A novel quantification method for retinal pigment epithelium phagocytosis using a very-long-chain polyunsaturated fatty acids-based strategy

Fangyuan Gao; Emily Tom; Stephanie A Lieffrig; Silvia C Finnemann; Dorota Skowronska-Krawczyk

doi:10.3389/fnmol.2023.1279457

A novel quantification method for retinal pigment epithelium phagocytosis using a very-long-chain polyunsaturated fatty acids-based strategy

Front Mol Neurosci. 2023 Oct 20:16:1279457. doi: 10.3389/fnmol.2023.1279457. eCollection 2023.

Authors

Fangyuan Gao¹, Emily Tom², Stephanie A Lieffrig³, Silvia C Finnemann³, Dorota Skowronska-Krawczyk^{1

2}

Affiliations

¹ Department of Ophthalmology, Center for Translational Vision Research, School of Medicine, UC Irvine, Irvine, CA, United States.
² Department of Physiology and Biophysics, Department of Ophthalmology, Center for Translational Vision Research, School of Medicine, UC Irvine, Irvine, CA, United States.
³ Center for Cancer, Genetic Diseases and Gene Regulation, Department of Biological Sciences, Fordham University, New York, NY, United States.

Abstract

Introduction: The vertebrate retinal pigment epithelium (RPE) lies adjacent to the photoreceptors and is responsible for the engulfment and degradation of shed photoreceptor outer segment fragments (POS) through receptor-mediated phagocytosis. Phagocytosis of POS is critical for maintaining photoreceptor function and is a key indicator of RPE functionality. Popular established methods to assess RPE phagocytosis rely mainly on quantifying POS proteins, especially their most abundant protein rhodopsin, or on fluorescent dye conjugation of bulk, unspecified POS components. While these approaches are practical and quantitative, they fail to assess the fate of POS lipids, which make up about 50% of POS by dry weight and whose processing is essential for life-long functionality of RPE and retina.

Methods: We have developed a novel very-long-chain polyunsaturated fatty acids (VLC-PUFA)-based approach for evaluating RPE phagocytic activity by primary bovine and rat RPE and the human ARPE-19 cell line and validated its results using traditional methods.

Results and discussion: This new approach can be used to detect in vitro the dynamic process of phagocytosis at varying POS concentrations and incubation times and offers a robust, unbiased, and reproducible assay that will have utility in studies of POS lipid processing.

Keywords: liquid chromatography-mass spectrometer; phagocytosis; quantification; retinal pigment epithelium; very-long-chain polyunsaturated fatty acids.

Abstract

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