Glial-neuronal signaling mechanisms underlying the neuroinflammatory effects of manganese

Abstract

Background: Exposure to increased manganese (Mn) causes inflammation and neuronal injury in the cortex and basal ganglia, resulting in neurological symptoms resembling Parkinson's disease. The mechanisms underlying neuronal death from exposure to Mn are not well understood but involve inflammatory activation of microglia and astrocytes. Expression of neurotoxic inflammatory genes in glia is highly regulated through the NF-κB pathway, but factors modulating neurotoxic glial-glial and glial-neuronal signaling by Mn are not well understood.

Methods: We examined the role of NF-κB in Mn-induced neurotoxicity by exposing purified microglia, astrocytes (from wild-type and astrocyte-specific IKK knockout mice), and mixed glial cultures to varying Mn concentrations and then treating neurons with the conditioned media (GCM) of each cell type. We hypothesized that mixed glial cultures exposed to Mn (0-100 μM) would enhance glial activation and neuronal death compared to microglia, wild-type astrocytes, or IKK-knockout astrocytes alone or in mixed cultures.

Results: Mixed glial cultures treated with 0-100 μM Mn for 24 h showed the most pronounced effect of increased expression of inflammatory genes including inducible nitric oxide synthase (Nos2), Tnf, Ccl5, Il6, Ccr2, Il1b, and the astrocyte-specific genes, C3 and Ccl2. Gene deletion of IKK2 in astrocytes dramatically reduced cytokine release in Mn-treated mixed glial cultures. Measurement of neuronal viability and apoptosis following exposure to Mn-GCM demonstrated that mixed glial cultures induced greater neuronal death than either cell type alone. Loss of IKK in astrocytes also decreased neuronal death compared to microglia alone, wild-type astrocytes, or mixed glia.

Conclusions: This suggests that astrocytes are a critical mediator of Mn neurotoxicity through enhanced expression of inflammatory cytokines and chemokines, including those most associated with a reactive phenotype such as CCL2 but not C3.

Keywords: Astrocyte; CCL2; Glial-glial communication; Glial-neuronal communication; Manganism; NF-κB; Neuroinflammation.

Fig. 1

Manganese-induced expression of neuroinflammatory genes in mixed glial cultures and in purified cultures of astrocytes and microglia. Inflammatory gene expression exhibited an overall dose-dependent increase in a mixed glia and b pure astrocytes and both dose-dependent increases in some genes and decreases in astrocyte-specific C3, Ccl2, and Ccr2 in c pure microglia. One-way ANOVA analyses performed. Data depicted as ± S.E.M. *P< 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (n ≥ 4 per treatment group; across ≥ 3 independent experiments)

Fig. 2

Conditioned media from Mn-treated glial cultures induces cell death in N2A neuronal cells. a N2A cells exposed to Mn-exposed GCM for 24 or 48 h showed a dose-dependent decrease in viability at 100 μM MnCl₂, more so at 48 h. b 48-h Mn-treated GCM caused a greater decrease in neuronal viability compared to either ACM or MCM. c, d (top) Flow cytometry analysis demonstrated that 48-h exposure to 100 μM MnCl₂-GCM increased apoptosis in N2A cells to a greater extent than ACM (c, d middle) or (c MCM, d bottom), based on the number of Annexin V+ and Propidium iodide+ cells. One-way ANOVA analyses performed for experiments comparing three or more treatment groups and t test in those comparing two treatment groups. Data depicted as ± S.E.M. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (n ≥ 4 per treatment group; across ≥ 3 independent experiments)

Fig. 3

Pharmacologic inhibition of NF-kB decreases inflammatory gene expression upon exposure to 100 μM MnCl₂. a 1 h pretreatment with the NF-κB inhibitor, Bay 11-7082 (Bay-11) [(E)- 3-(4-methylphenyl) sulfonylprop-2-enenitrile], suppressed expression of inflammatory genes in mixed glia following exposure to 100 μM MnCl₂. b MnCl₂ exposure in pure astrocytes was less than in mixed glia, as were the inhibitory effects of Bay-11, while the DMSO-vehicle control did not suppress inflammatory gene expression in either cell type. One-way ANOVA analyses performed. Data depicted as ± S.E.M. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (n ≥ 4 per treatment group; across ≥ 3 independent experiments)

