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.
Highlights
Autophagosome membranes and is itself degraded within autolysosomes
EDITORS’ PICK: A specific live-cell assay for native autophagic flux reflect increased autophagy induction or late-stage inhibition of autophagsome clearance; discriminating among these mechanisms is crucial for drug development, many assays are effectively unable to do so
A vector containing the Dendra2 open reading frame (ORF) flanked by 400 bp of homologous sequence 50 and 30 to the MAP1LC3B start codon was introduced to facilitate homology directed repair (HDR), thereby creating a sequence encoding Dendra2 fused to the N-terminus of LC3
Summary
Autophagosome membranes and is itself degraded within autolysosomes. For these reasons, it often serves as both a marker of autophagosomes and a representative autophagy substrate [3]. We labeled endogenous LC3 with Dendra using CRISPR/Cas editing, producing a novel cell line capable of assaying autophagic flux in living cells without the need for drug treatment, protein overexpression, or measurements of pathway intermediates, establishing a faithful reporter of native autophagy activity unadulterated by exogenous manipulations Leveraging this cell line for its unique perspective on autophagy and the opportunities it presents, we adapted Dendra2-LC3 cells for conducting high-content screens and identified several new and active autophagy modulators with promising therapeutic properties. We found that autophagy induction failed to consistently improve neuronal survival, instead displaying distinct effects that varied depending on the underlying pathogenic mutation
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