Functional Compartmentalization of Neurons and Gila

描述（由申请人提供）：在神经退行性疾病如阿尔茨海默病（AD）中，tau蛋白家族从其与微管的正常结合中被置换，并形成成对的螺旋丝（PHF），这是该疾病的标志性细胞骨架病理学。这种病理学的程度与AD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发病机制有关。此外，重要的是，编码人类tau蛋白的基因中的实际突变最近与各种遗传性痴呆有关，这些遗传性痴呆统称为与17号染色体相关的额颞叶痴呆伴帕金森综合征（FTDP-17）。这重新点燃了人们对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。tau蛋白在活神经元中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蛋白如何从其在神经元轴突中的正常浓度重新分布到被称为神经元缠结（NFT）的神经元索马中的病理性内含物。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。我们建议开发一个转基因模型系统，包括生殖系传输，研究人类tau蛋白的功能后果和贩运模式，无论是在与遗传性痴呆相关的位点上突变，还是在选择的翻译后修饰位点上改变，并在斑马鱼神经元中表达。分子生物学方案将用于制备在神经元特异性斑马鱼加塔-2启动子控制下的正常和突变的人tau绿色荧光蛋白（ht-GFP）构建体。这些构建体在显微注射到斑马鱼胚胎或发育后期鉴定的神经元中后表达。将检查细胞健康、发育、tau运输模式和细丝形成，并将其与所表达的tau突变类型以及所表达的tau磷酸化状态相关。总体指导假设是，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结构。该模型应证明是非常有用的，以及在未来的基因阵列检测，以确定特定的基因打开或关闭的关键时刻，在早期重组的FTDP-17突变体taus确定从真实的时间显微镜分析。此外，这里开发的方法将有广泛的用途，在研究的功能后果和潜在的遗传分析引入活的脊椎动物神经元参与神经退行性疾病的发病机制的其他分子。

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.