Phosphorylation Of Glycogen Synthase Kinase-3β Research Articles

Folic Acid Reduces Tau Phosphorylation by Regulating PP2A Methylation in Streptozotocin-Induced Diabetic Mice.

High incidence rate of Alzheimer’s disease (AD) is observed in patients with type 2 diabetes. Aggregated β-amyloid (Aβ) and hyperphosphorylated tau are the hallmarks of AD. Hyperphosphorylated tau has been detected in diabetic animals as well as in diabetic patients. Folates mediate the transfer of one carbon unit, required in various biochemical reactions. The effect of folate on tau phosphorylation in diabetic models still remains unknown. In this study, we investigated the effect and mechanism of folic acid on hyperphosphorylation of tau in streptozotocin (STZ)-induced diabetic mice. Diabetic mice induced by STZ, at the age of 10 weeks, were administered with three levels of folic acid: folic acid-deficient diet, diet with normal folic acid content, and 120 μg/kg folic acid diet for 8 weeks. Levels of serum folate and blood glucose were monitored. Tau phosphorylation, protein phosphatase 2A (PP2A) methylation, and Glycogen synthase kinase 3β (GSK-3β) phosphorylation were detected using Western blot. The S-adenosyl methionine:S-adenosyl homocysteine ratio (SAM:SAH) in brain tissues was also determined. DNA methyltransferase (DNMT) mRNA expression levels were detected using real-time PCR. Folic acid reduced tau hyperphosphorylation at Ser396 in the brain of diabetes mellitus (DM) mice. In addition, PP2A methylation and DNMT1 mRNA expression were significantly increased in DM mice post folic acid treatment. GSK-3β phosphorylation was not regulated by folic acid administration. Folic acid can reduce tau phosphorylation by regulating PP2A methylation in diabetic mice. These results support that folic acid can serve as a multitarget neuronal therapeutic agent for treating diabetes-associated cognitive dysfunction.

Melanocyte-protective effect of afzelin is mediated by the Nrf2-ARE signalling pathway via GSK-3β inactivation.

Vitiligo is an acquired condition characterized by depigmented, cutaneous lesions that result from the death of pigment-producing cells, melanocytes. The occurrence of oxidative stress has been proposed as a pathogenetic mechanism for melanocyte degeneration in vitiligo. Therefore, in this study, we investigated the cytoprotective effects of afzelin against oxidative stress and its mechanism of action in human epidermal melanocytes. We found that afzelin significantly inhibited hydrogen peroxide-induced cell death, cellular reactive oxygen species production and lipid peroxidation in melanocytes. In an antioxidant response element (ARE)-luciferase reporter assay, afzelin increased ARE-luciferase reporter activity in a concentration-dependent manner. Consistently, the expression of antioxidant genes, including NF-E2-related factor 2 (Nrf2), haem oxygenase-1 (HO-1) and catalase, was enhanced by afzelin treatment. Nuclear translocation of Nrf2 also increased in response to afzelin treatment. In addition, the phosphorylation of glycogen synthase kinase-3β (GSK-3β) was induced by afzelin treatment. The enhancement of HO-1 gene expression by afzelin treatment was reduced by Nrf2-siRNA expression. Furthermore, we found that the expression of Nrf2-siRNA significantly attenuated the cytoprotective effect of afzelin against hydrogen peroxide. These data suggest that the cytoprotective effects of afzelin against hydrogen peroxide may be mediated by Nrf2-ARE signalling via GSK-3β inactivation. Our data suggest the novel use of afzelin for the prevention of oxidative stress-induced damage in melanocytes and its potential as a therapeutic agent for vitiligo.

Extracellular cystatin SN and cathepsin B prevent cellular senescence by inhibiting abnormal glycogen accumulation

Cystatin SN (CST1), a known inhibitor of cathepsin B (CatB), has important roles in tumor development. Paradoxically, CatB is a member of the cysteine cathepsin family that acts in cellular processes, such as tumor development and invasion. However, the relationship between CST1 and CatB, and their roles in tumor development are poorly understood. In this study, we observed that the knockdown of CST1 induced the activity of senescence-associated β-galactosidase, a marker of cellular senescence, and expression of senescence-associated secretory phenotype genes, including interleukin-6 and chemokine (C-C motif) ligand 20, in MDA-MB-231 and SW480 cancer cells. Furthermore, CST1 knockdown decreased extracellular CatB activity, and direct CatB inhibition, using specific inhibitors or shCatB, induced cellular senescence. Reconstitution of CST1 restored CatB activity and inhibited cellular senescence in CST1 knockdown cells. CST1 knockdown or CatB inhibition increased glycogen synthase (GS) kinase 3β phosphorylation at serine 9, resulting in the activation of GS and the induction of glycogen accumulation associated with cellular senescence. Importantly, CST1 knockdown suppressed cancer cell proliferation, soft agar colony growth and tumor growth in a xenograft model. These results indicate that CST1-mediated extracellular CatB activity enhances tumor development by preventing cellular senescence. Our findings suggest that antagonists of CST1 or inhibitors of CatB are potential anticancer agents.

