Additional file 3 of APOE Christchurch enhances a disease-associated microglial response to plaque but suppresses response to tau pathology
Creators: name: Tran, Kristine M.
name: Kwang, Nellie E.
name: Butler, Claire A.
name: Gomez-Arboledas, Angela
name: Kawauchi, Shimako
name: Mar, Cassandra
name: Chao, Donna
name: Barahona, Rocio A.
name: Da Cunha, Celia
name: Tsourmas, Kate I.
name: Shi, Zechuan
name: Wang, Shuling
name: Collins, Sherilyn
name: Walker, Amber
name: Shi, Kai-Xuan
name: Alcantara, Joshua A.
name: Neumann, Jonathan
name: Duong, Duc M.
name: Seyfried, Nicholas T.
name: Tenner, Andrea J.
name: LaFerla, Frank M.
name: Hohsfield, Lindsay A.
name: Swarup, Vivek
name: MacGregor, Grant R.
name: Green, Kim N.
Publisher: name: figshare
Year: 2025
Subjects: subject: Medicine
subject: Neuroscience
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Mental Health

description: Supplementary Material 3. Supp. Figure 1: Sequence of the Apoeem1Aduci (ApoeCh) allele and off-target site analysis for crRNA TMF1648 on mouse chromosome 7. (a) Amino acid (aa) sequence alignment between human and mouse APOE and DNA sequence of the wildtype and ApoeCh alleles in the region of the APOE R136S (R128S in mouse mature APOE). Red-colored nucleotides denote the missense codon while the green nucleotides denote synonymous base changes introduced to prevent recutting of the targeted site. (b) Chromatograms of DNA sequence in wildtype and ApoeCh heterozygous mice, with colored asterisks corresponding to the colored nucleotides in the above sequence. (c-i) Chromatograms of N3F2 wildtype and ApoeCh homozygous offspring at six potential off-target sites on mouse chromosome 7. No difference was found in sequence between the B6J WT and ApoeChhomozygotes at each of the six potential off-target sites analyzed. The black underline denotes the crRNA target sequence while the blue underline denotes the NGG PAM site. Supp. Figure 2: Behavioral analysis of 4- and 12-mo-old WT, ApoeCh, 5xFAD, 5xFAD;ApoeCh mice. a,b Plasma triglyceride (a) and VLDL (b) level in 4 mo WT and ApoeCh HO mice. c,d Weight of 4-mo-old (c) and 12-mo-old (d) mice taken at euthanizing day. e, f Total time mice spent in the center of open field behavioral assay of 4-mo-old (e) and 12-mo-old (f) in the 5 min recording time. g,h Mean velocity mice traveled in the center of the open field in 5 min of 4-mo (g) and 12-mo-old mice (h). i, j Total time 4-mo (i) and 12-mo-old (j) mice spent in open arms of the elevated plus maze behavior test. n = 4-6 mice/sex/genotype. Data are represented as mean ± SEM. Student’s t-test, unpaired. Two-way ANOVA followed by Tukey’s post hoc tests to examine biologically relevant interactions. Statistical significance is denoted by *p&lt;0.05,**p&lt;0.01, ***p&lt;0.001, ****p&lt;0.0001. # denotes trending significance. Supp. Figure 3: Sex-specific differences in pathology in the 5xFAD mice. In each panel, sex of individual animals is denoted by pink (female) or blue (male) circles. a-c Sex-separated plasma cholesterol (a), triglycerides (b), and VLDL (c) in 4-mo-old WT and ApoeCh mice. d-e Weights of 4-mo (d) and 12-mo-old WT, ApoeCh, 5xFAD, and 5xFAD;ApoeCh mice separated by sex. f-i Quantification of Amyloglo+ plaque numbers in 4-mo (f) and 12-mo (h); and percent plaque coverage in 4-mo (g) and 12-mo (i) in whole brain hemisphere images of 5xFAD and 5xFAD;ApoeCh mice separated by sex. j-k Sex-separated LAMP1+ dystrophic neurites volume in 4-mo (j) and 12-mo (k) 5xFAD and 5xFAD;ApoeCh subiculum. l-m Plasma NfL levels of 4-mo (l) and 12-mo (m) WT, ApoeCh, 5xFAD, and 5xFAD;ApoeCh mice separated by sex. n = 4-6 mice/sex/genotype. Data are represented as mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc tests to examine biologically relevant interactions. In comparisons with 2 genotypes (i.e. 5xFAD, and 5xFAD;ApoeCh), multiple comparisons between all groups were performed. Comparisons with 4 genotypes (i.e. WT, ApoeCh, 5xFAD, and 5xFAD;ApoeCh), only comparisons between genotypes were analyzed and reported. Statistical significance is denoted by *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001, ****p&lt;0.0001. # denotes