12Autophagy is an evolutionarily conserved pathway mediating the breakdown of cellular 13 proteins and organelles. Emphasizing its pivotal nature, autophagy dysfunction 14 contributes to many diseases; nevertheless, development of effective autophagy 15 modulating drugs is hampered by fundamental deficiencies in available methods for 16 measuring autophagic activity, or flux. To overcome these limitations, we introduced the 17 photoconvertible protein Dendra2 into the MAP1LC3B locus of human cells via 18 CRISPR/Cas9 genome editing, enabling accurate and sensitive assessments of 19 autophagy in living cells by optical pulse labeling. High-content screening of 1,500 tool 20 compounds provided construct validity for the assay and uncovered many new 21 autophagy modulators. In an expanded screen of 24,000 diverse compounds, we 22 identified additional hits with profound effects on autophagy. Further, the autophagy 23 activator NVP-BEZ235 exhibited significant neuroprotective properties in a 24 neurodegenerative disease model. These studies confirm the utility of the Dendra2-LC3 25 assay, while simultaneously highlighting new autophagy-modulating compounds that 26 display promising therapeutic effects. 27 28 Introduction 29 Macroautophagy (hereafter referred to as autophagy) is an essential pathway for 30 protein homeostasis whereby cytoplasmic proteins and organelles are delivered to 31 lysosomes for degradation 1 . Through the coordinated action of a series of autophagy-32 related (ATG) proteins and cargo receptors including p62/SQSTM1, NBR1, and 33 optinuerin 2 , substrates are sequestered within double membrane vesicles called 34 autophagosomes. Autophagosomes mature as they traffic along microtubules and 35 eventually fuse with lysosomes to form autolysosomes, wherein hydrolases degrade 36 autophagic cargo. The protein LC3 (ATG8) is an obligate component of autophagosome 37 membranes and is itself degraded within autolysosomes. For these reasons, it often 38 2 serves as both a marker of autophagosomes and as a representative autophagy 39 substrate 3 . 40Underscoring the critical requirement of autophagy in cellular homeostasis, 41 deletion of core autophagy genes in mice results in embryonic lethality 4,5,6 . Accordingly, 42 dysfunctional autophagy is linked to a wide spectrum of human disease including 43 neurodegeneration, cancer, metabolic disorders, infectious and cardiovascular 44 diseases 7 . Often these conditions involve deficiencies in one or more steps of 45 autophagy, resulting in impaired clearance of potentially toxic cellular components, 46 and/or a failure to replenish amino acids required for anabolic processes. In these 47 instances, enhancing the rate of autophagic cargo clearance, commonly referred to as 48 flux, would be beneficial. Conversely, autophagy can promote tumor progression and 49 resistance to chemotherapy for some cancers 8,9,10,11 . Here, autophagy inhibition may 50 represent a more apt therapeutic strategy 7 . 51Autophagy is of particular importance in the central nervous system...