The BIN1 locus contains the second-most significant genetic risk factor for late-onset Alzheimer’s disease. BIN1 undergoes alternate splicing to generate tissue- and cell-type-specific BIN1 isoforms, which regulate membrane dynamics in a range of crucial cellular processes. Whilst the expression of BIN1 in the brain has been characterized in neurons and oligodendrocytes in detail, information regarding microglial BIN1 expression is mainly limited to large-scale transcriptomic and proteomic data. Notably, BIN1 protein expression and its functional roles in microglia, a cell type most relevant to Alzheimer’s disease, have not been examined in depth. Microglial BIN1 expression was analyzed by immunostaining mouse and human brain, as well as by immunoblot and RT-PCR assays of isolated microglia or human iPSC-derived microglial cells. Bin1 expression was ablated by siRNA knockdown in primary microglial cultures in vitro and Cre-lox mediated conditional deletion in adult mouse brain microglia in vivo. Regulation of neuroinflammatory microglial signatures by BIN1 in vitro and in vivo was characterized using NanoString gene panels and flow cytometry methods. The transcriptome data was explored by in silico pathway analysis and validated by complementary molecular approaches. Here, we characterized microglial BIN1 expression in vitro and in vivo and ascertained microglia expressed BIN1 isoforms. By silencing Bin1 expression in primary microglial cultures, we demonstrate that BIN1 regulates the activation of proinflammatory and disease-associated responses in microglia as measured by gene expression and cytokine production. Our transcriptomic profiling revealed key homeostatic and lipopolysaccharide (LPS)-induced inflammatory response pathways, as well as transcription factors PU.1 and IRF1 that are regulated by BIN1. Microglia-specific Bin1 conditional knockout in vivo revealed novel roles of BIN1 in regulating the expression of disease-associated genes while counteracting CX3CR1 signaling. The consensus from in vitro and in vivo findings showed that loss of Bin1 impaired the ability of microglia to mount type 1 interferon responses to proinflammatory challenge, particularly the upregulation of a critical type 1 immune response gene, Ifitm3. Our convergent findings provide novel insights into microglial BIN1 function and demonstrate an essential role of microglial BIN1 in regulating brain inflammatory response and microglial phenotypic changes. Moreover, for the first time, our study shows a regulatory relationship between Bin1 and Ifitm3, two Alzheimer’s disease-related genes in microglia. The requirement for BIN1 to regulate Ifitm3 upregulation during inflammation has important implications for inflammatory responses during the pathogenesis and progression of many neurodegenerative diseases.

中文翻译：

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BIN1 是小胶质细胞促炎和神经变性相关激活的关键调节因子

BIN1 基因座包含晚发性阿尔茨海默病的第二重要遗传风险因素。BIN1 经历交替剪接以产生组织和细胞类型特异性的 BIN1 亚型，其在一系列关键细胞过程中调节膜动力学。虽然 BIN1 在大脑中的表达已在神经元和少突胶质细胞中进行了详细表征，但有关小胶质细胞 BIN1 表达的信息主要限于大规模转录组学和蛋白质组学数据。值得注意的是，尚未深入研究 BIN1 蛋白表达及其在小胶质细胞（一种与阿尔茨海默病最相关的细胞类型）中的功能作用。通过对小鼠和人脑进行免疫染色，以及对分离的小胶质细胞或人 iPSC 衍生的小胶质细胞进行免疫印迹和 RT-PCR 分析，分析了小胶质细胞 BIN1 的表达。Bin1 表达通过体外原代小胶质细胞培养物中的 siRNA 敲低和体内成年小鼠大脑小胶质细胞中 Cre-lox 介导的条件性缺失而消融。使用 NanoString 基因面板和流式细胞术方法表征了 BIN1 在体外和体内对神经炎症小胶质细胞特征的调节。转录组数据通过计算机通路分析进行探索，并通过互补分子方法进行验证。在这里，我们描述了小胶质细胞 BIN1 在体外和体内的表达，并确定了小胶质细胞表达的 BIN1 亚型。通过在原代小胶质细胞培养物中沉默 Bin1 表达，我们证明 BIN1 调节小胶质细胞中促炎反应和疾病相关反应的激活，如通过基因表达和细胞因子产生所测量的。我们的转录组学分析揭示了关键的稳态和脂多糖 (LPS) 诱导的炎症反应途径，以及受 BIN1 调节的转录因子 PU.1 和 IRF1。体内小胶质细胞特异性 Bin1 条件性敲除揭示了 BIN1 在调节疾病相关基因表达同时抵消 CX3CR1 信号传导方面的新作用。体外和体内研究结果的共识表明，Bin1 的缺失损害了小胶质细胞对促炎挑战产生 1 型干扰素反应的能力，特别是关键的 1 型免疫反应基因 Ifitm3 的上调。我们的聚合发现提供了对小胶质细胞 BIN1 功能的新见解，并证明了小胶质细胞 BIN1 在调节脑炎症反应和小胶质细胞表型变化中的重要作用。而且，我们的研究首次显示 Bin1 和 Ifitm3（小胶质细胞中的两个阿尔茨海默病相关基因）之间存在调节关系。BIN1 在炎症过程中调节 Ifitm3 上调的要求对许多神经退行性疾病的发病机制和进展过程中的炎症反应具有重要意义。