Adiponectin improves amyloid-β 31-35-induced circadian rhythm disorder in mice

Adiponectin is an adipocyte-derived hormone, which is closely associated with the development of Alzheimer's disease (AD) and has potential preventive and therapeutic significance. In the present study, we explored the relationship between adiponectin and circadian rhythm disorder in AD, the effect of adiponectin on the abnormal expression of Bmal1 mRNA/protein induced by amyloid-β protein 31-35 (Aβ31-35), and the underlying mechanism of action. We found that adiponectin-knockout mice exhibited amyloid-β deposition, circadian rhythm disorders and abnormal expression of Bmal1. Adiponectin ameliorated the abnormal expression of the Bmal1 mRNA/protein caused by Aβ31-35 by inhibiting the activity of glycogen synthase kinase 3β (GSK3β). These results suggest that adiponectin deficiency could induce circadian rhythm disorders and abnormal expression of the Bmal1 mRNA/protein, whilst exogenous administration of adiponectin may improve Aβ31-35-induced abnormal expression of Bmal1 by inhibiting the activity of GSK3β, thus providing a novel idea for the treatment of AD.     

Interaction of SOCS3 with NonO attenuates IL-1beta-dependent MUC8 gene expression

The intracellular negatively regulatory mechanism which affects IL-1β-induced MUC8 gene expression remains unclear. We found that SOCS3 overexpression suppressed IL-1β-induced MUC8 gene expression in NCI-H292 cells, whereas silencing of SOCS3 restored IL-1β-induced MUC8 gene expression. Sequentially activated ERK1/2, RSK1, and CREB by IL-1β were not affected by SOCS3, indicating that SOCS3 has an independent mechanism of action. Using immunoprecipitaion and nano LC mass analysis, we found that SOCS3 bound NonO (non-POU-domain containing, octamer-binding domain protein) in the absence of IL-1β, whereas IL-1β treatment dissociated the direct binding of SOCS3 and NonO. A dominant-negative SOCS3 mutant (Y204F/Y221F) did not bind to NonO. Interestingly, SOCS3 overexpression dramatically suppressed MUC8 gene expression in cells transfected with wild-type or siRNA of NonO. Moreover, silencing of SOCS3 dramatically increased NonO-mediated MUC8 gene expression caused by IL-1β compared to NonO overexpression alone, suggesting that SOCS3 acts as a suppressor by regulating the action of NonO.     

Identification of novel TGF-β regulated genes with pro-migratory roles

Transforming growth factor-β (TGF-β) signaling plays fundamental roles in the development and homeostasis of somatic cells. Dysregulated TGF-β signaling contributes to cancer progression and relapse to therapies by inducing epithelial-to-mesenchymal transition (EMT), enriching cancer stem cells, and promoting immunosuppression. Although many TGF-β-regulated genes have been identified, only a few datasets were obtained by next-generation sequencing. In this study, we performed RNA-sequencing analysis of MCF10A cells and identified 1166 genes that were upregulated and 861 genes that were downregulated by TGF-β. Gene set enrichment analysis revealed that focal adhesion and metabolic pathways were the top enriched pathways of the up- and downregulated genes, respectively. Genes in these pathways also possess significant predictive value for renal cancers. Moreover, we confirmed that TGF-β induced expression of MICAL1 and 2, and the histone demethylase, KDM7A, and revealed their regulatory roles on TGF-β-induced cell migration. We also show a critical effect of KDM7A in regulating the acetylation of H3K27 on TGF-β-induced genes. In sum, this study identified novel effectors that mediate the pro-migratory role of TGF-β signaling, paving the way for future studies that investigate the function of MICAL family members in cancer and the novel epigenetic mechanisms downstream TGF-β signaling.     

Soluble oligomers of amyloid-β peptide disrupt membrane trafficking of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor contributing to early synapse dysfunction

β-Amyloid (Aβ), a peptide generated from the amyloid precursor protein, is widely believed to underlie the pathophysiology of Alzheimer disease (AD). Emerging evidences suggest that soluble Aβ oligomers adversely affect synaptic function, leading to cognitive failure associated with AD. The Aβ-induced synaptic dysfunction has been attributed to the synaptic removal of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors (AMPARs). However, the molecular mechanisms underlying the loss of AMPAR induced by Aβ at synapses are largely unknown. In this study we have examined the effect of Aβ oligomers on phosphorylated GluA1 at serine 845, a residue that plays an essential role in the trafficking of AMPARs toward extrasynaptic sites and the subsequent delivery to synapses during synaptic plasticity events. We found that Aβ oligomers reduce basal levels of Ser-845 phosphorylation and surface expression of AMPARs affecting AMPAR subunit composition. Aβ-induced GluA1 dephosphorylation and reduced receptor surface levels are mediated by an increase in calcium influx into neurons through ionotropic glutamate receptors and activation of the calcium-dependent phosphatase calcineurin. Moreover, Aβ oligomers block the extrasynaptic delivery of AMPARs induced by chemical synaptic potentiation. In addition, reduced levels of total and phosphorylated GluA1 are associated with initial spatial memory deficits in a transgenic mouse model of AD. These findings indicate that Aβ oligomers could act as a synaptic depressor affecting the mechanisms involved in the targeting of AMPARs to the synapses during early stages of the disease.     

