Regulation of Inflammatory Response in Neural Cells in Vitro by Thiadiazolidinones Derivatives through Peroxisome Proliferator-activated Receptor γ Activation

In most neurodegenerative disorders, including multiple sclerosis, Parkinson disease, and Alzheimer disease, a massive neuronal cell death occurs as a consequence of an uncontrolled inflammatory response, where activated astrocytes and microglia and their cytotoxic agents play a crucial pathological role. Current treatments for these diseases are not effective. In the present study we investigate the effect of thiadiazolidinone derivatives, which have been recently suggested to play a role in neurodegenerative disorders. We have found that thiadiazolidinones are potent neuroprotector compounds. Thiadiazolidinones inhibited inflammatory activation of cultured brain astrocytes and microglia by diminishing lipopolysaccharide-induced interleukin 6, tumor necrosis factor α, inducible nitric-oxide synthase, and inducible cyclooxygenase type 2 expression. In addition, thiadiazolidinones inhibited tumor necrosis factor-α and nitric oxide production and, concomitantly, protected cortical neurons from cell death induced by the cell-free supernatant from activated microglia. The neuroprotective effects of thiadiazolidinones are completely inhibited by the peroxisome proliferator-activated receptor γ antagonist GW9662. In contrast the glycogen synthase kinase 3β inhibitor LiCl did not show any effect. These findings suggest that thiadiazolidinones potently attenuate lipopolysaccharide-induced neuroinflammation and reduces neuronal death by a mechanism dependent of peroxisome proliferator-activated receptor γ activation. In most neurodegenerative disorders, including multiple sclerosis, Parkinson disease, and Alzheimer disease, a massive neuronal cell death occurs as a consequence of an uncontrolled inflammatory response, where activated astrocytes and microglia and their cytotoxic agents play a crucial pathological role. Current treatments for these diseases are not effective. In the present study we investigate the effect of thiadiazolidinone derivatives, which have been recently suggested to play a role in neurodegenerative disorders. We have found that thiadiazolidinones are potent neuroprotector compounds. Thiadiazolidinones inhibited inflammatory activation of cultured brain astrocytes and microglia by diminishing lipopolysaccharide-induced interleukin 6, tumor necrosis factor α, inducible nitric-oxide synthase, and inducible cyclooxygenase type 2 expression. In addition, thiadiazolidinones inhibited tumor necrosis factor-α and nitric oxide production and, concomitantly, protected cortical neurons from cell death induced by the cell-free supernatant from activated microglia. The neuroprotective effects of thiadiazolidinones are completely inhibited by the peroxisome proliferator-activated receptor γ antagonist GW9662. In contrast the glycogen synthase kinase 3β inhibitor LiCl did not show any effect. These findings suggest that thiadiazolidinones potently attenuate lipopolysaccharide-induced neuroinflammation and reduces neuronal death by a mechanism dependent of peroxisome proliferator-activated receptor γ activation. Inflammatory activation of neuronal and glial cells is believed to contribute to cell death and damage during neurological disease. One of the hallmarks of neurodegenerative and inflammatory pathologies is the increased number of activated astrocytes and microglia in response to the pathological stimulus (1Sheng J.G. Mrak R.E. Griffin W.S. Acta Neuropathol. 1997; 94: 1-5Crossref PubMed Scopus (161) Google Scholar, 2Arvin B. Neville L.F. Barone F.C. Feuerstein G.Z. Neurosci. Biobehav. Rev. 1996; 20: 445-452Crossref PubMed Scopus (352) Google Scholar). Under normal conditions, brain microglia, the functional equivalent of macrophages in the central nervous system (3Gonzalez-Scarano F. Baltuch G. Annu. Rev. Neurosci. 