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。

基本信息

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
项目类别：
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&apos 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

项目摘要

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.

概括我们建议扩大有关小型GTP-ase ARF6（ADP-核糖基化因子6）的发表工作及其控制缩回功能和胆固醇稳态。逆转录功能障碍和胆固醇Dyshomeostasis，尽管两者都建议是神经变性中最早的事件，包括在阿尔茨海默氏症中疾病（AD）尚未在神经元中机械上联系。我们最近提供了证据，表明成纤维细胞，ARF6消融导致磷脂酰肌醇-4-的内体池选择性增加磷酸盐（PI4P）。这引起了逆转录贩运的扰动，导致其货物的贩运者，包括非阳离子非依赖性的甘露糖-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，逆转录功能和胆固醇稳态实际上属于由ARF6调节的单个途径。我们将使用源自野生型（WT）小鼠或来自实验室中开发的ARF6 Flox/Flox（FL/FL）小鼠模型。我们将首先测试缩回功能是否功能使用对照shRA或一个感染的WT海马神经元控制胆固醇稳态（AIM1） shRNA针对vPS35，这是逆转录器复合物的核心亚基。我们将检查是否胆固醇使用胆固醇的荧光配体Filipin在VPS35耗尽的神经元中修饰分布。进一步提供机械洞察力，我们将测试逆转录货物CI-M6PR及其配体的贩运使用免疫细胞化学和共聚焦显微镜在耗尽VPS35的神经元中改变了NPC2。我们将进一步测试ARF6是否使用海马来控制缩回功能和胆固醇稳态（AIM2）源自ARF6 FL/FL小鼠并用对照或CRE慢病毒感染的神经元。我们将评估是否通过分析亚细胞定位和动力学，在没有ARF6的情况下，缩回功能受到损害使用共聚焦和旋转磁盘显微镜的逆转录子基以及其货物的命运。我们将还可以使用菲律宾检查对照和ARF6缺失的神经元中的胆固醇分布。最后，我们将确定 ARF6对缩回函数的效应子（AIM 3）在海马神经元中。我们将测试ARF6消融是否影响ARF6 - / - 神经元中PI4P的内体水平。另外，我们将表征酶效应器使用不同同工型的磷脂酰肌醇4-- WT神经元中的磷酸5-激酶，将PI4P磷酸化为Pi（4,5）P2。然后，我们将重新引入鉴定出野生型ARF6效应子 - 或其激酶死突变体 - ARF6 - / - 神经元中，并测试它是否营救了PI4P 内体积累，缩回功能障碍和胆固醇积累。如果成功的话，提案将提供机械证据，在神经元中，缩回功能障碍和胆固醇 dyshomeostasis均与神经变性有关，是由ARF6调节的单个途径的一部分因此，将保证在神经退行性疾病中对ARF6进行进一步研究，尤其是在负载中。