TGF-β induces global changes in DNA methylation during the epithelial-to-mesenchymal transition in ovarian cancer cells

A key step in the process of metastasis is the epithelial-to-mesenchymal transition (EMT). We hypothesized that epigenetic mechanisms play a key role in EMT and to test this hypothesis we analyzed global and gene-specific changes in DNA methylation during TGF-β-induced EMT in ovarian cancer cells. Epigenetic profiling using the Infinium HumanMethylation450 BeadChip (HM450) revealed extensive (P < 0.01) methylation changes after TGF-β stimulation (468 and 390 CpG sites altered at 48 and 120 h post cytokine treatment, respectively). The majority of gene-specific TGF-β-induced methylation changes occurred in CpG islands located in or near promoters (193 and 494 genes hypermethylated at 48 and 120 h after TGF-β stimulation, respectively). Furthermore, methylation changes were sustained for the duration of TGF-β treatment and reversible after the cytokine removal. Pathway analysis of the hypermethylated loci identified functional networks strongly associated with EMT and cancer progression, including cellular movement, cell cycle, organ morphology, cellular development, and cell death and survival. Altered methylation and corresponding expression of specific genes during TGF-β-induced EMT included CDH1 (E-cadherin) and COL1A1 (collagen 1A1). Furthermore, TGF-β induced both expression and activity of DNA methyltransferases (DNMT) -1, -3A, and -3B, and treatment with the DNMT inhibitor SGI-110 prevented TGF-β-induced EMT. These results demonstrate that dynamic changes in the DNA methylome are implicated in TGF-β-induced EMT and metastasis. We suggest that targeting DNMTs may inhibit this process by reversing the EMT genes silenced by DNA methylation in cancer.     

Reduced Smoothened level rescues Aβ-induced memory deficits and neuronal inflammation in animal models of Alzheimer's disease

Emerging evidence suggests that neuro-inflammation begins early and drives the pathogenesis of Alzheimer's disease(AD), and anti-inflammatory therapies are under clinical development. However,several anti-inflammatory compounds failed to improve memory in clinical trials, indicating that reducing inflammation alone might not be enough. On the other hand, neuro-inflammation is implicated in a number of mental disorders which share the same therapeutic targets. Based on these observations,we screened a batch of genes related with mental disorder and neuro-inflammation in a classical olfactory conditioning in an amyloid beta(Aβ) overexpression fly model. A Smoothened(SMO) mutant was identified as a genetic modifier of Aβ toxicity in 3-min memory and downregulation of SMO rescued Aβ induced 3-min and 1-h memory deficiency. Also, Aβ activated innate inflammatory response in fly by increasing the expression of antimicrobial peptides, which were alleviated by downregulating SMO.Furthermore, pharmaceutical administration of a SMO antagonist LDE rescued Aβ-induced upregulation of SMO in astrocytes of mouse hippocampus, improved memory in Morris water maze(MWM), and reduced expression of astrocyte secreting pro-inflammatory factors IL-1 b, TNFa and the microglia marker IBA-1 in an APP/PS1 transgenic mouse model. Our study suggests that SMO is an important conserved modulator of Aβ toxicity in both fly and mouse models of AD.     

