Tau phosphorylation by glycogen synthase kinase 3ß modulates enzyme acetylcholinesterase expression.

In Alzheimer's disease (AD), the enzyme acetylcholinesterase (AChE) co-localizes with hyperphosphorylated tau (P-tau) within neurofibrillary tangles. Having demonstrated that AChE expression is increased in the transgenic mouse model of tau Tg-VLW, here we examined whether modulating phosphorylated tau levels by over-expressing wild-type human tau and glycogen synthase kinase-3ß (GSK3ß) influences AChE expression. In SH-SY5Y neuroblastoma cells expressing higher levels of P-tau, AChE activity and protein increased by (20% ± 2%) and (440% ± 150%), respectively. Western blots and qPCR assays showed that this increment mostly corresponded to the cholinergic ACHE-T variant, for which the protein and transcript levels increased ~60% and ~23%, respectively. Moreover, in SH-SY5Y cells differentiated into neurons by exposure to retinoic acid (10 µM), over-expression of GSK3ß and tau provokes an imbalance in cholinergic activity with a decrease in the neurotransmitter acetylcholine in the cell (45 ± 10%). Finally, we obtained cerebrospinal fluid (CSF) from AD patients enrolled on a clinical trial of tideglusib, an irreversible GSK3ß inhibitor. In CSF of patients that received a placebo, there was an increase in AChE activity (35 ± 16%) respect to basal levels, probably because of their treatment with AChE inhibitors. However, this increase was not observed in tideglusib-treated patients. Moreover, CSF levels of P-tau at the beginning measured by commercially ELISA kits correlated with AChE activity. In conclusion, this study shows that P-tau can modulate AChE expression and it suggests that AChE may possibly increase in the initial phases of AD.

Thiadiazolidinone-8 Ameliorates Inflammation Associated with Experimental Colitis in Mice.

Thiadiazolidinone-8 (TDZD-8) is an effective thiadiazolidinone derivate that is able to suppress the expression of inflammatory cytokines; it also presents tissue protective actions by glycogen synthase kinase (GSK)-3ß inhibition, promoting thus an anti-inflammatory effect. Since inflammatory bowel disease is a chronic disease with reduced quality of life, where currently available therapies are only able to induce or maintain the patient in remission, it is crucial to investigate new pharmacological approaches. The main objective of this study was to evaluate the effect of TDZD-8 in 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis. Male CD-1 mice with TNBS-induced colitis were treated with a daily dose of TDZD-8 5 mg/kg/day IP during 4 days. The anti-inflammatory properties of TDZD-8 in the TNBS-induced colitis were confirmed by suppression of pro-inflammatory mediators, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and myeloperoxidase, as well as by the significant increase of the anti-inflammatory cytokine, IL-10. These treated mice also presented a reduction in fecal hemoglobin and alkaline phosphatase, suggesting a beneficial effect of TDZD-8. Furthermore, renal and hepatic biomarkers remained stabilized after treatment. In conclusion, TDZD-8 reduces the inflammatory response associated with TNBS-induced colitis in mice, and modulation of GSK-3ß seems to be an interesting pharmacological target in colitis.

AR-A014418, a glycogen synthase kinase-3ß inhibitor, mitigates lipopolysaccharide-induced inflammation in rat dental pulp stem cells via NLR family pyrin domain containing 3 inflammasome impairment.

