APOE4 Causes Widespread Molecular and Cellular Alterations Associated with Alzheimer's Disease Phenotypes in Human iPSC-Derived Brain Cell Types

Abstract

The apolipoprotein E4 (APOE4) variant is the single greatest genetic risk factor for sporadic Alzheimer's disease (sAD). However, the cell-type-specific functions of APOE4 in relation to AD pathology remain understudied. Here, we utilize CRISPR/Cas9 and induced pluripotent stem cells (iPSCs) to examine APOE4 effects on human brain cell types. Transcriptional profiling identified hundreds of differentially expressed genes in each cell type, with the most affected involving synaptic function (neurons), lipid metabolism (astrocytes), and immune response (microglia-like cells). APOE4 neurons exhibited increased synapse number and elevated Aβ₄₂ secretion relative to isogenic APOE3 cells while APOE4 astrocytes displayed impaired Aβ uptake and cholesterol accumulation. Notably, APOE4 microglia-like cells exhibited altered morphologies, which correlated with reduced Aβ phagocytosis. Consistently, converting APOE4 to APOE3 in brain cell types from sAD iPSCs was sufficient to attenuate multiple AD-related pathologies. Our study establishes a reference for human cell-type-specific changes associated with the APOE4 variant. VIDEO ABSTRACT.

Keywords: APOE; Alzheimer’s disease; Aβ; Aβ uptake; CRISPR/Cas9; cerebral organoids; cholesterol; early endosomes; iPSC; immune response.

Figure 1. Altered gene expression in APOE4 neural and glial cells is correlated with severe AD clinical phenotypes

(A) Schematics of RNA-seq with neuron, astrocytes and microglia-like cells derived from isogenic iPSCs homozygous for either APOE3 or APOE4. (B) Phylogenetic dendrogram of RNA-seq data. (C) Scatter plots of RNA-seq gene quantification values (log₁₀FPKM) of APOE3 and APOE4 groups in iPSC-derived brain cell types. DEGs are colored in red. (D) Dendrogram of RNA-seq co-expression gene network visualizing clustering of gene expression patterns in iPSC-derived cells. Color bars indicate different gene modules, with APOE module colored in pink. (E) Top six enriched canonical pathways of APOE gene module. (F) Expression changes of cell type signature genes in iPSC-derived corresponding APOE4 cell types were compared to APOE4 allele-associated eQTL effect sizes of those signature genes in postmortem human brain samples. The red dots in the plots showed overlap of significantly altered genes in both human brain samples and induced brain cell types. Among them, genes with concordant changes are pinpointed in the scatter plots. (G) Expression of the concordant genes in control and MCI brains were correlated with two AD phenotypical measurements: neocortical plaque density and clinical dementia rating (CDR). Statistical p-values of Spearman’s correlation are visualized in heatmaps, with blue or red colors indicating negative or positive correlation.

Figure 2. Increased number of synapses, early endosomes and Aβ₄₂ secretion in APOE4 neurons

(A) Immunocytochemistry with MAP2 and nestin antibodies in neurons. Scale bar = 10 μm. (B) Heatmaps of z-score values and barplots of enriched GO terms for DEGs between APOE3 and APOE4 neurons. (C) mEPSCs amplitude and frequency in iPSC-derived neurons. Scale bar = 20 pA and 200 ms. n=7~9 from three independent cultures. (D) Immunocytochemistry with synaptophysin and PSD-95 antibodies in neurons. Synaptophysin and PSD-95 signals were analyzed by IMARIS. n=9 from four independent cultures. Scale bar = 2 μm. (E) Secreted levels of Aβ₄₂ and Aβ₄₀ from iPSC-induced neurons were measured by ELISA and normalized to APOE3 neurons. n=6 per group. (F) Immunocytochemistry with EEA1 antibody in neurons. Scale bar = 10 μm. n=6 from three independent cultures. **P<0.01, ***P<0.001.

Figure 3. APOE4 astrocytes exhibited reduced APOE protein levels and impaired clearance of extracellular Aβ₄₂

