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IFN-γ-induced neuronal cell death in rat cortical neuron–glia cultures
Life Sciences 80 (2007) 1706 – 1712 www.elsevier.com/locate/lifescie
Protective effect of radicicol against LPS/IFN-γ-induced neuronal cell death in rat cortical neuron–glia cultures Mi-Jin Sohn, Hyun-Jeong Noh, Ick-Dong Yoo, Won-Gon Kim ⁎ Functional Metabolomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305–806, Republic of Korea Received 11 September 2006; accepted 31 January 2007
Abstract We investigated the protective activity of radicicol, an antifungal antibiotic, against inflammation-induced neurotoxicity in neuron–glia cultures. Radicicol potently prevented the loss of neuronal cell bodies and neurites from LPS/IFN-γ-induced neurotoxicity in rat cortical neuron– glia cultures with an EC50 value of 0.09 μM. Radicicol inhibited the LPS/IFN-γ-induced expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) in microglia. Additionally, radicicol decreased the LPS/IFN-γ-induced release of tumor necrosis factor-α (TNF-α) in the cultures. The inhibitory potency of radicicol against the production of NO and TNF-α was well correlated with the protection of neurons. These results suggest that the protective effect of radicicol against LPS/IFN-γ-induced neuronal cell death in neuron–glia cultures is mediated via the inhibition of TNF-α release, as well as the suppression of iNOS expression in microglia. © 2007 Elsevier Inc. All rights reserved. Keywords: Radicicol; Glia-mediated neurotoxicity; Inducible nitric oxide synthase; Microglia; Tumor necrosis factor-α
Introduction Inflammation in the central nervous system (CNS) has been associated with the pathogenesis of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, AIDS dementia complex, and amyotrophic lateral sclerosis, as well as post-ischemic brain injuries (Mcgeer et al., 1998; Raine 1994; Combs et al., 2000; Craft et al., 2006). The activation of microglia, the resident immune cells of the brain, is a histopathological hallmark of theses diseases. Activated microglia produce a variety of proinflammatory factors, including NO, TNF-α, interleukin-1β (IL-1β), and reactive oxygen species, which are believed to contribute to the neurodegenerative processes (Chao et al., 1992; Lee et al., 1993; Streit et al., 2005; Culbert et al., 2006). Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, contributes to bacterial meningitis and septic shock. The LPS has also been used as an immunostimulator of macrophages and microglia. In neuron– ⁎ Corresponding author. Tel.: +82 42 860 4298; fax: +82 42 860 4595. E-mail address: [email protected] (W.-G. Kim). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.01.054
glia cultures, treatment with LPS or a combination of LPS and IFN-γ activates microglia and produces NO and proinflammatory cytokines such as TNF-α and IL-1β. These cytotoxic factors ultimately cause neurotoxicity (Chao et al., 1992; Dawson et al., 1994; Jeohn et al., 2000; Block and Hong 2005). Radicicol, an antifungal antibiotic, has been reported to be a potent tranquilizer with low toxicity (McCapra et al., 1964). Radicicol induces the reversal of the tumor phenotype of srctransformed cells and inhibits in vivo angiogenesis (Kwon et al., 1992, 1995). The antitumor action of radicicol has been shown to result from inhibition of folding and activation of the protein tyrosine kinases through the disruption of the ATPase activity of the Hsp90 molecular chaperone (Sharma et al., 1998; Roe et al., 1999). Radicicol has also been reported to inhibit LPS-induced expression of iNOS by blocking the p38 kinase and NF-κB in macrophages (Jeon et al., 2000). Radicicol has been reported to rescue neurons from anti-cancer-induced neurotoxicity in cultured dorsal root ganglion (DRG) neurons and ischemia-induced hippocampal neurotoxicity in transient forebrain, although the mechanism of action was not clear (Sano 2001; Ohtsuki et al., 1996). The protective effect of radicicol against inflammationmediated neurotoxicity has not been reported previously.
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In the course of our screening for neuroprotective agents against immune-mediated neurotoxicity, we found that radicicol potently inhibits LPS/IFN-γ-induced neuronal cell death. In this study, we investigated the neuroprotective effect of radicicol on LPS/IFN-γinduced neuronal damage in rat cortical neuron–glia cultures in terms of the number and morphology of neuronal cells. We also examined its effect on LPS/IFN-γ-induced production of NO, iNOS expression, and release of TNF-α in neuron–glia cultures. Materials and methods Materials Radicicol and poly-D-lysine were obtained from Sigma (St. Louis, MO). Radicicol was dissolved and diluted with culture medium prior to use. LPS (E. coli 011:B4) was purchased from Calbiochem (La Jolla, CA). Minimal essential medium (MEM), penicillin, streptomycin, glutamine, sodium pyruvate, nonessential amino acid, and horse serum were obtained from Life Technologies (Gaithersburg, MD). IFN-γ was purchased from Biosource International (Camarillo, CA), while biotinylated secondary antibodies, ABC kits, and 3,3′-diaminobenzidine were from Vector Laboratories (Burlingame, CA). Cell culture Primary mixed neuron–glia cultures were prepared from the brains of embryonic day 16–17 rats (SD) as described previously (Kim et al., 2000). The whole brain was removed aseptically, and the blood vessels and meninges were discarded. The cortex regions were dissected, pooled, and mechanically dissociated by mild trituration in ice-cold calcium- and magnesium-free W3 buffer (15 mM HEPES, pH 7.4, 145 mM NaCl, 5.4 mM KCl, 1 mM NaH2PO4, and 11 mM glucose). 5 × 105 cells in 0.5 ml of culture medium were seeded to each poly-D-lysine-coated well of the 24-well culture plates. The cultures were maintained at 37 °C in an atmosphere of 5% CO2 and 95% air. The culture medium consisted of MEM supplemented with 10% heat-inactivated horse serum, 2 mM glutamine, 1 mM sodium pyruvate, 100 μM nonessential amino acid, 15 mM KCl, 50 U/ml penicillin, and 50 μg/ml streptomycin. The cultures were replenished with fresh media at 1 and 4 days after plating. At 8 days after plating, the cultures were treated with vehicle or LPS/IFN-γ. When antibodies against microtubule-associated protein-2 (MAP-2) or CR3 complement receptor, a marker for rat microglia, were used for immunostaining of neuron–glia cultures, the following composition was determined: 60% neurons, 3% microglia. The remaining cells were presumed to be astroglial cells.
