Murine Models of Presynaptic Neuromuscular Disease

描述（由申请人提供）：几种以骨骼肌无力为表现的人类神经肌肉疾病与乙酰胆碱（ACh）释放的突触前损伤有关。这些包括肉毒杆菌中毒，某些毒蛇和节肢动物的中毒以及几种人类先天性肌无力综合征。突触前神经肌肉疾病的最佳特征是兰伯特-伊顿肌无力综合征（LEMS），通常与燕麦细胞癌相关。LEMS骨骼肌无力被认为是由于自身免疫攻击运动神经末梢Ca通道功能复合物所致。本项目的目的是研究运动神经末梢乙酰胆碱释放中断的动物模型，以进一步了解LEMS等疾病的病因。运动神经末梢存在多种Ca通道亚型，如N-型、P/Q-型、R-型和l -型。P/ q型Ca通道主要负责控制哺乳动物神经肌肉连接处的乙酰胆碱释放，因此被认为是LEMS的主要自身免疫靶点。自身抗体被认为主要针对通道的a1亚基，尽管其他蛋白质也被认为是LEMS的抗原靶点。在小鼠LEMS的被动传递模式下，控制运动末端ACh释放的Ca通道表型发生改变，并表达了显著的l型成分，提示发生了代偿性改变，弥补了Ca通道正常P/Q补体的功能损失。在这个项目中，细胞内突触电位的微电极记录、神经末梢Ca通道活性的神经周围记录、FM1-43荧光记录和神经末梢Ca通道亚基免疫细胞化学记录将用于研究LEMS被动转移到P/ q型Ca通道的a1a亚基被基因改变（通过缺失或点突变）的小鼠后的神经肌肉传递。或者与P/ q型Ca通道Ca亚基共表达的正常亚基发生突变。目的是：1 . LEMS是否可以被动转移到缺乏功能或P/ q型Ca通道突变的转基因小鼠身上，它们的反应是否与野生型小鼠不同？这些小鼠是否正常表达l型Ca通道活性，在LEMS被动转移后是否发生改变？在缺少P/Q型Ca通道a1a基因的小鼠运动神经末梢，l型通道在调节乙酰胆碱释放方面是否发挥了更大的作用，还是a1a亚基发生了“摇摇”或“变瘦”等突变？在这两种情况下，l型钙通道是存在还是不存在？2. 哪些Ca通道参与缺乏P/ q型Ca通道或在成孔区发生突变的动物神经末梢神经诱发的乙酰胆碱释放。3. 钙通道0亚基在神经肌肉连接处乙酰胆碱释放中起什么作用？在04亚基中有突变（“嗜睡”）的动物是否容易诱导LEMS？本研究的结果将有助于更好地了解LEMS的病因以及LEMS患者运动神经末梢发生的后续变化。

DESCRIPTION (provided by applicant): Several human neuromuscular disorders which present as skeletal muscle weakness are associated with presynaptic impairment of release of acetycholine (ACh). These include botulism, envenomation by certain poisonous snakes and arthropods as well as several human congenital 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 autoimmune 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 the a1 subunit of the channel, although other proteins have also been postulated as antigenic targets in LEMS. In the passive transfer mode of LEMS in mice, the phenotype of Ca channel controlling ACh release from motor terminals changes, and expresses a significant L-type component, suggesting a compensatory change occurs to make up for functional loss of the normal P/Q 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 subunit which coexpresses with the P/Q-type Ca channel CA subunit is mutated. The aims are: I. 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 regulating ACh release in motor nerve terminals of mice missing the a1a gene of the P/Q- type Ca channel or in which mutations such as "tottering" or "leaner" occur in the a1a subunit? Do L-type Ca channels become present or unmasked under either of these conditions? 2. Which Ca channels are involved in nerve-evoked release of ACh from nerve terminals of animals lacking P/Q-type Ca channels, or having mutations in the pore-forming region. 3. What role does the Ca channel 0 subunit play in ACh release at neuromuscular junctions? Will animals having a mutation ("lethargic") in the 04 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.