ENHANCING LYSOSOME BIOGENESIS TO PREVENT AMYLOID PLAQUE PATHOGENESIS

增强溶酶体生物发生预防淀粉样斑块发病

• 批准号: 8724570
• 负责人: Jin-Moo Lee
• 金额: $ 22.57万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8724570
• 关键词: Alzheimer' s Disease; Amyloid; Amyloid Fibrils; Amyloid beta-Protein Precursor; Astrocytes; Attenuated; Autophagocytosis; Autophagosome; Biogenesis; Brain; Catabolism; Cell membrane; Cell model; Cells; Cherry - dietary; Chimera organism; Data; Degradation Pathway; Dementia; Deposition; Development; Disease; Endocytosis; Endosomes; Extracellular Space; Functional disorder; Gene Transfer; Generations; Glial Fibrillary Acidic Protein; Grant; Growth; Half-Life; Hippocampus (Brain); In Vitro; Individual; Integral Membrane Protein; Intercellular Fluid; Intervention; Lead; Lysosomes; Measures; Mediating; Metabolism; Microdialysis; Microglia; Microscopy; Mus; Neurons; Pathogenesis; Pathology; Pathway interactions; Phagocytosis; Pharmaceutical Preparations; Physiological; Prevalence; Process; Production; Proteolysis; Public Health; Role; Secondary to; Senile Plaques; Sirolimus; Slice; Testing; Tissues; Viral Genes; age related; aging population; amyloid peptide; amyloid precursor protein processing; cell type; clinically relevant; extracellular; in vivo; late endosome; lentivirally transduced; mouse model; neuroblastoma cell; peptide A; prevent; programs; promoter; protein metabolism; public health relevance; secretase; therapeutic target; trafficking; transcription factor; two-photon; uptake

DESCRIPTION (provided by applicant): The metabolism of amyloid precursor protein (APP) and amyloid-¿ peptide (A¿) are critical determinants of Alzheimer's disease (AD) pathogenesis. APP is a type-1 transmembrane protein which resides in the plasma membrane. A fraction of APP undergoes endocytosis and is trafficked to late endosomes, where proteolytic cleavage by ¿- and ?-secretase results in the liberation of A¿ which is released into the extracellular space (interstitial fluid, ISF), even in normal individuals. Elevated levels of ISF A¿ may promote aggregation into soluble oligomers and insoluble amyloid plaques, and subsequent development of AD pathology. In addition to production, A¿ degradation and clearance significantly influences ISF A¿ levels and plaque pathogenesis. It has been postulated that age-related and disease-specific lysosomal dysfunction drives AD pathogenesis. While the specific underlying causes of lysosomal dysfunction continue to be unraveled, the resultant disease- promoting mechanisms may depend upon the cell type. For example in neurons, where A¿ is produced, physiologic lysosomal proteolysis may favor complete, non-amyloidogenic APP processing and/or A¿ degradation prior to release. In astrocytes, lysosomal activity may be important for catabolism of extracellular A¿ (and possibly amyloid fibrils) taken up intracellularl; while in microglia, it may promote clearance of the phagocytosed amyloid deposits. Understanding the role of cell-type specific lysosomal dysfunction in AD pathogenesis will be critical for identifying potential targets for intervention. Ubiquitously expressed Transcription Factor EB (TFEB), has been recently identified as a master regulator of lysosome biogenesis, endocytosis, and autophagy. While drugs are currently available (e.g., rapamycin) that stimulate autophagy, the TFEB-regulated transcriptional program coordinately increases flux through multiple lysosomal degradative pathways; and is sufficient to alleviate abnormal substrate accumulation and pathology in various lysosome storage diseases. Our preliminary data demonstrate that exogenous TFEB expression decreased A¿ production/release by N2a-APP695 cells (a neuroblastoma cell model of APP processing) compared with controls. In addition, TFEB expression in N2a cells resulted in increased uptake and accelerated degradation of exogenously applied A¿. These data suggest that TFEB-induced lysosome biogenesis enhances APP and A¿ degradation through several cellular mechanisms. In this proposal, we hypothesize that enhancing lysosome biogenesis with exogenous expression of TFEB will suppress AD pathogenesis in a cell-type specific manner: in neurons, TFEB will facilitate complete proteolysis of APP and A¿ resulting in decreased A¿ generation and reduction in steady-state ISF A¿ levels; while in astrocytes, it will enhance A¿ uptake and degradation, resulting in reduced ISF A¿ half-life. Both mechanisms will attenuate amyloid plaque deposition. We will test this hypothesis in the following aims: 1. Determine the effect of TFEB-induced lysosomal biogenesis on APP processing and A¿ production in neurons. 2. Determine the effect of astrocytic expression of TFEB on A¿ and amyloid catabolism, and plaque growth.