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monoclonal antibody against the enzyme (Transduction Laboratories, Lexington, KY). In brief, the cultures were washed three times with phosphate-buffered saline (PBS), and were then treated for 20 min with blocking solution (1% skim milk, 0.4% Triton- X-100, and 4% normal serum in PBS). The cultures were incubated overnight at 4 °C with primary antibody diluted blocking solution (anti-MAP-2, 2.5 μg/ml; OX-42, 5 μg/ ml; anti-iNOS 0.25 μg/ml). The cells were rinsed three times for 10 min each in PBS, treated with 1% H2O2, and washed three times with PBS. The bound primary antibody was visualized using the avidin–biotin–peroxidase complex (ABC) method, followed by color development with 3,3′-diaminobenzidine. The number of MAP-2-positive neuronal cell bodies was determined by visually counting five representative fields from each well under the microscope. Nitrite and TNF-α assay The production of NO was assessed as the accumulation of nitrite in the culture supernatants; this was determined using a colorimetric reaction with Griess reagent (Green et al., 1982). The amount of TNF-α in the medium was measured with a rat TNF-α enzyme-linked immunosorbent assay kit (Biosource International, Camarillo, CA). Statistical analysis The data are expressed as mean ± SD. The treatment group was compared with the vehicle control with a Student two-tailed t-test. Results Radicicol protects neurons from LPS/IFN-γ-induced neurotoxicity in neuron–glia cultures Cortical neuron–glia cultures were treated for 48 h with the vehicle alone, LPS alone, or a combination of LPS and IFN-γ.
Immunocytochemistry Cell cultures were fixed with 3.7% formaldehyde and immunostained as described previously (Kim et al., 2000). Neurons were identified with a monoclonal antibody to MAP-2 (Boehringer Mannheim, Mannheim, Germany). Microglia were visualized by staining for the CR3 complement receptor using the monoclonal antibody, OX-42 (PharMingen, San Diego, CA). The cellular expression of iNOS was visualized with a
Fig. 1. Dependency on IFN-γ in LPS/IFN-γ-induced loss of MAP-2-positive neurons in neuron–glia cultures. Cultures were treated for 48 h with vehicle alone, 10 ng/ml LPS, or a combination of 10 ng/ml LPS and various concentrations of IFN-γ; cultures were then immunostained with an antibody to the neuronal cytoskeletal protein, MAP-2. The data are expressed as percentages of the total number (236 ± 14 cells per mm2) of MAP-2-IR cell bodies present in the vehicle-treated control cultures. The data represent the mean ± SD of at least three wells taken from two independent experiments. ⁎p b 0.01 and ⁎⁎ p b 0.005 compared with the group treated with only LPS.
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Fig. 2. Protective effect of radicicol in LPS/IFN-γ-induced loss of MAP-2-positive neurons in neuron–glia cultures. Cultures were treated for 48 h with a combination of 10 ng/ml LPS and 50 U/ml IFN-γ in the presence of various concentrations of radicicol and then immunostained with the MAP-2 antibody. A) Immunocytochemical analysis of MAP-2-positive neurons treated with vehicle alone, LPS/IFN-γ, LPS/IFN-γ + 1 μM radicicol, or 1 μM radicicol alone. Scale bar, 100 μm. B) Dosedependent protective effect of radicicol in the LPS/IFN-γ-induced loss of MAP-2-positive neurons in neuron–glia cultures. The data are expressed as percentages of the total number (267 ± 19 cells per mm2) of MAP-2-IR cell bodies present in the vehicle-treated control cultures. The data represent the mean ± SD of at least three wells taken from three independent experiments. ⁎p b 0.05, ⁎⁎p b 0.005, ⁎⁎⁎ p b 0.0001 compared with the group treated with LPS/IFN-γ alone.
Neurotoxicity was assessed by morphological analysis and counting of the number of neuronal cell bodies after immunostaining for MAP-2. Exposure of cortical neuron–glia cultures to LPS alone at concentrations up to 1 μg/ml did not alter the number of MAP-2-positive cells or the morphologies of these cells compared with vehicle-treated control cultures. In contrast, treatment of the cultures with a combination of LPS and IFN-γ markedly increased NO production and caused dramatic degeneration of MAP-2-IR cells within 48 h (Fig. 1). The loss of MAP-2-IR cells was dependent on the concentration of IFN-γ used. There was an 88.2% decrease in the number of MAP-2-positive neurons upon combined treatment with 10 ng/ ml LPS and 50 U/ml IFN-γ. The protective effect of radicicol against neuronal cell death induced by treatment with 10 ng/ml LPS and 50 U/ml IFN-γ in neuron–glia cultures was investigated. Radicicol strongly prevented the loss of neuronal cell bodies and neurites in a dosedependent manner, with an EC50 value of 0.09 μM (Fig. 2A and B). Radicicol at a concentration of 0.3 μM rescued over 90% of the
neurons. Radicicol itself, however, showed no toxic effect on neuronal cell bodies or neurites at 1 μM. Radicicol decreases LPS/IFN-γ-induced production of NO and expression of iNOS in neuron–glia cultures In agreement with previous studies which indicated that LPS-induced neurotoxicity was, at least in part, mediated via NO (Chao et al., 1992; Bronstein et al., 1995), the addition of the NO synthase inhibitor, NG-nitro-L-arginine-methyl ester (1 mM), to the cultures inhibited the accumulation of nitrite produced after treatment for 48 h with 10 ng/ml LPS and 50 U/ ml IFN-γ by 85.5%, and reduced the loss of MAP-2-IR cells by 88.1% (data not shown). Correlation between the protective effect of radicicol and the LPS/IFN-γ-induced production of NO was examined. Treatment of the cultures with 10 ng/ml LPS and 50 U/ml IFN-γ in neuron–glia cultures increased the accumulation of nitrite up to 27.7 μM within 48 h (Fig. 3). The addition of radicicol inhibited
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Fig. 3. Inhibitory effect of radicicol in LPS/IFN-γ-induced production of NO in neuron–glia cultures. Cultures were treated with a combination of 10 ng/ml LPS and 50 U/ml IFN-γ in the presence of various concentrations of radicicol, and the levels of NO production, assessed as the accumulation of nitrite, were quantified 24 or 48 h later. The data represent the mean ± SD of at least three wells taken from three independent experiments. ⁎p b 0.005 and ⁎⁎p b 0.0001 compared with the group treated with LPS/IFN-γ alone.
