ARF6, a new regulator of retromer function and cholesterol homeostasis in neurons.

ARF6，神经元中逆转录酶功能和胆固醇稳态的新调节剂。

• 批准号: 9808769
• 负责人: Catherine A Marquer
• 金额: $ 8.1万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9808769
• 关键词: ADP-ribosylation factor 6; Ablation; Affect; Alzheimer' s Disease; Amyloid; Amyloid beta-Protein Precursor; Applications Grants; Biological Models; Brain; Cell membrane; Cholesterol; Cholesterol Homeostasis; Cleaved cell; Complex; Confocal Microscopy; Data; Dementia; Disease Progression; Embryo; Endocytosis; Enzymes; Event; Fibroblasts; Filipin; Functional disorder; Grant; Hippocampus (Brain); IGF2R gene; Impairment; Knock-out; Late Onset Alzheimer Disease; Ligands; Link; Lysosomes; Microscopy; Molecular; Monomeric GTP-Binding Proteins; Mus; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Onset of illness; Organelles; PIP5K2A gene; Pathogenesis; Pathology; Pathway interactions; Phosphotransferases; Play; Protein Isoforms; Publishing; Recycling; Role; Severities; Subfamily lentivirinae; Testing; Therapeutic Intervention; Wild Type Mouse; Work; base; cholesterol control; immunocytochemistry; in vivo; insight; late endosome; mouse model; mutant; novel; phosphatidylinositol 4-phosphate; small hairpin RNA; targeted treatment; tau Proteins; trafficking

SUMMARY We propose to extend our published work on the small GTP-ase Arf6 (ADP-ribosylation factor 6) and its control of retromer function and cholesterol homeostasis. Retromer dysfunction and cholesterol dyshomeostasis, although both suggested to be some of the earliest events in neurodegeneration, including in Alzheimer's disease (AD), have not been mechanistically linked in neurons. We recently provided evidence that, in fibroblasts, Arf6 ablation resulted in a selective increase of an endosomal pool of phosphatidylinositol-4- phosphate (PI4P). This caused a perturbation of retromer trafficking that led to mistrafficking of its cargoes, including cation-independent mannose-6-phosphate receptor (CI-M6PR) and its ligand NPC2, and to an accumulation of cholesterol in the late endosomes/lysosomes (LE/Lys). Here, we postulate that, in neurons, retromer function and cholesterol homeostasis actually belong to a single pathway regulated by Arf6. We will test our hypothesis using primary cultures of hippocampal neurons derived from wild-type (WT) mice or from the Arf6 flox/flox (fl/fl) mouse model developed in the lab. We will first test whether retromer function controls cholesterol homeostasis (Aim1) using WT hippocampal neurons infected with a control shRNA or an shRNA directed against Vps35, the core subunit of the retromer complex. We will examine whether cholesterol distribution is modified in Vps35-depleted neurons using filipin, a fluorescent ligand of cholesterol. To further provide mechanistic insights, we will test whether trafficking of the retromer cargo CI-M6PR and its ligand NPC2 are altered in Vps35-depleted neurons, using immunocytochemistry and confocal microscopy. We will further test whether Arf6 controls retromer function and cholesterol homeostasis (Aim2) using hippocampal neurons derived from Arf6 fl/fl mice and infected with a control or a Cre lentivirus. We will assess whether retromer function is impaired in the absence of Arf6 by analyzing the subcellular localization and dynamics of the retromer subunits as well as the fate of its cargoes using confocal and spinning disk microscopies. We will also examine cholesterol distribution in control and Arf6-depleted neurons using filipin. Finally, we will identify the effectors of Arf6 on retromer function (Aim 3) in hippocampal neurons. We will test whether Arf6 ablation affects the endosomal levels of PI4P in Arf6-/- neurons. In addition, we will characterize the enzymatic effector of Arf6 on endosomal PI4P levels using a shRNA screen of the different isoforms of phosphatidylinositol-4- phosphate 5-kinases, which phosphorylate PI4P into PI(4,5)P2, in WT neurons. We will then reintroduce the identified wild-type Arf6 effector -or its kinase dead mutant- in Arf6-/- neurons and test if it rescues PI4P endosomal accumulation, retromer dysfunction and cholesterol accumulation. If successful, results from this proposal will provide mechanistic evidence that, in neurons, retromer dysfunction and cholesterol dyshomeostasis, both linked to neurodegeneration, are part of a single pathway regulated by Arf6 and will thus warrant the further study of Arf6 in neurodegenerative disorders, especially in LOAD.

