Dutta Somhrita, Rutkai Ibolya, Katakam Prasad V G, Busija David W
Neuroscience Program, Tulane University School of Science and Engineering, New Orleans, Louisiana, USA.
Department of Pharmacology, Tulane University School of Medicine, New Orleans, Louisiana, USA.
J Neurochem. 2015 Sep;134(5):845-56. doi: 10.1111/jnc.13181. Epub 2015 Jul 1.
We examined the role of the mechanistic target of rapamycin (mTOR) pathway in delayed diazoxide (DZ)-induced preconditioning of cultured rat primary cortical neurons. Neurons were treated for 3 days with 500 μM DZ or feeding medium and then exposed to 3 h of continuous normoxia in Dulbecco's modified eagle medium with glucose or with 3 h of oxygen-glucose deprivation (OGD) followed by normoxia and feeding medium. The OGD decreased viability by 50%, depolarized mitochondria, and reduced mitochondrial respiration, whereas DZ treatment improved viability and mitochondrial respiration, and suppressed reactive oxygen species production, but did not restore mitochondrial membrane potential after OGD. Neuroprotection by DZ was associated with increased phosphorylation of protein kinase B (Akt), mTOR, and the major mTOR downstream substrate, S6 Kinase (S6K). The mTOR inhibitors rapamycin and Torin-1, as well as S6K-targeted siRNA abolished the protective effects of DZ. The effects of DZ on mitochondrial membrane potential and reactive oxygen species production were not affected by rapamycin. Preconditioning with DZ also changed mitochondrial and non-mitochondrial oxygen consumption rates. We conclude that in addition to reducing reactive oxygen species (ROS) production and mitochondrial membrane depolarization, DZ protects against OGD by activation of the Akt-mTOR-S6K pathway and by changes in mitochondrial respiration. Ischemic strokes have limited therapeutic options. Diazoxide (DZ) preconditioning can reduce neuronal damage. Using oxygen-glucose deprivation (OGD), we studied Akt/mTOR/S6K signaling and mitochondrial respiration in neuronal preconditioning. We found DZ protects neurons against OGD via the Akt/mTOR/S6K pathway and alters the mitochondrial and non-mitochondrial oxygen consumption rate. This suggests that the Akt/mTOR/S6k pathway and mitochondria are novel stroke targets.
我们研究了雷帕霉素的作用机制靶点（mTOR）通路在二氮嗪（DZ）诱导的培养大鼠原代皮质神经元延迟预处理中的作用。将神经元用500μM DZ或维持培养基处理3天，然后在含有葡萄糖的杜氏改良 Eagle培养基中暴露于3小时持续常氧环境，或经历3小时氧糖剥夺（OGD），随后再进行常氧和维持培养基处理。OGD使细胞活力降低50%，使线粒体去极化，并降低线粒体呼吸作用，而DZ处理可改善细胞活力和线粒体呼吸作用，并抑制活性氧的产生，但在OGD后不能恢复线粒体膜电位。DZ的神经保护作用与蛋白激酶B（Akt）、mTOR以及mTOR主要下游底物S6激酶（S6K）的磷酸化增加有关。mTOR抑制剂雷帕霉素和Torin-1，以及靶向S6K的小干扰RNA（siRNA）消除了DZ的保护作用。雷帕霉素不影响DZ对线粒体膜电位和活性氧产生的作用。DZ预处理还改变了线粒体和非线粒体的氧消耗率。我们得出结论，除了减少活性氧（ROS）的产生和线粒体膜去极化外，DZ还通过激活Akt-mTOR-S6K通路以及改变线粒体呼吸作用来保护细胞免受OGD损伤。缺血性中风的治疗选择有限。二氮嗪（DZ）预处理可减少神经元损伤。利用氧糖剥夺（OGD），我们研究了神经元预处理中的Akt/mTOR/S6K信号传导和线粒体呼吸作用。我们发现DZ通过Akt/mTOR/S6K通路保护神经元免受OGD损伤，并改变线粒体和非线粒体的氧消耗率。这表明Akt/mTOR/S6k通路和线粒体是中风的新靶点。
