Research Paper Volume 13, Issue 8 pp 10866—10890

Pharmacologic activation of autophagy without direct mTOR inhibition as a therapeutic strategy for treating dry macular degeneration

Safety of confirmed autophagy inducers in primary hfRPE culture. (A) Tight-junction integrity, as measured by trans-epithelial electrical resistance (TEER), is a general marker of RPE health. Drug or vehicle (DMSO) replaced daily with measurement just before drug replacement. FLBZ shows enhanced TEER while all others, especially mTOR inhibitors Torin and GSK, demonstrate progressively lower tight-junction integrity. n=6. (B) Cytotoxicity as measured by percent of total possible LDH release into the apical supernate. Drug or vehicle (DMSO) replaced daily with supernate collected just before drug replacement. All compounds demonstrated slightly lower cytotoxicity than DMSO control. Note scale break on Y-axis, indicating all conditions, including vehicle, demonstrated minimal LDH release. n=6. (C) Outer segment (OS) phagocytosis efficiency, as measured by disappearance of rhodopsin, the primary protein in OS. Purified OS are fed to RPE cultures and Western blotting for rhodopsin (Rho) indicates undigested OS remaining, as elaborated in Methods. Direct mTOR inhibition (Torin, GSK) reduces phagocytosis efficiency, whereas D4476 and FLBZ have no effect on phagocytosis. Representative blot below each graph, with GAPDH bands demonstrating equal cell mass across Transwells used for the phagocytosis assays. Torin n=12, GSK n=4, FLBZ n=9, D4476 n=3. Uncropped blots for Figure 2 in Supplementary Figure 6. ns p > 0.05, *p .

Figure 2. Safety of confirmed autophagy inducers in primary hfRPE culture. (A) Tight-junction integrity, as measured by trans-epithelial electrical resistance (TEER), is a general marker of RPE health. Drug or vehicle (DMSO) replaced daily with measurement just before drug replacement. FLBZ shows enhanced TEER while all others, especially mTOR inhibitors Torin and GSK, demonstrate progressively lower tight-junction integrity. n=6. (B) Cytotoxicity as measured by percent of total possible LDH release into the apical supernate. Drug or vehicle (DMSO) replaced daily with supernate collected just before drug replacement. All compounds demonstrated slightly lower cytotoxicity than DMSO control. Note scale break on Y-axis, indicating all conditions, including vehicle, demonstrated minimal LDH release. n=6. (C) Outer segment (OS) phagocytosis efficiency, as measured by disappearance of rhodopsin, the primary protein in OS. Purified OS are fed to RPE cultures and Western blotting for rhodopsin (Rho) indicates undigested OS remaining, as elaborated in Methods. Direct mTOR inhibition (Torin, GSK) reduces phagocytosis efficiency, whereas D4476 and FLBZ have no effect on phagocytosis. Representative blot below each graph, with GAPDH bands demonstrating equal cell mass across Transwells used for the phagocytosis assays. Torin n=12, GSK n=4, FLBZ n=9, D4476 n=3. Uncropped blots for Figure 2 in Supplementary Figure 6. ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.