Protective Effects of Repetitive Transcranial Magnetic Stimulation Against Streptozotocin-Induced Alzheimer's Disease.

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive brain stimulation under investigation for treatment of a wide range of neurological disorders. In particular, the therapeutic application of rTMS for neurodegenerative diseases such as Alzheimer's disease (AD) is attracting attention. However, the mechanisms underlying the therapeutic efficacy of rTMS have not yet been elucidated, and few studies have systematically analyzed the stimulation parameters. In this study, we found that treatment with rTMS contributed to restoration of memory deficits by activating genes involved in synaptic plasticity and long-term memory. We evaluated changes in several intracellular signaling pathways in response to rTMS stimulation; rTMS treatment activated STAT, MAPK, Akt/p70S6K, and CREB signaling. We also systematically investigated the influence of rTMS parameters. We found an effective range of applications for rTMS and determined the optimal combination to achieve the highest efficiency. Moreover, application of rTMS inhibited the increase in cell death induced by hydrogen peroxide. These results suggest that rTMS treatment exerts a neuroprotective effect on cellular damage induced by oxidative stress, which plays an important role in the pathogenesis of neurological disorders. rTMS treatment attenuated streptozotocin (STZ)-mediated cell death and AD-like pathology in neuronal cells. In an animal model of sporadic AD caused by intracerebroventricular STZ injection, rTMS application improved cognitive decline and showed neuroprotective effects on hippocampal histology. Overall, this study will help in the design of stimulation protocols for rTMS application and presents a novel mechanism that may explain the therapeutic effects of rTMS in neurodegenerative diseases, including AD.

Anti-inflammatory effects of Allium cepa L. peel extracts via inhibition of JAK-STAT pathway in LPS-stimulated RAW264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Allium cepa L. (A. cepa) is one of the oldest cultivated plants in the world. A. cepa has been used in traditional folk medicine to treat inflammatory disease in several regions, such as Palestine and Serbia. A. cepa peel has a higher content of flavonoids, such as quercetin, than the edible parts. These flavonoids alleviate inflammatory diseases. However, the anti-inflammatory effects of A. cepa peel extract-obtained using various extraction methods-and their underlying mechanisms require further investigation. AIM OF THE STUDY: Although research to find safe anti-inflammatory substances in various natural products has been actively conducted for many years, it is important to continue identifying potential anti-inflammatory effects in natural materials. The purpose of this study was to investigate the ethnopharmacological properties of the A. cepa peel extract, whose efficacy when obtained through different extraction methods and underlying action mechanisms is not well known. The present study specifically aimed to observe the anti-inflammatory effects of the A. cepa peel extracts obtained using various extraction methods and the related detailed mechanisms of A. cepa peel extracts in lipopolysaccharide (LPS)-induced RAW264.7 cells. MATERIALS AND METHODS: The total flavonoid content of the A. cepa peel extracts was determined the diethylene glycol colorimetric method and measured using a calibration curve prepared using quercetin as a standard solution. The antioxidant activity was evaluated using the ABTS assay, and cytotoxicity was measured using the MTT assay. NO production was measured using Griess reagent. Protein levels were measured by western blotting, and mRNA expression was measured by RT-qPCR. Secreted cytokines were analyzed using ELISA or cytokine arrays. In the GSE160086 dataset, we calculated Z-scores for individual genes of interest and displayed using a heat map. RESULTS: Of the three A. cepa peel extracts obtained using different extraction methods, the A. cepa peel 50% EtOH extract (AP50E) was the most effective at inhibiting LPS-induced nitric oxide (NO) and inducible nitric oxide synthase (iNOS). Furthermore, AP50E significantly reduced the levels of pro-inflammation cytokines interleukin (IL)-1α, IL-1ß, IL-6, and IL-27. Additionally, AP50E directly inhibited the Janus kinase-signaling transducer and activator of transcription (JAK-STAT) pathway. CONCLUSIONS: These results showed that AP50E exhibited an anti-inflammatory effect in LPS-induced RAW264.7 mouse macrophages by directly inhibiting JAK-STAT signaling. Based on these findings, we propose AP50E as a potential candidate for the development of preventive or therapeutic agents against inflammatory diseases.

