Abstract
BackgroundExposure 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.MethodsWe 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.ResultsMixed 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.ConclusionsThis 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.
Highlights
Exposure to increased manganese (Mn) causes inflammation and neuronal injury in the cortex and basal ganglia, resulting in neurological symptoms resembling Parkinson’s disease
Nitric oxide synthase 2 (NOS2) and many other pro-inflammatory factors are highly regulated in glial cells by Nuclear factor kappa B (NF-κB), consistent with data reporting that the NF-κB-mediated pro-inflammatory cytokines C-C motif chemokine ligand 2 (CCL2), C-C motif chemokine ligand 5 (CCL5), and Tumor necrosis factor alpha (TNF) released by astrocytes are associated with Mn neurotoxicity in in vitro studies of murine glia [5, 15, 16]
The present data build on recently reported studies from our laboratory indicating that microglia amplify astrocyte activation during Mn exposure [6] and demonstrate that NF-κB in astrocytes stimulates the production of inflammatory cytokines and chemokines that cause neuronal cell death
Summary
Exposure to increased manganese (Mn) causes inflammation and neuronal injury in the cortex and basal ganglia, resulting in neurological symptoms resembling Parkinson’s disease. Data from our laboratory recently demonstrated that NF-κB signaling in microglia plays an essential role in inflammatory responses in Mn toxicity by regulating cytokines and chemokines that amplify the activation of astrocytes [6]. This demonstrates that nuclear factor kappa B (NF-κB) signaling in microglia is essential to inflammatory activation of astrocytes, subsequent effects of astrocytes on microglia and on neuronal cell death are less well understood. Previous studies from our laboratory and others demonstrated that expression of inducible nitric oxide synthase (iNOS/NOS2) and NO production in astrocytes causes injury to surrounding neurons in Mn-exposed mice [11,12,13,14]. NOS2 and many other pro-inflammatory factors are highly regulated in glial cells by NF-κB, consistent with data reporting that the NF-κB-mediated pro-inflammatory cytokines CCL2, CCL5, and TNF released by astrocytes are associated with Mn neurotoxicity in in vitro studies of murine glia [5, 15, 16]
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