The Role of Glial Cells in Neurobiology and Prion Neuropathology.

Prion diseases are rare and neurodegenerative diseases that are characterized by the misfolding and infectious spread of the prion protein in the brain, causing progressive and irreversible neuronal loss and associated clinical and behavioral manifestations in humans and animals, ultimately leading to death. The brain has a complex network of neurons and glial cells whose crosstalk is critical for function and homeostasis. Although it is established that prion infection of neurons is necessary for clinical disease to occur, debate remains in the field as to the role played by glial cells, namely astrocytes and microglia, and whether these cells are beneficial to the host or further accelerate disease. Here, we review the current literature assessing the complex morphologies of astrocytes and microglia, and the crosstalk between these two cell types, in the prion-infected brain.

Adipose-Derived Mesenchymal Stromal Cells Co-Cultured with Primary Mixed Glia to Reduce Prion-Induced Inflammation.

Mesenchymal stromal cells (MSCs) are potent regulators of inflammation through the production of anti-inflammatory cytokines, chemokines, and growth factors. These cells show an ability to regulate neuroinflammation in the context of neurodegenerative diseases such as prion disease and other protein misfolding disorders. Prion diseases can be sporadic, acquired, or genetic; they can result from the misfolding and aggregation of the prion protein in the brain. These diseases are invariably fatal, with no available treatments. One of the earliest signs of disease is the activation of astrocytes and microglia and associated inflammation, which occurs prior to detectable prion aggregation and neuronal loss; thus, the anti-inflammatory and regulatory properties of MSCs can be harvested to treat astrogliosis in prion disease. Recently, we showed that adipose-derived MSCs (AdMSCs) co-cultured with BV2 cells or primary mixed glia reduce prion-induced inflammation through paracrine signaling. This paper describes a reliable treatment using stimulated AdMSCs to decrease prion-induced inflammation. A heterozygous population of AdMSCs can easily be isolated from murine adipose tissue and expanded in culture. Stimulating these cells with inflammatory cytokines enhances their ability to both migrate toward prion-infected brain homogenate and produce anti-inflammatory modulators in response. Together, these techniques can be used to investigate the therapeutic potential of MSCs on prion infection and can be adapted for other protein misfolding and neuroinflammatory diseases.

Adipose-derived mesenchymal stromal cells decrease prion-induced glial inflammation in vitro.

Prion diseases are characterized by the cellular prion protein, PrPC, misfolding and aggregating into the infectious prion protein, PrPSc, which leads to neurodegeneration and death. An early sign of disease is inflammation in the brain and the shift of resting glial cells to reactive astrocytes and activated microglia. Few therapeutics target this stage of disease. Mesenchymal stromal cells produce anti-inflammatory molecules when exposed to inflammatory signals and damaged tissue. Here, we show that adipose-derived mesenchymal stromal cells (AdMSCs) migrate toward prion-infected brain homogenate and produce the anti-inflammatory molecules transforming growth factor ß (TGFß) and tumor necrosis factor-stimulated gene 6 (TSG-6). In an in vitro model of prion exposure of both primary mixed glia and BV2 microglial cell line, co-culturing with AdMSCs led to a significant decrease in inflammatory cytokine mRNA and markers of reactive astrocytes and activated microglia. This protection against in vitro prion-associated inflammatory responses is independent of PrPSc replication. These data support a role for AdMSCs as a beneficial therapeutic for decreasing the early onset of glial inflammation and reprogramming glial cells to a protective phenotype.

A Novel Glucocorticoid and Androgen Receptor Modulator Reduces Viral Entry and Innate Immune Inflammatory Responses in the Syrian Hamster Model of SARS-CoV-2 Infection.

