MOLECULAR BASES OF NEURONAL CONNECTIVITY

描述：虽然对成熟哺乳动物的视觉系统有损伤， 通常会导致永久性的功能丧失，最近的研究表明， CNS神经元可以被诱导再生其轴突，如果暴露于 合适的细胞和分子条件。 前两个目标 这项提案将检验一个假设，即刺激视网膜的一个因素， 神经节细胞再生轴突的关键是一种小分子AF-1。 在 金鱼，一种中枢神经系统神经元再生轴突的物种 我们发现视神经胶质细胞自发地分泌两种 诱导神经节细胞在培养中延伸长轴突的因子。 AF-1， 这两个因素中更有力的一个，对 大鼠的神经节细胞。 我们将从神经胶质细胞培养中获得AF-1和AF-2 并通过凝胶过滤、反相、 亲水亲和层析 根据序列数据，我们将 产生合成肽，测试这些肽的活性，并利用它们来 产生抗体。 我们将利用预测的核苷酸序列 和/或抗体来分离鱼和人的编码 推定的前体蛋白。 我们将研究AF-1是否能增强轴突生长， 在哺乳动物系统中的衍生物，从孤立的神经节 细胞与视网膜周围神经移植在体内。 我们将调查 AF-1是否与定义的神经营养因子BDNF协同作用， 检查它是否刺激涉及轴突的蛋白质的表达， 体内再生，例如，膜磷蛋白GAP-43。 目标3将 检验GAP-43的表达部分受 通过控制其mRNA的稳定性。 我们已经确定了 在GAP-43 mRNA的3'非翻译区内， 可能在调节mRNA稳定性中重要的蛋白质的位点，以及 已经确定了三种与这些区域结合的蛋白质。 使用PC 12细胞 作为一个模型系统，我们发现其中一种蛋白质的结合是 与GAP-43表达的增加平行。 我们将使用 各种分子生物学方法来检查的重要性， 鉴定的核苷酸结构域和相关的结合蛋白， 控制GAP-43 mRNA的稳定性。 总之，这些研究应该 大大增加了对控制细胞生长的分子机制的理解， 神经元的生长状态，并可能最终有助于发展 用于增强人体中受损连接的再生的方法 视觉系统

DESCRIPTION: Although injury to the visual system in mature mammals generally results in a permanent loss of function, recent studies show that CNS neurons can be induced to regenerate their axons if exposed to appropriate cellular and molecular conditions. The first two aims of this proposal will test the hypothesis that one factor that stimulates retinal ganglion cells to regenerate their axons is a small molecule, AF-1. In goldfish, a species in which CNS neurons regenerate their axons spontaneously, we have found that glia of the optic nerve secrete two factors that induce ganglion cells to extend long axons in culture. AF-1, the more potent of the two factors, exerts an equally dramatic effect on ganglion cells of the rat. We will obtain AF-1 and AF-2 from glial cultures and purify these to homogeneity by gel filtration, reverse-phase, and hydrophilic affinity chromatography. Based upon the sequence data, we will generate synthetic peptides, test these for activity and utilize them to generate antibodies. We will utilize the predicted nucleotide sequence and/or the antibodies to isolate the fish and human genes that encode the putative precursor proteins. We will examine whether AF-1 enhances axonal outgrowth in mammalian systems ranging in complexity from isolated ganglion cells to retinas with peripheral nerve grafts in vivo. We will investigate whether AF-1 works synergistically with the defined neurotrophin, BDNF, and examine whether it stimulates expression of proteins involved in axonal regeneration in vivo, e.g., the membrane phosphoprotein GAP-43. Aim 3 will examine the hypothesis that the expression of GAP-43 is regulated in part through controlling the stability of its mRNA. We have identified regions within the 3' untranslated region of GAP-43 mRNA which serve as binding sites for proteins that may be important in regulating mRNA stability, and have identified three proteins that bind to these regions. Using PC12 cells as a model system, we have found that the binding of one of the proteins is induced by NGF and parallels the increase in GAP-43 expression. We will use a variety of molecular biological approaches to examine the importance of the identified nucleotide domains and the associated binding proteins in controlling the stability of GAP-43 mRNA. Together, these studies should add considerably to understanding molecular mechanisms that control the neuron's growth state, and may ultimately contribute to the development of methods for enhancing regeneration of injured connections in the human visual system.