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

Abstract

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.

Keywords: Astrocyte reactivity; calcium; microglia; neuroinflammation; vascular.

Figure 1. HHcy diet and VIVIT treatment alter Iba1 and P2ry12 immunolabeling in sex dependent manner.

A, time-line of diet and AAV treatments. Male and female Mice received bilateral hippocampal injections of AAV vectors at approximately two months of age. One month following AAV injection, mice were started on a control (CT) diet or a HHcy inducing diet. At 12–15 weeks of diet treatment, mice were harvested and brains were sections for immunolabeling or scRNA seq analyses. B-C, low magnification photomicrographs of Iba1 immunolabeling in hippocampus across the four diet/AAV conditions in female (B) and male (C) mice. calibration bar is 1 mm. D, Iba1 immunolabeling photo micrograph at higher magnification. calibration bar is 100 μm. E-G, % area occupied by lba1 immunolabel across all mice (E), only females (F), or only males (G). H-I, Iow magnification photomicrographs of P2ry12 immunolabeling in hippocampus across the four diet/AAV conditions in female (H) and male (I) mice. calibration bar is 1 mm. J, P2ry12 immunolabeling photo micrograph at higher magnification. calibration bar is 100 μm. K-M, % area occupied by P2ry12 immunolabel across all mice (K), only females (L), or only males (M). HHcy had opposing effects on P2ry12 labeling in males and females (determined by three way ANOVA and Dunnett’s Multiple Comparison’s test. Each plot symbol represents an individual brain section.

Figure 2. Cell clusters in males and females based on canonical gene expression markers.

UMAPs show 15 unique cell clusters in female (A) and male (C) mice. The corresponding proportions of each unique cluster is shown in panel B (for females) and panel D for males.

Figure 3. Differentially expressed microglial genes and KEGG pathway mapping in females.

A, Volcano plot shows microglial DEGs that are upregulated (HHcy Up, red) and downregulated (HHcy Down, blue) in the HHcy-EGFP vs CT EGFP treatment conditions. B, KEGG pathways enriched for HHcy-sensitive (upregulated) DEGs. C, Volcano plot shows microglial DEGs that are upregulated (VIVIT Up, red) and downregulated (VIVIT Down, blue) by VIVIT in the CT-EGFP vs CT VIVIT treatment conditions. D, KEGG pathways enriched for VIVIT-sensitive (downregulated) DEGs in the CT EGFP vs CT VIVIT conditions. E, Volcano plot shows microglial DEGs that are upregulated (VIVIT Up, red) and downregulated (VIVIT Down, blue) by VIVIT in the HHcy-EGFP vs HHcy VIVIT treatment conditions. F, KEGG pathways enriched for VIVIT-sensitive (downregulated) DEGs in the HHcy EGFP vs HHcy VIVIT conditions. G, Venn diagrams showing DEGs that are sensitive to both HHcy (533 genes upregulated in HHcy EGFP vs CT EGFP comparison) and VIVIT (540 genes downregulated in HHcy VIVIT vs HHcy EGFP). Very little overlap (51 genes) was observed for HHcy and VIVIT sensitive genes.

Figure 4. Differentially expressed microglial genes and KEGG pathway mapping in males.

A, Volcano plot shows microglial DEGs that are upregulated (HHcy Up, red) and downregulated (HHcy Down, blue) in the HHcy-EGFP vs CT EGFP treatment conditions. B, KEGG pathways enriched for HHcy-sensitive (upregulated) DEGs. C, Volcano plot shows microglial DEGs that are upregulated (VIVIT Up, red) and downregulated (VIVIT Down, blue) by VIVIT in the CT-EGFP vs CT VIVIT treatment conditions. D, KEGG pathways enriched for VIVIT-sensitive (downregulated) DEGs in the CT EGFP vs CT VIVIT conditions. E, Volcano plot shows microglial DEGs that are upregulated (VIVIT Up, red) and downregulated (VIVIT Down, blue) by VIVIT in the HHcy-EGFP vs HHcy VIVIT treatment conditions. F, KEGG pathways enriched for VIVIT-sensitive (downregulated) DEGs in the HHcy EGFP vs HHcy VIVIT conditions. G, Venn diagrams showing DEGs that are sensitive to both HHcy (926 genes upregulated in HHcy EGFP vs CT EGFP comparison) and VIVIT (2402 genes downregulated in HHcy VIVIT vs HHcy EGFP). More than 90% of the HHcy sensitive genes (832 genes) were also sensitive to VIVIT treatment.

Figure 5. Microglial subclusters in female mice as a consequence of diet and AAV treatment.

A-B, UMAPs showing six distinct microglia clusters across diet and AAV conditions for females. C-D, Expression of common HSMG (Gpr34, P2ry12, Fcrls, and Tmem119) and DAM markers (Apoe, H2-D1, H2-K1, and B2m) across the entire microglial cluster. E, Clusters were collapsed into two categories (HSMG and DAM) based on the distribution of cellular markers in Cand D. F, Proportions of HSMG and DAM clusters (% total microglial (MG) population) across the diet and AAV treatment conditions. The DAM cluster was overrepresented in the HHcy conditions (especially HHcy VIVIT).

Figure 6. Microglial subclusters in male mice as a consequence of diet and AAV treatment.

A-B, UMAPs showing five distinct microglia clusters across diet and AAV conditions for males. C-D, Expression of common HSMG (Gpr34, P2ry12, Fcrls, and Tmem119) and DAM markers (Apoe, H2-D1, H2-K1, and B2m) across the entire microglial cluster. E, Clusters were collapsed into two categories (HSMG and DAM) based on the distribution of cellular markers in C and D. F, Proportions of HSMG and DAM clusters (% total microglial (MG) population) across the diet and AAV treatment conditions. HHcy and VIVIT treatment had opposing effects on the distribution of DAM and HSMG clusters. DAM clusters were overrepresented, while HSMG clusters were underrepresented in the HHcy EGFP condition. In contrast, HSMG clusters were overrepresented and DAM clusters underrepresented in the CT VIVIT condition. There was a roughly equal distribution of DAM and HSMG clusters in the CT EGFP and HHcy VIVIT conditions.