title: Concurrent detection of autolysosome formation and lysosomal degradation by flow cytometry in a high-content screen for inducers of autophagy.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Division of Theoretical Bioinformatics, German Cancer Research Center and Institute of Pharmacy and Molecular Biotechnology, Bioquant, University of Heidelberg, Heidelberg, Germany.
authors.forename: Phillip
authors.initials: P
authors.lastname: Hundeshagen
authors.forename: Anne
authors.initials: A
authors.lastname: Hamacher-Brady
authors.forename: Roland
authors.initials: R
authors.lastname: Eils
authors.forename: Nathan R
authors.initials: NR
authors.lastname: Brady
mesh_terms: D000818:Animals; D001343:Autophagy / Q000187:drug effects*; D002316:Cardiotonic Agents / Q000494:pharmacology*; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods; D006801:Humans; D008247:Lysosomes / Q000187:drug effects* / Q000378:metabolism
chemical_list: D002316:Cardiotonic Agents