Abstract Introduction Alzheimer's disease (AD) is a neurodegenerative disorder involving interactions between different cell types in the brain. Previous single‐cell and bulk expression Alzheimer's studies have reported conflicting findings about the key cell types and cellular pathways whose expression is primarily altered in this disease. We re‐analyzed these data in a uniform, coherent manner aiming to resolve and extend past findings. Our analysis sheds light on the observation that females have higher AD incidence than males. Methods We re‐analyzed three single‐cell transcriptomics datasets. We used the software Model‐based Analysis of Single‐cell Transcriptomics (MAST) to seek differentially expressed genes comparing AD cases to matched controls for both sexes together and each sex separately. We used the GOrilla software to search for enriched pathways among the differentially expressed genes. Motivated by the male/female difference in incidence, we studied genes on the X‐chromosome, focusing on genes in the pseudoautosomal region (PAR) and on genes that are heterogeneous across individuals or tissues for X‐inactivation. We validated findings by analyzing bulk AD datasets from the cortex in the Gene Expression Omnibus. Results Our results resolve a contradiction in the literature, showing that by comparing AD patients to unaffected controls, excitatory neurons have more differentially expressed genes than do other cell types. Synaptic transmission and related pathways are altered in a sex‐specific analysis of excitatory neurons. PAR genes and X‐chromosome heterogeneous genes, including, for example, BEX1 and ELK1, may contribute to the difference in sex incidence of Alzheimer's disease. GRIN1, stood out as an overexpressed autosomal gene in cases versus controls in all three single‐cell datasets and as a functional candidate gene contributing to pathways upregulated in cases. Discussion Taken together, these results point to a potential linkage between two longstanding questions concerning AD pathogenesis, involving which cell type is the most important and why females have a higher incidence than males. Highlights * By reanalyzing three, published, single‐cell RNAseq datasets, we resolved a contradiction in the literature and showed that when comparing AD patients to unaffected controls, excitatory neurons have more differentially expressed genes than do other cell types. * Further analysis of the published single‐cell datasets showed that synaptic transmission and related pathways are altered in a sex‐specific analysis of excitatory neurons. * Combining analysis of single‐cell datasets and publicly available bulk transcriptomics datasets revealed that X‐chromosome genes, such as BEX1, ELK1, and USP11, whose X‐inactivation status is heterogeneous may contribute to the higher incidence in females of Alzheimer's disease. Keywords: Alzheimer's disease, differential gene expression, excitatory neurons, sex difference, single‐cell transcriptomics 1. INTRODUCTION Alzheimer's disease (AD) is a progressive disease where neurons in brain regions involved in thinking, learning, and memory become damaged. In 2021, an estimated 6.2 million Americans 65 years of age and older were living with AD.[32] ^1 Alzheimer's is a complex physiological disease due to the involvement of many cell types, such as neurons, astrocytes, and microglia. We analyze published datasets of single‐cell and bulk gene expression in AD to investigate two questions regarding its pathogenesis: (1) what are the cell‐type–specific transcriptional alterations that are associated with AD and which cell type is the most transcriptionally altered; and (2), given the considerably larger AD incidence in females compared to males,[33] ^2 what are the underlying most notable sex‐specific, cell‐type–specific transcriptional alterations? Mathys et al. published the first single‐cell transcriptomics dataset from Alzheimer's patients and controls. They found that all cell types in the prefrontal cortex had transcriptional changes associated with AD.[34] ^3 They found neurons to have more differentially expressed genes (DEGs) downregulated, whereas other cell types such as astrocytes and microglia had more genes upregulated. They found excitatory neurons to have the most DEGs [35]^3 . However, in a second, single‐cell Alzheimer's dataset ([36]GSE157827), Lau et al. found that astrocytes have more DEGs compared to other cell types.[37] ^4 Aiming to resolve this apparent quandary, we reanalyze the Mathys and Lau datasets using consistent methods. For validation, we analyze a third single‐cell transcriptomics Alzheimer's dataset,[38] ^5 with the caveat that we cannot compare cell types, such as endothelial cells, that are not adequately sampled in all three datasets. In addition to the relative importance of different brain cell types, another longstanding puzzle about AD is the observation of its substantially higher incidence in females.[39] ^2 The biology of the sex differences in AD incidence is poorly understood, even after a mouse study targeting this question.[40] ^6 Mathys et al. did sex‐specific analysis and concluded that there are subtle differences in the transcriptomics between sexes in each cell type, with neurons and oligodendrocytes having the most extreme differences.[41] ^3 However, they did not look at specific genes or enriched pathways in a sex‐stratified way. To study the observed sex differences at a single‐cell resolution, Belonwu and colleagues reused the Mathys dataset to perform a sex‐stratified analysis to identify sex‐stratified cell‐type–specific perturbations in Alzheimer's patients. They found that neurons were more similar between males and females compared to glial cells, having more shared genes and pathways.[42] ^7 Their analysis was limited inadvertently to fewer than 200 genes. We instead perform a sex‐stratified analysis for most genes using the same differential expression method that we use to resolve the cell‐type contradiction. To validate the sex‐specific single‐cell findings, we additionally analyze three bulk expression datasets from the Gene Expression Omnibus (GEO; Methods). Notably, a recent article reported a study of the role of sex differences at the transcriptome level and how it influences complex traits analyzed in the most recent (v8) version of the Genotype‐Tissue Expression (GTEx) v8 project.[43] ^8 Oliva and colleagues identified sets of sex‐biased genes (genes whose expression levels differ significantly between males and females) for dozens of different tissues, which we further consider in our analysis. For our analysis of sex differences, we introduce standard terminology about X‐chromosome genes. Near the Xp telomere is the pseudoautosomal region (PAR) containing a handful of genes for which females express two copies on X, whereas males express one copy on X and one or more copies on Y. The gene copies on X and Y do not recombine and hence can diverge in evolution.[44] ^9 Among the non‐PAR X‐chromosome genes, most have their expression between males and females balanced by X‐chromosome inactivation in females,[45] ^10 which is regulated primarily by the RNA gene XIST.[46] ^11 We partition the non‐PAR genes into three categories according to their X‐inactivation status in females: always inactivated, always escaping X‐inactivation, and heterogeneous with respect to X‐inactivation using a published classification.[47] ^12 Heterogeneity of Xinactivation may be across individuals and/or across tissues. RESEARCH IN CONTEXT 1. Systematic Review: Literature search in PubMed and Google Scholar showed that females have a substantially higher incidence of Alzheimer's disease. Transcriptomics studies to investigate the sex difference have been limited. The authors searched in Synapse and GEO for single‐cell and bulk transcriptomics datasets. Six suitable datasets were identified. The analyses in references 3 and 4