A mouse model linking anesthetic sensitivity to mitochondrial function

项目摘要 挥发性麻醉剂(VAS)导致可逆性意识丧失和 使生物体对疼痛失去知觉仍然是150多年来医学上一个未解的谜团。我们 证明线粒体复合体I是线粒体的入口点和限速成分 电子传输链，控制整个动物王国的麻醉敏感性，从蠕虫到老鼠 对人类来说。这暗示着一种古老的机制就在眼前，它将线粒体功能与突触联系起来 VAS的静音功能。 Ndufs4(KO)小鼠缺乏线粒体复合体I的一个亚单位，这增加了该复合体的敏感性 异氟醚的抑制作用。对于异氟醚或氟烷，KO小鼠对尾巴挤压在 剂量比对照组低约3倍。Ndufs4(KO)也对VAS敏感，表现为翻正反射的丧失 作为终点。这些KO小鼠在哺乳动物中表现出最大的VA敏感性变化。 我们还发现，VA的敏感性完全由突变的谷氨酸能表达控制，没有 GABA能、胆碱能或星形胶质细胞表达的影响。我们测量了EC50的翻正反射损失 (LORR)KO小鼠的其他麻醉药，其靶点被很好地描述。令人惊讶的是，这些动物 实际上对氯胺酮的作用有抵抗力。Ndufs4(KO)的影响在以下方面是具体的 麻醉而不是单纯的广泛性中枢神经系统抑郁的结果。 我们希望在这项提案中了解脊髓缺陷导致的复杂程度 对VAS过敏。我们发现Ndufs4(KO)脊髓切片中的Trek-1通道是 对异氟醚过敏，这与其他人的观点一致，即小鼠的VA敏感性是依赖的 关于Trek-1功能。我们假设VA超敏反应是通过线粒体效应介导的。 TREK-1通道在前角神经元中的表达，目的是研究线粒体的潜在机制 诱导Trek-1对VAS敏感性的改变。我们将从细胞和分子目标转移到整体 动物行为的三个特定目的：1)调查哪些细胞线粒体功能肯定是有缺陷的 以产生Trek-I超敏；2)发现Ndufs4(KO)对 脊髓中的Trek-1通道，以及3)因为我们预测Trek-1的激活是VA的基础 我们的KO对尾夹的超敏反应，构建了缺乏Trek-1的Ndufs4(KO)，并测试了它的 VAS中的行为。 我们的首要目标是了解VAS的分子靶点。我们已经连接了线粒体 在蠕虫、小鼠和人的VAS行为中的作用，并提出线粒体代谢是一种新的但 VAS潜在影响的非常合理的机制。

PROJECT ABSTRACT The mechanism by which volatile anesthetics (VAs) produce reversible loss of consciousness and render an organism insensate to pain remains an unsolved mystery of medicine for over 150 years. We demonstrated that mitochondrial complex I, an entry point and rate limiting component of the mitochondrial electron transport chain, controls anesthetic sensitivity across the animal kingdom, from worms to mice to humans. The implication is that an ancient mechanism is at hand, linking mitochondrial function to synaptic silencing by VAs. Ndufs4(KO) mice lack a subunit of mitochondrial complex I, which increases sensitivity of the complex to isoflurane inhibition. For either isoflurane or halothane, the KO mice became unresponsive to a tail pinch at a dose ~3-fold lower than for controls. Ndufs4(KO) was also hypersensitive to VAs using loss of righting reflex as the endpoint. These KO mice display the greatest change in VA sensitivity described in a mammal. We also discovered that VA sensitivity was fully controlled by glutamatergic expression of the mutation, with no effect from GABAergic, cholinergic or astrocyte expression. We measured the EC50s for loss of righting reflex (LORR) of the KO mice for other anesthetics whose targets are well characterized. Surprisingly, the animals were actually resistant to the effects of ketamine. The effects of the Ndufs4(KO) are specific in terms of anesthetic and not simply the result of generalized CNS depression. We wish to understand, in this proposal how complex I defects in the spinal cord cause hypersensitivity to VAs. We discovered that TREK-1 channels in spinal cord slices from Ndufs4(KO) are hypersensitive to isoflurane, in agreement with others who have shown that VA sensitivity of mice is dependent on TREK-1 function. We hypothesize that the VA hypersensitivity is mediated through a mitochondrial effect on TREK-1 channels in ventral horn neurons, and aim to characterize the mechanisms underlying mitochondrially induced changes in TREK-1sensitivity to VAs. We will move from cellular and molecular targets to whole animal behaviors in 3 specific aims: 1) investigating which cells must be defective in mitochondrial function in order to produce TREK-I hypersensitivity; 2) discover the effects of Ndufs4(KO) on phosphorylation sites within TREK-1 channels in the spinal cord, and 3) since we predict that TREK-1 activation underlies the VA hypersensitivity of our KO response to tail clamp, construct an Ndufs4(KO) that lacks TREK-1, and test its behavior in VAs. Our overarching goal is to understand the molecular targets of VAs. We have linked mitochondrial function to behavior in VAs in worms, mice, and man, and propose that mitochondria metabolism is a novel but very plausible mechanism underlying effects of VAs.