Pathogenesis of neuromuscular synaptic dysfunction and transmission failure in organophosphate toxicity

有机磷毒性中神经肌肉突触功能障碍和传递失败的发病机制

• 批准号: MR/M024075/1
• 负责人: Richard Ribchester
• 金额: $ 85.36万
• 依托单位: University of Edinburgh
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FM024075%2F1
• 关键词: Pathogenesis; neuromuscular; synaptic; dysfunction; transmission

Every year, across the world, hundreds of thousands of people die following poisoning with organophosphorus (OP) pesticides. Other populations are at risk from terrorist use of OP nerve agents (as recently used in the Middle East). These compounds interrupt signalling in the brain and muscles, causing paralysis and death when breathing fails. However, if patients reach hospital in time, they can be treated: first with antidotes and then with assisted breathing, by mechanical ventilation, in an intensive care unit. Unfortunately and for unknown reasons, some patients are unable to breathe for themselves for several weeks, leaving them dangerously at risk of lethal complications, like pneumonia. We are trying to uncover the mechanism linking OP poisoning to paralysis and cessation of breathing. We started by setting up animal models of poisoning, focusing on a particular agricultural OP pesticide, dimethoate EC40. This pesticide contains two essential ingredients: dimethoate itself and an organic solvent, cyclohexanone. These substances are broken down in the body by metabolism: dimethoate is converted into omethoate in the liver; and cyclohexanone is converted to cyclohexanol in the stomach and intestines. Our investigations have so far led us to a surprising discovery: the two metabolites, omethoate and cyclohexanol, are much more toxic to the connections between motor neurones and muscle cells, at their nerve-muscle ("neuromuscular") junctions, than either of the main ingredients in the pesticide itself. Acetylcholine is the neurotransmitter molecule that carries signals from the motor nerve endings to the muscle at these neuromuscular junctions. Our data strongly suggest that omethoate and cyclohexanol act aggressively together, directly affecting the protein receptors for acetylcholine, located on the muscle side of neuromuscular junctions. In this project, we will home in on and investigate this theory in three, concerted ways. First of all, we will inject the human DNA code for the acetylcholine receptor proteins into cells in tissue culture. The cells will turn the DNA into working receptors, allowing us to make recordings using an exquisite electrical technique called patch-clamping. In this way we can test and measure directly how omethoate, cyclohexanol or both together affect the way the receptor molecules respond to acetylcholine. Secondly, we will use a new type of tissue culture assay, to measure the progressive toxicity of omethoate and cyclohexanol on transmission at neuromuscular junctions over a period of 24 hours, the same period over which patients typically deteriorate. For these measurements, we will take advantage of nerves and muscles in a breed of mouse (called WldS) whose neuromuscular junctions are normally quite resistant to nerve damage. This feature gives us the time we need to find out why pesticide and its metabolites cause muscles to become progressively paralysed. This assay also gives us the option of rhythmically stimulating the nerve (as occurs naturally in muscles we use for breathing) to test whether this activity actually makes the pesticide poisoning worse. Finally, we will combine electrical recording with an exciting new technology, called confocal endomicroscopy, and apply these to direct observation of living neuromuscular junctions. We will use this approach to assess the benefits of different kinds of drugs that protect acetylcholine receptors, which should prevent pesticide and their breakdown products from damaging the receptors. We will test these drugs in our DNA-injected cells, in our WldS mouse tissue cultures, and lastly in anaesthetised pigs, because we have found that pigs react to OP pesticides in a very similar way to humans. Treatments that work will then be tested in clinical trials, in patients poisoned by OP pesticides that we currently look after in hospitals in Sri Lanka. An effective treatment will save tens of thousands of lives every year.

