REGULATION OF CELL PHENOTYPE IN THE DEVELOPING NEOCORTEX

发育中新皮质细胞表型的调节

基本信息

批准号：
6354123
负责人：
Susan Hockfield
金额：
$ 10.17万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-03-01 至 2003-02-28
项目状态：
已结题

项目摘要

During neural development, a population of morphologically homogenous, mitotically active precursor cells gives rise to all the neuronal and glial cells of the adult cerebral cortex. Postmitotic neurons extend processes by which they migrate to their adult positions and begin the elaboration of axonal and dendritic arbors. To understand the molecular events that permit a cell to assume neuronal properties, we previously used 2- dimensional gel electrophoresis to identify 15 proteins that are differentially regulated over the course of corticogenesis. One of these, TOAD-64, is a 64,000 dalton protein that is up regulated nearly 7-fold over the course of late embryonic cortical development and is subsequently down regulated during postnatal life. The sequence of a full length cDNA of TOAD-64 is homologous to the unc-33 gene from C. elegans, mutations in which lead to defective patterns of axon outgrowth. Several lines of evidence suggest that TOAD-64 may, similarly, play a role in axonogenesis in the rodent. First, TOAD-64 is re-expressed in adult motor neurons as they extend axons following a peripheral nerve lesion. Second, TOAD-64 expression increases coincident with neuritogenesis in two cell lines, pC12 and p19. Finally, TOAD-64 protein is present at the most peripheral portions of the growth cone, in lamellipodia and filopodia. Together, these data support the possibility that TOAD-64 is part of the intracellular machinery by which growing neurons elaborate axons. The experiments proposed here are designed to further characterize TOAD-64 and will also address the possible functions of this new protein in neurite outgrowth. The first aim is to further characterize TOAD-64. A possible role for TOAD-64 in neuronal remodeling in the adult will be investigated. A new panel of antibodies to primate TOAD-64 will be generated to permit studies of early corticogenesis in the monkey. For studies of the mechanisms by which the TOAD-64 gene is regulated, a genomic clone will be isolated for future experiments to identify its regulatory sequences. The second aim is to determine the function of TOAD-64. We will explore the role TOAD-64 may play in axonogenesis by changing levels of protein expression in primary neuronal cultures and in two cell lines. To test whether TOAD-64 is required for a cell to elaborate neurites, antisense oligonucleotides will be used to reduce TOAD-64 expression in neurons in primary culture. We will also determine whether a reduction in TOAD-64 can alter the response of growth cones and neurites to repulsive substrates. To determine whether TOAD-64 is critical for the elaboration of neurites in PC12 and P19 cells, expression of TOAD-64 will be reduced or increased using stable transfection.

在神经发育过程中，一群形态上同质的，有丝分裂活跃的前体细胞产生所有神经元和神经胶质细胞成人大脑皮层的细胞。有丝分裂后神经元延长过程通过这种方式，他们迁移到成人的位置并开始阐述轴突和树突乔木。了解分子事件允许细胞呈现神经元特性，我们之前使用过 2- 三维凝胶电泳鉴定 15 种蛋白质在皮质生成过程中受到差异性调节。其中之一， TOAD-64 是一种 64,000 道尔顿的蛋白质，其上调近 7 倍胚胎晚期皮质发育过程，随后下降在产后生活中受到调节。全长 cDNA 的序列 TOAD-64 与线虫的 unc-33 基因同源，突变这导致轴突生长的模式有缺陷。几行有证据表明 TOAD-64 可能同样在轴突发生中发挥作用在啮齿动物中。首先，TOAD-64 在成年运动神经元中重新表达为它们在周围神经损伤后延长轴突。二、TOAD-64 在两个细胞系 pC12 中，表达增加与神经突发生同时发生和第19页。最后，TOAD-64 蛋白存在于最外周生长锥的部分，片状伪足和丝状伪足。一起，这些数据支持 TOAD-64 是生长中的神经元用来形成轴突的细胞内机制。这这里提出的实验旨在进一步表征 TOAD-64 和还将探讨这种新蛋白质在神经突中的可能功能生长出来的。第一个目标是进一步表征 TOAD-64。可能的角色为将研究 TOAD-64 在成人神经元重塑中的作用。一个新的将产生灵长类 TOAD-64 抗体组以进行研究猴子早期皮质发生的研究。对于机制的研究 TOAD-64 基因受到调节，将分离出基因组克隆未来的实验以确定其调控序列。第二个目标是确定 TOAD-64 的功能。我们将探索 TOAD-64 可能通过改变蛋白质水平在轴突发生中发挥作用在原代神经元培养物和两个细胞系中表达。测试细胞形成神经突是否需要 TOAD-64，反义寡核苷酸将用于减少神经元中 TOAD-64 的表达主流文化。我们还将确定 TOAD-64 的减少是否可以改变生长锥和神经突对排斥基质的反应。确定 TOAD-64 是否对于神经突的形成至关重要 PC12和P19细胞，TOAD-64的表达会减少或增加使用稳定转染。