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the LPS/IFN-γ-induced accumulation of nitrite in a concentration-dependent manner, with an EC50 value of 0.06 μM. At a concentration of 0.3 μM, radicicol inhibited the accumulation of nitrite by 82.6%. Importantly, the inhibitory potency of nitrite accumulation by radicicol occurred in parallel with the protection of neurons against LPS/IFN-γ-induced neurotoxicity. The inhibition of LPS/IFN-γ-induced iNOS expression by radicicol was also examined by immunostaining for iNOS. Upon treatment for 24 h with 10 ng/ml LPS and 50 U/ml IFN-γ, a number of iNOS-IR cells in neuron–glia cultures showed marked changes. The addition of radicicol (0.3 μM) to the cultures after treatment for 24 h with 10 ng/ml LPS and 50 U/ml IFN-γ decreased the number of iNOS-IR cells produced by 79.7%, which was well correlated with the proportion of MAP-2-IR cells lost, 93.0% (Fig. 4A). To determine whether the iNOS-IR cells were microglia, we then performed immunostaining with OX-42.
Fig. 4. Inhibitory effect of radicicol in LPS/IFN-γ-induced expression of iNOS in neuron–glia cultures. Cultures were treated for 24 h with a combination of 10 ng/ml LPS and 50 U/ml IFN-γ in the presence of 0.3 μM radicicol, and were then immunostained with antibody to iNOS and OX-42 for staining iNOS-positive cells and microglia, respectively. A) Immunocytochemical analysis of iNOS- or OX-42-positive cells treated with vehicle alone or LPS/IFN-γ. Scale bar, 100 μm. B) Inhibitory effect of radicicol on the number of iNOS-IR cells induced by LPS/IFN-γ. The data are expressed as the number of iNOS-IR cells per square millimeter and represent the mean ± SD of at least three wells. ⁎p b 0.0001 compared with the group treated with LPS/IFN-γ alone.
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Fig. 5. Inhibitory effect of radicicol in LPS/IFN-γ-induced release of TNF-α in neuron–glia cultures. Cultures were treated for 6 h with a combination of 10 ng/ ml LPS and 50 U/ml IFN-γ in the presence of various concentrations of radicicol, and the levels of TNF-α release were measured. The data represent the mean ± SD of at least three wells. ⁎p b 0.0001 compared with the group treated with LPS/ IFN-γ alone.
The iNOS-IR cells had a round or ramified morphology, which were the same as OX-42-IR cells (Fig. 4B). This result indicate that iNOS is expressed only in microglia, not in astrocytes, as reported in mice cortical neuron–glia cultures and in rat primary mixed astrocytes and microglial cell cultures (Kong et al., 1996; Jeohn et al., 2000; Possel et al., 2000). In addition, radicicol didn't show a significant cytotoxicity on basal and activated microglia. This result suggests that the protective activity of radicicol against LPS/IFN-γ-induced neuronal cell death is, at least in part, mediated by the inhibition of NO production, which results from the suppression of iNOS expression in microglia. Radicicol decreases LPS/IFN-γ-induced production of TNF-α in neuron–glia cultures To determine whether radicicol inhibits the production of the proinflammatory cytokine, TNF-α, from LPS/IFN-γ-stimulated neuron–glia cultures, levels of TNF-α in the culture medium were examined. Levels of TNF-α in the culture medium were measured at 6 h after LPS/IFN-γ treatment, a time at which the release of the cytokine is known to be near its maximal levels (Liu et al., 2000). Radicicol inhibited the LPS/IFN-γ-induced release of TNF-α in a concentration-dependent manner, with an EC50 value of 0.1 μM (Fig. 5). Upon treatment with radicicol at a concentration of 0.3 μM, TNF-α was reduced to its basal level. The dose-dependent decrease in the release of TNF-α was well correlated with the loss of MAP-2-IR cells. These results suggest that the protective activity of radicicol against LPS/IFNγ-induced neuronal cell death is mediated via the inhibition of TNF-α release, as well as the suppression of iNOS expression. Discussion Radicicol, originally isolated as an antifungal antibiotic, is well known to exhibit antitumor activity through its binding to the N-terminal ATP/ADP-binding domains of HSP90, which are essential for its function. Its oxime derivative has in vivo antitumor activity; geldanamycin, which has the same mechanism, is currently being used in clinical trials (Soga et al., 1999; Maloney and Workman 2002). Radicicol has also been reported to prevent neuronal apoptosis on anti-cancer-induced neurotoxicity in cultured dorsal root ganglion (DRG) neurons and
ischemia-induced hippocampal neurotoxicity in transient forebrain (Sano 2001; Ohtsuki et al., 1996). However, the protective effect of radicicol against inflammation-mediated neurotoxicity has not been reported. Inflammation in the CNS has been associated with the pathogenesis of neurodegenerative diseases. Activation of microglia, the resident immune cells of the brain, is believed to contribute to the neurodegenerative process through the release of proinflammatory factors such as TNF-α and IL-1, NO, reactive oxygen intermediates, and arachidonic metabolites. Production of these factors by microglia after exposure to LPS, the human immunodeficiency virus-1 coat protein (gp120), or β-amyloid has been well documented (Ii et al. 1996; Ralay Ranaivo et al., 2006). Using rat neuron–glia cultures, our study is the first to demonstrate that radicicol protects neurons from inflammationmediated neurotoxicity. Radicicol effectively prevents the loss of cell bodies and neurites due to LPS/IFN-r-induced neurotoxicity. Radicicol inhibited the LPS/IFN-γ-induced expression of iNOS, production of NO, and TNF-α in the cultures. Results of immunostaining showed that iNOS was expressed in