Murine Models of Presynaptic Neuromuscular Disease

描述（由申请方提供）：几种表现为骨骼肌无力的人类神经肌肉疾病与乙酰胆碱（ACh）释放的突触前损害有关。这些疾病包括肉毒杆菌中毒、某些毒蛇和节肢动物的毒刺以及几种人类先天性肌无力综合征。最具特征的突触前神经肌肉疾病是Lambert-Eaton肌无力综合征（LEMS），其通常与燕麦细胞癌相关。LEMS中的骨骼肌无力被认为是由运动神经末梢Ca通道功能复合体的自身免疫攻击引起的。 本项目的目的是研究运动神经末梢ACh释放中断的动物模型，以了解更多关于LEMS等疾病的病因。运动神经末梢中存在N型、P/Q型、R型和L型等多种钙通道亚型。P/Q型Ca通道主要负责控制哺乳动物神经肌肉接头处的ACh释放，因此是LEMS中假定的主要自身免疫靶点。 自身抗体被认为主要靶向通道的α 1亚基，尽管其他蛋白质也被假定为LEMS中的抗原靶点。在小鼠LEMS的被动传递模式中，控制ACh从运动终末释放的Ca通道表型发生变化，并表达显著的L型成分，提示发生代偿性变化以弥补Ca通道正常P/Q补体的功能丧失。 在本项目中，突触电位的细胞内微电极记录、神经末梢Ca通道活性的神经束膜记录、FM 1 -43荧光记录和神经末梢Ca通道亚单位免疫细胞化学将被用于研究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a1 a亚单位通过缺失或点突变进行遗传改变，或其中与P/Q型Ca通道CA亚基共表达的正常亚基发生突变。目标是：一。LEMS可以被动转移到缺乏功能或具有突变的P/Q型Ca通道的遗传改变小鼠中吗？它们的反应与野生型小鼠不同吗？ 这些小鼠是否正常表达L型钙通道活性，以及在被动转移LEMS后是否发生改变？ 在缺失P/Q型钙通道a1 a基因或a1 a亚基发生“摇摇欲坠”或“更瘦”突变的小鼠中，L型通道在调节运动神经末梢中ACh释放方面是否发挥更大作用？L型钙通道在这些条件下是否存在或暴露？ 2.缺乏P/Q型Ca通道或在孔形成区有突变的动物的神经末梢中，哪些Ca通道参与神经诱发的ACh释放。 3.钙通道0亚单位在神经肌肉接头ACh释放中起什么作用？ 在04亚基中具有突变（“昏睡”）的动物是否易受LEMS诱导？ 这项研究的结果应该提供一个更好的了解LEMS的病因和随后的变化，发生在运动神经末梢的LEMS患者。

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.