Expression of the oxygen-regulated protein ORP150 accelerates wound healing by modulating intracellular VEGF transport

Expression of angiogenic factors such as VEGF under conditions of hypoxia or other kinds of cell stress contributes to neovascularization during wound healing. The inducible endoplasmic reticulum chaperone oxygen-regulated protein 150 (ORP150) is expressed in human wounds along with VEGF. Colocalization of these two molecules was observed in macrophages in the neovasculature, suggesting a role of ORP150 in the promotion of angiogenesis. Local administration of ORP150 sense adenovirus to wounds of diabetic mice, a treatment that efficiently targeted this gene product to the macrophages of wound beds, increased VEGF antigen in wounds and accelerated repair and neovascularization. In cultured human macrophages, inhibition of ORP150 expression caused retention of VEGF antigen within the endoplasmic reticulum (ER), while overexpression of ORP150 promoted the secretion of VEGF into hypoxic culture supernatants. Taken together, these data suggest an important role for ORP150 in the setting of impaired wound repair and identify a key, inducible chaperone-like molecule in the ER. This novel facet of the angiogenic response may be amenable to therapeutic manipulation.     

Multiple, disparate roles for calcium signaling in apoptosis of human prostate and cervical cancer cells exposed to diindolylmethane

Diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous vegetables, causes growth arrest and apoptosis of cancer cells in vitro. DIM also induces endoplasmic reticulum (ER) stress, and thapsigargin, a specific inhibitor of the sarcoplasmic reticulum/ER calcium-dependent ATPase, enhances this effect. We asked whether elevated cytosolic free calcium [Ca2+]i is required for cytotoxicity of DIM and thapsigargin in two cancer cells lines (C33A, from cervix, and DU145, from prostate). [Ca2+]i was measured in real-time by FURA-2 fluorescence. We tested whether DIM, thapsigargin, and DIM + thapsigargin cause apoptosis, measured by nucleosome release, under conditions that prevented elevation of [Ca2+]i, using both cell-permeable and cell-impermeable forms of the specific calcium chelator BAPTA. DIM, like thapsigargin, rapidly mobilized ER calcium. C33A and DU145 responded differently to perturbations in Ca2+ homeostasis, suggesting that DIM induces apoptosis by different mechanisms in these two cell lines and/or that calcium mobilization also activates different survival pathways in C33A and DU145. Apoptosis in C33A was independent of increased [Ca2+]i, suggesting that depletion of ER Ca2+ stores may be sufficient for cell killing, whereas apoptosis in DU145 required elevated [Ca2+]i for full response. Inhibitor studies using cyclosporin A and KN93 showed that Ca2+ signaling is important for cell survival but the characteristics of this response also differed in the two cell lines. Our results underscore the complex and variable nature of cellular responses to disrupted Ca2+ homeostasis and suggest that alteration Ca2+ homeostasis in the ER can induce cellular apoptosis by both calcium-dependent and calcium-independent mechanisms.     

NMDA Receptor Stimulation Induces Reversible Fission of the Neuronal Endoplasmic Reticulum

With few exceptions the endoplasmic reticulum (ER) is considered a continuous system of endomembranes within which proteins and ions can move. We have studied dynamic structural changes of the ER in hippocampal neurons in primary culture and organotypic slices. Fluorescence recovery after photobleaching (FRAP) was used to quantify and model ER structural dynamics. Ultrastructure was assessed by electron microscopy. In live cell imaging experiments we found that, under basal conditions, the ER of neuronal soma and dendrites was continuous. The smooth and uninterrupted appearance of the ER changed dramatically after glutamate stimulation. The ER fragmented into isolated vesicles in a rapid fission reaction that occurred prior to overt signs of neuronal damage. ER fission was found to be independent of ER calcium levels. Apart from glutamate, the calcium ionophore ionomycin was able to induce ER fission. The N-methyl, D-aspartate (NMDA) receptor antagonist MK-801 inhibited ER fission induced by glutamate as well as by ionomycin. Fission was not blocked by either ifenprodil or kinase inhibitors. Interestingly, sub-lethal NMDA receptor stimulation caused rapid ER fission followed by fusion. Hence, ER fission is not strictly associated with cellular damage or death. Our results thus demonstrate that neuronal ER structure is dynamically regulated with important consequences for protein mobility and ER luminal calcium tunneling.     

