Sample Classification and Analytic Workflow
Samples were categorized into cases and controls based on tau tangle and Aβ plaque burdens, using Braak clinical staging and CERAD scores , respectively (AD: Braak stage EM C; CERAD score ge EMcontrol: Braak stage ≤ III; CERAD score e EM CSL_ resulted in snRNA-Seq datasets containing 17,723 genes expressed by 62,741 cells from the prefrontal cortex cohort (Table 1, Supplementary Table 1) and 10,846 genes expressed by 11,284 cells from the entorhinal cortex cohort (Table 2, Supplementary Table 2), which were acquired from different sets of individuals (Fig. 1). In both brain regions, a sex-stratified differential gene expression (DGE) analysis was performed comparing AD cases to controls, with APOE genotype as a covariate, in astrocytes (Ast), microglia (Mic), excitatory neurons (Ex), inhibitory neurons (In), undifferentiated neurons (Neu), oligodendrocytes (Oli), and OPCs (Supplementary Tables 3 and 4). For the entorhinal cortex cohort, data integration was performed and APOE genotype was included as a sole covariate in our DGE analysis to account for batch effects and avoid collinearity in our model. DEGs were determined using a BH-adjusted p value < 0.05 and absolute LFC > 0.25 as cutoffs. DEGs were passed as inputs for pathway enrichment analysis, which provided pathways to be used as inputs for subsequent network analysis. We examined gene expression and pathway networks in AD versus neurotypical cells to identify cell type- and brain region-specific and non-specific differences based on sex.
Sex-Stratified DGE Analysis in the Prefrontal Cortex Reveals Sex-Specific Disease-Related Changes in Glial Cell Types
Leveraging data from Mathys et al., from our sex-stratified DGE analysis, we identified DEGs meeting significance and LFC thresholds (Table 3) in all cell types except male inhibitory neurons when comparing AD to non-AD (Supplementary Table 5). We identified 73 DEGs across all cell types in the prefrontal cortex (Table 3, Supplementary Table 5). Of these DEGs, 36 were shared in both sexes, while 8 and 29 were specific to AD compared to control males and females, respectively. We also observed more shared DEGs in AD case versus control female signatures versus male signatures across the cell types (Fig. 2a), which is consistent with previous bulk tissue analysis . Some of the DEGs that overlap most across cell types within one sex or across sexes include LINGO1, a negative regulator of myelination [50, 51], which we found upregulated in all AD compared to control female cell types; SLC1A3, which encodes excitatory amino acid transporter 1 that transports glutamate in the synaptic cleft  and was perturbed in all female AD compared to control cell types except oligodendrocytes and OPCs; and SPP1, a protein involved in neuroinflammation also known as osteopontin  that we observed to be upregulated in AD versus control samples of both female and male excitatory neurons and microglia, as well as female astrocytes and inhibitory neurons. Also, clustering samples by AD compared to control pseudobulk cell type gene expression (Fig. 2b) showed samples to cluster by sex before cell type identity for all cell types except excitatory neurons.
In addition to identifying shared DEGs across cell types and sexes, we also observed a larger range of LFC in the analysis of female AD versus control ([− 0.423, 1.058], median = 0.314) compared to the analysis of male AD versus control ([− 0.370, 0.620], median = 0.343). Within each cell type, we observed DEGs, a number of which are relevant to and have been studied in AD (e.g., NRXN1 , SPP1 , DHFR , SGK1 , ERBB2IP ), meeting significance and LFC thresholds. These DEGs are shared by both sexes in AD versus control astrocytes, microglia, and excitatory neurons, with consistent directionality in both sexes (Fig. 2c, d, yellow color; Supplementary Fig. 3). Overall, in the prefrontal cortex, we identified sex-distinct disease-related transcriptomic changes in gene expression primarily among glial cells (Fig. 2d, brown color for female-distinct and blue color for male-distinct).
