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.

项目摘要/摘要 阿尔茨海默氏病（AD）的患病率是一种进行性神经退行性疾病，继续增加 以惊人的速度。当前的AD疗法可提供适度的症状缓解，从而需要疾病 - 修改疗法，这需要更好地了解AD基础机制。广告是 以刻板印象相关的刻板印象，其特征是tau病理通过大脑传播 随着疾病的进展。但是，仍然存在关键的需求，以了解 TAU在AD中的毒性，从而确定有希望的治疗靶标，以减慢和/或停止AD的进展。我们 以前表明，tau聚集在鱿鱼巨型轴突中的顺行快速轴突转运 激活蛋白质磷酸酶1（PP1） - 糖原合酶激酶3β（GSK3β）信号传导途径，该途径 导致通过驱动蛋白光的磷酸化从顺行运动蛋白风化蛋白释放货物 GSK3β的链。在tau聚集体中，称为磷酸酶激活的N末端表位的暴露 域（PAD）通过与鱿鱼巨人中PP1的相互作用和激活激活此信号通路 轴突。该途径的过度活动性导致异常货物释放，因此轴突运输在 乌贼。此外，我们最近证明了突变体P301L TAU与PP1结果之间的相互作用 在哺乳动物原发性神经元中的轴突转运中断，并在PP1γ上救出病理 击倒。 PAD暴露和轴突变性是AD和其他AD进展的早期证据 tauopathies。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病理在整个大脑中都有可能在 涉及细胞到细胞式播种的prion样过程。但是，种子的功能后果 内源性tau的聚集尚不完全了解。提出的实验旨在测试 AD脑衍生的Tau聚集体诱导tau种子哺乳动物的轴突变性的假设 神经元通过激活PAD-PP1-GSK3β信号通路。播种的病理后果 将在功能性活细胞轴突运输中确定AD衍生的TAU预先形成的原纤维（PFF-TAU） 测定。 PP1和GSK3β在PFF-TAU处理的神经元中的活性将使用生化测量 测定。此外，免疫荧光染色，共聚焦显微镜和立体测量 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tau在哺乳动物原发性神经元中是否会引起毒性 PAD-PP1-GSK3β途径的激活。这些新知识可以告知更多 AD的有效疗法。