Functional Compartmentalization of Neurons and Gila

描述（由申请人提供）：在阿尔茨海默病（AD）等神经退行性疾病中，tau蛋白家族从与微管的正常关联中移位，形成成对的螺旋细丝（PHF），这是该疾病的标志性细胞骨架病理学。这种病理程度与阿尔茨海默病的临床严重程度密切相关。tau蛋白的几种翻译后修饰，包括磷酸化，与AD的发病机制有关。此外，重要的是，编码人类tau基因的实际突变最近与各种遗传性痴呆有关，统称为与17号染色体相关的帕金森病额颞叶痴呆（FTDP-17）。这重新燃起了人们对tau蛋白在神经退行性疾病中的重要性的兴趣。尽管在tau生物学和神经退行性疾病领域取得了重大进展，但仍有几个重要问题尚未解决。活神经元中tau改变的早期功能后果尚不完全清楚，并且尚不清楚神经退行性疾病中的tau如何从其在神经元轴突中的正常浓度重新分布到神经元体细胞中的病理包涵体，即神经原纤维缠结（NFT）。造成这些知识差距的原因之一是研究这些过程的模型系统相对缺乏。我们建议建立一个包括种系传播在内的转基因模型系统，来研究人类tau蛋白的功能后果和运输模式，这些tau蛋白要么在遗传性痴呆相关位点发生突变，要么在特定的翻译后修饰位点发生改变，并在斑马鱼神经元中表达。在神经元特异性的斑马鱼GATA-2启动子控制下，分子生物学方案将用于制备正常和突变的人类tau绿色荧光蛋白（ht-GFP）构建物。这些构建体在注入斑马鱼胚胎或发育后期鉴定的神经元后表达。细胞健康，发育，tau运输模式和细丝形成将被检查，并与表达的tau突变类型以及表达的tau磷酸化状态相关。总的指导假设是，该模型允许解剖与疾病发展关键早期阶段相关的神经退行性疾病潜在机制相关的事件层次。该模型将被证明在未来的基因阵列分析中非常有用，可以通过实时显微镜分析鉴定出FTDP-17突变体在早期重组的关键时刻打开或关闭特定基因。此外，这里开发的方法将在向活体脊椎动物神经元引入与神经退行性疾病发病机制有关的其他分子的功能后果和潜在遗传分析的研究中具有广泛的用途。

DESCRIPTION (provided by applicant): In neurodegenerative conditions such as Alzheimer's disease (AD), the tau family of proteins are displaced from their normal association with microtubules and form into paired helical filaments (PHF) that are the hallmark cytoskeletal pathology of the disease. The degree of this pathology correlates closely with the clinical severity of AD. Several posttranslational modifications of tau, including phosphorylation, have been implicated in AD pathogenesis. In addition, and importantly, actual mutations in the genes encoding human tau have recently been implicated in a variety of hereditary dementias, collectively termed frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17). This has rekindled interest in the importance of tau in neurodegenerative diseases. Despite significant progress in the field of tau biology and neurodegenerative diseases, several important issues remain unresolved. The early functional consequences of tau alterations in living neurons is incompletely understood, and it is not clear how tau in neurodegenerative diseases becomes redistributed from its normal concentration in neuronal axons to pathological inclusions in neuronal soma known as neurofibrillary tangles (NFT). One of the reasons for these gaps in knowledge is the relative paucity of model systems to study these processes. We propose to develop a transgenic model system, including germ line transmission, to study the functional consequences and trafficking patterns of human tau either mutated on sites associated with hereditary dementias or altered at select posttranslational modification sites and expressed in zebrafish neurons. Molecular biological protocols will be used to prepare normal and mutated human tau green fluorescent protein (ht-GFP) constructs under control of the neuron specific zebrafish GATA-2 promoter. The constructs are expressed after microinjection into zebrafish embryos or identified neurons later in development. Cell health, development, tau trafficking patterns and filament formation will be examined and related to both the type of tau mutation being expressed as well as to the expressed tau phosphorylation status. The overall guiding hypothesis is that the model allows dissection of a hierarchy of events relevant to potential mechanisms of neurodegenerative diseases related to critical early stages in development of disease. The model should prove very useful as well in future gene array assays to identify specific genes turned on or off at critical times in the early reorganization of FTDP-17 mutant taus identified from real time microscopic analyses. In addition, the approaches developed here will have broad usefulness in the study of functional consequences and potential genetic analyses of introducing into living vertebrate neurons other molecules involved in the pathogenesis of neurodegenerative diseases.