Sex-Stratified DGE Analysis in the Entorhinal Cortex Reveals Sex-Specific Disease-Related Changes, Including Opposite Transcriptomic Changes Between Sexes
Leveraging data from Grubman et al., we identified DEGs (Table 4) comparing AD to non-AD in all cell types stratified by sex. We identified 232 DEGs across all cell types in the entorhinal cortex (Table 4, Supplementary Table 6). Of these DEGs, 211 were shared in both sexes, while 20 and 1 were specific to AD compared to control males and females, respectively. We observed shared DEGs across cell types when comparing AD versus control samples in both sexes (Fig. 3a). Some of the DEGs that overlap most across cell types within one sex or across sexes include CLU [9, 58], HSPA1A , RBFOX1 , and CST3 , which are relevant in AD progression. Clustering of samples by AD compared to control pseudobulk cell type-specific gene expression (Fig. 3b) showed samples to cluster by sex before cell type identity for every cell type and highlighted opposing gene expression patterns based on sex. Indeed, interestingly, 186 of the 211 DEGs shared between male and female AD were regulated in opposite directions with respect to controls, at least in some cell types.
When comparing the magnitude of gene expression changes across sexes in AD versus control samples, we found males to have a greater range of LFCs ([− 2.174, 3.461], median = 0.567) compared to females ([− 1.657, 2.649], median = − 0.436). We visualized these differences in DEGs such as LINGO1, which had a higher fold change difference in male astrocytes (3.415) compared to female astrocytes (0.4); GPM6A, which was upregulated in male oligodendrocytes and downregulated in female oligodendrocytes; CST3, which was upregulated in male neurons, male oligodendrocytes, and male and female OPCs, and downregulated in female neurons, female oligodendrocytes, and male and female astrocytes; and LINC00486, which was upregulated in all cell types of both sexes with an average LFC in males of 1.9 compared to 1.0 in females (Fig. 3c). Generally, directly comparing AD versus control DEGs within each cell type, we not only observe a subset of genes with directionally consistent changes among males and females (Fig. 3d, yellow color; Supplementary Fig. 3), but we also observed numerous changes in opposing directions across sexes (Fig. 3d, pink color; Supplementary Fig. 3) and a higher magnitude of disease-related changes in males compared to females.
Comparative Analysis Across Brain Regions Reveals More Shared Transcriptomic Sex Differences in the Entorhinal Cortex
We compared DEG results from the prefrontal and entorhinal cortices to determine whether changes in each sex were consistent across brain regions. Overall, we observed more overlaps across sex DEGs to be in the entorhinal cortex (Fig. 4a). Additionally, clustering samples by AD compared to control pseudobulk cell type gene expression (Fig. 4b) showed some clustering by brain region and sex.
Pathway and Network Analysis Reveals Sex-Specific Transcriptomic Perturbations in Glial Cells in the Prefrontal Cortex and Sex-Shared, but Flipped AD-Enriched Pathways in the Entorhinal Cortex.
Beyond identifying sex-dimorphic disease-associated genes, we performed a gene set enrichment analysis to elucidate potential biological mechanisms implicated in disease progression that are either shared or unique to each sex and to reveal the interconnections between disease-linked pathways within AD. The pathway enrichment was performed in g:Profiler , a web tool that performs functional enrichment analysis from a given gene list, using separate lists of upregulated and downregulated DEGs with an adjusted p value < 0.05 and relaxed absolute LFC above 0.1 in cell types of each sex as inputs. Significantly enriched biological pathways with an adjusted p value < 0.05 were applied to EnrichmentMap , a functional category grouping method from the Cytoscape software, to identify pathway network clusters annotated by associated biological processes (Fig. 5, Supplementary Figs. 3 and 4).
Female and male AD compared to control excitatory neurons of the prefrontal cortex shared six common enriched clusters of pathways (Fig. 5a), which were all perturbed in the same direction for both sexes. Two of these clusters (neurotransmitter glutamate/aspartate transmembrane activity and carboxylic acid biosynthetic process) were upregulated in disease in both sexes. Of the four downregulated pathway clusters, three were related to synaptic activity (modulation of the synaptic membrane, neurotransmitter release, and synapse assembly/cell junction organization), indicating a dysregulation of synaptic plasticity in AD excitatory neurons. The other downregulated pathway cluster was plasma membrane morphogenesis, which consisted of pathways including axonogenesis, cellular projection, and plasma membrane organization (Supplementary Tables 7 and 8).