(A) Immunocytochemistry with APOE antibody in astrocytes homozygous for either APOE3 or APOE4. Scale bar = 10 μm. n=6 per group. (B, C) Immunoblotting with APOE antibody in cell pellets (C) or cultured media (D) from APOE3 or APOE4 iPSC-induced astrocytes. Bar graphs represent relative immunoreactivity of APOE in each group normalized to APOE3 genotype. n=5~7 per group. The upper bands (arrow) in panel C likely correspond to the sialiated form of APOE. (D) Heatmaps of z-score values and barplots of enriched GO terms for DEGs between APOE3 and APOE4 astrocytes. (E) iPSC-derived astrocytes were fixed and incubated with filipin III for 1 hr followed by imaging. The bar graph represents the intensity of filipin III in the images. Scale bar = 10 μm. n=9 images from three independent cultures. (F) iPSC-derived astrocytes were sorted based on the intensity of filipin III. The bar graph represents the intensity of filipin III in sorted cells. n=6 per group. (G) iPSC-derived astrocytes were incubated with Aβ₄₂ oligomers for 2 days, then residual Aβ₄₂ were measured by ELISA. (Right) The Aβ₄₂ clearance index was calculated as described and normalized to APOE3 astrocytes. n=9 per group. (H) Immunocytochemistry with Aβ and GFAP antibodies in astrocytes. (Right) Relative immunoreactivity of Aβ overlapped with GFAP was normalized to APOE3 astrocytes. Scale bar = 10 μm. n=8 images from three independent cultures. *P<0.05, **P<0.01, ***P<0.001.

Figure 4. Altered process complexity and impaired clearance of extracellular Aβ in APOE4 microglia-like cells

(A) Immunocytochemistry with Iba1 antibody in APOE3 or APOE4 microglia-like cells. Bar graphs represent the number or length of process in microglia-like cells. Scale bar = 10 μm. n=7 images from four independent cultures. (B) Heatmaps of z-score values and barplots of enriched GO terms for DEGs between APOE3 and APOE4 microglia-like cells. (C) Induced microglia-like cells were treated with Aβ₄₂-555, and its uptake was monitored under the microscope for 1 hr. (Right) The intensity of cellular Aβ₄₂ in APOE3 and APOE4 microglia-like cells was plotted against time (min). (D) Schematics to determine Aβ clearance in APP^DP organoids by microglia-like cells. (Right) Three months APP^DP organoids cultured without microglia-like cells or with either APOE3 or APOE4 microglia-like cells were fixed and subjected to immunostaining with Iba1 and Aβ antibodies. Bar graph represents the number of Aβ puncta. Scale bar = 10 μm. n=4~6 organoids per group. *P<0.05, ***P<0.001.

Figure 5. Increased levels of Aβ and p-tau in APOE4 iPSC-derived cerebral organoids

(A) Schematics for generating cerebral organoids from iPSCs. (B) Six months organoids from APOE3 or APOE4 iPSCs were lysed and subjected to immunoblotting with APOE, Aβ, p-tau (AT-8) and GAPDH antibodies. Bar graphs represent relative immunoreactivity of APOE, Aβ and AT-8 normalized to GAPDH. n=3 per group. (C, D) Six months organoids from APOE3 or APOE4 iPSCs were fixed and subjected to immunostaining with Aβ, MAP2 and p-tau (AT-8) antibodies. Bar graphs represent the number and size of Aβ puncta (C) or immunoreactivity of AT-8 in organoids (D). Scale bar = 20 μm. n=15 images from 5~6 organoids. *P<0.05, **P<0.01, ***P<0.001.

Figure 6. Converting APOE4 to APOE3 attenuates AD-related phenotypes in sAD iPSC-derived neurons, glial and organoids

(A) mEPSCs amplitude (Left) and frequency (Right) in iPSC-derived neurons from APOE4 or APOE3 iPSCs. Scale bar = 20 pA and 200 ms. n=6~8 from three independent cultures. (B) Immunocytochemistry with synaptophysin and PSD-95 antibodies in neurons derived from APOE4 or APOE3 iPSCs. Synaptophysin and PSD-95 signals were analyzed by IMARIS. Scale bar = 2 μm. n=6 from three independent cultures. (C) Secreted levels of Aβ₄₂ and Aβ₄₀ from iPSC-induced neurons were measured by ELISA and normalized to APOE4 neurons. n=5 per group. (D) Relative immunoreactivity of APOE in induced APOE4 or APOE3 astrocytes was normalized to APOE4 astrocytes. n=5 per group. (E) iPSC-derived astrocytes were incubated with Aβ₄₂ oligomers for 2 days, then residual Aβ₄₂ were measured by ELISA. The Aβ₄₂ clearance index was calculated and normalized to APOE4 astrocytes. n=5~6 per group. (F) Immunocytochemistry with Aβ and Iba1 antibodies in APOE3 or APOE4 microglia-like cells. (Right) Relative immunoreactivity of Aβ overlapped with Iba1 was normalized to that of APOE4 microglia-like cells. Scale bar = 5 μm. n=8~14 images from four independent cultures. (G) Six months organoids from APOE4 or APOE3 iPSCs were subjected to immunostaining with Aβ and MAP2 antibodies. The bar graph represents the number of Aβ puncta in organoids. Scale bar = 20 μm. n=15 images from 5~6 organoids. *P<0.05, **P<0.01, ***P<0.001.