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（由适用提供）：神经疾病和精神病疾病影响美国人口的25％以上，年度医疗费用估计超过5580亿美元。数十年来已经注意到了对这些疾病的遗传敏感性。但是，其中许多疾病都来自多种因素，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。关于神经元回路的调节，对轴突初始段的生物学知之甚少。轴突初始段是1000 s神经元和最有可能起源电位来源的关键域。最近，建立了480 kDa ankyrin-g作为该领域的主要组织者。该蛋白是由包含替代剪接的巨型神经元特异性7.6kb外显子形成的，该外显子也被称为与各种神经系统和精神病有关的许多突变的部位，包括智力疾病，躁郁症，躁郁症，精神分裂症。使用新型技术，在神经元特异性7.6KB区域中，已显示480 kDa Ankyrin-G在高水平上被磷酸化。 S2417是这些高磷酸化的位点之一，对于募集其已知的结合伴侣β-4光谱蛋白与轴突初始段至关重要。该提案的总体目标是研究S2417在轴突初始段调节中的磷酸化作用。在证明S2417在体内磷酸化后，特定的目标1检验了S2417磷酸化的假设。首先，AIM1使用新型的转基因小鼠检查前脑发育和神经元活性对磷酸化的影响，该小鼠在CRE-结构酶的控制下表达了GFP，并使用480-KDA ANKYRIN-G的磷酸化磷酸化磷酸化体phossy Antibody对CRE-结构酶以及磷酸化的亚细胞定位。其次，AIM 1使用培养神经元中激酶的遗传操纵酪蛋白激酶2对S2417的磷酸化的贡献。接下来，AIM 2检验了以下假设：S2417的磷酸化通过测量生物物理特性，通过研究对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，使用神经元细胞培养测定法，AIM 2考试在轴突初始段中丧失了β-4光谱的下游后果。这个项目是创新的这是1）是对轴突初始段的分子调节的首次研究 480-kDa Ankyrin-g和2）这项研究利用了新技术和试剂。该培训计划有助于我在学术研究环境中成为独立科学家的长期目标。