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

阿尔茨海默病(AD)是一种进行性、致命性疾病，是最常见的神经退行性疾病 痴呆症的原因。除了年龄增长，还有一些已知的遗传风险因素与阿尔茨海默病有关。的 已确定的遗传风险因素，34 kDa蛋白，载脂蛋白(Apo)E4，具有重要意义，因为 ApoE4携带者占所有AD病例的65%-80%。虽然载脂蛋白E4在脂蛋白中起正常作用 转运，它是如何在AD发病机制中起作用的，目前尚不清楚。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)可以运输到细胞核，导致毒性和 炎症基因在BV2小胶质细胞中的表达。这项提案的目标是扩大这些发现 在体内，通过评估这种片段可能导致毒性、发育的机制 斑马鱼模型系统中的异常和行为缺陷。目标1中描述的实验 将严格检验nApoE41-151可能增加48例外源性治疗后死亡率的假设 受精后数小时(HPF)的斑马鱼胚胎。平行实验还将评估贩运 荧光共聚焦显微镜显示，nApoE41-151定位于神经系统内的细胞核。此外, 将根据明确定义的经验来源对发育异常进行评估 标准。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，包括 心血管系统。最后，使用该片段治疗后的tau病理检查可能 提供载脂蛋白E4与人类AD大脑中发现的一种标志性分子病理之间的联系。实验 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性影响 斑马鱼通过检查运动行为和记忆完整性。记忆和运动的行为测试 功能(通过甩尾和T迷宫评估)将在nApoE41-151治疗后进行评估。最后，在 目的3.我们将确定内源性napoE41-151在体内的潜在有害影响。 斑马鱼。为此，我们建议培育一种新的表达nApoE41-151的斑马鱼品系，以确定 低水平慢性表达是否会导致类似的发育和行为障碍 外源性治疗。为了实现这一目标，我们将使用Tol2转座酶方法， 将表达nApoE41-151片段。这些实验的目标将是产生一个永久性的 可永久繁殖的转基因斑马鱼品系，以充分研究其发育效应 胚胎和幼年斑马鱼，如果有必要，也可以在成年斑马鱼中继续 总体而言，我们正在提出一个体内模型系统来扩展我们的体外发现，并获得更强大的， 可以推断出包括人类在内的完整有机体的可靠数据。斑马鱼正在成为一种 有效的体内模型系统用于研究AD的原因有很多，包括他们的神经解剖学和 神经化学通路与人脑有很大的相似之处。此外，它们还表现出了一种相对 简单的神经系统和胚胎的光学透明度使器官系统和 大脑发育。为了加强我们对斑马鱼的研究，我们还将利用相对容易的 在斑马鱼中表达人类转基因基因，以评估在更生理的环境中潜在的长期影响 表达ApoE4的这个氨基末端片段。使用这样的模型，我们假设nApoE41- 151碎片将显示出显著的发育异常、运动障碍和记忆 这将进一步支持该片段在与AD相关的病因学中的新作用。

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.