Aberrant early endosome biogenesis mediates complement activation in the retinal pigment epithelium in models of macular degeneration

Abstract

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.

Oxidative Stress and Dysfunctional Intracellular Traffic Linked to an Unhealthy Diet Results in Impaired Cargo Transport in the Retinal Pigment Epithelium (RPE)

“…Although a majority of cargos are transported in this manner, there appears to be some variability, perhaps indicating a degree of flexibility which could help RPE to cope with stressful conditions. The importance of correct cargo trafficking and timely proteolytic degradation is demonstrated by evidence from diverse neurodegenerative and storage diseases which reveal impairment of these mechanisms in early stages of pathology . Our studies reveal divergent outcomes for POS trafficking under conditions of oxidative stress, impaired lysosomal function and disrupted autophagy ( Figure ), all of which can occur simultaneously in the senescent RPE .…”

“…Enlarged endosomes is a known feature of early stages of neuropathological conditions such as Alzheimer's disease and Down syndrome where misfolded proteins principally localize to Rab5 vesicles . Swollen endosomes are also reported in RPE of aged human donors and Abca4 −/− mouse model of Stargardt disease . The failure of POS cargos to reach terminal stages of the proteolytic pathway could lead to their accumulation in RPE cells.…”

Mitochondria-dependent phase separation of disease-relevant proteins drives pathological features of age-related macular degeneration

“…We and others have reported that the age-related and pathological accumulation of lipofuscin bisretinoids (composed of A2E and other vitamin A metabolites) in macular degenerations leads to a secondary accumulation of cholesterol in the RPE (13)(14)(15)52). We therefore asked if the increased mobility of ApoE3 and ApoE4 vesicles mitigates the cholesterol storage seen in RPE with A2E.…”

“…To avoid these potentially confounding issues, we chose to employ our well-characterized polarized adult primary porcine RPE cultures (40) transfected with fluorescently-tagged human ApoE2, E3 or E4 to investigate the functions of ApoE isoforms in the RPE. We have extensively used this model for live-cell imaging and have shown that it recapitulates critical disease phenotypes observed in human donor RPE and in mouse models of Stargardt inherited macular degeneration including lipofuscin-induced cholesterol accumulation, and consequent autophagic defects and complement-mediated mitochondrial injury (13)(14)(15). Pertinently, porcine APOE has a promoter polymorphism that decreases endogenous ApoE expression (41).…”

“…Our study elucidates how AMD risk variants act as tipping points to divert the RPE from normal aging towards AMD by disrupting critical metabolic functions, and identifies mitochondrial stress-mediated aberrant phase transitions as a novel mechanism of drusen biogenesis. which impairs autophagy and makes the RPE susceptible to complement-induced mitochondrial injury in models of macular degeneration (13)(14)(15). These studies underscore the need for strictly regulating RPE cholesterol, because it impacts several homeostatic mechanisms, including metabolism and inflammation, whose dysfunction contributes to AMD (7).…”