury in non-obese T2DM.

Background Gigantol is a pharmacologically active bibenzyl compound exerting potential anticancer activities. At non-toxic concentrations, it reduces cancer stem cell properties and tumorigenicity. The mechanisms of the effects of gigantol on cancer cell growth are largely unknown. This study aimed to unravel the molecular profile and identify the prominent molecular mechanism of the effects of gigantol in controlling lung cancer cell proliferation. Materials and methods Proteomics and bioinformatics analysis were used accompanied by experimental molecular pharmacology approaches. Results Gigantol exhibited antiproliferative effects on human lung cancer cells confirmed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide proliferation assay and colony growth assay. The protein profile in response to gigantol treatment associated with regulation of cell proliferation was analyzed to determine the prominent protein targets. Among the significant hub proteins, MYC, an important proto-oncogene and proliferation-promoting transcription factor, was down-regulated with the highest number of protein-protein interactions. MYC down-regulation was confirmed by western blot analysis. The up-stream regulator of MYC, Glycogen synthase kinase 3 beta (GSK3β) was found to be responsible for MYC destabilization mediated by gigantol. Gigantol facilitated GSK3β function and resulted in the increase of MYC-ubiquitin complex as evaluated by immunoprecipitation. Conclusion Gigantol was found to inhibit lung cancer proliferation through induction of GSK3β-mediated MYC ubiquitin-proteasome degradation. These data suggest gigantol to be a promising candidate for novel strategy in inhibition of lung cancer.

Both a diet rich in fructose and chronic stress exposure induce metabolic and cardiovascular disturbances. The aim of this study was to examine the effects of the fructose-rich diet and chronic stress, separately and in combination, on insulin signaling and molecules regulating glycogen synthesis and ion transport in the heart, and to reveal whether these effects coincide with changes in glucocorticoid receptor (GR) activation. Male Wistar rats were subjected to 10% fructose in drinking water and/or to chronic unpredictable stress for 9 weeks. Protein expression and/or phosphorylation of the insulin receptor (IR), protein tyrosine phosphatase 1B, insulin receptor substrate 1 (IRS1), protein kinase B (Akt), extracellular signal-regulated kinase 1/2 (ERK1/2), glycogen synthase kinase-3β (GSK-3β) and Na+/K+-ATPase α-subunits in cardiac tissue were analyzed by western blot. GR distribution between cytosolic and nuclear fractions was also analyzed. The fructose-rich diet decreased the level of pERK1/2 (Thr202/Tyr204) and pGSK-3β (Ser9) independently of stress, while chronic stress increased the IRS1 content and prevented the fructose diet-induced decrease of the pAkt (Ser473) level. The fructose-rich diet in combination with chronic stress reduced the protein content of cardiac IR and attenuated IRS1 upregulation. Separate treatments increased the protein content of Na+/K+-ATPase α1- and α2-subunits, while after combined treatment the α2 content was at the control level and the α1 content was lower than the control level. The effect of combined treatment on cardiac IR and α2-subunit expression could be mediated by increased GR nuclear accumulation. Our study provides new insights into the effects of chronic stress and a combination of the fructose diet and chronic stress on the studied molecules in the heart.

Introduction: Head and neck squamous cell cancer (HNSCC) is a common cancer worldwide. It has been associated with TP53 mutation and chronic inflammation. The control genes of inflammation, Tumor Necrosis Factor Receptor Superfamily (TNFRSF) in HNSCC has not been widely reported. The impact of the TNFRSF and survival and cell death regulation signalling (SCDRS) can be studied at protein, gene, mRNA and transcription level. In this manuscript, the association of mRNA of TNFRSF and SCDRS genes in treatment naive HNSCC with TP53 mutation is studied. Materials and Methods: TP53 mutation, tobacco use and mRNA levels of TNFRSF and SCDRS genes of 520 HNSCC cases were collated and analysed. Statistical and differential expression (DE) analysis was performed. Results: A total of 12 genes of the 51 genes studied were DE between TP53 subgroups. They were SCDRS genes (BAD, CASP9, GSK3B, NFKB2, TGFBR1, TGFBR2) and TNFRSF genes (TNFRSF10A/11B/14/25/6B/9). The network analysis and subsequent KEGG pathway analysis identified several key pathways including vital cancer pathways and transcriptional pathways in cancer. The key genes in the network that modulate TNFRSF and SCDRS mRNA expression in wild and mutant TP53 situation are presented. Conclusion: The present work identified certain key TNFRSF and SCDRS mRNAs that could differ based on TP53 status and count with tobacco use. Also this study identified certain pathways where the gene network could potentially alter the HNSCC progression, treatment response and prognosis. This adds to our knowledge of TP53 and inflammation in HNSCC carcinogenesis.

