Sigma Ligand-mediated Cytosolic Calcium Modulation in BV2 Microglia Cells

Abstract

While initially protective, prolonged neuroinflammation can lead to a disrupted brain microenvironment and neurodegenerative diseases. Microglia cells are the primary regulators of neuroinflammation, with anti- or pro-inflammatory phenotypes. Just as microglia are crucial in neuroinflammation, calcium is a crucial regulator in the activation of the microglia. Intracellular calcium dynamics can be modulated by small, membrane bound chaperone proteins called sigma receptors. Both sigma receptors 1 and 2, which are involved in a wide variety of cellular functions, have been implicated in neurodegenerative diseases, psychiatry, and cancer. We examined the use of sigma-receptor ligands to modulate cytosolic calcium levels in BV2 cells, an immortalized mouse microglia cell line. Immunofluorescence staining detected both sigma 1 and 2 receptors in perinuclear regions and the cell cytoplasm. Our selection of compounds was a mixture of commercially available sigma receptor ligands and compounds synthesized at Southern Illinois University Edwardsville. We used the Fluo-8-AM calcium probe to measure cytosolic calcium concentrations using flow cytometry after 15-, 25- and 35-minute ligand exposure. With 1 {micro}M ligand concentration, we found significantly increased cytosolic calcium levels in BV2 cells after 15-minute exposure. A moderate correlation was determined between sigma receptor 2 selectivity and calcium activity, suggesting a sigma-mediated calcium effect. In addition, we aimed to determine the location of calcium flux by pretreating cells with thapsigargin and EGTA to inhibit both the endoplasmic reticulum and extracellular space of suspected calcium entry. We found all four compounds tested, siramesine, PB-28, cis-8-OMe BBZI, and BN-IX-111-F1 promote calcium entry from the extracellular space, but only siramesine promotes calcium entry from both locations. We tested for a common drug-induced effect called phospholipidosis using HCS LipidTOX phospholipid detection reagent to determine a correlation between calcium activity and a membrane-mediated effect. Only two out of the five compounds tested, PB-28 and amiodarone, resulted in significant phospholipid accumulation at 10 {micro}M treatment, suggesting little to no correlation between calcium levels and phospholipidosis. Further investigations are required to target the exact mechanism of calcium flux and characterize the connection between phospholipidosis and sigma receptors after sigma ligand exposure. Nevertheless, our work emphasizes the importance of sigma ligand-modulated intracellular calcium dynamics as a potential route of therapy, specifically for modulation of neuroinflammation.