trending significance. Supp. Figure 4: Quantification of insoluble and soluble Aβ in micro-dissected hippocampi and cortices of 4-mo-old 5xFAD, 5xFAD;ApoeCh mice. In each panel, sex of individual animals is denoted by pink (female) or blue (male) circles. a, d Cortical and hippocampal soluble Aβ40 (a,b) and Aβ42 (c,d) level measured via MSD of 4-mo-old mice. e-h Cortical and hippocampal soluble Aβ42 (e,f) and Aβ42 (g,h) level measured via MSD of 4-mo-old mice. i-p Sex-separated data of 4-mo cortical and hippocampal Aβ levels from a-h. q-x Sex-separated data of 12-mo cortical and hippocampal Aβ levels from Figure 2f-m. n = 4-6 mice/sex/genotype. Data are represented as mean ± SEM. Student’s t-test, unpaired. Two-way ANOVA followed by Tukey’s post hoc tests to examine sex and biologically relevant interactions. Statistical significance is denoted by *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001, ****p&lt;0.0001. Supp. Figure 5: ApoeCh mutation induces no change in synapses of the 5xFAD mice. a Representative super-resolution images of Bassoon and Homer1 synaptic markers for WT, ApoeCh, 5xFAD, and 5xFAD;ApoeCh mice at 12 mo of age. Scale bar = 10µm. Insert scale bar = 1µm. b-d Quantification of Bassoon+ spots per µm3, Homer1+ spots per µm3 and colocalized Bassoon+/Homer1+ synaptic spots per µm3. e-g Quantification of Bassoon+ spots per µm3, Homer1+ spots per µm3 and colocalized Bassoon+/Homer1+ synaptic spots per µm3 separted by sex. Three images per mouse and n = 5-6 mice/sex/genotype. In each panel, sex of individual animals is denoted by pink (female) or blue (male) circles. Data are represented as mean ± SEM. Two-Way ANOVA followed by Tukey’s post hoc test to examine biologically relevant interactions. Comparisons with 4 genotypes (i.e. WT, ApoeCh, 5xFAD, and 5xFAD;ApoeCh), only comparisons between genotypes were analyzed and reported. Statistical significance is denoted *p&lt;0.05, **p&lt;0.001. Supp. Figure 6: Quantification of astrocyte and microglia in 4-mo-old WT, ApoeCh, 5xFAD, 5xFAD;ApoeCh mice. a, c Representative 20x confocal images of the subiculum stained for dense-core plaque with AmyloGlo (green) and immunolabeled for astrocyte with GFAP (red, a) and IBA1 for microglia (red, c). Scale bar=100µm. In each panel, sex of individual animals is denoted by pink (female) or blue (male) circles. b, d Total GFAP+ (b) and IBA1+ volume (d) in the subiculum per FOV of 4-mo-old WT, ApoeCh, 5xFAD, 5xFAD;ApoeCh mice. e, f Total number of IBA1+ cells per FOV of 4 mo (e) and 12 mo (f) WT, ApoeCh, 5xFAD, 5xFAD;ApoeCh mice in the subiculum. g-h Sex-separated 4-mo (g) and 12-mo (h) quantification of GFAP+ astrocyte volume in the subiculum. i-l Sex-separated 4-mo IBA1+ total volume (i) and cell number per FOV (j); and 12-mo IBA1+ total volume (k) and cell number per FOV (l) in the subiculum of WT, ApoeCh, 5xFAD, 5xFAD;ApoeCh mice. n = 4-6 mice/sex/genotype. Data are represented as mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc tests to examine biologically relevant interactions. For sex-stratified comparisons, only comparisons between genotypes were analyzed and reported. Statistical significance is denoted by *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001, ****p&lt;0.0001. # denotes trending significance. Supp. Figure 7: Cell segmentation and annotation in 5xFAD;ApoeCh spatial proteomics data. a Representative images of cell segmentation (teal outline) in cortex, dentate gyrus (i.e., high cell density region), and white matter tracts for 5xFAD;ApoeCh spatial proteomics dataset. DAPI nuclear stain is shown in light grey. White dotted line demarcating anatomical landmarks for dentate gyrus and white matter tracts. Scale bar = 50 µm. b 12 CELESTA cell types in XY space for all 12 hemibrains (n = 3/genotype). Supp. Figure 8: Cell segmentation and annotation in 5xFAD;ApoeCh spatial transcriptomics data. a Representative images of cell segmentation (teal outline) in white matter tracts, CA1, and cortex for 5xFAD;ApoeCh spatial transcriptomics dataset. DAPI nuclear stain shown in light grey, and histone marker shown in green. White dotted lines demarcating anatomical landmarks