Fig. 1

Identification and validation of small molecule autophagy-lysosome modulators that increase PGRN. a Schematic of the human GRN promoter dual reporter plasmid used for identifying compounds that affect PGRN expression at the transcriptional level. Secreted Gaussia Luciferase (GLuc) activity monitors GRN promoter activity while secreted Alkaline Phosphatase (SEAP) activity monitors transfection efficiency and is used for normalization. b Fold-change in reporter expression activity (GLuc/SEAP) after transfection into HEK293T cells followed by treatment with a custom library of autophagy-lysosome modulators. SAHA (1 μM), which increases GRN transcription, was used as a positive control for the reporter assay. Bafilomycin A1 (50 nM) and chloroquine (50 μM), which increase PGRN expression primarily via a post-transcriptional mechanism, were included as negative controls. Trehalose was used at 100 mM, as reported in the literature for inducing autophagy. All other compounds were used at 1 μM. The data presented are the average of two independent replicates of the drug screen. c Immunoblot of cell lysates and conditioned media (bottom) from human neuroglioma (H4) cells treated with vehicle, SAHA (1 μM), rapamycin (1 μM), PP242 (1 μM), Torin1 (1 μM), or trehalose (100 mM) for 24 h. Immunoblot of cell lysates from d human neuroblastoma cells (SH-SY5Y) cells and e mouse neuroblastoma cells (N2a) treated as in c. f Immunoblot of cell lysates from primary mouse cortical neurons (E18) treated for 24 h with vehicle, trehalose (100 mM), or PP242 (1 μM). In c-f, p62 and/or LC3-II were used to monitor autophagy induction and P-S6 and/or P-4EBP1 were used to assess mTOR inhibition. Immunoblot images are representative of at least two independent experiments