Despite significant research efforts, treatment options for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain limited. This is due in part to a lack of therapeutics that increase host defense to the virus. Replication of SARS-CoV-2 in lung tissue is associated with marked infiltration of macrophages and activation of innate immune inflammatory responses that amplify tissue injury. Antagonists of the androgen (AR) and glucocorticoid (GR) receptors have shown efficacy in models of COVID-19 and in clinical studies because the cell surface proteins required for viral entry, angiotensin converting enzyme 2 (ACE2) and the transmembrane protease, serine 2 (TMPRSS2), are transcriptionally regulated by these receptors. We postulated that the GR and AR modulator, PT150, would reduce infectivity of SARS-CoV-2 and prevent inflammatory lung injury in the Syrian golden hamster model of COVID-19 by down-regulating expression of critical genes regulated through these receptors. Animals were infected intranasally with 2.5 × 104 TCID50/ml equivalents of SARS-CoV-2 (strain 2019-nCoV/USA-WA1/2020) and PT150 was administered by oral gavage at 30 and 100 mg/Kg/day for a total of 7 days. Animals were examined at 3, 5 and 7 days post-infection (DPI) for lung histopathology, viral load and production of proteins regulating the progression of SARS-CoV-2 infection. Results indicated that oral administration of PT150 caused a dose-dependent decrease in replication of SARS-CoV-2 in lung, as well as in expression of ACE2 and TMPRSS2. Lung hypercellularity and infiltration of macrophages and CD4+ T-cells were dramatically decreased in PT150-treated animals, as was tissue damage and expression of IL-6. Molecular docking studies suggest that PT150 binds to the co-activator interface of the ligand-binding domain of both AR and GR, thereby acting as an allosteric modulator and transcriptional repressor of these receptors. Phylogenetic analysis of AR and GR revealed a high degree of sequence identity maintained across multiple species, including humans, suggesting that the mechanism of action and therapeutic efficacy observed in Syrian hamsters would likely be predictive of positive outcomes in patients. PT150 is therefore a strong candidate for further clinical development for the treatment of COVID-19 across variants of SARS-CoV-2.

NF-κB Signaling in Astrocytes Modulates Brain Inflammation and Neuronal Injury Following Sequential Exposure to Manganese and MPTP During Development and Aging.

Chronic exposure to manganese (Mn) is associated with neuroinflammation and extrapyramidal motor deficits resembling features of Parkinson's disease. Activation of astrocytes and microglia is implicated in neuronal injury from Mn but it is not known whether early life exposure to Mn may predispose glia to more severe inflammatory responses during aging. We therefore examined astrocyte nuclear factor kappa B (NF-κB) signaling in mediating innate immune inflammatory responses during multiple neurotoxic exposures spanning juvenile development into adulthood. MnCl2 was given in drinking water for 30-day postweaning to both wildtype mice and astrocyte-specific knockout (KO) mice lacking I kappa B kinase 2, the central upstream activator of NF-κB. Following juvenile exposure to Mn, mice were subsequently administered 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at 4 months of age. Animals were evaluated for behavioral alterations and brain tissue was analyzed for catecholamine neurotransmitters. Stereological analysis of neuronal and glial cell counts from multiple brain regions indicated that juvenile exposure to Mn amplified glial activation and neuronal loss from MPTP exposure in the caudate-putamen and globus pallidus, as well as increased the severity of neurobehavioral deficits in open field activity assays. These alterations were prevented in astrocyte-specific I kappa B kinase 2 KO mice. Juvenile exposure to Mn increased the number of neurotoxic A1 astrocytes expressing C3 as well as the number of activated microglia in adult mice following MPTP challenge, both of which were inhibited in KO mice. These results demonstrate that exposure to Mn during juvenile development heightens the innate immune inflammatory response in glia during a subsequent neurotoxic challenge through NF-κB signaling in astrocytes.

Genetic suppression of IKK2/NF-κB in astrocytes inhibits neuroinflammation and reduces neuronal loss in the MPTP-Probenecid model of Parkinson's disease.