In prefrontal cortex excitatory neurons, we also identified uniquely enriched disease pathway clusters for each sex (Fig. 5a). Female excitatory neurons showed upregulation of the HOXA5 factor, a DNA-binding transcription factor that regulates cell morphogenesis and tumor suppressor that inhibits proliferation and induces apoptosis , and downregulation of inflammatory-mediated cell to cell interaction through adhesion and molecule binding. Interestingly, a recent epigenome-wide association study examining samples in the prefrontal cortex and superior temporal gyrus observed elevated DNA methylation of the HOXA gene cluster to be associated with neuropathology in AD . In male excitatory neurons, we observed upregulation of axon regeneration and downregulation of distal axonal growth cone polarization. Interestingly, we also observed downregulation of tetrahydrobiopterin (BH4) synthesis, which is important for the production of essential neurotransmitters , and Rho GTPase activities in male AD compared to control excitatory neurons. Overall, excitatory neurons of the prefrontal cortex shared most case versus control differentially enriched pathways between male and females, the majority of which were downregulated in AD.
Like the enriched pathways in disease observed in excitatory neurons, the inhibitory neurons of the prefrontal cortex showed upregulation for glutamate/aspartate activities in both female and male AD inhibitory neurons compared to controls (Fig. 5b). Like male AD excitatory neurons, male AD inhibitory neurons also showed downregulation of axonal growth cone polarization and BH4 activities compared to controls. In addition, males specifically demonstrated upregulation in anterograde synaptic transmission and downregulation of nitric synthase, heat shock protein 90 (HSP90) complex, voltage potassium transporter, and kainite calcium-permeable receptor activities in AD. The ITGAV-ITGB-SPP1 complex, with known function in cell adhesion  and without previous links to AD, was uniquely upregulated in male inhibitory neurons. Of note, the pathway cluster neuronal projection was upregulated in females and downregulated in males, consistent with the enriched upregulated pathway clusters uniquely observed in females, which were modulation of spine morphogenesis and synaptic membranes. Lastly, the transcription factors, nuclear receptor TLX (essential for the regulation of self-renewal, neurogenesis, and maintenance in neuron stem cell)  and nuclear protein HOXB2 (involved in cellular development) , were upregulated only in AD female inhibitory neurons.
Unlike in neurons in the prefrontal cortex, we identified a variety of commonly enriched disease pathway networks in entorhinal cortex neurons that were regulated in opposite directions for the sexes (Fig. 5c). For instance, amyloid-beta binding/fibril formation, mitochondrial abnormality, coupled electron ATP metabolic process, demyelination/remyelination, cellular metabolism, extracellular organelle exosome vesicle, and cation transmembrane transport were among the clusters downregulated in females and upregulated in males. We did not observe any pathway networks unique to female neurons; however, for the AD male neurons in the entorhinal cortex, we identified pathways in maintaining cellular metabolism and homeostasis, through the upregulation of genes involved in axon myelination, regulation of the metabolic process, cell component locomotion, cytoskeleton organization, and intracellular ferritin complex (iron storage). In male neurons, we also observed synaptic activity deficiency, indicated by the downregulation of pathways in synaptic vesicle transport, presynaptic assembly at cell junction, synaptic membrane clustering, postsynaptic membrane morphogenesis, chemical regulation at the synapse, neuroligin family protein binding, and ionotropic receptor signaling. Additionally, male AD neurons compared to controls also showed downregulation in plasma membrane regulation, cell projection, and developmental process in differentiation. While sex differences are minimal in the neurons of the prefrontal cortex, we observed overwhelmingly shared but inversely regulated enrichment pathways in the neurons of the entorhinal cortex.
Microglia, the resident immune cells of the brain, have gained growing recognition as being critically involved in AD pathogenesis due to their key role contributing to neuroinflammation, a prominent feature of AD . Only a few significantly enriched disease pathways were observed in microglial cells of the prefrontal cortex, and none was shared across sexes (Fig. 5d). We observed upregulation of axon sprouting in response to injury in males, as well as an enriched upregulated pathway in axonogenesis regulation in females (Supplementary Table 8). Interestingly, a cluster of the PDE4B-DISC1 complex, with important functions in cAMP-regulated signal transduction and synaptic plasticity , was downregulated in females. The phosphodiesterase 4B (PDE4B) enzyme was previously shown to be pro-inflammatory in microglia and is currently under study as a therapeutic target for neuroinflammation and cognitive function impairment .
