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

ORP150是一种新的应激蛋白，定位于内质网(ER)。为了研究ORP150在迟发性神经细胞死亡中的作用，我们检测了其在沙土鼠脑缺血损伤后的表达。阻断颈总动脉后，在CA1区可观察到ORP150抗原及其转录本的表达，而预适应可使其表达增强。在培养的神经元中，无论是低氧还是谷氨酸，都能诱导ORP150的表达，用thapsigargin或breferdin-A处理也能观察到这一点，这表明谷氨酸和低氧都能引起内质网中的SHE(内质网应激)。在环己酰亚胺或携带ORP150反义结构的腺病毒感染后，神经元变得更容易受到这些压力的影响。在Cantrast中，腺病毒过表达orp150抑制了神经细胞的死亡，并且伴随着对钙离子…的抑制。谷氨酸诱导的更多的蛋白质和蛋白分解活性。此外，携带ORP150正义结构的腺病毒在CA1神经元过度表达ORP150可抑制缺血后迟发性神经细胞死亡。这些数据提示，ORP150可能是一种细胞内装置，参与了缺血耐受中的保护性反应。谷氨酸-受体相互作用引发的一系列事件扰乱了细胞内的稳态，导致细胞内游离钙升高和细胞死亡。受到这种环境变化影响的细胞表达应激蛋白，这些蛋白对维持代谢稳态和生存能力具有重要作用。我们发现150 kDa氧调节蛋白(ORP150)存在于内质网(ER)中，是一种可诱导的伴侣蛋白，在癫痫发作后的人脑和给予海人藻酸钾后的小鼠海马区均有表达。利用ORP150缺乏的杂合子小鼠，暴露于兴奋刺激导致海马神经元胞浆钙过度升高，伴随着μ-Calain和组织蛋白酶B的激活，以及在体外增加对谷氨酸诱导的细胞死亡的易感性，并降低在体内对红藻氨酸的存活率。相反，靶向神经元过表达ORP150抑制了这些事件中的每一个，并在降低癫痫强度的同时提高了神经元和动物的存活率。使用培养的海马神经元的研究表明，ORP150调节胞浆游离钙和蛋白分解途径的激活，导致兴奋性应激下神经元的细胞死亡。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，并准确地指出了一个关键的内质网伴侣蛋白ORP150，它协调对神经元生存至关重要的保护性应激反应。较少

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