Investigation of Anaphase Promoting Complex function in synaptic transmission

描述(申请人提供)：泛素信号系统，促进调节的蛋白质降解，是突触发育和突触传递所必需的。数以百计的酶具有在蛋白质中添加或移除泛素多肽的能力。人们对这些酶在神经元和突触中的功能和相关底物知之甚少。长期目标是确定泛素酶如何调节突触传递。鉴于泛素系统功能障碍与神经和神经退行性疾病有关，在这些疾病中发生异常的突触传递，这些信息可能会导致识别药物靶点，以帮助治疗这些疾病。最近的工作表明，后期促进复合体(APC)，一种控制细胞周期中蛋白质丰度的泛素连接酶，也调节神经元发育和谷氨酸能突触传递；然而，关于其作用机制和这些或其他突触的相关底物，仍有许多未知之处。本研究的目的是阐明APC在体内调节突触传递的分子机制。这将使用模式线虫秀丽线虫来完成，这种线虫在神经系统蛋白质和功能上与人类有很大的共同之处。初步实验表明，除了调节谷氨酸能突触外，APC还负向调节神经肌肉接头(NMJ)的传递，NMJ是线虫的三部分突触，突触后肌肉的收缩由兴奋性胆碱能和抑制性GABA能运动神经元的输入平衡控制。APC可能作用于任何或所有这些细胞类型，以影响肌肉活动量。更多的初步定位研究和行为分析导致了一个中心假设，即APC作用于突触前运动神经元，促进NMJ蛋白的泛素化和降解，NMJ蛋白对增加肌肉兴奋至关重要。在目标1中，将使用特定细胞类型的功能丧失和挽救实验、行为分析和定量成像来确定APC在NMJ信号转导过程中的作用部位和机制。更多的初步数据导致在NMJ发现了一种潜在的APC底物，G蛋白偶联受体FSHR-1。在目标2中，将利用遗传和行为分析以及成像和生化来表征FSHR-1在NMJ中的作用以及FSHR-1和APC之间的关系。在目标3中，将使用RNA干扰来鉴定更多的APC底物。这项研究将为如何调节兴奋性和抑制性传递的平衡提供新的见解--人类癫痫等癫痫综合征的平衡丧失。这些研究还将有助于了解泛素信号如何影响突触传递的全球图景，突触传递对学习记忆至关重要。鉴于泛素系统失调发生在阿尔茨海默病、帕金森病和亨廷顿病中，泛素酶的突变与几种神经遗传学疾病有关，阐明APC及其底物如何控制突触传递可能有助于相关治疗学的发展。 公共卫生相关性：该项目涉及确定后期促进复合体(APC)--一种泛素连接酶--调节体内突触传递的分子机制。神经元突触上的适当信号是学习和记忆所必需的，也是所有神经系统功能的基础。突触传递调节失调和泛素系统功能障碍在许多神经疾病中都会发生。因此，更全面地了解APC泛素连接酶的作用机制和相关底物将有助于了解突触传递和泛素酶活性的全球图景。

DESCRIPTION (provided by applicant): The ubiquitin signaling system, which promotes regulated protein degradation, is essential for synapse development and synaptic transmission. Hundreds of enzymes exist that have the ability to add or remove ubiquitin polypeptides from proteins. Less is known about the functions and relevant substrates of these enzymes in neurons and at synapses. The long-term goal is to determine how ubiquitin enzymes regulate synaptic transmission. Given that ubiquitin system dysfunction is linked to neurological and neurodegenerative diseases in which aberrant synaptic transmission occurs, such information may lead to the identification of drug targets to aid in treating these conditions. Recent work established that the Anaphase Promoting Complex (APC), an ubiquitin ligase that controls protein abundance in the cell cycle, also regulates neuronal development and glutamatergic synaptic transmission; yet much remains unknown regarding its mechanism of action and relevant substrates at these or other synapses. The objective of this proposal is to elucidate the molecular mechanisms by which the APC regulates synaptic transmission in vivo. This will be done using the model nematode, Caenorhabditis elegans, which shares significant conservation of nervous system proteins and function with humans. Preliminary experiments indicate that, in addition to regulating glutamatergic synapses, the APC negatively regulates transmission at the neuromuscular junction (NMJ), a tripartite synapse in C. elegans where contraction of postsynaptic muscle is controlled by a balance of inputs from excitatory cholinergic and inhibitory GABAergic motorneurons. The APC may act in any or all of these cell types to impact the amount of muscle activity. Additional preliminary localization studies and behavioral assays led to the central hypothesis that the APC acts in presynaptic motorneurons to promote ubiquitination and degradation of NMJ proteins important for increasing muscle excitation. In Aim 1, cell type-specific loss of function and rescue experiments, behavioral assays, and quantitative imaging will be used to determine the site and mechanism of APC action during NMJ signaling. Additional preliminary data led to the identification of one potential APC substrate at the NMJ, the G protein-coupled receptor, FSHR-1. In Aim 2, genetic and behavioral analyses, along with imaging and biochemistry, will be employed to characterize the role of FSHR-1 at the NMJ and the relationship between FSHR-1 and the APC. In Aim 3, RNA interference will be used to identify additional APC substrates. This research will provide new insight into how the balance of excitatory to inhibitory transmission is regulated-a balance lost i human seizure syndromes, such as epilepsy. These studies also will contribute to the global picture of how ubiquitin signaling impacts synaptic transmission, which is critical for learning an memory. Given that ubiquitin system misregulation occurs in Alzheimer's, Parkinson's, and Huntington's Diseases, and mutations in ubiquitin enzymes are linked to several neurogenetic disorders, elucidating how the APC and its substrates control synaptic transmission may facilitate the development of relevant therapeutics. PUBLIC HEALTH RELEVANCE: This project involves determining the molecular mechanism by which the Anaphase Promoting Complex (APC), an ubiquitin ligase enzyme, regulates synaptic transmission in vivo. Proper signaling at neuronal synapses is required for learning and memory and underlies all nervous system function. Misregulation of synaptic transmission and ubiquitin system dysfunction occur in many neurological disorders. Thus, a more complete understanding of the mechanism of action and relevant substrates of the APC ubiquitin ligase will contribute critical information to the global picture of synaptic transmission and ubiquitin enzyme activity.