Regulation of the DYRK1A kinase by the Down Syndrome Cell Adhesion Molecule DSCAM

摘要：候选与环境：福尔斯特实验室将进行遗传学、分子和形态研究 爱达荷大学与这项提案的目标相关的实验。福斯特实验室团队拥有广泛的 专业知识集中在神经发育的细胞生物学和遗传学方面。福尔斯特认为Dscam是第一个 调节神经元镶嵌组织的分子，并开发了许多用于 这些实验。在这里，我们将建立在我们研究DSCAM基因的经验的基础上来探索 下游信号网络在神经发育中的作用。研究建议：发育中的神经元 利用一系列分子线索组织成神经组织和器官。我们和其他人之前 据报道，唐氏综合征细胞黏附分子(Dscam)基因是糖尿病正常发育所必需的 神经组织。Dscam基因如何在单一细胞类型内调节广泛的细胞反应 一个活跃的研究领域。在我们的初步研究中，我们发现Dyrk1a的过度表达，其产物 与DSCAM发生生化相互作用，表观复制DSCAM功能丧失。此外，我们报告了一个重新分配的 细胞DYRK1A蛋白在DSCAM视网膜和脑功能丧失中的作用基于这些观察和 我们假设基因在促进或抑制发育细胞死亡中的已知拮抗作用 DSCAM在细胞表面的相互作用通过控制其亚细胞来拮抗DYRK1A的活性 本地化。我们从两个具体的目标来检验这一假设。目标1：先导遗传学研究表明， 减少Dscam剂量或增加Dyrk1a剂量会导致视网膜神经元突起的错误定位。一个 Ts65Dn三体小鼠模型中Dyrk1a表达的类似增加导致野生型靶向 然而，表型表明唐氏综合征临界区(DSCR)的一个或多个因素是 补偿Dyrk1a的过度表达。首先，我们将测试在Ts65Dn中是否增加Dyrk1a表达式 小鼠模型中其他DSCR基因的表达足以恢复Dyrk1a的过度表达 轴突靶向缺陷(目标1A)。第二，我们将测试是否将Dscam在Ts65Dn小鼠中的表达降低到野生 类型水平恢复Dyrk1a过表达表型(Aim 1B)。目标2：我们将扩大我们的试点研究 在视网膜中产生，进入整个大脑。我们将测试DYRK1A本地化是否在 大脑(目标2A)，如果DSCAM调节DYRK1A在大脑中的定位(目标2B)，如果Dscam的表达是 足以改变DYRK1A在体外的积累或定位(目标2C)。 长期目标：我们的长期目标是了解细胞表面的事件是如何传递到 产生不同的细胞反应。本R03将有助完成及发表一项先导研究 其结果将为实验提供信息，以了解DYRK1A本地化的调节如何影响其 在发育和疾病中的作用。 意义：了解为什么基因突变或过度表达与神经发育有关 能够呈现出各种体征和症状的人，也需要我们了解其功能 以开发临床干预措施为长期目标的受影响基因。在这里，我们将探索互动 唐氏综合症关键区两个相互作用的基因之间的相互作用，与研究和治疗相关 关注两个基因中的任何一个。了解表面蛋白利用的下游信号通路 受体将是帮助科学家和临床医生准确确定治疗人类疾病的策略的关键。

ABSTRACT: Candidate and Environment: The Fuerst Lab will conduct genetic, molecular and morphological experiments related to the Aims of this proposal at the University of Idaho. The Fuerst lab team has extensive expertise centered on the cell biology and genetics of neural development. Fuerst identified Dscams as the first molecules that regulate mosaic organization of neurons and developed many of the genetic reagents for use in these experiments. Here we will build on our foundation of experience studying Dscam genes to probe the function of downstream signaling networks in neural development. Research Proposal: Developing neurons utilize a range of molecular cues to organize into neural tissues and organs. We and others have previously reported that Down syndrome cell adhesion molecule (Dscam) genes are required for normal development of neural tissues. How Dscam genes mediate a wide range of cellular responses even within a single cell type is an active area of research. In our preliminary studies we found that overexpression of Dyrk1a, whose product interacts biochemically with DSCAM, phenocopies Dscam loss of function. Further we report a redistribution of cellular DYRK1A protein in the Dscam loss of function retina and brain. Based on these observations and the genes’ known antagonistic roles in either promoting or inhibiting developmental cell death, we hypothesize that DSCAM interactions on the cell surface antagonize DYRK1A activity by controlling its subcellular localization. We test this hypothesis in two specific Aims. Aim 1: Pilot genetic studies indicate that either decreasing Dscam dosage or increasing Dyrk1a dosage results in mistargeting of retinal neuron neurites. A similar increase in Dyrk1a expression in the Ts65Dn trisomic mouse models results in a wild type targeting phenotype, however, suggesting a factor or factors in the Down syndrome critical region (DSCR) are compensating for Dyrk1a overexpression. First, we will test if increasing Dyrk1a expression in the Ts65Dn mouse model above the expression of other DSCR genes is sufficient to restore Dyrk1a overexpression neurite targeting defects (Aim 1A). Second, we will test if reducing Dscam expression in Ts65Dn mice to wild type levels restores the Dyrk1a overexpression phenotype (Aim 1B). Aim 2: We will extend our pilot study generated in retina into the brain at large. We will test if DYRK1A localization is developmentally dynamic in the brain (Aim 2A), if DSCAM regulates DYRK1A localization in the brain (Aim 2B) and if Dscam expression is sufficient to modify DYRK1A accumulation or localization in vitro (Aim 2C). Long-term goals: Our long-term goal is to understand how events at the cell surface are transduced to generate different cellular responses. This R03 will facilitate completion and publication of a pilot study, the results of which will inform experiments to understand how regulation of DYRK1A localization impacts its function in development and disease. Significance: Understanding why mutation or overexpression of genes involved in neural development can present with a variety of signs and symptoms in people will require us to also understand the functions of the affected genes with the long-term goal of developing clinical interventions. Here we explore the interaction between two interacting genes in the Down Syndrome critical region, relevant to studies and treatments focusing on either single gene. Understanding the downstream signaling pathways utilized by surface receptors will be essential to help scientists and clinicians pinpoint strategies to treat human diseases.