Gollihue Jenna L, Aung Khine Zin, Rogers Colin B, Sompol Pradoldej, Katsumata Yuriko, Weekman Erica M, Wilcock Donna M, Morganti Josh M, Norris Christopher M
University of Kentucky.
Indidana University School of Medicine.
Res Sq. 2025 Mar 17:rs.3.rs-6198453. doi: 10.21203/rs.3.rs-6198453/v1.
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 ( 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.
高同型半胱氨酸血症（HHcy）诱导饮食可在小鼠中重现小脑血管疾病表型，包括脑血管病理/功能障碍、神经炎症、突触缺陷和认知能力下降。我们最近发现，星形胶质细胞通过钙调神经磷酸酶（CN）/活化T细胞核因子（NFATs）发出的信号在这些表型中起因果作用。在此，我们评估了星形胶质细胞信号对小胶质细胞的影响，小胶质细胞可设定大脑中的炎症基调。7至8周龄的雄性和雌性C57BL/6J小鼠接受海马内注射AAV2/5-Gfa2-EGFP（对照）或表达NFAT抑制剂VIVIT的腺相关病毒（AAV）（AAV2/5-Gfa2-VIVIT-EGFP）。然后给小鼠喂食对照饲料（CT）或缺乏B族维生素的饲料12周以诱导HHcy。免疫组织化学用于评估全小胶质细胞标志物Iba1和稳态小胶质细胞标志物P2ry12的表达。Iba1对饮食、AAV处理或性别几乎没有敏感性。相反，雄性小鼠中P2ry12的表达在HHcy饮食时降低，而雌性小鼠中则没有。用AAV-Gfa2-VIVIT处理雄性小鼠可防止P2ry12的丢失。接下来，我们进行了单细胞RNA测序（scRNAseq），以确定小胶质细胞基因和/或小胶质细胞聚类模式是否以性别依赖的方式对星形胶质细胞信号敏感。在雄性小鼠中，疾病相关的小胶质细胞基因和亚群在HHcy处理的小鼠中过度表达，而VIVIT促进了稳态小胶质细胞基因和聚类的出现。相比之下，雌性小鼠中的小胶质细胞基因对饮食和AAV处理不太敏感，尽管在HHcy条件下也观察到了类似疾病的基因表达模式。然而，雌性小鼠中对HHcy敏感的小胶质细胞基因很少受到VIVIT的影响。结果表明，在HHcy和小脑血管疾病背景下，星形胶质细胞信号对小胶质细胞表型具有性别差异影响。
