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有关 乙酰胆碱(ACh)释放障碍。这些包括肉毒杆菌中毒，某些毒蛇的中毒和 节肢动物以及几种人类先天性肌无力综合征。最好的特征是突触前 神经肌肉疾病是Lambert-Eaton肌无力综合征(LEMS)，常与燕麦细胞相关 癌症。肌萎缩侧索硬化症的骨骼肌无力被认为是对钙通道的自身免疫攻击所致 运动神经末梢的功能复合体。这个项目的目标是研究有干扰的动物模型。 运动神经末梢ACh的释放，以了解更多关于LEMS等疾病的病因。的多个子类型 运动神经末梢存在N-、P/Q-、R-和L等钙通道。P/Q型钙通道主要是 负责控制哺乳动物神经肌肉接头的ACh释放，因此被认为是主要的 LEMS中的自身免疫靶点。自身抗体被认为主要针对通道的CH亚单位，尽管其他 蛋白质也被认为是LEMS的抗原靶点。在小鼠被动转移LEMS模型中， 控制运动终末ACh释放的钙通道表型发生变化，并表达了新的补充 CA频道。在这个项目中，细胞内微电极记录突触电位，神经周围记录 神经末梢钙通道活性、FM1-43荧光和神经末梢钙通道亚单位的记录 免疫细胞化学将用于研究被动将LEMS移植到小鼠体内后的神经肌肉传递 其中P/Q型钙通道的A1a亚基被基因改变，无论是通过缺失或点突变，还是在 与P/Q型钙通道CH亚基共表达的正常(3)亚基发生突变。目标是：1. LEMS能否被动转移到缺乏功能或P/Q型突变的转基因小鼠 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，被动转移LEMS后会发生改变吗？L类经络在调节ACH中是否发挥更大作用 P/Q型Cachannel A1a基因缺失或突变的小鼠运动神经末梢释放 作为“摇摇欲坠”或“瘦身”发生在A1a亚基？L式的香榭丽舍是现身还是揭开面纱 这些条件？2.哪些钙通道参与神经诱发的动物神经终末ACh的释放 缺乏P/Q型钙通道，或在造孔区有突变？3.钙通道/3 亚单位在神经肌肉交界处的ACh释放中起作用？在j84亚单位发生突变(“昏昏欲睡”)的动物 对LEMS的诱导敏感吗？这项研究的结果应该能更好地理解糖尿病的病因。 LEMS患者运动神经末梢的LEM和随后的变化。

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.