Purchase this article with an account.
Aparna Lakkaraju, Jin Xu; Trafficking and Functions of Apolipoprotein E Isoforms in Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):886.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To investigate the intracellular trafficking and function of apolipoprotein E isoforms ApoE2, ApoE3 & ApoE4 in retinal pigment epithelial (RPE) cells. ApoE is a cholesterol transporter that occurs in three isoforms in humans. The E4 allele is a risk factor for Alzheimer's disease and the E2 allele is protective. Interestingly, the converse is true for age-related macular degeneration (AMD): ApoE2 is a risk factor and ApoE4 is protective. However, mice expressing human ApoE4 on a high fat diet exhibit many features of AMD. ApoE and cholesterol are major components of drusen, suggesting that they contribute to AMD pathogenesis. We have previously shown that the lipofuscin fluorophore A2E causes cholesterol accumulation in RPE cells. Here, we sought to investigate the intracellular itineraries and cholesterol transport functions of the three ApoE isoforms.
To determine the sub-cellular localization of endogenous ApoE, RPE cells were co-stained for ApoE and various organelle markers and imaged by confocal microscopy. GFP-tagged ApoE2, E3 or E4 were expressed in RPE cells using lipofection or nucleofection. Trafficking of tubules and vesicles carrying GFP-ApoE2, E3 or E4 were imaged in live RPE cells using spinning disk confocal microscopy. Cells were fixed and labeled with filipin to detect cellular cholesterol distribution. Cholesterol content in RPE cells expressing the three different ApoE isoforms was also measured biochemically.
Endogenous ApoE was in vesicular structures throughout the cytoplasm in RPE cells. A small fraction was in late endosomes and the Golgi. In hepatocytes, ApoE undergoes vesicular transport from the Golgi complex to the plasma membrane and other organelles such as late endosomes where it is stored before being secreted. Live imaging of GFP-tagged ApoE2, E3 or E4 in RPE cells showed vesicles and tubules undergoing vectorial transport with mean velocities of 3 µm/second. ApoE4 vesicles were larger and more numerous compared to ApoE2 or ApoE3 vesicles. Analysis of cholesterol distribution and measurement of cholesterol content in cells expressing different ApoE isoforms are ongoing.
Cholesterol and ApoE are common etiological factors in the pathogenesis of chronic diseases like Alzheimer’s disease, AMD and atherosclerosis. Understanding the role of ApoE2/3/4 in the RPE and retina is important because ApoE isoforms influence response to statins, which may be beneficial in AMD. ApoE2, E3 and E4 have differential lipoprotein binding preferences (VLDL vs LDL vs HDL), which could influence the delivery of dietary carotenoids to the retina and RPE.
This PDF is available to Subscribers Only