Examining the neurobehavioral and toxic effects of an amino-terminal fragment of ApoE4 in zebrafish

检查 ApoE4 氨基末端片段对斑马鱼的神经行为和毒性作用

基本信息

批准号：
10511272
负责人：
TROY T ROHN
金额：
$ 39.31万
依托单位：
BOISE STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-06-01 至 2025-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10511272
关键词：
Adolescent Adult Alleles Alzheimer&apos s Disease Alzheimer&apos s disease brain Alzheimer&apos s disease risk Amino Acids Animals Apolipoprotein E Behavior Behavioral Biological Biological Assay Biological Models Brain CRISPR/Cas technology Cardiovascular Diseases Cardiovascular system Cell Nucleus Cells Chronic Confocal Microscopy Data Defect Dementia Development Disease Progression Elderly Embryo Etiology Exhibits Fertilization Fishes Fluorescence Funding Gelatinase B Gene Expression Gene Transfer Techniques Genes Goals Growth Heart Heart Rate Hour Human Impairment In Vitro Inflammation Inflammatory Knock-in Late Onset Alzheimer Disease Learning Length Link Lipoproteins Locomotion Long-Term Effects Memory Memory impairment Methods Microglia Modeling Molecular Profiling Morphology Motor Mus Nervous system structure Neurodegenerative Disorders Neurofibrillary Tangles Neurologic Neurons Optics Organism PHF-1 Pathogenesis Pathology Pathway interactions Pattern Peptide Hydrolases Physiological Play Positioning Attribute Proteins Risk Risk Factors Rodent Role Scheme Spinal Cord Swimming Tail Testing Time Tissue-Specific Gene Expression Toxic effect Transgenes Transgenic Organisms Transposase Validation Zebrafish apolipoprotein E-4 base behavior test behavioral impairment body system cardiovascular risk factor cell motility dementia risk experimental study extracellular genetic risk factor hatching in vitro Assay in vivo in vivo Model molecular pathology mortality motor behavior motor disorder motor impairment neurobehavioral neurochemistry neuromuscular function novel tau Proteins tool trafficking young adult

项目摘要

Alzheimer’s disease (AD) is a progressive, fatal disorder neurodegenerative disease that is the most common cause of dementia. In addition to advancing age, there are known genetic risk factors associated with AD. Of the genetic risk factors identified, the 34 kDa protein, apolipoprotein (Apo) E4, is of significance importance as APOE4 carriers account for 65-80% of all AD cases. Although ApoE4 plays a normal role in lipoprotein transport, how it contributes to AD pathogenesis is currently unknown. Recent data from our lab suggests that, in vitro, a 151 amino-terminal fragment of ApoE4 (nApoE41-151) can traffic to the nucleus leading to toxicity and expression of inflammatory genes in BV2 microglia cells. The goal of this proposal is to expand those findings in vivo, by assessing the mechanisms by which this fragment may induce toxicity, developmental abnormalities, and behavior deficits in a model system consisting of zebrafish. Experiments described in Aim 1 will rigorously test the hypothesis that nApoE41-151 may increase mortality following exogenous treatment in 48 hours post-fertilization (hpf) zebrafish embryos. Parallel experiments will also assess whether trafficking of nApoE41-151 to the nucleus occurs within the nervous system by fluorescence confocal microscopy. In addition, an assessment of developmental abnormalities will be undertaken based on well-defined empirically derived criteria. We will determine if nApoE41-151 leads to toxicity or deformation of any other organ system including the cardiovascular system. Finally, examination for tau pathology following treatment with this fragment could provide a link between ApoE4 and a signature molecular pathology found in the human AD brain. Experiments outlined in Aim 2 will determine what sublethal effects the nApoE41-151 fragment may have on juvenile zebrafish by examining motor behavior as well as memory integrity. Behavior tests for memory and motor functions (assessed by tail flicking and T-maze) will be assessed following treatment of nApoE41-151. Finally, in Aim 3. we will determine the potential deleterious effects of endogenously generated the nApoE41-151 in zebrafish. In this Aim, we propose to generate a new zebrafish strain that expresses nApoE41-151 to determine whether low-level chronic expression leads to similar developmental and behavioral impairments as exogenous treatment. To accomplish this Aim, we will employ a Tol2 transposase method whereby the nApoE41-151 fragment will be expressed. The goal of these experiments will be to generate a permanent transgenic zebrafish line that can be bred in perpetuity in order to fully study the developmental effects in embryos and young juveniles but also continuing in adult zebrafish if warranted Overall, we are proposing an in vivo model system to extend our in vitro findings and to obtain more robust, reliable data that can be extrapolated to an intact organism including humans. Zebrafish are emerging as an effective in vivo model system to study AD for many reasons including the fact that their neuroanatomic and neurochemical pathways share strong similarities with the human brain. In addition, they exhibit a relatively simple nervous system and optical transparency of embryos permit for easy analyses of organ systems and brain development. To strengthen our zebrafish studies, we will also take advantage of the relative ease to express human transgenes in zebrafish to assess in a more physiological milieu the potential long-term effects of expressing this amino-terminal fragment of ApoE4. Using such a model, we hypothesize that the nApoE41- 151 fragment will demonstrate significant developmental abnormalities, motor dysfunction, and memory impairments that will further support a novel role of this fragment in the etiology associated with AD.

