Metabolomics of the Endosomal/Lysosomal System

描述（由申请人提供）：近年来，内体/溶酶体（E/L）系统的囊泡已成为调节许多细胞功能的关键位点。它们的生物学重要性通过许多溶酶体贮积病（LSD）的发生来例证，每种疾病都是由系统中单一蛋白质的缺乏引起的，表现为严重的表型，通常导致神经变性和早期死亡。这些单基因缺陷如何产生如此严重的表型尚不完全清楚;对E/L系统内发生的代谢变化的解剖应该为理解疾病的发病机制提供见解，并为筛查、早期诊断和监测治疗方法提供新的途径。LSD的疾病发病机制起源于E/L系统，这对患者代谢变化的表征提出了独特的挑战，因为循环生物液体不能提供这些变化的全面视图，并且定期获得组织样本是不可行的。我们将使用一种涉及外泌体的新方法来识别和表征LSD中发生的代谢变化。外泌体特别适合于这种类型的研究，因为它们由许多细胞类型分泌，并且在生物流体如血浆、尿液和脑脊液中发现，并且来源于晚期内体的膜。因此，它们含有通常在那里发现的蛋白质的子集，并且可以作为有用的源材料来表征由于疾病而在E/L系统内发生的变化。我们假设来源于疾病细胞的外泌体将反映疾病特异性的蛋白质和脂质变化。在这方面，外来体将提供每个LSD独特的“指纹”或“条形码”。在此，我们建议：1）检验来自人类疾病细胞的外来体具有独特的蛋白质和/或脂质标识符的假设，所述蛋白质和/或脂质标识符将它们与正常细胞的那些区别开，并揭示特定代谢途径的改变。我们将绘制这些通路并在体外和体内验证这些变化。2)检测葡萄糖代谢变化与NPC 1疾病严重程度相关的预测，并可用于监测疾病进展。简而言之，这种新方法是代谢分析的新范式，将有助于有效发现/表征改变的LSD代谢途径，并为我们了解溶酶体贮积病发病机制提供下一步。

DESCRIPTION (provided by applicant): During recent years, the vesicles of the endosomal/lysosomal (E/L) system have emerged as key sites for the regulation of many cellular functions. Their biological importance is exemplified by the occurrence of numerous lysosomal storage diseases (LSDs), each resulting from the deficiency of a single protein in the system, that manifest with severe phenotypes, usually leading to neurodegeneration and early death. How these single gene defects can produce such severe phenotypes is not entirely clear; dissection of the metabolic changes that occur within the E/L system should provide insights towards understanding disease pathogenesis and provide new avenues for screening, early diagnosis, and monitoring of therapeutic approaches. That disease pathogenesis of the LSDs originates in the E/L system presents unique challenges for the characterization of metabolic changes in patients, since circulating biological fluids do not offer a comprehensive view of these changes and obtaining tissue samples on a regular basis is not feasible. We will use a novel approach involving exosomes to identify and characterize the metabolic changes that occur in LSDs. Exosomes are uniquely suited for this type of study because they are secreted by many cell types and are found in biological fluids such as plasma, urine, and cerebrospinal fluid and are derived from the membranes of late endosomes. Thus, they contain a subset of proteins normally found there and can serve as useful source material to characterize the changes that occur within the E/L system as a result of disease. We hypothesize that exosomes derived from disease cells will reflect protein and lipid changes that are specific to the disease. In this respect, exosomes will provide a "fingerprint" or "barcode" unique to each LSD. Here, we propose to: 1) test the hypothesis that exosomes from human disease cells have unique protein and/or lipid identifiers that will distinguish them from those of normal cells and reveal alterations of specific metabolic pathways. We will map these pathways and validatelevaluate these changes in vitro and in vivo. 2) Test the prediction that changes in glucose metabolism correlate with NPC1 disease severity and can be used to monitor disease progression. In short, this new approach is a new paradigm in metabolic analysis and will facilitate the efficient discovery/characterization of altered LSD metabolic pathways and provide us with the next step in understanding lysosomal storage disease pathogenesis.