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Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Neuroscience

Supervisor

Lu, Wei-Yang

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.

Summary for Lay Audience

Microglia are the only immune cells residing in the brain. Their main roles are to ensure that neurons develop properly and remain healthy throughout life. When microglia fail to perform these roles, degenerative disorders such as Alzheimer’s disease or Parkinson’s disease may develop. Microglia carry out two specialized tasks that include removing cellular waste and/or pathogens through a process called phagocytosis and multiplying to increase their cellular numbers through a process called proliferation. In order to perform these specialized tasks, microglia take cues from changing calcium concentrations in their environments. However, the surrounding calcium can only enter microglia through specific channels on their cell surface that are formed from transient receptor potential (TRP) proteins. There are a variety of different TRP proteins, however they all act as a path for calcium to enter and activate microglia. When microglia become active, they produce a gas called nitric oxide. From previous studies, we know that nitric oxide influences microglia to carry out their specialized tasks such as phagocytosis and proliferation. However, it remains unknown whether nitric oxide is produced in microglia to directly regulate TRP channels and calcium entry, influencing their ability to perform these specialized tasks. In the following experiments, we demonstrated that nitric oxide does in fact regulate specific TRP channels on microglia to influence their ability to perform tasks. Specifically, in the presence of nitric oxide, the TRP vanilloid type 2 (TRPV2) channel, normally present within microglia, is transferred to the surface of microglia to cause calcium entry into microglia. We also demonstrated that nitric oxide restricted calcium influx through TRP canonical type 1 and 3 (TRPC1/3) channels on the surface of microglia. Importantly, we further demonstrated that the calcium influx from TRPV2 channels caused microglia to engulf more objects through phagocytosis, while simultaneously restricting their ability to multiply and proliferate. In many neurodegenerative disorders such as Alzheimer’s and Parkinson’s disease, microglia present with abnormal phagocytosis and proliferation. Therefore, studies such as the ones presented in this dissertation may be the first step to uncovering therapeutic targets for the neurodegenerative diseases that arise due to abnormal microglial activities.

Creative Commons License

Creative Commons Attribution-Noncommercial 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 4.0 License.

Available for download on Monday, August 01, 2022

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