Ceramide-mediated apical exosome release by the RPE triggers infiltration of microglia into the subretinal space in models of macular degeneration

Abstract

Purpose : We sought to understand the role of exosomes released by the retinal pigment epithelium (RPE) in macular degenerations and how this could be modulated by small molecule therapeutics. We built on our published studies showing that lipofuscin-mediated cholesterol accumulation activates acid sphingomyelinase (ASM). We hypothesized that the resulting increase in ceramide could drive abnormal exosome biogenesis in the RPE.

Methods : Polarized primary RPE cultures were treated with the lipofuscin bisretinoid A2E to induce cholesterol and ceramide accumulation (Toops et al. 2015; Tan et al. 2016; Kaur et al. 2018). Exosomes were purified via ultracentrifugation or size exclusion chromatography. NanoTracking analysis, STORM imaging, immunoblotting and LC-MS/MS were used to analyze exosome size distribution and protein cargo. Abca4^-/- mice were injected intraperitoneally with 10mg/kg desipramine, an ASM inhibitor. RPE flatmounts were processed for either electron microscopy or immunofluorescence.

Results : Excess ceramide in stressed RPE caused a two-fold increase in release of exosomes from the apical surface. This was corroborated in vivo, where increased numbers of intraluminal vesicles (ILVs) were observed in Abca4^-/- RPE compared to age-matched wildtypes. Desipramine treatment decreased the number of ILVs in Abca4^-/- RPE to that comparable to wildtypes. LC-MS/MS and immunoblotting revealed that exosomes released by bisretinoid-treated RPE had more pro-inflammatory proteins such as connexin 43 and fewer anti-inflammatory proteins when compared with exosomes from healthy RPE. Apical release of connexin43 can act as a chemoattractant for microglia to invade the subretinal space. Immunofluorescence of Abca4^-/- RPE flatmounts revealed significantly more Iba1+ve cells in the subretinal space when compared to wildtype mice.

Conclusions : Here we show that excess RPE ceramide leads to increased formation and release of pro-inflammatory exosomes at the apical surface of the RPE. These exosomes recruit Iba1+ve microglia into the subretinal space. Inhibiting ASM with desipramine decreases RPE ceramide and prevents aberrant exosome secretion. This research provides insight into the role of RPE-derived exosomes in retinal health and disease and identifies a potential therapeutic intervention in macular degenerations.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.