GROWTH OF RETINAL GANGLION CELL AXONS

视网膜神经节细胞轴突的生长

基本信息

批准号：
2159378
负责人：
Vance P Lemmon
金额：
$ 21.97万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-09-01 至 1998-06-30
项目状态：
已结题

项目摘要

Learning how axons of retinal ganglion cells grow from their origins in the retina to their targets in the brain is an important goal for understanding how the visual system develops. A number of cell adhesion molecules (CAMs) have been found in the optic fiber layer and optic nerve and are thought to be important in axon growth and guidance. A basic idea in the field has been that CAMs mediate cell-cell interactions via cell- cell binding. Over the past few years a more complex concept has evolved, i.e., that CAMs not only mediate cell adhesion but are also capable of signaling, perhaps by influencing intracellular second messenger systems. One CAM, called L1 (also referred to as NILE, Ng-CAM, 8D9 or G4) is present on axons of retinal ganglion cells. We have shown that L1 is a potent substrate for growth of retinal ganglion cell axons in vitro and that L1 binds to L1 in a homophilic manner. Using two different techniques, we have found that L1 is a strongly adhesive substrate and produces distinctive behavior by growth cones. Other labs have directly implicated L1 in regulating intracellular Ca++ and phosphorylation events, while we have identified a novel S6 kinase activity associated with L1. We have cloned and sequenced the human L1 cDNA and found that the cytoplasmic domain of L1 is highly conserved in mammals. The cytoplasmic domain of L1 in brain is 114 amino acids long and is identical in mice and humans. Using our cDNA clones, other laboratories have shown that human X-linked hydrocephalus (HSAS is due to a defect in L1 expression. Patients with this syndrome die around the time of birth or are severely retarded, have strabismus and other visual system defects. In at least one HSAS family this is doe to a mutation affecting the cytoplasmic domain of L1. L1 is an excellent CAM to study in order to understand how CAMs influence growth cone behavior by simultaneously mediating growth cone-substrate adhesion and influencing second messenger systems. The experiments in this proposal will use biochemical, immunological, molecular biological and cell biological experiments to focus on the cytoplasmic domain of L1. Site directed mutagenesis will be done on the cytoplasmic domain of L1 to define regions critical for L1-L1 homophilic binding and neurite outgrowth. We will also examine how phosphorylation of L1 is regulated and how phosphorylation of L1 influences L1 function. These experiments will provide new information about how cell adhesion modulates intracellular processes and also how changes in intracellular second messenger systems can alter cell adhesion. Finally, these experiments will provide a detailed description of how one cell adhesion molecule regulates growth cone behavior.

了解视网膜神经节细胞的轴突如何从其起源开始生长视网膜到大脑中的目标是一个重要目标了解视觉系统如何发育。细胞粘附数在光纤层和视神经中发现了分子（CAM）并被认为对轴突生长和引导很重要。一个基本想法在该领域，CAM 通过细胞介导细胞间相互作用细胞结合。在过去的几年里，一个更复杂的概念已经形成，也就是说，CAM 不仅介导细胞粘附，而且还能够信号传导，可能是通过影响细胞内第二信使系统。一个 CAM，称为 L1（也称为 NILE、Ng-CAM、8D9 或 G4）存在于视网膜神经节细胞的轴突上。我们已经证明 L1 是视网膜神经节细胞轴突体外生长的有效底物 L1 以同亲方式与 L1 结合。使用两种不同的技术，我们发现L1是一种强粘合基材，并且通过生长锥产生独特的行为。其他实验室直接 L1 参与调节细胞内 Ca++ 和磷酸化事件，同时我们已经确定了一种新的 S6 激酶活性相关与 L1。我们克隆并测序了人类 L1 cDNA，发现 L1 的胞质结构域在哺乳动物中高度保守。细胞质的大脑中 L1 结构域长 114 个氨基酸，与小鼠相同和人类。其他实验室使用我们的 cDNA 克隆表明人类 X 连锁脑积水（HSAS 是由于 L1 表达缺陷所致。患有这种综合征的患者在出生时死亡或病情严重弱智，有斜视等视觉系统缺陷。至少在一个 HSAS 家族，这是影响细胞质的突变的母鹿 L1 的域。 L1 是一个优秀的 CAM，可以通过研究来了解 CAM 如何影响通过同时介导生长锥-基质的生长锥行为粘附和影响第二信使系统。实验在该提案将使用生物化学、免疫学、分子生物学和细胞生物学实验重点关注L1的胞质结构域。将在 L1 的细胞质结构域上进行定点诱变定义对 L1-L1 同亲结合和神经突至关重要的区域生长出来的。我们还将研究 L1 的磷酸化是如何调节的以及 L1 磷酸化如何影响 L1 功能。这些实验将提供有关细胞粘附如何调节的新信息细胞内过程以及细胞内第二个过程如何变化信使系统可以改变细胞粘附。最后，这些实验将详细描述一种细胞粘附分子如何调节生长锥行为。