Neuroinflammatory activation of glia is considered a pathological hallmark of Parkinson's disease (PD) and is seen in both human PD patients and in animal models of PD; however, the relative contributions of these cell types, especially astrocytes, to the progression of disease is not fully understood. The transcription factor, nuclear factor kappa B (NFκB), is an important regulator of inflammatory gene expression in glia and is activated by multiple cellular stress signals through the kinase complex, IKK2. We sought to determine the role of NFκB in modulating inflammatory activation of astrocytes in a model of PD by generating a conditional knockout mouse (hGfapcre/Ikbk2F/F) in which IKK2 is specifically deleted in astrocytes. Measurements of IKK2 revealed a 70% deletion rate of IKK2 within astrocytes, as compared to littermate controls (Ikbk2F/F). Use of this mouse in a subacute, progressive model of PD through exposure to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTPp) revealed significant protection in exposed mice to direct and progressive loss of dopaminergic neurons in the substantia nigra (SN). hGfapcre/Ikbk2F/F mice were also protected against MPTPp-induced loss in motor activity, loss of striatal proteins, and genomic alterations in nigral NFκB gene expression, but were not protected from loss of striatal catecholamines. Neuroprotection in hGfapcre/Ikbk2F/F mice was associated with inhibition of MPTPp-induced astrocytic expression of inflammatory genes and protection against nitrosative stress and apoptosis in neurons. These data indicate that deletion of IKK2 within astrocytes is neuroprotective in the MPTPp model of PD and suggests that reactive astrocytes directly contribute the potentiation of dopaminergic pathology.

Glial-neuronal signaling mechanisms underlying the neuroinflammatory effects of manganese.

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.

Compensatory Expression of Nur77 and Nurr1 Regulates NF-κB-Dependent Inflammatory Signaling in Astrocytes.

Inflammatory activation of glial cells promotes loss of dopaminergic neurons in Parkinson disease. The transcription factor nuclear factor κB (NF-κB) regulates the expression of multiple neuroinflammatory cytokines and chemokines in activated glial cells that are damaging to neurons. Thus, inhibition of NF-κB signaling in glial cells could be a promising therapeutic strategy for the prevention of neuroinflammatory injury. Nuclear orphan receptors in the NR4A family, including NR4A1 (Nur77) and NR4A2 (Nurr1), can inhibit the inflammatory effects of NF-κB, but no approved drugs target these receptors. Therefore, we postulated that a recently developed NR4A receptor ligand, 1,1bis (3'indolyl) 1(pmethoxyphenyl) methane (C-DIM5), would suppress NF-κB-dependent inflammatory gene expression in astrocytes after treatment with 1-methyl-4-phenyl 1, 2, 3, 6-tetrahydropyridine (MPTP) and the inflammatory cytokines interferon γ and tumor necrosis factor α C-DIM5 increased expression of Nur77 mRNA and suppressed expression of multiple neuroinflammatory genes. C-DIM5 also inhibited the expression of NFκB-regulated inflammatory and apoptotic genes in quantitative polymerase chain reaction array studies and effected p65 binding to unique genes in chromatin immunoprecipitation next-generation sequencing experiments but did not prevent p65 translocation to the nucleus, suggesting a nuclear-specific mechanism. C-DIM5 prevented nuclear export of Nur77 in astrocytes induced by MPTP treatment and simultaneously recruited Nurr1 to the nucleus, consistent with known transrepressive properties of this receptor. Combined RNAi knockdown of Nur77 and Nurr1 inhibited the anti-inflammatory activity of C-DIM5, demonstrating that C-DIM5 requires these receptors to inhibit NF-κB. 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.

A novel diindolylmethane analog, 1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane, inhibits the tumor necrosis factor-induced inflammatory response in primary murine synovial fibroblasts through a Nurr1-dependent mechanism.

The progression of rheumatoid arthritis involves the thickening of the synovial lining due to the proliferation of fibroblast-like synoviocytes (FLS) and infiltration by inflammatory cells. Tumor necrosis factor alpha (TNFα) is a pro-inflammatory cytokine involved in progression of the disease. Under rheumatoid conditions, FLS express the tumor necrosis factor (TNF)-recognition complex (TNFR1, TNFR2, VCAM-1 and ICAM-1), which induces local macrophage activation and leads to downstream nuclear factor κB (NF-κB) signaling. The NF-κB-regulated inflammatory gene, cyclooxygenase (COX), increases synthesis of prostaglandins that contribute to the propagation of inflammatory damage within the joint. Because the nuclear orphan receptor, NR4A2 (Nurr1), can negatively regulate NF-κB-dependent inflammatory gene expression in macrophages, we postulated that activation of this receptor by the Nurr1 ligand 1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane (C-DIM12) would modulate inflammatory gene expression in synovial fibroblasts by inhibiting NF-κB. Treatment with C-DIM12 suppressed TNFα-induced expression of adhesion molecules and NF-κB regulated genes in primary synovial fibroblasts including vascular adhesion molecule 1 (VCAM-1), PGE2 and COX-2. Immunofluorescence studies indicated that C-DIM12 did not prevent translocation of p65 and stabilized nuclear localization of Nurr1 in synovial fibroblasts. Knockdown of Nurr1 expression by RNA interference prevented the inhibitory effects of C-DIM12 on inflammatory gene expression, indicating that the anti-inflammatory effects of this compound are Nurr1-dependent. Collectively, these data suggest that this receptor may be a viable therapeutic target in RA.

