Objective: To investigate metabolomic changes in SIM-A9 microglia in vitro following exposure to neuroinflammatory insult using high resolution mass spectrometry.

Background: Microglial cells play an essential role in the CNS, acting as the primary immunological response to stress within the cellular environment. As the macrophages of the CNS, microglial cells are activated during the progression of several neurodegenerative diseases. Microglial are either cytotoxic M1 or the neuroprotective M2 phenotype. In PD, change in polarization to the M1 phenotype is not well understood at the molecular level. We focus on deciphering the metabolic signature of microglial cells after activation by a neurotoxin, lipopolysaccharide (LPS) using LC-HRMS.

Methods: SIM-A9 microglial cells were passaged into T-80 flasks, at 10 million cells per flask in a 5% CO2 incubator at 37 °C for 24 hours. Cells were stimulated with LPS at 2.5 ng/mL and metabolite extraction was performed on cell pellet after an ammonium formate cell pellet wash using 80% ice-cold methanol in water, followed by homogenization on a bead beater. Cells were dried down with liquid nitrogen and reconstituted for subsequent mass spectrometric analysis. Samples were run via reversed-phase LC-HRMS using a Dionex UHPLC interfaced with a Thermo Scientific Q-Exactive mass spectrometer.

Results: Data analysis was performed using MZmine, with accurate mass and retention time matched to an internal laboratory library. After filtering, 955 features in positive ion mode and 698 in negative ion mode were used for analysis. Statistical analysis using MetaboAnalyst revealed noteworthy differences between activated and quiescent microglial cells. One elevated feature in LPS-induced cells was phosphocholine, an intermediate between choline and cytidine-diphosphocholine in the phosphatidylcholine biosynthetic pathway. Cytidine, a precursor for the biosynthesis of cytidine triphosphate (CTP), was also increased in cells induced with LPS. Further studies will focus on identifying other significant features in this dataset and determining the biological role they play during the LPS-induced activation of microglial cells, as well as analyzing the lipidomic profile of LPS activated cells to shed light on changes in lipid biosynthesis.

Conclusions: As the sentinels of the CNS, it is likely that microglia respond to changes in brain metabolism and disease state. To our knowledge, this is the first mass spectrometry based metabolomic profiling of activated microglial cells. This study will allow us to uncover links between bioenergetics factors and activation states of microglia.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/metabolomic-profiling-of-activated-microglial-cells/