Mechanistic Basis for Regulation of the Axon Initial Segment

轴突初始段调节的机制基础

• 批准号: 9122959
• 负责人: Kathryn Katsue Walder
• 金额: $ 3.42万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9122959
• 关键词: Action Potentials; Acute; Affect; Affinity; Age; Amino Acids; Ankyrins; Antibodies; Auditory; Axon; Binding; Biochemical; Biochemistry; Biological Assay; Biology; Bipolar Disorder; Brain; Breeding; Calcium/calmodulin-dependent protein kinase; Catalytic Domain; Cell Culture Techniques; Cells; Cellular biology; Clozapine; Code; Complementary DNA; Designer Drugs; Development; Disease; Environment; Epigenetic Process; Exons; Future; GTP-Binding Proteins; Gel Chromatography; Genes; Genetic; Genetic Predisposition to Disease; Goals; Health; Hippocampus (Brain); Human; Immune Sera; Immunohistochemistry; Intellectual functioning disability; Investigation; Laboratories; Length; Light; Literature; LoxP-flanked allele; Mass Spectrum Analysis; Measures; Medical; Mental disorders; Methods; Molecular; Mus; Mutation; Negative Staining; Neurologic; Neurons; Oxides; Peptides; Pharmaceutical Preparations; Phospho-Specific Antibodies; Phosphorylation; Phosphotransferases; Population; Post-Translational Protein Processing; Potassium Channel; Prosencephalon; Protein Isoforms; Proteins; RNA Splicing; Reagent; Recruitment Activity; Regulation; Reporting; Research; Role; Schizophrenia; Scientist; Sedimentation process; Signal Transduction; Site; Sodium Channel; Specificity; Spectrin; Stimulus; Synapses; System; Techniques; Testing; Therapeutic; Training; Transgenic Mice; Universities; Variant; Vertebrates; Work; Zebrafish; base; biophysical properties; calmodulin-dependent protein kinase II; casein kinase II; cell type; cost; developmental neurobiology; disability; genetic manipulation; in vivo; innovation; knockout animal; mimetics; nervous system disorder; neurofascin; neuron development; neuronal circuitry; novel; physical property; polypeptide; promoter; public health relevance; receptor; recombinase; research study; voltage

 DESCRIPTION (provided by applicant): Neurological diseases and psychiatric illnesses affect more than 25% of the US population and annual medical costs are estimated at an excess of $558 billion. Genetic susceptibility to these disabilities has been noted for decades; however, many of these disorders have contributions from multiple factors including genetics, epigenetics, and the environment. In order to continue the development of our understanding of these diseases, we must understand the basic biology of the neurological system. Little is known about regulation of neuronal circuits and even less is known about the biology of the axon initial segment. The axon initial segment is a critical domain for the integration of 1000s of neurons and the most likely origin of action potentials. Recently, 480-kDa Ankyrin-G was established as the master organizer of this domain. This protein is formed by the inclusion of an alternatively spliced giant neuron-specific 7.6kb exon which is also known to be the site of many mutations associated with various neurological and psychiatric diseases including intellectual disability, bipolar disorder, schizophrenia. Using novel techniques,480-kDa Ankyrin-G has been shown to be phosphorylated at high levels in the neuronal-specific 7.6kb region. S2417 is one of these sites of high phosphorylation, and is critical for recruiting its known binding partner beta-4 spectrin to the axon initial segment. The overall goal of this proposal is to investigate the role f phosphoregulation of S2417 in regulating the axon initial segment. Having demonstrated that S2417 is phosphorylated in vivo, Specific Aim 1 tests the hypothesis that S2417 phosphorylation is regulated. First, Aim1 examines the effects of development and neuronal activity on phosphorylation in the forebrain using novel transgenic mice that expresses GFP tagged Ankyrin-G polypeptides under the control of Cre-recombinase as well as the subcellular localization of phosphorylated 480-kDa Ankyrin-G using a phospho-specific antibody against S2417. Second, Aim 1 examines the contribution of Casein kinase 2 to the phosphorylation of S2417 using genetic manipulation of the kinase in cultured neurons. Next, Aim 2 tests the hypothesis that phosphorylation of S2417 changes the physical properties of the axon initial segment by investigating intramolecular changes to 480-kDa Ankyrin-G by measuring biophysical properties. Finally, Aim 2 examines downstream consequences from loss of beta-4 spectrin at the axon initial segment using neuronal cell culture assays. This project is innovative in that it 1) is the first study of molecular regulation of the axon initial segment by focusing on 480-kDa Ankyrin-G and 2) this study utilizes novel techniques and reagents. This training plan contributes to my long-term objective of being an independent scientist in an academic research setting.

 神经系统疾病和精神疾病影响超过25%的美国人口，每年的医疗费用估计超过5580亿美元。几十年来，人们一直注意到这些残疾的遗传易感性;然而，许多这些疾病都有多种因素的贡献，包括遗传学，表观遗传学和环境。为了继续发展我们对这些疾病的理解，我们必须了解神经系统的基本生物学。对神经元回路的调节知之甚少，对轴突起始段的生物学知之更少。轴突起始段是上千个神经元整合的关键区域，也是动作电位最可能的起源。最近，480-kDa锚蛋白-G被确定为该域的主组织者。这种蛋白质是通过包含一个选择性剪接的巨大神经元特异性7.6kb外显子而形成的，该外显子也是已知的与各种神经和精神疾病相关的许多突变的位点，包括智力残疾、双相情感障碍、精神分裂症。使用新的技术，480-kDa的锚蛋白-G已被证明是在神经元特异性的7.6kb区域的高水平磷酸化。S2417是这些高度磷酸化的位点之一，并且对于将其已知的结合伴侣β-4血影蛋白募集到轴突起始段至关重要。本研究的总体目标是研究S2417的磷酸化调节在调节轴突起始段中的作用。已经证明S2417在体内被磷酸化，特异性目的1测试了S2417磷酸化被调节的假设。首先，Aim 1检查的影响，发展和神经元活动的磷酸化前脑使用新的转基因小鼠，表达GFP标记的锚蛋白-G多肽的控制下的Cre重组酶，以及亚细胞定位的磷酸化480-kDa的锚蛋白-G使用磷酸特异性抗体对S2417。第二，目的1使用在培养的神经元中的激酶的遗传操作来检查酪蛋白激酶2对S2417的磷酸化的贡献。接下来，目的2测试的假设，即S2417的磷酸化改变轴突初始段的物理特性，通过研究分子内的变化，以480-kDa的锚蛋白-G通过测量生物物理特性。最后，目标2使用神经元细胞培养试验检查了轴突初始段β-4血影蛋白损失的下游后果。这个项目是创新的 因为它1）是第一个研究轴突起始段的分子调节， 480-kDa锚蛋白-G和2）这项研究利用新的技术和试剂。这个培训计划有助于我成为学术研究环境中的独立科学家的长期目标。