Chronological changes in the expression of phosphorylated tau and 5‑AMP‑activated protein kinase in the brain of senescence‑accelerated P8 mice.

Senescence-accelerated mouse prone 8 (SAMP8), a non‑transgenic animal model used for researching sporadic Alzheimer's disease (AD), presents AD pathologies and overall dysregulation in brain energy metabolism, which is one of the early pathogenic characteristics of AD. In the present study, the authors examined chronological changes in the expression patterns of phosphorylated tau and of proteins related to energy metabolism to evaluate the association of tau phosphorylation and metabolism, using young‑ (2‑months‑old), middle‑ (5‑months‑old) and old‑aged (10‑months‑old) SAMP8. The levels of phosphorylated 5'‑AMP activated protein kinase at Thr172 (p‑AMPK) and phosphorylated glycogen synthase kinase 3β (p‑GSK3βS9) in the cortex of SAMP8 at 2months were significantly higher than those in senescence‑accelerated mouse resistant 1 (SAMR1). The differences were not detected at 5 and 10months of age, which were concurrent with the changes in levels of phosphorylated tau at Ser396 (p‑tauS396), but not with p‑tauS262. The level of p‑tauS262 was considerably higher in the cortex of middle‑aged SAMP8 when compared with that of SAMR1 and sustained in old‑aged SAMP8, but not in the young cortex. The levels of cortical sirtuin1 (Sirt1) and insulin receptor substrate 1 (IRS‑1) expression of young SAMP8 were significantly lower, when compared with those in SAMR1. However, in the hippocampus of SAMP8, the patterns of chronological changes and levels of p‑tau, p‑AMPK, Sirt1 and IRS‑1 relative to SAMR1 were different from those in the cortex. Taken together, the results suggested that regulation of tau phosphorylation via the AMPK‑GSK3β pathway concurrent with dysregulation of energy metabolism may precede the pathological tau hyperphosphorylation in the cortex of SAMP8, and that the regulation of AMPK‑GSK3β‑mediated tau phosphorylation may be dependent on phosphor‑epitope in tau or the region of brain.

Methane rescues retinal ganglion cells and limits retinal mitochondrial dysfunction following optic nerve crush

Secondary degeneration is a common event in traumatic central nervous system disorders, which involves neuronal apoptosis and mitochondrial dysfunction. Exogenous methane exerts the therapeutic effects in many organ injury. Our study aims to investigate the potential neuroprotection of methane in a rat model of optic nerve crush (ONC). Adult male Sprague-Dawley rats were subjected to ONC and administrated intraperitoneally with methane-saturated or normal saline (10 ml/kg) once per day for one week after ONC. The retinal ganglion cells (RGCs) density was assessed by hematoxylin and eosin staining and Fluoro-Gold retrogradely labeling. Visual function was evaluated by flash visual evoked potentials (FVEP). The retinal apoptosis was measured by terminal-deoxy-transferase-mediated dUTP nick end labeling (TUNEL) assay and the expression of apoptosis-related factors, such as phosphorylated Bcl-2-associated death promoter (pBAD), phosphorylated glycogen synthase kinase-3β (pGSK-3β), Bcl-2 associated X protein (Bax) and Bcl-2 extra large (Bcl-xL). Retinal mitochondrial function was assessed by the mRNA expressions of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), nuclear respiratory factor 1 (NRF1) and mitochondrial transcription factor A (TFAM), the mitochondrial DNA (mtDNA) copy number, citrate synthase activity and ATP content. Methane treatment significantly improved the RGC loss and visual dysfunction following ONC. As expected, methane also remarkably inhibited the retinal neural apoptosis, such as the fewer TUNEL-positive cells in ganglion cell layer, accompanied by the up-regulations of anti-apoptotic factors (pGSK-3β, pBAD, Bcl-xL) and the down-regulation of pro-apoptotic factor (Bax). Furthermore, methane treatment suppressed up-regulations of critical mitochondrial components (PGC-1α, NRF1 and TFAM) mRNA and mtDNA copy number, as well as improved the reduction of functional mitochondria markers, including citrate synthase activity and ATP content, in retinas with ONC. Taken together, methane treatment promotes RGC survival and limits retinal mitochondrial dysfunction against ONC insult. Methane can be a potential neuroprotective agent for traumatic and glaucomatous neurodegeneration.

