内质网应激/未折叠蛋白反应在抑制脑胶质母细胞瘤中的研究进展

胶质母细胞瘤(GBM)是一种预后极差的中枢神经系统恶性肿瘤,具有手术难以切除、高侵袭性、增生性及强耐药性等特点。内质网(ER)应激会破坏细胞中的其他蛋白质稳态,从而导致未折叠的蛋白质响应(UPR)激活,这对于恢复这种平衡和细胞存活至关重要。除了作为一种适应性反应外,最近UPR还与肿瘤发生有关。肿瘤微环境中普遍存在的缺氧、活性氧和营养物质剥夺是众所周知的UPR诱因。在这里,笔者回顾目前对成人最具生命威胁的GBM中UPR的认识。患者样品显示出UPR的慢性激活,体外标准化学疗法和放射疗法通过加重ER应激导致细胞死亡而部分起作用。UPR与凋亡诱导剂如TRAIL和MDA-7的敏感性增强有关。最后,内质网应激被认为与GBM干细胞内稳态的维持有关。UPR中的IRE1与PERK传感器在原位GBM小鼠模型中的作用均有报道,但直接作用仍有待证明。综上所述,UPR似乎可通过抑制其活性成为GBM新的干预手段中一个有希望的治疗靶点。     

Involvement of endoplasmic reticulum stress in optic nerve degeneration following N-methyl-D-aspartate-induced retinal damage in mice

We evaluated time-dependent optic nerve degeneration and the role of endoplasmic reticulum (ER) stress in this process following retinal ganglion cell death in mice. Retinal damage was induced by intravitreal injection of N-methyl-D-aspartate (NMDA). Neurofilament heavy (NFH)- and phosphorylated NFH (pNFH)-positive axons were time-dependently decreased in optic nerves at 1, 3, 7, 14, and 28 days after NMDA injection. Expression of glial fibrillary acidic protein (GFAP)-positive astroglial cells and ionized calcium-binding adaptor molecule 1 (Iba1)-positive microglial cells showed a significant increase in the optic nerve at 7, 14, and 28 days after NMDA injection. In contrast, expression of myelin basic protein (MBP)-positive oligodendrocytes showed a significant decrease in the optic nerve at 7, 14, and 28 days after NMDA injection. In quantitative RT-PCR analysis, expressions of glucose-regulated protein 78 (Grp78)/BiP, Grp94, Calreticulin, C/EBP homologous protein (Chop), and the ER degradation enhancer mannosidase alpha-like 1 (Edem1) genes were increased in the optic nerve at 14 days after NMDA injection. In addition, the Grp94 gene was increased at 7 days after NMDA injection, and the Edem1 gene was increased at 3, 7, and 28 days after NMDA injection. GRP78 and CHOP proteins were colocalized with MBP in the optic nerve after NMDA injection. These findings suggest that the axonal degeneration is dramatic until 7 days after NMDA injection and that glial cells may play some role in the degeneration of the optic nerve. Furthermore, ER stress may play a pivotal role in the decrease of MBP-positive oligodendrocytes after NMDA-induced retinal damage.     

TorsinA in PC12 cells: localization in the endoplasmic reticulum and response to stress

Most cases of early-onset torsion dystonia are caused by deletion of GAG in the coding region of the DYT1 gene encoding torsinA. This autosomal dominant neurologic disorder is characterized by abnormal movements, believed to originate from neuronal dysfunction in the basal ganglia of the human brain. The torsins (torsinA and torsinB) are members of the "ATPases associated with a variety of cellular activities" (AAA(+)) superfamily of proteins that mediate chaperone and other functions involved in conformational modeling of proteins, protection from stress, and targeting of proteins to cellular organelles. In this study, the intracellular localization and levels of endogenous torsin were evaluated in rat pheochromocytoma PC12 cells following differentiation and stress. TorsinA, apparent MW 37 kDa, cofractionates with markers for the microsomal/endoplasmic reticulum (ER) compartment and appears to reside primarily within the ER lumen based on protease resistance. TorsinA immunoreactivity colocalizes with the lumenal ER protein protein disulfide isomerase (PDI) and extends throughout neurites. Levels of torsinA did not increase notably in response to nerve growth factor-induced differentiation. None of the stress conditions tested, including heat shock and the unfolded protein response, affected torsinA, except for oxidative stress, which resulted in an increase in the apparent MW of torsinA and redistribution to protrusions from the cell surface. These findings are consistent with a relatively rapid covalent modification of torsinA in response to oxidative stress causing a change in state. Mutant torsinA may interfere with and/or compromise ER functions, especially in dopaminergic neurons, which have high levels of torsinA and are intrinsically vulnerable to oxidative stress.     