Microglia in the entorhinal cortex had mostly downregulated pathway clusters in females and upregulated pathway clusters in males (Fig. 5e). Amyloid fibril formation, chaperone-mediated autophagy, protein folding, protein stability regulation, cell junction synapse, neurogenesis structure development, and cell body assembly were among the clusters shared by both sexes but downregulated in females and upregulated in males. Protein homeostasis was altered in disease for females, as shown by downregulation of tau protein kinase activity, tau protein binding, protein folding chaperone, and histone deacetylase binding. Protein degradation and secretion were also downregulated in females with AD compared to controls, as indicated through downregulation of lytic vacuole lysosome and secretory granule vesicle exocytosis respectively. Interestingly, nitric oxide synthase 3 (NOS3), which is involved in a complex cascade of events in oxidative stress that may induce cellular injury and accelerate neurodegenerative changes , and its chaperone, HSP90 , were downregulated in AD females compared to controls. In males, myelination in axon ensheathment, synaptic signaling transmission, and energy-coupled proton transport were upregulated. We also identified downregulation of two microRNA clusters, hsa-miR-190a and hsa-miR-3605, in AD males compared to healthy controls. These are potentially important findings because epigenetic modulation by microRNAs has the capacity to modify microglial behavior in physiological conditions, and dysregulation of microRNAs could mediate microglial hyper-activation and persistent neuroinflammation in neurological diseases . Overall, we observed extensive sex-specific pathway enrichments in microglial populations of AD compared to controls for both brain regions, but especially pronounced in entorhinal cortex.
Furthermore, astrocytes, oligodendrocytes, and OPCs also demonstrated sex-specific pathway perturbations in both prefrontal and entorhinal cortices (Supplementary Figs. 3 and 4). In astrocytes, which normally function to maintain overall brain homeostasis, we observed downregulated plasma and presynaptic membrane components and upregulated postsynaptic asymmetric synapse density in the prefrontal cortex of AD compared to controls in both sexes. In female AD astrocytes, we observed downregulation in pathways related to amino acid transport and vascular transport across the blood–brain barrier. Although the downregulation of these pathways was not observed in males, a related pathway cluster, presynaptic filopodia activities, was downregulated. These observed pathway networks suggest that the same biological process, regulation of synaptic activities, was disrupted in both sexes but via different mechanisms.
In oligodendrocytes, which provide support and insulation to axons in the brain, we observed downregulation in pathways related to regulation of synaptic activity in both female and male AD compared to controls, indicated by the downregulated clusters of cleft regulation, presynaptic assembly, and transmembrane transport channel in females, and neurotransmitter secretion, transmembrane ion transporter, and postsynaptic membrane potential regulation in males. Interestingly, pathways related to cell morphological changes and energy production were upregulated in males and downregulated in females, such as pathway clusters of neuron projection organization, cell migration/locomotion, cellular component organization, ATP coupled electron transport, and mitochondrial NADH dehydrogenase, suggesting oligodendrocyte responses were sex-specific when challenged by disease.
Lastly, we observed upregulation of membrane morphogenesis in female OPCs in the prefrontal cortex, as well as related pathway cluster, TROY-NGR-LINGO1-NGFR complex, which plays essential roles in the inhibition of axonal regeneration . In the entorhinal cortex, a few pathways were downregulated in female and male OPCs, including cell junction synapse assembly, glutamatergic synapse, and plasma membrane intrinsic component. The male OPCs of the entorhinal cortex were overwhelmingly enriched with upregulation in neuronal development, axon ensheathment, neuron myelination, and metabolic protein regulation, as well as ion and vesicle transport, with the exception that synaptic membrane adhesion molecules were downregulated. Although inconclusive due to the unbalanced numbers of significantly enriched pathways obtained in OPCs from both sexes, our observations suggest that AD female OPCs in the prefrontal cortex diverge more from controls compared to male OPCs, whereas in the entorhinal cortex, AD male OPCs were more perturbed by disease status compared to females.