Journal: bioRxiv

Article Title: Protective mechanisms against Alzheimer’s Disease in APOE3-Christchurch homozygous astrocytes

doi: 10.1101/2025.01.21.634115

Figure Lengend Snippet: (A) Schematic of the tau uptake assay in iPSC-derived astrocytes. (B) Representative overlaid histograms demonstrating the shift in fluorescence of cells with AF647 labeled tau (2N4R-P301L) aggregates (50nM, 3h) compared to cells treated with PBS. The dotted line represents the median AF647 intensity in the WT astrocytes treated with Tau. (C) Measurement of tau uptake by flow cytometry normalized to cells treated with PBS (n=3, 10000 cells each). (D) Measurement of tau uptake in cells treated with Tau (50nM, 3h) and various concentrations of heparin normalized to cells treated with PBS (n=3, 10000 cells each). Statistical significance is determined by two-way ANOVA with uncorrected Fisher’s LSD multiple comparison tests. Displayed is the multiple comparison of cells treated with 100 ug/ml heparin against cells with no treatment for each cell line. (E) Measurement of tau uptake in cells treated with Tau (50nM, 3h) and various concentrations of RAP normalized to cells treated with PBS (n=3, 10000 cells each). Statistical significance is determined by two-way ANOVA with uncorrected Fisher’s LSD multiple comparison tests. Displayed is the multiple comparison of cells treated with 500 nM RAP against cells with no treatment for each cell line. (F) Representative images of iPSC-derived astrocytes immunolabeled with anti-10E4, an HSPG marker. Green, 10E4; blue, Hoechst 33342; scale bar = 10 µm (G) Quantification of the expression of HSPGs using fluorescence intensity of anti-10E4 normalized to that of Hoechst per field of view (n=24 FOVs taken from 8 biological replicates, 3 FOVs per biological replicate) (H) Quantification of the expression of HSPG-related genes with RNA sequencing (n=3 for WT and n=6 for APOE3-Ch). Z-score of each HSPG gene is color coded and the size of the circle represents the mean expression. P-values were calculated using a Wald test for differential expression between groups (WT vs APOE3-Ch). Multiple testing correction was performed using the Benjamini-Hochberg method to control the false discovery rate (FDR). Genes with an adjusted p-value < 0.05 were considered statistically significant. Significantly upregulated genes were marked by red asterisks and significantly downregulated genes were marked by blue asterisks. (I) Representative images of iPSC-derived astrocytes immunolabeled with anti-LRP1. Green, LRP1; blue, Hoechst 33342; scale bar = 10 µm (J) Quantification of the expression of LRP1 using fluorescence intensity of anti-LRP1 normalized to that of Hoechst per field of view (n=12 FOVs taken from 4 biological replicates, 3 FOVs per biological replicate) All data are expressed as mean ± s.d. with individual data points shown. One-way ANOVA with Dunnett’s multiple comparison tests was performed to determine the significance unless otherwise specified. The results designated as “ns” are not significant; **p<0.01; ***p<0.001;****p<0.0001

Article Snippet: To probe for HSPGs, primary antibody mouse anti-10E4 (Amsbio, Cat. No. 370255-S) was applied at a 1:100 dilution in 5% normal goat serum.

Techniques: Derivative Assay, Fluorescence, Labeling, Flow Cytometry, Comparison, Immunolabeling, Marker, Expressing, RNA Sequencing Assay, Control