Retina and RPE lipid profile changes linked with ABCA4 associated Stargardt's maculopathy

Abstract

Stargardt maculopathy, caused predominantly by mutations in the ABCA4 gene, is characterized by an accumulation of non-degradable visual pigment derivative, lipofuscin, in the retinal pigment epithelium (RPE) - resulting in RPE atrophy. RPE is a monolayer tissue located adjacent to retinal photoreceptors and regulates their health and functioning; RPE atrophy triggers photoreceptor cell death and vision loss in Stargardt patients. Previously, ABCA4 mutations in photoreceptors were thought to be the major contributor to lipid homeostasis defects in the eye. Recently, we demonstrated that ABCA4 loss of function in the RPE leads to cell-autonomous lipid homeostasis defects. Our work underscores that an incomplete understanding of lipid metabolism and lipid-mediated signaling in the retina and RPE are potential causes for lacking treatments for this disease. Here we report altered lipidomic in mouse and human Stargardt models. This work provides the basis for therapeutics that aim to restore lipid homeostasis in the retina and the RPE.

Keywords: ABC transporters; ABCA4 loss of function; Lipid homeostasis; Lipidomic; Macular degeneration.

Figure 1:. ABCA4 loss of function altered major phospholipids/fatty acids lipids in the RPE.

LC-MS/MS analysis on the RPE/choroid and retina tissues from 11M WT and Abca4^−/− mice (n=16 eye/animal). LC-MS/MS analysis was performed in a Xevo TQ - S equipped with Acquity I class UPLC with a flow-through needle (Waters, Milford, MA, USA). A: A box plot showing relevant changes in the most common PC species (with a concentration > 4-13 nmol/eye) in the RPE. Note decreased levels of PC38:6 (DHA/16:0) and PC40:6 (DHA/18:0) and increased PC36:4 and PC38:4 in Abca4^−/− mice (*: p<0.05, ***: p<0.001). B: Box plot showing variations in the most frequent PE species (concentrations > 2-19 nmol/eye) in Abca4^−/− RPE. Note decreased PE (40:6) (DHA/18:0) and increased PE (36:1) and PE (38:7) in Abca4^−/− mice (*: p<0.05, ***: p<0.001). C: The most common phosphatidylserine(PS) species (concentrations > 1.5-10 nmol/eye) are shown using a box plot. Note decreased PS (46:12) and increased PS (38:5), PS (38:4) (18:0/20:4) in the Abca4^−/−(*: p<0.05). D: Box plots showing relevant changes in the most common PS species phospholipids (concentrations > 1.5-4.5 nmol/eye) in the retina (*: p<0.05, **: p<0.01). n represents independent samples from each group. The statistical analysis was performed using GraphPad Prism software (La Jolla, CA).

Figure 2:. ABCA4 loss of function increased cholesterol, its metabolite, and ceramide species in the Stargardt-iRPE cultures and Abca4^−/−-RPE/choroid.

LC-MS/MS analysis on t Stargardt-iRPE treated with POS for seven days (n=3) and the RPE/choroid tissues from 11M WT and Abca4^−/− mice (n=12 eye/animal). A: A box plot showing relevant changes in the ceramide species in the Stargardt-iRPE treated with POS. Note Increased levels of Cer 16:0 in the collected apical medium from Stargardt-iRPE ( ***: p<0.001). B: A box plot showing relevant changes in the cholesterol and its metabolite in the Stargardt-iRPE treated with POS. Note Increased levels of cholesterol and 5, 6-Epoxy Cholestanol in the collected medium from the apical and basal side in Stargardt-iRPE (*: p<0.05, **: p<0.01, ***: p<0.001). C: Box plot showing variations in the ceramide species in Abca4^−/− RPE. Note Increased levels of Cer 24:0 in the Abca4^−/− RPE ( **: p<0.01). D: Box plots showing relevant changes in the cholesterol and its metabolite in the Abca4^−/− RPE. Note Increased levels of cholesterol and 5, 6-Epoxy Cholestanol in the Abca4^−/− RPE ( **: p<0.01). n represents independent samples from each group.

Figure 3.. Schematic design of summarizing lipidomic change in Stargardt disease.

In Stargardt POS-dependent pathway, ABCA4 loss of function results in the ongoing intracellular accumulation of lipofuscin material, including A2E, which has been associated with lysosomal dysfunction, activating the complement system and autophagy down-regulation through elevated BMP, cholesterol, and ceramide level in the RPE cells. As shown in the figure, these defects can be restored by targeting cholesterol transport and the ceramide pathway. ABCA4 deficiency can also cause lipid handling defects cell autonomously in the RPE cells. In addition, the accumulated cholesterol and ceramide are associated with lysosome dysfunction in iRPE cells. Diagram created with BioRender.com.