Siah2: an E3 ligase organizes polarity and cytoskeletal links in neuron migration

Siah2：E3 连接酶在神经元迁移中组织极性和细胞骨架连接

基本信息

批准号：
10213851
负责人：
David Joseph Solecki
金额：
$ 39.27万
依托单位：
ST. JUDE CHILDREN'S RESEARCH HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-01 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10213851
关键词：
Actins Address Adhesions Affect Axon Biological Biological Assay Brain Cell Adhesion Cell Polarity Cells Cerebellum Complex Couples Crosslinker Cues Cytoplasmic Granules Cytoskeletal Modeling Cytoskeleton DCC gene Defect Dendrites Detection Development Disease Epigenetic Process Epilepsy Equilibrium Event Exocytosis Genes Genetic Genetic Epistasis Health Human Imaging Device Imaging technology Knowledge LIS1 protein Laboratories Light Link Logic Malignant Childhood Neoplasm Microscopy Microtubules Modeling Molecular Morphogenesis NTN1 gene Neuronal Differentiation Neuronal Migration Disorder Neurons Pathologic Pathway interactions Phenotype Positioning Attribute Process Radial Research Personnel Signal Pathway Signal Transduction Signaling Molecule Signaling Protein Site Slice Synapses Testing Therapeutic Transcriptional Regulation Ubiquitination Work adhesion receptor cell motility design drebrins gain of function improved in vivo insight junctional adhesion molecule lissencephaly live cell imaging live cell microscopy loss of function medulloblastoma migration nerve stem cell nervous system disorder neural circuit neurodevelopment neuron development neuronal circuitry newborn neuron novel prevent progenitor programs protein crosslink receptor response restraint stem cells trafficking ubiquitin-protein ligase