for white matter tracts and CA1 of hippocampus. Scale bar = 50 µm. b 33 cell types in XY space for all 12 sections (n = 3/genotype). Supp. Figure 9: Distribution of cell types across genotypes in 5xFAD;ApoeCh dataset. a-b Data quality is comparable across samples. a Number of unique transcripts per cell, grouped by sample. b Number of unique genes (features) per cell, grouped by sample. c UMAP of cells from 5xFAD;ApoeCh spatial transcriptomics dataset split by genotype (5xFAD;ApoeCh, 5xFAD, ApoeCh, WT). DAM (dark green) and DAA (dark orange) clusters are primarily present in 5xFAD;ApoeCh and 5xFAD genotypes, but not ApoeCh and WT genotypes. d Heatmap of the top five marker genes per major cell type. DGE analysis was performed between each major cell type compared to all other major cell types to identify the top 5 genes expressed for each major cell type. e Stacked bar plot of cell counts of all 33 clusters, split by genotype. Clusters are grouped by their major cell type (i.e., ODC 1-5 and OPC are grouped together on the bar plot), and then within each major cell type plotted by size in descending order. Supp. Figure 10: Differential gene expression analysis between 5xFAD vs. WT and 5xFAD;ApoeCh vs. 5xFAD across major cell types and all cell types. a Volcano plots showing DEGs between 5xFAD and WT across each cluster. Because DAMs and DAAs are present in 5xFAD but not in WT mice, DAMs and microglia were grouped together into “All microglia”, and DAAs and astrocytes were grouped together into “All astrocytes” for the 5xFAD vs. WT comparison. Each gene represents a point on the volcano plot. Red and blue colored points represent significantly up- and downregulated genes, respectively (i.e., padj &lt; 0.05 and absolute average difference &gt; 0.3). Black points represent unsignificant genes (i.e., padj&gt; 0.05 or absolute average difference &lt; 0.3). Only the top 20 genes are labelled for legibility. b Volcano plots showing DEGs between 5xFAD;ApoeCh and 5xFAD across each cluster. Dotted red outlines highlight clusters CA1 and L2/L3, which are shown to have the greatest (most negative) DD score in the 5xFAD;ApoeCh vs. 5xFAD comparison (Fig. 4i). c Volcano plots showing DEGs between 5xFAD and WT across each major cell type. DAMs and microglia are grouped into “All microglia”, and DAAs and astrocytes into “All astrocytes”. Supp. Figure 11: Differential gene expression changes in L2/L3 of cortex and CA1 of hippocampus. a Examples of high spatial specificity for dentate gyrus, CA1, and L2/L3 clusters in a 5xFAD;ApoeCh section. b Dot plot showing pseudo-bulk expression of the top DEGs in the 5xFAD;ApoeCh vs. 5xFAD comparison across all four genotypes in L2/L3 and CA1 clusters. Supp. Figure 12: Microglial subclustering in 5xFAD;ApoeCh dataset. a Feature plots for common microglial and DAM marker genes. b Example of cell segmentation and cell annotation surrounding an amyloid beta plaque in a 5xFAD;ApoeCh brain. (top) White arrows pointing to two cells annotated as amyloid beta plaques. Many cells in this plaque cluster were anucleate (i.e., did not contain histone marker). DAPI nuclear stain (light grey), histone marker (green). (bottom) Cell type annotation for all microglia. Cells annotated as Abeta plaques (red) are surrounded by 5 DAM-2 cells (turquoise), 2 DAM-1 cells (blue), and a Microglia-1 (magenta). c Heatmap of the top 10 marker genes for each microglial subcluster. DGE analysis was performed for each microglial subcluster compared to all other microglial subclusters to obtain the top 10 genes expressed in each microglial subcluster. Supp. Figure 13: Behavioral analysis of 5- and 9-mo-old WT, ApoeCh, PS19, PS19;ApoeCh mice. In each panel, sex of individual animals is denoted by pink (female) or blue (male) circles. a, b Weight of 5-mo-old (a) and 9-mo-old (b) mice taken at euthanizing day. c, d Violin plot of hindlimb clasping score of 4-mo-old (c) and 12-mo-old (d) mice. e, g Total time mice spent in the center of open field behavioral assay of 5-mo-old (e) and 9-mo-old (g) in the 5 min recording time. f, h Mean velocity mice traveled in the center of the open field in 5 mins of 5-mo (f) and 