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）：淀粉样蛋白前体蛋白（APP）和淀粉样蛋白肽（A¿）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1型跨膜蛋白，存在于质膜中。APP的一部分经历内吞作用并被运输到晚期的核内体，在那里通过¿-和？-分泌酶导致A - γ释放到细胞外空间（间质液，ISF），即使在正常人中也是如此。高水平的ISF A¿可能促进聚集成可溶性低聚物和不溶性淀粉样斑块，并随后发展为AD病理。除了产生外，A¿的降解和清除显著影响ISF A¿水平和斑块发病机制。人们一直认为，与年龄相关和疾病特异性的溶酶体功能障碍驱动AD的发病机制。虽然溶酶体功能障碍的具体潜在原因仍在继续揭示，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完整的、非淀粉样变性的APP加工和/或释放前的A¿降解。在星形胶质细胞中，溶酶体活性可能对细胞内摄取的细胞外A¿（可能还有淀粉样原纤维）的分解代谢很重要；而在小胶质细胞中，它可以促进被吞噬的淀粉样蛋白沉积物的清除。了解细胞型特异性溶酶体功能障碍在阿尔茨海默病发病机制中的作用对于确定潜在的干预靶点至关重要。泛在表达的转录因子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。虽然目前有药物（如雷帕霉素）刺激自噬，但tfeb调节的转录程序通过多种溶酶体降解途径协调增加通量；足以缓解各种溶酶体积存病的底物异常积累和病理。我们的初步数据表明，与对照组相比，外源性TFEB表达降低了N2a-APP695细胞（一种APP加工的神经母细胞瘤细胞模型）A¿的产生/释放。此外，TFEB在N2a细胞中的表达导致外源性A¿的摄取增加和降解加速。这些数据表明，tfeb诱导的溶酶体通过几种细胞机制增强了APP和A¿的降解。在这项研究中，我们假设通过外源性表达TFEB来增强溶酶体的生物发生将以一种细胞类型特异性的方式抑制AD的发病机制：在神经元中，TFEB将促进APP和a¿的完全蛋白水解，导致a¿的生成减少和稳态ISF a¿水平的降低；而在星形胶质细胞中，它会增强A的摄取和降解，导致ISF A的半衰期缩短。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APP加工和A¿产生的影响。2. 确定星形胶质细胞表达TFEB对A¿和淀粉样蛋白分解代谢以及斑块生长的影响。