Visual Abstract, Highlights • The reactive oxygen species–generating enzyme Nox2 is up-regulated in the leaflets of both rabbit and human with CAVD. • Nox2 is markedly induced in cultured porcine AVICs after osteogenic stimulation. Knockdown of endogenous Nox2 substantially suppressed AVIC calcification. • Celastrol, a natural compound capable of inhibiting Nox2 activity, significantly decreased AVIC calcification in vitro, and mitigated the severity of aortic valve fibrosis, calcification, and stenosis in a rabbit model of CAVD in vivo. • The protective effects of celastrol may, in part, involve the inhibition of Nox2-mediated glycogen synthase kinase 3 beta/β-catenin pathway., Summary This study sought to investigate whether reactive oxygen species (ROS)–generating reduced nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2) contributes to calcific aortic valve disease (CAVD) or whether celastrol, a natural Nox2 inhibitor, may provide potential therapeutic target for CAVD. CAVD is an active and cellular-driven fibrocalcific process characterized by differentiation of aortic valvular interstitial cells (AVICs) toward an osteogenic-like phenotype. ROS levels increase in calcified aortic valves, while the sources of ROS and their roles in the pathogenesis of CAVD are elusive. The roles of Nox2 and the effects of celastrol were studied using cultured porcine AVICs in vitro and a rabbit CAVD model in vivo. Nox2 proteins were significantly upregulated in human aortic valves with CAVD. In vitro, Nox2 was markedly induced upon stimulation of AVICs with osteogenic medium, along with the increases in ROS production and calcium nodule formation. Celastrol significantly decreased calcium deposition of AVICs by 35%, with a reduction of ROS generation. Knockdown of endogenous Nox2 substantially suppressed AVIC calcification by 39%, the inhibitory effect being similar to celastrol treatment. Mechanistically, either celastrol treatment or knockdown of Nox2 significantly inhibited glycogen synthase kinase 3 beta/β-catenin signaling, leading to attenuation of fibrogenic and osteogenic responses of AVICs. In a rabbit CAVD model, administration of celastrol significantly reduced aortic valve ROS production, fibrosis, calcification, and severity of aortic stenosis, with less left ventricular dilatation and better preserved contractile function. Upregulation of Nox2 is critically involved in CAVD. Celastrol is effective to alleviate CAVD, likely through the inhibition of Nox2-mediated glycogen synthase kinase 3 beta/β-catenin pathway in AVICs.

Background/Aim: Glycogen synthase kinase 3 beta (GSK3-β) acts either as a tumor suppressor or an oncogene in various human cancers. The present study aimed to investigate the expression and activity of p-GSK3-β (Ser9) in oral cancer patients. Materials and Methods: We investigated the levels of p-GSK3β in 152 oral cancer tissues by immunohistochemistry, and explored their prognostic impact. Results: To investigate the role of p-GSK3β (Ser9) in OSCC progression, we first analyzed the expression levels of protein p-GSK3β in normal and oral cancer tissues using immunohistochemical staining. p-GSK3β immunostaining was detected in 32 of 152 (21.1%) oral cancer specimens. High p-GSK3β expression was significantly associated with T (III/IV) stage. Kaplan-Meier survival analysis revealed that high levels of p-GSK3β were correlated with poor survival (p=0.001) in T stage (III/IV) OSCC patients. Multivariate analyses indicated that TN stage, AJCC tumor stage, tumor differentiation status and clinical therapy, but not p-GSK3β levels, were independent prognostic factors. Significant mortality risk was found in T stage (III/IV) oral cancer patients with high levels of p-GSK3β (p=0.0006). Conclusion: GSK3β inactivation is a key event in oral cancer patients and targeting GSK3β might be valuable in treating oral cancer patients.