Astragaloside IV attenuates lead acetate-induced inhibition of neurite outgrowth through activation of Akt-dependent Nrf2 pathway in vitro

Recently, oxidative stress is strongly associated with lead (Pb)-induced neurotoxicity. We reported previously that Astragaloside IV (AS-IV) possesses potent antioxidant properties. Here, we evaluate the hypothesis that AS-IV attenuates lead acetate (PbAc)-mediated inhibition of neurite outgrowth might mainly result from its antioxidant property via serine/threonine protein kinase (Akt)-dependent activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Interestingly, AS-IV attenuates PbAc-induced inhibition of neurite outgrowth and displayed potential antioxidant properties by inhibiting reactive oxygen species (ROS). Concomitantly, AS-IV enhanced phase II detoxifying enzymes such as heme oxygenase 1 (HO-1), thioredoxin reductase (TrxR), and glutamate cysteine ligase catalytic subunit (GCLc). Conversely, AS-IV had no effect on GCL modulatory subunit (GCLm) and superoxide dismutase (SOD) activity/expression. Furthermore, AS-IV evoked Akt phosphorylation, and subsequent induced phosphorylation of glycogen synthase kinase-3β (GSK-3β) at Ser9 (that is, inactivation), which stimulated Nrf2-mediated antioxidant response element (ARE)-containing activation. Importantly, Akt locates upstream of GSK-3β and regulates phase II detoxifying enzymes gene expression through Nrf2 nuclear accumulation in PC12 cells exposed to PbAc. Noteworthy, these results were further confirmed through signalling pathway inhibitors, dominant negative mutant and short hairpin RNA technology. Collectively, these in vitro findings suggest that AS-IV attenuates PbAc-induced inhibition of neurite outgrowth attributed to its antioxidant properties and may be a promising candidate for the treatment of lead developmental neurotoxicity.

CCL2 induces epithelial-mesenchyme transition in breast cancer MCF-7 cells

Objective To explore the effect and molecular mechanism of CC chemokine ligand 2 (CCL2) on epithelial-mesenchymal transition in human breast cancer cells. Methods Breast cancer Michigan cancer foundation-7 (MCF-7) cells were treated with 50 ng/ml CCL2. The abilities of invasion were detected by Transwell assay. The expression of epithelial-mesenchymal transition (EMT)-associated markers, E-cadherin and vimentin were detected by Western blot and quantitative real-time polymerase chain reaction (qRT-PCR). The expressions of Snail, protein kinase B (AKT), phosphorylated protein kinase B (p-AKT), phosphorylated glycogen synthase kinase 3β (p-GSK3β) and GSK3β were detected by Western blot. Snail nuclear localization was detected by immunoflurescence staining. Results We found that CCL2 treatment could induce morphological alteration of MCF-7 cells from epithelial morphology to mesenchymal morphology. CCL2 significantly increased the migration of MCF-7 cells, and increased the expression of mesenchymal maker vimentin and decreased epithelial marker E-cadherin. More important, treatment of CCL2 significantly increased the expression of Snail, and promoted the nuclear localization of Snail. Knockdown of Snail significantly reverse the effects of CCL2 on the EMT in MCF-7 cells. Moreover, treatment of CCL2 significantly increased the phosphorylation levels of p-AKT and p-GSK3β, and AKT inhibitor LY294002 significantly inhibited CCL2-induced Snail and p-GSK3β expression. Conclusion CCL2 might induce EMT in MCF-7 cells, by which mechanism is related to activate AKT/GSK3β Snail pathway. Key words: Chemokine CCL2; Breast neoplasms; Tumor cells, cultured; Biotransformation

P020 Epigenetic regulation of brain-derived neurotrophic factor (Bdnf) expression mediates the effects of anodal transcranial direct current stimulation (tDCS) on hippocampal synaptic plasticity and memory in mice

