ENHANCING LYSOSOME BIOGENESIS TO PREVENT AMYLOID PLAQUE PATHOGENESIS

描述(申请人提供)：淀粉样前体蛋白(APP)和淀粉样多肽(A？)的代谢是阿尔茨海默病(AD)发病的关键决定因素。APP是一种存在于质膜上的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小体，在那里被分泌酶的蛋白水解性裂解导致Aβ的释放，Aβ被释放到细胞外空间(间质液，ISF)，即使在正常人中也是如此。ISF A水平升高可能促进聚集成可溶性低聚体和不溶性淀粉样斑块，并随后发展为AD病理。除产生外，A？的降解和清除显著影响ISF？A？水平和斑块的发病机制。据推测，年龄相关和疾病特异性的溶酶体功能障碍推动了AD的发病。虽然溶酶体功能障碍的具体潜在原因仍在揭开，但由此产生的致病机制可能取决于细胞类型。例如，在产生A？的神经元中，生理性溶酶体蛋白分解可能有利于完整的、非淀粉样变性APP的处理和/或A？在释放之前的降解。在星形胶质细胞中，溶酶体的活性可能对细胞外Aβ(以及可能的淀粉样纤维)在细胞内的分解代谢起重要作用；而在小胶质细胞中，它可能促进吞噬的淀粉样沉积的清除。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。无处不在表达的转录因子EB(TFEB)，最近被发现是溶酶体生物发生、内吞和自噬的主要调节因子。虽然目前已有刺激自噬的药物(如雷帕霉素)，但TFEB调节的转录程序通过多条溶酶体降解途径协调增加通量；并足以缓解各种溶酶体储存疾病中的异常底物积累和病理。我们的初步数据表明，与对照组相比，外源TFEB表达减少了N2a-APP695细胞(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细胞中的表达增加了对外源A的摄取和加速了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促进APP和A的降解。在这个方案中，我们假设通过外源表达TFEB来促进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：在神经元中，TFEB将促进APP和A？的完全蛋白分解，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，它将促进A？的摄取和降解，导致ISF A？半衰期缩短。这两种机制都将减弱淀粉样斑块的沉积。我们将从以下几个方面对这一假说进行验证：1.确定TFEB诱导的溶酶体生物发生对APP加工和神经元A？产生的影响。2.检测星形细胞表达TFEB对Aβ和淀粉样蛋白分解代谢及斑块生长的影响。

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.