ALS IGG EFFECTS ON MOTORNEURON UNTRASTRUCTURE

肌萎缩侧索硬化症是一种毁灭性的神经肌肉疾病， 导致进行性虚弱，最终导致呼吸衰竭和 死亡。我们的长期目标是确定免疫学的作用 肌萎缩侧索硬化症的病因和发病机制。尽管病原学是 目前尚不清楚，最近的证据表明，免疫机制可能在 在引发运动神经元损伤和细胞死亡中的作用。体外试验结果 实验表明ALS免疫球蛋白与神经元电压门控相互作用 钙通道导致过多的钙内流和钙中介 神经元损伤。然而，人类的肌萎缩侧索硬化症是一种缓慢进展的病理 而ALS免疫球蛋白在体外具有快速杀伤力。我们的现在 研究的目的是描述超微结构的演变。 肌萎缩侧索硬化症免疫球蛋白对运动神经元钙水平的影响 在体内通过被动转移到小鼠体内。这类调查应有助于 在最早阶段定义神经元损伤，并可能填补这一空白； ALS的缓慢演变变化和模型实验的结果。 因此，具体目标是： (1)测定ALS-Ig G作用的时间/剂量依赖关系 小鼠运动神经元的超微结构和细胞内钙水平 注射ALS免疫球蛋白 (2)用肌萎缩侧索硬化症免疫球蛋白代替肌萎缩侧索硬化症免疫球蛋白以确定其特异性。 用电位膜预吸附肌萎缩侧索硬化症 靶点(纯化的钙通道)并通过拮抗它们的作用 钙通道阻滞剂。 (3)提供详细的电生理和超微结构研究 体内注射免疫球蛋白后递质释放和囊泡循环的研究 变成老鼠。 在这些研究中，小鼠将被注射不同的 肌萎缩侧索硬化症免疫球蛋白的剂量及超微结构的灌流固定 在不同的时间点进行分析。含有草酸根的离子 将应用戊二醛固定剂提供良好的超微结构 保存和同时保存原位钙分布。 形态和细胞内钙分布的变化将是 在运动神经末梢进行定量电子显微镜检测， 不同类型运动神经元的肌内轴突和胞体 对肌萎缩侧索硬化症的易感性和变化将与适当 控制。在平行实验中，肌萎缩侧索硬化症免疫球蛋白引起神经肌肉的变化 传输的特征将是测量微型终端的变化 通过追踪突触小泡的再循环 利用抗囊泡蛋白抗体的免疫组织化学方法。

Amyotrophic lateral sclerosis is a devastating neuromuscular disease which produces progressive weakness leading eventually to respiratory failure and death. Our long term objective is to define the role of immunological factors in the etiology and pathogenesis of ALS. Although the etiology is still unknown, recent evidence suggests that immune mechanisms may play a role in triggering motoneuron injury and cell death. Results of in vitro experiments suggest that ALS IgG interact with neuronal voltage gated calcium channels resulting in excess calcium influx and calcium mediated neuronal injury. However, ALS in man is a slowly progressive pathologic condition whereas ALS IgG in vitro has a fast killing potency. Our present investigations are aimed at characterizing the evolution of ultrastructural changes and alterations of calcium levels of motoneurons exposed to ALS IgG in vivo by passive transfer to mice. Such investigations should help to define neuronal injury at its earliest stage, and may fill the gap; between the slowly evolving changes in ALS and the result of model experiments. Accordingly, the specific aims are: (1) To determine the time/dose dependence of effects of ALS IgG on ultrastructure and intracellular calcium level of motoneurons of mice injected with ALS IgG (2) To determine the specificity of ALS IgG by substituting ALS IgG with disease control IgG, by preadsorption of ALS IgG with potential membrane targets (purified calcium channels) and by antagonizing their effect with Ca-channel blockers. (3) To provide a detailed electrophysiological and ultrastructural study of transmitter release and vesicle recycling after in vivo IgG injection into mice. For these studies, mice will be injected intraperitoneally with different dosages of ALS IgG followed by perfusion fixation for ultrastructural analysis at different time points. An oxalate-ion containing glutaraldehyde fixative will be applied to provide good ultrastructural preservation and simultaneous conservation of in situ calcium distribution. Morphological and intracellular calcium distributional changes will be assayed by quantitative electron microscopy at motor nerve terminals, intramuscular axons and cell bodies of motoneurons with different susceptibility to ALS and the changes will be related to appropriate controls. In parallel experiments ALS IgG induced changes in neuromuscular transmission will be characterized by measuring changes in miniature end- plate potential frequency and by tracking synaptic vesicle recycling with immunohistochemical methods utilizing antibodies against vesicle proteins.