microglia. It is well known that microglia are the major source of NO and TNF-α in mouse or rat cortical neuron– glia cultures (Kong et al., 1996; Jeohn et al., 2000; Possel et al., 2000). Interestingly, iNOS is also known to be expressed in astrocytes in astrocyte pure cultures such as rat primary astrocytes and astrocytoma cells, or in an ischemic human brain (Hsiao et al., 2007; Kim et al., 2006; Askalan et al., 2006). The inhibitory potency of radicicol against the production of NO and TNF-α was observed in parallel with the protection of neurons. Interestingly, radicicol at a concentration of 0.03 μM was able to show the protection of neurons (Fig. 2), which concentrations of radicicol however did not change NO and TNF-α production (Figs. 3 and 5). These results suggested that the protective effect of radicicol against LPS/IFN-γ-induced neuronal cell death is at least partially mediated via the inhibition of the release of TNF-α, as well as the suppression of iNOS expression in microglia. Radicicol has been reported to show both anti-proliferative and anti-apoptotic activity that is dependent on cell types, suggesting a possible role of heat-shock proteins (HSPs) in both the prevention and the development of apoptosis. It is well known that radicicol shows antitumor activity by binding the Nterminal pocket of HSP90 and destabilizing the HSP90 client proteins, which include receptor tyrosine kinases of the erbB family, Akt, and mutant p53, and are involved in signal transduction pathways that lead to the progression of cancer (Beliakoff et al., 2004). Conversely, although the mechanism of action was not clear, radicicol and geldanamycin have been known to prevent the neuronal apoptosis that is often induced by anti-cancer drugs such as vincristine, cisplatin, and taxol (Sano, 2001). Very low dose (20 nM) of radicicol promoted neurites and prevents apoptosis of DRG neurons in the absence of neutrophins. Radicicol suppressed actinomycin D-induced apoptosis in leukemia cells (Shino et al., 2002). Galea-Lauri et al. (1996) showed that an excess of HSP90 is associated with increased apoptosis in the U937 monoblastoid cell line
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following induction with TNF-α and cycloheximide. Unlike the effect of radicicol on cell survival, the anti-inflammatory activity of radicicol has not yet received much attention. Radicicol inhibited LPS-induced expression of iNOS by blocking the p38 kinase and NF-κB in macrophages (Jeon et al., 2000), and inhibited interleukin-8 production by the THP-1 human monocyte line in response to phorbol-12-myristate-13-acetate/LPS by blocking ERK 1/2 and p38 (Na et al., 2001). In conclusion, radicicol strongly protects against the loss of neuronal cell bodies and neurites from inflammation-mediated neurotoxicity in neuron–glia cultures. The protective effect of radicicol is mediated by the inhibition of the expression of iNOS and release of TNF-α in microglia, although the possibility that radicicol directly protects against neuronal apoptosis by suppressing the signal transduction pathway involving HSP90 cannot be ruled out. These results suggested that radicicol may be useful for the treatment of inflammation-mediated neurodegenerative diseases. Acknowledgement This work was supported by the 21C Frontier Microbial Genomics and Application Center Program, Ministry of Science and Technology (Grant MG05-0308-3-0), Republic of Korea. References Askalan, R., Deveber, G., Ho, M., Ma, J., Hawkins, C., 2006. Astrocyticinducible nitric oxide synthase in the ischemic developing human brain. Pediatric Research 60 (6), 687–692. Beliakoff, J., Whitesell, L., 2004. HSP90: an emerging target for breast cancer therapy. Anticancer Drugs 15 (7), 651–662. Block, M.L., Hong, J.-S., 2005. Microglia and inflammation-mediated neurodegeneration: multiple triggers with a common mechanism. Progress in Neurobiology 76, 77–98. Bronstein, D.M., Perez-Otano, I., Sun, V., Mullis Sawin, S.B., Chan, J., Wu, G.-C., Hudson, P.M., Kong, L.-Y., Hong, J.-S., McMillian, M.K., 1995. Glia dependent neurotoxicity and neuroprotection in mesencephalic cultures. Brain Research 704 (1), 112–116. Chao, C.C., Hu, S., Molitor, T.W., Shaskan, E.G., Peterson, P.K., 1992. Activated microglia mediate neuronal cell injury via a nitric oxide mechanism. Journal of Immunology 149 (8), 2736–2741. Combs, C.K., Johnson, D.E., Karlo, J.C., Cannady, S.B., Landreth, G.E., 2000. Inflammatory mechanism in Alzheimer's disease: inhibition of β-amyloidstimulated proinflammatory response and neurotoxicity by PPARγ agonists. Journal of Neurosience 20 (2), 558–567. Craft, J.M., Watterson, D.M., Van Eldik, L.J., 2006. Human amyloid β-induced neuroinflammation is early event in neurodegeneration. Glia 53 (5), 484–490. Culbert, A.A., Skaper, S.D., Howlett, D.R., Evans, N.A., Facci, L., Soden, P.E., Seymour, Z.M., Guillot, F., Gaestel, M., Richardson, J.C., 2006. MAPKactivated protein kinase 2 deficiency in microglia inhibits pro-inflammatory mediator release and resultant neurotoxicity: relevance to neuroinflammation in a transgenic mouse model of Alzheimer disease. Journal of Biological Chemistry 281 (33), 23658–23667. Dawson, V.L., Brahmbhatt, H.P., Mong, J.A., Dawson, T.M., 1994. Expression of inducible nitric oxide synthase causes delayed neurotoxicity in primary mixed neuronal-glial cortical cultures. Neurophamacology 33 (11), 1425–1430. Galea-Lauri, J., Richardson, A.J., Latchman, D.S., Katz, D.R., 1996. Increased heat shock protein 90(hsp90) expression leads to increased apoptosis in the monoblastoid cell line U937 following induction with TNF-α and cycloheximide. Journal of Immunology 157 (9), 4109–4118.