1999; 22: 219-240Crossref PubMed Scopus (898) Google Scholar), are involved in immune surveillance and host defense against infectious agents (4Gehrmann J. Matsumoto Y. Kreutzberg G.W. Brain Res. Brain Res. Rev. 1995; 20: 269-287Crossref PubMed Scopus (975) Google Scholar). However, in response to brain injury, infection, or inflammation, microglia readily become activated in a way similar to peripheral tissue macrophages. Now, there is a growing evidence that toxic mediators, including tumor necrosis factor-α (TNF-α), 1The abbreviations used are: TNF-α, tumor necrosis factor α; IL-6, interleukin-6; NO, nitric oxide; TDZDs, thiadiazolidinones; LPS, lipopolysaccharide; PPARγ, peroxisome proliferator-activated receptor gamma; RXR, retinoid X receptors; iNOS, inducible nitric-oxide synthase; COX-2, cyclooxygenase type 2; 15dPG-J2, 15-deoxy-Δ-12,14-prostaglandin J2; 9-cis-RA, 9-cis retinoic acid; PPRE, PPARγ response element; DAPI, 4,6-diamidino-2-phenylindole; GSK, glycogen synthase kinase. interleukin (IL)-6, and nitric oxide (NO), produced by activated microglial cells might be involved in the pathogenesis of various neurodegenerative diseases such as Parkinson disease, Alzheimer disease, multiple sclerosis, and AIDS dementia (3Gonzalez-Scarano F. Baltuch G. Annu. Rev. Neurosci. 1999; 22: 219-240Crossref PubMed Scopus (898) Google Scholar, 5Minghetti L. Levi G. Prog. Neurobiol. 1998; 54: 99-125Crossref PubMed Scopus (514) Google Scholar, 6McGeer P.L. McGeer E.G. Brain Res. Brain Res. Rev. 1995; 21: 195-218Crossref PubMed Scopus (1246) Google Scholar). Astrocytes, which are responsible for maintaining the homeostasis of the brain tissue, also participate to a large extend in the neuroimmune responses (7Genis P. Jett M. Bernton E.W. Boyle T. Gelbard H.A. Dzenko K. Keane R.W. Resnick L. Mizrachi Y. Volsky D.J. Epstein L.G. Gendelman H.E. J. Exp. Med. 1992; 176: 1703-1718Crossref PubMed Scopus (497) Google Scholar, 8Lassmann H. Rinner W. Hickey W.F. Neuropathol. Appl. Neurobiol. 1994; 20: 195-196PubMed Google Scholar). Hence, it is of great interest to find a means to modulate microglial activation in central nervous system inflammatory responses for the therapeutic intervention against these neurodegenerative diseases. Thiadiazolidinones (TDZDs) are small heterocyclic thiadiazolidinones, which were synthesized following a pathway that is based on the reactivity of N-alkyl-S-(N′-(chlorocarbonyl)amino) isothiocarbamoyl chlorides with isocyanates (9Ottman G. Hooks H. Angew Chem. Int. Ed. Engl. 1966; 5: 672-673Crossref Scopus (21) Google Scholar). They are small molecules with favorable ADME-Tox-drugable properties, such as oral bioavailability and blood-brain barrier penetration (10.Alonso, M., Dorronsoro, I., Castro, A., Rodriguez-Franco, M. I., Abellán, G., Boiani, M., Vericat, J. A., and Martinez, A. (2004) XVIII International Symposium on Medicinal Chemistry, Copenhagen, Denmark, August 15–19, 2004, pp. 337, CopenhagenGoogle Scholar) and they have been shown to be non-ATP competitive glycogen synthase kinase 3β (GSK-3β) inhibitors (11Martinez A. Alonso M. Castro A. Perez C. Moreno F.J. J. Med. Chem. 2002; 45: 1292-1299Crossref PubMed Scopus (425) Google Scholar). TDZDs have been postulated that could be of potential therapeutic use for the treatment of Alzheimer disease and other important unmet pathologies as diabetes type II, cancer, and chronic inflammatory processes (12Dorronsoro I. Castro A. Martinez A. Exp. Opin. Ther. Pat. 2002; 12: 1539-1544Crossref Scopus (59) Google Scholar, 13Martinez A. Castro A. Dorronsoro I. Alonso M. Med. Res. Rev. 2002; 22: 373-384Crossref PubMed Scopus (307) Google Scholar). Preliminary in vitro studies suggest potential neuroprotective effects of TDZDs against several insults, such as 6-OHDPA (14Chen G. Bower K.A. Ma C. Fang S. Thiele C.J. Luo J. FASEB J. 2004; 18: 1162-1164Crossref PubMed Scopus (184) Google Scholar), lipopolysaccharide (LPS) and glutamate (15Alonso M. Luna-Medina R. Fuertes A. Perez-Puerto M.J. Medina M. Perez-Castillo A. Martinez A. 8th International Montreal/Springfield Symposium on Advances in Alzheimer Therapy, Montreal, Canada, April 14–17, 2004. Madrid, Spain2004Google Scholar). The somehow chemical structure-related derivatives to TDZDs, and recently FDA approved thiazolidinediones, such as rosiglitazone, pioglitazone, and troglitazone, are known agonists of the peroxisome proliferator-activated receptor γ (PPARγ) (16Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3469) Google Scholar, 17Willson T.M. Brown P.J. Sternbach D.D. Henke B.R. J. Med. Chem. 2000; 43: 527-550Crossref PubMed Scopus (1706) Google Scholar). PPARs are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors, which includes retinoid, steroid, and thyroid hormone receptors, that regulate transcription of distinct genes through heterodimerization with the retinoid X receptors (RXR) (18Schoonjans K. Staels B. Auwerx J. Biochim. Biophys. Acta. 1996; 1302: 93-109Crossref PubMed Scopus (917) Google Scholar). The PPARγ receptor subtype seems to play a pivotal role in the regulation of cellular proliferation, differentiation, and inflammation. It has been recently shown that PPARγ ligands, including thiazolidinediones, have potent anti-inflammatory effects, such as the suppression of TNF-α and IL-1β, inducible nitric-oxide synthase (iNOS), and cyclooxygenase type 2 (COX-2) (19Jiang C. Ting A.T. Seed B. Nature. 1998; 391: 82-86Crossref PubMed Scopus (539) Google Scholar, 20Ricote M. Huang J.T. Welch J.S. Glass C.K. J. Leukocyte Biol. 1999; 66: 733-739Crossref PubMed Scopus (282) Google Scholar, 21Maggi Jr., L.B. Sadeghi H. Weigand C. Scarim A.L. Heitmeier M.R. Corbett J.A. Diabetes. 2000; 49: 346-355Crossref PubMed Scopus (116) Google Scholar, 22Bernardo A. Levi G. Minghetti L. Eur. J. Neurosci. 2000; 12: 2215-2223Crossref PubMed Scopus (223) Google Scholar, 23Subbaramaiah K. Lin D.T. Hart J.C. Dannenberg A.J. J. Biol. Chem. 2001; 276: 12440-12448Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar, 24Marx N. Mach F. Sauty A. Leung J.H. Sarafi M.N. Ransohoff R.M. Libby P. Plutzky J. Luster A.D. J. Immunol. 2000; 164: 6503-6508Crossref PubMed Scopus (298) Google Scholar, 25Han K.H. Chang M.K. Boullier A. Green S.R. Li A. Glass C.K. Quehenberger O. J. Clin. Investig. 2000; 106: 793-802Crossref PubMed Scopus (161) Google Scholar). Of relevance to central nervous system disease is that PPARγ agonists have been demonstrated to have similar anti-inflammatory effects on astrocytes and microglial cells (review in Ref. 26Kielian T. Drew P.D. J. Neurosci. Res. 2003; 71: 315-325Crossref PubMed Scopus (141) Google Scholar). Here we demonstrate a potent anti-inflammatory effect of two TDZD compounds, NP00111 and NP01138 (see Fig. 1), in primary cultures of cortical neurons, astrocytes, and microglia. Our results indicate that both compounds act broadly to inhibit the production of proinflammatory and neurotoxic products elaborated by LPS-stimulated astrocytes and microglial cells. Concomitantly, TDZDs also exert a neuroprotective effect in primary cultures of cortical neurons treated with cell-free supernatant from LPS-stimulated microglia. Our results suggest that the effects of TDZDs are dependent on PPARγ activation, as the PPARγ antagonist GW9662 abolished them. The capacity of TDZDs to suppress the expression of inflammatory cytokines and their protective action on neurons suggests that these agents may be of value in the treatment of Alzheimer disease or other inflammatory diseases. Primary Cell Cultures—Primary cortical neuronal cultures were prepared from the cerebral cortex of embryonic day 18 rats, according to published protocols (27Chowen J.A. Torres-Aleman I. Garcia-Segura L.M. Neuroendocrinology. 1992; 56: 895-901Crossref PubMed Scopus (92) Google Scholar). After removal of the meninges, the cerebral cortex was dissected and dissociated in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% fetal bovine serum (Invitrogen). After washing, cells were plated on and or and the cultures were in medium with In these conditions, the of the neuronal was as by and 2 were with were prepared from cerebral removal of the the cerebral cortex was and with for After the was with and the cells were plated on and in modified Eagle's medium containing 10% fetal bovine After the were on an for and the and microglial cells were After the were to any microglia cells and and a in In these conditions, the of the astrocytes was as by to microglial to and to Primary microglial cells were prepared from glial as glial cultures were on an for The supernatant was for and the cellular containing the microglial