Autophagy protects neuron from Aβ-induced cytotoxicity

Autophagy is a degradation pathway for the turnover of dysfunctional organelles or aggregated proteins in cells. Extracellular accumulation of β-amyloid peptide has been reported to be a major cause of Alzheimer's disease (AD) and large numbers of autophagic vacuoles accumulate in the brain of AD patient. However, how autophagic process is involved in Aβ-induced neurotoxicity and how Aβ peptide is transported into neuron and metabolized is still unknown. In order to study the role of autophagic process in Aβ-induced neurotoxicity, EGFP-LC3 was over-expressed in SH-SY5Y cells (SH-SY5Y/pEGFP-LC3). It was found that treatment with Aβ_25-35, Aβ_1-42 or serum-starvation induced strong autophagy response in SH-SY5Y/pEGFP-LC3. Confocal double-staining image showed that exogenous application of Aβ1-42 in medium caused the co-localization of Aβ_1-42 with LC3 in neuronal cells. Concomitant treatment of Aβ with a selective α7nAChR antagonist, α-bungarotoxin (α-BTX), enhanced Aβ-induced neurotoxicity in SH-SY5Y cells. On the other hand, nicotine (nAChR agonist) enhanced the autophagic process and also inhibited cell death following Aβ application. In addition, nicotine but not α-BTX increased primary hippocampal neuronal survival following Aβ treatment. Furthermore, using Atg7 siRNA to inhibit autophagosome formation in an early step or α7nAChR siRNA to knockdown α7nAChR significantly enhanced Aβ-induced neurotoxicity. Confocal double-staining image shows that nicotine treatment in the presence of Aβ enhanced the co-localization of α7nAChR with autophagosomes. These results suggest that α7nAChR may act as a carrier to bind with eAβ and internalize into cytoplasm and further inhibit Aβ-induced neurotoxicity via autophagic degradation pathway. Our results suggest that autophagy process plays a neuroprotective role against Aβ-induced neurotoxicity. Defect in autophagic regulation or Aβ-α7nAChR transport system may impair the clearance of Aβ and enhance the neuronal death.     

miR-6076 targets BCL6 in SH-SY5Y cells to regulate amyloid-β-induced neuronal damage

Amyloid-β_1-42 (Aβ_1-42) is strongly associated with Alzheimer's disease (AD). The aim of this study is to elucidate whether and how miR-6076 participates in the modulation of amyloid-β (Aβ)-induced neuronal damage. To construct the neuronal damage model, SH-SY5Y cells were treated with Aβ_1-42. By qRT-PCR, we found that miR-6076 is significantly upregulated in Aβ_1-42-treated SH-SY5Y cells. After miR-6076 inhibition, p-Tau and apoptosis levels were downregulated, and cell viability was increased. Through online bioinformatics analysis, we found that B-cell lymphoma 6 (BCL6) was a directly target of miR-6076 via dual-luciferase reporter assay. BCL6 overexpression mediated the decrease in elevated p-Tau levels and increased viability in SH-SY5Y cells following Aβ1-42 treatment. Our results suggest that down-regulation of miR-6076 could attenuate Aβ_1-42-induced neuronal damage by targeting BCL6, which provided a possible target to pursue for prevention and treatment of Aβ-induced neuronal damage in AD.     

Aβ stimulates microglial activation through antizyme-dependent downregulation of ornithine decarboxylase

Alzheimer's disease (AD) is one of the most prevalent neurodegenerative disorders. Its pathology is associated with the deposition of amyloid β (Aβ), an abnormal extracellular peptide. Moreover, its pathological progression is closely accompanied by neuroinflammation. Specifically, Aβ-associated microglial overactivation may have the central role in AD pathogenesis. Interestingly, arginine metabolism may contribute to the equilibrium between M1 and M2 microglia. However, little is known about the involvement of arginine metabolism in Aβ-induced microglial neuroinflammation and neurotoxicity. Moreover, the underlying mechanism by which Aβ induces the transition of microglia to the M1 phenotype remains unclear. In this study, we investigated the role of Aβ in mediating microglial activation and polarization both in vitro and in vivo. Our results demonstrated that under the Aβ treatment, ornithine decarboxylase (ODC), a rate-limiting enzyme in the regulation of arginine catabolism, regulates microglial activation by altering the antizyme (AZ) + 1 ribosomal frameshift. Furthermore, the restoration of ODC protein expression levels has profound effects on inhibition of Aβ-induced M1 markers and thus attenuates microglial-mediated cytotoxicity. Altogether, our findings suggested that Aβ may contribute to M1-like activation by disrupting the balance between ODC and AZ in microglia.     

Resveratrol protects rats from Aβ-induced neurotoxicity by the reduction of iNOS expression and lipid peroxidation

Alzheimer disease (AD) is an age-dependent neurodegenerative disease characterized by the formation of β-amyloid (Aβ)-containing senile plaque. The disease could be induced by the administration of Aβ peptide, which was also known to upregulate inducible nitric oxide synthase (iNOS) and stimulate neuronal apoptosis. The present study is aimed to elucidate the cellular effect of resveratrol, a natural phytoestrogen with neuroprotective activities, on Aβ-induced hippocampal neuron loss and memory impairment. On adult Sprague-Dawley rats, we found the injection of Aβ could result in a significant impairment in spatial memory, a marked increase in the cellular level of iNOS and lipid peroxidation, and an apparent decrease in the expression of heme oxygenase-1 (HO-1). By combining the treatment with Aβ, resveratrol was able to confer a significant improvement in spatial memory, and protect animals from Aβ-induced neurotoxicity. These neurological protection effects of resveratrol were associated with a reduction in the cellular levels of iNOS and lipid peroxidation and an increase in the production of HO-1. Moreover, the similar neurological and cellular response were also observed when Aβ treatment was combined with the administration of a NOS inhibitor, N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME). These findings strongly implicate that iNOS is involved in the Aβ-induced lipid peroxidation and HO-1 downregulation, and resveratrol protects animals from Aβ-induced neurotoxicity by suppressing iNOS production.     

