ENHANCING LYSOSOME BIOGENESIS TO PREVENT AMYLOID PLAQUE PATHOGENESIS

抽奖 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)的发病机制。APP是一种驻留在血浆中的1型跨膜蛋白 薄膜。一小部分APP经历内吞作用，并被运输到晚期内吞体内，在那里蛋白质降解 分泌酶的切割导致A的释放，A被释放到细胞外空间 (间质液体，ISF)，即使在正常人。ISF A的水平升高可能会促进聚集成 可溶性低聚体和不溶性淀粉样斑块，以及随后的AD病理发展。除了……之外 产生、降解和清除显著影响ISF A水平和斑块的发病机制。它 据推测，与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继续被揭开，但由此产生的疾病- 促进机制可能取决于细胞类型。例如，在神经元中，产生A？， 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的加工和/或A 在发布之前进行降解。在星形胶质细胞中，溶酶体的活性可能对细胞外的分解代谢很重要。 A？(可能还有淀粉样纤维)在细胞内摄取；而在小胶质细胞中，它可能促进 吞噬的淀粉样沉积物。了解细胞类型特异性溶酶体功能障碍在AD中的作用 发病机制将是确定干预的潜在靶点的关键。 无处不在表达的转录因子EB(TFEB)，最近被发现是一个主要的调节因子 溶酶体的生物发生、内吞作用和自噬。虽然目前有药物可用(如雷帕霉素) 刺激自噬，TFEB调节的转录程序通过 多种溶酶体降解途径；并足以减轻异常底物积累和 各种溶酶体贮积性疾病的病理学。我们的初步数据表明，外源TFEB 表达减少的N2a-APP695细胞(APP的神经母细胞瘤细胞模型)产生/释放A 处理)与对照进行比较。此外，TFEB在N2a细胞中的表达导致摄取增加 并加速了外源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促进APP和A的降解。在这项提案中，我们 外源表达TFEB促进溶酶体生物合成的假说 特定细胞类型的AD发病机制：在神经元中，TFEB将促进 APP和A导致A生成减少，稳态ISF A水平降低； 对于星形胶质细胞，它会促进A的摄取和降解，从而减少ISF A的半衰期。这两种机制 会减少淀粉样斑块的沉积。我们将从以下几个方面检验这一假说：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

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.