The Nurr1 Ligand,1,1-bis(3'-Indolyl)-1-(p-Chlorophenyl)Methane, Modulates Glial Reactivity and Is Neuroprotective in MPTP-Induced Parkinsonism.

The orphan nuclear receptor Nurr1 (also called nuclear receptor-4A2) regulates inflammatory gene expression in glial cells, as well as genes associated with homeostatic and trophic function in dopaminergic neurons. Despite these known functions of Nurr1, an endogenous ligand has not been discovered. We postulated that the activation of Nurr1 would suppress the activation of glia and thereby protect against loss of dopamine (DA) neurons after subacute lesioning with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Our previous studies have shown that a synthetic Nurr1 ligand, 1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane (C-DIM12), suppresses inflammatory gene expression in primary astrocytes and induces a dopaminergic phenotype in neurons. Pharmacokinetic analysis of C-DIM12 in mice by liquid chromatography-mass spectrometry demonstrated that approximately three times more compound concentrated in the brain than in plasma. Mice treated with four doses of MPTP + probenecid over 14 days were monitored for neurobehavioral function, loss of dopaminergic neurons, and glial activation. C-DIM12 protected against the loss of DA neurons in the substantia nigra pars compacta and DA terminals in the striatum, maintained a ramified phenotype in microglia, and suppressed activation of astrocytes. In vitro reporter assays demonstrated that C-DIM12 was an effective activator of Nurr1 transcription in neuronal cell lines. Computational modeling of C-DIM12 binding to the three-dimensional structure of human Nurr1 identified a high-affinity binding interaction with Nurr1 at the coactivator domain. Taken together, these data suggest that C-DIM12 is an activator of Nurr1 that suppresses glial activation and neuronal loss in vivo after treatment with MPTP, and that this receptor could be an efficacious target for disease modification in individuals with Parkinson's disease and related disorders.

Inflammatory Activation of Microglia and Astrocytes in Manganese Neurotoxicity.

Neurotoxicity due to excessive exposure to manganese (Mn) has been described as early as 1837 (Couper, Br Ann Med Pharm Vital Stat Gen Sci 1:41-42, 1837). Extensive research over the past two decades has revealed that Mn-induced neurological injury involves complex pathophysiological signaling mechanisms between neurons and glial cells. Glial cells are an important target of Mn in the brain, both for sequestration of the metal, as well as for activating inflammatory signaling pathways that damage neurons through overproduction of numerous reactive oxygen and nitrogen species and inflammatory cytokines. Understanding how these pathways are regulated in glial cells during Mn exposure is critical to determining the mechanisms underlying permanent neurological dysfunction stemming from excess exposure. The subject of this review will be to delineate mechanisms by which Mn interacts with glial cells to perturb neuronal function, with a particular emphasis on neuroinflammation and neuroinflammatory signaling between distinct populations of glial cells.

Microglia amplify inflammatory activation of astrocytes in manganese neurotoxicity.

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-1ß, 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.

The Nurr1 Activator 1,1-Bis(3'-Indolyl)-1-(p-Chlorophenyl)Methane Blocks Inflammatory Gene Expression in BV-2 Microglial Cells by Inhibiting Nuclear Factor κB.

