Abstract
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.
| Original language | English (US) |
|---|---|
| Article number | 101003 |
| Journal | Journal of Biological Chemistry |
| Volume | 297 |
| Issue number | 3 |
| DOIs | |
| State | Published - Sep 1 2021 |
Funding
This work was supported by the National Institutes of Health National Institute on Aging P30 AG053760, National Institute of Neurological Disorders and Stroke R01-NS097542 and R01-NS113943, the University of Michigan Protein Folding Disease Initiative, the Center for Chemical Genomics (CCG) at the University of Michigan Life Sciences Institute, Michigan Drug Discovery, the Robert Packard Center for ALS Research, and Ann Arbor Active Against ALS. We thank Dr Vincent Groppi of Michigan Drug Discovery for his continued support and insights; Dr Lois Weisman and Prof. Tamotsu Yoshimori for RFP-GFP-LC3 HeLa cells; and Dr Paula Gedraitis (Molecular Devices, LLC) for technical assistance. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Acknowledgments—This work was supported by the National Institutes of Health National Institute on Aging P30 AG053760, National Institute of Neurological Disorders and Stroke R01-NS097542 and R01-NS113943, the University of Michigan Protein Folding Disease Initiative, the Center for Chemical Genomics (CCG) at the University of Michigan Life Sciences Institute, Michigan Drug Discovery, the Robert Packard Center for ALS Research, and Ann Arbor Active Against ALS. We thank Dr Vincent Groppi of Michigan Drug Discovery for his continued support and insights; Dr Lois Weisman and Prof. Tamotsu Yoshimori for RFP-GFP-LC3 HeLa cells; and Dr Paula Gedraitis (Molecular Devices, LLC) for technical assistance. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
ASJC Scopus subject areas
- Molecular Biology
- Biochemistry
- Cell Biology
