Sigma receptors suppress multiple aspects of microglial activation

Abstract

During brain injury, microglia become activated and migrate to areas of degenerating neurons. These microglia release proinflammatory cytokines and reactive oxygen species causing additional neuronal death. Microglia express high levels of sigma receptors, however, the function of these receptors in microglia and how they may affect the activation of these cells remain poorly understood. Using primary rat microglial cultures, it was found that sigma receptor activation suppresses the ability of microglia to rearrange their actin cytoskeleton, migrate, and release cytokines in response to the activators adenosine triphosphate (ATP), monocyte chemoattractant protein 1 (MCP-1), and lipopolysaccharide (LPS). Next, the role of sigma receptors in the regulation of calcium signaling during microglial activation was explored. Calcium fluorometry experiments in vitro show that stimulation of sigma receptors suppressed both transient and sustained intracellular calcium elevations associated with the microglial response to these activators. Further experiments showed that sigma receptors suppress microglial activation by interfering with increases in intracellular calcium. In addition, sigma receptor activation also prevented membrane ruffling in a calcium-independent manner, indicating that sigma receptors regulate the function of microglia via multiple mechanisms.

Figure 1. Sigma receptor activation suppresses changes in microglia morphology in response to chemoattractant stimulation

Photomicrographs of primary rat microglia cultures exposed to vehicle (0.1% DMSO in DMEM) (A, D, G), 100 μM ATP for 5 min (B, H) and 10 min (E), or 100 μM ATP for 5 min (C, I) and 10 min (F) in the presence of 100 μM DTG. Microglia in A–F were treated in normal DMEM, whereas microglia in G–I were treated in Ca²⁺-free DMEM. All insets represent magnified image (2.7x). Scale bar = 2 μm.

Figure 2. Sigma receptor activation significantly decreases membrane ruffling in microglia stimulated with ATP

Degree of membrane ruffling assessed as described in the methods section. Microglia were preincubated with vehicle, or DTG (100 μM DTG) for 5 minutes then stimulated with DMEM or ATP (100 μM). Bars represent means ± SEM. Asterisks indicate significant differences between DMEM and ATP within Control (no DTG, p < 0.001) and DTG groups (p < 0.001). Pound symbol indicates significant difference between Control and DTG within the ATP group (p < 0.001). Significance was determined via two-way ANOVA with post-hoc Tukey Test.

Figure 3. The microglial migratory response to chemoattractant application is suppressed by sigma receptor activation

Microglial migration was assayed using a Boyden chamber fitted with a polycarbonate membrane containing 8μm pores. Microglia (500,000 cells) were placed in the upper chamber and control media, 100 μM ATP (A) or 10 nM MCP-1 (B) were added in the absence and presence of 300 μM DTG to the bottom chamber. Microglia were allowed to migrate for 4 hrs at 37 °C, then stained with DAPI and counted. Asterisks indicate a significant difference between ATP (A; p < 0.001, n = 6) and MCP-1 (B; p < 0.01, n = 4) from DMEM within the Control group (no DTG). Pound symbols indicate significant difference between Control and DTG within the ATP (A; p < 0.001) and MCP-1 (B; p < 0. 01) groups, respectively. In both cases significance was determined by two-way ANOVA followed by post-hoc Bonferroni tests.

Figure 4. The microglial inflammatory response is suppressed by sigma receptor activation

Primary microglial cultures were incubated with LPS (1 μg/ml) for 24 hrs and TNFα, IL10, and NO levels in the supernatant were measured by ELISA and the Greiss reaction, respectively. Data points represent mean ± SEM for TNFα, IL10, and NO levels as a function of DTG concentration (n=4 for all groups). Lines indicate best fits to the data using a Langmuir-Hill equation and yielded IC₅₀ values of 338.9, 109.6, and 166.0μM with Hill coefficients of 1.90, 1.94, and 1.4 for TNFα, IL10, and NO release, respectively.