Fig. 4

Pharmacologic inhibition of NF-κB in glia protects N2A neuronal cells from apoptosis induced by Mn-exposed glial conditioned media. Pharmacologic inhibition of NF-κB with Bay-11 in mixed glial cultures preserves N2A viability following treatment with 100 μM MnCl₂ GCM (a) and ACM (b). Direct treatment of N2A cells with Mn causes a dose-dependent decrease in viability (c). Flow cytometry analysis of apoptotic N2A cells indicated that direct treatment with 100 μM MnCl₂ (d, e) results in fewer Annexin V+ and PI+ cells compared to GCM (f, h) and ACM (g, i) and that pretreatment of glial cultures with Bay-11 prior to Mn exposure decreases the number of Annexin V+ and PI+ cells in both GCM- and ACM-treated N2A cells. One-way ANOVA analyses performed for experiments comparing three or more treatment groups and T test in those comparing two treatment groups. Data depicted as ± S.E.M. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (n ≥ 4 per treatment group; across ≥ 3 independent experiments)

Fig. 5

Gene deletion of IKK2 in astrocytes decreases inflammatory gene expression following exposure to 100 μM MnCl₂ in mixed glial cultures. Astrocyte-specific IKK2 knockout decreases inflammatory mRNA (a) and protein (b) expression of inflammatory genes in mixed glial cultures compared to corresponding WT-treated groups. Two-way ANOVA analyses performed. Data depicted as ± S.E.M. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (n ≥ 4 per treatment group; across ≥ 3 independent experiments)

Fig. 6

Loss of IKK2 expression in astrocytes reduces apoptosis in N2A cells following treatment with conditioned medium from mixed glial cultures. a GCM from mixed glial cultures containing WT astrocytes (black bars) treated with 100 μM MnCl₂ decreases N2A viability compared to control (~ 25%), whereas neuronal viability is largely preserved (indicated by lack of statistically significant loss) in N2A cells treated with GCM from mixed glial cultures containing IKK2 KO astrocytes (white bars) compared to control. b, c Mn-GCM (WT, black bars) causes an increase in Annexin V+ and PI+ N2A cells, which is reduced in N2A cells treated with GCM from mixed glial cultures containing IKK2 KO astrocytes (white bars). Two-way ANOVA analyses performed. Data depicted as ± S.E.M. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (n ≥ 4 per treatment group; across ≥ 3 independent experiments)

Fig. 7

Mixed glial cultures containing IKK2 knockout astrocytes fail to induce apoptosis in primary cortical neurons following exposure to MnCl₂. The number of apoptotic neurons is statistically decreased in response to Mn-treated IKK2 KO GCM compared to WT GCM for Annexin V+ cells (a, b) and caspase 3/7+ cells (c, d). Necrotic neurons (PI+; e, f) are similarly, statistically decreased in neurons treated with IKK2 KO GCM compared to WT GCM. Two-way ANOVA analyses performed. Data depicted as ± S.E.M. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (100–200 cells per group from four biological replicates across ≥ 3 independent experiments)

Fig. 8

Selective knockdown of the astrocyte-specific chemokine, Ccl2, with siRNA in mixed glia does not inhibit Mn-induced inflammatory gene expression but prevents neuronal cell death. a Ccl2 KD does not inhibit mRNA expression of inflammatory genes in Mn-exposed mixed glia compared to mixed glia treated with Mn in the presence of control siRNA. b CCL2 protein levels in GCM are reduced in Ccl2 KD mixed glial cultures. c Viability of N2A cells is preserved following treatment with Mn-GCM from Ccl2 KD mixed glia compared to those treated with control siRNA Mn-GCM. d (left two panels) CCL2 is released into medium in both mixed glia (GCM) and pure astrocytes (ACM) following treatment with MnCl₂. d (right two panels) Pharmacologic inhibition of NF-κB with Bay-11 in both mixed glia and pure astrocyte cultures inhibits release of CCL2, whereas CCL2 levels are statistically increased compared to control in both Mn-GCM and Mn-ACM treated with vehicle control (DMSO). e C3 KD does not inhibit mRNA expression of inflammatory genes in Mn-exposed mixed glia compared to mixed glia treated with Mn in the presence of control siRNA. f N2A viability is decreased in cells exposed to C3 KD Mn-GCM compared to those treated with siRNA control Mn-GCM. One-way ANOVA analyses performed for experiments comparing three or more treatment groups and t test in those comparing two treatment groups. Data depicted as ± S.E.M. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 (≥ 4 per treatment group; across ≥ 3 independent experiments)