Significance COVID-19 is taking a major toll on personal health, healthcare systems, and the global economy. With three betacoronavirus epidemics in less than 20 y, there is an urgent need for therapies to combat new and existing coronavirus outbreaks. Our analysis of clinical data from over 300,000 patients in three major health systems demonstrates a 50% reduced risk of COVID-19 in patients taking lithium, a direct inhibitor of glycogen synthase kinase-3 (GSK-3). We further show that GSK-3 is essential for phosphorylation of the SARS-CoV-2 nucleocapsid protein and that GSK-3 inhibition blocks SARS-CoV-2 infection in human lung epithelial cells. These findings suggest an antiviral strategy for COVID-19 and new coronaviruses that may arise in the future., The coronaviruses responsible for severe acute respiratory syndrome (SARS-CoV), COVID-19 (SARS-CoV-2), Middle East respiratory syndrome-CoV, and other coronavirus infections express a nucleocapsid protein (N) that is essential for viral replication, transcription, and virion assembly. Phosphorylation of N from SARS-CoV by glycogen synthase kinase 3 (GSK-3) is required for its function and inhibition of GSK-3 with lithium impairs N phosphorylation, viral transcription, and replication. Here we report that the SARS-CoV-2 N protein contains GSK-3 consensus sequences and that this motif is conserved in diverse coronaviruses, raising the possibility that SARS-CoV-2 may be sensitive to GSK-3 inhibitors, including lithium. We conducted a retrospective analysis of lithium use in patients from three major health systems who were PCR-tested for SARS-CoV-2. We found that patients taking lithium have a significantly reduced risk of COVID-19 (odds ratio = 0.51 [0.35–0.74], P = 0.005). We also show that the SARS-CoV-2 N protein is phosphorylated by GSK-3. Knockout of GSK3A and GSK3B demonstrates that GSK-3 is essential for N phosphorylation. Alternative GSK-3 inhibitors block N phosphorylation and impair replication in SARS-CoV-2 infected lung epithelial cells in a cell-type–dependent manner. Targeting GSK-3 may therefore provide an approach to treat COVID-19 and future coronavirus outbreaks.

Diabetic foot ulcers are seriously endangering the physical and mental health of patients. Due to the long duration of inflammation, the treatment of nonhealing wounds in diabetes is one of the most prominent healthcare problems in the world. The nuclear factor kappa B (NF-κB) signaling pathway, a classical pathway that triggers inflammatory response, is regulated by many regulators, such as glycogen synthase kinase 3 beta (GSK-3β). Noncoding RNAs, a large class of molecules that regulate gene expression at the posttranscriptional or posttranslational level, play an important role in various stages of wound healing, especially in the stage of inflammation. Herein, we summarized the roles of noncoding RNAs in the NF-κB/GSK-3β signaling, which might provide new ideas for the treatment of diabetic wound healing.