Background/purpose: Cell pyroptosis and gingival inflammation have been implicated in periodontitis progression. Our previous study revealed that AR-A014418, a pharmacological inhibitor of glycogen synthase kinase-3ß (GSK-3ß), can enhance the migratory and osteogenic differentiation abilities of rat dental pulp stem cells (rDPSCs). The present study aimed to explore the effect of AR on the inflammation of rDPSCs. Materials and methods: The primary rDPSCs were isolated and identified by flow cytometry, as well as Oil red O and Alizarin Red S staining. The rDPSCs were cultured and exposed to lipopolysaccharide (LPS) before treating them with different concentrations of AR-A014418. The cell viability was detected using the CCK-8 assay. The generation and secretion of pro-inflammatory cytokines (IL-18, TNF-α, L-1ß, and IL-6) were examined by qPCR and ELISA, respectively. To investigate the activation of the NLRP3 inflammasome, the expression levels of pro-caspase 1, cleaved caspase 1, as well as NLRP3 were analyzed by western blotting and immunofluorescence, respectively. Results: In the rDPSCs, LPS prohibited cell viability and enhanced the generation and secretion of pro-inflammatory cytokines. LPS upregulated NLRP3 and cleaved caspase-1 protein levels and promoted ASC speck formation in the rDPSCs. AR-A014418 administration effectively blocked the LPS-induced inflammation of the rDPSCs in a dose-dependent way. Mechanistically, AR-A014418 significantly restrained the up-regulation of NLRP3 and cleaved caspase-1 in LPS-treated rDPSCs. Conclusion: Collectively, our findings suggest that AR-A014418 significantly mitigates LPS-induced inflammation of rDPSCs by blocking the activation of the NLRP3 inflammasome.

Glycogen synthase kinase-3ß inhibitor promotes the migration and osteogenic differentiation of rat dental pulp stem cells via the ß-catenin/PI3K/Akt signaling pathway.

Background/purpose: Glycogen synthase kinase-3ß (GSK3ß) inhibitor enhances bone formation, while dental pulp stem cells (DPSC) are potentially used to repair bone defects. The present study aimed to investigate the effect of AR-A014418 (AR, a specific glycogen synthase kinase-3ß inhibitor) on the migration and osteogenic differentiation of rat-derived dental pulp stem cells (rDPSCs), and further explore the underlying mechanism. Materials and methods: rDPSCs were isolated from rats, and then cultured with different concentrations of AR with or without LY294002 (a PI3K inhibitor). Then, cell viability, migration, osteogenic differentiation, and the involvement of PI3K pathway were detected by CCK-8 assay, Transwell assay, Alizarin Red S Staining, Alkaline phosphatase (ALP) assay, Western blot, and RT-PCR, respectively. Results: Our present study demonstrated that AR of various concentrations (1 µM, 2.5 µM, and 5 µM) not only promoted the rDPSC proliferation and migration, but also increased calcium deposition, the activity of alkaline phosphatase (ALP), and levels of osteogenic markers (RUNX2, OPN, OCN, and OSX) in rDPSCs. It was also found that the administration of AR resulted in an increase in the expression level of p-GSK3ß (Ser), ß-catenin, p-PI3K, and p-Akt, and a reduction in p-GSK3ß (Tyr216). Furthermore, PI3K inhibitor LY294002 abrogated the enhanced cell migration and osteogenic differentiation of rDPSCs induced by AR. Conclusion: Our results provide evidence that AR significantly promotes migration and osteogenic differentiation of rDPSCs by activating ß-catenin/PI3K/Akt signaling pathway.

Immobilization of Glycogen Synthase Kinase-3ß Inhibitor on 316L Stainless Steel via Polydopamine to Accelerate Endothelialization.

The coverage of stents with healthy endothelium is crucial to the success of cardiovascular stent implantation. Immobilizing bioactive molecules on stents is an effective strategy to generate such stents. Glycogen synthase kinase-3ß inhibitor (GSKi) is a bioactive molecule that can effectively accelerate vascular endothelialization. In this work, GSKi was covalently conjugated on 316L stainless steel through polydopamine to develop a stable bioactive surface. Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and water contact angle results revealed the successful introduction of GSKi onto 316L stainless steel. The GSKi coating did not obviously affect the hemocompatibility of plates. The adhesion and proliferation of human coronary artery endothelial cells (HCAECs) on stainless steel was significantly promoted by the addition of GSKi. In summary, this work provides a universal and stable strategy of immobilizing GSKi on the stent surface. This method has the potential for widespread application in the modification of vascular stents.