Question Understanding the cellular and molecular mechanisms underlying tDCS action is critical for a rationale use of this technique in clinical settings. Here we investigated the effects of tDCS on hippocampal synaptic plasticity and memory focusing on epigenetic mechanisms affecting the expression of plasticity-related genes. Methods Electrophysiological, behavioral and molecular indices of hippocampal plasticity were investigated following 20-min anodal tDCS delivered to awake mice. Results Hippocampal slices from tDCS-mice showed greater long-term potentiation (LTP) at CA3-CA1 synapses compared to sham-stimulated controls. Enhanced LTP was associated with improved hippocampal-dependent learning and memory assessed by the Morris water maze and novel object recognition tests. Remarkably, all these effects persisted 1 week after tDCS. Real-time PCR, Western Blotting and chromatin immunoprecipitation experiments revealed that tDCS effects were due to an intracellular signaling cascade including: (i) increased phosphorylation of cAMP response element-binding protein (CREB); (ii) enhanced CREB binding to the Bdnf promoter I; and, (iii) recruitment of the histone acetyltransferase CBP (CREB-binding protein) leading to enhanced histone 3 acetylation on Bdnf promoter I. As a consequence, the expression of Bdnf exons I and IX mRNAs was increased in the hippocampi of tDCS mice along with Bdnf protein levels. Accordingly, molecular, electrophysiological and behavioral effects of tDCS were prevented by mice treatment with either the acetylation inhibitor, curcumin, or the Bdnf receptor TrkB antagonist, ANA-12. Remarkably, we found that tDCS enhanced glycogen synthase kinase-3β(GSK-3β) phosphorylation at Ser9 that was prevented by ANA-12, thus suggesting that GSK-3βinhibition plays a role in the tDCS-induced increase of hippocampal plasticity. Conclusion Our findings show that anodal tDCS increases hippocampal LTP and memory via chromatin remodeling of Bdnf regulatory sequences leading to increased expression of this gene. These results lend support to the use of tDCS for prevention and treatment of brain diseases associated with impaired neuroplasticity.

Involvement of glycogen synthase kinase-3β in liver ischemic conditioning induced cardioprotection against myocardial ischemia and reperfusion injury in rats.

Remote ischemic conditioning has been convincingly shown to render the myocardium resistant to a subsequent more severe sustained episode of ischemia. Compared with other organs, little is known regarding the effect of transient liver ischemic conditioning. We proposed the existence of cardioprotection induced by remote liver conditioning. Male Sprague-Dawley rats were divided into sham-operated control (no further hepatic intervention) and remote liver ischemic conditioning groups. For liver ischemic conditioning, three cycles of 5 min of liver ischemia-reperfusion stimuli were conducted before-(liver preconditioning), post-myocardial ischemia (liver postconditioning), or in combination of both (liver preconditioning + liver postconditioning). Rats were exposed to 45 min of left anterior descending coronary artery occlusion, followed by 3 h of reperfusion thereafter. ECG and hemodynamics were measured throughout the experiment. The coronary artery was reoccluded at the end of reperfusion for infarct size determination. Blood samples were taken for serum lactate dehydrogenase and creatine kinase-MB test. Heart tissues were taken for apoptosis measurements and Western blotting. Our data demonstrate that liver ischemic preconditioning, postconditioning, or a combination of both, offered strong cardioprotection, as evidenced by reduction in infarct size and cardiac tissue damage, recovery of cardiac function, and inhibition of apoptosis after ischemia-reperfusion. Moreover, liver ischemic conditioning increased cardiac (not hepatic) glycogen synthase kinase-3β (GSK-3β) phosphorylation. Accordingly, inhibition of GSK-3β mimicked the cardioprotective action of liver conditioning. These results demonstrate that remote liver ischemic conditioning protected the heart against ischemia and reperfusion injury via GSK-3β-dependent cell-survival signaling pathway.NEW & NOTEWORTHY Remote ischemic conditioning protects hearts against ischemia and reperfusion (I/R) injury. However, it is unclear whether ischemic conditioning of visceral organs such as the liver, the largest metabolic organ in the body, can produce cardioprotection. This is the first study to show the cardioprotective effect of remote liver ischemic conditioning in a rat model of myocardial I/R injury. We also, for the first time, demonstrated these protective properties are associated with glycogen synthase kinase-3β-dependent cell-survival signaling pathway.

Artesunate Protects Against the Organ Injury and Dysfunction Induced by Severe Hemorrhage and Resuscitation.

