Quantifying autophagy using novel LC3B and p62 TR-FRET assays

PLoS One. 2018 Mar 19;13(3):e0194423. doi: 10.1371/journal.pone.0194423. eCollection 2018.

Abstract

Autophagy is a cellular mechanism that can generate energy for cells or clear misfolded or aggregated proteins, and upregulating this process has been proposed as a therapeutic approach for neurodegenerative diseases. Here we describe a novel set of LC3B-II and p62 time-resolved fluorescence resonance energy transfer (TR-FRET) assays that can detect changes in autophagy in the absence of exogenous labels. Lipidated LC3 is a marker of autophagosomes, while p62 is a substrate of autophagy. These assays can be employed in high-throughput screens to identify novel autophagy upregulators, and can measure autophagy changes in cultured cells or tissues after genetic or pharmacological interventions. We also demonstrate that different cells exhibit varying autophagic responses to pharmacological interventions. Overall, it is clear that a battery of readouts is required to make conclusions about changes in autophagy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Autophagosomes / metabolism*
  • Autophagy / physiology*
  • Fluorescence Resonance Energy Transfer / methods*
  • HEK293 Cells
  • Humans
  • Microtubule-Associated Proteins / metabolism*
  • Rats
  • Sequestosome-1 Protein / metabolism*

Substances

  • LC3 protein, rat
  • Microtubule-Associated Proteins
  • Sequestosome-1 Protein
  • Sqstm1 protein, rat

Grant support

CHDI Foundation is a privately-funded not-for-profit biomedical research organization exclusively dedicated to discovering and developing therapeutics that slow the progression of Huntington’s disease. CHDI Foundation conducts research in a number of different ways; for the purposes of this manuscript, research was conducted at the contract research organization IRBM Science Park and Evotec under a fee-for-service agreement and in collaboration with IRBM Promidis. The authors listed all contributed to the conception, study design, data collection and analysis, decision to publish, and preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.