Complestatin is a noncompetitive peptide antagonist of N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptors: secure blockade of ischemic neuronal death

Complestatin, a peptide derived from Streptomyces, was found to protect cultured cortical neurons from excitotoxicity induced by N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or kainate. This neuroprotective behavior of complestatin was attributed to a blockade of Ca2+ ion entry and accumulation, after the activation of NMDA and AMPA/kainate receptors. Complestatin reversibly interfered with NMDA- and AMPA-mediated excitatory synaptic transmission. Complestatin also protected cortical neurons from prolonged deprivation of oxygen and glucose, more effectively than combined antagonists of NMDA and AMPA/kainate receptors. Neurotoxicity, evolving within 1 to 2 days after continuous exposure to combined NMDA and AMPA/kainate antagonists, was not observed in cortical cell cultures that were exposed to complestatin. Finally, complestatin dose dependently prevented neuronal death evolving within the inner nuclear and ganglion cell layers, after transient retinal ischemia. We conclude that complestatin possesses novel pharmacological properties that effectively prevent excitotoxicity under certain pathological conditions.     

癫痫发作后神经元的损伤:凋亡与坏死

目的癫痫能导致神经元的损伤,并增加其后癫痫发作的危险性.分析癫痫发作后神经元损伤的可能机制,以期为预防及减轻癫痫发作后脑损伤提供理论依据.资料来源应用计算机检索PubMed数据库1995/2004-06相关文章,检索词为"epilepsy"neuron damage"necrosis"和"apoptosis",分别组合进行检索,限定语言种类为英文.资料选择对资料进行初审,选取包括癫痫及其发作后神经元损伤的有关人类及动物实验的文献,筛除其他非相关资料,对剩余的文献开始查找全文.资料提炼共收集到14篇有关癫痫发作后神经元损伤的随机对照实验,4篇有关中枢神经元兴奋毒性损伤的非随机对照实验,另有3篇关于神经元损伤的相关文献.共21篇文献,全部符合入选标准.资料综合14个随机对照实验均选用化学点燃或电点燃的方法诱导癫痫模型,观测指标包括神经元及细胞器超微结构的改变及凋亡相关因子的表达变化.4篇非随机对照实验采用中枢神经元其他缺血缺氧模型,用电镜直接观察不同损伤形式的神经元中不同凋亡因子的表达,为中枢神经元的兴奋毒性损伤的机制提供直接依据.另3篇相关文献介绍了神经元损伤的途径和损伤相关因子的表达.结论癫痫发作后神经元的死亡形式与损伤的强度和线粒体的功能状态有关,线粒体构成了神经元存亡的控制中心.细胞色素C的释放和半胱氨酸天冬氨酸蛋白酶的激活是神经元损伤的最后共同通路.     

Hippocampal mossy fiber denervation induces a supersensitivity to cholecystokinin of CA3 pyramidal neurons in the guinea pig but not in the rat

Immunohistochemical studies have revealed the presence of cholecystokinin (CCK) in the guinea pig hippocampal mossy fiber projections, but this peptide appears to be absent in this system in the rat. However, in both species the mossy fiber system shows a strong opiate-like immunoreactivity. The present electrophysiological studies were undertaken to determine, in the two species, the effect of a unilateral colchicine-induced mossy fiber denervation, by comparing the responsiveness of target pyramidal neurons to Met-enkephalin, CCK and the nonpeptidic excitatory agents acetylcholine, kainate, quisqualate and ibotenate, on the intact and on the lesioned side. In both species, the colchicine lesion induced an increased responsiveness to Metenkephalin in the CA1 area, whereas no change was found in the neuronal responsiveness to the other excitatory agents tested. In the rat, the responsiveness of CA3 pyramidal neurons to kainate was reduced by 90%, those to the other excitatory agents were unchanged. In the guinea pig, the mossy fiber denervation induced a 10-fold increase of the responsiveness of CA3 pyramidal neurons to CCK, but did not modify their response to Met-enkephalin, kainate, quisqualate, ibotenate and acetylcholine. These results are consistent with the lack of CCK-like immunoreactivity in the mossy fiber projection to the CA3 region of the rat and with previous reports suggesting the presynaptic location of kainate receptors in this region. They provide novel evidence for the physiological role of CCK in the hippocampal mossy fiber projection in the guinea pig.     

