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: 10464286
• 负责人: Rebecca Lynn Mueller
• 金额: $ 3.89万
• 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/10464286
• 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; Genes; Goals; Hand; Healthcare; Hippocampus (Brain); 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; Seeds; Signal Pathway; Squid; Stains; Synapses; Tauopathies; Techniques; Tertiary Protein Structure; Testing; Therapeutic; Toxic effect; Training; Viral Vector; Virus; Work; axonal degeneration; design; effective therapy; experimental study; 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.

项目总结/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疗法提供了适度的症状缓解，创造了对疾病的需求- 修改疗法，这需要更好地了解AD的潜在机制。AD是 其特征在于tau病理学以一种刻板的模式通过大脑传播， 随着疾病的发展然而，仍然存在一个关键的需要，以了解分子基础的 研究AD中tau毒性，从而鉴定有希望的治疗靶点以减缓和/或停止AD进展。我们 先前的研究表明，tau聚集体通过以下方式破坏鱿鱼巨轴突中的顺行快速轴突运输： 激活蛋白磷酸酶1（PP 1）-糖原合成酶激酶3β（GSK 3 β）信号通路， 通过驱动蛋白光的磷酸化， GSK3β。在tau蛋白聚集体中，称为磷酸酶激活蛋白的N-末端表位的暴露， PAD结构域（PAD）通过与巨型鱿鱼中PP 1的相互作用和激活来激活这一信号通路 轴突该途径的过度活性导致异常的货物释放，从而导致轴突运输缺陷。 鱿鱼。此外，我们最近证明，突变P301 L tau和PP 1之间的相互作用， 在哺乳动物原代神经元轴突运输中断，病理学是拯救后PP 1 γ 击倒。PAD暴露和轴突变性在AD进展的早期和其他疾病中是明显的。 tau蛋白病因此，了解PAD暴露的潜在毒性效应与以下因素直接相关： 疾病越来越多的证据表明，tau蛋白病理学可能在大脑中传播， 朊病毒样过程涉及细胞间tau接种。然而，种子的功能性后果 内源性tau的聚集尚未完全了解。所提出的实验旨在测试 AD脑源性tau聚集体在tau种子哺乳动物中诱导轴突变性假说 PAD-PP 1-GSK 3 β信号通路的激活。播种的病理后果 与AD衍生的tau预形成的原纤维（PFF-tau）将被确定在功能活细胞轴突运输 分析。PFF-tau处理的神经元中PP 1和GSK 3 β的活性将使用生物化学方法测量。 测定。此外，免疫荧光染色，共聚焦显微镜和体视学测量， 技术将用于评估轴突和突触变性。在完成这个项目后，我们将 更好地了解tau蛋白在哺乳动物原代神经元中的播种是否会通过以下方式引起毒性： PAD-PP 1-GSK 3 β通路的激活。这些新知识可以为开发更多 有效治疗AD。