The Effect of Nitric Oxide on Microglia Function and Activity: Implications on Transient Receptor Potential Channels
Abstract
Microglia proliferation and phagocytosis is critical for proper development and maintenance of the central nervous system (CNS), whereas dysregulated proliferation and phagocytosis contributes to various CNS pathologies. Specifically, in response to pathology or tissue injury, microglia transform to an activated state characterized by elevated inducible nitric oxide synthase (iNOS) expression, increased nitric oxide (NO) production, active phagocytosis, and decreased proliferation. Importantly, microglial phagocytosis and proliferation are tightly regulated by intracellular calcium levels. As non-excitable cells, microglia rely on calcium entry through transient receptor potential (TRP) channels to carry out phagocytosis and proliferation. However, the role of iNOS/NO signaling in regulating TRP channel mediated calcium dynamics in microglia is not entirely understood. We hypothesized that NO signaling regulates the activity of TRP channels in microglia to influence phagocytosis and proliferation. Using pharmacology, immuno-blots, cellular patch-clamp electrophysiology, calcium-imaging, polymerase chain reaction, and assays for phagocytosis and proliferation, this work determined that NO critically regulates microglial calcium homeostasis. We demonstrated that NO regulates the calcium dynamics of TRP vanilloid type 2 (TRPV2) and TRP canonical 1 and 3 (TRPC1/3) channels to enhance microglial phagocytosis and inhibit proliferation. Specifically, iNOS/NO signaling in microglia restricted calcium influx through TRPC1/3 channels independent of protein kinase G (PKG) signaling, while a simultaneous enhancement in the plasma membrane expression and calcium influx through TRPV2 channels occurred in a PKG-dependent mechanism. The plasma membrane expression of TRPV2 was negatively associated with cell-cycle progression in microglia cultures. Specifically, increased production of NO, plasma membrane expression of TRPV2, and calcium influx was observed in non-dividing microglia. The calcium influx through TRPV2 channels caused nuclear translocation of the transcription factor nuclear factor of activating T-cells cytoplasmic 2 and increased mRNA expression of the cyclin dependent kinase inhibitor p21 in murine microglia. The work from this thesis identified NO as a crucial regulator of TRP channel activity and calcium homeostasis within microglia. Future studies can apply these findings to better understand CNS pathologies that display altered microglial proliferation or phagocytosis such as Alzheimer’s disease, Parkinson’s disease, stroke recovery, or cancer progression.