Sustained IRE1 and ATF6 signaling is important for survival of melanoma cells undergoing ER stress

Apoptosis triggered by endoplasmic reticulum (ER) stress is associated with rapid attenuation of the IRE1α and ATF6 pathways but persistent activation of the PERK branch of the unfolded protein response (UPR) in cells. However, melanoma cells are largely resistant to ER stress-induced apoptosis, suggesting that the kinetics and durations of activation of the UPR pathways are deregulated in melanoma cells undergoing ER stress. We show here that the IRE1α and ATF6 pathways are sustained along with the PERK signaling in melanoma cells subjected to pharmacological ER stress, and that this is, at least in part, due to increased activation of the MEK/ERK pathway. In contrast to an initial increase followed by rapid reduction in activation of IRE1α and ATF6 signaling in control cells that were relatively sensitive to ER stress-induced apoptosis, activation of IRE1α and ATF6 by the pharmacological ER stress inducer tunicamycin (TM) or thapsigargin (TG) persisted in melanoma cells. On the other hand, the increase in PERK signaling lasted similarly in both types of cells. Sustained activation of IRE1α and ATF6 signaling played an important role in protecting melanoma cells from ER stress-induced apoptosis, as interruption of IRE1α or ATF6 rendered melanoma cells sensitive to apoptosis induced by TM or TG. Inhibition of MEK partially blocked IRE1α and ATF6 activation, suggesting that MEK/ERK signaling contributed to sustained activation of IRE1α and ATF6. Taken together, these results identify sustained activation of the IRE1α and ATF6 pathways of the UPR driven by the MEK/ERK pathway as an important protective mechanism against ER stress-induced apoptosis in melanoma cells.     

Rapamycin decreases tau phosphorylation at Ser214 through regulation of cAMP-dependent kinase

Preventing or reducing tau hyperphosphorylation is considered to be a therapeutic strategy in the treatment of Alzheimer’s disease (AD). Rapamycin may be a potential therapeutic agent for AD, because the rapamycin-induced autophagy may enhance the clearance of the hyperphosphorylated tau. However, recent rodent studies show that the protective effect of rapamycin may not be limited in the autophagic clearance of the hyperphosphorylated tau. Because some tau-related kinases are targets of the mammalian target of rapamycin (mTOR), we assume that rapamycin may regulate tau phosphorylation by regulating these kinases. Our results showed that in human neuroblastoma SH-SY5Y cells, treatment with rapamycin induced phosphorylation of the type IIα regulatory (RIIα) subunit of cAMP-dependent kinase (PKA). Rapamycin also induced nuclear translocation of the catalytic subunits (Cat) of PKA and decreases in tau phosphorylation at Ser214 (pS214). The above effects of rapamycin were prevented by pretreatment with the mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor U0126. In addition, these effects of rapamycin might not depend on the level of tau expression, because similar results were obtained in both the non-tau-expressing wild type human embryonic kidney 293 (HEK293) cells and HEK293 cells stably transfected with the longest isoform of recombinant human tau (tau441; HEK293/tau441). These findings suggest that rapamycin decreases pS214 via regulation of PKA. Because tau phosphorylation at Ser214 may prime tau for further phosphorylation by other kinases, our findings provide a novel possible mechanism by which rapamycin reduces or prevents tau hyperphosphorylation.     

Pin1-dependent prolyl isomerization modulates the stress-induced phosphorylation of high molecular weight neurofilament protein

Aberrant phosphorylation of neuronal cytoskeletal proteins is a key pathological event in neurodegenerative disorders such as Alzheimer disease (AD) and amyotrophic lateral sclerosis, but the underlying mechanisms are still unclear. Previous studies have shown that Pin1, a peptidylprolyl cis/trans-isomerase, may be actively involved in the regulation of Tau hyperphosphorylation in AD. Here, we show that Pin1 modulates oxidative stress-induced NF-H phosphorylation. In an in vitro kinase assay, the addition of Pin1 substantially increased phosphorylation of NF-H KSP repeats by proline-directed kinases, Erk1/2, Cdk5/p35, and JNK3 in a concentration-dependent manner. In vivo, dominant-negative (DN) Pin1 and Pin1 small interfering RNA inhibited epidermal growth factor-induced NF-H phosphorylation. Because oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, we studied the role of Pin1 in stressed cortical neurons and HEK293 cells. Both hydrogen peroxide (H(2)O(2)) and heat stresses induce phosphorylation of NF-H in transfected HEK293 cells and primary cortical cultures. Knockdown of Pin1 by transfected Pin1 short interference RNA and DN-Pin1 rescues the effect of stress-induced NF-H phosphorylation. The H(2)O(2) and heat shock induced perikaryal phospho-NF-H accumulations, and neuronal apoptosis was rescued by inhibition of Pin1 in cortical neurons. JNK3, a brain-specific JNK isoform, is activated under oxidative and heat stresses, and inhibition of Pin1 by Pin1 short interference RNA and DN-Pin1 inhibits this pathway. These results implicate Pin1 as a possible modulator of stress-induced NF-H phosphorylation as seen in neurodegenerative disorders like AD and amyotrophic lateral sclerosis. Thus, Pin1 may be a potential therapeutic target for these diseases.     

