Decreased lipidated ApoE-receptor interactions confer protection against pathogenicity of ApoE and its lipid cargoes in lysosomes

Abstract

While apolipoprotein E (APOE) is the strongest genetic modifier for late-onset Alzheimer's disease (LOAD), the molecular mechanisms underlying isoform-dependent risk and the relevance of ApoE-associated lipids remain elusive. Here, we report that impaired low-density lipoprotein (LDL) receptor (LDLR) binding of lipidated ApoE2 (lipApoE2) avoids LDLR recycling defects observed with lipApoE3/E4 and decreases the uptake of cholesteryl esters (CEs), which are lipids linked to neurodegeneration. In human neurons, the addition of ApoE carrying polyunsaturated fatty acids (PUFAs)-CE revealed an allelic series (ApoE4 > ApoE3 > ApoE2) associated with lipofuscinosis, an age-related lysosomal pathology resulting from lipid peroxidation. Lipofuscin increased lysosomal accumulation of tau fibrils and was elevated in the APOE4 mouse brain with exacerbation by tau pathology. Intrahippocampal injection of PUFA-CE-lipApoE4 was sufficient to induce lipofuscinosis in wild-type mice. Finally, the protective Christchurch mutation also reduced LDLR binding and phenocopied ApoE2. Collectively, our data strongly suggest decreased lipApoE-LDLR interactions minimize LOAD risk by reducing the deleterious effects of endolysosomal targeting of ApoE and associated pathogenic lipids.

Keywords: Christchurch; PUFA; apolipoprotein E; arachidonate; cholesterol; high-density lipoprotein; induced pluripotent stem cells; lipofuscin; low-density lipoprotein.

Figure 1.. ApoE isoform-dependent LDLR binding results in differential cellular uptake

(A and B) Confocal images and quantification of FITC-labeled POPC-lipidated ApoE (10 μg/mL) bound to surface of 293T cells overexpressing LDLR after 1 h incubation at 4°C. Scale bar in (A): 10 μm. (C) HTRF showing POPC-lipidated ApoE binding to LDLR ECD (3 independent experiments.). (D) SPR profiles for POPC-lipidated ApoE isoform binding to biotinylated LDLR ECD. Red: Experimental data. Black: Curve fit using a 1:1 kinetic binding model. Each curve represents binding at one concentration (0.5, 1, and 2 μM for lipApoE2; 0.0156, 0.03125, and 0.0625 μM for lipApoE3/E4). (E and G) Confocal images of pHrodo green-labeled POPC-lipidated ApoE (10 μg/mL) in WT, LDLR KO or LRP1 KO HAP1 cells (E) and H4 cells with or without co-treatment of 20 μg/mL LDLR ECD or 2.5 μg/mL heparin (G) after 1 d incubation. Scale bars: 10 μm. (F and H) Quantification of pHrodo green signals in (E) and (G), respectively. For (B), one-way ANOVA was performed with Dunnett’s multiple comparisons. For (F) and (H), two-way ANOVA was performed with Holm-Sidak’s multiple comparisons. See also Figure S1.

Figure 2.. Lipidated ApoE3 and ApoE4 alter cellular distribution of LDLR and impair LDL uptake

(A and C) Confocal images of cell surface LDLR staining in H4 cells and APOE KO iAstrocytes, respectively, after 1 d incubation with 10 μg/mL POPC-lipidated ApoE. Scale bars: 10 μm. (B and D) Quantification of surface LDLR signals in (A) and (C), respectively. (E and F) Confocal images of total LDLR staining for H4 cells after 1 d incubation with 10 μg/mL POPC-lipidated ApoE and quantification of total LDLR signals and spot density (i.e., spot number/spot area). Scale bar in (E): 10 μm. (G) Confocal images showing colocalization of internalized ApoE4 with LDLR in H4 cells. Scale bar: 10 μm. (H) Super-resolution confocal images showing co-immunostaining of LAMP1, ApoE, and LDLR in APOE KO iAstrocytes after 1 d treatment with 10 μg/mL POPC-lipidated ApoE4. Scale bar: 2.5 μm. Insets: higher-magnification images; scale bar: 0.5 μm. (I) Quantification of internalized lipApoE signals and LAMP1-positive vesicles containing ApoE immunoreactivity in (H). (J and K) Confocal images of DiI-LDL signals in H4 cells after 3–4 h co-treatment of 10 μg/mL POPC-lipidated ApoE and 10 μg/mL DiI-LDL (J) or after 1 d pre-treatment with 10 μg/mL POPC-lipidated ApoE followed by 3–4 h incubation with DiI-LDL. Scale bars in (K): 10 μm. (L) Quantification of DiI-LDL signals in H4 cells in (J) and (K). For (B), (D), (F) and (I), one-way ANOVA was performed with Sidak’s multiple comparisons. For (L), two-way ANOVA was performed with Tukey’s multiple comparisons. See also Figure S2.