Light pollution has become a potential health risk factor worldwide. Chronic exposure to constant light (CCL) leads to depressive-like behavior, yet the mechanism remains unclear. In this study, mice exposed to CCL for 3 weeks exhibited depression-like behaviors, with decreased melatonin in plasma and increased oxidative stress in hippocampus. Meanwhile, CCL-exposed mice showed elevated plasma corticosterone (CORT) levels and diminished glucocorticoid receptor (GR) phosphorylation in hippocampus. Concurrently, glycogen synthase kinase 3 beta (GSK3β) was inactivated with increased phosphorylation at Ser9. The interrelationship of GSK3β and GR was clarified in mouse hippocampal neuron (HT-22) cells. GSK3β inhibitor CHIR-99021 induced GR inhibition with diminished phosphorylation, while GR inhibitor RU486 did not affect GSK3β expression or phosphorylation. Furthermore, GSK3β-mediated GR inhibition was reproduced in vitro in HT-22 cells treated with melatonin receptor antagonist luzindole and H2O2 in combination. Finally, melatonin reversed GSK3β-mediated GR inhibition in hippocampus and improved CCL-induced depression-like behavior in mice. These results indicate that CCL induces melatonin deficiency and oxidative stress in hippocampus, which in turn leads to GSK3β-mediated GR inhibition and depression-like behavior in mice.

The aim of this study was to investigate the effects of the GSK-3β/NF-κB pathway on integrin-associated protein (CD47) expression after myocardial infarction (MI) in rats. An MI Sprague Dawley rat model was established by ligating the left anterior descending coronary artery. The rats were divided into three groups: Sham, MI, and SB + MI (SB216763) groups. Immunohistochemistry was used to observe the changes in cardiac morphology. A significant reduction in the sizes of fibrotic scars was observed in the SB + MI group compared to that in the MI group. SB216763 decreased the mRNA and protein expression of CD47 and NF-κB during MI. Primary rat cardiomyocytes (RCMs) and the H9c2 cell line were used to establish in vitro hypoxia models. Quantitative real-time PCR and western blotting analyses were conducted to detect mRNA and protein expression levels of CD47 and NF-κB and apoptosis-related proteins, respectively. Apoptosis of hypoxic cells was assessed using flow cytometry. SB216763 reduced the protein expression of CD47 and NF-κB in RCMs and H9c2 cells under hypoxic conditions for 12 h, and alleviated hypoxia-induced apoptosis. SN50 (an NF-κB inhibitor) also decreased CD47 protein expression in RCMs and H9c2 cells under hypoxic conditions for 12 h and protected cells from apoptosis. GSK-3β upregulates CD47 expression in cardiac tissues after MI by activating NF-κB, which in turn leads to myocardial cell damage and apoptosis.

Objectives: The aim of the present work is evaluating the special effects of Urtica Dioica and Lamium Album on the serum level of K-Ras and GSK-3 beta in diabetic rats. Methods: In the present experimental study, 32 male Wistar rats randomly divided into 4 groups (Group I: normal control rats; receiving daily PBS, Group 2: diabetic control rats; receiving single dose of streptozotocin (60 mg/kg) and daily PBS, Group 3: Diabetic rats treated with 100 mg/kg of hydroalcoholic extract of the U. dioi ca , Group 4: Diabetic rats treated with 100 mg/kg of hydroalcoholic extract of L. Album. Diabetes-induced by an intraperitoneal injection of streptozotocin (60 mg/ kg). On the 14 th day of treatment, the weight, fasting blood sugar (FBS) and on 28 th day blood glucose, K-Ras and GSK3 beta was measured. Results: In diabetic group blood GSK- 3 beta increase in comparison to control group (P < 0.05), also blood K-Ras decrease in the diabetic group (P < 0.05). Both extracts reduced GSK-3 beta level, however, this reduction was only statistically significant by U.dioica (P < 0.05). Compared to diabetic group, blood K-Ras level increased by both extract (P < 0.05). Also diabetes induction increase blood glucose levels and both extracts decrease its level significantly (P < 0.05).there is no significant differences among both extract effects on blood glucose, and K-Ras. Conclusion: For the first time shown that both extracts by regulating GSK-3 beta and K-Ras improve blood glucose level. More studies are needed to determine all the effects of these herbs.

