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

摘要 我们建议扩展我们发表的关于小GTP酶Arf6(ADP核糖化因子6)及其调控的工作 逆转聚体功能和胆固醇动态平衡。逆转录功能障碍和胆固醇代谢紊乱， 尽管这两个事件都是神经退化的早期事件，包括阿尔茨海默氏症 疾病(AD)，还没有在神经元中有机械性的联系。我们最近提供的证据表明，在 成纤维细胞，Arf6消融导致磷脂酰肌醇-4-内体池选择性增加。 磷酸盐(PI4P)。这造成了回溯贩运的扰动，导致了其货物的错误抽奖， 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 胆固醇在晚期内切/溶酶体内的积聚(LE/Lys)。在这里，我们假设，在神经元中， 逆转聚体功能与胆固醇稳态实际上属于Arf6调控的单一途径。 我们将使用来自野生型(WT)小鼠的海马神经元的原代培养来验证我们的假设 来自实验室开发的Arf6 Flox/Flox(f1/fl)小鼠模型。我们将首先测试逆转录病毒是否起作用 利用感染对照shRNA或AN的WT海马神经元控制胆固醇稳态(Aim1) 针对VPS35的shRNA，VPS35是逆聚体复合体的核心亚单位。我们将检查胆固醇是否 在VPS35耗竭的神经元中，使用胆固醇的荧光配体菲立磷脂可以改变其分布。为了进一步 提供机械洞察，我们将测试逆转录货物CI-M6PR及其配体的贩运 利用免疫细胞化学和共聚焦显微镜，NPC2在VPS35耗竭的神经元中发生改变。我们会 进一步利用海马区测试Arf6是否控制逆转聚体功能和胆固醇稳态(AIM2) 来自Arf6 fl/fl小鼠的神经元，并感染了对照或Cre慢病毒。我们将评估是否 通过分析逆转录酶的亚细胞定位和动力学，在没有Arf6的情况下逆转录酶功能受损 用共聚焦显微镜和旋转圆盘显微镜观察逆转录病毒亚基及其货物的去向。我们会 还可以使用非利培酮检查对照神经元和Arf6耗竭神经元中的胆固醇分布。最后，我们将确定 Arf6对海马神经元逆转录功能(Aim 3)的影响。我们将测试Arf6消融是否 影响Arf6-/-神经元内体PI4P的水平。此外，我们还将对酶效应器进行表征 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 在WT神经元中，磷酸5-激酶将PI4P磷酸化为PI(4，5)P2。然后我们将重新介绍 在Arf6-/-神经元中鉴定野生型Arf6效应器或其激酶死亡突变体，并测试其是否拯救PI4P 内体积聚、逆转录功能障碍和胆固醇积聚。如果成功，则结果为 该提案将提供机制证据，证明在神经元中，逆转录功能障碍和胆固醇 代谢紊乱，两者都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，Arf6在神经退行性疾病中的进一步研究将是必要的，尤其是在负荷中。

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.