脑溢安对谷氨酸致神经元损伤的保护作用

目的：探讨中医平肝熄风，凉血泻火治法的抗神经损伤机理。方法：建立谷氨酸致体外培养的大鼠海马神经元兴奋毒性损伤模型，并用脑溢安含药血清、细胞外调节蛋白激酶（ERK）阻滞剂PD98059进行干预，分别检测各组神经元活化的ERK、cjun氨基末端激酶（JNK）水平及上清液LDH活性。结果：脑溢安含药血清能上调谷氨酸损伤后的大鼠海马神经元ERK水平，下调JNK水平，PD98059能阻断脑溢安对受损经元的保护作用。结论：以平肝熄风、凉血泻火为主要治法的脑溢安含药血清对谷氨酸致培养大鼠海马神经元损伤后的保护作用与MAPK信号转导途径有关，脑溢安上调ERK表达，下调JNK表达，促进细胞生存。     

Calcium/calmodulin-dependent protein kinase II links ER stress with Fas and mitochondrial apoptosis pathways

ER stress–induced apoptosis is implicated in various pathological conditions, but the mechanisms linking ER stress–mediated signaling to downstream apoptotic pathways remain unclear. Using human and mouse cell culture and in vivo mouse models of ER stress–induced apoptosis, we have shown that cytosolic calcium resulting from ER stress induces expression of the Fas death receptor through a pathway involving calcium/calmodulin-dependent protein kinase IIγ (CaMKIIγ) and JNK. Remarkably, CaMKIIγ was also responsible for processes involved in mitochondrial-dependent apoptosis, including release of mitochondrial cytochrome c and loss of mitochondrial membrane potential. CaMKII-dependent apoptosis was also observed in a number of cultured human and mouse cells relevant to ER stress–induced pathology, including cultured macrophages, endothelial cells, and neuronal cells subjected to proapoptotic ER stress. Moreover, WT mice subjected to systemic ER stress showed evidence of macrophage mitochondrial dysfunction and apoptosis, renal epithelial cell apoptosis, and renal dysfunction, and these effects were markedly reduced in CaMKIIγ-deficient mice. These data support an integrated model in which CaMKII serves as a unifying link between ER stress and the Fas and mitochondrial apoptotic pathways. Our study also revealed what we believe to be a novel proapoptotic function for CaMKII, namely, promotion of mitochondrial calcium uptake. These findings raise the possibility that CaMKII inhibitors could be useful in preventing apoptosis in pathological settings involving ER stress–induced apoptosis.     

Death of cultured hippocampal pyramidal neurons induced by pathological activation of N-methyl-D-aspartate receptors is reduced by monosialogangliosides.

Glutamate-induced delayed neurotoxicity has been proposed to account for the selective loss of hippocampal pyramidal neurons after an ischemic period. We have studied the effects of exogenous glutamate and combined oxygen-glucose deprivation on the survival of hippocampal pyramidal neurons cultured from rat fetuses. Acute glutamate neurotoxicity (20 min, 23-25 degrees C) occurred in a concentration-dependent manner (LD50: 50 microM), destroying virtually all neurons 24 hr later. Such injury was prevented by N-methyl-D-aspartate (NMDA), but not non-NMDA, antagonists. Hippocampal cell death induced by removal of oxygen and glucose showed a similar pharmacological profile, indicating a role for NMDA receptor activation in neuronal loss associated with this energy crisis situation. Monosialogangliosides such as GM1 were effective in protecting against neurodegeneration induced by either direct glutamate exposure or oxygen and glucose deprivation. The selective action of gangliosides in disrupting the pathological consequences of glutamate receptor activation may provide a new therapeutic tool for excitatory amino acid-related brain injury processes.     