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Protective effect of radicicol against LPS/IFN-γ-induced neuronal cell death in rat cortical neuron–glia cultures Mi-Jin Sohn, Hyun-Jeong Noh, Ick-Dong Yoo, Won-Gon Kim ⁎ Functional Metabolomics Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305–806, Republic of Korea Received 11 September 2006; accepted 31 January 2007
Abstract We investigated the protective activity of radicicol, an antifungal antibiotic, against inflammation-induced neurotoxicity in neuron–glia cultures. Radicicol potently prevented the loss of neuronal cell bodies and neurites from LPS/IFN-γ-induced neurotoxicity in rat cortical neuron– glia cultures with an EC50 value of 0.09 μM. Radicicol inhibited the LPS/IFN-γ-induced expression of inducible nitric oxide synthase (iNOS) and production of nitric oxide (NO) in microglia. Additionally, radicicol decreased the LPS/IFN-γ-induced release of tumor necrosis factor-α (TNF-α) in the cultures. The inhibitory potency of radicicol against the production of NO and TNF-α was well correlated with the protection of neurons. These results suggest that the protective effect of radicicol against LPS/IFN-γ-induced neuronal cell death in neuron–glia cultures is mediated via the inhibition of TNF-α release, as well as the suppression of iNOS expression in microglia. © 2007 Elsevier Inc. All rights reserved. Keywords: Radicicol; Glia-mediated neurotoxicity; Inducible nitric oxide synthase; Microglia; Tumor necrosis factor-α
Introduction Inflammation in the central nervous system (CNS) has been associated with the pathogenesis of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, AIDS dementia complex, and amyotrophic lateral sclerosis, as well as post-ischemic brain injuries (Mcgeer et al., 1998; Raine 1994; Combs et al., 2000; Craft et al., 2006). The activation of microglia, the resident immune cells of the brain, is a histopathological hallmark of theses diseases. Activated microglia produce a variety of proinflammatory factors, including NO, TNF-α, interleukin-1β (IL-1β), and reactive oxygen species, which are believed to contribute to the neurodegenerative processes (Chao et al., 1992; Lee et al., 1993; Streit et al., 2005; Culbert et al., 2006). Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, contributes to bacterial meningitis and septic shock. The LPS has also been used as an immunostimulator of macrophages and microglia. In neuron– ⁎ Corresponding author. Tel.: +82 42 860 4298; fax: +82 42 860 4595. E-mail address: [email protected] (W.-G. Kim). 0024-3205/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2007.01.054
glia cultures, treatment with LPS or a combination of LPS and IFN-γ activates microglia and produces NO and proinflammatory cytokines such as TNF-α and IL-1β. These cytotoxic factors ultimately cause neurotoxicity (Chao et al., 1992; Dawson et al., 1994; Jeohn et al., 2000; Block and Hong 2005). Radicicol, an antifungal antibiotic, has been reported to be a potent tranquilizer with low toxicity (McCapra et al., 1964). Radicicol induces the reversal of the tumor phenotype of srctransformed cells and inhibits in vivo angiogenesis (Kwon et al., 1992, 1995). The antitumor action of radicicol has been shown to result from inhibition of folding and activation of the protein tyrosine kinases through the disruption of the ATPase activity of the Hsp90 molecular chaperone (Sharma et al., 1998; Roe et al., 1999). Radicicol has also been reported to inhibit LPS-induced expression of iNOS by blocking the p38 kinase and NF-κB in macrophages (Jeon et al., 2000). Radicicol has been reported to rescue neurons from anti-cancer-induced neurotoxicity in cultured dorsal root ganglion (DRG) neurons and ischemia-induced hippocampal neurotoxicity in transient forebrain, although the mechanism of action was not clear (Sano 2001; Ohtsuki et al., 1996). The protective effect of radicicol against inflammationmediated neurotoxicity has not been reported previously.
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In the course of our screening for neuroprotective agents against immune-mediated neurotoxicity, we found that radicicol potently inhibits LPS/IFN-γ-induced neuronal cell death. In this study, we investigated the neuroprotective effect of radicicol on LPS/IFN-γinduced neuronal damage in rat cortical neuron–glia cultures in terms of the number and morphology of neuronal cells. We also examined its effect on LPS/IFN-γ-induced production of NO, iNOS expression, and release of TNF-α in neuron–glia cultures. Materials and methods Materials Radicicol and poly-D-lysine were obtained from Sigma (St. Louis, MO). Radicicol was dissolved and diluted with culture medium prior to use. LPS (E. coli 011:B4) was purchased from Calbiochem (La Jolla, CA). Minimal essential medium (MEM), penicillin, streptomycin, glutamine, sodium pyruvate, nonessential amino acid, and horse serum were obtained from Life Technologies (Gaithersburg, MD). IFN-γ was purchased from Biosource International (Camarillo, CA), while biotinylated secondary antibodies, ABC kits, and 3,3′-diaminobenzidine were from Vector Laboratories (Burlingame, CA). Cell culture Primary mixed neuron–glia cultures were prepared from the brains of embryonic day 16–17 rats (SD) as described previously (Kim et al., 2000). The whole brain was removed aseptically, and the blood vessels and meninges were discarded. The cortex regions were dissected, pooled, and mechanically dissociated by mild trituration in ice-cold calcium- and magnesium-free W3 buffer (15 mM HEPES, pH 7.4, 145 mM NaCl, 5.4 mM KCl, 1 mM NaH2PO4, and 11 mM glucose). 5 × 105 cells in 0.5 ml of culture medium were seeded to each poly-D-lysine-coated well of the 24-well culture plates. The cultures were maintained at 37 °C in an atmosphere of 5% CO2 and 95% air. The culture medium consisted of MEM supplemented with 10% heat-inactivated horse serum, 2 mM glutamine, 1 mM sodium pyruvate, 100 μM nonessential amino acid, 15 mM KCl, 50 U/ml penicillin, and 50 μg/ml streptomycin. The cultures were replenished with fresh media at 1 and 4 days after plating. At 8 days after plating, the cultures were treated with vehicle or LPS/IFN-γ. When antibodies against microtubule-associated protein-2 (MAP-2) or CR3 complement receptor, a marker for rat microglia, were used for immunostaining of neuron–glia cultures, the following composition was determined: 60% neurons, 3% microglia. The remaining cells were presumed to be astroglial cells.