9-mo-old mice (h). i, j Total time 5-mo (i) and 12-mo-old (j) mice spent in open arms of the elevated plus maze behavior test. k, l Measurement of plasma NfL level in WT, ApoeCh, PS19, and PS19;ApoeCh mice at 5- (k) and 9-mo (l). n = 4-10 mice/sex/genotype. Data are represented as mean ± SEM. Two-way ANOVA followed by Tukey’s post hoc tests to examine biologically relevant interactions. Statistical significance is denoted by *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001,****p&lt;0.0001. # denotes trending significance. Supp. Figure 14: Immunohistochemical and biochemical analyses of tau in 5 and 9-mo-old WT, ApoeCh, PS19, PS19;ApoeCh mice. In each panel, sex of individual animals is denoted by pink (female) or blue (male) circles. a Representative 20x confocal images of the dentate gyrus (top) and piriform cortex (bottom) immunolabeled with AT8 for phosphorylated tau (green). Scale bar = 100µm. b, c Percent coverage of AT8+ area per FOV in the dentate gyrus (b) and piriform cortex (c). d Representative whole hemisphere stitched images of MC1 immunolabeled 9-mo WT, PS19, and PS19;ApoeCh mice with zoomed-in inset of MC1+ inclusion in the cortex. Scale bar = 500µm e Quantification of MC1+ inclusions number in the hemisphere stitched image of 9-mo PS19 and PS19;ApoeCh mice. f-k Measurement of total tau in RAB, RIPA, and formic acid fraction of the micro-dissected hippocampi (f-h) and cortices (i-k) of 9-mo-old WT, ApoeCh, PS19, and PS19;ApoeCh mice. n = 4-6 mice/sex/genotype. Data are represented as mean ± SEM. Student’s t-test, unpaired comparison. Two-way ANOVA followed by Tukey’s post hoc tests to examine biologically relevant interactions. Statistical significance is denoted by *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001, ****p&lt;0.0001. Supp. Figure 15: Western blot images of total tau and p-tau in cortical soluble protein fraction of 9-mo PS19 mice. a-b Whole western blot images from left to right: total protein stain, total tau detected by Dako antibody, p-tau detected by AT8, merged blot of Dako- and AT8-detected protein. c-d Whole western blot images from left to right: total protein stain, total tau detected by HT7 antibody, p-tau detected by pThr231, merged blot of HT7- and pThr231-detected protein. Sample genotypes on blots: 1= WT Female, 2= PS19 Female, 3= PS19;ApoeCh Female, 4= PS19 Male, 5= PS19;ApoeCh Male, 6= PS19 Female, 7= PS19;ApoeCh Female, 8= PS19 Male, 9= PS19;ApoeCh Male, 10 = WT Male, 11= MAPT KO Female. Red arrows highlight rows of bands of interest. White boxes highlight portions of blots used as represented images in Figure 5k-p. n = 4 mice/sex/genotype. Supp. Figure 16: Immunohistochemical analysis of 5 and 9-mo-old WT, ApoeCh, PS19, PS19;ApoeCh mice. a, c Representative 20x confocal images of the dentate gyrus immunolabeled with IBA1 for microglia (red, a) and GFAP for astrocytes (blue, c). Scale bar = 100µm. In each panel, sex of individual animals is denoted by pink (female) or blue (male) circles. b, d Total volume of IBA1+ (b) and GFAP+ (d) cells per FOV of the dentate gyrus. e, f IBA1+ microglia number per FOV of 5-mo-old (e) and 9-mo-old (f) mice in the dentate gyrus. g Representative whole hemisphere stitched images immunostained for CD68. Scale bar = 500µm. h Percent CD68+ area covered in the whole hemisphere brain sections of 9-mo-old WT, ApoeCh, PS19, and PS19;ApoeCh mice. n = 4-6 mice/sex/genotype. Data are represented as mean ± SEM. Student’s t-test, unpaired. Two-way ANOVA followed by Tukey’s post hoc tests to examine biologically relevant interactions. Statistical significance is denoted by *p&lt;0.05, **p&lt;0.01, ***p&lt;0.001, ****p&lt;0.0001. Supp. Figure 17: Cell segmentation and annotation in PS19;ApoeCh spatial proteomics data. a Representative images of cell segmentation (teal outline) in dentate gyrus, choroid plexus, and cortex for PS19;ApoeCh spatial proteomics dataset. DAPI nuclear stain is shown in light grey. White dotted line demarcates anatomical landmarks for dentate gyrus. Scale bar = 50 µm. b CELESTA cell types in XY space for 11 hemibrains (n = 3/genotype, except n = 2 for ApoeCh). Supp. Figure 18: Cell segmentation