Abnormally enlarged early endosomes (EEs) are pathological features of neurodegenerative diseases, yet insight into the mechanisms and consequences of EE expansion remains elusive. Here, we report swollen apical EEs in the retinal pigment epithelium (RPE) of aged human donors and in the pigmented mouse model of Stargardt early-onset macular degeneration. Using high-resolution live-cell imaging, we show that age-related and pathological accumulation of lipofuscin bisretinoids increases ceramide at the apical surface of the RPE, which promotes inward budding and homotypic fusion of EEs. These enlarged endosomes internalize the complement protein C3 into the RPE, resulting in the intracellular generation of C3a fragments. Increased C3a in turn activates the mechanistic target of rapamycin (mTOR), a regulator of critical metabolic processes such as autophagy. The antidepressant desipramine, which decreases ceramide levels by inhibiting acid sphingomyelinase, corrects EE defects in the RPE of mice. This prevents C3 internalization and limits the formation of C3a fragments within the RPE. Although uncontrolled complement activation is associated with macular degenerations, how complement contributes to pathology in a progressive disease is not well understood. Our studies link expansion of the EE compartment with intracellular complement generation and aberrant mTOR activation, which could set the stage for chronic metabolic reprogramming in the RPE as a prelude to disease. The pivotal role of ceramide in driving EE biogenesis and fusion in the mice RPE suggests that therapeutic targeting of ceramide could be effective in Stargardt disease and other macular degenerations.

Age-related macular degeneration (AMD) damages the retinal pigment epithelium (RPE), the tissue that safeguards photoreceptor health, leading to irreversible vision loss. Polymorphisms in cholesterol and complement genes are implicated in AMD, yet mechanisms linking risk variants to RPE injury remain unclear. We sought to determine how allelic variants in the apolipoprotein E cholesterol transporter modulate RPE homeostasis and function. Using live-cell imaging, we show that inefficient cholesterol transport by the AMD risk-associated ApoE2 increases RPE ceramide, leading to autophagic defects and complement-mediated mitochondrial damage. Mitochondrial injury drives redox state-sensitive cysteine-mediated phase separation of ApoE2, forming biomolecular condensates that could nucleate drusen. The protective ApoE4 isoform lacks these cysteines and is resistant to phase separation and condensate formation. In Abca4 -/-Stargardt macular degeneration mice, mitochondrial dysfunction induces liquid-liquid phase separation of p62/SQSTM1, a multifunctional protein that regulates autophagy. Drugs that decrease RPE cholesterol or ceramide prevent mitochondrial injury and phase separation in vitro and in vivo. In AMD donor RPE, mitochondrial fragmentation correlates with ApoE and p62 condensates. Our studies demonstrate that major AMD genetic and biological risk pathways converge upon RPE mitochondria, and identify mitochondrial stress-mediated protein phase separation as an important pathogenic mechanism and promising therapeutic target in AMD.

Procollagen C-proteinase enhancer 1 (PCPE-1) has been characterized as a protein capable of enhancing the activity of bone morphogenetic protein 1/tolloid-like proteinases (BTPs) in the biosynthetic processing of C-propeptides from procollagens I–III. This processing step is thought necessary to the formation of collagen I–III monomers capable of forming fibrils. Thus, PCPE-1 is predicted to play an important role in scarring, as scar tissue is predominantly composed of fibrillar collagen. Corneal scarring is of great clinical importance, as it leads to loss of visual acuity and, in severe cases, blindness. Here, we have investigated a possible role for PCPE-1 in corneal scarring. Although differences in corneal opacity associated with scarring following injury of Pcolce−/− and WT mice using full-thickness excision or alkali burn models of corneal injury were not grossly apparent, differences in procollagen I processing levels between Pcolce−/− and WT primary corneal keratocytes were consistent with a role for PCPE-1 in corneal collagen deposition. An unexpected finding was that neoangiogenesis, which follows alkali burn cornea injury, was strikingly increased in Pcolce−/− cornea, compared to WT. A series of aortic ring assays confirmed the anti-angiogenic effects of PCPE-1. Another unexpected finding was of abnormalities of epithelial basement membrane and of re-epithelialization following Pcolce−/− corneal injury. Thus, PCPE-1 appears to be of importance as an anti-angiogenic factor and in re-epithelialization following injury in cornea, and perhaps in other tissues as well.