Inhibition of astrocyte signaling leads to sex-specific changes in microglia phenotypes in a diet-based model of small cerebral vessel disease

Hyperhomocysteinemia (HHcy)-inducing diets recapitulate small cerebral vessel disease phenotypes in mice including cerebrovascular pathology/dysfunction, neuroinflammation, synaptic deficits, and cognitive decline. We recently showed that astrocyte signaling through calcineurin(CN)/nuclear factor of activated T cells (NFATs) plays a causative role in these phenotypes. Here, we assessed the impact of astrocytic signaling on microglia, which set inflammatory tone in brain. Seven-to-eight-week-old male and female C57BL/6J mice received intrahippocampal injections of AAV2/5-Gfa2-EGFP (control) or adeno-associated virus (AAV) expressing the NFAT inhibitor VIVIT ( i.e. , AAV2/5-Gfa2-VIVIT-EGFP). Mice were then fed with control chow (CT) or B-vitamin-deficient chow for 12 weeks to induce HHcy. Immunohistochemistry was used to assess the expression of the pan-microglial marker Iba1 and the homeostatic microglial marker P2ry12. Iba1 showed little sensitivity to diet, AAV treatment, or sex. Conversely, P2ry12 expression was reduced with HHcy diet in males, but not females. Treatment of males with AAV-Gfa2-VIVIT prevented the loss of P2ry12. We next conducted single-cell RNA sequencing (scRNAseq) to determine if microglial genes and/or microglial clustering patterns were sensitive to astrocyte signaling in a sex-dependent manner. In males, disease-associated microglial genes and subclusters were overrepresented in HHcy-treated mice, while VIVIT promoted the appearance of homeostatic microglial genes and clusters. In contrast, microglial genes in females were less sensitive to diet and AAV treatments, though disease-like patterns in gene expression were also observed in the HHcy condition. However, very few of the HHcy-sensitive microglial genes in females were affected by VIVIT. The results suggest a sexually dimorphic influence of astrocyte signaling on microglial phenotypes in the context of HHcy and small cerebral vessel disease.