Katanin p80: A Role for Regulation of Microtubule Severing in Cortical Developmen

Katanin p80：微管切断在皮质发育中的调节作用

• 批准号: 8524549
• 负责人: Wen Fan Hu
• 金额: $ 3.39万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-05-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8524549
• 关键词: 5' Splice Site; Affect; Alleles; Apoptotic; Architecture; Biochemical; Biological; Biological Assay; Brain; Bromodeoxyuridine; C-terminal; Catalytic Domain; Cell Culture Techniques; Cell Cycle; Cell Cycle Arrest; Cell Cycle Progression; Cell Line; Cell physiology; Cells; Centrosome; Cerebral cortex; Cilia; Co-Immunoprecipitations; Cortical Malformation; Coupled; DNA; DNA Sequence; Data; Development; Embryo; Embryonic Development; Enzymes; Exhibits; Exons; Family; Flagella; Gene Targeting; Genes; Genetic; Goals; Histology; Homologous Gene; Immunohistochemistry; Intellectual functioning disability; Knockout Mice; Lead; Length; Location; Meiosis; Messenger RNA; Microcephaly; Microtubules; Mitosis; Mitotic; Mitotic spindle; Mus; Mutant Strains Mice; Mutation; N-terminal; Neurons; Palestinian; Pathway interactions; Patients; Phenotype; Physiologic pulse; Play; Proteins; RNA Interference; RNA Splicing; Regulation; Research; Reverse Transcriptase Polymerase Chain Reaction; Role; Series; Spliced Genes; Staining method; Stains; Stem cells; Surface; Testing; Time; Transcript; Ventricular; Western Blotting; brain size; cell fate specification; cognitive function; exome; genetic linkage analysis; in vivo; insight; katanin; kinetosome; lissencephaly; loss of function; lymphoblast; migration; mouse model; mutant; nerve stem cell; nervous system disorder; novel; paralogous gene; postnatal; premature; progenitor; protein p80; public health relevance; research study

DESCRIPTION (provided by applicant): The long term goals of the proposed research are to elucidate the mechanisms governing neuronal proliferation and determination of brain size. Mutations in genes causing microcephaly encode proteins that localize to the centrosome and pericentriolar matrix, suggesting that this is the seat of an important common pathway that regulates neuronal progenitor mitosis. However, the precise mechanism by which these proteins affect proliferation is unclear. Our lab has recently identified a novel mutation in a centrosomal protein in a consanguineous Palestinian family affected by severe microcephaly, lissencephaly and intellectual disability. The major goal of this proposal is to study the role of this protein i regulating neuronal progenitor mitosis and cell cycle progression in the developing mammalian cerebral cortex. Specific Aim 1 will characterize the novel mutation using RT-PCR and Western blot analysis to assay for abnormal mRNA transcripts and protein products in patient lymphoblasts. Preliminary mini- gene splicing assays show the mutation generates an mRNA transcript that skips an in-frame exon, producing a smaller, less stable protein product. Specific Aim 2 will study the cell biological role of the protein and test the hypotheses that its loss of function will cause mitotic arrest, either by disrupting spindle architecture or ciliary resorption and that this may represent a common effector pathway for other microcephaly genes, such as NDE1, which exhibits a strikingly similar phenotype. This will accomplished by RNAi knockdown in cell culture to assay for prolonged cell cycle length, abnormal mitotic spindle organization, changes in ciliary length and functional and biochemical interaction with Nde1. Finally, Specific Aim 3 will study the effect of loss of function in vivo using a knockout mouse model. Mutant mice will be analyzed for changes in brain histology using immunohistochemistry for progenitor, neuronal subtype and cortical layer specific markers, and apoptotic markers, at various time points during embryonic and early postnatal development; and for premature progenitor cell cycle exit using BrdU pulse experiments, coupled with Ki67 staining. Together, these experiments will provide further insights into the mechanisms regulating neuronal progenitor proliferation during normal cortical development and how their deregulation causes developmental neurological disorders.

描述(由申请人提供)：拟议研究的长期目标是阐明神经元增殖的机制和大脑大小的确定。导致小头症的基因突变编码定位于中心体和中心周围基质的蛋白质，这表明这是调节神经元前体有丝分裂的重要共同途径的所在地。然而，这些蛋白质影响增殖的确切机制尚不清楚。我们的实验室最近在一个患有严重小头畸形、无脑畸形和智力残疾的近亲巴勒斯坦家庭中发现了一种新的中心体蛋白突变。这项建议的主要目的是研究该蛋白I在哺乳动物大脑皮质发育中调节神经前体细胞有丝分裂和细胞周期进展的作用。具体目的1将利用RT-PCR和Western印迹分析来鉴定新的突变，以检测患者淋巴母细胞中异常的mRNA转录本和蛋白产物。初步的迷你基因剪接分析表明，该突变产生了一种跳过框架内外显子的mRNA转录本，产生了更小、更不稳定的蛋白质产物。具体目标2将研究该蛋白的细胞生物学作用，并测试其功能丧失将导致有丝分裂停止的假设，无论是通过破坏纺锤体结构还是纤毛吸收，这可能代表了其他小头畸形基因的共同效应途径，例如表现出惊人相似表型的NDE1。这将通过在细胞培养中敲除RNAi来检测细胞周期长度延长、有丝分裂纺锤体组织异常、纤毛长度变化以及与NDE1的功能和生化相互作用。最后，特殊目标3将使用基因敲除的小鼠模型在体内研究功能丧失的影响。突变小鼠将在胚胎和出生后早期发育的不同时间点通过免疫组织化学分析祖细胞、神经元亚型和皮质层特异性标记以及凋亡标记的变化；并使用BrdU脉冲实验结合Ki67染色分析过早的祖细胞周期退出。总之，这些实验将为调节正常皮质发育期间神经前体细胞增殖的机制以及它们的放松调控如何导致发育神经疾病提供进一步的见解。