Figure 3.. ApoE isoform- and LDLR-dependent lipid uptake impacts lipid homeostasis

(A and C)Confocal images and quantification of pHrodo signals in H4 cells after 1 d incubation with 10 μg/mL pHrodo red-labeled HDL pre-complexed with 10 μg/mL ApoE with and without co-treatment of 20 μg/ml LDLR ECD. Scale bar in (A): 10 μm. (B and D) Confocal images and quantification of BODIPY signals in APOE KO iAstrocytes after 1 d incubation with 10 μg/mL BODIPY-CE pre-complexed with 10 μg/mL HDL and 10 μg/mL ApoE with and without co-treatment of 20 μg/ml LDLR ECD. Scale bar: 10 μm. (E) Relative expression of 6 genes quantified by qPCR for APOE KO iAstrocytes after 1 d treatment with 10 μg/mL CE(18:1) pre-complexed with 10 μg/mL HDL and 10 μg/mL ApoE, with and without co-treatment of 20 μg/ml LDLR ECD. (F)-(G) Lipidomic analysis for APOE KO iAstrocytes after overnight treatment with 10 μg/mL CE(18:1) pre-complexed with 10 μg/mL HDL and 10 μg/mL ApoE, with and without co-treatment of 20 μg/ml LDLR ECD or 500nM ACAT1 inhibitor K604. (F) A heatmap for the top 25 lipids showing the greatest differences among the 3 isoform treatment conditions. (G) A subset of lipid species showing ApoE isoform-dependent changes. Two-way ANOVA was performed with Tukey’s (C and D) or Sidak’s (E) multiple comparisons; for (G), one-way ANOVA was performed with Sidak’s multiple comparisons. See also Figure S3.

Figure 4.. Differential lipid burden modulates inflammatory responses and transcription of microglia

(A and B) Confocal images and quantification of BODIPY signals in APOE KO iMg after 1 d incubation with 10 μg/mL BODIPY-CE pre-complexed with 10 μg/mL HDL and 10 μg/mL ApoE, with and without co-treatment of 20 μg/ml LDLR ECD, in medium containing 100 ng/mL LPS. Scale bar for (A): 20 μm. (C) Relative expression of 5 genes quantified by qPCR for APOE KO iMg after live imaging shown in (A). (D) Normalized concentrations of 5 cytokines measured in conditioned media of APOE KO iMg after live imaging shown in Figure S4A. (E) – (F) Results from RNA sequencing performed on APOE KO iMg after live imaging shown in Figure S4A. (E) Volcano plots showing significantly different individual genes with adjusted p value < 0.1, comparing ApoE4 vs. ApoE2 and ApoE4 vs. ApoE4 + LDLR ECD treatment conditions. (F) Volcano plots showing Hallmark pathways that are the most altered for different pairs of comparisons. One-way ANOVA was performed with Holm-Sidak’s multiple comparisons. See also Figure S4.

Figure 5.. ApoE isoforms carrying PUFA-CE differentially induce lipofuscin in cultured cells

(A and B) Confocal images and quantification of lipofuscin (488nm channel) in NGN2 iNeurons after 21 d incubation with 20 μg/mL CE(20:4)/POPC-lipidated ApoE isoforms. Scale bar in (A): 10 μm. (C) Super-resolution confocal images demonstrating predominant localization of lipofuscin in LAMP1-positive vesicles in NGN2 iNeurons after 14 d treatment with 20 μg/mL CE(20:4)/POPC-lipidated ApoE4. Scale bar: 2.5 μm. Insets: higher-magnification images for single or merged channels; scale bar: 0.5 μm. (D and E) Confocal images and quantification of 4-HNE immunoreactivities in NGN2 iNeurons after the same treatment conditions as in (A). Scale bar in (D): 10 μm. Yellow arrows point to examples of punctate perikaryal 4-HNE signals being quantified. (F), (H), and (J) Confocal images of lipofuscin (488nm channel) in NGN2 iNeurons (F) or in H4 cells (H and J) after 3 d incubation with 20 μg/mL CE(20:4)/POPC-lipidated ApoE with or without 20 μg/mL LDLR ECD, or with 20 μg/mL CE(18:1)/POPC-lipidated ApoE4. Scale bars: 10 μm. (G), (I), and (K) Quantifications of lipofuscin in (F), (H), and (K), respectively. (L-N) Confocal images and quantification of lipofuscin (488nm channel) in H4 cells after 1 d treatment of 20 μg/mL CE(20:4)/POPC-lipidated ApoE3 with 25 μM Vitamin E (in methanol) or 25 μM Lalistat 2 (Lal-2, in DMSO) compared with respective vehicle control. Scale bars: 10 μm. (O) Non-reduced SDS-PAGE analysis for supernatant (S) and pellet (P) fractions from ultracentrifugation of CE(20:4)/POPC-lipidated ApoE after 4 d incubation at 37°C at pH 7.4 or pH 4.5. (P) Quantification of percentage of ApoE recovered in the pellet fraction for experiments shown in (O) using 2 different batches of CE(20:4)/POPC-lipidated ApoE (densitometry of the 34 kDa band is shown). (Q) Quantification of relative aggregation for CE(20:4)/POPC-lipidated ApoE isoforms after 4 d incubation at 37°C at pH 4.5, with percentage of pelletable ApoE for each isoform normalized by mean pelletable ApoE in each experiment (combined results from 3 independent experiments for 2 batches of preparations). Except for (P), one-way ANOVA was performed with Holm-Sidak’s multiple comparisons. See also Figure S5.