Aberrant neuronal differentiation and inhibition of dendrite outgrowth resulting from endoplasmic reticulum stress

Neural stem cells (NSCs) play an essential role in development of the central nervous system. Endoplasmic reticulum (ER) stress induces neuronal death. After neuronal death, neurogenesis is generally enhanced to repair the damaged regions. However, it is unclear whether ER stress directly affects neurogenesis‐related processes such as neuronal differentiation and dendrite outgrowth. We evaluated whether neuronal differentiation and dendrite outgrowth were regulated by HRD1, a ubiquitin ligase that was induced under mild conditions of tunicamycin‐induced ER stress. Neurons were differentiated from mouse embryonic carcinoma P19 cells by using retinoic acid. The differentiated cells were cultured for 8 days with or without tunicamycin and HRD1 knockdown. The ER stressor led to markedly increased levels of ER stress. ER stress increased the expression levels of neuronal marker βIII‐tubulin in 8‐day‐differentiated cells. However, the neurites of dendrite marker microtubule‐associated protein‐2 (MAP‐2)‐positive cells appeared to retract in response to ER stress. Moreover, ER stress markedly reduced the dendrite length and MAP‐2 expression levels, whereas it did not affect the number of surviving mature neurons. In contrast, HRD1 knockdown abolished the changes in expression of proteins such as βIII‐tubulin and MAP‐2. These results suggested that ER stress caused aberrant neuronal differentiation from NSCs followed by the inhibition of neurite outgrowth. These events may be mediated by increased HRD1 expression. © 2014 The Authors Journal of Neuroscience Research Published by Wiley Periodicals, Inc.     

Involvement of endoplasmic reticulum stress in optic nerve degeneration after chronic high intraocular pressure in DBA/2J mice

DBA/2J mice are one of several animal strains used for experimental models of both intraocular hypertension and glaucoma. This study investigates the relationship between endoplasmic reticulum (ER) stress and optic nerve degeneration in DBA/2J mice. Intraocular pressure (IOP) was measured in DBA/2J mice between the ages of 6 and 15 months. Optic nerve damage was assessed at 15 months of age. The nerve was immunostained with antibodies to either neurofilament heavy chain (NFH) or phosphorylated NFH (pNFH), and optic nerve damage was assessed by performing NFH‐ and pNFH‐positive axon counts. Expression levels of the ER stress proteins 78‐kDa glucose‐regulated protein, also known as binding immunoglobulin protein, and C/EBP homologous protein were assayed with Western blotting. We also investigated ER stress localization in the optic nerve by double immunostaining with antibodies to ionized calcium‐binding adaptor molecule 1, myelin basic protein, and glial fibrillary acidic protein (GFAP). In DBA/2J mice, IOP began to rise at 8 months of age, and retinal degeneration was detected at 15 months of age. DBA/2J mice had fewer axons than controls at 15 months of age. ER stress‐related protein levels were higher in the optic nerves of DBA/2J mice and were colocalized with GFAP‐positive astrocytes. Our findings suggest that ER stress plays a role in optic nerve degeneration during chronic ocular hypertension. Furthermore, ER stress may be related in some way to astrocyte activation. © 2015 Wiley Periodicals, Inc.     

