UCSF

The retinal pigment epithelium (RPE) is the primary site of injury in age-related macular degeneration (AMD), the leading cause of permanent central vision loss in the elderly. Our work seeks to understand the mechanisms driving RPE atrophy to identify novel therapeutic targets for AMD. Using super-resolution and high-speed live imaging, along with a mouse model of disease and AMD human donor tissue, this thesis examines how mitochondrial integrity regulates cellular pathways involved in macular degeneration.

Cholesterol regulates essential cellular processes, but its dysregulation in the RPE can lead to disease. Our lab has demonstrated that complement and cholesterol pathways, which are major genetic and biological risk pathways for AMD, collaborate to damage RPE mitochondria. We demonstrated how allelic variants of apolipoprotein E, a cholesterol transporter implicated in AMD, regulate cholesterol homeostasis in the RPE. The AMD-risk ApoE2 accumulates cholesterol, leading to trafficking defects and mitochondrial injury, whereas the AMD-protective ApoE4 efficiently transports cholesterol and maintains mitochondrial health. ApoE and cholesterol are primary components of drusen, a hallmark of AMD. We identified a novel mechanism linking RPE mitochondrial injury with drusen biogenesis: fragmented mitochondria drive redox state-related liquid-liquid phase separation (LLPS) of ApoE2, the AMD risk isoform.

This thesis also explores how mitochondrial integrity is regulated and how these processes are disrupted in AMD. We report excess oxidative stress and fragmented mitochondria in models of macular degeneration, suggesting a loss of mitochondrial quality control (MQC). Mitochondria undergo repeated cycles of fusion and fission, as well as degradation through mitophagy, to maintain their integrity. However, how the MQC machinery activates to induce mitochondrial fragmentation in diseased RPE remains unclear. Here, we identified three potential mechanisms driving mitochondrial fragmentation in diseased RPE: 1) defects in mitochondrial fusion proteins: mitofusin 2 (MFN2) and optic atrophy 1 (OPA1); 2) increased expression of a mitochondrial fission regulator, mitochondrial fission process 1 (MTFP1); 3) phase separation of a mitochondrial dynamics regulator, mitochondrial rho GTPase 1 (Miro1).

This thesis further investigates mechanisms regulating drusen deposition in the RPE. Ceramide accumulation impairs intracellular trafficking, leading to endosomal abnormalities that activate exocytosis machinery. We observed an upregulation of proteins involved in different aspects of exocytosis, including Rab11, Rab27a, and the endosomal sorting protein HRS.

Overall, these studies highlight the crucial role of mitochondrial health in RPE function and retinal health. Our research reveals that targeting upstream pathologies like excess cholesterol and ceramide to protect RPE mitochondria can prevent drusen formation and RPE atrophy in models of macular degeneration.