Abstract
Purpose:
Damage to the retinal pigment epithelium (RPE) contributes to inherited and age-related macular degenerations (AMD). Two features that promote RPE dysfunction are inefficient autophagy and chronic inflammation. We recently showed that lipofuscin bisretinoids in Abca4-/- mice activate acid sphingomyelinase (ASMase), which hydrolyzes sphingomyelin to ceramide. Excess ceramide increases microtubule acetylation and prevents autophagosome trafficking. Treatment with an FDA-approved ASMase inhibitor restores autophagy in the RPE. Here, we first investigated how microtubule acetylation impacts endo-lysosome trafficking, which mediates inflammatory responses; next we evaluated the potential of ASMase inhibitors in promoting debris removal and reducing inflammation in the RPE.
Methods:
Cell surface delivery of the complement-regulatory protein CD59, which inhibits membrane attack complex (MAC) assembly, was measured by live imaging using spinning disk microscopy of adult primary polarized RPE. Membrane repair by lysosome exocytosis after MAC attack was monitored in real-time by total internal reflection fluorescence (TIRF) microscopy. Pro-inflammatory protein expression was assessed by immunohistochemistry of mouse retinal cryosections, immunoblotting and cytokine ELISAs. We used desipramine to inhibit ASMase in the RPE.
Results:
Live imaging and immunofluorescence data show that membrane CD59 and robust lysosome exocytosis protect the RPE from MAC attack. RPE with bisretinoids have less cell surface CD59 and are defective in lysosome exocytosis, making them vulnerable to complement-mediated damage. RPE from Abca4-/- mice or primary RPE with bisretinoids secrete more pro-inflammatory proteins (e.g., MHC class II) compared to healthy RPE. Desipramine treatment restores cell surface CD59, corrects lysosome exocytosis defects and decreases pro-inflammatory responses.
Conclusions:
There is a critical need for novel therapies that target the earliest stages of RPE dysfunction to prevent vision loss in macular degenerations. Our mechanism-based approach identified ASMase as a promising drug target that tackles the twin problems of cellular clearance and inflammation, which are key mediators of RPE damage in AMD. We are currently evaluating the efficacies of a panel of FDA-approved ASMase inhibitors in maintaining multiple aspects of cellular homeostasis in the RPE.