RIG-CAA: Myelin Dependent Structuring of Axoplasm requires Phosphorylation of NF-M

RIG-CAA：轴浆的髓磷脂依赖性结构需要 NF-M 的磷酸化

• 批准号: 0544602
• 负责人: Michael Garcia
• 金额: $ 15万
• 依托单位: University of Missouri-Columbia
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-15 至 2009-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0544602&HistoricalAwards=false
• 关键词: RIG; CAA; Myelin; Dependent; Structuring

This is a Research Initiation Grant to Broaden Participation in the Biological Sciences (Program Solicitation NSF 05-581). Intellectual Merit: Neuronal myelination is necessary for establishing the mature axonal diameters that, along with saltatory conduction, are essential for rapid impulse transmission. Axonal diameter is regulated through accumulation and modification of the neuronal specific intermediate filaments (neurofilaments). A series of nerve grafting and genetic experiments has led to the proposal of a myelin derived "outside-in" signal that results in stoichiometrically phosphorylated neurofilaments. Loss of neurofilaments or mature myelin results in axons that fail to achieve mature diameters and display reduced conduction velocities. Axons respond to "outside-in" signals via neurofilament carboxy terminal phosphorylation, resulting in increased axonal diameter through phosphorylation dependent formation of carboxy-terminal crossbridges. Recent work has established that an essential target for myelin-derived signals exists within the carboxy terminal 426 amino acids of so-called "neurofilament medium" (NF-M), one of three neurofilament component polypeptides that combine as heteropolymers to form neurofilaments (the other two are "neurofilament light" and "neurofilament heavy"). The precise identity of the essential amino acids within the NF-M tail domain that serve as this target have yet to be elucidated. Identification of the putative signaling cascade and intra-axonal targets will establish the basic mechanism of cell-cell communication that is required to establish and maintain cellular volume during nervous system development. The general hypothesis of this project is that phosphorylation of serine residues within lysine-serine-proline (KSP) motifs in the NF-M tail domain is the mechanism of myelin-dependent regulation of axonal diameter in both the central and peripheral nervous systems. Toward that end, Dr. Garcia plans to use gene replacement in mice to: mutate all known phosphorylation sites within NF-M's tail to alanine, thereby preventing phosphorylation, to determine which of these sites are the essential target within NF-M's tail domain for the outside-in signaling cascade; to mutate the phosphorylation sites to glutamate, to mimic the charge associated with phosphorylation to determine the consequences to axonal growth and organization associated with chronic phosphorylation; and to expand the number of KSP motifs of the NF-M tail domain by generating a chimeric protein consisting of mouse and bovine NF-M to determine if the number of available phosphorylation sites within NF-M's tail domain regulates axonal caliber. He will also use existing neurofilament modified mice to determine if phosphorylation of NF-M tail domain KSP motifs is essential for radial growth of CNS axons. These experiments will establish the mechanism of axonal response to myelination by identifying the amino acids within the NF-M tail domain and the method of post-translational modification that together with myelination facilitate radial axonal growth in both central and peripheral nervous systems. Moreover, this constitutes the first necessary step in elucidating the signaling cascade that derives from myelinating cells resulting in radial axonal growth through neurofilament modification. Elucidation of the signaling cascade and intra-axonal targets are crucial to understanding the mechanism of cell-cell communication required to establish mature axonal diameters during nervous system development.Broader Impacts: Dr. Garcia will host undergraduate students in his laboratory through his participation in established undergraduate research programs at the University of Missouri, including the EXPRESS (Exposure to Research for Science Students) program, that provide opportunities for undergraduates who are members of underrepresented groups to work in faculty research laboratories during the academic years. In addition, because he himself is a member of an underrepresented minority group, he serves as a role model for minority students who aspire to careers in science.

这是扩大对生物科学的参与的研究启动赠款（计划招标NSF 05-581）。 智力优点：神经元髓鞘化对于建立成熟的轴突直径是必要的，这些轴突直径与盐传导一起对于快速脉冲传播至关重要。 通过积累和修饰神经元特异性中间丝（神经丝）来调节轴突直径。 一系列神经嫁接和遗传实验导致了髓磷脂衍生的“外部”信号的建议，该信号导致静态磷酸化的神经丝。 神经丝或成熟的髓磷脂的损失导致轴突无法实现成熟的直径并显示出降低的传导速度。 轴突通过神经丝羧末端磷酸化响应“外部”信号，从而通过碳二末端跨桥的磷酸化依赖性形成而导致轴突直径增加。最近的工作表明，在所谓的“神经丝中培养基”（NF-M）的426个氨基酸氨基酸（NF-M）中存在一个基本目标，这是三种神经丝组件多肽之一，它们是形成神经纤维的杂物，是神经纤维的杂物（其他两种）（其他两种是神经纤维纤维的浅色亮度光明）。 NF-M尾域中必需氨基酸的确切身份尚未阐明。 识别推定的信号级联和轴内靶标将建立细胞 - 细胞通信的基本机制，这些机制是在神经系统发育过程中建立和维持细胞体积所需的基本机制。 该项目的一般假设是，NF-M尾域中赖氨酸 - 丝氨酸 - 丙烯（KSP）基序中丝氨酸残基的磷酸化是中枢和外周神经系统中轴突直径调节的机理。 为此，Garcia博士计划在小鼠中使用基因替换，以突变NF-M尾巴内的所有已知磷酸化位点至丙氨酸，从而防止磷酸化，以确定这些位点是NF-M的尾部域内的哪些位点，用于外部信号级联的NF-M尾部结构域中；为了突变磷酸化位点至谷氨酸，以模仿与磷酸化相关的电荷，以确定与慢性磷酸化相关的轴突生长和组织的后果；并通过产生由小鼠和牛NF-M组成的嵌合蛋白来扩大NF-M尾域的KSP基序数量，以确定NF-M的尾部结构域中可用的磷酸化位点的数量是否调节轴突纤维。 他还将使用现有的神经丝修饰小鼠来确定NF-M尾域KSP基序的磷酸化是否对于CNS轴突的径向生长至关重要。 这些实验将通过鉴定NF-M尾巴内的氨基酸以及翻译后修饰的方法来确定轴突反应的机制，这些方法与髓鞘形成促进了中央和周围神经系统中的径向轴突生长。此外，这构成了阐明源自髓鞘细胞的信号传导级联反应的第一步，从而导致通过神经丝修饰导致径向轴突生长。 阐明信号级联和轴内目标对于理解在神经系统发展过程中建立成熟的轴突直径所需的细胞室沟通机制至关重要。BodRoader的影响。在学年中，代表性不足的小组在教师研究实验室工作。 此外，由于他本人是代表性不足的少数群体的成员，因此他是渴望从事科学职业的少数族裔学生的榜样。