The Molecular roles of Filamin A and Arfgef2 in cortical development

Filamin A 和 Arfgef2 在皮质发育中的分子作用

• 批准号: 7676848
• 负责人: JASON B NEAL
• 金额: $ 2.11万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7676848
• 关键词: A kinase anchoring protein; Actins; Address; Affect; Antibodies; Apical; Axon; Binding; Brain; Bromodeoxyuridine; Cell Death; Cell Proliferation; Cells; Childhood; Complex; Cortical Malformation; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoskeleton; Data; Defect; Dendrites; Development; Developmental Delay Disorders; Disease; Dyslexia; Embryo; Epilepsy; F-Actin; Genes; Goals; Golgi Apparatus; Impairment; In Situ; In Vitro; Individual; Link; Membrane; Mental Retardation; Microcephaly; Molecular; Morphology; Mus; Mutation; N-Cadherin; Neuroepithelial; Neuroepithelial Cells; Neuronal Differentiation; Neurons; Nodule; Nuclear; Pathway interactions; Phosphorylation; Phosphorylation Site; Phosphotransferases; Proteins; Radial; Regulation; Role; Scaffolding Protein; Signal Transduction; Signaling Molecule; Silver; Site; Staging; Staining method; Stains; Terminator Codon; Testing; Thick; Transport Vesicles; Tyrosine; Ventricular; Vimentin; beta catenin; filamin; genetic regulatory protein; in vivo; lateral ventricle; migration; nerve stem cell; neuroepithelium; neurofilament; periventricular heterotopia; progenitor; relating to nervous system; subventricular zone; tau-1

DESCRIPTION (provided by applicant): Malformations of cortical development (MCD) are recognized as a significant cause of epilepsy, developmental delay, and mental retardation. Periventricular heterotopia (PH) is one such MCD characterized by 1) ectopic neuronal nodule formation along the lateral ventricles of the brain suggesting defects in neuronal migration 2) microcephaly, suggesting defects in neural proliferation 3) dyslexia, suggesting abberent cortical connectivity. The most common form of PH is X-linked and due to mutations in the Filamin A (encodes FLNA) gene. A rarer autosomal recessive form of PH with microcephaly has been attributed to mutations in the vesicle transport gene Arfgef2 (encodes BIG2). The overall goal of this proposal is to 1) understand the pathogenic mechanisms giving rise to the various developmental abnormalities seen in affected individuals with PH and 2) address any shared cellular and molecular pathways between the two known causative genes Filamin A and Arfgef2, in giving rise to this disorder. Specific Aim I will test the hypothesis that FLNA regulates neuroepithelial cell proliferation, migration, and differentiation during cortical development. We will use a null FLNA mouse (Dilp2) to assess impairments in: 1.1) neuroepithelial cell proliferation (BrdU, PHS, Ki-67), thickness of cortical plate, intermediate and ventricular/subventricular zones (Dapi), 1.2) neural migration by BrdU birthdating, and neuroepithelial lining integrity by staining for apical (Palsl, aPKC, Par3/6) and basolateral (B-catenin, N-cadherin) proteins, 1.3) neural differentiation in situ by staining for axonal organization (neurofilament, golgi stain) and in vitro by examining neuritogenesis (F-actin), axonogenesis (MAP2), and dendritogenesis (Tau-1). Specific Aim II will test the hypothesis that FLNA-BIG2 binding regulates BIG2 subcellular localization and BIG2-dependent Sec7 activity. FLNA is a scaffolding protein whose PKA-phosphorylation (S2152) regulates its distribution to the membrane. Our data shows that FLNA binds BIG2 and is responsible for PKA- dependent localization of BIG2 to membrane ruffles. BIG2 is an A-kinase anchoring protein (AKAP) whose Sec7 function is dependent on PKA. Our binding studies implicate FLNA in the regulation of BIG2-Sec7 activity by PKA. Specific Aim II will address whether 2.1) FLNA regulates BIG2 distribution, 2.2) PKA phosp- horylation sites on BIG2 (S614 and S1678) regulate FLNA-BIG2 binding and consequent Sec7 function. Malformations of cortical development represent up to 40% of pediatric epilepsy cases. Many of the genes causal for these disorders have been identified, yet their functions are not yet clear. This study provides a framework with which to understand the role of PH-associated genes in cortical development.

描述（由申请人提供）：皮质发育畸形（MCD）被认为是癫痫、发育迟缓和智力低下的重要原因。脑室周围异位 (PH) 就是这样一种 MCD，其特征为 1) 沿大脑侧脑室形成异位神经元结节，表明神经元迁移缺陷 2) 小头畸形，表明神经增殖缺陷 3) 阅读障碍，表明皮质连接异常。最常见的 PH 形式是 X 连锁的，是由于 Filamin A（编码 FLNA）基因突变所致。一种罕见的常染色体隐性遗传性 PH 伴小头畸形，归因于囊泡转运基因 Arfgef2（编码 BIG2）的突变。该提案的总体目标是 1) 了解在 PH 受影响的个体中引起各种发育异常的致病机制，以及 2) 解决两个已知致病基因 Filamin A 和 Arfgef2 之间的任何共享细胞和分子途径，从而给予上升到这种障碍。具体目标我将检验 FLNA 在皮质发育过程中调节神经上皮细胞增殖、迁移和分化的假设。我们将使用无效 FLNA 小鼠 (Dilp2) 来评估以下方面的损伤：1.1) 神经上皮细胞增殖 (BrdU、PHS、Ki-67)、皮质板厚度、中间区和心室/心室/心室下区 (Dapi)、1.2) 神经迁移通过顶端（Palsl、aPKC、Par3/6）和基底外侧（B-连环蛋白、 N-钙粘蛋白）蛋白，1.3）通过轴突组织染色（神经丝、高尔基体染色）进行原位神经分化，并通过检查神经突发生（F-肌动蛋白）、轴突发生（MAP2）和树突发生（Tau-1）进行体外神经分化。具体目标 II 将检验 FLNA-BIG2 结合调节 BIG2 亚细胞定位和 BIG2 依赖性 Sec7 活性的假设。 FLNA 是一种支架蛋白，其 PKA 磷酸化 (S2152) 可调节其在膜上的分布。我们的数据表明，FLNA 结合 BIG2，并负责 BIG2 依赖于 PKA 的膜皱褶定位。 BIG2 是一种 A 激酶锚定蛋白 (AKAP)，其 Sec7 功能依赖于 PKA。我们的结合研究表明 FLNA 参与 PKA 对 BIG2-Sec7 活性的调节。具体目标 II 将解决 2.1) FLNA 是否调节 BIG2 分布，2.2) BIG2 上的 PKA 磷酸化位点（S614 和 S1678）是否调节 FLNA-BIG2 结合以及随后的 Sec7 功能。皮质发育畸形占小儿癫痫病例的 40%。许多导致这些疾病的基因已被识别，但它们的功能尚不清楚。这项研究提供了一个框架来了解 PH 相关基因在皮质发育中的作用。