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M. W. Davies, M. B. Rozanowska, M. E. Boulton; Modelling Lipofuscin Formation in vitro Using Photoreceptor Outer Segments and Mitochondria as Substrates. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3038.
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RPE lipofuscin is believed to accumulate as the result of the incomplete degradation of both photoreceptor outer segment tips and spent intracellular organelles. In this study we have compared photoreceptor outer segments (POS) and mitochondria as substrates for the formation of lipofuscin-like fluorophores in vitro.
Lysosomes and mitochondria were isolated from bovine liver and POS from light adapted bovine retina. POS or mitochondria were incubated with lysosomes at either pH 4.5 or 7.0 for 4 months at 37°C. POS, mitochondria or lysosomes alone acted as controls. In some experiments POS and mitochondria were peroxidised by exposure to UV radiation. The preparations were monitored by appearance, absorption spectroscopy, fluorescence spectroscopy and photoreactivity. The preparations were extracted using Folch’s reagent and the chloroform soluble fraction analysed by TLC and the insoluble interfacial material by proteomics.
Both mitochondria and POS with lysosomes developed a golden-yellow granular precipitate by 4 months incubation which resembled lipofuscin. The "yellowing" of the samples was greatest in samples maintained at pH 7 compared to pH 4.5. Both preparations demonstrated increasing absorbance toward the shorter wavelengths and excitation and emission spectra with similarities to lipofuscin granules isolated from RPE cells. Both preparations demonstrated photoreactivity which was weaker than freshly isolated lipofuscin granules but significantly greater than either POS or mitochondria alone. TLC demonstrated predominantly blue fluorophores and none of the orange-yellow fluorophores associated with lipofuscin. Proteomic analysis of the lipofuscin-like material showed that samples contained a range of proteins that derived from POS, mitochondria and the lysosomes and that many of these were oxidatively modified.
These models highlight the roles of material acquired through both phagocytosis and autophagy in the formation of retinal lipofuscin and the importance of peroxidation in this process.
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