Lack of correlation between the spatial distribution of A2E and lipofuscin fluorescence in the human retinal pigment epithelium.

PURPOSE The accumulation of lipofuscin in the RPE is a hallmark of aging in the eye. The best characterized component of lipofuscin is A2E, a bis-retinoid byproduct of the normal retinoid visual cycle, which exhibits a broad spectrum of cytotoxic effects in vitro. The purpose of our study was to correlate the distribution of lipofuscin and A2E across the human RPE. METHODS Lipofuscin fluorescence was imaged in flat-mounted RPE from human donors of various ages. The spatial distributions of A2E and its oxides were determined using matrix-assisted laser desorption-ionization imaging mass spectrometry (MALDI-IMS) on flat-mounted RPE tissue sections and retinal cross-sections. RESULTS Our data support the clinical observations of strong RPE fluorescence, increasing with age, in the central area of the RPE. However, there was no correlation between the distribution of A2E and lipofuscin, as the levels of A2E were highest in the far periphery and decreased toward the central region. High-resolution MALDI-IMS of retinal cross-sections confirmed the A2E localization data obtained in RPE flat-mounts. Singly- and doubly-oxidized A2E had distributions similar to A2E, but represented <10% of the A2E levels. CONCLUSIONS This report to our knowledge is the first description of the spatial distribution of A2E in the human RPE by imaging mass spectrometry. These data demonstrate that the accumulation of A2E is not responsible for the increase in lipofuscin fluorescence observed in the central RPE with aging.

[1]  J. D. Bronson,et al.  Role of Photoreceptor-specific Retinol Dehydrogenase in the Retinoid Cycle in Vivo* , 2005, Journal of Biological Chemistry.

[2]  S. Jockusch,et al.  Formation of a Nonaoxirane from A2E, a Lipofuscin Fluorophore related to Macular Degeneration, and Evidence of Singlet Oxygen Involvement , 2002 .

[3]  L. D. Del Priore,et al.  Age-related changes in human RPE cell density and apoptosis proportion in situ. , 2002, Investigative ophthalmology & visual science.

[4]  K. Nakanishi,et al.  The lipofuscin fluorophore A2E mediates blue light-induced damage to retinal pigmented epithelial cells. , 2000, Investigative ophthalmology & visual science.

[5]  Gerald McGwin,et al.  Human chorioretinal layer thicknesses measured in macula-wide, high-resolution histologic sections. , 2011, Investigative ophthalmology & visual science.

[6]  A. Cuervo,et al.  A2E, a pigment of RPE lipofuscin, is generated from the precursor, A2PE by a lysosomal enzyme activity. , 2008, Advances in experimental medicine and biology.

[7]  Yiannis Koutalos,et al.  Lipofuscin and N-Retinylidene-N-Retinylethanolamine (A2E) Accumulate in Retinal Pigment Epithelium in Absence of Light Exposure , 2012, The Journal of Biological Chemistry.

[8]  S. Davies,et al.  Photodamage to human RPE cells by A2-E, a retinoid component of lipofuscin. , 2000, Investigative ophthalmology & visual science.

[9]  Kazunori Yamamoto,et al.  The bisretinoids of retinal pigment epithelium , 2012, Progress in Retinal and Eye Research.

[10]  G. Travis,et al.  Biosynthesis of a major lipofuscin fluorophore in mice and humans with ABCR-mediated retinal and macular degeneration. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[11]  C K Dorey,et al.  In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelium lipofuscin characteristics. , 1995, Investigative ophthalmology & visual science.

[12]  J. Simon,et al.  Spectroscopic and morphological studies of human retinal lipofuscin granules , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[13]  C. Wadelius,et al.  Enhanced accumulation of A2E in individuals homozygous or heterozygous for mutations in BEST1 (VMD2). , 2007, Experimental eye research.

[14]  Bogdan Serban,et al.  Retinal carotenoids can attenuate formation of A2E in the retinal pigment epithelium. , 2009, Archives of biochemistry and biophysics.

