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.

在老化的大脑中维持正常的CNS功能需要不断供应源自成人大脑中存在的神经干细胞/祖细胞的从头星形胶质细胞。成体室管膜下区的星形胶质细胞具有干细胞的功能并可分化为神经元。这些过程是如何管理的，目前还不清楚。识别控制胚胎神经胶质细胞生成和分化的因素有助于理解成年过程的调节。核蛋白g-sept在胚胎神经胶质形成和分化期间表达（Dobi等人，2000）。在胚胎神经胶质细胞培养物中阻断g-sept与其DNA位点的结合导致GFAP+的数量减少和巢蛋白+增殖（BrdU+）细胞的数量增加。我们使用DNA亲和层析纯化了g-sept蛋白。序列分析表明，g-sept与核内异质性核糖核蛋白A（hnRNP A）家族成员具有同源性。该建议的假设是，g-sept调节神经胶质的形成和/或分化。本提案的目的是在分子水平上表征g-sept，并确定其在神经胶质形成/分化中的功能意义。具体目标是：1。g-sept的分子表征：将使用来自胚胎大鼠前脑cDNA文库的简并引物通过PCR以及通过5'和3' RACE获得全长g-sept cDNA;将确定g-sept cDNA的一级结构，并将确定其与已知蛋白质的关系;将表达g-sept，并将通过Southwestern分析和EMSA表征重组蛋白。这些实验将提供有关g-sept的一级结构的信息，也为功能研究提供试剂。2.确定g-sept在神经胶质形成中的功能意义。g-sept的可用性将通过反义处理和/或通过在胚胎神经胶质培养物中过表达该蛋白而改变;在处理后的不同时间点，将通过免疫组织化学和BrdU掺入分析培养物的巢蛋白和GFAP表达以及增殖;通过原位杂交组织化学测定胚胎（E18）和成年大鼠脑中g-sept表达的模式。这些研究将提供有关g-sept在神经胶质形成中的表达及其作用的基础信息。结果将用于设计详细的研究，重点是在老化的大脑神经胶质细胞的调控。