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monoclonal antibody against the enzyme (Transduction Laboratories, Lexington, KY). In brief, the cultures were washed three times with phosphate-buffered saline (PBS), and were then treated for 20 min with blocking solution (1% skim milk, 0.4% Triton- X-100, and 4% normal serum in PBS). The cultures were incubated overnight at 4 °C with primary antibody diluted blocking solution (anti-MAP-2, 2.5 μg/ml; OX-42, 5 μg/ ml; anti-iNOS 0.25 μg/ml). The cells were rinsed three times for 10 min each in PBS, treated with 1% H2O2, and washed three times with PBS. The bound primary antibody was visualized using the avidin–biotin–peroxidase complex (ABC) method, followed by color development with 3,3′-diaminobenzidine. The number of MAP-2-positive neuronal cell bodies was determined by visually counting five representative fields from each well under the microscope. Nitrite and TNF-α assay The production of NO was assessed as the accumulation of nitrite in the culture supernatants; this was determined using a colorimetric reaction with Griess reagent (Green et al., 1982). The amount of TNF-α in the medium was measured with a rat TNF-α enzyme-linked immunosorbent assay kit (Biosource International, Camarillo, CA). Statistical analysis The data are expressed as mean ± SD. The treatment group was compared with the vehicle control with a Student two-tailed t-test. Results Radicicol protects neurons from LPS/IFN-γ-induced neurotoxicity in neuron–glia cultures Cortical neuron–glia cultures were treated for 48 h with the vehicle alone, LPS alone, or a combination of LPS and IFN-γ.
Immunocytochemistry Cell cultures were fixed with 3.7% formaldehyde and immunostained as described previously (Kim et al., 2000). Neurons were identified with a monoclonal antibody to MAP-2 (Boehringer Mannheim, Mannheim, Germany). Microglia were visualized by staining for the CR3 complement receptor using the monoclonal antibody, OX-42 (PharMingen, San Diego, CA). The cellular expression of iNOS was visualized with a
Fig. 1. Dependency on IFN-γ in LPS/IFN-γ-induced loss of MAP-2-positive neurons in neuron–glia cultures. Cultures were treated for 48 h with vehicle alone, 10 ng/ml LPS, or a combination of 10 ng/ml LPS and various concentrations of IFN-γ; cultures were then immunostained with an antibody to the neuronal cytoskeletal protein, MAP-2. The data are expressed as percentages of the total number (236 ± 14 cells per mm2) of MAP-2-IR cell bodies present in the vehicle-treated control cultures. The data represent the mean ± SD of at least three wells taken from two independent experiments. ⁎p b 0.01 and ⁎⁎ p b 0.005 compared with the group treated with only LPS.
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Fig. 2. Protective effect of radicicol in LPS/IFN-γ-induced loss of MAP-2-positive neurons in neuron–glia cultures. Cultures were treated for 48 h with a combination of 10 ng/ml LPS and 50 U/ml IFN-γ in the presence of various concentrations of radicicol and then immunostained with the MAP-2 antibody. A) Immunocytochemical analysis of MAP-2-positive neurons treated with vehicle alone, LPS/IFN-γ, LPS/IFN-γ + 1 μM radicicol, or 1 μM radicicol alone. Scale bar, 100 μm. B) Dosedependent protective effect of radicicol in the LPS/IFN-γ-induced loss of MAP-2-positive neurons in neuron–glia cultures. The data are expressed as percentages of the total number (267 ± 19 cells per mm2) of MAP-2-IR cell bodies present in the vehicle-treated control cultures. The data represent the mean ± SD of at least three wells taken from three independent experiments. ⁎p b 0.05, ⁎⁎p b 0.005, ⁎⁎⁎ p b 0.0001 compared with the group treated with LPS/IFN-γ alone.
Neurotoxicity was assessed by morphological analysis and counting of the number of neuronal cell bodies after immunostaining for MAP-2. Exposure of cortical neuron–glia cultures to LPS alone at concentrations up to 1 μg/ml did not alter the number of MAP-2-positive cells or the morphologies of these cells compared with vehicle-treated control cultures. In contrast, treatment of the cultures with a combination of LPS and IFN-γ markedly increased NO production and caused dramatic degeneration of MAP-2-IR cells within 48 h (Fig. 1). The loss of MAP-2-IR cells was dependent on the concentration of IFN-γ used. There was an 88.2% decrease in the number of MAP-2-positive neurons upon combined treatment with 10 ng/ ml LPS and 50 U/ml IFN-γ. The protective effect of radicicol against neuronal cell death induced by treatment with 10 ng/ml LPS and 50 U/ml IFN-γ in neuron–glia cultures was investigated. Radicicol strongly prevented the loss of neuronal cell bodies and neurites in a dosedependent manner, with an EC50 value of 0.09 μM (Fig. 2A and B). Radicicol at a concentration of 0.3 μM rescued over 90% of the
neurons. Radicicol itself, however, showed no toxic effect on neuronal cell bodies or neurites at 1 μM. Radicicol decreases LPS/IFN-γ-induced production of NO and expression of iNOS in neuron–glia cultures In agreement with previous studies which indicated that LPS-induced neurotoxicity was, at least in part, mediated via NO (Chao et al., 1992; Bronstein et al., 1995), the addition of the NO synthase inhibitor, NG-nitro-L-arginine-methyl ester (1 mM), to the cultures inhibited the accumulation of nitrite produced after treatment for 48 h with 10 ng/ml LPS and 50 U/ ml IFN-γ by 85.5%, and reduced the loss of MAP-2-IR cells by 88.1% (data not shown). Correlation between the protective effect of radicicol and the LPS/IFN-γ-induced production of NO was examined. Treatment of the cultures with 10 ng/ml LPS and 50 U/ml IFN-γ in neuron–glia cultures increased the accumulation of nitrite up to 27.7 μM within 48 h (Fig. 3). The addition of radicicol inhibited
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Fig. 3. Inhibitory effect of radicicol in LPS/IFN-γ-induced production of NO in neuron–glia cultures. Cultures were treated with a combination of 10 ng/ml LPS and 50 U/ml IFN-γ in the presence of various concentrations of radicicol, and the levels of NO production, assessed as the accumulation of nitrite, were quantified 24 or 48 h later. The data represent the mean ± SD of at least three wells taken from three independent experiments. ⁎p b 0.005 and ⁎⁎p b 0.0001 compared with the group treated with LPS/IFN-γ alone.
