Transcriptomic Profiling Across Age, APOE and Sex for Models of Alzheimer’s Disease at a Single Cell Resolution

Abstract

AbstractBackgroundAdvanced age, apolipoprotein E4(APOE4), and female sex are well‐established risk factors for Alzheimer’s Disease(AD). Almost two‐thirds of people with AD are women. The APOE4 genotype, the strongest genetic risk factor for AD, is associated with higher AD risk in women than in men between ages 55 to 70. We propose to study the interplay between these risk factors by analyzing single‐nucleus RNA‐sequencing(snRNA‐seq) data of the hippocampus of human APOE4 and APOE3 knock‐in(KI) female and male mice across age.MethodsA sex‐balanced cohort of APOE3‐KI and APOE4‐KI homozygous mice were sacrificed at 6 (young), 12 (adult), and 18 (old) months of age(Figure1A). Single nuclei were prepared from the hippocampus from each mouse and sequenced. Demultiplexed fastq files were aligned to a custom reference genome mm10‐1.2.0, using Cellranger v2.0.1. Quality assurance, count normalization, clustering, cell type identification, and differentially expressed gene(DEG) analysis were performed using Seurat4.0.ResultsOur dataset consists of a total of 527,395 cells covering 27,153 genes. A total of 36 cell clusters were determined with six major brain cell types including astrocytes, microglia, oligodendrocytes, oligodendrocyte precursor cells, and excitatory and inhibitory neurons (Figure1B). We investigated the abundance of each cell type and found that cellular composition is consistent across ages, APOE genotypes, and sexes for all cell types except astrocytes. Astrocyte abundance is significantly higher in old females compared to old males(padj = 0.025, Tukey’s multiple comparison test) and in APOE4 young males compared to APOE3 young males (padj = 0.042)(Figure1C).Sex and age stratified differential gene expression analysis comparing APOE4 versus APOE3 astrocytes reveals many shared DEGs between young and adult males but not with old males, while females have few consistent DEGs across the three age groups(Figure1D). We identified several DEGs associated with plasma membrane assembling and post‐synaptic transmission and signaling that are upregulated in APOE4 old females versus APOE3 old females while downregulated in APOE4 young females, young and adult male compared to their APOE3 counterparts.ConclusionWe identify clear interplay between age, APOE genotype, and sex in mouse models of AD and investigate the interaction and convergence of these risk factors at a single cell resolution.