Sexual diversity of the choroid plexus: from single-cell transcriptional landscape to interplay between circadian clock and energy metabolism pathways

The choroid plexus (ChP) is a specialized epithelial tissue that serves as a critical interface between the peripheral circulation and the central nervous system. The ChP also harbors one of the most robust intrinsic circadian oscillators in the brain. The influence of biological sex and the estrous cycle on its cellular organization and circadian and metabolic regulation has not been studied. To address this knowledge gap, we combined single-cell RNA sequencing (scRNA-seq) of male and female mouse fourth ventricle ChP (4VChP) with ex vivo bioluminescence recordings of organotypic ChP explants and in vivo RT-qPCR profiling of circadian clock gene expression across the estrous cycle. scRNA-seq revealed sex-specific transcriptional programs in ChP epithelial cells, including differential enrichment of pathways associated with circadian clock regulation, endoplasmic reticulum protein processing, and carbohydrate metabolism. Expression of genes related to metabolic and biosynthetic activity was higher in female than in male 4VChP, whereas cell-to-cell communication between epithelial and stromal compartments was lower in females. Sex and estrous cycle affected sensitivity of the 4VChP clock to estrogen and glucose, as well as downstream NAMPT-SIRT1 signaling, indicating that the female 4VChP clock is more tightly coupled to metabolic and NAD+ signaling and may be more vulnerable to metabolic perturbations due to low energy supply. Collectively, these findings suggest that biological sex exerts a significant influence on the transcriptional, circadian, and metabolic architecture of the ChP, establishing this tissue as a sexually dimorphic and hormonally and metabolically dynamic regulator of brain homeostasis.