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the LPS/IFN-γ-induced accumulation of nitrite in a concentration-dependent manner, with an EC50 value of 0.06 μM. At a concentration of 0.3 μM, radicicol inhibited the accumulation of nitrite by 82.6%. Importantly, the inhibitory potency of nitrite accumulation by radicicol occurred in parallel with the protection of neurons against LPS/IFN-γ-induced neurotoxicity. The inhibition of LPS/IFN-γ-induced iNOS expression by radicicol was also examined by immunostaining for iNOS. Upon treatment for 24 h with 10 ng/ml LPS and 50 U/ml IFN-γ, a number of iNOS-IR cells in neuron–glia cultures showed marked changes. The addition of radicicol (0.3 μM) to the cultures after treatment for 24 h with 10 ng/ml LPS and 50 U/ml IFN-γ decreased the number of iNOS-IR cells produced by 79.7%, which was well correlated with the proportion of MAP-2-IR cells lost, 93.0% (Fig. 4A). To determine whether the iNOS-IR cells were microglia, we then performed immunostaining with OX-42.
Fig. 4. Inhibitory effect of radicicol in LPS/IFN-γ-induced expression of iNOS in neuron–glia cultures. Cultures were treated for 24 h with a combination of 10 ng/ml LPS and 50 U/ml IFN-γ in the presence of 0.3 μM radicicol, and were then immunostained with antibody to iNOS and OX-42 for staining iNOS-positive cells and microglia, respectively. A) Immunocytochemical analysis of iNOS- or OX-42-positive cells treated with vehicle alone or LPS/IFN-γ. Scale bar, 100 μm. B) Inhibitory effect of radicicol on the number of iNOS-IR cells induced by LPS/IFN-γ. The data are expressed as the number of iNOS-IR cells per square millimeter and represent the mean ± SD of at least three wells. ⁎p b 0.0001 compared with the group treated with LPS/IFN-γ alone.
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Fig. 5. Inhibitory effect of radicicol in LPS/IFN-γ-induced release of TNF-α in neuron–glia cultures. Cultures were treated for 6 h with a combination of 10 ng/ ml LPS and 50 U/ml IFN-γ in the presence of various concentrations of radicicol, and the levels of TNF-α release were measured. The data represent the mean ± SD of at least three wells. ⁎p b 0.0001 compared with the group treated with LPS/ IFN-γ alone.
The iNOS-IR cells had a round or ramified morphology, which were the same as OX-42-IR cells (Fig. 4B). This result indicate that iNOS is expressed only in microglia, not in astrocytes, as reported in mice cortical neuron–glia cultures and in rat primary mixed astrocytes and microglial cell cultures (Kong et al., 1996; Jeohn et al., 2000; Possel et al., 2000). In addition, radicicol didn't show a significant cytotoxicity on basal and activated microglia. This result suggests that the protective activity of radicicol against LPS/IFN-γ-induced neuronal cell death is, at least in part, mediated by the inhibition of NO production, which results from the suppression of iNOS expression in microglia. Radicicol decreases LPS/IFN-γ-induced production of TNF-α in neuron–glia cultures To determine whether radicicol inhibits the production of the proinflammatory cytokine, TNF-α, from LPS/IFN-γ-stimulated neuron–glia cultures, levels of TNF-α in the culture medium were examined. Levels of TNF-α in the culture medium were measured at 6 h after LPS/IFN-γ treatment, a time at which the release of the cytokine is known to be near its maximal levels (Liu et al., 2000). Radicicol inhibited the LPS/IFN-γ-induced release of TNF-α in a concentration-dependent manner, with an EC50 value of 0.1 μM (Fig. 5). Upon treatment with radicicol at a concentration of 0.3 μM, TNF-α was reduced to its basal level. The dose-dependent decrease in the release of TNF-α was well correlated with the loss of MAP-2-IR cells. These results suggest that the protective activity of radicicol against LPS/IFNγ-induced neuronal cell death is mediated via the inhibition of TNF-α release, as well as the suppression of iNOS expression. Discussion Radicicol, originally isolated as an antifungal antibiotic, is well known to exhibit antitumor activity through its binding to the N-terminal ATP/ADP-binding domains of HSP90, which are essential for its function. Its oxime derivative has in vivo antitumor activity; geldanamycin, which has the same mechanism, is currently being used in clinical trials (Soga et al., 1999; Maloney and Workman 2002). Radicicol has also been reported to prevent neuronal apoptosis on anti-cancer-induced neurotoxicity in cultured dorsal root ganglion (DRG) neurons and
ischemia-induced hippocampal neurotoxicity in transient forebrain (Sano 2001; Ohtsuki et al., 1996). However, the protective effect of radicicol against inflammation-mediated neurotoxicity has not been reported. Inflammation in the CNS has been associated with the pathogenesis of neurodegenerative diseases. Activation of microglia, the resident immune cells of the brain, is believed to contribute to the neurodegenerative process through the release of proinflammatory factors such as TNF-α and IL-1, NO, reactive oxygen intermediates, and arachidonic metabolites. Production of these factors by microglia after exposure to LPS, the human immunodeficiency virus-1 coat protein (gp120), or β-amyloid has been well documented (Ii et al. 1996; Ralay Ranaivo et al., 2006). Using rat neuron–glia cultures, our study is the first to demonstrate that radicicol protects neurons from inflammationmediated neurotoxicity. Radicicol effectively prevents the loss of cell bodies and neurites due to LPS/IFN-r-induced neurotoxicity. Radicicol inhibited the LPS/IFN-γ-induced expression of iNOS, production of NO, and TNF-α in the cultures. Results of immunostaining showed that iNOS was expressed in microglia. It is well known that microglia are the major source of NO and TNF-α in mouse or rat cortical neuron– glia cultures (Kong et al., 1996; Jeohn et al., 2000; Possel et al., 2000). Interestingly, iNOS is also known to be expressed in astrocytes in astrocyte pure cultures such as rat primary astrocytes and astrocytoma cells, or in an ischemic human brain (Hsiao