UCLA

Recessive Stargardt disease (STGD1) is an inherited macular degeneration that presents within the first two decades of life. Currently there are no suitable treatments for STGD1, resulting in progressive central vision loss in patients. A key pathological feature of STGD1 is the deposition of autofluorescent lipofuscin in the RPE cells. These lipofuscin granules contain toxic vitamin A dimers (bisretinoids), lipids, and protein aggregates. STGD1 is caused by mutations in the ATP-binding cassette subfamily A member 4 (ABCA4) gene. Traditionally, ABCA4 was thought to be exclusively expressed in membrane discs of photoreceptors outer segments (OS). ABCA4 functions as an outwardly directing flippase, from lumen to cytoplasmic side of disc membranes, for the conjugate of retinaldehyde and phosphatidylethanolamine, N-retinylidene-phosphatidylethanolamine (N-ret-PE). Additionally, ABCA4 is known to transport phosphatidylethanolamine (PE) alone as well. Recent evidence showed that ABCA4 is endogenously expressed and functional in the membranes of RPE cells. In the RPE, ABCA4 localizes within the endo-lysosomes and facilitates recycling of free retinaldehyde released from rhodopsin proteolysis following daily phagocytosis. This prevents build-up of reactive retinaldehydes and formation of bisretinoids that are cytotoxic. This novel finding supports the idea that the RPE is a key cellular player in STGD1 pathogenesis, a topic that is introduced in Chapter 1. This thesis builds upon this ground-breaking discovery to explore the molecular mechanisms contributing to cell autonomous RPE dysfunction specific to STGD1. The RPE cells are particularly sensitive to changes in endo-lysosomal function because of their high phagocytic activity. To prevent buildup of photooxidative damage, photoreceptors undergo a daily renewal process. RPE cells phagocytose 10% of photoreceptor OS, which are enriched in lipids and proteins. Each RPE cell provides support to ~50-100 photoreceptors, resulting in phagocytosis of an immense amount of material which must be promptly cleared via the endo-lysosomal pathway to prevent accumulation of toxic aggregates in lipofuscin deposits. Maintaining RPE cell integrity and function requires proper clearance of engulfed phagosomal material. Convergence of this huge degradative burden and ABCA4 localization to the endo-lysosomal compartment underscores the importance to study components of the endo-lysosomal system in the pathophysiology of STGD1. The two main goals of this dissertation were to (i) understand how loss of ABCA4 flippase activity affects lipid handling in the RPE and (ii) molecular mechanism leading to protein aggregates found in lipofuscin. In this study, we utilized a newly developed ‘disease-in-a-dish’ RPE model derived from three patients, clinically and molecularly diagnosed with STGD1, and the established STGD1 mouse model (Abca4-/-). Chapter 2 of this dissertation presents the premiere changes in lipid composition overtime in the RPE lacking functional ABCA4. These studies provide further support that ABCA4 plays a role in modulating lipid homeostasis in the RPE. Here, I show evidence that PE membrane composition is altered and abnormally accumulates in the RPE of STGD1 patient-derived cells and Abca4-/- mice. Furthermore, lipidomics and proteomics analysis reveal significant changes in lipid composition and proteins involved in metabolic pathways in RPE cells lacking functional ABCA4. These results indicate that the ABCA4 PE-moiety flippase activity is crucial in maintaining lipid homeostasis and the availability of lipids as a source of energy in the RPE. Additionally, these findings have directed current research to investigate lipid-mediated bioenergetic changes involving the mitochondria in the RPE of STGD1 models. Chapter 3 of this thesis identifies Cathepsin D (CatD), a lysosomal protease, as a key molecular player contributing to endo-lysosomal dysfunction in STGD1 pathology. Protein degradation in the RPE is largely mediated by CatD. Its maturation and activity depend on a narrow range of acidic pH within the endo-lysosomes. Elevation of lysosomal pH is evidenced in STGD1 patient derived RPE cells. We also show that in both RPE cells from STGD1 patients and Abca4-/- mice, CatD activity and protein maturation is impaired. STGD1 RPE cells have reduced degradation rates of phagocytosed OS compared to normal. However, re-acidification of lysosomal pH can rescue CatD deficiencies in the human-derived RPE cells, suggesting modulation of CatD activity as a potential therapeutic target for ABCA4-mediated retinopathies. In summary, we provide further evidence that RPE dysfunction is the initiator for STGD1 pathogenesis due to loss of ABCA4 in the RPE. This work identifies two new pathways in the RPE responsible for STGD1 involving (i) a shift in lipid homeostasis and metabolism due to changes in membrane lipid composition and (ii) impaired protein degradation resulting from CatD deficiencies. These findings are encouraging for future research aimed at targeting the RPE cells for treatment in STGD1.