项目摘要

A spectrum of human neurological disorders including epilepsies, Lissencephaly or pediatric cancer is due in part to defective neuronal motility or germinal zone (GZ) exit and the resultant errors in neuronal circuit formation. To design strategies to prevent or treat such disorders, the field has sought to clarify the molecular mechanisms regulating neuronal motility and migration initiation. Despite advances implicating various genes essential for neuronal migration, a key gap in our knowledge is to discover how disparate cytoskeletal or signaling molecules cooperatively execute complex neuronal motility programs, such as GZ exit or nucleokinesis. My laboratory has tackled this challenge by dissecting neuronal polarity pathways impacting neuronal differentiation, nucleokinesis and adhesion control during GZ exit using the evolutionarily conserved Partitioning Defective, or Pard, polarity signaling complex as a molecular entry point. We gained insights into the regulatory logic controlling polarity during cerebellar granule neuron (CGN) development by discovering that the Seven in Absentia 2 (Siah2) E3 ubiquitin ligase is a Pard, complex antagonist. Siah2 is heavily expressed in CGN progenitors (GNPs); but not postmitotic CGNs, where Siah2-targeting of Pard3 for degradation constitutes an active pathway for progenitor polarity inhibition. We are uniquely positioned to discover new cellular mechanisms that control the onset of neuronal polarity since others overlooked inhibitory pathways in the past. In preliminary studies, we characterized new Siah2 targets relevant to GZ exit and radial migration: the Deleted in Colorectal Carcinoma (DCC) Netrin-1 (Ntn1) receptor and drebrin microtubule-actin crosslinking protein. Preliminary analysis shows: 1) Ntn1 stimulates CGN GZ exit and that Siah2 inhibits, while Pard3 promotes Ntn1-induced DCC receptor exocytosis, suggesting a hypothesis that Siah2-Pard3 antagonism regulates CGN sensitivity to Ntn1 GZ repulsion possibly through a link between DCC and junctional adhesion molecule-C (JAM-C), an adhesion receptor exocytosed in a Pard3-dependent manner. 2) Drebrin links actin- microtubule dynamics that are in turn regulated by Siah2 ubiquitination, suggesting a hypothesis that Siah2- drebrin antagonism governs the onset of nucleokinesis via microtubule-actin interactions. Remarkably, Siah2 expression is enhanced in Lissencephaly 1 (Lis1) deficient CGNs and Siah2 loss of function (LOF) or drebrin gain of function (LOF) rescues Lis1LOF migration phenotypes, suggesting that altering the balance of microtubule-actin interactions could have therapeutic value in classic neuronal migration disorders. We will build on our expertise examining polarity signaling in neuronal migration to combine in vivo genetics and ex vivo mechanistic studies with the power of transformative imaging technologies like Lattice Light Sheet Microscopy (LLSM) live-cell imaging to explore the following aims: Aim1: Define how Siah2 and Pard3 regulate DCC-dependent Ntn1 GZ repulsion. Challenge: Our current understanding of how guidance cues are interpreted in conjunction with cell adhesion and neuronal polarity pathways is limited. The findings that Siah2-Pard3 antagonism regulates DCC trafficking through a link to JAM-C adhesion sites presents an unique opportunity to test the premise that an adhesion and guidance receptor coincidence detection circuit control migration initiation. Approach: We will ablate DCC and Ntn1 in GNPs to confirm GZ repulsion in vivo. We will use epistasis in ex vivo slices or Ntn1 gradients in combination with mechanistic live-cell imaging, including LLSM, to assess how Ntn1 sensitivity couples to migration path selection via Siah2 antagonism of Pard3, DCC and JAM-C. Impact: Aim 1 will provide a new conceptual model for how polarity inhibition regulates GNP GZ occupancy and how relief of inhibition in postmitotic CGN stimulates GZ exit through adhesion and guidance receptors. Aim 2: Determine how Siah2 regulates microtubule-actin interactions during neuronal differentiation and Lis1-deficient CGNs. Challenge: Our current understanding of how cytoskeletal systems cooperate to execute radial migration or how these interactions fail in migration disorders is poor. Our findings that Siah2 regulates drebrin-dependent microtubule-actin interactions and Siah2LOF rescues Lis1LOF migration defects presents an opportunity to test two premises: 1) enhanced cytoskeletal interactions during CGN differentiation regulate the onset of classic nucleokinesis and 2) enhanced microtubule-actin restore migration in Lis1-deficient CGNs. Approach: We will use in vivo genetics, an ex vivo epistatis screen and live-cell imaging assays to assess how Siah2 mechanistically controls microtubule-actin interactions in normal or pathologic forms of CGN migration. Impact: Aim 2 could provide a new conceptual model of how the cytoskeletal interactions that drive neuronal motility are elaborated when neuronal progenitors transition to postmitotic neurons and may open the potential to exploit these mechanisms to further understand neuronal migration disorders. The proposed studies will also provide key new insights into addition questions: 1) what are the cell biological pathways that work in parallel to classic cell polarity signaling pathway during neural development and 2) how are rapid cell biological responses during brain controlled by post-translational processes like ubiquitination.