概括 我们建议扩展我们已发表的关于小 GTP 酶 Arf6（ADP-核糖基化因子 6）及其控制的工作 逆转录酶功能和胆固醇稳态。逆转录酶功能障碍和胆固醇动态平衡， 尽管两者都被认为是神经退行性疾病（包括阿尔茨海默病）中最早的一些事件 疾病（AD），在神经元中尚未有机械联系。我们最近提供的证据表明，在 成纤维细胞中，Arf6 消融导致磷脂酰肌醇-4-内体库选择性增加 磷酸盐（PI4P）。这引起了逆转录酶贩运的扰动，导致其货物被误贩运， 包括阳离子非依赖性甘露糖-6-磷酸受体 (CI-M6PR) 及其配体 NPC2，以及 晚期内体/溶酶体 (LE/Lys) 中胆固醇的积累。在这里，我们假设，在神经元中， 逆转录酶功能和胆固醇稳态实际上属于 Arf6 调节的单一途径。 我们将使用源自野生型 (WT) 小鼠或小鼠的海马神经元的原代培养物来检验我们的假设。 来自实验室开发的 Arf6 flox/flox (fl/fl) 小鼠模型。我们首先测试一下retromer是否有功能 使用感染对照 shRNA 或 shRNA 针对 Vps35（Retromer 复合物的核心亚基）。我们将检查胆固醇是否 使用 filipin（胆固醇的荧光配体）在 Vps35 耗尽的神经元中修改分布。为了进一步 提供机制见解，我们将测试逆转录酶货物 CI-M6PR 及其配体的运输是否 使用免疫细胞化学和共聚焦显微镜，NPC2 在 Vps35 耗尽的神经元中发生改变。我们将 使用海马进一步测试 Arf6 是否控制逆转录酶功能和胆固醇稳态 (Aim2) 源自 Arf6 fl/fl 小鼠并感染对照或 Cre 慢病毒的神经元。我们将评估是否 通过分析 Arf6 的亚细胞定位和动态，逆转录酶功能在缺乏 Arf6 的情况下受损 使用共聚焦和转盘显微镜观察逆转录酶亚基及其货物的命运。我们将 还使用 filipin 检查对照神经元和 Arf6 耗尽的神经元中的胆固醇分布。最后，我们将识别 Arf6 对海马神经元逆转录酶功能（目标 3）的效应器。我们将测试Arf6是否消融 影响 Arf6-/- 神经元中 PI4P 的内体水平。此外，我们将表征酶效应子 使用不同亚型磷脂酰肌醇-4-的shRNA筛选Arf6对内体PI4P水平的影响 磷酸 5-激酶，在 WT 神经元中将 PI4P 磷酸化为 PI(4,5)P2。然后我们将重新引入 在 Arf6-/- 神经元中鉴定出野生型 Arf6 效应子 - 或其激酶死亡突变体 - 并测试它是否可以拯救 PI4P 内体积累、逆转录酶功能障碍和胆固醇积累。如果成功，结果如下 该提案将提供机制证据，证明在神经元、逆转录酶功能障碍和胆固醇中 体内平衡失调均与神经退行性变有关，是 Arf6 和 Arf6 调节的单一途径的一部分 因此，需要进一步研究 Arf6 在神经退行性疾病中的作用，特别是在 LOAD 中。