Sepsis is a systemic inflammatory response syndrome resulting from infection. This study aimed at exploring the role of microRNA-140 (miR-140) in septic mice. Wnt family member 11 (WNT11) was verified to be a target gene of miR-140 after bioinformatic prediction and dual luciferase reporter gene assay. Importantly, miR-140 negatively regulated WNT11. We initially induced the model of sepsis by endotoxin, and then ectopic expression and knockdown experiments were performed to explore the functional role of miR-140 in sepsis. Additionally, cross-sectional areas of muscle fiber, lactic acid production, 3-methylhistidine (3-MH) and tyrosine (Tyr) production in extensor digitorium longus (EDL) muscles, and serum levels of inflammatory factors were examined. The effect of miR-140 on the expression of WNT signaling pathway-related and apoptosis-related factors in skeletal muscle tissue was determined. The experimental results indicated that upregulated miR-140 or silenced WNT11 increased cross-sectional areas of muscle fiber while decreasing lactic acid production, skeletal muscle cell apoptosis [corresponding to downregulated B cell lymphoma 2 (Bcl-2)-associated X protein (Bax) and caspase-3 and upregulated Bcl-2], and the proteolytic rate of Tyr and 3-MH. Also, overexpressed miR-140 or silenced WNT11 reduced inflammation as reflected by decreased serum levels of IL-6, IL-10, and TNF-α. Furthermore, overexpression of miR-140 was shown to suppress the activation of the WNT signaling pathway, accompanied by decreased expression of WNT11, β-catenin, and GSK-3β. Taken together, upregulation of miR-140 could potentially inhibit skeletal muscle lactate release, an indirect measure of glycolysis, and atrophy in septic mice through suppressing the WNT signaling pathway via inhibiting WNT11 expression.

Alzheimer's disease (AD) is a multifactorial syndrome resulting in profound misery and poses a substantial burden on human health, economy, and society throughout the world. Based on the numerous AD-related targets in the disease network, multi-target design strategy is a crucial direction to seek for enhanced therapy, and multi-target drugs have the ability to regulate more targets than single-target drugs, affecting the disease network with more potency. Herein, we highlight nine major targets associated with AD, which are acetylcholine esterase (AChE), beta-site amyloid precursor protein cleaving enzyme 1 (?-secretase, BACE-1), glycogen synthase kinase 3 beta (GSK-3?), monoamine oxidases (MAOs), metal ions in the brain, N-methyl-D-aspartate (NMDA) receptor, 5-hydroxytryptamine (5-HT) receptors, the third subtype of histamine receptor (H3 receptor), and phosphodiesterases (PDEs), and their respective relationship to the disease network. Furthermore, eleven multi-target design strategies classified by the involvement of AChE and related promising compounds for improved therapy of AD in recent years are described based on the nine major targets.

Transplanted endothelial progenitor cells (EPCs) repair blood vessels and exert regenerative effects on disorders such as lower limb ischemia. EPCs serve as a model for pathophysiological and pharmacokinetic studies, which is important for drug discovery. However, primary human EPCs are phenotypically unstable, which limits their clinical utility. Therefore, we employed human induced pluripotent stem (iPS) cells to circumvent this problem. Here we focused on human iPS cell-derived sac-like structures (iPS-sacs), which contain endothelial lineage cells and hematopoietic lineage cells. Previous studies isolated only hematopoietic lineage cells from iPS-sacs. Therefore, here we attempted to isolate EPCs. However, iPS-sacs generated by a published protocol did not contain sufficient EPCs. Therefore, to generate iPS-sacs highly enriched in EPCs, we added the glycogen synthase kinase 3 beta (GSK3β) inhibitor CHIR-99021 to the culture medium early during differentiation. The cells rapidly differentiated into mesoderm to yield abundant EPCs, and CHIR-99021 increased the proportion of EPCs contained in iPS-sacs. EPCs, which were purified using anti-platelet endothelial cell adhesion molecule (PECAM1) antibody-conjugated beads, expressed markers of immature endothelial cells. Purified EPCs formed tube-like structures and incorporated acetylated low density lipoprotein (Ac-LDL), reflecting endothelial phenotypes. The simple method described here will likely improve regenerative medicine and facilitate basic studies on the endothelial lineage.

