In Vitro Screening Platforms for Identifying Autophagy Modulators in Mammalian Cells

Methods Mol Biol. 2019;1880:389-428. doi: 10.1007/978-1-4939-8873-0_26.

Abstract

Autophagy is a vital homeostatic pathway essential for cellular survival and human health. It primarily functions as an intracellular degradation process for the turnover of aggregation-prone proteins and unwanted organelles. Dysregulation of autophagy underlying diverse human diseases reduces cell viability, whereas stimulation of autophagy is cytoprotective in a number of transgenic disease models including neurodegenerative disorders. Thus, therapeutic exploitation of autophagy is considered a potential treatment strategy in certain human diseases, and therefore, chemical inducers of autophagy have tremendous biomedical relevance. In this review, we describe the in vitro screening platforms to identify autophagy modulators in mammalian cells using various methodologies including fluorescence and high-content imaging, flow cytometry, fluorescence and luminescence detection by microplate reader, immunoblotting, and immunofluorescence. The commonly used autophagy reporters in these screening platforms are either based on autophagy marker like LC3 or autophagy substrate such as aggregation-prone proteins or p62/SQSTM1. The reporters and assays for monitoring autophagy are evolving over time to become more sensitive in measuring autophagic flux with the capability of high-throughput applications for drug discovery. Here we highlight these developments and also describe the stringent secondary autophagy assays for characterizing the autophagy modulators arising from the primary screen. Since autophagy is implicated in myriad human physiological and pathological conditions, these technologies will enable identifying novel chemical modulators or genetic regulators of autophagy that will be of biomedical and fundamental importance to human health.

Keywords: Aggregation-prone protein; Autophagy; Autophagy drug discovery; Autophagy modulators; Autophagy substrates; Chemical screen; LC3; Screening platform; p62.

Publication types

  • Research Support, Non-U.S. Gov't
  • Review

MeSH terms

  • Animals
  • Autophagy / physiology*
  • Autophagy-Related Proteins / metabolism*
  • Biological Assay / instrumentation
  • Biological Assay / methods*
  • Cell Culture Techniques / instrumentation
  • Cell Culture Techniques / methods*
  • Cell Line
  • Flow Cytometry / instrumentation
  • Flow Cytometry / methods
  • Genes, Reporter / genetics
  • Humans
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / genetics
  • Microscopy, Fluorescence / instrumentation
  • Microscopy, Fluorescence / methods
  • Microtubule-Associated Proteins
  • Sequestosome-1 Protein / metabolism
  • Transfection / instrumentation
  • Transfection / methods

Substances

  • Autophagy-Related Proteins
  • Luminescent Proteins
  • Microtubule-Associated Proteins
  • Sequestosome-1 Protein