促红细胞生成素对缺血性脑损伤的神经保护作用

促红细胞生成素(Epo)是一种主要影响红细胞生成的体液因子,随着在神经细胞膜及星形胶质细胞内发现EpoR及Epo,Epo与神经系统的关系受到人们的重视,越来越多的资料显示,Epo在缺血性脑损伤中抗凋亡、抗兴奋性氨基酸神经毒性、抗自由基、抗氧化,减轻脑缺血后继发生性丘脑损害中起了重要的神经保护作用。     

Mefloquine-induced disruption of calcium homeostasis in mammalian cells is similar to that induced by ionomycin

In previous studies, we have shown that mefloquine disrupts calcium homeostasis in neurons by depletion of endoplasmic reticulum (ER) stores, followed by an influx of external calcium across the plasma membrane. In this study, we explore two hypotheses concerning the mechanism(s) of action of mefloquine. First, we investigated the possibility that mefloquine activates non-N-methyl-d-aspartic acid receptors and the inositol phosphate 3 (IP3) signaling cascade leading to ER calcium release. Second, we compared the disruptive effects of mefloquine on calcium homeostasis to those of ionomycin in neuronal and nonneuronal cells. Ionomycin is known to discharge the ER calcium store (through an undefined mechanism), which induces capacitative calcium entry (CCE). In radioligand binding assays, mefloquine showed no affinity for the known binding sites of several glutamate receptor subtypes. The pattern of neuroprotection induced by a panel of glutamate receptor antagonists was dissimilar to that of mefloquine. Both mefloquine and ionomycin exhibited dose-related and qualitatively similar disruptions of calcium homeostasis in both neurons and macrophages. The influx of external calcium was blocked by the inhibitors of CCE in a dose-related fashion. Both mefloquine and ionomycin upregulated the IP3 pathway in a manner that we interpret to be secondary to CCE. Collectively, these data suggest that mefloquine does not activate glutamate receptors and that it disrupts calcium homeostasis in mammalian cells in a manner similar to that of ionomycin.     

Hypoglycemic neuronal death is triggered by glucose reperfusion and activation of neuronal NADPH oxidase

Hypoglycemic coma and brain injury are potential complications of insulin therapy. Certain neurons in the hippocampus and cerebral cortex are uniquely vulnerable to hypoglycemic cell death, and oxidative stress is a key event in this cell death process. Here we show that hypoglycemia-induced oxidative stress and neuronal death are attributable primarily to the activation of neuronal NADPH oxidase during glucose reperfusion. Superoxide production and neuronal death were blocked by the NADPH oxidase inhibitor apocynin in both cell culture and in vivo models of insulin-induced hypoglycemia. Superoxide production and neuronal death were also blocked in studies using mice or cultured neurons deficient in the p47(phox) subunit of NADPH oxidase. Chelation of zinc with calcium disodium EDTA blocked both the assembly of the neuronal NADPH oxidase complex and superoxide production. Inhibition of the hexose monophosphate shunt, which utilizes glucose to regenerate NADPH, also prevented superoxide formation and neuronal death, suggesting a mechanism linking glucose reperfusion to superoxide formation. Moreover, the degree of superoxide production and neuronal death increased with increasing glucose concentrations during the reperfusion period. These results suggest that high blood glucose concentrations following hypoglycemic coma can initiate neuronal death by a mechanism involving extracellular zinc release and activation of neuronal NADPH oxidase.     

Kainate receptor trafficking: physiological roles and molecular mechanisms

Recently, there has been intense interest in the mechanisms regulating the trafficking and synaptic targeting of kainate receptors in neurons. This topic is still in its infancy when compared with studies of trafficking of other ionotropic glutamate receptors; however, it is already clear that mechanisms exist for subunit- and splice variant-specific trafficking of kainate receptors. There is also enormous diversity of kainate receptor targeting, with the best-studied neurons in this regard being hippocampal CA3 pyramidal neurons and CA1 GABAergic interneurons. This review summarizes the current state of knowledge on this topic, focusing on the molecular mechanisms of kainate receptor trafficking and the potential for these mechanisms to regulate neuronal kainate receptor function.     