[15]  J. Havel,et al.  Laser desorption ionization of red phosphorus clusters and their use for mass calibration in time-of-flight mass spectrometry. , 2009, Rapid communications in mass spectrometry : RCM.

[16]  K. Palczewski,et al.  Primary amines protect against retinal degeneration in mouse models of retinopathies , 2011, Nature chemical biology.

[17]  M. Boulton,et al.  Retinal Pigment Epithelium Lipofuscin Proteomics*S , 2008, Molecular & Cellular Proteomics.

[18]  K. Nakanishi,et al.  The Biosynthesis of A2E, a Fluorophore of Aging Retina, Involves the Formation of the Precursor, A2-PE, in the Photoreceptor Outer Segment Membrane* , 2000, The Journal of Biological Chemistry.

[19]  J. Simon,et al.  Compositional studies of human RPE lipofuscin: mechanisms of molecular modifications. , 2011, Journal of mass spectrometry : JMS.

[20]  A. Harman,et al.  Development and aging of cell topography in the human retinal pigment epithelium. , 1997, Investigative ophthalmology & visual science.

[21]  K. Nakanishi,et al.  Isolation and one-step preparation of A2E and iso-A2E, fluorophores from human retinal pigment epithelium. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[22]  J. Jonas,et al.  Retinal pigment epithelial cell count, distribution, and correlations in normal human eyes. , 1996, American journal of ophthalmology.

[23]  J. Weiter,et al.  The topography and age relationship of lipofuscin concentration in the retinal pigment epithelium. , 1978, Investigative ophthalmology & visual science.

[24]  E. Rodriguez-Boulan,et al.  The lipofuscin fluorophore A2E perturbs cholesterol metabolism in retinal pigment epithelial cells , 2007, Proceedings of the National Academy of Sciences.

[25]  A. Pande,et al.  Novel Lipofuscin Bisretinoids Prominent in Human Retina and in a Model of Recessive Stargardt Disease* , 2009, The Journal of Biological Chemistry.

[26]  T Michael Redmond,et al.  Effect of Rpe65 knockout on accumulation of lipofuscin fluorophores in the retinal pigment epithelium. , 2001, Investigative ophthalmology & visual science.

[27]  Joan E. Roberts,et al.  Comparison of the Aerobic Photoreactivity of A2E with its Precursor Retinal ¶ , 2003, Photochemistry and photobiology.

[28]  K. Tomer,et al.  Blue light induced A2E oxidation in rat eyes — experimental animal model of dry AMD , 2010, Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology.

[29]  Koji Nakanishi,et al.  Ocular Age Pigment "A2-E": An Unprecedented Pyridinium Bisretinoid , 1996 .

[30]  C K Dorey,et al.  Age-related accumulation and spatial distribution of lipofuscin in RPE of normal subjects. , 2001, Investigative ophthalmology & visual science.

[31]  C. Drea,et al.  Relationship between dietary retinol and lipofuscin in the retinal pigment epithelium , 1986, Mechanisms of Ageing and Development.

[32]  K. Nakanishi,et al.  A2E, a fluorophore of RPE lipofuscin, can destabilize membrane. , 2006, Advances in experimental medicine and biology.

[33]  Y. Koutalos,et al.  Spatial localization of A2E in the retinal pigment epithelium. , 2011, Investigative ophthalmology & visual science.

[34]  Danielle B. Gutierrez,et al.  Mass spectrometry provides accurate and sensitive quantitation of A2E , 2010, Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology.

[35]  K. Palczewski,et al.  Limited roles of Rdh8, Rdh12, and Abca4 in all-trans-retinal clearance in mouse retina. , 2009, Investigative ophthalmology & visual science.

[36]  L. Feeney.  Lipofuscin and melanin of human retinal pigment epithelium. Fluorescence, enzyme cytochemical, and ultrastructural studies. , 1978, Investigative ophthalmology & visual science.