To evaluate the effects of artesunate on organ injury and dysfunction associated with hemorrhagic shock (HS) in the rat. HS is still a common cause of death in severely injured patients and is characterized by impairment of organ perfusion, systemic inflammatory response, and multiple organ failure. There is no specific therapy that reduces organ injury/dysfunction. Artesunate exhibits pharmacological actions beyond its antimalarial activity, such as anticancer, antiviral, and anti-inflammatory effects. Rats were submitted to HS. Mean arterial pressure was reduced to 30 mm Hg for 90 minutes, followed by resuscitation. Rats were randomly treated with artesunate (2.4 or 4.8 mg/kg i.v.) or vehicle upon resuscitation. Four hours later, parameters of organ injury and dysfunction were assessed. Artesunate attenuated the multiple organ injury and dysfunction caused by HS. Pathway analysis of RNA sequencing provided good evidence to support an effect of artesunate on the Akt-survival pathway, leading to downregulation of interleukin-1 receptor-associated kinase 1. Using Western blot analysis, we confirmed that treatment of HS rats with artesunate enhanced the phosphorylation (activation) of Protein kinase B (Akt) and endothelial nitric oxide synthase and the phosphorylation (inhibition) of glycogen synthase kinase-3β (GSK-3β). Moreover, artesunate attenuated the HS-induced activation of nuclear factor kappa B and reduced the expression of proinflammatory proteins (inducible nitric oxide synthase, tumor necrosis factor-α, and interleukin 6). Artesunate attenuated the organ injury/dysfunction associated with HS by a mechanism that involves the activation of the Akt-endothelial nitric oxide synthase survival pathway, and the inhibition of glycogen synthase kinase-3β and nuclear factor kappa B. A phase II clinical trial evaluating the effects of good manufacturing practice-artesunate in patients with trauma and severe hemorrhage is planned.

Regulatory role of tumor necrosis factor receptor-associated factor 6 in breast cancer by activating the protein kinase B/glycogen synthase kinase 3β signaling pathway.

Tumor necrosis factor receptor-associated factor6 (TRAF6) is an endogenous adaptor of innate and adaptive immune responses, and serves a crucial role in tumor necrosis factor receptor and toll‑like/interleukin‑1 receptor signaling. Although studies have demonstrated that TRAF6 has oncogenic activity, its potential contributions to breast cancer in human remains largely uninvestigated. The present study examined the expression levels and function of TRAF6 in breast carcinoma (n=32) and adjacent healthy (n=25) tissue samples. Compared with adjacent healthy tissues, TRAF6 protein expression levels were significantly upregulated in breast cancer tissues. Reverse transcription‑quantitative polymerase chain reaction analysis revealed a significant upregulation of the cellular proliferative marker Ki‑67 and proliferation cell nuclear antigen expression levels in breast carcinoma specimens. Furthermore, protein expression levels of the accessory molecule, transforming growth factor β‑activated kinase 1 (TAK1), were significantly increased in breast cancer patients, as detected by western blot analysis. As determined by MTT assay, TRAF6 exerted profoundly proliferative effects in the MCF‑7 breast cancer cell line; however, these detrimental effects were ameliorated by TAK1 inhibition. Notably, protein kinase B (AKT)/glycogen synthase kinase (GSK)3β phosphorylation levels were markedly upregulated in breast cancer samples, compared with adjacent healthy tissues. In conclusion, an altered TRAF6‑TAK1 axis and its corresponding downstream AKT/GSK3β signaling molecules may contribute to breast cancer progression. Therefore, TRAF6 may represent a potential therapeutic target for the treatment of breast cancer.

Elevating CXCR7 Improves Angiogenic Function of EPCs via Akt/GSK-3β/Fyn-Mediated Nrf2 Activation in Diabetic Limb Ischemia.

Endothelial progenitor cells (EPCs) respond to stromal cell-derived factor 1 (SDF-1) through chemokine receptors CXCR7 and CXCR4. Whether SDF-1 receptors involves in diabetes mellitus-induced EPCs dysfunction remains unknown. To determine the role of SDF-1 receptors in diabetic EPCs dysfunction. CXCR7 expression, but not CXCR4 was reduced in EPCs from db/db mice, which coincided with impaired tube formation. Knockdown of CXCR7 impaired tube formation of EPCs from normal mice, whereas upregulation of CXCR7 rescued angiogenic function of EPCs from db/db mice. In normal EPCs treated with oxidized low-density lipoprotein or high glucose also reduced CXCR7 expression, impaired tube formation, and increased oxidative stress and apoptosis. The damaging effects of oxidized low-density lipoprotein or high glucose were markedly reduced by SDF-1 pretreatment in EPCs transduced with CXCR7 lentivirus but not in EPCs transduced with control lentivirus. Most importantly, EPCs transduced with CXCR7 lentivirus were superior to EPCs transduced with control lentivirus for therapy of ischemic limbs in db/db mice. Mechanistic studies demonstrated that oxidized low-density lipoprotein or high glucose inhibited protein kinase B and glycogen synthase kinase-3β phosphorylation, nuclear export of Fyn and nuclear localization of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), blunting Nrf2 downstream target genes heme oxygenase-1, NAD(P)H dehydrogenase (quinone 1) and catalase, and inducing an increase in EPC oxidative stress. This destructive cascade was blocked by SDF-1 treatment in EPCs transduced with CXCR7 lentivirus. Furthermore, inhibition of phosphatidylinositol 3-kinase/protein kinase B prevented SDF-1/CXCR7-mediated Nrf2 activation and blocked angiogenic repair. Moreover, Nrf2 knockdown almost completely abolished the protective effects of SDF-1/CXCR7 on EPC function in vitro and in vivo. Elevated expression of CXCR7 enhances EPC resistance to diabetes mellitus-induced oxidative damage and improves therapeutic efficacy of EPCs in treating diabetic limb ischemia. The benefits of CXCR7 are mediated predominantly by a protein kinase B/glycogen synthase kinase-3β/Fyn pathway via increased activity of Nrf2.