Pharmacological anti-tumor effects of natural Chamaecyparis obtusa (siebold & zucc.) endl. Leaf extracts on breast cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Chamaecyparis obtusa (C. obtusa, cypress species) is a plant that grows mainly in the temperate Northern Hemisphere and has long been used as a traditional anti-inflammatory treatment in East Asia. C. obtusa contains phytoncides, flavonoids, and terpenes, which have excellent anti-cancer effects and have been reported to prevent the progression of various cancers. However, the detailed mechanisms underlying the anti-cancer effects of C. obtusa extracts are unknown. AIM OF THE STUDY: We sought to confirm the anti-cancer effects of C. obtusa leaf extracts and to reveal the mechanism of action, with the possibility of its application in the treatment or prevention of cancer. MATERIAL &METHODS: The cytotoxicity of C. obtusa leaf extracts was confirmed using an MTT assay. Intracellular changes in protein levels were measured by immunoblotting, and mRNA levels were measured with qRT-PCR. Wound healing assay and transwell migration assay were used to evaluate the metastatic potential of breast cancer cells. The extract-induced apoptosis was observed using IncuCyte Annexin V Red staining analysis. A syngeneic breast cancer mouse model was established by injecting 4T1-Luc mouse breast cancer cells into the fat pad of female BALB/c mice, and the extract was administered orally. Luciferin solution was injected intraperitoneally to assess primary tumor development and metastasis by bioluminescence. RESULTS: C. obtusa leaf extracts were extracted with boiling water, 70% EtOH, and 99% EtOH. Among the extracts, the 99% EtOH extract of C. obtusa leaf (CO99EL) most clearly inhibited the tyrosine phosphorylation of Signal Transducer and Activator of Transcription 3 (pY-STAT3) in MDA-MB-231 breast cancer cells at a concentration of 25 and 50 µg/mL. In addition, CO99EL strongly inhibited not only endogenous pY-STAT3 levels but also IL-6-induced STAT3 activation in various types of cancer cells, including breast cancer. CO99EL inhibited metastatic potential by downregulating the expression of N-cadherin, fibronectin, TWIST, MMP2, and MMP9 in MDA-MB-231 breast cancer cells. CO99EL also induced apoptotic cell death by increasing cleaved caspase-3 and decreasing anti-apoptotic proteins Bcl-2 and Bcl-xL. In an in vivo syngeneic breast cancer mouse model, 100 mg/kg CO99EL suppressed tumor growth and induced apoptosis of cancer cells. Moreover, CO99EL significantly inhibited lung metastasis from primary breast cancer. CONCLUSIONS: Our study demonstrated that 100 mg/kg CO99EL has potent anti-tumor effects against breast cancer, thus suggesting that 100 mg/kg CO99EL has potential applications in the treatment and prevention of breast cancer.

Novel Activity of ODZ10117, a STAT3 Inhibitor, for Regulation of NLRP3 Inflammasome Activation.

