Morin hydrate attenuates CSE-induced lipid accumulation,ER stress,and oxidative stress in RPE cells: implications for age-related macular degeneration

Oxidative stress has a key role in the pathogenesis of age-related macular degeneration (AMD). Cigarette smoking is known to the one of the main risk factors of AMD through oxidative stress-mediated endoplasmic reticulum (ER) stress and lipid accumulation in human retinal pigment epithelium (RPE) cells. A number of studies have investigated the benefits of antioxidants in the AMD. However, previous studies have not shown that efficacy of antioxidant in the treatment of AMD. Recent studies demonstrated that morin hydrate (MH) has antioxidant properties, anti-inflammatory, and antiapoptosis effects, however, the protective effects of MH against cigarette smoke extract (CSE)-induced AMD have not been studied in detail. We tested the potential effect of MH against the CSE-induced lipid accumulation in RPE cells and mice RPE layer. Herein, we observed that expose of RPE cells to CSE reduced cell viability, increased the lipid accumulation, ER stress, and oxidative stress. Concomitantly, CSE treatment to mice induced AMD associated histopathological changes, lipid accumulation, ER stress and oxidative stress in RPE layer. MH significantly attenuated cytotoxicity, lipid accumulation, ER stress, and oxidative stress via activated AMPK-Nrf2 signaling pathway in RPE cells and mice RPE layer. In addition, AMPK inhibition reversed MH-induced RPE cell protection against CSE. Thus, we conclude that MH protects RPE cells from CSE through reduced oxidative stress, ER stress, and lipid accumulation via activated AMPK-Nrf2-HO-1 signaling pathway. These findings suggest that MH treatment may be exploited in effective strategy against CSE-induced AMD.     

α - synuclein and Parkinson's disease: the first roadblock

α-synuclein gene mutations are major underlying genetic defects known in familial juvenile onset Parkinson's disease (PD), and α-synuclein is a major constituent of Lewy Bodies, the pathological hallmark of PD. The normal cellular function of α-synuclein has been elusive, and its exact etiological mechanism in causing dopaminergic neuronal death in PD is also not clearly understood. Very recent reports now indicate that mutant or simply over-expressed α-synuclein could cause damage by interfering with particular steps of neuronal membrane traffic. α-synuclein selectively blocks endoplamic reticulum-to-Golgi transport, thus causing ER stress. A screen in a yeast revealed that α-synuclein toxicity could be suppressed by over-expression of the small GTPase Ypt1/Rab1, and that over-expression of the latter rescues neuron loss in invertebrate and mammalian models of α-synuclein-induced neurodegeneration. α-synuclein may also serve a chaperone function for the proper folding of synaptic SNAREs that are important for neurotransmitter release. We discuss these recent results and the emerging pathophysiological interaction of α-synuclein with components of neuronal membrane traffic.     

Production of a recombinant full-length collagen type I α-1 and of a 45-kDa collagen type I α-1 fragment in barley seeds

Recombinant DNA technology can be used to design and express collagen and gelatin-related proteins with predetermined composition and structure. Barley seed was chosen as a production host for a recombinant full-length collagen type I α1 (rCIa1) and a related 45-kDa rCIa1 fragment. The transgenic barley seeds were shown to accumulate both the rCIa1 and the 45-kDa rCIa1 fragment. Even when the amount of the rCIa1 was just above the detection threshold, this work using rCIa1 as a model demonstrated for the first time that barley seed can be used as a production system for collagen-related structural proteins. The 45-kDa rCI1a fragment expression, targeted to the endoplasmic reticulum, was controlled by three different promoters (a constitutive maize ubiquitin , seed endosperm-specific rice glutelin and germination-specific barley α - amylase fusion) to compare their effects on rCIa1 accumulation. Highest accumulation of the 45-kDa rCIa1 was obtained with the glutelin promoter (140 mg/kg seed), whereas the lowest accumulation was obtained with the α - amylase promoter. To induce homozygosity for stable 45-kDa rCIa1 production in the transgenic lines, doubled haploid (DH) progeny was generated through microspore culture. The 45-kDa rCIa1 expression levels achieved from the best DH lines were 13 mg/kg dry seeds under the ubiquitin promoter and 45 mg/kg dry seeds under the glutelin promoter. Mass spectroscopy and amino acid composition analysis of the purified 45-kDa rCIa1 fragment revealed that a small percent of prolines were hydroxylated with no additional detectable post-translational modifications.     