and annotation in PS19;ApoeCh spatial transcriptomics data. a Representative images of cell segmentation (teal outline) in cortex, dentate gyrus, and white matter tracts for PS19;ApoeCh spatial transcriptomics dataset. DAPI nuclear stain (light grey), histone marker (green). White dotted line demarcates anatomical landmarks for dentate gyrus, white matter tracts, and CA1 of hippocampus. Scale bar = 50 µm. b 40 clusters in XY space for all 11 sections (n = 3/genotype, except n = 2 for ApoeCh). Supp. Figure 19: Distribution of cell types across genotypes in PS19;ApoeCh dataset. a-b Data quality is comparable across samples. a Number of unique transcripts per cell, grouped by sample. b Number of unique genes (features) per cell, grouped by sample. c UMAP of cells from PS19;ApoeCh spatial transcriptomics dataset split by genotype (PS19;ApoeCh, PS19, ApoeCh, and WT). DAM (dark green) cluster is present in PS19 genotypes, but not in PS19;ApoeCh, ApoeCh, or WT genotypes. d Heatmap of the top 5 marker genes per major cell type. DGE analysis was performed between each major cell type compared to all other major cell types to identify the top 5 genes expressed for each major cell type. e Stacked bar plot of each of the 40 clusters, split by genotype. Clusters are grouped by their major cell type (e.g., Astrocyte 1-3 and DAA are grouped together) and plotted in order from largest to smallest cluster. Supp. Figure 20: Differential gene expression analysis between PS19 vs. WT and PS19;ApoeCh vs. PS19 across major cell types and all cell types. a Volcano plots of DEGs between PS19 and WT for all clusters. Because DAA and DAM clusters are present in very low numbers in WT mice, DAA and astrocytes are combined into “All astrocytes”, and DAM and microglia are combined into “All microglia”. b Volcano plots of DEGs between PS19;ApoeCh and PS19 for all 40 clusters. c Volcano plots of DEGs between PS19 and WT for each major cell type. All astrocytes and all microglia are grouped together. Supp. Figure 21: Differential gene expression between PSEN1-E280A;APOECh vs. PSEN1-E280A in human snRNA-seq data. a UMAP of integrated human snRNA-seq data comprising 13,643 cells by 975 genes. Cell type annotations were derived from metadata from the publicly available datasets. b Heatmap of the top 5 marker genes in each cell type. c DEGs between PSEN1-E280A;APOE3Ch vs. PSEN1-E280A in astrocytes. d Scatterplot of log-2 fold-change of the average expression between the two groups for all statistically significant genes (padj &lt; 0.05) in the human comparison (PSEN1-E280A;APOE3Ch vs. PSEN1-E280A; x-axis) vs. 5xFAD comparison (5xFAD;ApoeCh vs. 5xFAD; y-axis) in all microglia. Point color represents direction of correlation; positive (blue) and negative (yellow). Regression line in blue. e Scatterplot of log2FC of average expression for human comparison vs. PS19 comparison (PS19;ApoeCh vs. PS19; y-axis) in all microglia. d-e While the magnitude of difference appears greater for the human data, it should be noted that the raw transcript counts obtained from imaging-based single-cell spatial transcriptomics (hybridization-based) are on a different scale from those obtained from snRNA-seq (sequencing-based. Thus, interpretation is limited to the direction and statistical significance of these changes. All positively correlated genes (blue) from d and e are listed in the intersections of the Venn diagram in Fig. 9k. Supp. Figure 22: Unbiased proteomics show differential effects of ApoeCh in 5xFAD versus PS19 mice. a-d These results complement Fig. 10g-j by presenting the opposite regulation patterns. a Pathway analysis of all significantly downregulated proteins in the 5xFAD;ApoeCh vs. 5xFAD comparison. b Pathway analysis of all significantly upregulated proteins in the PS19;ApoeCh vs. PS19 comparison. c Protein-protein interaction (PPI) network of all significantly downregulated proteins in the 5xFAD;ApoeCh vs. 5xFAD comparison. The thickness of the line connecting two nodes indicates the degree of confidence in the prediction of the PPI. d PPI network of all significantly upregulated proteins in the PS19;ApoeCh vs. PS19 comparison.