Development of Zebrafish Taupoathy Models for Translational Research

描述(由申请人提供)：这项工作的长期目标是开发有效的治疗进行性核上性瘫痪(PSP)和皮质基底膜变性(CBD)的方法。这些常见的神经退行性疾病与显著的运动和认知异常有关，预后较差(中位生存期6-8年)，因为目前的治疗方法无法阻止疾病的进展。这两种疾病的特征都是神经元丢失和微管相关蛋白Tau(4R-Tau)4重复亚型的异常聚集在整个中枢神经系统的神经元中积累。人们认为4R-Tau在这些疾病的发病机制中起中心作用：(1)编码Tau的MAPT基因突变(如P301L)可导致PSP和CBD表型；(Ii)PSP和CBD与MAPT基因上的遗传变异密切相关。为了解决在开发靶向Tau积聚的药物及其在体内神经元中的后果方面取得进展的关键障碍，我们建议开发针对药物发现努力、假定治疗药物的快速比较测试以及识别新治疗靶点的研究而优化的斑马鱼共济失调模型。我们的初步研究表明，在中枢神经系统神经元中表达人4R-Tau基因的转基因斑马鱼出现了进行性的神经表型、脑萎缩、Tau过度磷酸化和嗜银沉积，类似于人类的疾病。在这些初步研究中，由于结构性转基因表达，选择了具有早发性表型的转基因品系作为对照。在这个探索性的提案中，我们将使用条件表达式方法来开发增强的斑马鱼共济失调模型，该模型的表型异常发生得足够早，最大限度地有助于药物发现工作和多孔板格式的比较疗效研究。在目标1中，我们将使用基于Gal4/UAS的两部分系统来允许4R-Tau在中枢神经系统神经元中高水平表达，而不会选择建立具有早发性表型的系。我们将建立WT或P301L突变的人4R-Tau在UAS增强子下表达的转基因动物，并使用我们构建的新型Gal4驱动系在中枢神经系统广泛反式激活Tau的表达。在目标2中，我们将确定表达人Tau的斑马鱼幼虫是否有条件地表现出生存能力下降、运动或眼球运动障碍，以及这些表型是否可以用于药物发现测试。在目标3中，我们将确定表达人Tau的斑马鱼幼虫是否有条件地表现出Tau过度磷酸化、神经纤维缠结形成或神经元死亡的证据，以及这是否可以在体内成像。这些新的转基因株系将成为翻译研究的有价值的工具，以开发PSP和CBD的新疗法，以及潜在的其他转化性疾病，如阿尔茨海默病和慢性头部损伤。我们未来的工作将利用这些模型进行药物发现和开发，包括早期发现努力和后来的优化研究，将这些疾病的新治疗方法带入神经学临床。

DESCRIPTION (provided by applicant): The long-term aim of this work is to develop effective treatments for progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). These common neurodegenerative diseases are associated with prominent motor and cognitive abnormalities and have a poor prognosis (median survival 6 - 8 years) because current treatments do not prevent disease progression. Both diseases are characterized by neuronal loss and accumulation of abnormal aggregates of the 4-repeat isoform of the microtubule-associated protein Tau (4R- Tau) in neurons throughout the CNS. It is thought that 4R-Tau is central to the pathogenesis of these disorders: (i) mutations (e.g. P301L) in the MAPT gene encoding Tau can give rise to PSP and CBD phenocopies; (ii) PSP and CBD are strongly associated with genetic variants at the MAPT locus. In order to address a critical barrier to progress in developing drugs to target Tau accumulation and its consequences in neurons in vivo, we propose to develop zebrafish Tauopathy models that are optimized for drug discovery efforts, rapid comparative testing of putative therapeutic agents, and studies to identify novel therapeutic targets. Our preliminary studies showed that transgenic zebrafish expressing a human 4R-Tau transgene in CNS neurons developed progressive neurological phenotypes, brain atrophy, Tau hyperphosphorylation and argyrophilic deposits, similar to the human diseases. In these preliminary studies, transgenic lines with early- onset phenotypes were selected against, because of constitutive transgene expression. In this exploratory proposal, we will employ a conditional expression approach to develop enhanced zebrafish Tauopathy models with phenotypic abnormalities occurring sufficiently early to be maximally useful for drug discovery efforts and comparative efficacy studies in multiwell plate formats. In Aim 1, we will use a bipartite system based on Gal4/UAS to allow expression of 4R-Tau at high levels in CNS neurons, without selecting against the establishment of lines with early-onset phenotypes. We will generate transgenic animals in which WT or P301L mutant human 4R-Tau is expressed under a UAS enhancer, and use novel Gal4 driver lines we have constructed to trans-activate Tau expression widely in the CNS. In Aim 2, we will determine whether zebrafish larvae expressing human Tau conditionally show impaired survival, motor or oculomotor deficits, and whether these phenotypes could be used as drug discovery assays. In Aim 3, we will determine whether zebrafish larvae expressing human Tau conditionally show evidence of Tau hyper-phosphorylation, neurofibrillary tangle formation or neuronal death, and whether this can be imaged in vivo. These novel transgenic lines will be a valuable tool for translational studies to develop new therapies for PSP and CBD, and potentially other Tauopathies such as Alzheimer's disease and chronic head injury. Our future work will exploit these models for drug discovery and development, both early-phase discovery efforts and later optimization studies, to bring new treatments for these disorders into the neurology clinic.