The Interplay of Apoes with Syndecans in Influencing Key Cellular Events of Amyloid Pathology

Abstract

Apolipoprotein E (ApoE) isoforms exert intricate effects on cellular physiology beyond lipid transport and metabolism. ApoEs influence the onset of Alzheimer's disease (AD) in an isoform-dependent manner: ApoE4 increases AD risk, while ApoE2 decreases it. Previously we demonstrated that syndecans, a transmembrane proteoglycan family with increased expression in AD, trigger the aggregation and modulate the cellular uptake of amyloid beta (Aβ). Utilizing our previously established syndecan-overexpressing cellular assays, we now explore how the interplay of ApoEs with syndecans contributes to key events, namely uptake and aggregation, in Aβ pathology. The interaction of ApoEs with syndecans indicates isoform-specific characteristics arising beyond the frequently studied ApoE-heparan sulfate interactions. Syndecans, and among them the neuronal syndecan-3, increased the cellular uptake of ApoEs, especially ApoE2 and ApoE3, while ApoEs exerted opposing effects on syndecan-3-mediated Aβ uptake and aggregation. ApoE2 increased the cellular internalization of monomeric Aβ, hence preventing its extracellular aggregation, while ApoE4 decreased it, thus helping the buildup of extracellular plaques. The contrary effects of ApoE2 and ApoE4 remained once Aβ aggregated: while ApoE2 reduced the uptake of Aβ aggregates, ApoE4 facilitated it. Fibrillation studies also revealed ApoE4's tendency to form fibrillar aggregates. Our results uncover yet unknown details of ApoE cellular biology and deepen our molecular understanding of the ApoE-dependent mechanism of Aβ pathology.

Keywords: ApoE; amyloid beta; endocytosis; neurodegeneration; protein aggregation; syndecans.

Figure 1

Cellular uptake of ApoEs into WT K562 cells and SDC transfectants. WT K562 cells and SDC transfectants were treated with either of the FITC-labeled ApoE isoforms for 3 h at 37 °C. Cellular uptake of ApoE isoforms was then measured with flow cytometry and confocal microscopy. (A) Flow cytometry histograms representing intracellular fluorescence WT K562 cells and SDC transfectants treated with FITC-labeled ApoEs. (B,C) Detected intracellular and cellular fluorescence intensities were normalized to FITC-ApoE2-treated WT K562 cells as standards. The bars represent the mean ± SEM of six independent experiments. Statistical significance vs. standards (i.e., FITC-ApoE2-treated WT K562 cells) was assessed with analysis of variance (ANOVA). * p < 0.05; ** p < 0.01; *** p < 0.001. (D) Confocal microscopic visualization of ApoE uptake into K562 cells and SDC transfectants. The nuclei of cells were stained with DAPI. Representative images of three independent experiments are shown. Scale bar = 10 μm.

Figure 2

Colocalization of ApoEs and SDCs. SDC transfectants were treated with either of the FITC-ApoEs for 3 h at 37 °C. After incubation, the cells were permeabilized and treated with the respective APC-labeled SDC antibody. Nuclei of cells were stained with DAPI and colocalization was then analyzed with confocal microscopy. (A–C) Confocal microscopic images of SDC transfectants treated either of the FITC-ApoEs and the respective APC-labeled SDC antibody. Representative images of three independent experiments are shown. Scale bar = 10 μm. MOC or PCC ± SEM for the colocalization of SDCs with FITC-ApoEs was calculated by analyzing 18 images with ~7 cells in each image (from three separate samples). (D) SDS-PAGE showing FITC-labeled ApoEs immunoprecipitated with SDC3 from extracts of SDC3 transfectants. Lanes 1–3: 0.5 µg of FITC-ApoEs. Lane 4: molecular weight (MW) marker. Lanes 5–7: immunoprecipitates of SDC3 transfectants treated with either of the FITC-ApoEs. Lanes 8-10: immunoprecipitates of untreated SDCs transfectants. Standard protein size markers are indicated on the left. Image acquisition was carried out with UVITEC Alliance Q9 Advanced Imager.

Figure 3

Colocalization of ApoEs with SDC3 in SH-SY5Y cells. (A,B) Undifferentiated (A) or differentiated (B) SH-SY5Y cells were treated with either of the FITC-ApoEs for 3 h at 37 °C. After incubation, the cells were permeabilized and treated with the APC-labeled SDC3 antibody. Nuclei of cells were stained with DAPI and colocalization was then analyzed with confocal microscopy. Representative images of three independent experiments are shown. Scale bar = 20 μm. MOCand PCC ± SEM, for the colocalization of SDCs with FITC-ApoEs, was calculated by analyzing 21 images with ~7 cells in each image (from three separate samples). (C) SDS-PAGE showing FITC-labeled ApoEs immunoprecipitated with SDC3 from extracts of differentiated SH-SY5Y. Lane 1: MW marker. Lanes 2–4: 0.5 µg of FITC-ApoE2,3 and 4, respectively. Lanes 5–7: immunoprecipitate of SH-SY5Y cells treated with either of the FITC-ApoEs. Lanes 8–10: immunoprecipitate of untreated SH-SY5Y cells. Standard protein size markers are indicated on the left. Image acquisition was carried out with UVITEC Alliance Q9 Advanced Imager.

