Mechanistic Basis for Regulation of the Axon Initial Segment

 描述(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，这些疾病中的许多都有多种因素造成，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更是知之甚少。轴突起始段是整合上千个神经元的关键区域，也是最有可能的动作电位来源。最近，480 kDa Ankyrin-G被确定为该领域的主组织者。这种蛋白质是由一个可选择性剪接的巨型神经元特异的7.6kb外显子形成的，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置，包括智力残疾、双相情感障碍、精神分裂症。使用新技术，480 kDa Ankyrin-G已被证明在神经元特异的7.6kb区域高度磷酸化。S2417是这些高度磷酸化的位点之一，对于招募其已知的结合伙伴β-4血影蛋白到轴突起始片段至关重要。本研究的总体目标是研究S2417的磷酸调节在调节轴突起始节段中的作用。在证明S2417在体内被磷酸化后，特殊目的1检验了S2417磷酸化受到调控的假设。首先，Aim1使用在Cre重组酶控制下表达GFP标记的Ankyrin-G多肽的新型转基因小鼠，以及使用针对S2417的磷酸特异性抗体对磷酸化的480 kDa Ankyrin-G的亚细胞定位，研究了发育和神经元活动对前脑磷酸化的影响。第二，Aim 1通过对培养神经元中酪蛋白激酶2的遗传操作，研究了酪蛋白激酶2对S2417磷酸化的影响。接下来，Aim 2通过测量生物物理特性来研究480 kDa Ankyrin-G的分子内变化，从而验证S2417的磷酸化改变轴突起始片段的物理特性的假设。最后，Aim 2使用神经细胞培养分析，研究了轴突起始段β-4血影蛋白缺失对下游的影响。该项目具有创新性。 这是第一次对轴突起始节段的分子调控进行研究。 480 kDa Ankyrin-G和2)本研究使用了新的技术和试剂。这一培训计划有助于我在学术研究环境中成为一名独立科学家的长期目标。

 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.