Investigating the functional consequences of seeding in mammalian primary neurons with Alzheimer’s Disease brain-derived tau filaments

研究在哺乳动物原代神经元中接种阿尔茨海默病脑源性 tau 丝的功能后果

• 批准号: 10649456
• 负责人: Rebecca Lynn Mueller
• 金额: $ 1.97万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-18 至 2023-11-17
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10649456
• 关键词: Address; Alzheimer' s Disease; Alzheimer' s disease brain; Alzheimer' s disease therapy; Automobile Driving; Axon; Axonal Transport; Biochemical; Biological Assay; Biology; Brain; Cells; Confocal Microscopy; Dependence; Development; Disease; Disease Progression; Epitopes; Exhibits; Experimental Designs; Genes; Goals; Hand; Healthcare; Hippocampus; Human; Immunologics; Impairment; In Vitro; Kinesin; Knock-in; Knock-in Mouse; Knowledge; Light; Measurement; Measures; Mediating; Microscopy; Modeling; Molecular; Molecular Conformation; Molecular Motors; Motor; N-terminal; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurons; Pathologic; Pathology; Pathway interactions; Pattern; Phosphoric Monoester Hydrolases; Phosphorylation; Play; Prevalence; Process; Protein phosphatase; Proteins; Role; Sampling; Signal Pathway; Squid; Stains; Synapses; Tauopathies; Techniques; Tertiary Protein Structure; Testing; Therapeutic; Toxic effect; Training; Viral Vector; Virus; Work; axonal degeneration; effective therapy; fast axonal transport; glycogen synthase kinase 3 beta; insight; knock-down; mouse model; mutant; non-demented; novel; pre-formed fibril; prion-like; protein activation; protein aggregation; small hairpin RNA; squid axon; tau Proteins; tau aggregation; therapeutic target; tool

Project Summary/Abstract The prevalence of Alzheimer’s disease (AD), a progressive neurodegenerative disease, continues to increase at a staggering rate. Current AD therapies provide modest symptomatic relief, creating a need for disease- modifying therapies, which requires a better understanding of the mechanisms underlying AD. AD is characterized by the spread of tau pathology through the brain in a stereotypical pattern that correlates well with the disease progression. However, a critical need still exists to understand the molecular underpinnings of tau toxicity in AD and thereby identify promising therapeutic targets to slow and/or halt AD progression. We previously showed that tau aggregates disrupt anterograde fast axonal transport in the squid giant axon by activating the protein phosphatase 1 (PP1)- glycogen synthase kinase 3β (GSK3β) signaling pathway which causes the release of cargo from the anterograde motor protein kinesin through phosphorylation of kinesin light chains by GSK3β. In tau aggregates, the exposure of an N-terminal epitope called the phosphatase activating domain (PAD) activates this signaling pathway via interaction with and activation of PP1 in the squid giant axon. Overactivity of this pathway results in aberrant cargo release and thus axonal transport deficits in the squid. Furthermore, we recently demonstrated that interactions between the mutant P301L tau and PP1 results in disrupted axonal transport in mammalian primary neurons, and the pathology is rescued upon PP1γ knockdown. PAD exposure and axonal degeneration are evident early in the progression of AD and in other tauopathies. Therefore, understanding the potentially toxic effects of PAD exposure is directly relevant to disease. Mounting evidence suggests that tau pathology is propagated throughout the brain potentially in a prion-like process involving cell-to-cell tau seeding. However, the functional consequences of seeded aggregation of endogenous tau is not fully understood. The proposed experiments are designed to test the hypothesis that AD brain-derived tau aggregates induce axonal degeneration in tau-seeded mammalian neurons via activation of the PAD-PP1-GSK3β signaling pathway. The pathological consequences of seeding with AD-derived tau pre-formed fibrils (PFF-tau) will be determined in functional live-cell axonal transport assays. The activity of PP1 and GSK3β in PFF-tau-treated neurons will be measured using biochemical assays. Furthermore, immunofluorescent staining, confocal microscopy and stereological measurement techniques will be used to assess axonal and synaptic degeneration. Upon completion of this project, we will have a better understanding of whether tau seeding in mammalian primary neurons causes toxicity through activation of the PAD-PP1-GSK3β pathway. This new knowledge could inform the development of more effective therapies for AD.

项目概要/摘要 阿尔茨海默病（AD）是一种进行性神经退行性疾病，其患病率持续增加 以惊人的速度。目前的 AD 疗法可以适度缓解症状，从而产生了对疾病的需求 修改疗法，这需要更好地了解 AD 的潜在机制。广告是 其特点是 tau 蛋白病理学以一种刻板模式在大脑中传播，这种模式与相关性良好 随着病情的进展。然而，仍然迫切需要了解其分子基础 tau 蛋白在 AD 中的毒性，从而确定有希望的治疗靶点来减缓和/或阻止 AD 进展。我们 先前表明，tau 蛋白聚集体通过破坏鱿鱼巨轴突的顺行快速轴突运输 激活蛋白磷酸酶 1 (PP1)-糖原合酶激酶 3β (GSK3β) 信号通路， 通过驱动蛋白光的磷酸化，导致顺行运动蛋白驱动蛋白释放货物 GSK3β 链。在 tau 聚集体中，称为磷酸酶激活的 N 末端表位的暴露 结构域 (PAD) 通过与巨型鱿鱼中的 PP1 相互作用并激活 PP1 来激活该信号通路 轴突。该通路的过度活跃会导致异常的货物释放，从而导致轴突运输缺陷 乌贼。此外，我们最近证明突变体 P301L tau 和 PP1 之间的相互作用导致 哺乳动物原代神经元轴突运输中断，PP1γ 可以挽救病理学 击倒。 PAD 暴露和轴突变性在 AD 和其他疾病进展的早期就很明显 tau蛋白病。因此，了解 PAD 暴露的潜在毒性作用与 疾病。越来越多的证据表明，tau 蛋白病理学可能以以下方式在整个大脑中传播： 涉及细胞间 tau 播种的类似朊病毒的过程。然而，种子的功能后果 内源性 tau 蛋白的聚集尚不完全清楚。所提出的实验旨在测试 假设 AD 脑源性 tau 聚集物会诱导 tau 种子哺乳动物的轴突变性 神经元通过激活 PAD-PP1-GSK3β 信号通路。播种的病理后果 AD 衍生的 tau 预形成原纤维 (PFF-tau) 将在功能性活细胞轴突运输中测定 化验。 PFF-tau 处理的神经元中 PP1 和 GSK3β 的活性将使用生化测量 化验。此外，免疫荧光染色、共焦显微镜和体视测量 技术将用于评估轴突和突触变性。该项目完成后，我们将 更好地了解哺乳动物原代神经元中的 tau 蛋白播种是否会导致毒性 PAD-PP1-GSK3β 通路的激活。这些新知识可以为更多的开发提供信息 AD 的有效治疗方法。