Maturation and Maintenance of the Postsynaptic Apparatus

描述（由申请人提供）：突触前和突触后元件在突触形成时交换重要的发育信号。骨骼神经肌肉接点（NMJ）的简单、大小和可及性使其成为研究这些信号及其使用的细胞内信号转导机制的绝佳准备。我们和其他人之前定义了NMJ突触后分化初始步骤的基本途径，其中蛋白聚糖z-agrin是神经源性信号，MuSK是其主要受体，rapsyn是导致乙酰胆碱受体聚集的关键细胞内效应物。在此基础上，我们将分析胚胎NMJ突触后结构在出生后早期发生的巨大变化。其中包括卵形斑块的拓扑重塑为复杂的椒盐卷饼状分支阵列；分子结构的改变；膜细分成不同的结构域；以及生物物理特性和代谢稳定性的变化。使用一种新的培养系统，其中斑块向椒盐卷转变发生在动脉瘤中，我们将分析两种主要的神经来源信号，agrin和乙酰胆碱，形成突触后膜的方式。使用新生成的条件突变小鼠，我们将确定agrin是否不仅需要NMJs的形成，还需要它们的成熟或维持。通过体内靶向诱变和培养肌管的延时成像，我们将分析lpha-dystrobrevin和LL5_这两种突触相关蛋白在突触后成熟中的作用。总之，这些关于关键突触信号（agrin和神经递质）和细胞内介质（α -抗strobrevin和LL5_）的研究将为理解突触后成熟是如何发生和调控提供一个框架。重要的是，所有这些成分都存在于中枢突触，因此结果将直接增强我们对出生后大脑突触重塑的理解。人们对这种重塑进行了深入的研究，因为它是关键时期和成年人可塑性的基础，而且因为调节它的过程中的缺陷似乎可能是自闭症和成瘾等多种疾病的基础。对NMJ突触形成早期事件的研究已经让我们了解了不易接近的神经元突触，我们相信突触成熟和维持的研究将同样广泛适用。与公共健康相关：神经细胞在称为突触的连接处相互交流。突触是正常神经系统中信息处理和可塑性的部位，也是许多神经和行为障碍的缺陷部位。在过去的几年里，许多突触形成的早期步骤已经被描述过，但是对于最初形成的突触是如何在出生后成熟过程中被重塑的，我们知之甚少。我们建议在骨骼肌神经接点（NMJ）研究这些过程。这种突触的简单性、大小和可接近性使其成为研究调节突触成熟的信号及其使用的细胞内信号转导机制的极好准备。此外，在出生后的早期生活中，胚胎NMJ的突触后装置发生了戏剧性的变化。其中包括卵形斑块的拓扑重塑为复杂的椒盐卷饼状分支阵列；分子结构的改变；膜细分成不同的结构域；以及生物物理特性和代谢稳定性的变化。具体来说，我们将利用NMJ分析两种神经源性信号——agrin和acetylcholine，以及两种肌内信号分子——α -肌营养不良蛋白和LL5_的作用，这两种信号分子都与突触后发育有关。使用一种新的培养系统，其中斑块到椒盐卷饼的转变发生动脉瘤，我们将分析这些分子如何形成突触后膜。从这些研究中得出的假设将用基因工程突变小鼠进行检验。重要的是，所有这些成分都存在于中枢突触，因此结果将直接增强我们对出生后大脑突触重塑的理解。人们对这种重塑进行了深入的研究，因为它是关键时期和成年人可塑性的基础，而且因为调节它的过程中的缺陷似乎可能是自闭症和成瘾等多种疾病的基础。对NMJ突触形成早期事件的研究已经让我们了解了不易接近的神经元突触，我们相信突触成熟和维持的研究将同样广泛适用。

DESCRIPTION (provided by applicant): Pre- and postsynaptic elements exchange developmentally important signals as synapses form. The simplicity, size and accessibility of the skeletal neuromuscular junction (NMJ) make it an excellent preparation for studying these signals and the intracellular signal transduction mechanisms they use. We and others previously defined a rudimentary pathway for initial steps of postsynaptic differentiation at the NMJ in which the proteoglycan z-agrin is a nerve-derived signal, MuSK is its main receptor, and rapsyn is a critical intracellular effector leading to aggregation of acetylcholine receptors. We will now build on this foundation to analyze the dramatic alterations that transform the postsynaptic apparatus at the embryonic NMJ during early postnatal life. These include topological remodeling of the ovoid plaque to a complex pretzel-shaped array of branches; alterations in molecular architecture; subdivision of the membrane into distinct domains; and changes in biophysical properties and metabolic stability. Using a novel culture system in which the plaque to pretzel transition occurs aneurally, we will analyze ways in which two main nerve-derived signals, agrin and acetylcholine, shape the postsynaptic membrane. Using newly generated conditional mutant mice, we will determine whether agrin is required not only for the formation of NMJs but also for their maturation or maintenance. Using targeted mutagenesis in vivo and time-lapse imaging of cultured myotubes, we will analyze roles of lpha-dystrobrevin and LL5_, two synapse-associated proteins that we have already implicated in postsynaptic maturation. Together, these studies on key transsynaptic signals (agrin and neurotransmitter) and intracellular mediators (alpha-dystrobrevin and LL5_) will provide a framework for understanding how postsynaptic maturation occurs and how it is regulated. Importantly, all of these components are present at central synapses, so results will directly enhance our understanding of synaptic remodeling in the postnatal brain. Such remodeling has been studied intensively, because it underlies plasticity during the critical period and in adults, and because defects in processes that regulate it seem likely to underlie conditions as diverse as autism and addiction. Studies of early events in synapse formation at the NMJ have already informed our understanding of less accessible neuron-neuron synapses, and we believe that studies of synaptic maturation and maintenance will be equally broadly applicable. PUBLIC HEALTH RELEVANCE: Nerve cells communicate with each other at junctions called synapses. Synapses are the sites of information processing and plasticity in the normal nervous system, and the sites of defects believed to underlie many neurological and behavioral disorders. Many of the early steps in synapse formation have been described over the past several years, but less is known about how the initially-formed synapse is remodeled during postnatal maturation. We propose to study these processes at the skeletal neuromuscular junction (NMJ). The simplicity, size and accessibility of this synapse make it an excellent preparation for studying signals that regulate synaptic maturation and the intracellular signal transduction mechanisms they use. Moreover, dramatic alterations transform the postsynaptic apparatus at the embryonic NMJ during early postnatal life. These include topological remodeling of the ovoid plaque to a complex pretzel-shaped array of branches; alterations in molecular architecture; subdivision of the membrane into distinct domains; and changes in biophysical properties and metabolic stability. Specifically, we will use the NMJ to analyze roles of two nerve-derived signals, agrin and acetylcholine, and two intramuscular signaling molecules, alpha-dystrobrevin and LL5_, all of which have already been implicated in postsynaptic development. Using a novel culture system in which the plaque to pretzel transition occurs aneurally, we will analyze how these molecules shape the postsynaptic membrane. Hypotheses derived from these studies will be tested using genetically engineered mutant mice. Importantly, all of these components are present at central synapses, so results will directly enhance our understanding of synaptic remodeling in the postnatal brain. Such remodeling has been studied intensively, because it underlies plasticity during the critical period and in adults, and because defects in processes that regulate it seem likely to underlie conditions as diverse as autism and addiction. Studies of early events in synapse formation at the NMJ have already informed our understanding of less accessible neuron-neuron synapses, and we believe that studies of synaptic maturation and maintenance will be equally broadly applicable.