Development of a specific live-cell assay for native autophagic flux

J Biol Chem. 2021 Sep;297(3):101003. doi: 10.1016/j.jbc.2021.101003. Epub 2021 Jul 23.


Autophagy is an evolutionarily conserved pathway mediating the breakdown of cellular proteins and organelles. Emphasizing its pivotal nature, autophagy dysfunction contributes to many diseases; nevertheless, development of effective autophagy modulating drugs is hampered by fundamental deficiencies in available methods for measuring autophagic activity or flux. To overcome these limitations, we introduced the photoconvertible protein Dendra2 into the MAP1LC3B locus of human cells via CRISPR/Cas9 genome editing, enabling accurate and sensitive assessments of autophagy in living cells by optical pulse labeling. We used this assay to perform high-throughput drug screens of four chemical libraries comprising over 30,000 diverse compounds, identifying several clinically relevant drugs and novel autophagy modulators. A select series of candidate compounds also modulated autophagy flux in human motor neurons modified by CRISPR/Cas9 to express GFP-labeled LC3. Using automated microscopy, we tested the therapeutic potential of autophagy induction in several distinct neuronal models of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In doing so, we found that autophagy induction exhibited discordant effects, improving survival in disease models involving the RNA binding protein TDP-43, while exacerbating toxicity in neurons expressing mutant forms of UBQLN2 and C9ORF72 associated with familial ALS/FTD. These studies confirm the utility of the Dendra2-LC3 assay, while illustrating the contradictory effects of autophagy induction in different ALS/FTD subtypes.

Keywords: ALS; FTD; autophagy; high-content; high-throughput; neurodegeneration; photoconvertible; repurposing; screen; stem cell.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Amyotrophic Lateral Sclerosis / genetics
  • Amyotrophic Lateral Sclerosis / therapy
  • Autophagy* / drug effects
  • Autophagy-Related Proteins / genetics
  • C9orf72 Protein / genetics
  • CRISPR-Cas Systems
  • DNA-Binding Proteins / genetics
  • Drug Screening Assays, Antitumor
  • Frontotemporal Dementia / genetics
  • Frontotemporal Dementia / therapy
  • HEK293 Cells
  • High-Throughput Screening Assays
  • Humans
  • Luminescent Proteins / genetics
  • Microtubule-Associated Proteins / genetics
  • Models, Biological
  • Motor Neurons / metabolism
  • Mutation


  • Adaptor Proteins, Signal Transducing
  • Autophagy-Related Proteins
  • C9orf72 Protein
  • C9orf72 protein, human
  • DNA-Binding Proteins
  • Dendra2 protein, Dendronephthya
  • Luminescent Proteins
  • MAP1LC3B protein, human
  • Microtubule-Associated Proteins
  • TARDBP protein, human
  • UBQLN2 protein, human