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: 7546911
• 负责人: JASON B NEAL
• 金额: $ 3.17万
• 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/7546911
• 关键词: A kinase anchoring protein; Actins; Address; Affect; Antibodies; Apical; Axon; Binding; 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; Localized; Membrane; Mental Retardation; Microcephaly; Molecular; Morphology; Mus; Mutation; N-Cadherin; Neuroepithelial; Neuroepithelial Cells; Neuronal Differentiation; Neurons; Nodule; Nuclear; Numbers; 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; 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（MCD）的畸形被认为是癫痫，发育延迟和智力低下的重要原因。 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. pH的最常见形式是X连锁的，并且由于丝蛋白A（编码FLNA）基因的突变。具有小头畸形的pH的稀有常染色体隐性形式归因于囊泡转运基因ArfGeF2（编码BIG2）中的突变。该提案的总体目标是1）了解患有pH值的受影响个体的各种发育异常的致病机制和2）解决两种已知的病因基因A和ARFGEF2之间的任何共享的细胞和分子途径，从而引起这种疾病。具体目的我将检验FLNA调节皮质发育过程中神经上皮细胞增殖，迁移和分化的假设。 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).具体目标II将检验以下假设：FLNA-BIG2结合调节BIG2亚细胞定位和BIG2依赖性SEC7活性。 FLNA是一种脚手架蛋白，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BIG2与膜荷叶边的PKA依赖性定位。 BIG2是一种A-激酶锚定蛋白（AKAP），其SEC7功能取决于PKA。我们的结合研究暗示了FLNA在PKA对BIG2-SEC7活性的调节中。具体目标II将解决2.1）FLNA是否调节BIG2分布，2.2）BIG2（S614和S1678）上的PKA phosp- horylation位点调节FLNA-BIG2结合及SEC7功能。皮质发育的畸形占小儿癫痫病例的40％。这些疾病的许多因果因素已被鉴定出来，但它们的功能尚不清楚。这项研究提供了一个框架，以了解pH相关基因在皮质发育中的作用。