Murine Models of Presynaptic Neuromuscular Disease

几种表现为骨骼肌无力的人类神经肌肉疾病与突触前神经元损伤有关。 乙酰胆碱（ACh）释放受损。这些包括肉毒杆菌中毒，某些毒蛇的毒液， 节肢动物以及几种人类先天性肌无力综合征。突触前的最佳特征 神经肌肉疾病是Lambert-Eaton肌无力综合征（LEMS），其通常与燕麦细胞 carcinoma. LEMS中的骨骼肌无力被认为是由于对Ca通道的自身免疫攻击 运动神经末梢的功能复合体。本项目的目的是研究动物模型与中断 运动神经末梢ACh的释放，以了解更多有关LEMS等疾病的病因。多个亚型的 运动神经末梢存在N型、P/Q型、R型和L型钙通道。P/Q型钙通道主要是 负责控制哺乳动物神经肌肉接头处的ACh释放，因此被认为是主要的 自身免疫靶点自身抗体被认为主要靶向通道的CH亚基，尽管其他抗体也可以靶向通道的CH亚基。 蛋白质也被假定为LEMS中的抗原靶。在小鼠LEMS被动转移模型中， 控制ACh从运动末梢释放的Ca通道的表型改变，并表达一种新的补体， 钙通道。在该项目中，细胞内微电极记录突触电位、神经束膜记录 神经末梢Ca通道活性，FM 1 - 43荧光和神经末梢Ca通道亚单位的记录 免疫细胞化学将用于研究LEMS被动转移到小鼠后的神经肌肉传递， 其中P/Q型Ca通道的α 1A亚基通过缺失或点突变而被遗传改变，或 其中与P/Q型Ca通道CH亚基共表达的正常β亚基被突变。目标是：1. LEMS是否可以被动转移到缺乏功能性或具有突变的P/Q型Ca 通道，它们的反应与野生型小鼠不同吗？这些小鼠是否表达L型钙通道活性 正常情况下，它是改变后，被动转移LEMS？L型通道在调节ACh中是否发挥更大的作用 在缺失P/Q型Cachanel的a1A基因的小鼠的运动神经末梢中的释放，或者在其中突变， "摇摇欲坠"或"瘦"发生在a1A亚基？L型缓存通道是否在以下任一情况下出现或暴露 这些条件？2.哪些钙通道参与了动物神经末梢乙酰胆碱的释放 缺乏P/Q型钙通道，或者在成孔区有突变？3. Ca通道/3在细胞内的作用是什么？ 亚单位在神经肌肉接头ACh释放中作用？在j84亚基中具有突变（"昏睡"）的动物 易受LEMS的诱导？这项研究的结果应该提供了一个更好的了解的病因， LEMS和LEMS患者运动神经末梢发生的后续变化。

Several human neuromuscular disorders which present as skeletal muscle weakness are associated with presynaptic impairment of release of acetylcholine (ACh). These include botulism, envenomation by certain poisonous snakes and arthropods as well as several human congential myasthenic syndromes. The best characterized of the presynaptic neuromuscular disorders is Lambert-Eaton Myasthenic Syndrome (LEMS) which often associates with oat cell carcinoma. Skeletal muscle weakness in LEMS is thought to result from autoimmunic attack on the Ca channel functional complex at the motor nerve terminal. The objective of this project is to study animal models with disruptions of motor nerve terminal ACh release to learn more about the etiology of diseases such as LEMS. Multiple subtypes of Ca channels such as the N-, P/Q-, R- and L-type exist in motor nerve terminals. P/Q-type Ca channels are primarily responsible for controlling ACh release at mammalian neuromuscular junctions, and hence are the presumed primary autoimmune target in LEMS. Autoantibodies are thought to target primarily to CH subunit of the channel, although other proteins have also been postulated as antigenic targets in LEMS. In the passive transfer model of LEMS in mice, the phenotype of Ca channel controlling ACh release from motor terminals changes, and expresses a new complement of Ca channels. In this project, intracellular microelectrode recordings of synaptic potentials, perineurial recordings of nerve terminal Ca channel activity, recordings of FM1-43 fluorescence and nerve terminal Ca channel subunit immunocytochemistry will be used to study neuromuscular transmission following passive transfer of LEMS to mice in which the a1A subunit of the P/Q-type of Ca channel is genetically-altered, either by deletion or point mutation, or in which the normal (3 subunit which coexpresses with the P/Q-type Ca channel CH subunit is mutated. The aims are: 1. Can LEMS be passively transferred to genetically-altered mice lacking functional or having mutated P/Q-type Ca channels and do they respond differently than do wildtype mice? Do these mice express L-type Ca channel activity normally, and is it altered after passive transfer of LEMS? Do L-type channels play a greater role in regulatingACh release in motor nerve terminals of mice missing the a1A gene of the P/Q-type Cachannel or in which mutations such as "tottering" or "leaner" occur in the a1A subunit? Do L-type Cachannels become present or unmasked uner either of these condidtions? 2. Which Ca channels are involved in nerve-evoked release of ACh from nerve termianls of animals lacking P/Q-type Ca channels, or having mutations in the pore-forming region? 3. What role does the Ca channel /3 subunit play in ACh release at neuromuscularjunctions? Will animals having a mutation ("lethargic") in the j84 subunit be susceptible to induction of LEMS? Results of this study should provide a better understanding of the etiology of LEMS and subsequent changes that occur at motor nerve terminals in patients with LEMS.