Project description:Microglia, the brain’s principal immune cells, have been implicated in the pathogenesis of Alzheimer’s disease (AD), a condition shown to affect more females than males. Although sex differences in microglial function and transcriptomic programming have been described across development and in disease models of AD, no studies have comprehensively identified the sex divergences that emerge in the aging mouse hippocampus. Further, existing models of AD generally develop pathology (amyloid plaques and tau tangles) early in life and fail to recapitulate the aged brain environment associated with late-onset AD. Here, we examined and compared the transcriptomic and translatomic sex effects in young and old murine hippocampal microglia. Hippocampal tissue from C57BL6/N and microglial NuTRAP mice of both sexes were collected at young (5-6 month-old [mo]) and old (22-25 mo) ages. Cell sorting and affinity purification techniques were used to isolate the microglial transcriptome and translatome for RNA-sequencing and differential expression analyses. Flow cytometry, qPCR, and imaging approaches were used to confirm the transcriptomic findings. There were marginal sex differences identified in the young hippocampal microglia, with most differentially expressed genes (DEGs) restricted to the sex chromosomes. Both sex chromosomally- and autosomally-encoded sex differences emerged with aging. These sex DEGs identified at old age were primarily female-biased and enriched in senescent and disease-associated microglial signatures. Pathway analyses identified upstream regulators induced to a greater extent in females than in males, including inflammatory mediators IFNG, TNF, and IL1B, as well as AD-risk genes TREM2 and APP. These data suggest that female microglia adopt disease-associated and senescent phenotypes in the aging mouse hippocampus, even in the absence of disease pathology, to a greater extent than males. This sexually divergent microglial phenotype may explain the difference in susceptibility and disease progression in the case of AD pathology. Future studies will need to explore sex differences in microglial heterogeneity in response to AD pathology and determine how sex-specific regulators (i.e., sex chromosomal or hormonal) elicit these sex effects.

Project description:Alzheimer’s disease (AD), the most common age-related neurodegenerative disease, is closely associated with and manifested by neuroinflammation, yet the alteration of immune landscape in AD is largely unknown, preventing a deeper mechanistic understanding of neuroinflammation in AD. Two thirds of AD patients are females, and women have a higher risk of developing AD. Women with AD have more extensive brain histological changes than men with AD, more severe cognitive symptoms, and more severe neurodegeneration, suggesting that the disease affects female and male brains differentially. Despite evident AD sex variance, mechanisms and pathways are still poorly understood. Thus, a focus on sex differences in AD is essential to move the field towards effective interventions and to develop sex-specific therapies. To understand the cellular heterogeneity and disease-associated cellular changes in human AD brain, we performed unbiased massively parallel single nucleus RNA sequencing with postmortem frozen human brain tissues of middle temporal gyrus, a brain cortical region strongly affected by AD. From 12 individuals with and without AD, we isolated brain nuclei by sucrose gradient ultracentrifugation, generated single nucleus libraries with 10x Chromium platform (10x Genomics), and sequenced on NovaSeq6000 S4 sequencer (Illumina). We integrated snRNA-seq data of human brains from these 12 individuals of both Alzheimer’s Disease (AD, Braak Stage V/VI, n = 6) and age-matched healthy controls (HC, Braak Stage I/II, n = 6) by single nucleus analysis using Seurat. After quality control filtering, we profiled and analyzed 64,845 single nucleus transcriptomes, clustered all the cells jointly across the 12 donors that include 6 females and 6 males, and identified and annotated the major cell types of the human brain by interrogating the expression patterns of known gene markers, including neurons, excitatory neurons, inhibitory neurons, astrocytes, microglia, oligodendrocytes, oligodendrocyte precursor cells, endothelial cells, and pericytes.

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