Figure 6.. ApoE4 induces lipofuscin in mouse brain and perturbs tau pff degradation in neurons

(A and B) Confocal images of lipofuscin (545nm channel) in the hippocampus of 9.5-month-old APOE KO (EKO), APOE3 KI (E3), APOE4 KI (E4), P301S MAPT/APOE KO (Tau/EKO), P301S MAPT/APOE3 KI (Tau/E3), and P301S MAPT/APOE4 KI (Tau/E4) mice with quantification (12 mice/group). Scale bar in (A): 20 μm. (C) Confocal images of lipofuscin co-labeled with neuronal (NeuN) and microglial (Iba1) markers in the hippocampus of 9.5-month-old E4 and Tau/E4 mice. White arrows point to examples of neuronal lipofuscin adjacent to NeuN staining. Yellow arrows point to examples of lipofuscin that appears inside Iba1-positive microglia. (D and E) Confocal images of lipofuscin (545nm channel) in the hippocampus of 9.5-month-old E4 or Tau/E4 mice after 3.5-month treatment with vehicle (Ctrl) or LXR agonist GW3965 (LXR) with quantification (12 mice/group). Scale bar in D: 20 μm. (F) Images of thresholded lipofuscin in the brain of 6-month-old WT mice at 2 weeks after injection of PBS or CE(20:4)/POPC-lipidated ApoE4 into each side of the hippocampus. Arrows denote the approximate injection sites. Scale bars: 1 mm for the whole-hemisphere images; 500 μm for the zoom-in images. (G) Quantification of thresholded lipofuscin around the coronal plane of injection but with the injection tract excluded, for the experiment shown in (F) (4 mice, one-tailed paired t-test). (H) Confocal images of pHrodo signals detected in NGN2 iNeurons after 14 d treatment with 20 μg/mL CE(20:4)/POPC-lipidated ApoE followed by 13 d incubation with 2.5 μg/mL pHrodo red-labeled tau pffs. Scale bar: 10 μm. (I) Quantification of pHrodo signals in (H) for all neurons or only among neurons classified as lipofuscin-positive (LF +ve) (3 independent experiments, one-way ANOVA, Holm-Sidak’s multiple comparisons). (J-M) Confocal images and quantification of lipofuscin (J-K) and pHrodo signals (L-M) detected in NGN2 iNeurons after 14 d treatment with 20 μg/mL CE(20:4)/POPC-lipidated ApoE4 or CE(18:1)/POPC-lipidated ApoE4 followed by 14 d incubation with 2.5 μg/mL pHrodo red-labeled tau pffs. Scale bars: 10 μm. For (K) and (M), each dot represents one independent experiment, with two-tailed unpaired test performed. For (B) and (E), one-way ANOVA was performed with Sidak’s multiple comparisons. See also Figure S6.

Figure 7.. Christchurch mutation reduces LDLR binding, lipid uptake and lipofuscin

(A) Competitive HTRF demonstrating inhibition of tagged lipApoE4-LDLR ECD binding in the presence of different concentrations of untagged lipidated ApoE variants (n = 3 independent experiments). No inhibitor: no addition of untagged lipidated ApoE. The first panel shows the complete dose response. The second and third panels show the % inhibition at 125 nM and 250 nM, respectively. (B and C) Confocal images and quantification of pHrodo green-labeled POPC-lipidated ApoE isoforms (10 μg/mL) in H4 cells after 1 d incubation. Scale bar in (B): 10 μm. (D and E) Confocal images and quantification of pHrodo signals detected in H4 cells after 1 d incubation with 10 μg/mL pHrodo green-labeled HDL pre-complexed with 10 μg/mL ApoE isoforms. Scale bar in (D): 10 μm. (F and G) Confocal images and quantification of lipofuscin in H4 cells after 3 d incubation with 20 μg/mL CE(20:4)/POPC-lipidated ApoE isoforms. Scale bar in (F): 10 μm. One-way ANOVA was performed with Holm-Sidak’s multiple comparisons. See also Figure S7.