Altered calcium/calmodulin kinase II activity changes calcium homeostasis that underlies epileptiform activity in hippocampal neurons in culture

Epilepsy is characterized by the occurrence of spontaneous recurrent epileptiform discharges (SREDs) in neurons. A decrease in calcium/calmodulin-dependent protein kinase II (CaMK-II) activity has been shown to occur with the development of SREDs in a hippocampal neuronal culture model of acquired epilepsy, and altered calcium (Ca(2+)) homeostasis has been implicated in the development of SREDs. Using antisense oligonucleotides, this study was conducted to determine whether selective suppression of CaMK-II activity, with subsequent induction of SREDs, was associated with altered Ca(2+) homeostasis in hippocampal neurons in culture. Antisense knockdown resulted in the development of SREDs and a decrease in both immunocytochemical staining and enzyme activity of CaMK-II. Evaluation of [Ca(2+)](i) using Fura indicators revealed that antisense-treated neurons manifested increased basal [Ca(2+)](i), whereas missense-treated neurons showed no change in basal [Ca(2+)](i). Antisense suppression of CaMK-II was also associated with an inability of neurons to restore a Ca(2+) load. Upon removal of oligonucleotide treatment, CaMK-II suppression and Ca(2+) homeostasis recovered to control levels and SREDs were abolished. To our knowledge, the results demonstrate the first evidence that selective suppression of CaMK-II activity results in alterations in Ca(2+) homeostasis and the development of SREDs in hippocampal neurons and suggest that CaMK-II suppression may be causing epileptogenesis by altering Ca(2+) homeostatic mechanisms.     

Mechanisms of ischemic cerebral injury

Normal compensatory mechanisms protect the central nervous system (CNS) from moderate hypoxia and ischemia; however, after more severe ischemia progressive brain hypoperfusion ensues and irreversible damage occurs. Ischemic brain injury remains greatly significant clinically and elucidating the determinants of ischemic neuronal injury and death continues to challenge researchers. Although altered perfusion and decreased energy charge may contribute to the production of irreversible damage, the distribution of lesions seen after insult does not correspond with the degree of ischemic blood flow impairment, nor can neuronal energy deprivation explain the cell damage. Other factors, such as derangements in astrocyte function, calcium homeostasis, free radical metabolism, acid-base regulation and excitatory neurotransmitters also probably mediate ischemic neuronal death. Continued investigation to establish the cellular pathophysiology of cerebral ischemia can guide rational research and therapeutic strategies.     

维生素E对鼠海马神经元细胞的抗氧化损伤作用

目的观察维生素E对老年痴呆(Alzheimer'sdisease)的治疗作用,研究氧化损伤对鼠海马神经元细胞的损伤作用及维生素E对其氧化损伤的保护作用。方法用氧化(H2O2)、维生素E处理后氧化的方法对鼠海马神经细胞(HT-22)进行分组处理。用二维电泳法、蛋白银染法及特殊氧化蛋白免疫染色法探测被氧化的蛋白。结果氧化处理12h后出现细胞生存率明显下降31.19%;经维生素E处理后再氧化处理的细胞生存率几乎达到正常组水平。经H2O2氧化处理的鼠海马神经元细胞的氧化蛋白数目增加了,而维生素E提前处理过的那组没有增加。结论维生素E对鼠神经元细胞的氧化损伤起保护作用,是有效的抗氧化治疗剂。     

Cotargeting stress-activated hsp27 and autophagy as a combinatorial strategy to amplify endoplasmic reticular stress in prostate cancer