ER signaling in unfolded protein response

Abnormally folded proteins are susceptible to aggregation and accumulation in cells, ultimately leading to cell death. To protect cells against such dangers, expression of various genes including molecular chaperones can be induced and ER-associated protein degradation (ERAD) activated in response to the accumulation of unfolded protein in the endoplasmic reticulum (ER). This is known as the unfolded protein response (UPR). ERAD requires retrograde transport of unfolded proteins from the ER back to the cytosol via the translocon for degradation by the ubiquitin-proteasome system. Hrd1p is a UPR-induced ER membrane protein that acts as a ubiquitin ligase (E3) in the ERAD system. Hrd3p interacts with and stabilizes Hrd1p. We have isolated and identified human homologs (HRD1 and SEL1/HRD3) of Saccharomyces cerevisiae Hrd1p and Hrd3p. Human HRD1 and SEL1 were up-regulated in response to ER stress and overexpression of human IRE1 and ATF6, which are ER stress-sensor molecules in the ER. HEK293T cells overexpressing HRD1 showed resistance to ER stress-induced cell death. These results suggest that HRD1 and SEL1 are up-regulated by the UPR and contribute to protection against the ER stress-induced cell death by degrading unfolded proteins accumulated in the ER.     

抑制聚ADP-核糖聚合酶-1活性降低HEK293/tau441细胞tau蛋白的磷酸化

为了研究聚ADP-核糖聚合酶-1 fpoly(ADP-ribose)polymerase-1,PARP-1]对微管相关蛋白tau磷酸化水平的影响,本实验分别用不同剂量(0.5,1,2,4 mmol/L)的PARP-1的抑制剂3-氨基苯甲酰胺(3-aminobenzamide,3-AB)处理稳定表达tau441蛋白的HE...     

Stress-induced hyperphosphorylation of tau in the mouse brain

We previously showed that starvation causes reversible hyperphosphorylation of tau in the mouse brain. To explore possible involvement of stress in tau hyperphosphorylation quantitative analysis of phosphorylated tau in four brain regions of mice subjected to cold water stress (CWS) was made by immunoblot analyses using phosphorylation-dependent antibodies directed to eight sites on tau known to be hyperphosphorylated in the brain of Alzheimer's disease (AD) patients. Ser199, Ser202/Thr205, Thr231/Ser235 were hyperphosphorylated 20 and 40 min after CWS. The response was pronounced in the hippocampus and cerebral hemisphere, but weak in the cerebellum in parallel with the regional vulnerability in AD. Among the regulatory phosphorylation of protein kinases studied, a transient phosphorylation of tau protein kinase I/glycogen synthase kinase 3beta at Ser9 was most conspicuous.     

Crocin and Quercetin protect HCT116 and HEK293 cells from Zearalenone-induced apoptosis by reducing endoplasmic reticulum stress

Mycotoxins are considered to be significant contaminants of food and animal feed. Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by several species of Fusarium in cereals and agricultural products. ZEN has been shown to be cytotoxic, genotoxic, and mutagenic in different cell types. In the present study, we investigated the involvement of endoplasmic reticulum (ER) stress in ZEN-mediated toxicity in human intestine (HCT116) and kidney (HEK293) cells and evaluated the effects of the two common dietary compounds Quercetin (QUER) and Crocin (CRO). We show that ZEN treatment induces ER stress and activates the unfolded protein response (UPR) as evidenced by XBP1 mRNA splicing and upregulation of GRP78, ATF4, GADD34, PDIA6, and CHOP. Activation of the ER stress response is associated with activation of the mitochondrial pathway of apoptosis. This apoptotic process is characterized by an increase in ROS generation and lipid peroxidation, a loss of mitochondrial transmembrane potential (ΔΨm), and an activation of caspases and DNA damages. We also demonstrate that the antioxidant properties of QUER and CRO help to prevent ER stress and reduce ZEN-induced apoptosis in HCT116 and HEK293 cells. Our results suggest that antioxidant molecule might be helpful to prevent ZEN-induced ER stress and toxicity.     

