Role of VRK2 Kinase in Brain Development and Function

项目总结/摘要 本项目的目的是研究Vrk 2激酶在斑马鱼中的作用。基因组研究 将VRK 2与精神分裂症、双相情感障碍、睡眠、重度抑郁症等联系起来。这些众多 相关性表明它可能是神经精神疾病易感性的一个关键因素。但它 目前还不清楚VRK 2在脊椎动物大脑中的功能。初步研究导致了创造和 Vrk 2斑马鱼突变体的初步表征。这些突变体改变了睡眠行为， 活动，特别是在涉及睡眠的下丘脑区域。对于目标1，我们将扩大 这些突变体的细胞，发育和行为差异的表征。我们将进一步 探索他们的睡眠异常，如评估剥夺后的稳态和昼夜节律反应， 如黑暗条件下的挑战。确定vrk 2动作的时间和位置， 行为结果是目标1的第二个目标。我们假设vrk 2突变表型是由于 缺乏其直接靶点的磷酸化。因此，在目标2中，我们建议试行几项战略， 在斑马鱼中发现这些目标。方法的选择将取决于我们的调查， 当正常行为和大脑活动需要vrk 2功能时（目标1），并且包括多个备份 因此，磷酸化蛋白质组学可能具有挑战性。建议的方法包括开发一种类似物- Vrk 2的敏感变体仅明确标记Vrk 2的直接底物用于随后的免疫沉淀。 变异体和ATP类似物将被注射到单细胞胚胎中或暴露于纯化的斑马鱼中 大脑裂解物，如果vrk 2在早期胚胎中发挥作用，影响后来的大脑发育，或者如果我们得出结论， 可能会直接诱导参与睡眠信号传导的蛋白质磷酸化。这些目标将提供洞察力， vrk 2调控的分子、细胞、发育和行为过程。定义的功能 增加神经精神疾病风险的基因将为了解其原因提供基础 并开发新的诊断和治疗方法。

PROJECT SUMMARY/ABSTRACT The goal of this project is to investigate the roles of Vrk2 kinase in zebrafish. Genomic studies have linked VRK2 to schizophrenia, bipolar disorder, sleep, major depression, and more. These numerous associations indicate that it may be a key factor underlying predisposition to neuropsychiatric disorders. Yet it remains unclear how VRK2 is functioning in the vertebrate brain. Preliminary studies led to the creation and initial characterization of vrk2 zebrafish mutants. These mutants had altered sleep behavior and reduced brain activity, particularly in areas of the hypothalamus that are implicated in sleep. For Aim 1, we will expand the characterization of the cellular, developmental, and behavioral differences in these mutants. We will further explore their sleep abnormalities, such as assessing homeostasis after deprivation and circadian response to challenges such as dark-only conditions. Identifying the timing and location of vrk2 action that drive these behavioral outcomes is the second goal of Aim 1. We hypothesize that vrk2 mutant phenotypes result from absent phosphorylation of its direct targets. Therefore, in Aim 2 we propose to pilot several strategies for discovering these targets in zebrafish. The choice of method will depend on our investigation of where and when vrk2 function is required for normal behavior and brain activity (Aim 1) and includes multiple backup strategies, as phosphoproteomics can be challenging. Proposed approaches include developing an analog- sensitive variant of Vrk2 to explicitly label only direct substrates of Vrk2 for subsequent immunoprecipitation. The variant and ATP analog will either be injected into single-cell embryos or exposed to purified zebrafish brain lysate, if vrk2 has a role in early embryos that influences later brain development or if we conclude that it likely induces direct phosphorylation of proteins involved in sleep signaling. These aims will provide insight into the molecular, cellular, developmental and behavioral processes regulated by vrk2. Defining the function of genes that increase risk for neuropsychiatric disorders will provide the foundation to understand their causes and develop new diagnostics and therapies.