Exploring the sex and estrous cycle-dependent differences in microglia response to amyloid-beta pathology in female and male C57BL6/J (WT) and 5xFAD using bulk mRNAseq of isolated microglia.

Alzheimer's disease (AD) presents with a sex bias where women are at higher risk and exhibit worse cognitive decline and brain atrophy compared to men. Microglia play a significant role in the pathogenesis and progression of AD and have been shown to be sexually differentiated in health and disease. Whether and how microglia contribute to the sex differences in AD remains to be elucidated. Herein, we characterize the sex differences in microglial transcriptomic changes that occur in females and males in response to amyloid-beta (Aß). In females, we focus on two hormonally distinct stages of the rodent estrous cycle: proestrus and diestrus. Our transcriptomic data revealed that female microglia are not overtly different at the proestrus or diestrus stages. However, we found stark sex differences between female and male microglia transcriptomes in the 5xFAD brains, where female 5xFAD microglia upregulated genes involved in glycolytic metabolism, antigen presentation, disease-associated microglia, and microglia neurodegenerative phenotype (DAM/MGnD) compared to male 5xFAD microglia. Interestingly, a significant proportion of differentially expressed genes (DEGs) between female and male 5xFAD microglia were interferon-stimulated genes (ISGs), all of which were upregulated in females. Overall design: RNAseq profiling of diestrus and proestrus female and male C57BL6/J (BL6) and 5xFAD microglia at 5.5 months of age. Vaginal smears (started 2 weeks prior to sacrifice) and uterus weight (at the time of sacrifice) was used to group females into proestrus (Pro) and diestrus (Die). Cortex was microdissected and homogenized to make a single cell suspension. Myelin was removed using a Percoll density gradient and microglia were stained with fluorescently-conjugated antibodies. Micoglia were isolated via fluorescence-activated cell sorting on the basis of CD11b+CD45low-intermediate expression. TMEM119 and P2RY12 were used to cofirm microglial identity.