Dynamics of AChE in Normal and Mutant Synapses

描述(由申请人提供)： 本研究的目的是研究活体小鼠神经肌肉接头(NMJ)突触间隙中细胞外基质乙酰胆碱酯酶(AChE)的动态(移除、插入和移动)。特别是，我们将研究哪些因素可能调节它的动态行为。我们将侧重于突触活性的作用，突触活性是细胞外基膜(层粘连蛋白α4)和突触后蛋白中的关键分子，以及dystrophin糖蛋白复合体。我们的第二个目标是研究突触后膜上乙酰胆碱受体(AChR)的代谢稳定性是否受AchE功能的影响。最后，我们将研究AChE和AChR在单个突触上的突触动力学变化。为了解决这些问题，我们将使用NMJ作为体内突触的模型。高分辨率共聚焦显微镜、体内荧光成像分析和我们最近发展的光解结合技术的使用将有助于我们更好地了解AChE和AChR密度是如何在活的突触中维持和调节的。通过研究这些问题，我们应该更好地理解突触变化是如何发生在较难到达的中央突触中的。此外，许多神经肌肉疾病对神经肌肉交界处AChRs和疼痛的密度有主要或继发性的影响，如重症肌无力。通过了解这些关键突触分子的调节，我们希望定义新的方法，可能被用来开发这些毁灭性疾病的有效疗法。

DESCRIPTION (provided by applicant): The goal of this proposal is to study the dynamics (the removal, the insertion, and the mobility) of the extracellular matrix acetylcholinesterase (ACHE) in the synaptic cleft in the neuromuscular junction (NMJ) of living mice. In particular, we will study what factors that might regulate its dynamic behavior. We will focus on the role of synaptic activity, a key molecule in the extracellular basal lamina (laminin alpha 4) and in postsynaptic proteins, and the dystrophin glycoprotein complex. Our second goal is to investigate whether the metabolic stability of acetylcholine receptors (AChR) in the postsynaptic membrane is influenced by AChE function. Finally we will study changes in synaptic dynamics of both AChE and AChR at a single synapse. To address these questions we will use the NMJ as a model of synapses in vivo. The use of high-resolution confocal microscopy, in vivo fluorescence imaging assay and the photo-unbinding technique that we developed recently will help us to better understand how the densities of AChE and AChR are maintained and regulated in living synapses. By investigating such questions, we should gain a better understanding of how synaptic alterations occur in less accessible central synapses. Additionally many neuromuscular diseases have either primarily or secondarily major impact on the density of AChRs and AChEs at neuromuscular junctions such as myasthenia gravis. By understanding the regulation of these key synaptic molecules, we expect to define new approaches that might be used to develop effective therapies for these devastating diseases.