Hsp27 is a stress-activated multifunctional chaperone that inhibits treatment-induced apoptosis and causes treatment resistance in prostate and other cancers. We previously showed that targeted suppression of Hsp27 sensitizes cancer cells to hormone and chemotherapy. However, mechanisms by which Hsp27 confers cell treatment resistance are incompletely defined. Here, we report that Hsp27 protects human prostate cancer cells against proteotoxic stress induced by proteasome inhibition, and that Hsp27 silencing using siRNA or antisense (OGX-427) induced both apoptosis and autophagy through mechanisms involving reduced proteasome activity and induction of endoplasmic reticulum (ER) stress. We found that autophagy activation protected against ER stress-induced cell death, whereas inhibition of autophagy activation following Hsp27 silencing using either pharmacologic inhibitors or atg3 silencing enhanced cell death. Importantly, cotargeting Hsp27 and autophagy by combining OGX-427 with the autophagy inhibitor, chloroquine, significantly delayed PC-3 prostate tumor growth in vivo. These findings identify autophagy as a cytoprotective, stress-induced adaptive pathway, activated following disruption of protein homeostasis and ER stress induced by Hsp27 silencing. Combinatorial cotargeting cytoprotective Hsp27 and autophagy illustrates potential benefits of blocking activation of adaptive pathways to improve treatment outcomes in cancer. Mol Cancer Ther; 11(8); 1661-71. ?2012 AACR.     

Reduction of ER stress via a chemical chaperone prevents disease phenotypes in a mouse model of primary open angle glaucoma

Mutations in myocilin (MYOC) are the most common genetic cause of primary open angle glaucoma (POAG), but the mechanisms underlying MYOC-associated glaucoma are not fully understood. Here, we report the development of a transgenic mouse model of POAG caused by the Y437H MYOC mutation; the mice are referred to herein as Tg-MYOC(Y437H) mice. Analysis of adult Tg-MYOC(Y437H) mice, which we showed express human MYOC containing the Y437H mutation within relevant eye tissues, revealed that they display glaucoma phenotypes (i.e., elevated intraocular pressure [IOP], retinal ganglion cell death, and axonal degeneration) closely resembling those seen in patients with POAG caused by the Y437H MYOC mutation. Mutant myocilin was not secreted into the aqueous humor but accumulated in the ER of the trabecular meshwork (TM), thereby inducing ER stress in the TM of Tg-MYOC(Y437H) mice. Furthermore, chronic and persistent ER stress was found to be associated with TM cell death and elevation of IOP in Tg-MYOC(Y437H) mice. Reduction of ER stress with a chemical chaperone, phenylbutyric acid (PBA), prevented glaucoma phenotypes in Tg-MYOC(Y437H) mice by promoting the secretion of mutant myocilin in the aqueous humor and by decreasing intracellular accumulation of myocilin in the ER, thus preventing TM cell death. These results demonstrate that ER stress is linked to the pathogenesis of POAG and may be a target for treatment in human patients.     

Regulation of seizure spreading by neuroserpin and tissue-type plasminogen activator is plasminogen-independent

Tissue-type plasminogen activator (tPA) is a highly specific serine proteinase expressed in the CNS during events that require neuronal plasticity. In this study we demonstrate that endogenous tPA mediates the progression of kainic acid-induced (KA-induced) seizures by promoting the synchronization of neuronal activity required for seizure spreading, and that, unlike KA-induced cell death, this activity is plasminogen-independent. Specifically, seizure induction by KA injection into the amygdala induces tPA activity and cell death in both hippocampi, and unilateral treatment of rats with neuroserpin, a natural inhibitor of tPA in the brain, enhances neuronal survival in both hippocampi. Inhibition of tPA within the hippocampus by neuroserpin treatment does not prevent seizure onset but instead markedly delays the progression of seizure activity in both rats and wild-type mice. In tPA-deficient mice, seizure progression is significantly delayed, and neuroserpin treatment does not further delay seizure spreading. In contrast, plasminogen-deficient mice show a pattern of seizure spreading and a response to neuroserpin that is similar to that of wild-type animals. These findings indicate that tPA acts on a substrate other than plasminogen and that the effects of neuroserpin on seizure progression and neuronal cell survival are mediated through the inhibition of tPA.