A potent mechanism-inspired O-GlcNAcase inhibitor that blocks phosphorylation of tau in vivo

Pathological hyperphosphorylation of the microtubule-associated protein tau is characteristic of Alzheimer's disease (AD) and the associated tauopathies. The reciprocal relationship between phosphorylation and O-GlcNAc modification of tau and reductions in O-GlcNAc levels on tau in AD brain offers motivation for the generation of potent and selective inhibitors that can effectively enhance O-GlcNAc in vertebrate brain. We describe the rational design and synthesis of such an inhibitor (thiamet-G, K(i) = 21 nM; 1) of human O-GlcNAcase. Thiamet-G decreased phosphorylation of tau in PC-12 cells at pathologically relevant sites including Thr231 and Ser396. Thiamet-G also efficiently reduced phosphorylation of tau at Thr231, Ser396 and Ser422 in both rat cortex and hippocampus, which reveals the rapid and dynamic relationship between O-GlcNAc and phosphorylation of tau in vivo. We anticipate that thiamet-G will find wide use in probing the functional role of O-GlcNAc in vertebrate brain, and it may also offer a route to blocking pathological hyperphosphorylation of tau in AD.     

T-cadherin attenuates the PERK branch of the unfolded protein response and protects vascular endothelial cells from endoplasmic reticulum stress-induced apoptosis

Endoplasmic reticulum (ER) stress activated by perturbations in ER homeostasis induces the unfolded protein response (UPR) with chaperon Grp78 as the key activator of UPR signalling. The aim of UPR is to restore normal ER function; however prolonged or severe ER stress triggers apoptosis of damaged cells to ensure protection of the whole organism. Recent findings support an association of ER stress-induced apoptosis of vascular cells with cardiovascular pathologies. T-cadherin (T-cad), an atypical glycosylphosphatidylinositol-anchored member of the cadherin superfamily is upregulated in atherosclerotic lesions. Here we investigate the ability of T-cad to influence UPR signalling and endothelial cell (EC) survival during ER stress. EC were treated with a variety of ER stress-inducing compounds (thapsigargin, dithiothereitol, brefeldin A, tunicamycin, A23187 or homocysteine) and induction of ER stress validated by increases in levels of UPR signalling molecules Grp78 (glucose-regulated protein of 78 kDa), phospho-eIF2α (phosphorylated eukaryotic initiation factor 2α) and CHOP (C/EBP homologous protein). All compounds also increased T-cad mRNA and protein levels. Overexpression or silencing of T-cad in EC respectively attenuated or amplified the ER stress-induced increase in phospho-eIF2α, Grp78, CHOP and active caspases. Effects of T-cad-overexpression or T-cad-silencing on ER stress responses in EC were not affected by inclusion of either N-acetylcysteine (reactive oxygen species scavenger), LY294002 (phosphatidylinositol-3-kinase inhibitor) or SP6000125 (Jun N-terminal kinase inhibitor). The data suggest that upregulation of T-cad on EC during ER stress attenuates the activation of the proapoptotic PERK (PKR (double-stranded RNA-activated protein kinase)-like ER kinase) branch of the UPR cascade and thereby protects EC from ER stress-induced apoptosis.     

Tau phosphorylation and μ-calpain activation mediate the dexamethasone-induced inhibition on the insulin-stimulated Akt phosphorylation

Evidence has suggested that insulin resistance (IR) or high levels of glucocorticoids (GCs) may be linked with the pathogenesis and/or progression of Alzheimer's disease (AD). Although studies have shown that a high level of GCs results in IR, little is known about the molecular details that link GCs and IR in the context of AD. Abnormal phosphorylation of tau and activation of μ-calpain are two key events in the pathology of AD. Importantly, these two events are also related with GCs and IR. We therefore speculate that tau phosphorylation and μ-calpain activation may mediate the GCs-induced IR. Akt phosphorylation at Ser-473 (pAkt) is commonly used as a marker for assessing IR. We employed two cell lines, wild-type HEK293 cells and HEK293 cells stably expressing the longest human tau isoform (tau-441; HEK293/tau441 cells). We examined whether DEX, a synthetic GCs, induces tau phosphorylation and μ-calpain activation. If so, we examined whether the DEX-induced tau phosphorylation and μ-calpain activation mediate the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation. The results showed that DEX increased tau phosphorylation and induced tau-mediated μ-calpain activation. Furthermore, pre-treatment with LiCl prevented the effects of DEX on tau phosphorylation and μ-calpain activation. Finally, both LiCl pre-treatment and calpain inhibition prevented the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation. In conclusion, our study suggests that the tau phosphorylation and μ-calpain activation mediate the DEX-induced inhibition on the insulin-stimulated Akt phosphorylation.     

