AGRIN GENE EXPRESSION

确定化学物质形成的基础机制 在发育过程中的突触，以及它们在成人中的修改，是 对了解神经系统功能至关重要。很多什么 已知的这些过程来自于对神经肌肉的研究 连接，在那里人们相信由马达供应的蛋白集聚蛋白 神经元，指导突触后突触的形成和维持 仪器。最近已经证实，成人体内的许多神经元 大鼠中枢神经系统也表达集聚蛋白，增加了集聚蛋白发挥作用的可能性 在神经元间突触形成中的作用与在 神经肌肉接头。这里提出的实验的目的是为了测试 关于集聚蛋白指导组织和维持 中枢神经系统中的神经元突触。原位杂交和聚合酶链- 将使用聚合酶链式反应(PCR)技术来确定集聚蛋白的水平 发育过程中不同脑区的mRNA表达及其意义 结果与突触形成的时间进程相关。四 或者，剪接的集聚蛋白mRNAs在大脑中表达，编码 诱导突触后突触能力不同的蛋白质 专门研究培养的肌肉纤维。由于可选剪接 代表了一种机制，通过这种机制，不同的集聚蛋白水平 功能特性可能受到调节，集聚模式的变化 研发过程中的RNA剪接也将受到监测。要确定 特殊聚集素异构体的细胞起源--单细胞聚合酶链式反应技术 将使用允许识别的细胞的集聚蛋白mRNA图谱 建立并在神经元的情况下，与其 神经递质表型。阿格林在神经肌肉连接处的位置 是表明agrin在神经肌肉中的作用的关键证据 突触发生。因此，在研究集聚蛋白mRNA的同时， 集聚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技术来确定突触 显微镜技术。作为对集聚蛋白在神经元中作用的直接测试 突触形成，抗集聚蛋白抗体阻断的能力 细胞内形成的突触上神经递质受体的组织 将对中枢神经系统神经元间的培养进行检测。最后，初步研究 研究表明，癫痫样发作会引起脑脊液中胆碱能水平的显著变化 表明集聚蛋白基因的表达可能是 受突触活动的调节。这项研究将扩大到包括 癫痫发作后集聚蛋白基因表达变化的时程观察 级别和可选的拼接。这里概述的实验是 首先研究集聚蛋白的作用和集聚蛋白基因的调控 在中枢神经系统中的表达。他们将为我们提供对 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，它们可以预期会产生影响 论癫痫和学习障碍的治疗和预防 以及与唐氏综合症和阿尔茨海默氏症等疾病相关的记忆 疾病。

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.