PHOSPHORYLATION CONTROL--TAU NEUROTOXICITY IN DROSOPHILA

磷酸化控制--TAU在果蝇中的神经毒性

• 批准号: 6819671
• 负责人: MEL B FEANY
• 金额: $ 14.32万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6819671
• 关键词: Alzheimer' s disease; Drosophilidae; SDS polyacrylamide gel electrophoresis; dementia; fluorescence resonance energy transfer; genetic screening; genetically modified animals; green fluorescent proteins; immunocytochemistry; in situ hybridization; neural degeneration; neurofibrillary tangles; neurotoxicology; neurotoxins; phosphoprotein phosphatase; phosphorylation; polymerase chain reaction; progressive supranuclear palsy; protein kinase; protein structure function; site directed mutagenesis; tau proteins; terminal nick end labeling; western blottings

Tau is the major protein component of the characteristic intracellular protein aggregate of Alzheimer's disease and related disorders, the neurofibrillary tangle. Tau deposited in neurofibrillary tangles is markedly hyperphosphorylated, but the role of phosphorylation in influencing tau neurotoxicity has been difficult to dissect in conventional experimental systems. In preliminary experiments, we provide two lines of evidence supporting a critical role for phosphorylation in controlling tau toxicity in a transgenic Drosophila model of tauopathy. First, kinases and phosphatases comprise the major category of genetic modifiers identified in a forward genetic screen for modifiers of tau toxicty. These kinase and phosphatase modifiers include several enzymes known to phosphorylate or dephosphorylate tau in vitro, as well as novel kinases. Selective candidate testing also reveals that overexpression of the known tau kinases MARK, protein kinase A, and CDK5 enhances tau toxicity. Second, mutation of 14 proline-directed serine or threonine phosphorylation sites on tau, including the AT8, AT100, TG3, and PHF-1 sites, markedly attenuates tau toxicity. We now propose to investigate the role of proline-directed phosphorylation in determining tau neurotoxicity in detail. In Specific Aim 1 we will define the phosphorylation sites required for tau neurotoxicity by mutating sites individually or in small groups. These experiments will reveal if any individual phosphorylation site exerts a significant influence on tau toxicity, or if sites work primarily in concert. When the phosphorylation site(s) controlling tau toxicity have been defined, we will confirm the role of phosphorylation in determining toxicity by altering these site(s) to negatively charged amino acids to mimic phosphorylation. In Specific Aim 2 we will determine if the kinase modifiers we have identified through our genetic screens and candidate testing act on specific phosphorylation sites by determining if kinase modifiers are able to alter the toxicity of phosphorylation site mutant tau. Finally, in Specific Aim 3 we will investigate the role of our novel kinase modifiers in human neurodegenerative diseases characterized by deposition of abnormally phosphorylated and aggregated tau, including Alzheimer's disease, Pick's disease, and progressive supranuclear palsy.

Tau 蛋白是阿尔茨海默病和相关疾病的特征性细胞内蛋白聚集体（神经原纤维缠结）的主要蛋白成分。沉积在神经原纤维缠结中的 tau 蛋白明显过度磷酸化，但磷酸化在影响 tau 神经毒性中的作用在传统实验系统中很难剖析。在初步实验中，我们提供了两条证据，支持磷酸化在转基因果蝇 tau 病模型中控制 tau 毒性中的关键作用。首先，激酶和磷酸酶构成了遗传修饰剂的主要类别。 tau 毒性修饰因子的正向遗传筛选。这些激酶和磷酸酶修饰剂包括几种已知可在体外磷酸化或去磷酸化 tau 的酶，以及新型激酶。选择性候选测试还表明，已知 tau 激酶 MARK、蛋白激酶 A 和 CDK5 的过度表达会增强 tau 毒性。其次，tau 上 14 个脯氨酸定向的丝氨酸或苏氨酸磷酸化位点（包括 AT8、AT100、TG3 和 PHF-1 位点）的突变可显着减弱 tau 毒性。我们现在建议详细研究脯氨酸定向磷酸化在确定 tau 神经毒性中的作用。在具体目标 1 中，我们将通过单独或小组突变位点来定义 tau 神经毒性所需的磷酸化位点。这些实验将揭示是否有任何单个磷酸化位点发挥了 对 tau 毒性有重大影响，或者位点是否主要协同工作。当控制 tau 毒性的磷酸化位点被确定后，我们将通过将这些位点改变为带负电荷的氨基酸来模拟磷酸化，从而确认磷酸化在确定毒性中的作用。在具体目标 2 中，我们将通过确定激酶修饰剂是否能够改变磷酸化位点突变 tau 的毒性来确定我们通过遗传筛选和候选测试鉴定的激酶修饰剂是否作用于特定磷酸化位点。最后，在具体目标 3 中，我们将研究新型激酶修饰剂在以异常磷酸化沉积为特征的人类神经退行性疾病中的作用。 tau 蛋白聚集，包括阿尔茨海默病、皮克病和进行性核上性麻痹。