ENHANCING LYSOSOME BIOGENESIS TO PREVENT AMYLOID PLAQUE PATHOGENESIS

描述（申请人提供）：淀粉样前体蛋白（APP）和淀粉样肽（A）的代谢是阿尔茨海默病（AD）发病机制的关键决定因素。APP是存在于质膜中的1型跨膜蛋白。APP的一小部分经历内吞作用，并被贩运到晚期内体，在那里蛋白水解裂解的？分泌酶导致A？的释放，A？被释放到细胞外空间（间质液，ISF），即使在正常个体中。升高的ISF A？水平可能促进聚集成可溶性寡聚体和不溶性淀粉样斑块，以及随后AD病理学的发展。除了产生，A？降解和清除显著影响ISF A？水平和斑块发病机制。据推测，年龄相关的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潜在原因继续被解开，但所产生的疾病促进机制可能取决于细胞类型。 例如，在产生A ²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进行完全的、非淀粉样蛋白生成的APP加工和/或A ²降解。在星形胶质细胞中，溶酶体活性可能对细胞外A？（以及可能的淀粉样纤维）在细胞内摄取的催化作用很重要;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靶点至关重要。普遍表达的转录因子EB（TFEB）最近被鉴定为溶酶体生物发生、内吞作用和自噬的主要调节因子。虽然药物目前可用（例如，由于TFEB调节的转录程序（例如雷帕霉素）刺激自噬，TFEB调节的转录程序协调地增加通过多种溶酶体降解途径的通量;并且足以减轻各种溶酶体贮积病中的异常底物积累和病理学。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-APP 695细胞（APP加工的神经母细胞瘤细胞模型）的A？产生/释放。此外，TFEB在N2a细胞中的表达导致外源性应用的A？的吸收增加和降解加速。这些数据表明，TFEB诱导的溶酶体生物合成通过几种细胞机制增强APP和A？降解。在该提议中，我们假设通过TFEB的外源性表达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 <$水平降低;而在星形胶质细胞中，它将增强A <$摄取和降解，导致ISF A <$半衰期降低。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.确定星形胶质细胞表达TFEB对A？和淀粉样蛋白降解以及斑块生长的影响。

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.