ANALYSIS OF MOLECULAR MECHANISMS UNDERLYING CORTICAL ALIGNMENT

本研究的目的是为了更好地了解皮质发育的细胞和分子机制，特别是reelin信号级联与皮质细胞排列的相互作用。神经系统突变小鼠纺机产生了各种类型的皮层神经元，并在正常时间内完成了迁移，但在径向维度上的细胞模式发生了扭曲，即皮层层压大致倒转。我们通过实验发现，Reelin是reeler基因（Reln）的产物，由Cajal-Retzius细胞分泌，为迁移的皮层神经元在发育中的皮层的适当位置排列提供了指导信号。利用一种独特的识别Reelin的抗体CR-50，我们发现Reelin相互结合形成一个巨大的蛋白质复合物，并表明这种组装形成对其功能至关重要。在缺乏mdab1基因的yotari突变小鼠中也观察到reeler表型，mdab1基因编码更多的信号转导相关蛋白Dab1。我们发现，yotari突变是由dab1基因序列被一个长穿插核元件（L1）片段取代引起的。与Reelin不同，Dab1在皮质板神经元中表达，存在于细胞质中，并与Src家族的酪氨酸激酶相互作用。此外，在皮质细胞培养中加入Reelin后，Dab1的磷酸化降低，但增加。这些观察结果表明，在皮质板神经元的细胞膜上存在Reelin受体，它感知细胞外的Reelin，与酪氨酸激酶偶联，并通过Dab1将该信号传递给细胞。Yagi小组先前利用Fyn结合活性从小鼠大脑中分离出一种定位于皮质神经元细胞膜上的cadherin相关神经元受体（CNR）家族蛋白，表明Fyntyrosine激酶在构建脑网络中具有重要的分子功能。与Yagi小组合作，我们确定了一个CNR家族作为Reelin受体的候选。除了CNRs外，两种膜蛋白apoER2和VLDLR也被鉴定为Reelan受体。我们提供了针对这些受体蛋白的抗体，并表明细胞外Reelin蛋白和这些受体蛋白在皮质板神经元突起上共定位。我们现在正在研究这些受体如何作为Reelin受体的组成部分协同作用。少

The present research aim is to provide better understanding the cellular and molecular mechanisms of cortical development, especially the interplay of reelin signal cascade with cortical cell's alignment. In the neurological mutant mice reeler, the various classes of cortical neurons are generated and complete their migration at the normal time, however, the cellular pattern in the radial dimension is distorted, i.e., the cortical lamination is roughly inverted. We showed experimentally that Reelin, the product of the reeler gene (Reln) and secreted from the Cajal-Retzius cells, provides an instructive signal for migrating cortical neurons to align at appropriate location ill developing cortex. Using a unique antibody CR-50 which recognizes Reelin, we showed that Reelin binds each other to form a huge protein complex and suggested that this assembly formation is critical for its function. The reeler phenotype is also observed in yotari mutant mice lacking the mdab1 gene which encodes s … More ignal transduction-associated protein Dab1. We have found that the yotari mutation is caused by a replacement of dab1 gene sequence with a long interspersed nuclear element (L1) fragment. Unlike Reelin, Dab1 is expressed in the cortical plate neurons, resides in the cytoplasm and interacts with tyrosine kinases of the Src family. In addition, phosphorylation of Dab1 is decreased in reeler, but increased when Reelin is added to cortical cell cultures. These observations indicated the presence of the Reelin receptors in the cell membrane of the cortical plate neurons that sense the extracellular Reelin, couple with tyrosine kinases and relay this signal to the cell via Dab1. Yagi group had previously showed that Fyntyrosine kinase has a crucial molecular function in building brain network and isolated a novel cadherin-related neuronal receptor (CNR) family proteins localized on the cell membrane of the cortical neurons using Fyn binding activity from the mouse brain. Collaborating with Yagi group, we identified a CNR family as a candidate of Reelin receptors. In addition to CNRs, two membrane proteins, apoER2 and VLDLR, have been identified as Reelan receptors. We provided antibodies against these receptor proteins and showed that extracellular Reelin proteins and these receptor proteins are co-localized on the processes of cortical plate neurons. We are now investigating how these receptors act in concert as components of the Reelin receptors. Less