Characteristics of Chinese herbal medicine usage in ischemic heart disease patients among type 2 diabetes and their protection against hydrogen peroxide-mediated apoptosis in H9C2 cardiomyoblasts.

Evidence for long-term use of Chinese herbal medicine (CHM) as an adjuvant treatment in patients with type 2 diabetes (T2D) remains limited. This study aimed to assess the frequency of use, utilization patterns, and therapeutic effects of adjuvant CHM for ischemic heart disease (IHD) in patients with T2D in Taiwan. We identified 4620 IHD patients with T2D. After matching for age, gender, and insulin use, 988 subjects each were allocated to a CHM group and a non-CHM group. There were no differences in baseline characteristics except for comorbidities. The CHM group contained more cases with chronic obstructive pulmonary disease, hepatitis, ulcer disease, and hyperlipidemia. The cumulative survival probability was higher in CHM users than in matched non-CHM users aged 60 years or older (P < .0001, log rank test) regardless of gender (P = .0046 for men, P = .0010 for women, log rank test). Among the top 12 CHM combinations, Shu-Jing-Huo-Xue-Tang and Shao-Yao-Gan-Cao-Tang (13.6%) were the most common. This dual combination improved antiapoptotic activity in H2O2-exposed H9C2 cells by enhancing phosphorylation of glycogen synthase kinase-3β and p38 mitogen-activated protein kinase and could increase the survival of myocardial cells. Our study suggests that adjuvant CHM therapy may increase the survival probability and provides a comprehensive list for future investigations of the safety and efficacy of CHM for IHD patients with T2D.

Adult neural stem cell dysfunction in the subventricular zone of the lateral ventricle leads to diabetic olfactory defects.

Sensitive smell discrimination is based on structural plasticity of the olfactory bulb, which depends on migration and integration of newborn neurons from the subventricular zone. In this study, we examined the relationship between neural stem cell status in the subventricular zone and olfactory function in rats with diabetes mellitus. Streptozotocin was injected through the femoral vein to induce type 1 diabetes mellitus in Sprague-Dawley rats. Two months after injection, olfactory sensitivity was decreased in diabetic rats. Meanwhile, the number of BrdU-positive and BrdU+/DCX+ double-labeled cells was lower in the subventricular zone of diabetic rats compared with age-matched normal rats. Western blot results revealed downregulated expression of insulin receptor β, phosphorylated glycogen synthase kinase 3β, and β-catenin in the subventricular zone of diabetic rats. Altogether, these results indicate that diabetes mellitus causes insulin deficiency, which negatively regulates glycogen synthase kinase 3β and enhances β-catenin degradation, with these changes inhibiting neural stem cell proliferation. Further, these signaling pathways affect proliferation and differentiation of neural stem cells in the subventricular zone. Dysfunction of subventricular zone neural stem cells causes a decline in olfactory bulb structural plasticity and impairs olfactory sensitivity in diabetic rats.

Δ9-Tetrahydrocannabinol Experience Influences ΔFosB and Downstream Gene Expression in Prefrontal Cortex.

Introduction: Repeated administration of abused drugs, including Δ9-tetrahydrocannabinol (THC), induces the stable transcription factor ΔFosB in dopaminergic terminal field regions of the mesolimbic system. These studies investigated the effect of prior repeated THC treatment on THC-induced ΔFosB expression and regulation of downstream targets in the forebrain.Methods: Mice received THC (10 mg/kg) or vehicle twice daily for 13 days, and then half of each group received a single injection of THC or vehicle 45 min before brain collection. ΔFosB messenger RNA (mRNA) and protein were measured by polymerase chain reaction and immunoblotting, respectively. Potential downstream targets of ΔFosB induction were measured by immunoblot.Results: THC injection in mice with a history of repeated THC treatment enhanced ΔFosB expression as compared with vehicle in the prefrontal cortex (PFC), nucleus accumbens (NAc), and amygdala. This change occurred concomitantly with an increase in ΔFosB mRNA in the PFC and NAc. THC injection in mice with a history of repeated THC treatment increased expression of cyclin-dependent kinase 5 (Cdk5) and its regulatory protein p35 only in the PFC. This increase in Cdk5 and p35 expression in PFC was also found in mice that had only received repeated THC administration, suggesting that this effect might be due to induction of ΔFosB. Extracellular signal-regulated kinase (ERK) phosphorylation was increased in PFC after THC injection in repeated THC-treated mice. Phosphorylation of glycogen synthase kinase-3β (GSK3β), a Cdk5 target, was reduced in PFC after repeated THC treatment regardless of THC history, and phosphorylation of dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) at the Cdk5-regulated threonine 75 site was unchanged.Conclusion: These results suggest that a history of repeated THC administration primes THC-mediated induction of ΔFosB in the NAc and PFC, and that expression of both downstream targets of ΔFosB (e.g., Cdk5 and p35) and upstream activators (e.g., pERK) in the PFC is dependent on THC history, which might have functional implications in addiction and neuropsychiatric disease.