et al., 2007; Kim et al., 2006; Askalan et al., 2006). The inhibitory potency of radicicol against the production of NO and TNF-α was observed in parallel with the protection of neurons. Interestingly, radicicol at a concentration of 0.03 μM was able to show the protection of neurons (Fig. 2), which concentrations of radicicol however did not change NO and TNF-α production (Figs. 3 and 5). These results suggested that the protective effect of radicicol against LPS/IFN-γ-induced neuronal cell death is at least partially mediated via the inhibition of the release of TNF-α, as well as the suppression of iNOS expression in microglia. Radicicol has been reported to show both anti-proliferative and anti-apoptotic activity that is dependent on cell types, suggesting a possible role of heat-shock proteins (HSPs) in both the prevention and the development of apoptosis. It is well known that radicicol shows antitumor activity by binding the Nterminal pocket of HSP90 and destabilizing the HSP90 client proteins, which include receptor tyrosine kinases of the erbB family, Akt, and mutant p53, and are involved in signal transduction pathways that lead to the progression of cancer (Beliakoff et al., 2004). Conversely, although the mechanism of action was not clear, radicicol and geldanamycin have been known to prevent the neuronal apoptosis that is often induced by anti-cancer drugs such as vincristine, cisplatin, and taxol (Sano, 2001). Very low dose (20 nM) of radicicol promoted neurites and prevents apoptosis of DRG neurons in the absence of neutrophins. Radicicol suppressed actinomycin D-induced apoptosis in leukemia cells (Shino et al., 2002). Galea-Lauri et al. (1996) showed that an excess of HSP90 is associated with increased apoptosis in the U937 monoblastoid cell line
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following induction with TNF-α and cycloheximide. Unlike the effect of radicicol on cell survival, the anti-inflammatory activity of radicicol has not yet received much attention. Radicicol inhibited LPS-induced expression of iNOS by blocking the p38 kinase and NF-κB in macrophages (Jeon et al., 2000), and inhibited interleukin-8 production by the THP-1 human monocyte line in response to phorbol-12-myristate-13-acetate/LPS by blocking ERK 1/2 and p38 (Na et al., 2001). In conclusion, radicicol strongly protects against the loss of neuronal cell bodies and neurites from inflammation-mediated neurotoxicity in neuron–glia cultures. The protective effect of radicicol is mediated by the inhibition of the expression of iNOS and release of TNF-α in microglia, although the possibility that radicicol directly protects against neuronal apoptosis by suppressing the signal transduction pathway involving HSP90 cannot be ruled out. These results suggested that radicicol may be useful for the treatment of inflammation-mediated neurodegenerative diseases. Acknowledgement This work was supported by the 21C Frontier Microbial Genomics and Application Center Program, Ministry of Science and Technology (Grant MG05-0308-3-0), Republic of Korea. References Askalan, R., Deveber, G., Ho, M., Ma, J., Hawkins, C., 2006. Astrocyticinducible nitric oxide synthase in the ischemic developing human brain. Pediatric Research 60 (6), 687–692. Beliakoff, J., Whitesell, L., 2004. HSP90: an emerging target for breast cancer therapy. Anticancer Drugs 15 (7), 651–662. Block, M.L., Hong, J.-S., 2005. Microglia and inflammation-mediated neurodegeneration: multiple triggers with a common mechanism. Progress in Neurobiology 76, 77–98. Bronstein, D.M., Perez-Otano, I., Sun, V., Mullis Sawin, S.B., Chan, J., Wu, G.-C., Hudson, P.M., Kong, L.-Y., Hong, J.-S., McMillian, M.K., 1995. Glia dependent neurotoxicity and neuroprotection in mesencephalic cultures. Brain Research 704 (1), 112–116. Chao, C.C., Hu, S., Molitor, T.W., Shaskan, E.G., Peterson, P.K., 1992. Activated microglia mediate neuronal cell injury via a nitric oxide mechanism. Journal of Immunology 149 (8), 2736–2741. Combs, C.K., Johnson, D.E., Karlo, J.C., Cannady, S.B., Landreth, G.E., 2000. Inflammatory mechanism in Alzheimer's disease: inhibition of β-amyloidstimulated proinflammatory response and neurotoxicity by PPARγ agonists. Journal of Neurosience 20 (2), 558–567. Craft, J.M., Watterson, D.M., Van Eldik, L.J., 2006. Human amyloid β-induced neuroinflammation is early event in neurodegeneration. Glia 53 (5), 484–490. Culbert, A.A., Skaper, S.D., Howlett, D.R., Evans, N.A., Facci, L., Soden, P.E., Seymour, Z.M., Guillot, F., Gaestel, M., Richardson, J.C., 2006. MAPKactivated protein kinase 2 deficiency in microglia inhibits pro-inflammatory mediator release and resultant neurotoxicity: relevance to neuroinflammation in a transgenic mouse model of Alzheimer disease. Journal of Biological Chemistry 281 (33), 23658–23667. Dawson, V.L., Brahmbhatt, H.P., Mong, J.A., Dawson, T.M., 1994. Expression of inducible nitric oxide synthase causes delayed neurotoxicity in primary mixed neuronal-glial cortical cultures. Neurophamacology 33 (11), 1425–1430. Galea-Lauri, J., Richardson, A.J., Latchman, D.S., Katz, D.R., 1996. Increased heat shock protein 90(hsp90) expression leads to increased apoptosis in the monoblastoid cell line U937 following induction with TNF-α and cycloheximide. Journal of Immunology 157 (9), 4109–4118.
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Shino, Y., Fujita, Y., Oka, S., Yamazaki, Y., 2002. ATPase inhibitors suppress actinomycin D-induced apoptosis in leukemia cells. Anticancer Research 22 (5), 2907–2911. Soga, S., Neckers, L.M., Schulte, T.W., Shiotsu, Y., Akasaka, K., Narumi, H., Agatsuma, T., Ikuina, Y., Murakata, C., Tamaoki, T., Akinaga, S., 1999. KF25706, a novel oxime derivative of radicicol, exhibits in vivo antitumor activity via selective depletion of Hsp90 binding signaling molecules. Cancer Research 59 (12), 2931–2938. Streit, W.J., Conde, J.R., Fendrick, S.E., Flanary, B.E., Mariani, C.L., 2005. Role of microglia in the central nervous system's immune response. Neurological Research 27 (7), 685–691.