每年，全世界有数十万人死于有机磷（OP）农药中毒。其他人群面临恐怖分子使用OP神经毒剂的风险（如最近在中东使用的）。这些化合物会干扰大脑和肌肉中的信号，导致呼吸衰竭时瘫痪和死亡。然而，如果患者及时到达医院，他们可以得到治疗：首先使用解毒剂，然后在重症监护室通过机械通气进行辅助呼吸。不幸的是，由于未知的原因，一些患者无法呼吸几个星期，使他们处于致命并发症的危险之中，如肺炎。我们正试图揭示OP中毒与瘫痪和呼吸停止之间的联系机制。我们首先建立了动物中毒模型，重点是一种特殊的农业有机磷农药，乐果EC 40。这种杀虫剂含有两种基本成分：乐果本身和一种有机溶剂环己酮。这些物质在体内通过新陈代谢分解：乐果在肝脏中转化为氧乐果;环己酮在胃和肠中转化为环己醇。到目前为止，我们的研究使我们有了一个令人惊讶的发现：氧化乐果和环己醇这两种代谢物对运动神经元和肌肉细胞之间的神经-肌肉（“神经肌肉”）连接处的毒性比杀虫剂本身的任何一种主要成分都大。乙酰胆碱是一种神经递质分子，它将信号从运动神经末梢传递到这些神经肌肉接头处的肌肉。我们的数据强烈表明，氧乐果和环己醇一起积极行动，直接影响乙酰胆碱的蛋白质受体，位于神经肌肉接头的肌肉侧。在这个项目中，我们将以三种协调一致的方式来研究这个理论。首先，我们将乙酰胆碱受体蛋白的人类DNA代码注入组织培养的细胞中。这些细胞将把DNA转化为工作受体，使我们能够使用一种称为膜片钳的精密电子技术进行记录。通过这种方式，我们可以直接测试和测量氧乐果、环己醇或两者一起如何影响受体分子对乙酰胆碱的反应。其次，我们将使用一种新型的组织培养试验，以测量氧乐果和环己醇在24小时内对神经肌肉接头传递的进行性毒性，在此期间，患者通常会恶化。对于这些测量，我们将利用一种小鼠（称为WldS）的神经和肌肉，其神经肌肉接头通常对神经损伤具有很强的抵抗力。这一特征给了我们时间来找出为什么杀虫剂及其代谢物会导致肌肉逐渐瘫痪。这种分析也让我们可以选择有节奏地刺激神经（就像我们用于呼吸的肌肉中自然发生的那样），以测试这种活动是否真的会使农药中毒变得更糟。最后，我们将结合联合收割机电记录与一个令人兴奋的新技术，所谓的共聚焦显微内镜，并应用这些直接观察活的神经肌肉接头。我们将使用这种方法来评估不同种类的保护乙酰胆碱受体的药物的益处，这些药物应该防止农药及其分解产物破坏受体。我们将在我们的DNA注射细胞中测试这些药物，在我们的WldS小鼠组织培养中，最后在麻醉的猪中测试，因为我们发现猪对OP农药的反应与人类非常相似。有效的治疗方法将在临床试验中进行测试，在我们目前在斯里兰卡医院治疗的OP农药中毒患者中进行测试。有效的治疗每年将挽救成千上万的生命。

项目成果

Antagonistic postsynaptic and presynaptic actions of cyclohexanol on neuromuscular synaptic transmission and function

• DOI: 10.1113/jp281921
• 发表时间: 2021-11-26
• 期刊: JOURNAL OF PHYSIOLOGY-LONDON
• 影响因子: 5.5
• 作者: Dissanayake，Kosala N.;Margetiny，Filip;Ribchester，Richard R.
• 通讯作者: Ribchester，Richard R.

Relationship between alcohol co-ingestion and outcome in profenofos self-poisoning - A prospective case series.

• DOI: 10.1371/journal.pone.0200133
• 发表时间: 2018
• 期刊: PloS one
• 影响因子: 3.7
• 作者: Dhanarisi HKJ;Gawarammana IB;Mohamed F;Eddleston M
• 通讯作者: Eddleston M

"Calcium bombs" as harbingers of synaptic pathology and their mitigation by magnesium at murine neuromuscular junctions.

• DOI: 10.3389/fnmol.2022.937974
• 发表时间: 2022
• 期刊: FRONTIERS IN MOLECULAR NEUROSCIENCE
• 影响因子: 4.8
• 作者: Dissanayake, Kosala N.;Redman, Robert R.;Mackenzie, Harry;Eddleston, Michael;Ribchester, Richard R.
• 通讯作者: Ribchester, Richard R.

Circadian and Brain State Modulation of Network Hyperexcitability in Alzheimer's Disease.

• DOI: 10.1523/eneuro.0426-17.2018
• 发表时间: 2018-03
• 期刊: eNeuro
• 影响因子: 3.4
• 作者: Brown R;Lam AD;Gonzalez-Sulser A;Ying A;Jones M;Chou RC;Tzioras M;Jordan CY;Jedrasiak-Cape I;Hemonnot AL;Abou Jaoude M;Cole AJ;Cash SS;Saito T;Saido T;Ribchester RR;Hashemi K;Oren I
• 通讯作者: Oren I

A pilot clinical study of the neuromuscular blocker rocuronium to reduce the duration of ventilation after organophosphorus insecticide poisoning.

神经肌肉阻滞剂罗库溴铵减少有机磷杀虫剂中毒后通气时间的初步临床研究。

• DOI: 10.1080/15563650.2019.1643467
• 发表时间: 2020
• 期刊: Clinical toxicology (Philadelphia, Pa.)
• 作者: Dhanarisi J