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

神经元迁移对发育中的大脑的形态发生至关重要，而迁移缺陷会导致严重的发育和认知障碍，如缺脑症导致癫痫和智力迟钝。为了设计预防或治疗这些疾病的策略，有必要了解调节神经元运动和迁移起始的分子机制。最近，7 in Absentia同源物（Siah） E3泛素连接酶已被确定为分区缺陷（PAR）蛋白的新调节剂。通过酵母- 2杂交筛选分离出Siah作为par结合蛋白，并通过降解PAR3抑制小脑颗粒神经元的生发区退出和迁移。一项进一步的Siah靶点的计算机和功能筛选发现了22种具有Siah退化的蛋白质和神经元迁移的潜在影响。该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，并测试它们是否足以和/或必要的生发区退出和神经元迁移。候选蛋白的Siah依赖性降解将在异种表达系统（HEK293细胞）中进行测试，其中Siah和候选蛋白将同时表达。之后，新发现的siah降解蛋白与小脑颗粒神经元的生发区退出和径向迁移的相关性将在器官型小脑培养中进行测试。随后，调节生发区退出和/或神经元迁移的蛋白质信号通路将通过荧光标记的粘附受体确定。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.