The NLRP3 inflammasome serves as a host defense mechanism against various pathogens, but there is growing evidence linking its activation in sterile condition to diverse inflammatory diseases. Therefore, the identification of specific inhibitors that target NLRP3 inflammasome activation is meaningful and important for novel therapies for NLRP3 inflammasome-associated diseases. In this study, we identified a chemical compound, namely ODZ10117 (ODZ), that showed NLRP3 inflammasome-targeting anti-inflammatory effects during the screening of a chemical library for anti-inflammatory activity. Although ODZ was initially discovered as a STAT3 inhibitor, here we found it also has inhibitory activity on NLRP3 inflammasome activation. ODZ inhibited the cleavage of caspase-1 and IL-1ß-induced canonical NLRP3 inflammasome triggers, but had no effect on those induced by AIM2 or NLRC4 triggers. Mechanistically, ODZ impairs NLRP3 inflammasome activation through the inhibition of NLRP3-NEK7 interaction that is required for inflammasome formation. Moreover, the results obtained from the in silico docking experiment suggested that ODZ targets NLRP3 protein, which provides evidence for the specificity of ODZ to the NLRP3 inflammasome. Furthermore, ODZ administration significantly reduced MSU-induced IL-1ß release and the mortality rate of mice with LPS-induced sepsis. Collectively, these results demonstrate a novel effect of ODZ10117 in regulating NLRP3 inflammasome activation both in vitro and in vivo, making it a promising candidate for the treatment of NLRP3-inflammasome-associated immune disorders and cancer.

Loss of phospholipase Cγ1 suppresses hepatocellular carcinogenesis through blockade of STAT3-mediated cancer development.

Phospholipase C gamma 1 (PLCγ1) plays an oncogenic role in several cancers, alongside its usual physiological roles. Despite studies aimed at identifying the effect of PLCγ1 on tumors, the pathogenic role of PLCγ1 in the tumorigenesis and development of hepatocellular carcinoma (HCC) remains unknown. To investigate the function of PLCγ1 in HCC, we generated hepatocyte-specific PLCγ1 conditional knockout (PLCγ1f/f ; Alb-Cre) mice and induced HCC with diethylnitrosamine (DEN). Here, we identified that hepatocyte-specific PLCγ1 deletion effectively prevented DEN-induced HCC in mice. PLCγ1f/f ; Alb-Cre mice showed reduced tumor burden and tumor progression, as well as a decreased incidence of HCC and less marked proliferative and inflammatory responses. We also showed that oncogenic phenotypes such as repressed apoptosis, and promoted proliferation, cell cycle progression and migration, were induced by PLCγ1. In terms of molecular mechanism, PLCγ1 regulated the activation of signal transducer and activator of transcription 3 (STAT3) signaling. Moreover, PLCγ1 expression is elevated in human HCC and correlates with a poor prognosis in patients with HCC. Our results suggest that PLCγ1 promotes the pathogenic progression of HCC, and PLCγ1/STAT3 axis was identified as a potential therapeutic target pathway for HCC.

The acidic tumor microenvironment enhances PD-L1 expression via activation of STAT3 in MDA-MB-231 breast cancer cells.

Tumor acidosis, a common phenomenon in solid cancers such as breast cancer, is caused by the abnormal metabolism of cancer cells. The low pH affects cells surrounding the cancer, and tumor acidosis has been shown to inhibit the activity of immune cells. Despite many previous studies, the immune surveillance mechanisms are not fully understood. We found that the expression of PD-L1 was significantly increased under conditions of extracellular acidosis in MDA-MB-231 cells. We also confirmed that the increased expression of PD-L1 mediated by extracellular acidosis was decreased when the pH was raised to the normal range. Gene set enrichment analysis (GSEA) of public breast cancer patient databases showed that PD-L1 expression was also highly correlated with IL-6/JAK/STAT3 signaling. Surprisingly, the expression of both phospho-tyrosine STAT3 and PD-L1 was significantly increased under conditions of extracellular acidosis, and inhibition of STAT3 did not increase the expression of PD-L1 even under acidic conditions in MDA-MB-231 cells. Based on these results, we suggest that the expression of PD-L1 is increased by tumor acidosis via activation of STAT3 in MDA-MB-231 cells.

Targeted drug release system based on pH-responsive PAA-POSS nanoparticles.

An amphipathic PAA-POSS@DOX drug delivery system that responds sensitively to pH changes in the cancer microenvironment has been developed using a nanoparticle based on inorganic polyhedral oligomeric silsesquioxane (POSS). POSS was introduced to the carboxylic acid group of polyacrylic acid to which doxorubicin anticancer drug was loaded to prepare 480 ± 192 nm self-assembled nanoparticles. PAA-POSS had a high loading efficiency of over 75% and doxorubicin was quickly released to the target area responding sensitively to weakly acidic conditions. The possibility of employing PAA-POSS as a targeted drug delivery system has been confirmed by observing the death of cells of the MDA-MB-231 breast cancer line.

