ENHANCING LYSOSOME BIOGENESIS TO PREVENT AMYLOID PLAQUE PATHOGENESIS

摘要 淀粉样前体蛋白（APP）和淀粉样肽（A <$）的代谢是淀粉样变性的关键决定因素。 阿尔茨海默病（AD）的发病机制。APP是存在于血浆中的1型跨膜蛋白 膜的APP的一部分经历内吞作用并被运输到晚期内体，在晚期内体中蛋白水解。 通过分泌酶和分泌酶的切割导致A <$的释放，A <$被释放到细胞外空间 （间质液，ISF），即使在正常个体中。ISF A？水平升高可能会促进聚集成 可溶性低聚物和不溶性淀粉样蛋白斑块，以及随后AD病理学的发展。除了 A的产生、降解和清除显著影响ISF A水平和斑块发病机制。它 已经假定年龄相关的和疾病特异性的溶酶体功能障碍驱动AD发病机制。 虽然溶酶体功能障碍的具体潜在原因继续被解开，由此产生的疾病- 促进机制可能取决于细胞类型。例如，在神经元中，A？产生的地方， 生理性溶酶体蛋白水解可能有利于完全的、非淀粉样蛋白生成的APP加工和/或A？ 在释放前降解。在星形胶质细胞中，溶酶体活性可能对细胞外基质的释放很重要。 A？（可能还有淀粉样纤维）在细胞内被吸收;而在小胶质细胞中，它可能促进 吞噬淀粉样沉积物。了解细胞类型特异性溶酶体功能障碍在AD中的作用 发病机制对于确定潜在的干预目标至关重要。 普遍表达的转录因子EB（TFEB），最近已被确定为主调节因子 溶酶体生物发生、内吞作用和自噬作用。虽然药物目前可用（例如，雷帕霉素） 刺激自噬，TFEB调节的转录程序协调增加通量，通过 多个溶酶体降解途径;并且足以减轻异常底物积累， 各种溶酶体贮积病的病理学。我们的初步数据表明，外源性TFEB 表达减少N2a-APP 695细胞（APP的神经母细胞瘤细胞模型）的A？产生/释放 处理）与对照组相比。此外，N2a细胞中TFEB的表达导致摄取增加， 加速了外源A的降解。这些数据表明TFEB诱导的溶酶体 生物发生通过几种细胞机制增强APP和A？降解。在本提案中，我们 假设用TFEB外源性表达增强溶酶体生物发生将抑制 AD发病机制以细胞类型特异性的方式：在神经元中，TFEB将促进 APP和A <$导致A <$生成减少和稳态ISF A <$水平降低;而 星形胶质细胞，它会增强A <$摄取和降解，导致减少ISF A <$半衰期。两种机制 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 β和淀粉样蛋白catenase的影响，以及斑块生长。

ABSRACT 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 intracellularly; 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.