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、发育迟缓和智力低下的重要原因。脑室周围异位症(PH)是一种MCD，其特征是1)在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，提示神经增殖缺陷；3)阅读障碍，提示皮质连接障碍。最常见的PH是X连锁的，是由于丝氨酸A(编码FLNA)基因的突变所致。一种罕见的常染色体隐性形式的PH合并小头畸形被归因于囊泡运输基因Arfgef2(编码BIG2)的突变。这项建议的总体目标是：1)了解引起受影响的PH患者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之间的任何共同的细胞和分子途径。具体目的：我将测试这一假设，即Flna在皮质发育过程中调节神经上皮细胞的增殖、迁移和分化。我们将使用一只空的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，中间区和室/室下区(DAPI)，1.2)BrdU出生时的神经迁移，以及通过顶端(Pals1，aPKC，Par3/6)和基底外侧(B-catenin，N-cadherin)蛋白染色检测神经上皮衬里完整性，1.3)通过轴突组织(神经丝，Golgi染色)原位和通过检测神经发生(F-肌动蛋白)，轴突发生(MAP2)和树突发生(Tau-1)来评估神经分化。特殊目的II将检验这一假设，即FLNA-BIG2结合调节BIG2亚细胞定位和BIG2依赖的Sec7活性。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与BIG2结合，并负责依赖PKA将BIG2定位到膜褶皱上。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参与了PKA对BIG2-Sec7活性的调节。特殊目的II将讨论2.1)Flna是否调节BIG2的分布，2.2)BIG2上的PKA磷酸化位点(S614和S1678)是否调节Flna-BIG2的结合和随后的Sec7功能。皮质发育畸形占儿童癫痫病例的40%。许多导致这些疾病的基因已经被识别出来，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.