The Molecular roles of Filamin A and Arfgef2 in cortical development

描述（由申请人提供）：皮质发育畸形（MCD）被认为是癫痫、发育迟缓和智力迟钝的重要原因。脑室周围异位（PH）是一种MCD，其特征是1)沿脑侧脑室形成异位神经元结节，提示神经元迁移缺陷；2)小头畸形，提示神经增生缺陷；3)阅读障碍，提示皮质连通性缺失。PH最常见的形式是x连锁的，是由丝蛋白A（编码FLNA）基因突变引起的。一种罕见的常染色体隐性PH伴小头畸形归因于囊泡转运基因Arfgef2（编码BIG2）的突变。本提案的总体目标是：1)了解导致PH患者出现各种发育异常的致病机制；2)解决导致这种疾病的两个已知致病基因Filamin A和Arfgef2之间共享的细胞和分子途径。我将验证FLNA在皮层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，中间和心室/室下区（Dapi）， 1.2)神经迁移（BrdU出生测定），神经上皮内膜完整性（通过对顶端（Palsl, aPKC, Par3/6）和基底外侧（B-catenin, N-cadherin）蛋白进行染色），1.3)神经分化(轴突组织染色（神经丝，高尔基染色），体外检测神经细胞发生（F-actin），轴突发生（MAP2），和树突发生（Tau-1）。特异性Aim II将验证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）调节其在膜上的分布。我们的数据表明，FLNA结合BIG2并负责PKA依赖的BIG2定位到膜褶边。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通过PKA调控BIG2-Sec7活性。Specific Aim II将探讨2.1)FLNA是否调控BIG2分布，2.2)BIG2上PKA磷酸化位点（S614和S1678）是否调控FLNA-BIG2结合及其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确定，但它们的功能尚不清楚。这项研究为理解ph相关基因在皮质发育中的作用提供了一个框架。

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.