FUNCTIONAL ROLES OF THE NF-H NEUROFILAMENT PROTEIN

NF-H 神经丝蛋白的功能作用

• 批准号: 3760755
• 负责人: DON W CLEVELAND
• 金额: --
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3760755
• 关键词: antisense nucleic acid; axon; crosslink; electron microscopy; gel electrophoresis; gene expression; genetically modified animals; laboratory mouse; microscopy; molecular cloning; neurofilament; neurofilament proteins; neurons; peripheral nervous system; phosphorylation; polymerase chain reaction; protein structure function; site directed mutagenesis; tissue /cell culture; transfection; vimentin

The cytoarchitecture of nerve cells is comprised principally of two structural components: microtubules and neurofilaments (NF). While the function of microtubules in neurite extension and as substrates for transport of particles into the axon is well accepted, no companion function for NF, the intermediate filaments of neurons, is proven. However, we have found that the level of synthesis of the subunit proteins of NF directly correlates with the diameters of mammalian nerve fibers. This has lead us to propose that NF gene expression is a principal determinator of axonal caliber, which itself specifies conduction velocity of the axon. We now propose to use a combination of in vitro and in vivo molecular genetics to test this putative function of NF. Our approach will focus on the NF-H polypeptide, the largest of the three NF subunits. While the other subunits (NF-L and NF-M) are more abundant, the long carboxy-terminal tail domain OF NF-H has long been speculated to cross link adjacent NFs and perhaps to determine the lateral spacing of NFs. Starting from the cloned mouse NF-H gene, we shall use DNA transfection to determine the assembly properties of NF-H when expressed alone or in combination with NF-L and NF-M. Since in axons NF-H is heavily phosphorylated and its carboxy-terminal domain carries >50 repeats of a putative phosphorylation site, we will also perform similar experiments with mutant NF-H genes deleted in this and other domains. Finally, the in vivo properties of such mutant NF-H polypeptides will be assessed by producing transgenic mice whose genomes contain genes specifically engineered to increase or decrease NF-H expression.

神经细胞的细胞结构主要由两部分组成 结构成分：微管和神经丝。而当 微管在神经突起延伸中的作用及其作为基质的作用 颗粒进入轴突的运输是被广泛接受的，没有同伴 神经纤维是神经元的中间细丝，其功能已得到证实。 然而，我们发现，亚基的合成水平 神经营养因子的蛋白与哺乳动物神经的直径直接相关 纤维。这导致我们提出，核因子基因的表达是一种 轴突口径的主要决定因素，它本身规定 轴突的传导速度。我们现在建议结合使用 体外和体内分子遗传学来验证这一推测的功能 NF型。我们的方法将重点放在核因子-H多肽上，它是 三个核因子亚基。而其他亚基(核因子-L和核因子-M)是 更丰富的是，NF-H的长羧基末端尾部结构域 被推测为与相邻的NFS交叉链接，可能是为了确定 NFS侧向间距。从克隆的小鼠核因子-H基因开始，我们 应使用DNA转染法确定核因子-H的组装性质 单独表达或与核因子-L、核因子-M联合表达时。自年起 轴突NF-H高度磷酸化，其羧基末端结构域 携带一个假定的磷酸化位点的&gt；50重复，我们还将 对这两个基因中缺失的突变的NF-H基因进行类似的实验 其他域。最后，对该突变体NF-H的体内特性进行了研究 多肽将通过生产转基因小鼠进行评估，转基因小鼠的基因组 含有专门设计来增加或减少核因子-H的基因 表情。