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.

阿尔茨海默病 (AD) 是一种进行性、致命性的神经退行性疾病，是最常见的疾病除了年龄增长之外，还有已知的与 AD 相关的遗传风险因素。已确定的遗传风险因素，即 34 kDa 蛋白、载脂蛋白 (Apo) E4，具有重要意义尽管 ApoE4 在脂蛋白中发挥正常作用，但 APOE4 携带者占所有 AD 病例的 65-80%。我们实验室的最新数据表明，它如何促进 AD 发病机制目前尚不清楚。在体外，ApoE4 的 151 个氨基末端片段 (nApoE41-151) 可以运输到细胞核，导致毒性和 BV2 小胶质细胞中炎症基因的表达该提案的目标是扩展这些发现。在体内，通过评估该片段可能诱导毒性、发育的机制目标 1 中描述的由斑马鱼组成的模型系统中的异常和行为缺陷。将严格检验 nApoE41-151 可能会增加 48 例患者接受外源性治疗后死亡率的假设受精后数小时（hpf）斑马鱼胚胎的平行实验还将评估是否存在贩运。通过荧光共聚焦显微镜观察，nApoE41-151 进入细胞核发生在神经系统内。将基于明确定义的经验得出的发育异常评估我们将确定 nApoE41-151 是否会导致任何其他器官系统的毒性或变形。最后，用该片段治疗后可检查 tau 病理学。提供 ApoE4 与人类 AD 大脑实验中发现的标志性分子病理学之间的联系。目标 2 中概述的将确定 nApoE41-151 片段可能对青少年产生什么亚致死影响通过检查运动行为以及记忆完整性来对斑马鱼进行记忆和运动行为测试。最后，将在 nApoE41-151 处理后评估功能（通过甩尾和 T 迷宫评估）。目标 3. 我们将确定内源性产生的 nApoE41-151 的潜在有害影响在此目标中，我们建议生成表达 nApoE41-151 的新斑马鱼品系以确定低水平的慢性表达是否会导致类似的发育和行为障碍为了实现这一目标，我们将采用 Tol2 转座酶方法，从而 nApoE41-151 片段将被表达，这些实验的目标是产生永久的。可以永久繁殖的转基因斑马鱼品系，以便充分研究斑马鱼的发育影响胚胎和幼鱼，如果有必要的话，也可以在成年斑马鱼中继续进行总的来说，我们提出了一个体内模型系统来扩展我们的体外研究结果并获得更稳健的、可以推断到包括人类在内的完整生物体的可靠数据正在出现。有效的体内模型系统研究 AD 的原因有很多，包括它们的神经解剖学和神经化学途径与人脑有很强的相似性，此外，它们表现出相对的特征。简单的神经系统和胚胎的光学透明度可以轻松分析器官系统和为了加强我们对斑马鱼的研究，我们还将利用相对容易的优势在斑马鱼中表达人类转基因，以在更生理的环境中评估潜在的长期影响使用这样的模型，我们捕获了 nApoE41- 的氨基末端片段。 151片段将表现出明显的发育异常、运动功能障碍和记忆力损伤将进一步支持该片段在 AD 相关病因学中的新作用。