LAMININS AND NEUROMUSCULAR SYNAPSE FORMATION

描述（来自申请人摘要）：我们的长期目标是了解 脊椎动物突触形成的分子基础。当前没有任何 靶源性信号已经被牢固地确立为神经调节器 脊椎动物的末端形成。该应用程序的重点是肌肉来源的 在神经肌肉接头处控制神经末梢形成的线索， 研究最多的突触以前的研究发现，在基底细胞中， 衬于突触间隙的层，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 层粘连蛋白-11，突触基底层的肌源性成分。层粘连蛋白-11 是异源三聚体，由三个同源层粘连蛋白“链”组成：a5，b2，和 g1。通过靶向缺失b2（s-层粘连蛋白）而缺乏层粘连蛋白-11的小鼠死于 在一个月大时出现神经肌肉衰竭。运动神经末梢 b2基因敲除的细胞分化差，切断的轴突不能再神经支配 b2缺乏的突触部位。纯化的层粘连蛋白-11（而非其他 层粘连蛋白）对培养的运动神经突起到“停止”信号的作用，这表明 层粘连蛋白-11在体内直接调节轴突。以前，层粘连蛋白-b2被认为 以包含层粘连蛋白-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，a2 b2 g1） 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， b2基因敲除小鼠的突触前缺陷主要是由于伴随的 在B2-缺陷突触处层粘连蛋白-A5的损失。我们建议检验这一假设 有三个方面我们将确定层粘连蛋白-a5是否是突触形成所必需的 通过表征层粘连蛋白A5-敲除小鼠形成。我们将确定是否 层粘连蛋白-a5直接调节轴突，使用体外轴突生长测定 在含有纯化的层粘连蛋白三聚体的基底上，所述层粘连蛋白三聚体含有A5（但不含B2）。 最后，由于神经末梢周围的雪旺细胞也受到调节， 通过层粘连蛋白-11，我们将确定层粘连蛋白-A5是否直接调节神经 通过测定神经-肌肉共培养物中的突触发生， 雪旺氏细胞可以被清除。这些研究将提供深入了解 神经元靶点培育和控制的机制， 他们接受神经支配，这些见解将促进治疗方法， 神经肌肉疾病和损伤。

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.