间歇低氧大鼠淋巴细胞促进脑血管内皮细胞凋亡的机制

目的 探讨间歇低氧大鼠淋巴细胞促进脑血管内皮细胞内质网应激凋亡作用及机制。方法 取SD雄性大鼠30只,分为正常对照组、间歇低氧组、抗低氧组,每组各10只; 抗低氧组、间歇低氧组建立间歇低氧大鼠模型,并分别于间歇低氧前半小时腹腔注射100 mg/kg超氧阴离子清除剂和等量生理盐水,1次/d,然后间歇低氧干预8 h/d; 正常对照组维持常氧量,干预6周; 各组干预后分离淋巴细胞,测定CD4⁺、CD8⁺ T细胞凋亡率; 取对数期内皮细胞RBE4,与各组分离淋巴细胞共培养,命名为正常对照RBE4组、间歇低氧RBE4组、抗低氧RBE4组; 4 h后测定培养基上清液中超氧化物歧化酶(Superoxide dismutase,SOD)及丙二醛(Malonaldehyde,MDA)水平、内皮细胞凋亡率、内皮细胞中C增强子结合蛋白同源蛋白(Cenhancer binding protein homologous protein,CHOP)、葡萄糖调节蛋白78(Glucose regulatory protein 78,GRP78)、半胱氨酸天冬氨酸蛋白水解酶-3(Cysteine containing aspartate-specific proteases-3,Caspase-3)mRNA及蛋白水平。结果 与正常对照组比较,间歇低氧组、抗低氧组CD4⁺、CD8⁺ T细胞凋亡率降低,且间歇低氧组低于抗低氧组(P<0.05); 与正常对照RBE4组比较,间歇低氧RBE4组、抗低氧RBE4组上清液中SOD水平降低、MDA水平增高、RBE4凋亡率增高、CHOP,Caspase-3 mRNA及蛋白水平升高、GRP78 mRNA及蛋白水平降低(P均<0.05); 与抗低氧RBE4组比较,间歇低氧组SOD水平降低、MDA水平增高、RBE4凋亡率增高、CHOP,Caspase-3 mRNA及蛋白水平升高、GRP78 mRNA及蛋白水平降低(P均<0.05)。结论 间歇低氧大鼠淋巴细胞促进脑血管内皮细胞凋亡可能通过激发内质网应激而发挥作用。     

内质网应激在大鼠脑缺血再灌注损伤中的作用研究

目的 观察生长停滞及DNA损伤诱导基因153(GADD153)和caspase-12在脑缺血再灌注损伤大鼠脑组织中的动态改变,探讨内质网应激在脑缺血再灌注损伤中的作用. 方法 42只大鼠按随机数字表法分为正常对照组(3只)、假手术组(3只)和脑缺血再灌注损伤组(36只);脑缺血再灌注损伤组又分为脑缺血2h再灌注6h、12h、24 h、72 h组,每组各9只.采用线栓法建立大鼠大脑中动脉缺血再灌注损伤模型.采用免疫组化染色、免疫荧光双标染色、Westem blotting检测各组大鼠脑组织中GADD153和caspase-12的表达. 结果 免疫组化染色和Western blotting结果均显示正常对照组和假手术组大鼠脑组织中GADD 153和caspase-12表达为阴性;再灌注6h组GADD153表达增加,并逐渐增高,持续至72 h时较再灌注6h组明显增高,差异均有统计学意义(P＜0.05);再灌注6h组caspase-12表达增加,至24 h达高峰,72 h时仍维持在较高水平,与再灌注6h组比较差异有统计学意义(P＜0.05).免疫荧光双标染色结果显示,再灌注6h组可见少量双标阳性细胞,12h、24 h时双标阳性细胞数均较再灌注6h组明显增多,差异有统计学意义(P＜0.05),至72 hcaspase-12单标阳性细胞数减少,GADD 153单标阳性细胞数仍较多,双标阳性细胞数减少. 结论 GADD153和caspase-12在脑缺血再灌注损伤大鼠脑组织中的表达随时间呈动态改变,表明内质网应激参与了脑缺血再灌注损伤的病理过程.     

The signaling mechanisms of hippocampal endoplasmic reticulum stress affecting neuronal plasticity-related protein levels in high fat diet-induced obese rats and the regulation of aerobic exercise

High fat diet (HFD)-induced obesity has been shown to reduce the levels of neuronal plasticity-related proteins, specifically brain-derived neurotrophic factor (BDNF) and synaptophysin (SYN), in the hippocampus. However, the underlying mechanisms are not fully clear. Endoplasmic reticulum stress (ERS) has been reported to play a key role in regulating gene expression and protein production by affecting stress signaling pathways and ER functions of protein folding and post-translational modification in peripheral tissues of obese rodent models. Additionally, HFD that is associated with hyperglycemia could induce hippocampal ERS, thus impairing insulin signaling and cognitive health in HFD mice. One goal of this study was to determine whether hyperglycemia and hyperlipidemia could cause hippocampal ERS in HFD-induced obese SD rats, and explore the potential mechanisms of ERS regulating hippocampal BDNF and SYN proteins production. Additionally, although regular aerobic exercise could reduce central inflammation and elevate hippocampal BDNF and SYN levels in obese rats, the regulated mechanisms are poorly understood. Nrf2-HO-1 pathways play roles in anti-ERS, anti-inflammation and anti-apoptosis in peripheral tissues. Therefore, the other goal of this study was to determine whether aerobic exercise could activate Nrf2-HO-1 in hippocampus to alleviate obesity-induced hippocampal ERS, which would lead to increased BDNF and SYN levels.Male SD rats were fed on HFD for 8 weeks to establish the obese model. Then, 8 weeks of aerobic exercise treadmill intervention was arranged for the obese rats. Results showed that HFD-induced obesity caused hyperglycemia and hyperlipidemia, and significantly promoted hippocampal glucose transporter 3 (GLUT3) and fatty acid transport protein 1 (FATP1) protein expression. These results were associated with the activation of hippocampal ERS and ERS-mediated apoptosis. At the same time, we found that excessive hippocampal ERS not only significantly decreased proBDNF—the precursor of mature BDNF, but also attenuated p38/ERK-CREB signaling pathways and activated NLRP3-IL-1β pathways in obese rats. These results were associated with reduced BDNF and SYN protein production. However, these adverse changes were obviously reversed by aerobic exercise intervention through activating the Nrf2-HO-1 pathways.These results suggest that dietary obesity could induce hippocampal ERS in male SD rats, and excessive hippocampal ERS plays a critical role in decreasing the levels of BDNF and SYN. Moreover, aerobic exercise could activate hippocampal Nrf2 and HO-1 to relieve ERS and heighten BDNF and SYN production in obese rats.     

