Nuclear transport as a molecular and cellular vulnerability in AD

核运输是 AD 中分子和细胞的脆弱性

• 批准号: 10213341
• 负责人: Timothy J Mitchison
• 金额: $ 48.06万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-15 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10213341
• 关键词: Address; Age; Aging; Alzheimer' s Disease; Alzheimer' s disease related dementia; Binding; Biological Assay; Biological Models; Brain; Cell Nucleus; Cell fusion; Cell model; Cells; Cellular biology; Cytoplasm; Defect; Diffusion; Drug Targeting; Elements; Evaluation; Exportins; Exposure to; Family; Future; Gel; Gene Expression; Generations; Genetic; Glycine; Goals; Health; Heart; Human; Hydrogen Bonding; Hydrophobic Interactions; Hydrophobicity; Image Analysis; Immunoassay; Impairment; Importins; Interphase Cell; Karyopherins; Kinetics; Light; Link; Measurement; Measures; Microscopy; Modeling; Modification; Molecular; Movement; Mus; Nerve Degeneration; Neurobiology; Neurodegenerative Disorders; Neuronal Differentiation; Neurons; Nuclear; Nuclear Envelope; Nuclear Export; Nuclear Import; Nuclear Pore; Nuclear Pore Complex; Nuclear Proteins; Optical reporter; Pathology; Pathway interactions; Patients; Peptides; Pharmaceutical Preparations; Pharmacology; Phenylalanine; Pore Proteins; Protein Glycosylation; Proteins; Proteomics; Proxy; Publications; Regulation; Research; Resolution; Signal Transduction; Techniques; Testing; aging brain; alpha Karyopherins; base; cell type; drug candidate; drug development; drug discovery; drug testing; experimental study; exportin 1 protein; glycosylation; heterokaryon; hydrophilicity; in vivo; induced pluripotent stem cell; knock-down; microscopic imaging; misfolded protein; mouse model; nerve stem cell; novel; nucleocytoplasmic transport; optogenetics; prevent; programs; relating to nervous system; success; tau Proteins; tau mutation; therapeutic candidate; tool; trafficking

Abstract Molecular trafficking between the nucleus and the cytoplasm is essential for cellular health and is tightly regulated in all cell types including those in the brain. Recent publications demonstrated nuclear transport defects in neurons in Alzheimer’s disease (AD) and related dementias (ADRDs). AD/ADRDs are caused, in part, by misfolded tau protein, suggesting misfolded tau may impair nuclear transport by aberrantly interacting with nuclear pore proteins. We propose that late-onset neurodegenerative disease, such as AD, reflects two vulnerabilities: (i) At the cellular level, intrinsic loss of nuclear import efficiency during the neural differentiation program sensitizes the neurons to damages associated with misfolded tau. (ii) At the molecular level, nuclear pore complexes (NPCs) are selectively vulnerable to disruption by misfolded proteins because their activity depends on exposed hydrophobic phenylalanine-glycine (FG) repeats that are easily disrupted by misfolded AD/ADRD-tau and turn over very slowly. To test these hypothesis, we developed novel optogenetic nuclear transport assays, based on photo-activatable NLS/NES elements. We now propose to combine Mitchison group’s expertise in advanced microscopy and image analysis with Song group’s expertise in neuron cell biology and pathology models to measure rates of nuclear import and export in living neurons and test the effects of neural differentiation, misfolded tau, and drug candidates that may alleviate the effects of misfolded tau on nuclear transport. We will (i) characterize the change in nuclear transport rates during neural differentiation, (ii) compare the sensitivity of nuclear transport to AD/ADRD-related misfolded tau challenges (e.g. G272V-tau and P301S-tau) in neurons and neural progenitors, and (iii) investigate the underlying molecular mechanisms using super-resolution microscopy and immunoassays. The transport assays will also enable future translational programs aimed at rescuing nuclear transport in aging neurons. As a test case, we will characterize drugs that increase O-linked b-N-acetylglucosamine (O-GlcNAc) modification of intracellular proteins. This modification is thought to inhibit aggregation of misfolded proteins such as tau. However, FG repeat in NPC are among the most O-GlcNAc modified proteins. We propose that the function of this druggable modification is to protect the intrinsic vulnerability of NPCs to damage by misfolded proteins. Success on this R21 pilot will set the stage for moving our optical reporter strategy into mouse models of brain aging and degeneration, and for identifying drug targets and testing candidate therapeutic molecules in high- content assay formats.

抽象的 细胞核和细胞质之间的分子运输对于细胞健康至关重要，并且紧密相关 在包括大脑在内的所有细胞类型中受到调节。最近的出版物展示了核运输 阿尔茨海默病（AD）和相关痴呆症（ADRD）中的神经元缺陷。 AD/ADRD 是由 部分由错误折叠的 tau 蛋白引起，表明错误折叠的 tau 蛋白可能通过异常相互作用损害核运输 与核孔蛋白。我们认为迟发性神经退行性疾病，例如 AD，反映了两个方面 漏洞：（i）在细胞水平上，神经分化过程中核输入效率的内在损失 该程序使神经元对与错误折叠的 tau 蛋白相关的损伤敏感。 (ii) 在分子水平上，核 孔复合体 (NPC) 选择性地容易受到错误折叠蛋白质的破坏，因为它们的活性 取决于暴露的疏水性苯丙氨酸-甘氨酸 (FG) 重复序列，这些重复序列很容易被错误折叠破坏 AD/ADRD-tau 并非常缓慢地翻转。为了检验这些假设，我们开发了新型光遗传学核 基于光激活 NLS/NES 元件的转运测定。我们现在建议合并 Mitchison 团队在先进显微镜和图像分析方面的专业知识与宋团队在神经元细胞方面的专业知识 生物学和病理学模型来测量活神经元的核输入和输出率并测试 神经分化、错误折叠 tau 蛋白的影响以及可能减轻错误折叠影响的候选药物 tau 蛋白对核运输的影响。我们将（i）描述神经过程中核转运速率的变化 分化，(ii) 比较核运输对 AD/ADRD 相关错误折叠 tau 挑战的敏感性 （例如 G272V-tau 和 P301S-tau）在神经元和神经祖细胞中，以及 (iii) 研究潜在的 使用超分辨率显微镜和免疫分析的分子机制。运输分析也将 启用旨在拯救衰老神经元核运输的未来转化计划。作为一个测试用例，我们 将表征增加细胞内 O-连接 b-N-乙酰氨基葡萄糖 (O-GlcNAc) 修饰的药物 蛋白质。这种修饰被认为可以抑制错误折叠蛋白质（如 tau）的聚集。然而，FG NPC 中的重复序列是 O-GlcNAc 修饰最多的蛋白质之一。我们建议这个功能 可药物修饰是为了保护 NPC 固有的脆弱性，使其免受错误折叠蛋白质的损害。 R21 试点的成功将为将我们的光学报告策略转移到小鼠大脑模型中奠定基础 老化和退化，以及识别药物靶点和测试候选治疗分子的高 内容分析格式。