Similar molecules spatially correlate with lipofuscin and N-retinylidene-N-retinylethanolamine in the mouse but not in the human retinal pigment epithelium

Abstract

The accumulation of lipofuscin in the retinal pigment epithelium (RPE) has been implicated in the development of age-related macular degeneration (AMD) in humans. The exact composition of lipofuscin is not known but its best characterized component is N-retinylidene-N-retinylethanolamine (A2E), a byproduct of the retinoid visual cycle. Utilizing our recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS)-based technique to determine the spatial distribution of A2E, this study compares the relationships of lipofuscin fluorescence and A2E in the murine and human RPE on representative normal tissue. To identify molecules with similar spatial patterns, the images of A2E and lipofuscin were correlated with all the individual images in the MALDI-IMS dataset. In the murine RPE, there was a remarkable correlation between A2E and lipofuscin. In the human RPE, however, minimal correlation was detected. These results were reflected in the marked distinctions between the molecules that spatially correlated with the images of lipofuscin and A2E in the human RPE. While the distribution of murine lipofuscin showed highest similarities with some of the known A2E-adducts, the composition of human lipofuscin was significantly different. These results indicate that A2E metabolism may be altered in the human compared to the murine RPE.

Keywords: A2E; Human; Imaging; Lipofuscin; MALDI; Mass spectrometry; Mouse; Profiling; RPE; Retinoids.

Figure 1. Lipofuscin fluorescence and MALDI images of A2E in murine eye

Lipofuscin fluorescence image (A) was stitched together from individual fields taken with a fluorescent microscope (λ_exc = 488 nm) from a left eye of a 6 month old Sv129 mouse. The optic nerve head is indicated by asterisk to the right. The MALDI image of A2E (B) was collected in the same tissue at 150 µm resolution and was interpolated to improve optical resolution. It represents integrated signal from a ± 0.5 m/z mass window around m/z 592.4. The images are oriented: dorsal – top; ventral – bottom; nasal – right; temporal – left. The intensity is coded by the indicated false color scale. Bar = 1 mm. C, Normalized cross-sectional intensity profiles of lipofuscin fluorescence (dashed blue line, derived from A) and A2E (red line, derived from B). The intensity profiles were extracted along the path of the white arrows from interpolation-free intensity data corresponding to A and B respectively and were smoothed. The optic nerve head is indicated by asterisk. There is a good agreement between lipofuscin and A2E intensity distributions.

Figure 2. Lipofuscin fluorescence and MALDI images of A2E in human eye

Lipofuscin fluorescence image (A) was acquired in a Xenogen IVIS 200 bioluminescence imaging system (λ_exc = 450–490 nm, λ_em = 575 – 650 nm) at a final resolution of 350 µm in an 88 year right human eye. The macular area is indicated by a dashed circle and the optic nerve head area is indicated by asterisk to the top left. The MALDI image of A2E (B) was collected in the same tissue at 300 µm resolution and was interpolated to improve optical resolution. It represents integrated signal from a ± 0.5 m/z mass window around m/z 592.4. The images are oriented: dorsal – top; ventral – bottom; nasal – left; temporal – right. The intensity is coded by the indicated false color scale. Bar = 1 cm. C, Normalized cross-sectional intensity profiles of lipofuscin fluorescence (dashed blue line, derived from A) and A2E (red line, derived from B). The intensity profiles were extracted along the path of the white arrows from interpolation-free intensity data corresponding to A and B respectively and were smoothed. The macular area is indicated by # sign below and the optic nerve head by an asterisk to the right. There is little agreement between lipofuscin and A2E intensity distributions.

Figure 3. Molecules spatially correlating with lipofuscin and A2E in murine eyes

A, Schematic representation of the murine image stack, indicating three random images at m/z 555, m/z 727, and m/z 811. B, Average mass spectrum (MALDI profile) from the 6 month old Sv129 tissue in the m/z 550–950 range. The spectrum represents the molecules present in the entire dataset. The positions of A2E (m/z 592) and singly-oxidized A2E (m/z 608) are indicated. C, The interpolation-free MALDI image of A2E (comparable to Fig. 1B but at the resolution of the imaging dataset) used to determine which molecules spatially correlate with A2E. The intensity is coded by the indicated color scale. D, The array of Pearson correlation coefficients between the image of A2E (panel C) and every individually collected molecular component (indicated by their respective m/z value) shown in the m/z 550–950 range. E, The lipofuscin fluorescence image used to determine which molecules spatially correlate with lipofuscin (comparable to Fig. 1A but at the resolution of the imaging dataset). F, The array of Pearson correlation coefficients between the image of lipofuscin fluorescence (panel E) and each images in the stack (indicated by their respective m/z value) shown in the m/z 550–950 range.

Figure 4. Molecules spatially correlating with lipofuscin and A2E in human eyes

A, Schematic representation of the human image stack, indicating three random example molecular images at m/z 570, m/z 696, and m/z 783. B, Average mass spectrum (MALDI profile) from the 88 year old human tissue in the m/z 550–950 range. The spectrum represents the molecules present in the entire dataset. The position of A2E (m/z 592) is indicated. C, The interpolation-free MALDI image of A2E (comparable to Fig. 2B) used to determine which molecular masses spatially correlate with A2E. The intensity is coded by the indicated color scale. D, The array of Pearson correlation coefficients between the image of A2E (panel C) and each image in the stack (indicated by their respective m/z value) shown in the m/z 550–950 range. The location of singly-oxidized A2E (m/z 608) is indicated. E, The lipofuscin fluorescence image used to determine which molecules co-localize with lipofuscin (comparable to Fig. 2A). F, The array of Pearson correlation coefficients between the image of lipofuscin fluorescence (panel E) and each image in the stack (indicated by their respective m/z value) shown in the m/z 550–950 range.