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.

项目摘要/摘要 阿尔茨海默病(AD)是一种进行性神经退行性疾病，其患病率持续上升 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- 修改治疗方法，这需要更好地了解AD的发病机制。广告是 以tau病理通过大脑以刻板印象的模式传播为特征，这种模式与 随着疾病的发展。然而，仍有迫切需要了解其分子基础。 Tau对AD的毒性，从而确定减缓和/或阻止AD进展的有希望的治疗靶点。我们 先前研究表明，tau聚集体通过以下方式扰乱鱿鱼巨大轴突的顺行快速轴突运输 激活蛋白磷酸酶1-糖原合成酶激酶3β(β)信号通路 通过光的运动蛋白的磷酸化导致顺行运动蛋白释放货物 链由GSK3β提供。在tau聚集体中，N端表位的暴露称为磷酸酶激活 在鱿鱼中，结构域(PAD)通过与PP1的相互作用和激活来激活这一信号通路 轴突。这一途径的过度活动导致异常的货物释放，从而导致轴突运输缺陷。 鱿鱼。此外，我们最近证明了突变体P301L tau和PP1之间的相互作用结果 在哺乳动物初级神经元的轴突运输中断中，病理通过pp1γ得以挽救 击倒对手。垫暴露和轴突变性在AD进展的早期和其他疾病中明显 紧张症。因此，了解接触护垫的潜在毒性直接关系到 疾病。越来越多的证据表明tau的病理在整个大脑中传播，可能是在 与细胞间tau播种有关的类普里子过程。然而，种子的功能后果 内源性tau的聚集性还不完全清楚。拟议的实验旨在测试 AD脑源性tau聚集体诱导tau种子哺乳动物轴突变性的假说 通过激活Pad-Pp1-Gsk3β信号通路来激活神经元。播种的病理后果 有了AD来源的tau，预先形成的纤维(pff-tau)将在功能活细胞轴突运输中被确定 化验。PfTau处理的神经元中pp1和gsk3β的活性将用生化方法测定。 化验。免疫荧光染色、共聚焦显微镜和体视学测量 这些技术将用于评估轴突和突触退行性变。在这个项目完成后，我们将 更好地了解tau在哺乳动物初级神经元中的种植是否通过 激活Pad-Pp1-Gsk3β通路。这一新的知识可以为更多的 阿尔茨海默病的有效疗法。