Maturation and Maintenance of the Postsynaptic Apparatus

描述(由申请人提供)：突触前和突触后的元素以突触的形式交换发育重要的信号。骨骼神经肌肉接头(NMJ)的简单、大小和可达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极好准备。我们和其他人以前在NMJ为突触后分化的初始步骤定义了一条基本途径，其中蛋白多糖z-agrin是神经来源的信号，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器。我们现在将在此基础上分析在出生后早期改变胚胎NMJ突触后装置的戏剧性变化。这些包括卵形斑块的拓扑重塑为复杂的椒盐卷饼形状的分支阵列；分子结构的改变；膜细分为不同的区域；以及生物物理性质和代谢稳定性的变化。使用一种新的培养系统，其中斑块到椒盐卷饼的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--集聚蛋白和乙酰胆碱--塑造突触后膜的方式。利用新产生的条件突变小鼠，我们将确定集聚蛋白是否不仅是NMJ形成所必需的，而且是它们成熟或维持所必需的。利用体内靶向突变和培养肌管的时间推移成像，我们将分析lpha-dystrobrevin和LL5_蛋白的作用，这两个蛋白是我们已经在突触后成熟中涉及的两个突触相关蛋白。总之，这些对关键的跨突触信号(集聚蛋白和神经递质)和细胞内介质(α-dystrobrevin和LL5_)的研究将为理解突触后成熟的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央突触，因此结果将直接增强我们对出生后大脑突触重构的理解。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，而且调节它的过程中的缺陷似乎可能是自闭症和成瘾等各种疾病的基础。对NMJ突触形成早期事件的研究已经为我们对较难获得的神经元-神经元突触的理解提供了信息，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通信。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基础缺陷部位。在过去的几年里，突触形成的许多早期步骤已经被描述，但关于最初形成的突触在出生后成熟期间是如何重塑的，人们知之甚少。我们建议研究骨骼神经肌肉接头(NMJ)的这些过程。这种突触的简单、大小和可访问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，在出生后早期，巨大的变化改变了胚胎NMJ的突触后装置。这些包括卵形斑块的拓扑重塑为复杂的椒盐卷饼形状的分支阵列；分子结构的改变；膜细分为不同的区域；以及生物物理性质和代谢稳定性的变化。具体地说，我们将利用NMJ来分析两个神经衍生信号--集聚蛋白和乙酰胆碱，以及两个肌肉内信号分子--α-dystrobrevin和LL5_-的作用，它们都已经与突触后发育有关。使用一种新的培养系统，在该系统中，斑块向椒盐卷饼的转变发生在动脉瘤中，我们将分析这些分子如何塑造突触后膜。从这些研究中得出的假说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央突触，因此结果将直接增强我们对出生后大脑突触重构的理解。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，而且调节它的过程中的缺陷似乎可能是自闭症和成瘾等各种疾病的基础。对NMJ突触形成早期事件的研究已经为我们对较难获得的神经元-神经元突触的理解提供了信息，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.