Connexin43 is an important modulator of many signaling pathways in bone. β-Catenin, a key regulator of the osteoblast differentiation and function, is among the pathways downstream of connexin43-dependent intercellular communication. There are striking overlaps between the functions of these two proteins in bone cells. However, differential effects of connexin43 on β-catenin activity have been reported. Here, we examined how connexin43 influenced both Wnt-dependent and Wnt-independent activation of β-catenin in osteoblasts in vitro. Our data show that loss of connexin43 in primary osteoblasts or connexin43 overexpression in UMR106 cells regulated active β-catenin and phospho-Akt levels, with loss of connexin43 inhibiting and connexin43 overexpression increasing the levels of active β-catenin and phospho-Akt. Increasing connexin43 expression synergistically enhanced Wnt3a-dependent activation of β-catenin protein and β-catenin transcriptional activity, as well as Wnt-independent activation of β-catenin by prostaglandin E2 (PGE2). Finally, we show that the activation of β-catenin by PGE2 required signaling through the phosphatidylinositol 3-kinase (PI3K)/Akt/glycogen synthase kinase 3 beta (GSK3β) pathway, as the PI3K inhibitor, LY-294002, disrupted the synergy between connexin43 and PGE2. These data show that connexin43 regulates Akt and β-catenin activity and synergistically enhances both Wnt-dependent and Wnt-independent β-catenin signaling in osteoblasts.

Background Stress-induced gastric ulcer (SGU) is one of the most common visceral complications after trauma. Restraint water-immersion stress (RWIS) can cause serious gastrointestinal dysfunction and has been widely used to study the pathogenesis of SGU to identify medications that can cure the disease. The mediodorsal thalamic nucleus (MD) is the centre integrating visceral and physical activity and contributes to SGU induced by RWIS. Hence, the role of the MD during RWIS needs to be studied. Aim To screen for differentially expressed proteins in the MD of the RWIS rats to further elucidate molecular mechanisms of SGU. Methods Male Wistar rats were selected randomly and divided into two groups, namely, a control group and an RWIS group. Gastric mucosal lesions of the sacrificed rats were measured using the erosion index and the proteomic profiles of the MD were generated through isobaric tags for relative and absolute quantitation (iTRAQ) coupled with two-dimensional liquid chromatography and tandem mass spectrometry. Additionally, iTRAQ results were verified by Western blot analysis. Results A total of 2853 proteins were identified, and these included 65 dysregulated (31 upregulated and 34 downregulated) proteins (fold change ratio ≥ 1.2). Gene Ontology (GO) analysis showed that most of the upregulated proteins are primarily related to cell division, whereas most of the downregulated proteins are related to neuron morphogenesis and neurotransmitter regulation. Ingenuity Pathway Analysis revealed that the dysregulated proteins are mainly involved in the neurological disease signalling pathways. Furthermore, our results indicated that glycogen synthase kinase-3 beta might be related to the central mechanism through which RWIS gives rise to SGU. Conclusion Quantitative proteomic analysis elucidated the molecular targets associated with the production of SGU and provides insights into the role of the MD. The underlying molecular mechanisms need to be further dissected.

Dysregulated microRNAs (miRNAs) play crucial roles in the regulation of cancer stem cells (CSCs), and CSCs are closely associated with tumor initiation, metastasis, and recurrence. Here we found that miR-150-5p was significantly downregulated in CSCs of non-small-cell lung cancer (NSCLC) and its expression level was negatively correlated with disease progression and poor survival in patients with NSCLC. Inhibition of miR-150-5p increased the CSC population and sphere formation of NSCLC cells in vitro and stimulated NSCLC cell tumorigenicity and metastatic colonization in vivo. In contrast, miR-150-5p overexpression potently inhibited sphere-formed NSCLC cell tumor formation, metastatic colonization, and recurrence in xenograft models. Furthermore, we identified that miR-150-5p significantly inhibited wingless (Wnt)-β-catenin signaling by simultaneously targeting glycogen synthase kinase 3 beta interacting protein (GSKIP) and β-catenin in NSCLC cells. miR-150-5p also targeted high mobility group AT-hook 2 (HMGA2), another regulator of CSCs, and Wnt-β-catenin signaling. The restoration of HMGA2 and β-catenin blocked miR-150-5p overexpression-induced inhibition of CSC traits in NSCLC cells. These findings suggest that miR-150-5p functions as a CSC suppressor and that overexpression of miR-150-5p may be a novel strategy to inhibit CSC-induced metastasis and recurrence in NSCLC. Keywords: miR-150-5p, cancer stem cell, metastasis, recurrence, NSCLC