一系列人类神经系统疾病，包括癫痫、无脑畸形或小儿癌症部分原因是神经元运动或生发区 (GZ) 出口缺陷以及神经元回路中由此产生的错误形成。为了设计预防或治疗此类疾病的策略，该领域试图阐明分子机制调节神经元运动和迁移起始的机制。尽管进展涉及多种基因对于神经元迁移至关重要，我们知识中的一个关键差距是发现不同的细胞骨架或信号分子协同执行复杂的神经元运动程序，例如 GZ 退出或核分裂。我的实验室通过剖析影响神经元极性的通路来应对这一挑战使用进化保守的 GZ 退出过程中的神经元分化、核分裂和粘附控制将缺陷极性信号复合体或 Pard 分区作为分子入口点。我们深入了解了通过发现小脑颗粒神经元（CGN）发育过程中控制极性的调节逻辑 the Seven in Absentia 2 (Siah2) E3 泛素连接酶是 Pard 复合物拮抗剂。 Siah2 大量表达于 CGN 祖细胞 (GNP)；但不是有丝分裂后 CGN，其中 Siah2 靶向 Pard3 进行降解构成祖细胞极性抑制的主动途径。我们处于独特的地位，可以发现新的细胞由于过去其他人忽视了抑制途径，因此控制神经元极性发生的机制。在初步研究中，我们描述了与广州退出和径向迁移相关的新 Siah2 目标：结直肠癌 (DCC) Netrin-1 (Ntn1) 受体和 Drebrin 微管-肌动蛋白交联中删除蛋白质。初步分析显示：1）Ntn1刺激CGN GZ退出，Siah2抑制，而Pard3 促进 Ntn1 诱导的 DCC 受体胞吐作用，提示 Siah2-Pard3 拮抗作用的假设可能通过 DCC 和连接粘附之间的联系调节 CGN 对 Ntn1 GZ 排斥的敏感性分子-C (JAM-C)，一种以 Pard3 依赖性方式胞吐的粘附受体。 2）Drebrin链接actin- 微管动力学反过来又受到 Siah2 泛素化的调节，这表明 Siah2- drebrin 拮抗作用通过微管-肌动蛋白相互作用控制核分裂的发生。值得注意的是，Siah2 在 Lissencephaly 1 (Lis1) 缺陷的 CGN 和 Siah2 功能丧失 (LOF) 或 drebrin 中表达增强功能获得（LOF）拯救了 Lis1LOF 迁移表型，表明改变平衡微管-肌动蛋白相互作用可能对经典的神经元迁移障碍具有治疗价值。我们将利用我们的专业知识来检查神经元迁移中的极性信号传导，以结合体内遗传学以及利用晶格光片等变革性成像技术的力量进行离体机制研究显微镜 (LLSM) 活细胞成像旨在探索以下目标：目标 1：定义 Siah2 和 Pard3 如何调节 DCC 依赖性 Ntn1 GZ 排斥。挑战：我们目前对如何结合细胞粘附解释引导线索的理解并且神经元极性通路是有限的。 Siah2-Pard3 拮抗作用调节 DCC 贩运的发现通过 JAM-C 粘附位点的链接提供了一个独特的机会来测试粘附力和引导受体重合检测电路控制迁移启动。方法：我们将消融 GNP 中的 DCC 和 Ntn1，以确认体内的 GZ 排斥。我们将在 ex 中使用上位性体内切片或 Ntn1 梯度与机械活细胞成像（包括 LLSM）相结合，以评估如何 Ntn1 敏感性通过 Siah2 对 Pard3、DCC 和 JAM-C 的拮抗作用与迁移路径选择相结合。影响：目标 1 将为极性抑制如何调节 GNP GZ 占用提供新的概念模型以及有丝分裂后 CGN 抑制的缓解如何通过粘附和引导受体刺激 GZ 退出。目标 2：确定 Siah2 在神经元分化过程中如何调节微管-肌动蛋白相互作用和 Lis1 缺陷的 CGN。挑战：我们目前对细胞骨架系统如何合作执行径向迁移或这些相互作用如何在迁移障碍中失败尚不清楚。我们发现 Siah2 调节 Drebrin 依赖性微管-肌动蛋白相互作用和 Siah2LOF 拯救 Lis1LOF 迁移缺陷提供了测试机会两个前提：1）CGN 分化过程中增强的细胞骨架相互作用调节经典的发作核分裂和 2) 增强的微管肌动蛋白恢复 Lis1 缺陷的 CGN 中的迁移。方法：我们将使用体内遗传学、离体上位筛选和活细胞成像分析来评估如何 Siah2 在正常或病理形式的 CGN 迁移中机械地控制微管-肌动蛋白相互作用。影响：目标 2 可以提供一个新的概念模型，说明细胞骨架相互作用如何驱动神经元当神经元祖细胞转变为有丝分裂后神经元时，运动性会得到阐述，并可能打开潜力利用这些机制来进一步了解神经元迁移障碍。拟议的研究还将为其他问题提供关键的新见解：1）什么是细胞生物学在神经发育过程中与经典细胞极性信号传导途径并行工作的途径以及 2) 如何是大脑中由泛素化等翻译后过程控制的快速细胞生物反应。