Glial signaling mechanisms in the progression of neuroinflammatory injury

2018 Summer.Includes bibliographical references.The response of glial cells to foreign and endogenous stress signals is extensive. As a result, release of inflammatory factors as means of cellular communication and innate immune function, or neuroinflammation, can contribute to neurodegeneration and increased activation of surrounding glia, often associated with Parkinson's disease (PD). The identification of glial activation as an early event in the progression of neurodegenerative disease that precedes neuronal cell death presents an opportunity for better diagnostic markers, as well as new pathways that could be targeted therapeutically. The transcription factor, Nuclear Factor-kappa B (NF-κB), regulates the expression of multiple neuroinflammatory cytokines and chemokines in activated glial cells but the signaling factors modulating glial-glial and glial-neuronal signaling during neurotoxic injury are poorly understood. Thus, inhibition of NF-κB signaling in glial cells could be a promising therapeutic strategy for the prevention of neuroinflammatory injury. Recently, it was found that selected orphan nuclear receptors in the NR4A family (nerve growth factor-induced-β/NGFI-β), including NR4A1 (Nur77) and NR4A2 (Nurr1), can inhibit the inflammatory effects of NF-κB but there are no approved drugs that target these receptors. In the current studies, we utilized several experimental approaches to target neuroinflammation in cellular models of PD and manganese neurotoxicity in primary glia and in animal models. One of these studies demonstrated that a novel ligand of NR4A1 and NR4A2, 1,1-bis (3'-indolyl) -1-(p-methoxyphenyl) methane (C-DIM5), suppressed NF-κB-dependent inflammatory gene expression in astrocytes following treatment with 1-methyl-4-phenyl-1, 2, 3,6-tetrahydropyridine (MPTP) and the inflammatory cytokines, IFN-γ and TNF-α. These data were further supported by previous studies from our laboratory, which examined efficacy of multiple C-DIM compounds in PD animal and cellular models, including one (C-DIM12) identified as a modulator of Nurr1 activity that also inhibited NF-kB-dependent gene expression in glial cells. Collectively, these data demonstrate that NR4A1/Nur77 and NR4A2/Nurr1 dynamically regulated inflammatory gene expression in glia by modulating the transcriptional activity of NF-κB. An additional study examined the role of NF-κB in manganese (Mn)-induced neurotoxicity by exposing purified microglia, astrocytes (from both wild-type and an astrocyte- specific NF-kB (IKK2) knock-out (KO) mouse) and mixed glial cultures to varying Mn concentrations and then treated neurons with the conditioned media (GCM) of each cell type. In doing so, we showed that mixed glial cultures exposed to Mn enhanced glial activation and neuronal death compared to microglia, wild type astrocytes or IKK2-knockout astrocytes alone or in mixed cultures suggesting that astrocytes are a critical mediator of Mn neurotoxicity through enhanced expression of inflammatory cytokines and chemokines, including those most associated with reactive phenotype such as C3 and CCL2. Thus, these studies elucidate key mechanisms associated with neuroinflammation and present potential therapeutic targets in glial cells that regulate the progression of neuroinflammatory injury

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

Suppression of 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-Induced Nitric-Oxide Synthase 2 Expression in Astrocytes by a Novel Diindolylmethane Analog Protects Striatal Neurons against ApoptosisS⃞

The progressive debilitation of motor functions in Parkinson's disease (PD) results from degeneration of dopaminergic neurons within the substantia nigra pars compacta of the midbrain. Long-term inflammatory activation of microglia and astrocytes plays a central role in the progression of PD and is characterized by activation of the nuclear factor-κB (NF-κB) signaling cascade and subsequent overproduction of inflammatory cytokines and nitric oxide (NO). Suppression of this neuroinflammatory phenotype has received considerable attention as a potential target for chemotherapy, but there are no currently approved drugs that sufficiently address this problem. The data presented here demonstrate the efficacy of a novel anti-inflammatory diindolylmethane class compound, 1,1-bis(3′-indolyl)-1-(p-t-butylphenyl)methane (DIM-C-pPhtBu), in suppressing NF-κB-dependent expression of inducible nitric-oxide synthase (NOS2) and NO production in astrocytes exposed to the parkinsonian neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) through a mechanism distinct from that described for the thiazolidinedione-class compound, rosiglitazone. Chromatin immunoprecipitations revealed that micromolar concentrations of DIM-C-pPhtBu prevented association of the p65 subunit of NF-κB with enhancer elements in the Nos2 promoter but had little effect on DNA binding of either peroxisome proliferator-activated receptor-γ (PPAR-γ) or the nuclear corepressor NCoR2. Treatment with DIM-C-pPhtBu concomitantly suppressed NO production and protein nitration in MPTP-activated astrocytes and completely protected cocultured primary striatal neurons from astrocyte-dependent apoptosis. These data demonstrate the efficacy of DIM-C-pPhtBu in preventing the activation of NF-κB-dependent inflammatory genes in primary astrocytes and suggest that this class of compounds may be effective neuroprotective anti-inflammatory agents in vivo

Microglia amplify inflammatory activation of astrocytes in manganese neurotoxicity

Abstract Background As the primary immune response cell in the central nervous system, microglia constantly monitor the microenvironment and respond rapidly to stress, infection, and injury, making them important modulators of neuroinflammatory responses. In diseases such as Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, and human immunodeficiency virus-induced dementia, activation of microglia precedes astrogliosis and overt neuronal loss. Although microgliosis is implicated in manganese (Mn) neurotoxicity, the role of microglia and glial crosstalk in Mn-induced neurodegeneration is poorly understood. Methods Experiments utilized immunopurified murine microglia and astrocytes using column-free magnetic separation. The effect of Mn on microglia was investigated using gene expression analysis, Mn uptake measurements, protein production, and changes in morphology. Additionally, gene expression analysis was used to determine the effect Mn-treated microglia had on inflammatory responses in Mn-exposed astrocytes. Results Immunofluorescence and flow cytometric analysis of immunopurified microglia and astrocytes indicated cultures were 97 and 90% pure, respectively. Mn treatment in microglia resulted in a dose-dependent increase in pro-inflammatory gene expression, transition to a mixed M1/M2 phenotype, and a de-ramified morphology. Conditioned media from Mn-exposed microglia (MCM) dramatically enhanced expression of mRNA for Tnf, Il-1β, Il-6, Ccl2, and Ccl5 in astrocytes, as did exposure to Mn in the presence of co-cultured microglia. MCM had increased levels of cytokines and chemokines including IL-6, TNF, CCL2, and CCL5. Pharmacological inhibition of NF-κB in microglia using Bay 11-7082 completely blocked microglial-induced astrocyte activation, whereas siRNA knockdown of Tnf in primary microglia only partially inhibited neuroinflammatory responses in astrocytes. Conclusions These results provide evidence 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