Maternal separation causes depression and anxiety. Exercise ameliorates maternal separation-induced depression. In this study, we investigated the effect of treadmill exercise on anxiety-like behavior in relation with glycogen synthase kinase 3 beta (GSK3β)/β-catenin pathway using maternal separation rat pups. For this study, elevated plus maze test, immunohistochemistry for serotonin (5-hydroxytryptamine, 5-HT), tryptophan hydroxylase (TPH), and western blot for total GSK3β (t-GSK3β), phosphorylated GSK3β (p-GSK3β), total β-catenin (t-β-catenin), and phosphorylated β-catenin (p-β-catenin) were conducted. The rat pups in the exercise groups were scheduled to run on a treadmill for 30 min once a day for 10 days, starting on postnatal day 21. For the rat pups in the fluoxetine-treated group, fluoxetine was orally administrated once a day for 10 consecutive days, starting on postnatal day 21. Anxiety-like behavior was appeared in the rat pups by maternal separation. Maternal separation suppressed 5-HT and TPH expression in the dorsal raphe. Maternal separation suppressed phosphorylation of GSK3β and increased phosphorylation of β-catenin in the hippocampus. However, treadmill exercise and fluoxetine treatment alleviated anxiety and increased 5-HT and TPH expression in the dorsal raphe. Treadmill exercise and fluoxetine treatment also enhanced GSK3β phosphorylation and suppressed β-catenin phosphorylation in the hippocampus. In this study, alleviating effect of treadmill exercise on maternal separation-induced anxiety appeared through enhancing 5-HT expression and GSK3β phosphorylation, and then inhibiting β-catenin phosphorylation. These results showed that treadmill exercise relieves anxiety through GSK3β/β-catenin pathway. Treadmill exercise showed similar ameliorating effect on anxiety-like behavior as fluoxetine.

GSK3B has been an interesting drug target in the pharmaceutical industry. Its dysfunctional expression has prognostic significance in the top 3 cause of death associated with non-communicable diseases (cancer, Alzheimer's disease and type 2 diabetes). Previous studies have shown clearly that inhibiting GSK3B has proven therapeutic significance in Alzheimer's disease, but its contribution to various cancers has not been clearly resolved. In this study we report the contribution and prognostic significance of GSK3B to two breast cancer subtypes; ductal carcinoma in-situ (DCIS) and invasive ductal carcinoma (IDC) using the Oncomine platform. We performed high throughput screening using molecular docking. We identified BT-000775, a compound that was subjected to further computational hit optimization protocols. Through computational predictions, BT-000775 is a highly selective GSK3B inhibitor, with superior binding affinity and robust ADME profiles suitable for the patho-physiological presentations.

Many cellular processes in cancer are attributed to kinase signaling networks. V-akt murine thymoma viral oncogene homolog (AKT) plays a major role in the PI3K/AKT signaling pathways. AKT is activated by PI3K or phosphoinositide-dependent kinases (PDK) as well as growth factors, inflammation, and DNA damage. Signal transduction occurs through downstream effectors such as mTOR, glycogen synthase kinase 3 beta (GSK3β), or forkhead box protein O1 (FOXO1). The abnormal overexpression or activation of AKT has been observed in many cancers, including ovarian, lung, and pancreatic cancers, and is associated with increased cancer cell proliferation and survival. Therefore, targeting AKT could provide an important approach for cancer prevention and therapy. In this review, we discuss the rationale for targeting AKT and also provide details regarding synthetic and natural AKT-targeting compounds and their associated studies.