September 2016
Volume 57, Issue 12
Open Access
ARVO Annual Meeting Abstract  |   September 2016
Protective mechanisms against sub-lytic complement attack in the retinal pigment epithelium
Author Affiliations & Notes
  • Li Xuan Tan
    Ophthalomology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States
    Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Kimberly A Toops
    Ophthalomology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Aparna Lakkaraju
    Ophthalomology and Visual Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States
    Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States
  • Footnotes
    Commercial Relationships   Li Xuan Tan, P140282US02 (P); Kimberly Toops, P140282US02 (P); Aparna Lakkaraju, P140282US02 (P)
  • Footnotes
    Support  NIH grants (R01EY023299, P30EY016665), Research to Prevent Blindness, BrightFocus Foundation (M2015350), Wisconsin Partnership Program New Investigator Program, Reeves Foundation, Macular Society UK, Retina Research Foundation, Rebecca Meyer Brown Professorship and gifts from the Schuette-Kraemer family and the Christenson estate for macular degeneration research
Investigative Ophthalmology & Visual Science September 2016, Vol.57, 5783. doi:
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    • Get Citation

      Li Xuan Tan, Kimberly A Toops, Aparna Lakkaraju; Protective mechanisms against sub-lytic complement attack in the retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2016;57(12):5783.

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

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Abstract

Purpose : Abnormal activation of the complement pathway is implicated in inherited and age-related macular degenerations. Assembly of sub-lytic membrane attack complexes (MAC) on the surface of retinal pigment epithelial (RPE) cells forms pores that compromise membrane integrity and promotes inflammation. Here we asked how the RPE limits the assembly of MAC and how MAC pores are sealed to prevent complement-mediated injury.

Methods : Polarized monolayers of primary porcine RPE were exposed to 10% normal human serum as the source of complement for 10 min to induce the assembly of MAC on the RPE membrane. Recycling of the membrane-bound complement regulator CD59 was followed by immunofluorescence microscopy. Membrane repair and mitochondrial fragmentation were monitored by live-cell imaging and biochemical assays. Immunohistochemistry and immunoblotting were used to determine complement activation and mitochondrial health in wild-type and Abca4−/− mice.

Results : Complement exposure causes a dramatic acceleration of CD59 recycling to the apical surface in healthy RPE cells. Live imaging showed that RPE lysosomes fused with the plasma membrane within seconds of complement exposure to close MAC pores and preserve cell integrity. In RPE from Abca4-/- mice or primary RPE with vitamin A dimers, excess cholesterol derailed organelle traffic and this caused a significant reduction in cell surface CD59 and the efficiency of lysosome-mediated membrane repair. Persistent MAC pores in RPE with vitamin A dimers resulted in mitochondrial fragmentation and oxidative stress. Removal of excess cholesterol restored protective mechanisms and maintained mitochondrial homeostasis in Abca4-/- mice RPE.

Conclusions : To date, approaches to target the complement pathway in macular degenerations have all focused on directly inhibiting complement proteins. Our studies move beyond this narrow focus by providing valuable insight into how the RPE deals with complement at a cellular level, and by identifying drugs that can help strengthen innate mechanisms essential for preserving RPE health and function.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

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