Static magnetic fields promote osteoblastic/cementoblastic differentiation in osteoblasts, cementoblasts, and periodontal ligament cells.

PurposeAlthough static magnetic fields (SMFs) have been used in dental prostheses and osseointegrated implants, their biological effects on osteoblastic and cementoblastic differentiation in cells involved in periodontal regeneration remain unknown. This study was undertaken to investigate the effects of SMFs (15 mT) on the osteoblastic and cementoblastic differentiation of human osteoblasts, periodontal ligament cells (PDLCs), and cementoblasts, and to explore the possible mechanisms underlying these effects.MethodsDifferentiation was evaluated by measuring alkaline phosphatase (ALP) activity, mineralized nodule formation based on Alizarin red staining, calcium content, and the expression of marker mRNAs assessed by reverse transcription polymerase chain reaction (RT-PCR). Signaling pathways were analyzed by western blotting and immunocytochemistry.ResultsThe activities of the early marker ALP and the late markers matrix mineralization and calcium content, as well as osteoblast- and cementoblast-specific gene expression in osteoblasts, PDLCs, and cementoblasts were enhanced. SMFs upregulated the expression of Wnt proteins, and increased the phosphorylation of glycogen synthase kinase-3β (GSK-3β) and total β-catenin protein expression. Furthermore, p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK), and nuclear factor-κB (NF-κB) pathways were activated.ConclusionsSMF treatment enhanced osteoblastic and/or cementoblastic differentiation in osteoblasts, cementoblasts, and PDLCs. These findings provide a molecular basis for the beneficial osteogenic and/or cementogenic effect of SMFs, which could have potential in stimulating bone or cementum formation during bone regeneration and in patients with periodontal disease.

Fexaramine Inhibits Receptor Activator of Nuclear Factor-κB Ligand-induced Osteoclast Formation via Nuclear Factor of Activated T Cells Signaling Pathways.

BackgroundOsteoclasts are bone resorbing cells and are responsible for bone erosion in diseases as diverse as osteoporosis, periodontitis, and rheumatoid arthritis. Fexaramine has been developed as an agonist for the farnesoid X receptor (FXR). This study investigated the effects of fexaramine on receptor activator of nuclear factor (NF)-κB ligand (RANKL)-induced osteoclast formation and signaling pathways.MethodsOsteoclasts were formed by culturing mouse bone marrow-derived macrophages (BMMs) with macrophage colony-stimulating factor (M-CSF) and RANKL. Bone resorption assays were performed using dentine slices. The mRNA expression level was analyzed by real-time polymerase chain reaction. Western blotting assays were conducted to detect the expression or activation level of proteins. Lipopolysaccharide-induced osteoclast formation was performed using a mouse calvarial model.ResultsFexaramine inhibited RANKL-induced osteoclast formation, without cytotoxicity. Furthermore, fexaramine diminished the RANKL-stimulated bone resorption. Mechanistically, fexaramine blocked the RANKL-triggered p38, extracellular signal-regulated kinase, and glycogen synthase kinase 3β phosphorylation, resulting in suppressed expression of c-Fos and NF of activated T cells (NFATc1). Consistent with the in vitro anti-osteoclastogenic effect, fexaramine suppressed lipopolysaccharide-induced osteoclast formation in the calvarial model.ConclusionsThe present data suggest that fexaramine has an inhibitory effect on osteoclast differentiation and function, via downregulation of NFATc1 signaling pathways. Thus, fexaramine could be useful for the treatment of bone diseases associated with excessive bone resorption.

