Graduate Program of Neuroscience, The University of Western Ontario, London, Canada.
Department of Molecular Medicine, Robarts Research Institute, The University of Western Ontario, London, Canada.
Glia. 2019 Dec;67(12):2294-2311. doi: 10.1002/glia.23685. Epub 2019 Aug 27.
Microglia phagocytosis is critical for central nervous system development, and dysregulation of phagocytosis may contribute to a variety of neurological disorders. During initial stages of phagocytosis, microglia display increased nitric oxide (NO) production via inducible nitric oxide synthase (iNOS) activity and amplified calcium entry through transient receptor potential vanilloid type 2 (TRPV2) channels. The present study investigated the regulatory role of iNOS/NO signaling in microglial phagocytosis and TRPV2 channel activation using phagocytosis assay, calcium imaging, patch clamp electrophysiology, immunocytochemistry, and immunoblot assays. Results showed that primary microglia from iNOS-knockout (iNOS ) mice exhibited substantial deficits in phagocytic capacity and TRPV2 channel activity relative to wild-type (WT) controls. Specifically, iNOS microglia displayed a lower level of TRPV2 protein localized on the plasma membrane (PM) without any significant change in the mRNA levels of Fc-gamma receptors and TRPV2. In addition, iNOS microglia, unlike their WT controls, failed to elicit a calcium influx in response to application of the TRPV2-agonist 2-aminoethoxydiphenyl borate (2APB). Importantly, the phagocytic capacity and the PM expression and activity of TRPV2 in iNOS microglia were largely corrected by pretreatment with NO-donors. Accordingly, the 2APB-evoked calcium influx and the PM expression of TRPV2 in WT microglia were significantly decreased by selective inhibition of iNOS, protein kinase-G (PKG), or phosphoinositide-3-kinase (PI3K), respectively. Together, results from this study indicated that iNOS/NO signaling upregulates microglial phagocytosis and increases TRPV2 trafficking to the PM via PKG/PI3K dependent pathway(s).
小胶质细胞的吞噬作用对于中枢神经系统的发育至关重要，而吞噬作用的失调可能导致多种神经紊乱。在吞噬作用的初始阶段，小胶质细胞通过诱导型一氧化氮合酶（iNOS）活性增加一氧化氮（NO）的产生，并通过瞬时受体电位香草醛亚型 2（TRPV2）通道放大钙内流。本研究使用吞噬作用测定、钙成像、膜片钳电生理学、免疫细胞化学和免疫印迹分析，探讨了 iNOS/NO 信号在小胶质细胞吞噬作用和 TRPV2 通道激活中的调节作用。结果表明，与野生型（WT）对照相比，iNOS 敲除（iNOS）小鼠的原代小胶质细胞在吞噬能力和 TRPV2 通道活性方面表现出明显缺陷。具体而言，iNOS 小胶质细胞显示出较低水平的位于质膜（PM）上的 TRPV2 蛋白，而 Fc-γ 受体和 TRPV2 的 mRNA 水平没有任何显著变化。此外，与 WT 对照不同，iNOS 小胶质细胞未能对 TRPV2 激动剂 2-氨基乙氧基二苯硼酸盐（2APB）的应用引起钙内流。重要的是，NO 供体预处理可显著纠正 iNOS 小胶质细胞的吞噬能力以及 TRPV2 在 PM 上的表达和活性。相应地，选择性抑制 iNOS、蛋白激酶-G（PKG）或磷脂酰肌醇-3-激酶（PI3K）分别显著降低了 WT 小胶质细胞中 2APB 诱导的钙内流和 TRPV2 在 PM 上的表达。总之，本研究结果表明，iNOS/NO 信号通过 PKG/PI3K 依赖的途径上调小胶质细胞的吞噬作用，并增加 TRPV2 向 PM 的转运。
