Nuclear transport as a molecular and cellular vulnerability in AD

摘要 细胞核和细胞质之间的分子运输对细胞健康是必不可少的，并且紧密地 在所有类型的细胞中都有调节，包括大脑中的那些。最近的出版物显示了核运输 阿尔茨海默病(AD)和相关痴呆(ADRD)的神经元缺陷。AD/ADRD是在 部分是由于错误折叠的tau蛋白，这表明错误折叠的tau可能通过异常相互作用而损害核运输。 核孔蛋白。我们认为迟发性神经退行性疾病，如阿尔茨海默病，反映了两种 脆弱性：(1)在细胞水平上，神经分化过程中固有的核输入效率丧失 程序使神经元对与错误折叠的tau相关的损害敏感。(Ii)在分子水平上，核 孔道复合体(Npc)选择性地容易被错误折叠的蛋白质破坏，因为它们的活性。 依赖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 Add/ADRD-tau并非常缓慢地翻转。为了验证这些假说，我们开发了新型的光遗传核 转运分析，基于可光激活的NLS/NES元素。我们现在提议将米奇森 集团在高级显微镜和图像分析方面的专长与宋集团在神经细胞方面的专长 生物学和病理学模型，以测量活神经元的核进出口速率，并测试 神经分化、tau错误折叠和可能减轻错误折叠影响的候选药物的影响 关于核运输的问题。我们将(I)描述神经过程中核转运率的变化 差异，(2)比较核运输对AD/ADRD相关的错误折叠tau挑战的敏感性 (例如，G272V-tau和P301S-tau)在神经元和神经前体细胞中的表达，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。运输化验也将 启动未来旨在挽救老化神经元中核运输的翻译计划。作为测试用例，我们 将表征增加O-连接b-N-乙酰氨基葡萄糖(O-GlcNAc)细胞内修饰的药物 蛋白质。这种修饰被认为可以抑制错误折叠的蛋白质，如tau的聚集。但是，最终聚集 鼻咽癌中的重复序列是O-GlcNAc修饰最多的蛋白质之一。我们认为，这一功能 可药物修饰是为了保护NPC固有的易受错误折叠蛋白质损伤的能力。 R21试验的成功将为将我们的光学记者策略转移到小鼠大脑模型奠定基础 衰老和退化，以及识别药物靶点和测试高密度脂蛋白的候选治疗分子 内容分析格式。

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.