GABAergic Neuron Differentiation in C. elegans

描述(由申请人提供)：突触发生的分子描述仍然是了解神经系统发育和功能的关键研究目标。通过描述线虫在突触形成中起作用的基因，我们的工作有助于发现几条信号通路，指导突触前分化的不同方面。这一应用的核心是rpm-1基因(用于突触前形态的调节)。Rpm-1功能的丧失会导致突触前结构紊乱，并以神经元类型特有的方式扰乱轴突模式。RPM-1是保守的PHR蛋白家族成员，包括哺乳动物Pam和Phr1，以及果蝇Highwire。PhR蛋白是含有多个功能结构域的大分子，包括RCC1鸟嘌呤交换因子同源结构域和Ring-inger E3泛素连接酶结构域。在目前的资助期间，我们证明了RPM-1作为保守的MAP激酶DLK-1的E3泛素连接酶发挥作用。DLK-1激活两个下游的激酶，一个是MAPKK MKK-4，一个是p38MAPK PMK-3。RPM-1下调这种MAPK级联对于正常突触的形成是必不可少的。在遗传方法的同时，我们使用生化方法鉴定了RPM-1相关蛋白，发现RPM-1通过Rabgef GLO-4正向调节晚期溶酶体运输。本申请的总体目标是定义RPM-1/MAPK级联的目标，并了解级联是如何调节的。我们已经确定了两个新基因，mak-2，线虫MAP激酶激活蛋白2的同源基因，以及uev-3，一种含有非活性泛素结合酶结构域的蛋白。在这两个基因中，功能丧失的表现类似于MAPK级联失活。我们的初步研究表明MAK-2和UEV-3在DLK-1和MKK-4的下游起作用。在AIM-1中，我们将研究MAP激酶对MAK-2的调控，并确定MAK-2的靶点。在AIM-2中，我们将分析UEV-3与MAP激酶之间的相互作用。在AIM-3中，我们将探索突触发育的一条新途径。单个突触发生通路的功能丧失对突触的发育和功能的影响较小，表明突触信号中存在高度的功能冗余。使用遗传修饰物筛选寻找其他突触发育基因，我们已经确定了SydN-1(Syd Enhancer)基因，它似乎定义了一条核途径，可能以突触发生依赖的方式调节轴突修剪。我们将研究SydN-1及其候选相互作用基因的细胞和分子功能。成功完成我们的目标将阐明PHR/MAPK通路如何发挥作用。突触稳定性的调节是与痴呆和衰老相关的主要发病事件之一。这项研究将有助于理解建立和维持突触的基本机制，也可能为突触功能障碍的发病机制提供见解。公共卫生相关性：本应用研究突触发育过程中p38蛋白激酶的信号转导途径。它将确定MAPKAP蛋白和含有UEV结构域的蛋白与MAP激酶级联的分子、生化和细胞相互作用。它将为控制突触稳定性的基本机制和疾病中突触功能障碍的发病过程提供深入的见解。

DESCRIPTION (provided by applicant): A molecular description of synaptogenesis remains a key research goal in understanding the development and function of the nervous system. By characterizing C. elegans genes that function in synapse formation, our work has contributed to the discovery of several signaling pathways instructing different aspects of presynaptic differentiation. Central to this application is the rpm-1 gene (for regulator of presynaptic morphology). Loss of function in rpm-1 causes disorganized presynaptic architecture and disrupts axonal patterning in a neuron-type specific manner. RPM-1 is a member of the conserved PHR protein family that includes mammalian Pam and Phr1, and Drosophila Highwire. PHR proteins are large molecules containing multiple functional domains, including an RCC1 guanine exchange factor homology domain and a Ring-finger E3 ubiquitin ligase domain. During the current funding period, we demonstrated that RPM-1 functions as an E3 ubiquitin ligase for the conserved MAP kinase kinase kinase DLK-1. DLK-1 activates two downstream kinases, a MAPKK MKK-4 and a p38 MAPK PMK-3. Down-regulation of this MAP kinase cascade by RPM-1 is essential for normal synapse formation. In parallel to the genetic approaches, we used biochemical methods to identify RPM-1 associated proteins, and discovered that RPM-1 positively regulates late endo-lysosomal trafficking via the RabGEF GLO-4. The overall goals of the present application are to define the targets of the RPM-1/MAPK cascade, and to understand how the cascade is regulated. We have identified two new genes, mak-2, the C. elegans ortholog of MAP kinase activated kinase 2, and uev-3, a protein containing an inactive ubiquitin conjugating enzyme domain. Loss of function in either gene behaves in a manner similar to that of inactivating the MAPK cascade. Our preliminary studies suggest that MAK-2 and UEV-3 act downstream of DLK-1 and MKK-4. In aim-1, we will examine the regulation of MAK-2 by the MAP kinases and identify the targets of MAK-2. In aim- 2, we will analyze the interaction between UEV-3 and the MAP kinases. In aim-3, we will explore a novel pathway in synapse development. Loss of function in individual synaptogenic pathways has mild effects on synaptic development and function, indicating a high degree of functional redundancy in synaptic signaling. Using genetic modifier screens to search for other synapse development genes, we have identified the gene sydn-1 (for syd enhancer), which appears to define a nuclear pathway that may regulate axonal pruning in a synaptogenesis dependent manner. We will investigate the cellular and molecular functions of SYDN-1 and its candidate interacting genes. Successful completion of our aims will elucidate how the PHR/MAPK pathway functions. Regulation of synapse stability is one of the major pathogenesis events associated with dementia and ageing. This study will contribute to the understanding of the basic mechanisms that build and maintain synapses, and may also provide insights into the pathogenesis of synapse dysfunction. PUBLIC HEALTH RELEVANCE: This application investigates the signal transduction pathway of a p38 MAP kinase in synapse development. It will determine the molecular, biochemical, and cellular interactions of a MAPKAP protein and a UEV-domain containing protein with the MAP kinase cascade. It will provide insights into the basic mechanisms controlling synapse stability and into the pathogenesis process underlying synapse dysfunction in diseases.