Endoplasmic reticulum stress-induced apoptosis in the penumbra aggravates secondary damage in rats with traumatic brain injur y

Neuronal apoptosis is mediated by intrinsic and extrinsic signaling pathways such as the membrane-mediated, mitochondrial, and endo-plasmic reticulum stress pathways. Few studies have examined the endoplasmic reticulum-mediated apoptosis pathway in the penumbra after traumatic brain injury, and it remains unclear whether endoplasmic reticulum stress can activate the caspase-12-dependent apoptotic pathway in the traumatic penumbra. Here, we established rat models of lfuid percussion-induced traumatic brain injury and found that protein expression of caspase-12, caspase-3 and the endoplasmic reticulum stress marker 78 kDa glucose-regulated protein increased in the traumatic penumbra 6 hours after injury and peaked at 24 hours. Furthermore, numbers of terminal deoxynucleotidyl transferase-mediat-ed dUTP nick end labeling-positive cells in the traumatic penumbra also reached peak levels 24 hours after injury. These ifndings suggest that caspase-12-mediated endoplasmic reticulum-related apoptosis is activated in the traumatic penumbra, and may play an important role in the pathophysiology of secondary brain injury.     

Smooth endoplasmic reticulum dilation and degeneration in Purkinje neuron dendrites of aging ethanol-fed female rats

The effects of chronic ethanol consumption on the extensive Purkinje neuron (PN) dendritic arbor of male rats include dilation of the smooth endoplasmic reticulum (SER) and dendritic regression. The purpose of the present study was to examine the molecular layer of female rats for the presence of ethanol-related SER dilation and evidence of degeneration within the PN dendritic arbor. Twenty-one 12-month-old Fischer 344 female rats (n = 7/treatment group) received a liquid ethanol, liquid control, or rat chow diet for a period of 40 weeks. Ethanol-fed rats received 35% of their dietary calories as ethanol. Pair-fed rats received a liquid control diet that was isocaloric to the ethanol diet. Chow-fed rats received standard laboratory rat chow ad libitum. At the end of treatment, tissues from the anterior and posterior lobes of the cerebellar vermis were viewed and photographed with the electron microscope. The diameters of SER profiles were measured and the density of degenerating bodies within the PN dendritic arbor was quantitated. In the posterior lobe, ethanol-related SER dilation was apparent. In the anterior lobe, the density of degenerating bodies within PN dendritic shafts was significantly increased but SER dilation in PN dendritic shafts was absent. These results confirm that SER dilation and dendritic degeneration in PN dendrites may precede and contribute to ethanol-related regression in female rats. In addition, comparison of these results with data obtained in male rats from a previous study suggests that PN dendrites in females may be more sensitive to the effects of ethanol.     

Fructus Broussonetae extract improves cognitive function and endoplasmic reticulum stress in Alzheimer's disease models

This study investigated the effects and possible targets of Fructus Broussonetiae extract,a traditional Chinese medicinal herb,on a model of Alzheimer’s disease induced by beta-amyloid peptide 25-35 and D-galactose.The results revealed that intragastric administration of Fructus Broussonetiae significantly increased the expression of immunoglobulin-binding protein,a key factor in the endoplasmic reticulum stress-signaling pathway in rat hippocampus.In contrast,the treatment significantly decreased expression levels of PKR-like endoplasmic reticulum kinase and C/EBP homologous protein,and substantially improved learning,memory and spatial recognition dysfunction in rats.This evidence indicates that Fructus Broussonetiae extract improves spatial learning and memory abilities in rats by affecting the regulation of hippocampal endoplasmic reticulum stress and activation of the apoptosis pathway.     

