A mouse model linking anesthetic sensitivity to mitochondrial function

将麻醉敏感性与线粒体功能联系起来的小鼠模型

基本信息

批准号：
9922910
负责人：
Margaret Mary Sedensky
金额：
$ 54.48万
依托单位：
SEATTLE CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-05-01 至 2022-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9922910
关键词：
Agreement Anesthetics Animal Behavior Animals Anterior Horn Cells Astrocytes Behavior Biology Caenorhabditis elegans Cells Characteristics Child Clinical Closure by clamp Complex Conscious Control Animal Data Defect Dependence Dose Drug Design Electron Transport Electrophysiology (science)Exposure to Functional disorder Future Genes Glutamates Goals Halothane Hand Human Hypersensitivity Invertebrates Investigation Isoflurane Ketamine Knock-out Knockout Mice Knowledge Laboratories Lead Link Mammals Measurement Measures Mediating Medicine Mental Depression Mitochondria Modeling Modern Medicine Molecular Molecular Target Monitor Mus Mutation Nematoda Neurons Organism Pain Phenotype Phosphorylation Phosphorylation Site Post-Translational Protein Processing Potassium Potassium Channel Reflex action Resistance Safety Secondary to Slice Spinal Cord Synapses Tail Technology Testing Unconscious State Ventral Horn of the Spinal Cord Work cellular targeting cholinergic cholinergic neuron improved inhibitor/antagonist interest knockout animal man mitochondrial dysfunction mitochondrial metabolism mouse model mutant neurotoxicity novel potassium channel protein TREK-1 response side effect

项目摘要

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.

项目摘要挥发性麻醉剂 (VA) 产生可逆性意识丧失的机制 150多年来，如何使生物体对疼痛失去知觉一直是医学界未解之谜。我们证明线粒体复合物 I，线粒体的入口点和限速成分电子传输链，控制从蠕虫到小鼠的整个动物界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联系起来 VA 沉默。 Ndufs4(KO) 小鼠缺乏线粒体复合物 I 的亚基，这增加了该复合物的敏感性对异氟烷有抑制作用。对于异氟烷或氟烷，KO 小鼠在剂量比对照低约 3 倍。 Ndufs4(KO) 也因翻正反射丧失而对 VA 过敏作为端点。这些 KO 小鼠表现出哺乳动物中 VA 敏感性的最大变化。我们还发现 VA 敏感性完全由突变的谷氨酸表达控制，没有 GABA 能、胆碱能或星形胶质细胞表达的影响。我们测量了翻正反射丧失的 EC50 (LORR) KO 小鼠用于其他麻醉剂，其靶标已得到充分表征。令人惊讶的是，动物们实际上对氯胺酮的作用有抵抗力。 Ndufs4(KO) 的效果具体体现在：麻醉，而不仅仅是全身中枢神经系统抑制的结果。我们希望了解，在这个提案中，复杂的 I 型脊髓缺陷是如何引起的对VA过敏。我们发现 Ndufs4(KO) 脊髓切片中的 TREK-1 通道是对异氟烷过敏，与其他人的观点一致，表明小鼠的 VA 敏感性取决于 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 在腹角神经元中形成通道，旨在表征线粒体的潜在机制诱导 TREK-1 对 VA 敏感性的变化。我们将从细胞和分子目标转向整体动物行为有 3 个具体目标：1）调查哪些细胞的线粒体功能一定有缺陷为了产生TREK-I超敏反应； 2) 发现Ndufs4(KO)对磷酸化位点的影响脊髓中的 TREK-1 通道，3) 因为我们预测 TREK-1 激活是 VA 的基础我们的 KO 反应对尾夹的超敏反应，构建缺乏 TREK-1 的 Ndufs4(KO)，并测试其 VA 中的行为。我们的首要目标是了解 VA 的分子靶标。我们已经将线粒体联系起来线虫、小鼠和人的 VA 行为发挥作用，并提出线粒体代谢是一种新颖但 VAs 影响的潜在机制非常合理。