Developing a Quantitative Method for Determining Lipofuscin Content in Mouse Retinal Pigment Epithelium: An Age Comparison of Wild Type and Immunoproteasome Knockout Mice

Abstract

Aging of post-mitotic cells is associated with the accumulation of lipofuscin, which can lead to deleterious changes in the body and increased susceptibility to certain diseases. This study focused on measuring lipofuscin in the aging retina and determining whether the absence of immunoproteasome affected this age-related process. We hypothesize that lipofuscin increased with aging and the absence of immunoproteasomes. To test our hypothesis, RPE cells from mice of different ages, including wild type and immunoproteasome knockout strains, were used. The cells were processed for lipid extracts containing lipofuscin. The lipid extracts were then used to measure lipofuscin content using fluorescence spectroscopy. Here we developed an optimal method for measuring lipofuscin, including homogenization with PBS buffer, and extraction under dark condition. It was found that fluorescence intensity increases with age in knockout mice, but decreases with age in wild type mice. The intensity was also observed to be higher in knockout compared with wild type. Intensity-average-emission-maximum (IAEM) values were found to vary within different age groups. Our method of quantifying lipofuscin could detect differences in content between retinas from mice of different ages and between strains. The higher content of lipofuscin in KO mice supports our hypothesis. Varied IAEM suggests different fluorescent species developed with aging. This study is important as it might contribute to the aspect of aging theory and, thus, provide insight to studies associated with degenerative diseases, such as age macular degeneration (AMD).