Mechanistic Basis for Regulation of the Axon Initial Segment

轴突初始段调节的机制基础

基本信息

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
项目类别：
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

项目摘要

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.

描述（由申请人提供）：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 5,580 亿美元，但几十年来人们已经注意到这些疾病的遗传易感性；疾病受到多种因素的影响，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。关于轴突起始段的生物学知之甚少。轴突起始段是数千个神经元整合的关键域，也是动作电位最可能的起源。最近，480 kDa Ankyrin-G 被确定为轴突起始段。该蛋白由可变剪接的巨神经元特异性 7.6kb 外显子形成，该外显子也被认为是与各种神经和精神疾病（包括智力疾病）相关的许多突变的位点。使用新技术，480-kDa Ankyrin-G 已被证明在神经元特异性 7.6kb 区域中被高水平磷酸化，是这些高磷酸化位点之一，并且对于招募至关重要。其已知的结合伙伴 β-4 血影蛋白与轴突起始段的结合本提案的总体目标是研究 S2417 的磷酸调节在调节轴突初始段中的作用。证明 S2417 在体内被磷酸化后，Specific Aim 1 测试了 S2417 磷酸化受到调节的假设。首先，Aim1 使用表达 GFP 标记的锚蛋白的新型转基因小鼠检查了发育和神经元活动对前脑磷酸化的影响。 Cre重组酶控制下的-G多肽以及磷酸化的亚细胞定位使用针对 S2417 的磷酸化特异性抗体进行 480-kDa 锚蛋白-G 其次，目标 1 通过对培养神经元中的激酶进行基因操作来检查酪蛋白激酶 2 对磷酸化的贡献。 S2417 通过测量 480-kDa Ankyrin-G 的分子内变化来改变轴突初始段的物理特性最后，目标 2 使用神经细胞培养测定检查轴突起始段 β-4 血影蛋白丢失的下游后果。因为它 1) 是第一个通过关注轴突起始段分子调控的研究 480-kDa Ankyrin-G 和 2) 这项研究采用了新颖的技术和试剂。该培训计划有助于实现我成为学术研究环境中的独立科学家的长期目标。