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LAMININS AND NEUROMUSCULAR SYNAPSE FORMATION

层粘连蛋白和神经肌肉突触的形成

基本信息

批准号：
6702288
负责人：
BRUCE L PATTON
金额：
$ 30.2万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-02-09 至 2006-01-31
项目状态：
已结题

项目摘要

DESCRIPTION (From Applicant's Abstract): Our long-term goal is to understand the molecular basis of synapse formation in vertebrates. Currently, no target-derived signals have been firmly established as regulators of nerve terminal formation in vertebrates. This application focuses on a muscle-derived cue that controls nerve terminal formation at the neuromuscular junction, the most-studied synapse. Previous studies identified an activity in the basal lamina lining the synaptic cleft that induces presynaptic differentiation in reinnervating axons. The best candidate for mediating that activity is laminin-11, a muscle-derived component of the synaptic basal lamina. Laminin-11 is a heterotrimer, composed of three homologous laminin "chains": a5, b2, and g1. Mice lacking laminin-11 through targeted deletion of b2 (s-laminin) die of neuromuscular failure at one month of age. Motor nerve terminals in the b2-knock-out are poorly differentiated, and cut axons fail to reinnervate b2-deficient synaptic sites properly. Purified laminin-11 (but not other laminins) acts as a "stop" signal to cultured motor neurites, suggesting laminin-11 directly regulates axons in vivo. Previously, laminin-b2 was thought to contain the active sites in laminin-11. However, this simple hypothesis is now insufficient, because another b2-containing laminin (laminin-3, a2b2g1) was found to promote rather than stop neurite outgrowth. We now hypothesize that laminin-a5 bears much of the inhibitory activity of laminin-11, and that presynaptic defects in b2-knock-out mice are largely due to the accompanying loss of laminin-a5 at b2-deficient synapses. We propose to test this hypothesis in three ways. We will determine if laminin-a5 is required in vivo for synapse formation by characterizing laminin a5-knock-out mice. We will determine if laminin-a5 directly regulates axons, using in vitro assays of neurite outgrowth on substrates containing purified laminin trimers that contain a5 (but not b2). Finally, because Schwann cells that surround nerve terminals are also regulated by laminin-11, we will determine whether laminin-a5 directly regulates nerve terminal formation by assaying synaptogenesis in nerve-muscle co-cultures, where Schwann cells can be eliminated. These studies will provide insight into the mechanisms by which neuronal targets foster and control the pattern of innervation they receive, and these insights will advance methods of treating neuromuscular disorders and injuries.

描述（来自申请人的摘要）：我们的长期目标是了解脊椎动物突触形成的分子基础。目前，没有目标衍生信号已被牢固确立为神经调节剂脊椎动物的末端形成。该应用程序重点关注肌肉衍生的控制神经肌肉接头神经末梢形成的线索研究最多的突触。先前的研究确定了基础中的一种活动突触间隙内衬的层，诱导突触前分化重新支配轴突。调解该活动的最佳候选人是 laminin-11，突触基底层的肌肉来源成分。层粘连蛋白11 是一个异源三聚体，由三个同源层粘连蛋白“链”组成：a5、b2 和 g1。通过定向删除 b2（s-层粘连蛋白）而缺乏层粘连蛋白-11 的小鼠会死于一个月大时出现神经肌肉衰竭。运动神经末梢位于 b2-knock-out 分化差，并且切断的轴突无法重新受神经支配 b2 缺陷突触位点正确。纯化的层粘连蛋白 11（但不是其他层粘连蛋白）充当培养的运动神经突的“停止”信号，表明 laminin-11 直接调节体内轴突。此前，层粘连蛋白-b2被认为包含层粘连蛋白 11 中的活性位点。然而，这个简单的假设是现在还不够，因为另一种含 b2 的层粘连蛋白（层粘连蛋白-3，a2b2g1）发现促进而不是阻止神经突生长。我们现在假设层粘连蛋白-a5 具有层粘连蛋白-11 的大部分抑制活性，并且 b2 敲除小鼠的突触前缺陷很大程度上是由于伴随的 b2 缺陷突触处层粘连蛋白 a5 丢失。我们建议检验这个假设以三种方式。我们将确定体内突触是否需要层粘连蛋白-a5 通过表征层粘连蛋白 a5 敲除小鼠的形成。我们将确定是否使用神经突生长的体外测定，层粘连蛋白-a5 直接调节轴突在含有纯化层粘连蛋白三聚体且含有 a5（但不含 b2）的底物上。最后，因为神经末梢周围的雪旺细胞也受到调节通过laminin-11，我们将确定laminin-a5是否直接调节神经通过分析神经-肌肉共培养物中的突触发生来确定末端形成，施万细胞可以被消除。这些研究将提供深入了解神经元靶点促进和控制模式的机制他们接受的神经支配，这些见解将推进治疗方法神经肌肉疾病和损伤。