An hnRNP A-like Protein in Neural Stem Cells

神经干细胞中的 hnRNP A 样蛋白

• 批准号: 6401171
• 负责人: Denes V. Agoston
• 金额: $ 7.41万
• 依托单位: HENRY M. JACKSON FDN FOR THE ADV MIL/MED
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6401171
• 关键词: DNA binding protein; cell differentiation; cell proliferation; embryonic stem cell; gel mobility shift assay; genetic library; glial fibrillary acidic protein; heterogeneous nuclear ribonucleoprotein; immunocytochemistry; in situ hybridization; laboratory rat; neurogenesis; prosencephalon; protein structure function

The maintenance of normal CNS functions in the aging brain requires constant supply of de novo astrocytes that originate from neural stem cells/progenitors present in the adult brain. Astrocytes of the adult subventricular zone can function as stem cells and differentiate into neurons. How these processes are regulated is currently not well known. Identification of factors that control embryonic neurogliagenesis and differentiation could help to understand the regulation of the adult process. The nuclear protein g-sept is expressed during embryonic neurogliagenesis and differentiation (Dobi et al, 2000). Blocking g-sept binding to its DNA site in embryonic neuroglia cultures results in a decreased number of GFAP+ and an increased number of nestin+ proliferating (BrdU+) cells. We have purified the g-sept protein using DNA-affinity chromatography. Sequence analysis showed that g-sept share homology with members of the heterogeneous nuclear ribonucleoprotein A (hnRNP A) family. The hypothesis of this proposal is that g-sept regulates neurogliagenesis and/or differentiation. The objective of this proposal is to characterize g-sept at the molecular level and to determine its functional significance in neurogliagenesis / differentiation. The Specific Aims are: 1. Molecular characterization of g-sept: full length g-sept cDNA will be obtained by PCR using degenerate primers from an embryonic rat forebrain cDNA library and by 5' and 3' RACE; the primary structure of g-sept cDNA will be determined and its relationship to known proteins will be determined; g-sept will be expressed and the recombinant protein will be characterized by Southwestern analysis and by EMSA. These experiments will provide information about the primary structure of g-sept and also reagents for functional studies. 2. Determine the functional significance of g-sept in neurogliagenesis. The availability of g-sept will be altered by antisense treatment and/or by overexpression of the protein in embryonic neuroglia cultures; at various time points following treatments, cultures will be analyzed for nestin and GFAP expression and for proliferation by immunohistochemistry and by BrdU incorporation; the pattern of g-sept expression in the embryonic (E18) and in the adult rat brain will determined by in situ hybridization histochemistry. These studies will provide basic information about the expression and about the role of g-sept in neurogliagenesis. Results will be used to design detailed studies focusing on the regulation of neurogliagenesis in the aging brain.

维持正常的中枢神经系统功能在衰老的大脑中需要持续的从头星形胶质细胞供应，这些星形胶质细胞源自成人大脑中存在的神经干细胞/祖细胞。成年室室内区的星形胶质细胞可以作为干细胞起作用，并分化为神经元。这些过程的调节方式目前尚不广为人知。鉴定控制胚胎神经化和分化的因素可以有助于了解成人过程的调节。核蛋白G-nept在胚胎神经分离和分化过程中表达（Dobi等，2000）。阻断胚胎神经蛋白培养物中的G-9与其DNA位点的结合导致GFAP+数量减少，巢蛋白+增殖（BRDU+）细胞数量增加。我们已经使用DNA亲和力色谱纯化G-9月份蛋白。序列分析表明，G-SEPT与异质性核核糖核蛋白A（HNRNP A）家族的成员共享同源性。该提议的假设是G-9S调节神经激素和/或分化。该提案的目的是表征分子水平的G-nept，并确定其在神经分散性 /分化中的功能意义。具体目的是：1。g-sept的分子表征：全长g-sept cDNA将通过PCR使用来自胚胎大鼠前脑cDNA文库的简并底漆以及5'和3'种族获得。将确定G-9月cDNA的主要结构，并确定其与已知蛋白质的关系； g-nept将被表达，重组蛋白将通过西南分析和EMSA来表征。这些实验将提供有关G-9的主要结构以及功能研究的试剂的信息。 2。确定g-sept在神经落细菌中的功能意义。反义治疗和/或蛋白质中蛋白质中的蛋白质培养物的可用性将改变。在处理后的各个时间点，将分析培养物的Nestin和GFAP表达，以及通过免疫组织化学和BRDU掺入来分析培养物。胚胎（E18）和成年大鼠大脑中G-9月表达的模式将通过原位杂交组织化学确定。这些研究将提供有关g-9月在神经激活中的表达和作用的基本信息。结果将用于设计详细的研究，重点是调节衰老大脑中神经激素。