Concurrent detection of autolysosome formation and lysosomal degradation by flow cytometry in a high-content screen for inducers of autophagy.
Concurrent detection of autolysosome formation and lysosomal degradation by flow cytometry in a high-content screen for inducers of autophagy.
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DOI：
10.1186/1741-7007-9-38
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发表时间：
2011-06-02
期刊：
影响因子：
5.4
通讯作者：
Brady NR
中科院分区：
文献类型：
--
作者：
Hundeshagen P;Hamacher-Brady A;Eils R;Brady NR
自噬介导胞质成分的溶酶体降解。最近的工作将自噬功能障碍与病理联系起来，包括癌症和心血管疾病。迄今为止，由于缺乏能够精确报告自噬活性的标准化检测方法，调节自噬的临床适用药物的鉴定受到阻碍。我们开发并实施了一种基于流式细胞术的高内涵筛选方法，用于快速、精确和定量地测量自噬的药物控制。我们的测定允许在基础和激活自噬条件下对自溶酶体形成和降解以及内溶酶体活性进行时间分辨的单独测量。作为概念验证，我们分析了传统的自噬调节剂，包括心脏保护化合物氨基咪唑甲酰胺核糖核苷酸 (AICAR)、雷帕霉素和白藜芦醇，并揭示了雷帕霉素和自噬抑制剂 3-甲基腺嘌呤 (3-MA) 的惊人条件依赖性。为了识别具有转化潜力的新型自噬调节剂，我们筛选了 Prestwick 化学库中的 1,120 种美国食品和药物管理局 (FDA) 批准的化合物，以了解其对自溶酶体形成的影响。总共有 38 种化合物被鉴定为潜在的自噬激活剂，36 种化合物被鉴定为潜在的自噬抑制剂。值得注意的是，自噬增强剂中有强心苷，我们从中选择了地高辛、旋花花苷和地高辛，通过标准生化和成像技术进行验证。我们报告了这些强心苷诱导自噬通量，以及允许特定增强自噬活性而不影响内溶酶体活性的浓度。我们对自噬和内溶酶体活性的系统分析优于传统的自噬测定，并强调了药物对自噬影响的复杂性。我们证明了现有监管机构的条件依赖性。此外，我们还发现了新的自噬调节剂，并将强心苷描述为自噬流的新型有效诱导剂。
Autophagy mediates lysosomal degradation of cytosolic components. Recent work has associated autophagic dysfunction with pathologies, including cancer and cardiovascular disease. To date, the identification of clinically-applicable drugs that modulate autophagy has been hampered by the lack of standardized assays capable of precisely reporting autophagic activity. We developed and implemented a high-content, flow-cytometry-based screening approach for rapid, precise, and quantitative measurements of pharmaceutical control over autophagy. Our assay allowed for time-resolved individual measurements of autolysosome formation and degradation, and endolysosomal activities under both basal and activated autophagy conditions. As proof of concept, we analyzed conventional autophagy regulators, including cardioprotective compounds aminoimidazole carboxamide ribonucleotide (AICAR), rapamycin, and resveratrol, and revealed striking conditional dependencies of rapamycin and autophagy inhibitor 3-methyladenine (3-MA). To identify novel autophagy modulators with translational potential, we screened the Prestwick Chemical Library of 1,120 US Food and Drug Administration (FDA)-approved compounds for impact on autolysosome formation. In all, 38 compounds were identified as potential activators, and 36 as potential inhibitors of autophagy. Notably, amongst the autophagy enhancers were cardiac glycosides, from which we selected digoxin, strophanthidin, and digoxigenin for validation by standard biochemical and imaging techniques. We report the induction of autophagic flux by these cardiac glycosides, and the concentrations allowing for specific enhancement of autophagic activities without impact on endolysosomal activities. Our systematic analysis of autophagic and endolysosomal activities outperformed conventional autophagy assays and highlights the complexity of drug influence on autophagy. We demonstrate conditional dependencies of established regulators. Moreover, we identified new autophagy regulators and characterized cardiac glycosides as novel potent inducers of autophagic flux.
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4
作者：
Jäger, S;Bucci, C;Eskelinen, EL
通讯作者：
Eskelinen, EL
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