Figure 4

SDC3 overexpression increases ApoE uptake into SH-SY5Y cells. SDC3-overexpressing clones created in differentiated SH-SY5Y cells were selected by measuring SDC3 expression with flow cytometry. (A) Flow cytometry histograms representing SDC3 expression levels and intracellular fluorescence of FITC-ApoE-treated differentiated WT SH-SY5Y cells and SDC3 transfectants, created in differentiated SH-SY5Y cells. SDC3 transfectants and WT SH-SY5Y cells were treated with FITC-labeled ApoEs at 37 °C for 3 h and then processed for uptake studies. (B) Detected intracellular fluorescence intensities were normalized to FITC-ApoE2-treated WT SH-SY5Y cells as standards. The bars represent the mean ± SEM of three independent experiments. Statistical significance vs. standards was assessed with ANOVA. (C) Fold change in SDC3 expression, along with ApoE uptake following SDC3 overexpression in differentiated SH-SY5Y cells. The bars represent the mean ± SEM of three independent experiments. The statistical significance vs. ApoE2-treated WT SH-SY5Y cells as standards was assessed with ANOVA. * p < 0.05; ** p < 0.01.

Figure 5

The effect of ApoEs on Aβ1-42 fibrillation in WT and SDC3-overexpressing differentiated SH-SY5Y cells. WT and SDC3-overexpressing differentiated SH-SY5Y cells were incubated with either of the ApoE isoforms at 37 °C. Thirty minutes later, some of the cells were treated with Aβ1–42 and incubated for either 3 h or 18 h (A,B). After incubation, fibrillation was analyzed with either ThT staining or electron microscopy. (A,C) Results of ThT fluorescence assays after 3 h and 18 h of incubation with Aβ1–42 in the presence or absence of ApoEs (A) or the ApoE isoforms alone (C). Amyloid fluorescence is expressed as fold change over background ThT fluorescence. The bars represent the mean ± SEM of four independent experiments. In the case of Aβ1–42 treatment (A), statistical significance was assessed vs. Aβ1–42-only treated cells with ANOVA. For cells receiving ApoE treatment only (B), statistical significance was assessed vs. untreated controls. * p < 0.05; ** p < 0.01; *** p < 0.001. (B,D) Scanning electron microscope visualization of WT and SDC3-overexpressing differentiated SH-SY5Y cells at 3 h and 18 h of incubation with Aβ1–42 in the presence or absence of ApoEs (B) or the ApoE isoforms alone (D). Representative images of three independent experiments are shown. Scale bar = 1 μm.

Figure 6

The effects of ApoEs on Aβ1–42 uptake in WT and SDC3-overexpressing differentiated SH-SY5Y cells. WT and SDC3-overexpressing differentiated SH-SY5Y cells were preincubated with different ApoEs for 30 min at 37 °C. The cells were then treated with FITC-labeled or, in case of fibrillation studies, unlabeled Aβ1–42 for either 3 or 18 h. After incubation, the cellular uptake of Aβ1–42 was analyzed. (A,B) Flow cytometry histograms representing the intracellular fluorescence of differentiated WT and SDC3-overexpressing differentiated SH-SY5Y cells treated with FITC-labeled Aβ1–42 for 3 (A) and 18 h (B) in the absence or presence of ApoEs. (C,D) Detected intracellular fluorescence intensities were normalized to FITC-Aβ1–42-only treated WT SH-SY5Y cells as standards. The bars represent the mean ± SEM of four independent experiments. Statistical significance vs. standards was assessed with ANOVA. * p < 0.05; ** p < 0.01. (E,F) Confocal microscopic visualization of ThT-labeled Aβ1–42 fibrils in WT and SDC3-overexpressing differentiated SH-SY5Y cells at 3 h (E) and 18 h (F) of incubation. The nuclei of cells were stained with DRAQ5. Representative images of three independent experiments are shown. Scale bar = 20 μm.

Figure 7

The colocalization of ApoE4 and Aβ1–42 fibrils. Differentiated SH-SY5Y cells were preincubated with FITC-ApoE4 and then treated with Aβ1–42 for 18 h. After incubation, Aβ1–42 fibrils were stained with Congo Red (CR), while the nuclei of cells were stained with DAPI. The colocalization of ApoE4 with CR-stained Aβ1–42 fibrils was then analyzed with confocal microscopy. Representative images of three independent experiments are shown. Scale bar = 20 μm. MOC and PCC ± SEM, for the colocalization of ApoE4 with CR-stained Aβ1–42 fibrils, was calculated by analyzing 21 images with ~5 cells in each image (from three separate samples). Scale bar = 20 μm.