June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Novel mechanisms of RPE injury and drusen nucleation mediated by AMD genetic risk variants
Author Affiliations & Notes
  • Aparna Lakkaraju
    Dept of Ophthalmology School of Medicine, University of California, San Francisco, San Francisco, California, United States
  • Nilsa LaCunza
    Dept of Ophthalmology School of Medicine, University of California, San Francisco, San Francisco, California, United States
  • Li Xuan Tan
    Dept of Ophthalmology School of Medicine, University of California, San Francisco, San Francisco, California, United States
  • Colin Germer
    Dept of Ophthalmology School of Medicine, University of California, San Francisco, San Francisco, California, United States
  • Footnotes
    Commercial Relationships   Aparna Lakkaraju, None; Nilsa LaCunza, None; Li Xuan Tan, None; Colin Germer, None
  • Footnotes
    Support  NIH grant EY023299; BrightFocus Foundation; Macular Society UK
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 1424. doi:
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    • Get Citation

      Aparna Lakkaraju, Nilsa LaCunza, Li Xuan Tan, Colin Germer; Novel mechanisms of RPE injury and drusen nucleation mediated by AMD genetic risk variants. Invest. Ophthalmol. Vis. Sci. 2020;61(7):1424.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Allelic variants in cholesterol transporters and complement proteins are associated with age-related macular degeneration (AMD), which is characterized by dysfunction of the retinal pigment epithelium (RPE) and formation of drusen. We know little about how genetic risk variants drive RPE injury and drusen biogenesis. Drusen are rich in cholesterol and the cholesterol transporter apolipoprotein E (ApoE), which is implicated in AMD: of the three ApoE isoforms expressed by humans (ApoE2, E3 and E4), ApoE2 increases AMD risk and ApoE4 is protective. Here we investigated how ApoE alleles regulate cholesterol trafficking, autophagy and complement-mediated mitochondrial injury in the RPE, and examined how they dictate drusen nucleation.

Methods : Polarized monolayers of primary porcine RPE were transfected with mCherry-ApoE2, E3 or E4 and cholesterol content was measured biochemically. Trafficking of ApoE vesicles and LC3-labeled autophagosomes were examined by high-speed live imaging using spinning disk microscopy. Mitochondrial integrity was assessed by live imaging after complement exposure. Normal and AMD donor retinal tissues were stained for ApoE aggregates and mitochondrial integrity.

Results : Vesicles with ApoE2 (the AMD risk allele) are not dynamic and therefore least efficient in transporting cholesterol in the RPE, compared to ApoE3 or ApoE4 vesicles. Excess cholesterol in RPE expressing ApoE2 prevents autophagosome trafficking and makes the RPE susceptible to complement-induced mitochondrial injury. Recent biophysical studies suggest that compromised proteostasis and mitochondrial function induce aberrant phase transitions of intrinsically disordered proteins like ApoE, leading to their aggregation in the form of biomolecular condensates. In agreement with this, we observed significantly more aggregates in ApoE2 RPE, compared to RPE expressing ApoE3 or ApoE4. Drugs that decrease cholesterol restore autophagy and mitochondrial health, and prevent aggregate formation in ApoE2-expressing RPE.

Conclusions : These studies provide insight into how major AMD risk variants mediate multiple levels of crosstalk between complement, lipid, and metabolic pathways, and thus regulate RPE homeostasis. Our data on the role of cytosolic phase separation and biomolecular condensates in nucleating drusen-like aggregates in ApoE2 RPE could form the basis for a personalized approach to target these deposits in AMD.

This is a 2020 ARVO Annual Meeting abstract.

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