A mouse model linking anesthetic sensitivity to mitochondrial function

项目摘要 挥发性麻醉剂（VA）产生可逆性意识丧失的机制， 使有机体对疼痛无感觉是150多年来医学上未解之谜。我们 证明了线粒体复合物I，线粒体的进入点和速率限制组分， 电子传递链，控制从蠕虫到老鼠的动物王国的麻醉敏感性 对人类这意味着一种古老的机制即将到来，将线粒体功能与突触连接起来。 沉默的VAs Ndufs 4（KO）小鼠缺乏线粒体复合物I的亚基，这增加了复合物的敏感性 异氟烷抑制。对于异氟烷或氟烷，KO小鼠在24小时内对夹尾反应迟钝。 剂量比对照组低约3倍。Ndufs 4（KO）也对VA过敏，使用翻正反射丧失 作为终点。这些KO小鼠显示出在哺乳动物中描述的VA敏感性的最大变化。 我们还发现，VA敏感性完全受突变的谷氨酸能表达控制， GABA能、胆碱能或星形胶质细胞表达的影响。我们测量了翻正反射丧失的EC 50 KO小鼠对靶点已充分表征的其他麻醉剂的LORR。令人惊讶的是，动物 对氯胺酮有抵抗力Ndufs 4（KO）的作用在以下方面是特异性的： 麻醉，而不仅仅是普遍的中枢神经系统抑制的结果。 我们希望了解，在这个提案中，如何复杂的I脊髓缺陷导致 对VA过敏。我们发现来自Ndufs 4（KO）的脊髓切片中的TREK-1通道是 对异氟醚过敏，与其他人一致，他们已经表明小鼠的VA敏感性依赖于 TREK-1功能。我们假设VA超敏反应是通过线粒体对 TREK-1通道在腹角神经元，并旨在表征潜在的机制， 诱导TREK-1对VA敏感性的改变。我们将从细胞和分子靶点转向整体靶点， 动物行为有3个具体目标：1）研究哪些细胞在线粒体功能上有缺陷， 为了产生TREK-I超敏反应; 2）发现Ndufs 4（KO）对细胞内磷酸化位点的影响， 脊髓中的TREK-1通道，以及3）由于我们预测TREK-1激活是VA的基础， 我们的KO应答对尾夹的超敏性，构建缺乏TREK-1的Ndufs 4（KO），并测试其 行为在VA。 我们的首要目标是了解VA的分子靶点。我们将线粒体 在蠕虫，小鼠和人的VA中起作用，并提出线粒体代谢是一种新的，但 VA的潜在作用机制非常合理。

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.