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于细胞健康是必不可少的，并且与细胞的生长密切相关。 在所有类型的细胞中都受到调节，包括大脑中的细胞。最近的出版物表明核运输 阿尔茨海默病（AD）和相关痴呆（ADRD）中的神经元缺陷。AD/ADRD是由 部分通过错误折叠的tau蛋白，提示错误折叠的tau蛋白可能通过异常相互作用损害核转运 核孔蛋白。我们认为，迟发性神经退行性疾病，如AD，反映了两个 脆弱性：（i）在细胞水平，在神经分化过程中核输入效率的内在损失 程序使神经元对与错误折叠的tau相关的损伤敏感。(ii)在分子水平上， 孔复合物（NPC）选择性地易受错误折叠蛋白质的破坏，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，其容易被错误折叠破坏 AD/ADRD-tau和非常缓慢地翻身。为了验证这些假设，我们开发了新的光遗传学核 基于可光活化的NLS/内斯元件的转运测定。我们现在提议将联合收割机米奇森 Song Group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中的核输入和输出速率，并测试 神经分化的作用、错误折叠的tau蛋白和可以减轻错误折叠的tau蛋白的作用的药物候选物 Tau核运输。我们将（i）描述神经细胞核转运速率的变化 （ii）比较核转运对AD/ADRD相关错误折叠tau挑战的敏感性 (e.g. G272 V-tau和P301 S-tau）在神经元和神经祖细胞中的表达，以及（iii）研究潜在的 分子机制，使用超分辨率显微镜和免疫测定。运输试验还将 使未来的翻译计划，旨在挽救老化神经元的核运输。作为测试案例，我们 将表征增加细胞内O-连接的b-N-乙酰葡糖胺（O-GlcNAc）修饰的药物， proteins.这种修饰被认为可以抑制错折叠蛋白质如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。我们认为，这一功能 药物修饰是为了保护NPC对错误折叠蛋白质的内在脆弱性。 这项R21试验的成功将为我们的光学报告策略进入小鼠大脑模型奠定基础。 老化和退化，并用于确定药物靶点和测试候选治疗分子在高- 含量测定格式。

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.