NR4A family orphan nuclear receptors are an important class of transcription factors for development and homeostasis of dopaminergic neurons that also inhibit expression of inflammatory genes in glial cells. The identification of NR4A2 (Nurr1) as a suppressor of nuclear factor κB (NF-κB)-related neuroinflammatory genes in microglia and astrocytes suggests that this receptor could be a target for pharmacologic intervention in neurologic disease, but compounds that promote this activity are lacking. Selected diindolylmethane compounds (C-DIMs) have been shown to activate or inactivate nuclear receptors, including Nurr1, in cancer cells and also suppress astrocyte inflammatory signaling in vitro. Based upon these data, we postulated that C-DIM12 [1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane] would suppress inflammatory signaling in microglia by a Nurr1-dependent mechanism. C-DIM12 inhibited lipopolysaccharide (LPS)-induced expression of NF-κB-regulated genes in BV-2 microglia including nitric oxide synthase (NOS2), interleukin-6 (IL-6), and chemokine (C-C motif) ligand 2 (CCL2), and the effects were attenuated by Nurr1-RNA interference. Additionally, C-DIM12 decreased NF-κB activation in NF-κB-GFP (green fluorescent protein) reporter cells and enhanced nuclear translocation of Nurr1 primary microglia. Chromatin immunoprecipitation assays indicated that C-DIM12 decreased lipopolysaccharide-induced p65 binding to the NOS2 promoter and concurrently enhanced binding of Nurr1 to the p65-binding site. Consistent with these findings, C-DIM12 also stabilized binding of the Corepressor for Repressor Element 1 Silencing Transcription Factor (CoREST) and the Nuclear Receptor Corepressor 2 (NCOR2). Collectively, these data identify C-DIM12 as a modulator of Nurr1 activity that results in inhibition of NF-κB-dependent gene expression in glial cells by stabilizing nuclear corepressor proteins, which reduces binding of p65 to inflammatory gene promoters.

Novel para-phenyl substituted diindolylmethanes protect against MPTP neurotoxicity and suppress glial activation in a mouse model of Parkinson's disease.

The orphan nuclear receptor NR4A2 (Nurr1) constitutively regulates inflammatory gene expression in glial cells by suppressing DNA binding activity of NF-κB. We recently reported that novel 1,1-bis(3'-indolyl)-1-(p-substitutedphenyl)methane (C-DIM) compounds that activate NR4A family nuclear receptors in cancer lines also suppress inflammatory gene expression in primary astrocytes and prevent loss of dopaminergic neurons in mice exposed to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid (MPTPp). In this study, we postulated that the basis for this neuroprotection involves blockade of glial activation and subsequent expression of NF-κB-regulated inflammatory genes. To examine this mechanism, we treated transgenic NF-κB/EGFP reporter mice with MPTPp for 7 days (MPTPp7d) followed by daily oral gavage with either vehicle (corn oil; MPTPp14d) or C-DIMs containing p-methoxyphenyl (C-DIM5), p-hydroxyphenyl (C-DIM8), or p-chlorophenyl (C-DIM12) groups. Each compound conferred significant protection against progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc), even when given after 7 days of dosing with MPTPp. C-DIM12 had the greatest neuroprotective activity in MPTPp-treated mice, and was also the most potent compound in suppressing activation of microglia and astrocytes, expression of cytokines and chemokines in quantitative polymerase chain reaction (qPCR) array studies, and in reducing expression of NF-κB/EGFP in the SN. C-DIM12 prevented nuclear export of Nurr1 in dopaminergic neurons and enhanced expression of the Nurr1-regulated proteins tyrosine hydroxylase and the dopamine transporter. These data indicate that NR4A-active C-DIM compounds protect against loss of dopamine neurons in the MPTPp model of PD by preventing glial-mediated neuronal injury and by supporting a dopaminergic phenotype in TH-positive neurons in the SNpc.

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 apoptosis.

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-kappaB (NF-kappaB) 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-kappaB-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-kappaB with enhancer elements in the Nos2 promoter but had little effect on DNA binding of either peroxisome proliferator-activated receptor-gamma (PPAR-gamma) 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-kappaB-dependent inflammatory genes in primary astrocytes and suggest that this class of compounds may be effective neuroprotective anti-inflammatory agents in vivo.