MOLECULAR BASIS OF TROPHIC INTERACTION IN SYNAPTOGENESIS

突触发生中营养相互作用的分子基础

• 批准号: 3086951
• 负责人: DOUGLAS L. FALLS
• 金额: $ 5.67万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-04-01 至 1992-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3086951
• 关键词: acetylcholine; affinity chromatography; antibody; bioassay; brain; chemical structure function; chick embryo; enzyme linked immunosorbent assay; gel electrophoresis; genetic manipulation; glycoproteins; immunofluorescence technique; immunologic techniques; laboratory mouse; laboratory rabbit; laboratory rat; membrane proteins; messenger RNA; molecular weight; monoclonal antibody; myogenesis; neurogenesis; neuromuscular junction; nicotinic receptors; protein sequence; synapses; tissue /cell culture

Understanding the mechanisms of synaptic development will be central to understanding the effects of genetic and environmental factors (including drugs) on the fetus and young child. The development of the nerve-muscle synapse in vivo and in tissue culture is a valuable model of CNS synaptic development. A striking feature of this nerve-muscle synapse development is the formation of an aggregate of nicotinic acetylcholine receptors (AChR) in the postsynaptic membrane. In at least some species, an increase in the rate of insertion of AChRs into the membrane at the site of the nascent synapse makes an important contribution to the formation of this aggregate. A 42kD protein has now been purified (Usdin and Fischbach, 1986) from the chicken brain which, when present at a concentration on the order of 0.5ng/ml, causes an increase in the rate of AChR incorporation into the membrane of cultured chick myotubes and also in the number of AChR aggregates. This protein might be released by the spinal motor neuron at the site of nerve-muscle contact and induce synaptic specializations. The purpose of this project is to investigate the biological role of this protein and its mechanism of action. The specific aims are: 1) Purify enough of the 42kD acetylcholine receptor inducing activity (42kD ARIA) to provide amounts needed for production of antibodies, determination of amino-terminal sequence, and studies of the biological properties of this protein. 2) Raise monoclonal and polyclonal antibodies directed against this 42kD ARIA. 3) Use these antibody reagents to investigate the biological role of this protein. A key question is whether or not antibodies directed against this protein block the formation of synaptic specializations at sites of nerve-muscle contact. This will be investigated in vitro and in vivo. The regional distribution of the 42kD ARIA will be studied using immunofluorescence, and an ELISA assay will be developed to quantitate levels of the 42kD ARIA. The effect of focal application of this protein onto myotubes will also be examined. 4) Long-term goals include determining the full amino acid sequence of the 42kD ARIA and studying its affect on the rate of synthesis of AChR subunits and on subunit mRNA levels.

了解突触发育的机制将是 对了解遗传和环境的影响至关重要 因素(包括药物)对胎儿和幼儿的影响。这个 神经-肌肉突触在体内和组织内的发育 文化是中枢神经系统突触发育的有价值的模型。一个 这种神经-肌肉突触发育的显著特征是 烟碱型乙酰胆碱受体聚集体的形成 (Achr)在突触后膜。至少在某些物种中， AChRs插入细胞膜的速度增加 在新生突触的位置产生了一个重要的 对这种集合体的形成做出了贡献。42kD蛋白 (Usdin和Fischbach，1986) 其中的鸡脑，出现时集中在一份订单上 0.5 ng/ml时，AChR参入率增加 进入培养的雏鸡肌管的膜中，也在 AChR聚合数。这种蛋白质可能通过以下方式释放 神经-肌肉接触部位的脊髓运动神经元和 诱导突触特化。这个项目的目的是 探讨该蛋白的生物学作用及其机制 行动的一部分。具体目标是： 1)对诱导的42kD乙酰胆碱受体进行足够的纯化 提供生产所需数量的活动(42kD ARIA) 抗体、氨基末端序列的测定和研究 这种蛋白质的生物学特性。 2)制备针对人的单抗和多克隆抗体 这个42kD的ARIA。 3)利用这些抗体试剂研究其生物学作用。 这种蛋白质的。一个关键问题是抗体是否 针对这种蛋白阻断突触的形成 神经-肌肉接触部位的专门化。这将是 体外和体内研究。人口数量的地区分布 42kD ARIA将使用免疫荧光进行研究，并 将建立定量检测42kD水平的酶联免疫吸附试验 艾瑞亚。该蛋白在水稻上的聚焦应用效果 肌管也将被检查。 4)长期目标包括确定完整的氨基酸 42kD ARIA基因序列及其对死亡率的影响 AChR亚基的合成和亚单位的mRNA水平。