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

这是一个研究启动补助金，brokenet参与生物科学（计划征求NSF 05-581）。 智力优势：神经元髓鞘形成对于建立成熟的轴突直径是必要的，而成熟的轴突直径沿着跳跃式传导对于快速脉冲传递是必不可少的。 轴突直径通过神经元特异性中间丝（神经丝）的积累和修饰来调节。 一系列的神经移植和遗传学实验已经提出了髓磷脂衍生的“由外向内”信号，其导致化学计量的磷酸化神经丝。 神经丝或成熟髓鞘的缺失导致轴突不能达到成熟的直径并显示出降低的传导速度。 轴突通过神经丝羧基末端磷酸化对“由外向内”信号作出反应，通过羧基末端横桥的磷酸化依赖性形成导致轴突直径增加。最近的工作已经确定，髓磷脂衍生信号的必需靶标存在于所谓的“神经丝介质”（NF-M）的羧基末端426个氨基酸内，所述“神经丝介质”是三种神经丝组分多肽之一，所述三种神经丝组分多肽联合收割机作为杂聚物组合以形成神经丝（另外两种是“神经丝轻”和“神经丝重”）。作为该靶标的NF-M尾结构域内的必需氨基酸的精确身份尚未阐明。 推定的信号级联和轴突内靶点的鉴定将建立细胞-细胞通信的基本机制，所述细胞-细胞通信是在神经系统发育期间建立和维持细胞体积所需的。 该项目的一般假设是NF-M尾域中赖氨酸-丝氨酸-脯氨酸（KSP）基序内的丝氨酸残基的磷酸化是中枢和外周神经系统中轴突直径的髓鞘依赖性调节的机制。 为了达到这个目的，Garcia博士计划在小鼠中使用基因替换：将NF-M尾部内所有已知的磷酸化位点突变为丙氨酸，从而阻止磷酸化，以确定这些位点中的哪些是NF-M尾部结构域内由外向内信号级联的重要靶点;使磷酸化位点突变为谷氨酸，模拟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;以及通过产生由小鼠和牛NF-M组成的嵌合蛋白来扩大NF-M尾结构域的KSP基序的数目，以确定NF-M尾结构域内可用的磷酸化位点的数目是否调节轴突口径。 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CNS轴突的径向生长至关重要。 这些实验将通过鉴定NF-M尾结构域内的氨基酸和翻译后修饰的方法来建立轴突对髓鞘形成的反应机制，所述翻译后修饰与髓鞘形成一起促进中枢和外周神经系统中的径向轴突生长。此外，这构成了阐明源自髓鞘形成细胞的信号级联的第一个必要步骤，该信号级联通过神经丝修饰导致径向轴突生长。 阐明信号级联和轴突内靶点对于理解神经系统发育过程中建立成熟轴突直径所需的细胞间通讯机制至关重要。加西亚博士将通过参与密苏里州大学的本科生研究项目，包括EXPRESS，在他的实验室接待本科生（暴露于科学学生的研究）计划，提供机会，为本科生谁是代表性不足的群体的成员在学年期间在教师研究实验室工作。 此外，由于他本人是一个代表性不足的少数群体的成员，他作为一个榜样，少数民族学生谁渴望在科学事业。

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.