Effect of Denosumab on the Change of Osteoclast Precursors Compared to Zoledronate Treatment in Postmenopausal Women with Osteoporosis.

BACKGROUND: A rapid increase in bone turnover and bone loss has been observed in response to the discontinuation of denosumab. It led to an acute increase in the fracture risk, similar to that observed in the untreated patients. We aimed to investigate the effect of denosumab on osteoclast (OC) precursor cells compared to that of zoledronate. METHODS: The study compared the effects of denosumab (60 mg/24-week) and zoledronate (5 mg/48-week) over 48 weeks in postmenopausal women with osteoporosis. From patients' peripheral mononuclear cells, CD14+/CD11b+/vitronectin receptor (VNR)- and CD14+/CD11b+/VNR+ cells were isolated using fluorescent-activated cell sorting, representing early and late OC precursors, respectively. The primary endpoint was the changes in OC precursors after 48 weeks of treatment. RESULTS: Among the 23 patients, 11 were assigned to the denosumab group and 12 to the zoledronate group (mean age, 69 years). After 48 weeks, the changes in OC precursors were similar between and within the groups. Serum C-terminal telopeptide of type I collagen levels were inversely correlated with OC precursor levels after denosumab treatment (r=-0.72, P<0.001). Lumbar spine, femur neck, and total hip bone mineral density (BMD) increased in both groups. Lumbar spine BMD increased more significantly in the denosumab group than in the zoledronate group. CONCLUSIONS: Denosumab and zoledronate treatments induced similar changes in OC precursors. During denosumab treatment, old age and suppressed bone turnover were associated with increased OC precursor cell populations. Further validation studies with prospective designs are required.

STAT3 activation in microglia increases pericyte apoptosis in diabetic retinas through TNF-ɑ/AKT/p70S6 kinase signaling.

Diabetic retinopathy (DR) is one of the vascular complications associated with diabetes mellitus. Pericyte loss is an early characteristic phenomenon in DR. However, the mechanism by which pericyte apoptosis occurs in DR is not fully understood. We have focused on the increased STAT3 activation in diabetic retinas because STAT3 activation is associated with inflammation, and persistent chronic inflammation is closely related to retinal lesions. In this study, we demonstrated that STAT3 was activated by IFN-γ and IL-6 that highly expressed in diabetic retinas. We identified TNF-α as a potent inducer of pericyte apoptosis in diabetic retinas from the gene expression analysis and found that STAT3 activation in microglia increased TNF-α expression in the diabetic retinas. We also demonstrated that increased TNF-α expression in microglia caused pericyte apoptosis through downregulating AKT/p70S6 kinase signaling. Moreover, we took advantage of mice lacking STAT3 in microglia and demonstrated that STAT3 ablation in microglia reduced the pericyte apoptosis and TNF-α expression in the diabetic retinas. These results suggest that STAT3 activation in microglia plays an important role in pericyte apoptosis in the diabetic retinas through increased TNF-α expression and provide STAT3 activation in microglia as a potential therapeutic target for preventing pericyte loss in DR.

