AGRIN GENE EXPRESSION

AGRIN 基因表达

• 批准号: 2271865
• 负责人: MARTIN A SMITH
• 金额: $ 16.68万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2271865
• 关键词: RNA splicing; antibody; developmental neurobiology; electron microscopy; epilepsy; gene expression; glutamate receptor; hippocampus; immunocytochemistry; in situ hybridization; laboratory rat; messenger RNA; nerve /myelin protein; neuromuscular junction; neurotransmitter receptor; polymerase chain reaction; protein biosynthesis; synapses; synaptogenesis; tissue /cell culture

Identification of the mechanisms that underlie the formation of chemical synapses during development, and their modification in the adult, is of critical importance to understanding nervous system function. Much of what is known about theses processes comes from studies of the neuromuscular junction, where it is believed that the protein agrin, supplied by motor neurons, directs the formation and maintenance of the postsynaptic apparatus. It has recently been established that many neurons in the adult rat CNS also express agrin, raising the possibility that agrin plays a similar role in synapse formation between neurons as it does at the neuromuscular junction. The aim of experiments proposed here is to test the hypothesis that agrin direct the organization and maintenance of neuronal synapses in the CNS. In situ hybridization and polymerase chain- reaction (PCR) techniques will be used to determine the level of agrin mRNA expression in different brain regions during development and the results correlated with the time course of synapse formation. Four alternatively spliced agrin mRNAs are expressed in the brain that encode proteins that differ in their ability to induce postsynaptic specializations in cultured muscle fibers. Since alternative splicing represents a mechanism whereby the levels of agrin proteins with different functional properties might be regulated, changes in the pattern of agrin RNA splicing during development will also be monitored. To determine the cellular origin of specific agrin isoforms, a single cell PCR technique will be employed that allows the agrin mRNA profile of an identified cell to be established and, in the case of a neuron, correlated with its neurotransmitter phenotype. Agrin's location at the neuromuscularjunction was a critical piece of evidence pointing to agrin's role in neuromuscular synaptogenesis. Therefore, in parallel with studies of agrin mRNA, the distribution of agrin protein in the brain and its localization at synapses will be determined, using immunohistochemical and electron microscopy techniques. As a direct test of agrin's role in neuronal synapse formation, the ability of antiagrin antibodies to block organization of neurotransmitter receptors at synapses formed in cell culture between CNS neurons will be examined. Finally, preliminary studies have shown that epileptiform seizures induce marked changes in the levels of agrin mRNA in the brain, suggesting that agrin gene expression may be regulated by synaptic activity. This study will be extended to include examination of the time course of seizure induced changes in agrin mRNA levels and alternative splicing. The experiments outlined here are the first to examine the role of agrin and the regulation of agrin gene expression in the CNS. They will provide insights into the mechanisms of synapse formation between neurons and the cellular changes associated with epilepsy and synaptic plasticity. They can be expected therefore to impact upon the treatment and prevention of epilepsy, and disorders of learning and memory associated with such conditions as Down syndrome and Alzheimer disease.

确定化学物质形成的基本机制 发育过程中的突触，以及它们在成年后的变化， 对了解神经系统功能至关重要。的很多东西 对这些过程的了解来自于对神经肌肉的研究， 连接处，据信由马达供应的蛋白聚集蛋白 神经元，指导突触后的形成和维持， 设备.最近的研究表明，成年人的许多神经元 大鼠中枢神经系统也表达聚集蛋白，增加了聚集蛋白在中枢神经系统中起作用的可能性。 在神经元之间的突触形成中起着类似的作用， 神经肌肉接头实验的目的是测试 关于聚集蛋白指导植物组织和维持的假说， 中枢神经系统中的神经元突触。原位杂交和聚合酶链- 将使用PCR技术来确定聚集蛋白的水平 发育过程中不同脑区mRNA的表达及其与发育的关系 结果与突触形成的时间过程相关。四 选择性剪接的聚集蛋白mRNA在大脑中表达， 不同的蛋白质在它们的能力，以诱导突触后 在培养的肌肉纤维中的特殊化。由于选择性剪接 代表了一种机制，即具有不同的聚集蛋白水平， 功能特性可能受到调节，聚集蛋白模式的变化 还将监测发育期间的RNA剪接。确定 特异性聚集蛋白异构体的细胞起源，单细胞PCR技术 将采用允许鉴定的细胞的聚集蛋白mRNA谱 建立，并在神经元的情况下，与其 神经递质表型Agrin在神经肌肉连接处的位置 是一个关键的证据，指向agrin的作用， 突触发生因此，在研究聚集蛋白mRNA的同时， 聚集蛋白在脑内的分布及其在脑内的定位 将使用免疫组织化学和电子显微镜来确定突触。 显微镜技术。作为对聚集蛋白在神经细胞中作用的直接测试， 突触形成，抗聚集蛋白抗体阻断突触形成的能力， 神经递质受体在细胞内形成的突触处的组织 将检查CNS神经元之间的培养。最后，初步研究 已经表明癫痫样发作会引起 在大脑中聚集蛋白mRNA的表达，这表明聚集蛋白基因表达可能是 由突触活动调节。这项研究将扩大到包括 检查癫痫发作诱导的聚集蛋白mRNA变化的时间过程 水平和可变剪接。这里概述的实验是 第一次研究聚集蛋白的作用和聚集蛋白基因的调控 在CNS中的表达。他们将提供深入了解的机制， 神经元之间的突触形成和与之相关的细胞变化 癫痫和突触可塑性。因此，可以预期， 治疗和预防癫痫和学习障碍 与唐氏综合症和老年痴呆症等病症相关的记忆力 疾病