Attenuation of Oxidative Stress-Induced Osteoblast Apoptosis by Curcumin is Associated with Preservation of Mitochondrial Functions and Increased Akt-GSK3β Signaling

Background: Osteoblast apoptosis induced by oxidative stress plays a crucial role in the development and progression of osteoporosis. Curcumin, a natural antioxidant isolated from Curcuma longa, has highly protective effects against osteoporosis. However, the effects of curcumin on oxidative stress-induced osteoblast apoptosis remain unclear. This study aimed to explore the effect of curcumin on hydrogen peroxide (H₂O₂) induced osteoblast apoptosis and the underlying mechanisms. Methods: An osteoblastic cell line (Saos-2) was exposed to various concentrations of H₂O₂ with or without curcumin treatment. Cell viability was evaluated by MTT assays. The apoptosis rate was analyzed by flow cytometry and TUNEL assays. Mitochondrial ROS and membrane potential were determined using a fluorescence microscope. Mitochondrial respiratory enzyme activity was measured using a spectrophotometer. Protein levels were detected by western blotting. Results: Curcumin was cytoprotective because it greatly improved the viability of Saos-2 cells exposed to H₂O₂ and attenuated H₂O₂-induced apoptosis. Curcumin treatment also preserved the mitochondrial redox potential, decreased the mitochondrial oxidative status, and improved the mitochondrial membrane potential and functions. Furthermore, curcumin treatment markedly increased levels of phosphorylated protein kinase B (Akt) and phosphorylated glycogen synthase kinase-3β (GSK3β). Conclusion: Curcumin administration ameliorates oxidative stress-induced apoptosis in osteoblasts by preserving mitochondrial functions and activation of Akt-GSK3β signaling. These data provide experimental evidence supporting the clinical use of curcumin for prevention or treatment of osteoporosis.

The mechanism of Naringin-enhanced remyelination after spinal cord injury.

Our previous study revealed that intragastric administration of naringin improved remyelination in rats with spinal cord injury and promoted the recovery of neurological function of the injured spinal cord. This study sought to reveal the mechanisms by which naringin improves oligodendrocyte precursor cell differentiation and maturation, and promotes remyelination. Spinal cord injury was induced in rats by the weight-drop method. Naringin was intragastrically administered daily (20, 40 mg/kg) for 4 weeks after spinal cord injury induction. Behavioral assessment, histopathological staining, immunofluorescence spectroscopy, ultrastructural analysis and biochemical assays were employed. Naringin treatment remarkably mitigated demyelination in the white matter, increased the quality of myelinated nerve fibers and myelin sheath thickness, promoted oligodendrocyte precursor cell differentiation by upregulating the expression of NKx2.2 and 2'3'-cyclic nucleotide 3'-phosphodiesterase, and inhibited β-catenin expression and glycogen synthase kinase-3β (GSK-3β) phosphorylation. These findings indicate that naringin treatment regulates oligodendrocyte precursor cell differentiation and promotes remyelination after spinal cord injury through the β-catenin/GSK-3β signaling pathway.

Prohibitin confers cytoprotection against ISO-induced hypertrophy in H9c2 cells via attenuation of oxidative stress and modulation of Akt/Gsk-3β signaling.

Numerous hypertrophic stimuli, including β-adrenergic agonists such as isoproterenol (ISO), result in generation of reactive oxygen species (ROS) and alteration in the mitochondrial membrane potential (Δψ) leading to oxidative stress. This process is well associated with phosphorylation of thymoma viral proto-oncogene Akt (Ser473) and glycogen synthase kinase-3β (Gsk-3β) (Ser9), with resultant inactivation of Gsk-3β. In the present study, we found that the protective defensive role of prohibitin (PHB) against ISO-induced hypertrophic response in rat H9c2 cells is via attenuation of oxidative stress-dependent signaling pathways. The intracellular levels of mitochondrial membrane potential along with cellular ROS levels and mitochondrial superoxide generation were determined. In order to understand the regulation of Akt/Gsk-3β signaling pathway, we carried out immmunoblotting for key proteins of the pathway such as PTEN, PI3K, phosphorylated, and unphosphorylated forms of Akt, Gsk-3β, and immunofluorescence experiments of p-Gsk-3β. Enforced expression of PHB in ISO-treated H9c2 cells suppressed cellular ROS production with mitochondrial superoxide generation and enhanced the mitochondrial membrane potential resulting in suppression of oxidative stress which likely offered potent cellular protection, led to the availability of more healthy cells, and also, significant constitutive activation of Gsk-3β via inactivation of Akt was observed. Knockdown of PHB expression using PHB siRNA in control H9c2 cells reversed these effects. Overall, our results demonstrate that PHB confers cytoprotection against oxidative stress in ISO-induced hypertrophy and this process is associated with modulation of Akt/Gsk-3β signaling mechanisms as evident from our PHB overexpression and knockdown experiments.