内质网应激对七氟醚麻醉术后认知功能障碍大鼠脑海马组织及神经元的影响

目的探讨七氟醚通过影响内质网应激对麻醉术后认知功能障碍(POCD)大鼠脑海马组织、神经元的影响及内质网应激抑制剂的干预作用.方法选择清洁级SD雄性大鼠24只,随机分为3组:A组(对照组)、B组(七氟醚组)、C组(七氟醚+内质网应激抑制剂4-苯基丁酸(4-PBA)组),每组8只.采用Morris水迷宫实验检测大鼠认知功能,采用苏木精-伊红染色观察各组大鼠脑海马组织病理形态学改变,采用流式细胞术检测脑海马神经元细胞凋亡数,采用ELISA法检测脑海马组织中脑源性神经营养因子(BDNF)、神经元特异性烯醇化酶(NSE)的表达水平,采用Western blot和qRT-PCR法检测脑组织Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2(Bcl-2)、葡萄糖调节蛋白78(GRP78)、葡萄糖调节蛋白94(GRP94)、C/EBP同源蛋白(CHOP)、X-盒结合蛋白1(XBP1)表达水平.结果苏木精-伊红染色结果表明,与A组细胞,B组大鼠脑海马组织中细胞明显减少,层次不完整,分布不均匀,细胞排列紊乱;C组大鼠脑海马组织中细胞较B组增加,海马细胞排列紊乱现象有所改善.与A组相比,B组和C组大鼠逃避潜伏期长,平台所在象限停留时间及穿越平台次数显著减少(P<0.05);海马神经元细胞凋亡数显著增加,脑组织Bax、NSE、GRP78、GRP94、CHOP、XBP1表达水均显著升高而Bcl-2、BDNF表达水平显著降低(P<0.05).与B组相比,C组大鼠逃避潜伏期明显缩短,平台所在象限停留时间及穿越平台次数显著增加,海马神经元细胞凋亡数显著减少(P<0.05),其余各指标均有不同程度的改善.结论七氟醚可通过促进内质网应激引起大鼠脑海马组织损伤及神经元细胞凋亡水平增加,从而引起大鼠认知功能障碍;抑制内质网应激可改善七氟醚诱导的大鼠认知功能障碍.     

Ulinastatin suppresses endoplasmic reticulum stress and apoptosis in the hippocampus of rats with acute paraquat poisoning

Lung injury is the main manifestation of paraquat poisoning. Few studies have addressed brain damage after paraquat poisoning. Ulinastatin is a protease inhibitor that can effectively stabilize lysosomal membranes, prevent cell damage, and reduce the production of free radicals. This study assumed that ulinastatin would exert these effects on brain tissues that had been poisoned with paraquat. Rat models of paraquat poisoning were intraperitoneally injected with ulinastatin. Simultaneously, rats in the control group were administered normal saline. Hematoxylin-eosin staining showed that most hippocampal cells were contracted and nucleoli had disappeared in the paraquat group. Fewer cells in the hippocampus were concentrated and nucleoli had disappeared in the ulinastatin group. Western blot assay showed that expressions of GRP78 and cleaved-caspase-3 were significantly lower in the ulinastatin group than in the paraquat group. Immunohistochemical findings showed that CHOP immunoreactivity was significantly lower in the ulinastatin group than in the paraquat group. Terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling staining showed that the number of apoptotic cells was reduced in the paraquat and ulinastatin groups. These data confirmed that endoplasmic reticular stress can be induced by acute paraquat poisoning. Ulinastatin can effectively inhibit this stress as well as cell apoptosis, thereby exerting a neuroprotective effect.     

Endoplasmic reticulum stress is involved in okadaic acid-induced tau phosphorylation and neurotoxic effects of okadaic acid

The role of the endoplasmic reticulum in Alzheimer’s disease pathogenesis remains poorly understood. The present study investigated the correlation of okadaic acid-induced tau hyperphosphorylation and neurotoxicity with endoplasmic reticulum stress. Following co-culture of various concentrations of okadaic acid (25, 50, 100, and 200 nmol/L) and human neuroblastoma SH-SY5Y cells, okadaic acid, a selective protein phosphatase (PP-1, PP-2A) inhibitor, reduced cell viability in a concentration-dependent manner,...