Rescue of Neuronal Cell Death by ER-stress protein over expression

ORP150是一种定位于内质网（ER）的新型应激蛋白。为了研究ORP150在延迟性神经元细胞死亡中的作用，我们检测了其在沙鼠脑缺血损伤后的表达。在颈总动脉病变闭塞后，在CA1区观察到ORP150抗原及其转录本的表达，并通过预处理增强了这种表达。在培养的神经元中，暴露于缺氧或谷氨酸均可诱导ORP150的表达，并且在用thapsigargin或breferdina处理培养时也观察到这一点，表明谷氨酸和缺氧均可引起内质网应激（ER stress）。在环苄胺治疗或携带ORP150反义结构的腺病毒感染后，神经元对这些应激变得更脆弱。相比之下，腺腺腺过多表达ORP150抑制了神经元细胞的死亡，并伴有谷氨酸诱导的Ca2+升高和蛋白水解活性的抑制。此外，携带ORP150感觉结构的腺病毒在CA1神经元中过表达ORP150可抑制缺血后延迟的神经元细胞死亡。这些数据提示ORP150可能作为一种细胞内装置，参与缺血耐受的保护反应。谷氨酸受体相互作用引发的一系列事件扰乱细胞稳态，导致细胞内游离钙升高和细胞死亡。受到这种环境变化的细胞表达应激蛋白，这对维持代谢稳态和生存能力起重要作用。我们发现一种存在于内质网（ER）的诱导伴侣，150 kDa的氧调节蛋白（ORP150）在癫痫发作后的人脑和给药后的小鼠海马中都有表达。在ORP150缺失的小鼠中，暴露于兴奋性刺激下，海马神经元显示胞质钙的过度升高，并伴有μ-calpain和组织蛋白酶B的激活，以及体外对谷氨酸诱导的细胞死亡的易变性增加，体内对盐酸盐的存活率降低。相比之下，ORP150的靶向神经元过表达抑制了这些事件，并在降低癫痫发作强度的同时提高了神经元和动物的存活率。对培养海马神经元的研究表明，ORP150调节兴奋性应激下神经元胞质游离钙和蛋白水解途径的激活，导致细胞死亡。我们的数据强调了内质网应激在谷氨酸毒性中的关键作用，并指出了一个关键的内质网伴侣ORP150，它协调了对神经元存活至关重要的保护性应激反应。少

ORP150 is a novel stress protein localized in the endoplasmic reticulum (ER). To investigate the role of ORP150 in delayed neuronal cell death, we have examined its expression in the gerbil brain after the ischemic insult. The expression of ORP150 antigen, as well as its transcripts, was observed in the CA1 region after the occlusion of the common carotid altety, and this was enhanced by the preconditioning. In cultured neurons, exposure to either hypoxia or glutamate induced the expression of ORP150, and this was also observed by treating the culture with either thapsigargin or breferdin-A, indicating that both glutamate and hypoxia can cause shess in the ER (ER stress). Neurons became more vulnerable to these stresses following treatment of either cyclcheximide or the infection with an adenovirus carrying ORP150 antisense structure. In cantrast, the overexpression of ORP150 by adenovinus suppressed the neuronal cell death, and this was accompanied by the suppression of the Ca2+ eleva … More tion and proteolytic activity induced by glutamate. Further, overexpressicn of ORP150 in CA1 neurons by the adenovirus carrying ORP150-sense struciture suppressed delayed neuronal cell death after ischemia. These data suggest a possible function of ORP150 as an intracellular apparatus, which participates in a protective response in ischemic tolerance.A series of events initiated by glutamate-receptor interaction perturbs cellular homeostasis resulting in elevation of intracellular free calcium and cell death. Cells subject to such environmental change express stress proteins, which contribute importantly to maintenance of metabolic homeostasis and viability. We show that an inducible chaperone present in endoplasmic reticulum (ER), the 150 kDa oxygen-regulated protein (ORP150) is expressed both in human brain after seizure attack and in mice hippocampus after kainite administration. Using mice heterozygous for ORP150 deficiency, exposure to excitatory stimuli caused hippocampal neurons to display exaggerated elevation of cytosolic calcium accompanied by activation of μ-calpain and cathepsin B, as well as increased vulnerability to glutamate-induced cell death in vitro and decreased survival to kainate in vivo. In contrast, targeted neuronal overexpression of ORP150 suppressed each of these events, and enhanced neuronal and animal survival in parallel with diminished seizure intensity. Studies using cultured hippocampal neurons showed that ORP150 regulates cytosolic free calcium and activation of proteolytic pathways causing cell death in neurons subject to excitatory stress. Our data underscore a pivotal role for ER stress in glutamate toxicity, and pinpoint a key ER chaperone, ORP150, which orchestrates the protective stress response critical for neuronal survival. Less