Figure 5. Transient intracellular calcium signaling evoked by ATP was suppressed by sigma receptor activation

Representative traces of [Ca²⁺]_i as a function of time recorded during ATP (300 μM) stimulation in the absence (Control) and presence of DTG (100 μM), without (A) or with (B) Metaphit (50μM, 1 hr, room temperature). C, Mean change in peak [Ca²⁺]_i (± SEM) measured in the above treatment groups. Asterisk denotes significant difference (p < 0.05, n = 25), as measured by one-way ANOVA with post-hoc Dunn’s test using ATP as control.

Figure 6. Basal intracellular calcium increases are suppressed by sigma receptor activation

Microglia, plated on poly-D-Lysine coverslips, were stimulated for 24 hours with LPS (1μg/ml) in the absence (Vehicle) and presence of 300 μM DTG (DTG). Mean basal calcium levels (± SEM) measured using fura-2 calcium imaging. Values represent measurements from three pooled experiments each having a minimum of 30 cells per group. Asterisk denotes statistical difference (p < 0.05, n = 31) between Media and LPS treatment groups within the Vehicle group, and pound symbol denotes significant difference between Vehicle and DTG within the LPS treatment group (p < 0.05, n=31). Statistical difference was determined using a two-way ANOVA with post-hoc Bonferroni test.

Figure 7. Increases in intracellular calcium induce membrane ruffling in microglia

Micrographs of phalloidin stained microglia treated with vehicle (DMSO, A) or 1 μM ionomycin for 5 minutes (B). C, Bar graph of degree of membrane ruffling determined for the indicated conditions. Bars represent means ± SEM. Asterisks indicate a significant difference between DMEM and Ionomycin treatment groups (p < 0.001). Significance was determined via two-way ANOVA with post-hoc Tukey Test. All insets represent magnified image (2.7x). Scale bar = 2 μm.

Figure 8. Microglial migration is independent of calcium release from internal stores but requires La³⁺-sensitive calcium influx

A, Microglial chemotaxis in response to ATP (100 μM) in the absence and presence of DTG (300μM) and DTG with ionomycin (1 μM). Data were analyzed via two-way ANOVA with post-hoc Tukey test. Asterisk indicates a statistical difference from respective controls (p <0.001 for all), pound symbol indicates a statistical difference from ATP alone (p <0.001 for all), and dagger indicates statistical significance from ATP+DTG (p <0.05 for all). B, Microglia migration induced by 300 μM ATP without and with pretreatment of the cells with 10 μM thapsigargin for 30 min (37 °C) (Thapsigargin). Asterisk indicates a statistical difference from control (p <0.05). Bar represent means ± SEM, n = 3. C, Microglia migration evoked by 300 μM ATP in the absence (Control) and presence of 50 μM La³⁺. Asterisk indicates a statistical difference from Control (p < 0.001), pound symbol indicates a statistical difference from ATP alone (p < 0.01). Bar represent means ± SEM, n = 3. Statistical significance was determined by two-way ANOVA followed by post-hoc Bonferroni multiple comparison tests. D, Relative ATP induced migration in the absence (Control) and presence of 50 μM La³⁺. Asterisk denotes statistical difference (p < 0.05 by student’s T test).

Figure 9. Calcium influx restores nitric oxide production following sigma receptor activation in LPS stimulated microglia

Relative levels of NO produced by microglia under control condition (DMEM), in the presence of 300 μM DTG (DTG), and following application of 1 μM ionomycin alone (Iono) and with 300 μM DTG (Iono + DTG). Microglia were also treated for 24 hrs with LPS (1 μg/ml) alone (LPS), LPS and 300 μM DTG (LPS+DTG), LPS and 1 μM ionomycin, and with a combination of the three compounds (LPS+DTG+IONO). Bars represent mean fluorescent intensities from DAF-FM loaded cells. Asterisk indicates statistical difference from DMEM treated cells, pound symbol denotes statistical significance from LPS treated cells, and dagger indicates significance from LPS+DTG treated cells. In all cases p < 0.001, as determined by three-way ANOVA with post-hoc Tukey test (n ≥ 400 cells for all samples).