Identification and validation of proteins with Siah-degron-motifs that are essential for neuronal migration.

神经元迁移对于发育中的大脑的形态发生是必不可少的，而缺陷迁移会导致严重的发育和认知障碍，如导致癫痫和智力低下的无脑。为了设计预防或治疗这种疾病的策略，有必要了解调控神经元运动和迁移启动的分子机制。最近，七缺失同系物(Siah)E3泛素连接酶被确定为一种新的分割缺陷(PAR)蛋白的调节因子。SIah是通过酵母双杂交筛选得到的一种PAR结合蛋白，通过降解PAR3来抑制小脑颗粒神经元生发区的退出和迁移。对进一步的Siah靶点的计算机和功能筛查发现了22种带有Siah退化的蛋白质和在神经元迁移中的潜在含义。拟议项目的目的是验证这些蛋白质是否确实以Siah依赖的方式降解，并测试它们是否对生发区退出和神经元迁移是充分和/或必要的。候选蛋白依赖于Siah的降解将在异源表达系统(HEK293细胞)中进行测试，在该系统中，Siah和候选蛋白将同时表达。之后，将在器官型小脑培养中测试新发现的Siah降解蛋白与生发区退出和小脑颗粒神经元的径向迁移的相关性。随后，调节生发区退出和/或神经元迁移的蛋白质的信号通路将通过荧光标记的黏附受体来确定。综上所述，该项目将极大地增加对控制生发区退出和神经元迁移的分子机制的了解，并可能为发现神经元定位障碍的新诊断和潜在治疗方法提供一条途径。

Neuronal migration is essential for the morphogenesis of the developing brain and defective migration leads to profound developmental and cognitive disorders, such as lissencephalies with resulting epilepsy and mental retardation. In order to design strategies to prevent or treat such disorders, it is necessary to understand the molecular mechanisms that regulate neuronal motility and migration initiation. Recently, the Seven in Absentia homolog (Siah) E3 ubiquitin ligase has been identified as a novel regulator of the partitioning defective (PAR) proteins. Siah was isolated as a PAR-binding protein by a yeast-two hybrid screen and inhibits the germinal zone exit and migration of cerebellar granule neurons through the degradation of PAR3. An in silico and functional screen for further Siah targets revealed 22 proteins with Siah degrons and potential implications in neuronal migration. The aims of the proposed project are to validate whether these proteins are indeed degraded in a Siah-dependent manner and to test whether they are sufficient and/or necessary for germinal zone exit and neuronal migration. Siah-dependent degradation of the candidate proteins will be tested in a heterologous expression system (HEK293 cells) where Siah and a candidate protein will be expressed simultaneously. Afterwards the relevance of newly identified Siah-degraded proteins for germinal zone exit and radial migration of cerebellar granule neurons will be tested in organotypic cerebellar cultures. Subsequently the signaling pathways of the proteins that regulate germinal zone exit and/or neuronal migration will be determined via fluorescence-tagged adhesion receptors. Taken together this project will increase the knowledge about molecular mechanisms that control germinal zone exit and neuronal migration tremendously and could provide an avenue to discover new diagnostics and potential treatments for neuronal positioning disorders.