The molecular mechanism of PICALM-dependent endosomal trafficking

PICALM依赖性内体运输的分子机制

• 批准号: 10017851
• 负责人: Zhen Zhao
• 金额: $ 20.63万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-15 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10017851
• 关键词: 3-Dimensional; Abeta synthesis; Alzheimer' s Disease; Alzheimer' s disease related dementia; Amyloid; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Antisense Oligonucleotides; Apolipoprotein E; Autophagocytosis; Binding; Bioinformatics; Biological Assay; Biology; Blood; Blood - brain barrier anatomy; Brain; Cell Surface Receptors; Cells; Clathrin; Clathrin Adaptors; Complex; Data; Defect; Development; Early Endosome; Endocytosis; Endosomes; Endothelium; Ensure; Event; Excision; Functional disorder; Growth; Health; Homeostasis; Human; In Vitro; Insulin; Ligands; Maps; Mediating; Mitotic; Modeling; Molecular; Mus; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Outcome; Pathogenesis; Pathogenicity; Pathway interactions; Peptides; Phosphatidylinositols; Protein Deficiency; Proteins; Regulation; Research Priority; Role; Sorting - Cell Movement; Spatial Distribution; Specific qualifier value; System; Testing; Transferrin; Validation; Vesicle; amyloid peptide; amyloid precursor protein processing; base; beta secretase; brain endothelial cell; brain metabolism; gamma secretase; in vivo; insight; internal control; knock-down; novel; receptor; receptor function; risk variant; secretase; tau Proteins; trafficking; transcytosis; uptake; wasting

ABSTRACT Dysregulation of endocytosis and endosomal trafficking pathways contributes to the pathogenesis of Alzheimer’s disease and related dementia (ADRD). Beside controlling amyloid precursor processing and β-amyloid (Aβ) production in neuronal cells, endocytosis and endosomal trafficking pathways also exist in brain endothelial cells and govern the Aβ clearance cross Blood-brain barrier (BBB) through receptor mediated transport (RMT). A properly functioning RMT is highly selective due to the spatial distribution of the receptors and specific interaction with their ligands, which ensures the exclusive entry of essential peptides and proteins into the brain and effective clearance of toxic waste from brain to blood in maintaining CNS health and functions. However, our understanding of this unique RMT system within the BBB remains very limited. RMT is tightly regulated by products of AD risk genes, such as Apolipoprotein E and phosphatidylinositol-binding clathrin assembly protein (PICALM). PICALM, a highly validated risk gene for Alzheimer’s disease, is also an endosomal protein and a key component of the RMT machinery at the BBB. PICALM controls the RMT transcytosis across the BBB by facilitating the clathrin-mediated endocytosis and intracellular trafficking of cell surface receptors and their ligands. PICALM deficiency in mice results in defected transferrin trafficking and diminished brain clearance of Alzheimer’s amyloid-β peptides (Aβ) across the BBB. Therefore, delineate the molecular mechanism of PICALM-mediated transcytosis events offers new opportunities in advancing our understanding of the BBB RMT system, as well as its role in AD pathogenesis. Through in-depth analysis of PICALM’s interactome and functional target validations using an in vitro BBB model, we found that PICALM interacting mitotic regulator (PIMREG) is a novel functional partner for PICALM in the brain endothelial cells and is required for the later steps of RMT transcytosis. Therefore, we hypothesize that PIMREG is an integral component of the RMT machinery, and teams up with PICALM and other proteins in controlling the intracellular trafficking and transcytosis of cargo vesicles, which is essential for brain homeostasis and Aβ clearance. To test our hypothesis, we propose to determine the function of PICALM-PIMREG complex in controlling endosomal trafficking events in primary brain endothelial cells (AIM 1), understand the role PIMREG in regulating the PICALM-mediated RMT transcytosis of different ligands across the in vitro BBB model (AIM 2), and probe PIMREG’s function in vivo for PICALM-dependent Aβ clearance across the BBB using antisense oligonucleotides (ASOs) (AIM 3). We expect to generate unique new insights into the biology and molecular mechanism of RMT transcytosis at the BBB, and provide first-hand evidence of a novel component of PICALM- dependent Aβ clearance across the BBB, which will expand our understanding of RMT and its roles in BBB dysfunction and consequent neurodegeneration in ADRD.

摘要 内吞和内体运输途径的失调有助于阿尔茨海默病的发病机制 疾病和相关痴呆症（ADRD）。除了控制淀粉样蛋白前体加工和β-淀粉样蛋白（Aβ） 在神经元细胞中产生，内吞和内体运输途径也存在于脑内皮细胞中 并通过受体介导的转运（RMT）控制Aβ穿过血脑屏障（BBB）的清除。一 由于受体的空间分布和特异性相互作用，功能正常的RMT具有高度选择性 与它们的配体，这确保了必需的肽和蛋白质进入大脑和有效的排他进入， 清除有毒废物从大脑到血液，维持中枢神经系统的健康和功能。但我们的 对BBB内这种独特的RMT系统的理解仍然非常有限。 RMT受到AD风险基因产物的严格调控，如载脂蛋白E和磷脂酰肌醇结合蛋白。 网格蛋白组装蛋白（PICALM）。PICALM是一种高度有效的阿尔茨海默病风险基因，也是一种 内体蛋白和BBB的RMT机制的关键组成部分。PICALM控制RMT 通过促进网格蛋白介导的细胞内吞和细胞内运输， 表面受体及其配体。小鼠PICALM缺陷导致转铁蛋白运输缺陷， 减少阿尔茨海默氏淀粉样β肽（Aβ）穿过BBB的脑清除率。因此，描绘出 PICALM介导的转胞吞作用的分子机制为我们的研究提供了新的机会。 了解BBB RMT系统及其在AD发病机制中的作用。通过深入分析 使用体外BBB模型验证PICALM的相互作用组和功能靶点，我们发现PICALM 有丝分裂相互作用调节因子（PIMREG）是脑内皮细胞中PICALM的一种新的功能伴侣， 是RMT转胞吞作用的后续步骤所必需的。因此，我们假设PIMREG是一个积分 RMT机制的组成部分，并与PICALM和其他蛋白质合作控制细胞内的 运输和转运的货物囊泡，这是必不可少的脑稳态和Aβ清除。 为了验证我们的假设，我们建议确定PICALM-PIMREG复合物在控制 原代脑内皮细胞（AIM 1）内体运输事件，了解PIMREG在调节 PICALM介导的不同配体跨体外BBB模型（AIM 2）的RMT转胞吞作用，并探测 PIMREG在体内对PICALM依赖性Aβ通过BBB的清除的功能，使用反义寡核苷酸 寡核苷酸（ASO）（AIM 3）。我们希望能对生物学和分子生物学产生独特的新见解， RMT在BBB转胞吞作用的机制，并提供PICALM的新组分的第一手证据- 依赖性Aβ清除通过BBB，这将扩大我们对RMT及其在BBB中作用的理解 ADRD中的功能障碍和随之发生的神经变性。