Chamaecyparis obtusa (Siebold & Zucc.) Endl. leaf extracts prevent inflammatory responses via inhibition of the JAK/STAT axis in RAW264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Chamaecyparis obtusa (Siebold & Zucc.) Endl. (C. obtusa) has been used as folk medicine in East Asia and has been reported to alleviate inflammatory diseases. However, the detailed mechanisms for the anti-inflammatory effects of C. obtusa remain unclear. AIM OF THE STUDY: Although the anti-inflammatory mechanisms of natural products have been studied for decades, it is still important to identify the potential anti-inflammatory effects of natural sources. In this study, we investigated the anti-inflammatory effects and underlying mechanism of C. obtusa leaf extracts. MATERIAL &METHODS: The cell viability was determined by MTT and crystal violet staining. NO production in the supernatant was measured using Griess reagent. The cell lysates were analyzed by immunoblotting and RT-qPCR. Secreted cytokines were analyzed using ELISA kit and cytokine array kit. mRNA expression from the GSE9632 database set. Z-scores were calculated for each gene and visualized by heat map. RESULTS: Among the extracts of C. obtusa obtained with different extraction methods, the 99% ethanol leaf extract (CO99EL) strongly inhibited lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression and Janus kinase/signaling transducer and activator of transcription (JAK/STAT) phosphorylation in RAW264.7 cells. In addition, CO99EL strongly inhibited LPS-induced interleukin (IL)-1ß, IL-6, IL-27, and C-C motif chemokine ligand (CCL)-1 production and directly inhibited LPS-induced JAK/STAT phosphorylation in RAW264.7 cells. CONCLUSIONS: These findings demonstrate that CO99EL significantly prevents LPS-induced macrophage activation by inhibiting the JAK/STAT axis. Therefore, we suggest the use of C. obtusa extracts as therapeutic approach for inflammatory diseases.

Novel cancer stem cell marker MVP enhances temozolomide-resistance in glioblastoma.

The resistance of highly aggressive glioblastoma multiforme (GBM) to chemotherapy is a major clinical problem resulting in a poor prognosis. GBM contains a rare population of self-renewing cancer stem cells (CSCs) that proliferate, spurring the growth of new tumors, and evade chemotherapy. In cancer, major vault protein (MVP) is thought to contribute to drug resistance. However, the role of MVP as CSCs marker remains unknown and whether MVP could sensitize GBM cells to Temozolomide (TMZ) also is unclear. We found that sensitivity to TMZ was suppressed by significantly increasing the MVP expression in GBM cells with TMZ resistance. Also, MVP was associated with the expression of other multidrug-resistant proteins in tumorsphere of TMZ-resistant GBM cell, and was highly co-expressed with CSC markers in tumorsphere culture. On the other hands, knockdown of MVP resulted in reduced sphere formation and invasive capacity. Moreover, high expression of MVP was associated with tumor malignancy and survival rate in glioblastoma patients. Our study describes that MVP is a potentially novel maker for glioblastoma stem cells and may be useful as a target for preventing TMZ resistance in GBM patients.

Insulin smart drug delivery nanoparticles of aminophenylboronic acid-POSS molecule at neutral pH.

Self-regulated "smart" insulin administration system that mimic pancreatic endocrine function would be highly desirable for diabetes management. Here, a glucose-responsive continuous insulin delivery system is developed, where novel polyhedral oligosilsesquioxane (POSS) modified with 3-aminophenylboronic acid (APBA) were used to encapsulate insulin (insulin entrapment efficiency: 73.2%) to prepare a fast response, high stability, good distribution, and excellent biocompatible system. Due to the strong hydrophobicity of POSS, the POSS moiety is located at the core in aqueous solution and combines with the boronic group of APBA and the diol generated in PEG-insulin to form a nanomicelle structure, that is, nanoparticles naturally. Micelles self-assembled from these molecules possess glucose-responsiveness at varying glucose concentrations. The interaction of the PBA and diol containing insulin via boronate ester bond and its interchange with glucose was investigated by FT-IR, 1H NMR and XPS. Furthermore, the successful glucose-triggered release of insulin from the POSS-APBA micelles was investigated at neutral pH. A linear graph was plotted with the measured released insulin vs glucose concentrations, with a linear correlation coefficient (R2) value close to 1. Circular dichroism (CD) spectroscopy analysis was performed to measure insulin activity by comparing secondary structures of insulin, PEG-Insulin, and POSS-APBA@insulin. When confirming intracellular apoptosis signaling, cleaved caspase 3 and caspase 9 were not increased by 640 µg/ml POSS-APBA and POSS-APBA@insulin in HeLa, HDF and HUVE cells. Application in the biomedical field for controlled delivery of insulin appear to be promising.