阿尔茨海默氏病（AD）是一种进行性致命疾病神经退行性疾病，是最常见的痴呆症原因。除了增长年龄外，还有与AD相关的已知遗传危险因素。的鉴定出34 kDa蛋白载脂蛋白（APO）E4的遗传危险因素具有重要意义 APOE4载体占所有广告案例的65-80％。尽管APOE4在脂蛋白中起正常的作用运输，目前未知对AD发病机理的贡献。我们实验室的最新数据表明，在体外，APOE4的151个氨基末端片段（Napoe41-151）可以传播到核导致毒性和 BV2小胶质细胞中炎症基因的表达。该提议的目的是扩大这些发现在体内，通过评估该碎片可能诱导毒性的机制，发育异常和行为在由斑马鱼组成的模型系统中定义。 AIM 1中描述的实验将严格检验以下假设：napoe41-151可能会在48中增加外源治疗后死亡率施肥后小时（HPF）斑马鱼胚胎。并行实验还将评估贩运 Napoe41-151到核通过荧光共聚焦显微镜在神经系统内发生。此外，根据定义明确的经验得出的发育异常评估标准。我们将确定Napoe41-151是否导致任何其他器官系统的毒性或变形心血管系统。最后，对使用此片段治疗后的tau病理学检查可以在人类AD大脑中发现的APOE4与APOE4和一个签名分子病理之间提供联系。实验在AIM 2中概述将决定Napoe41-151片段对少年有什么影响斑马鱼通过检查运动行为以及记忆完整性。记忆和电机的行为测试在治疗NAPOE41-151后，将评估功能（通过尾部闪烁和T迷宫评估）。最后，在 AIM 3。我们将确定内生产生Napoe41-151的潜在有害影响斑马鱼。在此目的中，我们建议生成一种新的斑马鱼菌株，该斑马鱼表达napoe41-151以确定低级慢性表达是否会导致类似的发育和行为障碍外源治疗。为了实现这一目标，我们将采用一种TOL2转座酶方法 Napoe41-151片段将被表达。这些实验的目的是生成永久性可以永久繁殖的转基因斑马鱼线，以便充分研究胚胎和年轻少年，但如果有必要总体而言，我们提出了一个体内模型系统，以扩展我们的体外发现并获得更健壮的发现，可靠的数据可以推断到包括人类在内的完整生物。斑马鱼正在作为一个有效的体内模型系统研究AD的原因有很多，包括其神经解剖学和神经化学途径与人脑有着强烈的相似性。此外，他们暴露了一个相对的简单的神经系统和胚胎的光学透明度允许可轻松分析器官系统和大脑发育。为了加强我们的斑马鱼研究，我们还将利用相对轻松的在斑马鱼中表达人类转基因，以在更物理的环境中评估潜在的长期影响表达APOE4的这种氨基末端片段。使用这样的模型，我们假设Napoe41- 151片段将显示出明显的发育异常，运动功能障碍和记忆力将进一步支持该片段在与AD相关的病因中的新作用的障碍。