Nitric oxide induces tau hyperphosphorylation via glycogen synthase kinase-3beta activation

Nitric oxide is associated with neurofibrillary tangle, which is composed mainly of hyperphosphorylated tau in the brain of Alzheimer's disease (AD). However, the role of nitric oxide in tau hyperphosphorylation is unclear. Here we show that nitric oxide produced by sodium nitroprusside (SNP), a recognized donor of nitric oxide, induces tau hyperphosphorylation at Ser396/404 and Ser262 in HEK293/tau441 cells with a simultaneous activation of glycogen synthase kinase-3beta (GSK-3beta). Pretreatment of the cells with 10 mM lithium chloride (LiCl), an inhibitor of GSK-3, 1 h before SNP administration inhibits GSK-3beta activation and prevents tau from hyperphosphorylation. This is the first direct evidence demonstrating that nitric oxide induces AD-like tau hyperphosphorylation in vitro, and GSK-3beta activation is partially responsible for the nitric oxide-induced tau hyperphosphorylation. It is suggested that nitric oxide may be an upstream element of tau abnormal hyperphosphorylation in AD.     

Tau phosphorylation at threonine-175 leads to fibril formation and enhanced cell death: implications for amyotrophic lateral sclerosis with cognitive impairment

Although amyotrophic lateral sclerosis (ALS) can be associated with cognitive impairment (ALSci) as a reflection of frontotemporal lobar degeneration, the basis of this process is unknown. The observation of neuronal and extraneuronal tau deposition in ALSci in addition to a unique tau phosphorylation at Thr175 has suggested that ALSci can be associated with alterations in tau metabolism. We have examined the association between phosphorylation at Thr175 and tau fibril formation. Both soluble and insoluble tau was purified from control, patients with Alzheimer's disease (AD), ALS without cognitive impairment, and ALSci and the tendency to fibril formation assayed ex vivo using the thioflavin S fluorescence assay. The extent of fibril formation was significantly greater in tau derived from ALSci, with ALS-derived tau being intermediate between control and AD-derived tau. Using both Neuro2A and human embryonic kidney (HEK293T) cells, we expressed full-length tau constructs harboring either a pseudophosphorylation at Thr175 (Thr175-Asp-tau), inhibition of Thr175 phosphorylation (Thr175-Ala-tau) or intact tau (wild-type tau). Both tau fibril formation and cell death were significantly enhanced in the presence of Thr175-Asp-tau, regardless of the tau isoform, suggesting that phosphorylation of Thr175 is associated with tau fibril formation in ALSci.     

Estradiol Attenuates Tau Hyperphosphorylation Induced by Upregulation of Protein Kinase-A

Protein kinase A (PKA) plays a crucial role in tau hyperphosphorylation, an early event of Alzheimer disease (AD), and 17β-estradiol replacement in aging women forestalls the onset of AD. However, the role of estradiol in PKA-induced tau hyperphosphorylation is not known. Here, we investigated the effect of 17β-estradiol on cAMP/PKA activity and the PKA-induced tau hyperphosphorylation in HEK293 cells stably expressing tau441. We found that 17β-estradiol effectively attenuated forskolin-induced overactivation of PKA and elevation of cAMP, and thus prevented tau from hyperphosphorylation. These data provide the first evidence that 17β-estradiol can inhibit PKA overactivation and the PKA-induced tau hyperphosphorylation, implying a preventive role of 17β-estradiol in AD-like tau pathology. Xin-An Liu and Ling-Qiang Zhu contributed equally to this work.     

Quercetin Protects against Okadaic Acid-Induced Injury via MAPK and PI3K/Akt/GSK3β Signaling Pathways in HT22 Hippocampal Neurons

Increasing evidence shows that oxidative stress and the hyperphosphorylation of tau protein play essential roles in the progression of Alzheimer’s disease (AD). Quercetin is a major flavonoid that has anti-oxidant, anti-cancer and anti-inflammatory properties. We investigated the neuroprotective effects of quercetin to HT22 cells (a cell line from mouse hippocampal neurons). We found that Okadaic acid (OA) induced the hyperphosphorylation of tau protein at Ser199, Ser396, Thr205, and Thr231 and produced oxidative stress to the HT22 cells. The oxidative stress suppressed the cell viability and decreased the levels of lactate dehydrogenase (LDH), superoxide dismutase (SOD), mitochondria membrane potential (MMP) and Glutathione peroxidase (GSH-Px). It up-regulated malondialdehyde (MDA) production and intracellular reactive oxygen species (ROS). In addition, phosphoinositide 3 kinase/protein kinase B/Glycogen synthase kinase3β (PI3K/Akt/GSK3β) and mitogen activated protein kinase (MAPK) were also involved in this process. We found that pre-treatment with quercetin can inhibited OA-induced the hyperphosphorylation of tau protein and oxidative stress. Moreover, pre-treatment with quercetin not only inhibited OA-induced apoptosis via the reduction of Bax, and up-regulation of cleaved caspase 3, but also via the inhibition of PI3K/Akt/GSK3β, MAPKs and activation of NF-κB p65. Our findings suggest the therapeutic potential of quercetin to treat AD.