Apolipoprotein E Interferes with IAPP Aggregation and Protects Pericytes from IAPP-Induced Toxicity

Abstract

Apolipoprotein E (ApoE) has become a primary focus of research after the discovery of its strong linkage to Alzheimer's disease (AD), where the ApoE4 variant is the highest genetic risk factor for this disease. ApoE is commonly found in amyloid deposits of different origins, and its interaction with amyloid-β peptide (Aβ), the hallmark of AD, is well known. However, studies on the interaction of ApoEs with other amyloid-forming proteins are limited. Islet amyloid polypeptide (IAPP) is an amyloid-forming peptide linked to the development of type-2 diabetes and has also been shown to be involved in AD pathology and vascular dementia. Here we studied the impact of ApoE on IAPP aggregation and IAPP-induced toxicity on blood vessel pericytes. Using both in vitro and cell-based assays, we show that ApoE efficiently inhibits the amyloid formation of IAPP at highly substoichiometric ratios and that it interferes with both nucleation and elongation. We also show that ApoE protects the pericytes against IAPP-induced toxicity, however, the ApoE4 variant displays the weakest protective potential. Taken together, our results suggest that ApoE has a generic amyloid-interfering property and can be protective against amyloid-induced cytotoxicity, but there is a loss of function for the ApoE4 variant.

Keywords: IAPP amyloid; Thioflavin T; apolipoprotein E; cytotoxicity; pericytes.

Figure 1

Thioflavin-T (ThT) binding kinetics of 5 µM islet amyloid polypeptide (IAPP) in the presence of the three apolipoprotein E (ApoE) variants: ApoE2 (a), ApoE3 (b), and ApoE4 (c). The ApoE variants were used at the concentrations indicated in the figure.

Figure 2

TEM images of IAPP samples incubated in the presence or absence of ApoE. Fibrils produced from 5 µM IAPP alone (a); from IAPP in the presence of 16 nM ApoE2, ApoE3, and ApoE4 (b,c,d); and from IAPP in the presence of 1 µM ApoE2, ApoE3, and ApoE4 (e,f,g). The scale bar is 100 nm. Larger fields of the images are shown in Figure S3.

Figure 3

Effect of ApoEs on the elongation of IAPP during ThT binding kinetics. A total of 500 nM ApoE was added to the aggregating 5 µM IAPP at the indicated time points of the reaction. ApoE2 (a), ApoE3 (b), and ApoE4 (c).

Figure 4

Influence of ApoEs on IAPP-induced cytotoxicity in human blood vessel pericytes. The viability of the cells treated with 10 µM IAPP alone or in combination with ApoE variants in the concentration range 1 µM–16 nM was analyzed after 48, 72, and 144 h of co-incubation with the cells. Control cells represent the samples treated with 1 µM ApoE only. The percentage of the viability is calculated based on untreated cells having 100% viability. The key statistical values are presented on the graphs. p < 0.05 (*); p < 0.01 (**); p < 0.001 (***). Red and blue colored asterisks represent comparison with ApoE2 and ApoE3, respectively.

Figure 5

Morphology of the pericytes imaged under the phase-contrast microscope after 72 h treatment (scale bar is 100 µm). The upper panel represents controls (untreated cells) and cells treated with 1 µM ApoE only (here is the representative image from sample treated with ApoE4) and 10 µM IAPP only. Below are the images of the cells treated with 1 µM IAPP in combination with the indicated ApoE variants at the highest (1 µM) and the lowest (16 nM) concentrations used in the experiment.