In vitro activity: AR-7 (Atypical Retinoid 7) is a potent and selective retinoic acid receptor α (RARα) antagonist and an enhancer of the chaperone-mediated autophagy (CMA). CMA is contributory to cellular quality control and the cellular response to stress through the selective degradation of cytosolic proteins in lysosomes. A decrease in CMA activity occurs in aging and in age-related disorders such as neurodegenerative diseases and diabetes. Signaling through RARα inhibits CMA. AR7 was identified to significantly activates CMA activity in mouse fibroblasts. A marked increase in CMA-activating potency is found when AR7 and GR1 are combined, supporting their cooperative effect. Treatment with the transcriptional repressor Actinomycin D partially reduces the stimulatory effect of AR7 on CMA, consistent with transcriptional changes contributing to the upregulation of CMA. The chemical enhancement of CMA protects cells from oxidative stress and from proteotoxicity, supporting a potential therapeutic opportunity when reduced CMA contributes to cellular dysfunction and disease.

Kinase Assay: A RAR-responsive luciferase construct was utilized to monitor the activity of RARα in cultured cells. Cells were co-transfected with pCMX-Gal-L-hRARα and a construct encoding the firefly luciferase reporter gene under the control of a minimal promoter and tandem repeats of the retinoic-acid response element (tk-px3-luc). Co-transfection with a renilla luciferase reporter plasmid was performed to control for efficiency of transfection. RXR activity was measured by similar procedures using co-transfection with pCMX-mRXR and tk-apoA1-luc. Compounds were added 24h after transfection and 48h later cells were lysed and assayed for luciferase activity using the Dual-Luciferase Reporter Assay System (Progmega). Luciferase values were normalized to the renilla luciferase reporter.

Cell Assay: Mouse fibroblasts (NIH3T3) from the American Type Culture Collection were cultured in Dulbecco’s Modified Eagle’s Medium (Sigma) in the presence of 10% newborn calf serum. Serum removal was performed by thoroughly washing the cells with Hanks’ Balanced Salt Solution (Invitrogen) and placing them in serum-free complete medium. Where indicated, cells were treated with the macroautophagy inhibitor 3-methyladenine (Sigma) at a final concentration of 10mM or with 20mM NH4Cl and 100μM leupeptin (Fisher BioReagents) to inhibit lysosomal proteolysis. Where indicated, paraquat was added directly to the culture media to induce oxidative stress. Stable knock-down of LAMP-2A, LAMP-2B or RARα was obtained using vector-mediated stable RNA interference (RNAi) directed specifically against the LAMP-2A or LAMP-2B exon as described previously or against the two following regions of RARα: GAAAGTCTACGTCCGGAAA and GCAGCAGTTCCGAAGAGAT. The plasmid encoding for mCherry-GFP-LC3 was from Addgene and for α-synuclein was a generous gift from Dr. Esther Wong (Nanyang Technological University, Singapore). Sources of chemicals and antibodies were as described previously. ATRA was purchased from Sigma, BMS614 and AM580 were from Tocris Bioscience and the antibody against RARα was from Cell Signaling.