内质网应激与心血管疾病研究概述

内质网应激是继死亡受体信号途径和线粒体途径之后新近发现的一条细胞凋亡通路,适度的应激可通过未折叠蛋白反应(UPR)产生细胞保护作用,但当应激过强或长时间不缓解时则会触发CHOP、ASK1/JNK及Caspases等通路诱导细胞凋亡。近年来研究发现内质网应激参与多种心血管疾病的发生发展,通过对相关通路的干预可以产生心肌细胞的保护作用,这有望成为防治心脏疾病的新靶点。     

Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions

Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells.     

Deficiency in the mitochondrial apoptotic pathway reveals the toxic potential of autophagy under ER stress conditions

Endoplasmic reticulum (ER) stress-induced cell death is normally associated with activation of the mitochondrial apoptotic pathway, which is characterized by CYCS (cytochrome c, somatic) release, apoptosome formation, and caspase activation, resulting in cell death. In this study, we demonstrate that under conditions of ER stress cells devoid of CASP9/caspase-9 or BAX and BAK1, and therefore defective in the mitochondrial apoptotic pathway, still undergo a delayed form of cell death associated with the activation of caspases, therefore revealing the existence of an alternative stress-induced caspase activation pathway. We identified CASP8/caspase-8 as the apical protease in this caspase cascade, and found that knockdown of either of the key autophagic genes, ATG5 or ATG7, impacted on CASP8 activation and cell death induction, highlighting the crucial role of autophagy in the activation of this novel ER stress-induced death pathway. In line with this, we identified a protein complex composed of ATG5, FADD, and pro-CASP8 whose assembly coincides with caspase activation and cell death induction. Together, our results reveal the toxic potential of autophagy in cells undergoing ER stress that are defective in the mitochondrial apoptotic pathway, and suggest a model in which the autophagosome functions as a platform facilitating pro-CASP8 activation. Chemoresistance, a common problem in the treatment of cancer, is frequently caused by the downregulation of key mitochondrial death effector proteins. Alternate stress-induced apoptotic pathways, such as the one described here, may become of particular relevance for tackling the problem of chemoresistance in cancer cells.     

Induction of ER stress in response to oxygen-glucose deprivation of cortical cultures involves the activation of the PERK and IRE-1 pathways and of caspase-12

Disturbance of calcium homeostasis and accumulation of misfolded proteins in the endoplasmic reticulum (ER) are considered contributory components of cell death after ischemia. However, the signal-transducing events that are activated by ER stress after cerebral ischemia are incompletely understood. In this study, we show that caspase-12 and the PERK and IRE pathways are activated following oxygen-glucose deprivation (OGD) of mixed cortical cultures or neonatal hypoxia–ischemia (HI). Activation of PERK led to a transient phosphorylation of eIF2α, an increase in ATF4 levels and the induction of gadd34 (a subunit of an eIF2α-directed phosphatase). Interestingly, the upregulation of ATF4 did not lead to an increase in the levels of CHOP. Additionally, IRE1 activation was mediated by the increase in the processed form of xbp1, which would be responsible for the observed expression of edem2 and the increased levels of the chaperones GRP78 and GRP94. We were also able to detect caspase-12 proteolysis after HI or OGD. Processing of procaspase-12 was mediated by NMDA receptor and calpain activation. Moreover, our data suggest that caspase-12 activation is independent of the unfolded protein response activated by ER stress.     