Both type I and II IFN induce insulin resistance by inducing different isoforms of SOCS expression in 3T3-L1 adipocytes

Although elevation of the blood glucose level is a causal adverse effect of treatment with interferon (IFN), the precise underlying molecular mechanism is largely unknown. We examined the effects of type I and type II IFN (IFN-β and IFN-γ) on insulin-induced metabolic signaling leading to glucose uptake in 3T3-L1 adipocytes. IFN-β suppressed insulin-induced tyrosine phosphorylation of IRS-1 without affecting its expression, whereas IFN-γ reduced both the protein level and tyrosine phosphorylation. Although both IFNs stimulated phosphorylation of STAT1 (at Tyr(701)) and STAT3 (at Tyr(705)) after treatment for 30 min, subsequent properties of induction of the SOCS isoform were different. IFN-β preferentially induced SOCS1 rather than SOCS3, whereas IFN-γ strongly induced SOCS3 expression alone. In addition, adenovirus-mediated overexpression of either SOCS1 or SOCS3 inhibited insulin-induced tyrosine phosphorylation of IRS-1, whereas the reduction of IRS-1 protein was observed only in SOCS3-expressed cells. Notably, IFN-β-induced SOCS1 expression and suppression of insulin-induced tyrosine phosphorylation of IRS-1 were attenuated by siRNA-mediated knockdown of STAT1. In contrast, adenovirus-mediated expression of a dominant-negative STAT3 (F-STAT3) attenuated IFN-γ-induced SOCS3 expression, reduction of IRS-1 protein, and suppression of insulin-induced glucose uptake but did not have any effect on the IFN-β-mediated SOCS1 expression and inhibition of insulin-induced glucose uptake. Interestingly, pretreatment of IFN-γ with IL-6 synergistically suppressed insulin signaling, even when IL-6 alone had no significant effect. These results indicate that type I and type II IFN induce insulin resistance by inducing distinct SOCS isoforms, and IL-6 synergistically augments IFN-γ-induced insulin resistance by potentiating STAT3-mediated SOCS3 induction in 3T3-L1 adipocytes.     

Accelerated epithelial cell senescence in IPF and the inhibitory role of SIRT6 in TGF-β-induced senescence of human bronchial epithelial cells

Reepithelialization of remodeled air spaces with bronchial epithelial cells is a prominent pathological finding in idiopathic pulmonary fibrosis (IPF) and is implicated in IPF pathogenesis. Recent studies suggest that epithelial senescence is a risk factor for development of IPF, indicating such reepithelialization may be influenced by the acceleration of cellular senescence. Among the sirtuin (SIRT) family, SIRT6, a class III histone deacetylase, has been demonstrated to antagonize senescence. We evaluated the senescence of bronchiolization in association with SIRT6 expression in IPF lung. Senescence-associated β-galactosidase staining and immunohistochemical detection of p21 were performed to evaluate cellular senescence. As a model for transforming growth factor (TGF)-β-induced senescence of abnormal reepithelialization, we used primary human bronchial epithelial cells (HBEC). The changes of SIRT6, p21, and interleukin (IL)-1β expression levels in HBEC, as well as type I collagen expression levels in fibroblasts, were evaluated. In IPF lung samples, an increase in markers of senescence and SIRT6 expression was found in the bronchial epithelial cells lining cystically remodeled air spaces. We found that TGF-β induced senescence in primary HBEC by increasing p21 expression, and, whereas TGF-β also induced SIRT6, it was not sufficient to inhibit cellular senescence. However, overexpression of SIRT6 efficiently inhibited TGF-β-induced senescence via proteasomal degradation of p21. TGF-β-induced senescent HBEC secreted increased amounts of IL-1β, which was sufficient to induce myofibroblast differentiation in fibroblasts. These findings suggest that accelerated epithelial senescence plays a role in IPF pathogenesis through perpetuating abnormal epithelial-mesenchymal interactions, which can be antagonized by SIRT6.