Spectroscopic and morphological studies of human retinal lipofuscin granules

Abstract

The emission properties of ocular lipofuscin granules isolated from human retinal pigment epithelial cells are examined by using steady-state fluorescence spectroscopy and spectrally resolved confocal microscopy. The shape of the emission spectrum of a thick sample of lipofuscin granules dried on glass varies with excitation energy. The polarization of this emission is wavelength-dependent, exhibiting significant polarization near the excitation wavelength and becoming mostly depolarized over the majority of the emission spectrum. These results show that the yellow-emitting fluorophores [e.g., A2E (2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]-pyridinium)] are excited as a result of energy transfer within the granules and therefore are not the dominant blue-absorbing chromophores within lipofuscin granules. Atomic force microscopy images show lipofuscin granules to be an aggregated structure. Bulk and in vivo emission measurements must therefore take into account the effect of Raleigh scattering. When corrected for scattering, the emission spectrum of a thick lipofuscin deposit or intracellular lipofuscin resembles that for A2E. The sum of the emission spectra of a collection of individual granules also resembles the emission spectrum of A2E, but the spectrum of individual granules varies significantly. This result suggests that the agreement between the emission spectra of lipofuscin and A2E is fortuitous, and the collective data indicate the presence of several blue-absorbing chromophores in lipofuscin and show A2E is not the dominant yellow-emitting fluorophore in many of the granules studied.L ipofuscin (LF) is a common morphological result of the aging process and is manifested as a heterogeneous complex of fluorescent, lipid-protein aggregates found in the cytoplasm of postmitotic cells (1-5). In the retinal pigment epithelium (RPE) of the human eye, the formation of LF is attributed to the accumulation of indigestible end-products from the phagocytosis of photoreceptor outer segments (1, 6-8). LF accumulates in RPE cells as clusters of granules and can occupy Ϸ20% of the cytoplasmic space by 80 yr of age (9). LF exhibits a yellow fluorescence ( max Ϸ 600 nm) upon blue-light excitation (10). In vitro experiments also show that blue-light excitation of cultured RPE cells ʈ fed LF generates a variety of reactive oxygen intermediates (including hydrogen peroxide, singlet oxygen, and superoxide radical anion), which renders LF phototoxic to cultured RPE cells (11)(12)(13).Determining the molecule(s) responsible for the aerobic photoreactivity and emissive properties of LF is the focus of current research. In a groundbreaking paper in 1988, Eldred and Katz (14) analyzed chloroform:methanol (2:1, vol͞vol) extracts of RPE cells and separated several emissive bands by using TLC. A variety of yellow-emitting fluorophores were found. To date, only two isomers of a pyridinium bis-retinoid, A...

Retinal Pigment Epithelium Lipofuscin Proteomics

“…Morphometric analyses showed little difference in the diameter, circularity, and roundness of the granules from purified or crude preparations with the average diameter (ϳ0.75 Ϯ 0.22 m) agreeing well with the 0.7 m value reported from atomic force microscopy (30). Removal of the extragranular debris was accompanied by loss of granuleassociated membranes, but this was accomplished without reduction in the mass (size) of the fluorescent granules.…”

Involvement of a Novel Q-SNARE, D12, in Quality Control of the Endomembrane System

“…6A). This feature is consistent with the presence of lipofuscin granules, which are manifested as heterogeneous complexes of fluorescent lipidprotein aggregates in the cytosol (42,43). It is well known that lipofuscin occasionally appears in cells when the culture condition has deteriorated.…”

Characterization of Native Retinal Fluorophores Involved in Biosynthesis of A2E and Lipofuscin-associated Retinopathies

“…2A, dashed trace) is comparable with the spectra of A2E-laden human RPE cells (20) and human lipofuscin granules (21). In contrast, the fluorescence spectrum from retina explants demonstrated lower intensity relative to RPE/eyecups and an emission maximum of ϳ625 nm ( Fig.…”