钙网蛋白与肿瘤免疫

钙网蛋白是内质网分子伴侣,负责糖蛋白的折叠及维持细胞内Ca2＋平衡。新近研究发现,某些凋亡刺激能诱导肿瘤细胞内的钙网蛋白快速转位到肿瘤细胞膜上,这种表面包被有大量钙网蛋白的肿瘤细胞,能被抗原呈递细胞有效识别和吞噬,由此激发抗同种肿瘤的特异性免疫杀伤效应,提示钙网蛋白在肿瘤免疫治疗中的潜在应用价值。对钙网蛋白在肿瘤免疫中的研究进展作一综述。     

Salinomycin induces cell death with autophagy through activation of endoplasmic reticulum stress in human cancer cells

Salinomycin is perhaps the first promising compound that was discovered through high throughput screening in cancer stem cells. This novel agent can selectively eliminate breast and other cancer stem cells, though the mechanism of action remains unclear. In this study, we found that salinomycin induced autophagy in human non-small cell lung cancer (NSCLC) cells. Furthermore, we demonstrated that salinomycin stimulated endoplasmic reticulum stress and mediated autophagy via the ATF4-DDIT3/CHOP-TRIB3-AKT1-MTOR axis. Moreover, we found that the autophagy induced by salinomycin played a prosurvival role in human NSCLC cells and attenuated the apoptotic cascade. We also showed that salinomycin triggered more apoptosis and less autophagy in A549 cells in which CDH1 expression was inhibited, suggesting that the inhibition of autophagy might represent a promising strategy to target cancer stem cells. In conclusion, these findings provide evidence that combination treatment with salinomycin and pharmacological autophagy inhibitors will be an effective therapeutic strategy for eliminating cancer cells as well as cancer stem cells.     

A novel cellular stress response characterised by a rapid reorganisation of membranes of the endoplasmic reticulum

Canonical endoplasmic reticulum (ER) stress, which occurs in many physiological and disease processes, results in activation of the unfolded protein response (UPR). We now describe a new, evolutionarily conserved cellular stress response characterised by a striking, but reversible, reorganisation of ER membranes that occurs independently of the UPR, resulting in impaired ER transport and function. This reorganisation is characterised by a dramatic redistribution and clustering of ER membrane proteins. ER membrane aggregation is regulated, in part, by anti-apoptotic BCL-2 family members, particularly MCL-1. Using connectivity mapping, we report the widespread occurrence of this stress response by identifying several structurally diverse chemicals from different pharmacological classes, including antihistamines, antimalarials and antipsychotics, which induce ER membrane reorganisation. Furthermore, we demonstrate the potential of ER membrane aggregation to result in pathological consequences, such as the long-QT syndrome, a cardiac arrhythmic abnormality, arising because of a novel trafficking defect of the human ether-a-go-go-related channel protein from the ER to the plasma membrane. Thus, ER membrane reorganisation is a feature of a new cellular stress pathway, clearly distinct from the UPR, with important consequences affecting the normal functioning of the ER.     

bFGF attenuates endoplasmic reticulum stress and mitochondrial injury on myocardial ischaemia/reperfusion via activation of PI3K/Akt/ERK1/2 pathway

Extensive research focused on finding effective strategies to prevent or improve recovery from myocardial ischaemia/reperfusion (I/R) injury. Basic fibroblast growth factor (bFGF) has been shown to have therapeutic potential in some heart disorders, including ischaemic injury. In this study, we demonstrate that bFGF administration can inhibit the endoplasmic reticulum (ER) stress and mitochondrial dysfunction induced in the heart in a mouse model of I/R injury. In vitro, bFGF exerts a protective effect by inhibiting the ER stress response and mitochondrial dysfunction proteins that are induced by tert‐Butyl hydroperoxide (TBHP) treatment. Both of these in vivo and in vitro effects are related to the activation of two downstream signalling pathways, PI3K/Akt and ERK1/2. Inhibition of these PI3K/Akt and ERK1/2 pathways by specific inhibitors, LY294002 and PD98059, partially reduces the protective effect of bFGF. Taken together, our results indicate that the cardioprotective role of bFGF involves the suppression of ER stress and mitochondrial dysfunction in ischaemic oxidative damage models and oxidative stress‐induced H9C2 cell injury; furthermore, these effects underlie the activation of the PI3K/Akt and ERK1/2 signalling pathways.