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

这是一项研究启动助学金，旨在扩大对生物科学的参与(计划征集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，而轴突直径的跳跃传导对于快速的脉冲传递是必不可少的。轴突直径是通过神经元特定的中间丝(神经丝)的积累和修饰来调节的。一系列神经移植和基因实验导致了髓鞘来源的“自外而内”信号的提出，这种信号导致了化学计量的磷酸化神经细丝。神经细丝或成熟髓鞘的丢失会导致轴突无法达到成熟的直径，并显示出传导速度减慢。轴突通过神经丝羧基末端的磷酸化对“由外而内”的信号作出反应，通过依赖于磷酸化的羧基末端交叉桥的形成而导致轴突直径的增加。最近的研究表明，髓鞘信号的一个重要靶点存在于所谓的神经丝介质(NF-M)的羧基末端426个氨基酸中，NF-M是三种神经丝成分多肽之一，它们以杂聚体的形式结合形成神经丝(另外两种是神经丝轻和神经丝重)。作为这一靶点的核因子-M尾部结构域中的必需氨基酸的确切身份尚未阐明。识别可能的信号级联和轴突内靶点将建立神经系统发育过程中建立和维持细胞体积所需的细胞-细胞通信的基本机制。该项目的一般假设是，核因子-M尾部结构域中赖氨酸-丝氨酸-脯氨酸(KSP)基序中丝氨酸残基的磷酸化是中枢和外周神经系统对轴突直径的髓鞘依赖性调节机制。为此，加西亚博士计划在小鼠中使用基因替换来：将NF-M尾部所有已知的磷酸化位点突变为丙氨酸，从而防止磷酸化，以确定这些位点中的哪些是NF-M尾部结构域中由外向内信号级联的基本靶点；将这些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；并通过产生由小鼠和牛NF-M组成的嵌合蛋白来扩大NF-M尾部结构域的KSP基序的数量，以确定NF-M尾部结构域中可用的磷酸化位点的数量是否调节轴突口径。他还将使用现有的神经丝修饰小鼠来确定核因子-M尾部结构域KSP基序的磷酸化是否对中枢神经系统轴突的放射状生长至关重要。这些实验将通过鉴定核因子-M尾部结构域中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，翻译后修饰的方法与髓鞘形成一起促进中枢和周围神经系统的放射状轴突生长。此外，这是阐明髓鞘细胞通过神经丝修饰导致放射状轴突生长的信号级联反应的第一个必要步骤。阐明信号级联和轴突内目标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-细胞通信机制至关重要。广泛影响：加西亚博士将通过参与密苏里大学既定的本科生研究项目在他的实验室接待本科生，其中包括EXPRESS(科学学生研究暴露)计划，该计划为代表不足群体的本科生提供在学年期间在教职员工研究实验室工作的机会。此外，由于他自己是一个少数族裔群体的成员，他是渴望从事科学事业的少数族裔学生的榜样。

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.