描述（由申请人提供）：淀粉样前体蛋白（APP）和淀粉样肽（AQ）的代谢是阿尔茨海默病（AD）发病机制的关键决定因素。 APP 是一种存在于质膜中的 1 型跨膜蛋白。一小部分 APP 经历内吞作用并被运输到晚期内体，其中 ¿- 和 β-分泌酶的蛋白水解裂解导致 A¿ 的释放，释放到细胞外空间（间质液，ISF），即使在正常个体中也是如此。 ISF A 水平升高可能促进聚集成可溶性低聚物和不溶性淀粉样斑块，以及 AD 病理的后续发展。除了产生之外，A¿ 的降解和清除也显着影响 ISF A 水平和斑块发病机制。据推测，年龄相关和疾病特异性的溶酶体功能障碍驱动 AD 发病机制。虽然溶酶体功能障碍的具体根本原因仍待阐明，但由此产生的疾病促进机制可能取决于细胞类型。 例如，在产生 A 的神经元中，生理性溶酶体蛋白水解可能有利于在释放之前完全的、非淀粉样蛋白形成的 APP 加工和/或 A 的降解。在星形胶质细胞中，溶酶体活性可能对于细胞内吸收的细胞外 A（以及可能的淀粉样原纤维）的分解代谢很重要；而在小胶质细胞中，它可能促进吞噬淀粉样沉积物的清除。了解细胞类型特异性溶酶体功能障碍在 AD 发病机制中的作用对于确定潜在的干预目标至关重要。普遍表达的转录因子 EB (TFEB) 最近被确定为溶酶体生物发生、内吞作用和自噬的主要调节因子。虽然目前有刺激自噬的药物（例如雷帕霉素），但 TFEB 调节的转录程序可协调增加通过多种溶酶体降解途径的通量；并足以缓解各种溶酶体贮积病中的异常底物积累和病理。我们的初步数据表明，与对照相比，外源 TFEB 表达减少了 N2a-APP695 细胞（APP 处理的神经母细胞瘤细胞模型）的 A¿ 产生/释放。此外，N2a 细胞中的 TFEB 表达导致外源 A¿ 的吸收增加并加速降解。这些数据表明 TFEB 诱导的溶酶体生物发生通过多种细胞机制增强 APP 和 A¿ 的降解。在本提案中，我们假设通过外源表达 TFEB 来增强溶酶体生物合成将以细胞类型特异性方式抑制 AD 发病机制：在神经元中，TFEB 将促进 APP 和 A¿ 的完全蛋白水解，导致 A¿ 生成减少和稳态 ISF A¿ 水平降低；而在星形胶质细胞中，它会增强 A¿ 的摄取和降解，导致 ISF A 半衰期缩短。这两种机制都会减弱淀粉样斑块的沉积。我们将在以下目标中检验这一假设： 1. 确定 TFEB 诱导的溶酶体生物发生对神经元中 APP 加工和 A¿ 产生的影响。 2. 确定 TFEB 星形胶质细胞表达对 A¿ 和淀粉样蛋白分解代谢以及斑块生长的影响。