Inhibition of mTORC1 through ATF4-induced REDD1 and Sestrin2 expression by Metformin.

BACKGROUND: Although the major anticancer effect of metformin involves AMPK-dependent or AMPK-independent mTORC1 inhibition, the mechanisms of action are still not fully understood. METHODS: To investigate the molecular mechanisms underlying the effect of metformin on the mTORC1 inhibition, MTT assay, RT-PCR, and western blot analysis were performed. RESULTS: Metformin induced the expression of ATF4, REDD1, and Sestrin2 concomitant with its inhibition of mTORC1 activity. Treatment with REDD1 or Sestrin2 siRNA reversed the mTORC1 inhibition induced by metformin, indicating that REDD1 and Sestrin2 are important for the inhibition of mTORC1 triggered by metformin treatment. Moreover, REDD1- and Sestrin2-mediated mTORC1 inhibition in response to metformin was independent of AMPK activation. Additionally, lapatinib enhances cell sensitivity to metformin, and knockdown of REDD1 and Sestrin2 decreased cell sensitivity to metformin and lapatinib. CONCLUSIONS: ATF4-induced REDD1 and Sestrin2 expression in response to metformin plays an important role in mTORC1 inhibition independent of AMPK activation, and this signalling pathway could have therapeutic value.

3D cell culture using a clinostat reproduces microgravity-induced skin changes.

Exposure to microgravity affects human physiology in various ways, and astronauts frequently report skin-related problems. Skin rash and irritation are frequent complaints during space missions, and skin thinning has also been reported after returning to Earth. However, spaceflight missions for studying the physiological changes in microgravity are impractical. Thus, we used a previously developed 3D clinostat to simulate a microgravity environment and investigate whether physiological changes of the skin can be reproduced in a 3D in vitro setting. Our results showed that under time-averaged simulated microgravity (taSMG), the thickness of the endothelial cell arrangement increased by up to 59.75%, indicating skin irritation due to vasodilation, and that the diameter of keratinocytes and fibroblast co-cultured spheroids decreased by 6.66%, representing skin thinning. The α1 chain of type I collagen was upregulated, while the connective tissue growth factor was downregulated under taSMG. Cytokeratin-10 expression was significantly increased in the taSMG environment. The clinostat-based 3D culture system can reproduce physiological changes in the skin similar to those under microgravity, providing insight for understanding the effects of microgravity on human health before space exploration.

STAT3 activation in microglia exacerbates hippocampal neuronal apoptosis in diabetic brains.

Diabetes mellitus (DM) characterized by hyperglycemia leads to a variety of complications, including cognitive impairment or memory loss. The hippocampus is a key brain area for learning and memory and is one of the regions that is most sensitive to diabetes. However, the pathogenesis of diabetic neuronal lesion is not yet completely understood. We focused on the association of microglia activation and brain lesions in diabetes. In this study, we investigated whether and how signal transducer and activator of transcription 3 (STAT3) activation in microglia affects neuronal lesions in diabetic brains. Using a streptozotocin-induced type 1 DM model, we showed enhanced hippocampal neuronal apoptosis that was associated with increased STAT3 activation. We found that hyperglycemia increased the expression of inflammatory cytokines such as interferon-γ (IFN-γ) and interleukin-6, in the diabetic hippocampus. In particular, IFN-γ induced autocrine activation of microglia, and STAT3 activation is important for this process. We also demonstrated that STAT3 activation in microglia increased tumor necrosis factor-α (TNF-α) expression; subsequently, TNF-α increased neuronal apoptosis by increasing reactive oxygen species (ROS) levels in the neuronal cells. We also took advantage of mice lacking STAT3 in microglia and demonstrated that depletion of microglial STAT3 reduced neuronal apoptosis in the diabetic hippocampus. Taken together, these results suggest that STAT3 activation in microglia plays an important role in hyperglycemia-induced neuronal apoptosis in the diabetic hippocampus and provide a potential therapeutic benefit of STAT3 inhibition in microglia for preventing diabetic neuronal lesions.

