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.

抽象的 内吞作用和内体贩运途径的失调有助于阿尔茨海默氏症的发病机理 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）之外 神经元细胞，内吞作用和内体运输途径的产生也存在于脑内皮细胞中 并通过受体介导的转运（RMT）控制Aβ清除横脑屏障（BBB）。一个 由于接收器的空间分布和特定的相互作用，正确的功能RMT具有高度选择性 凭借其配体，可确保必需的辣椒和蛋白质的独家进入大脑且有效 在维持CNS健康和功能方面，从大脑到血液的有毒废物清除。但是，我们的 了解BBB中这种独特的RMT系统仍然非常有限。 RMT受AD风险基因的产物（例如载脂蛋白E和磷脂酰肌醇结合）的严格调节 网格蛋白组装蛋白（PICALM）。 PICALM是阿尔茨海默氏病高度验证的风险基因，也是一种 内体蛋白质和BBB RMT机械的关键组成部分。 PICALM控制RMT 通过支撑网格蛋白介导的内吞作用和细胞内贩运细胞的跨BBB的转胞细胞增多 表面受体及其配体。小鼠的皮卡缺乏会导致转铁蛋白运输和 在BBB上，阿尔茨海默氏症淀粉样蛋白β肽（Aβ）的大脑清除率降低。因此，描述 皮基介导的转单胞菌病的分子机制为推进我们的 了解BBB RMT系统及其在AD发病机理中的作用。通过深入分析 PICALM的Interactome和功能目标验证使用体外BBB模型，我们发现Picalm 相互作用的有丝分裂调节剂（PIMREG）是脑内皮细胞中皮层的新功能伙伴， RMT转介症的后期步骤是必需的。因此，我们假设Pimreg是一个不可或缺的 RMT机械的组成部分，并与PICALM和其他蛋白质合作控制细胞内 货物蔬菜的运输和跨经细胞增多症，这对于大脑稳态和Aβ清除率至关重要。 为了检验我们的假设，我们建议确定Picalm-Pimreg复合物在控制中的功能 原代脑内皮细胞中的内体运输事件（AIM 1），了解调节中的作用 跨体外BBB模型的多种配体的皮基介导的RMT转介症（AIM 2），并证明 使用反义，PIMREG在BBB跨BBB的PICALM依赖性Aβ间隙的体内功能 寡核苷酸（ASOS）（AIM 3）。我们期望对生物学和分子产生独特的新见解 RMT转胞膜在BBB上的机制，并提供了一方面的证据，证明了皮基的新成分 跨BBB的依赖Aβ清除率，这将扩大我们对RMT及其在BBB中的作用的理解 ADRD中的功能障碍和随之而来的神经变性。