Chromatin accessibility of circulating CD8+ T cells predicts treatment response to PD-1 blockade in patients with gastric cancer.

Although tumor genomic profiling has identified small subsets of gastric cancer (GC) patients with clinical benefit from anti-PD-1 treatment, not all responses can be explained by tumor sequencing alone. We investigate epigenetic elements responsible for the differential response to anti-PD-1 therapy by quantitatively assessing the genome-wide chromatin accessibility of circulating CD8+ T cells in patients' peripheral blood. Using an assay for transposase-accessible chromatin using sequencing (ATAC-seq), we identify unique open regions of chromatin that significantly distinguish anti-PD-1 therapy responders from non-responders. GC patients with high chromatin openness of circulating CD8+ T cells are significantly enriched in the responder group. Concordantly, patients with high chromatin openness at specific genomic positions of their circulating CD8+ T cells demonstrate significantly better survival than those with closed chromatin. Here we reveal that epigenetic characteristics of baseline CD8+ T cells can be used to identify metastatic GC patients who may benefit from anti-PD-1 therapy.

Ablation of STAT3 in Purkinje cells reorganizes cerebellar synaptic plasticity in long-term fear memory network.

Emotional memory processing engages a large neuronal network of brain regions including the cerebellum. However, the molecular and cellular mechanisms of the cerebellar cortex modulating the fear memory network are unclear. Here, we illustrate that synaptic signaling in cerebellar Purkinje cells (PCs) via STAT3 regulates long-term fear memory. Transcriptome analyses revealed that PC-specific STAT3 knockout (STAT3PKO) results in transcriptional changes that lead to an increase in the expression of glutamate receptors. The amplitude of AMPA receptor-mediated excitatory postsynaptic currents at parallel fiber (PF) to PC synapses was larger in STAT3PKO mice than in wild-type (WT) littermates. Fear conditioning induced long-term depression of PF-PC synapses in STAT3PKO mice while the same manipulation induced long-term potentiation in WT littermates. STAT3PKO mice showed an aberrantly enhanced long-term fear memory. Neuronal activity in fear-related regions increased in fear-conditioned STAT3PKO mice. Our data suggest that STAT3-dependent molecular regulation in PCs is indispensable for proper expression of fear memory.

Tubulosine selectively inhibits JAK3 signalling by binding to the ATP-binding site of the kinase of JAK3.

Gain- or loss-of-function mutations in Janus kinase 3 (JAK3) contribute to the pathogenesis of various haematopoietic malignancies and immune disorders, suggesting that aberrant JAK3 signalling is an attractive therapeutic target to treat these disorders. In this study, we performed structure-based computational database screening using the 3D structure of the JAK3 kinase domain and the National Cancer Institute diversity set and identified tubulosine as a novel JAK3 inhibitor. Tubulosine directly blocked the catalytic activity of JAK3 by selective interacting with the JAK3 kinase domain. Consistently, tubulosine potently inhibited persistently activated and interleukin-2-dependent JAK3, and JAK3-mediated downstream targets. Importantly, it did not affect the activity of other JAK family members, particularly prolactin-induced JAK2/signal transducer and activator of transcription 5 and interferon alpha-induced JAK1-TYK2/STAT1. Tubulosine specifically decreased survival and proliferation of cancer cells, in which persistently active JAK3 is expressed, by inducing apoptotic and necrotic/autophagic cell death without affecting other oncogenic signalling. Collectively, tubulosine is a potential small-molecule compound that selectively inhibits JAK3 activity, suggesting that it may serve as a promising therapeutic candidate for treating disorders caused by aberrant activation of JAK3 signalling.