cells in modified Eagle's medium containing 10% fetal bovine serum and a of on were to for 2 and the medium was to and medium containing factor was The of microglia by was as by with the of and were to the medium to and cells were for tissue medium was and the cells were for iNOS, or expression. In the of neuronal medium from and microglia was to cortical cell which were for an cells were and for neuronal cell death and expression of to and were from was from was from and and were from were with an against a for were by for the receptor with and astrocytes were with an against an is to the of the treatment the on in cell were with for with and with for After a with the primary cells were with and with an The for was a The were a of cell of cells was the to be were and were of the of cell cortical neuronal cultures were treated or not with NP00111 or with glutamate or cell-free supernatant from LPS-stimulated microglia, and on the of cells was by with TNF-α in astrocytes and microglial cultures was by TNF-α as the and TNF-α as the production of was by the of of the products of by a based on the of by the of astrocytes and microglia cells with or neuronal cultures with medium from microglia, in the or of TDZDs, of were with of in and for The was on a neuronal cell was and in Dulbecco's modified Eagle's medium containing 10% fetal bovine and 2 and cultures were with the containing PPARγ of a with PPARγ and expression according to the cells of of PPARγ, and of and were treatment with NP00111 NP01138 9-cis retinoic for of and was in shown are the means of for cells with treatments were the with NP00111 and NP01138 Inflammatory of potential anti-inflammatory of TDZDs was by the production of inflammatory from cultured astrocytes and microglial cells. we a of production of by astrocytes and microglia cells. and microglia were with of TDZDs for and cells were cultured for with shown in Fig. NP00111 and NP01138 inhibited the production of and the expression of induced by The effects of TDZDs were not by a of cell as the of astrocytes and microglial cells to NP00111 or NP01138 did not cell not The effects of both TDZDs were and, we used We the effects of TDZDs on LPS-stimulated primary cultures of primary astrocytes were with we a of production and also of TNF-α in the medium and which was inhibited by NP00111 and NP01138 treatment study the effect of NP00111 and NP01138 on activation, we these compounds the of TNF-α and proinflammatory In addition, we also the of inducible and The is responsible for the of P. J. S. H. H. K. Res. 2001; PubMed Scopus Google Scholar), and of in activated microglia and astrocytes central to We by by shown in Fig. and the of TNF-α and were increased treatment of astrocytes, and treatment of the cultures with NP00111 or NP01138 completely effect. In addition, in conditions, and were in and their was induced The of NP00111 and NP01138 inhibited and expression. similar cellular is present in as by not results were with microglia. cultures treated with an in the of and TNF-α and with and, a in and TNF-α was in microglia cultures treated with NP00111 or of TDZD compounds to the medium of microglial cells inhibited the expression of the two and TNF-α, as as and and similar cellular is present in as by not these results suggest that NP00111 and NP01138 inhibit inflammatory activation of cultured glial cells. NP00111 and NP01138 from the effect of NP00111 and NP01138 on neuronal cell death induced by cell-free supernatant from LPS-stimulated microglia embryonic cortical neurons were with supernatant from microglia in the or of TDZD compounds. The treatment of cultures with cell-free from LPS-stimulated microglia in a in the number of cells with the cultures The of NP00111 or NP01138 to neuronal cultures treated with medium from microglial cells neuronal cell that TDZDs could their neuroprotective effects by the effects of activated cytotoxic induced by medium from microglia was with an in neuronal expression of TNF-α, IL-6, iNOS, and 6, and in a similar to the in glial cells treated with In addition, as in astrocytes and microglia, the TDZD compounds NP00111 and NP01138 completely the of these genes in neuronal similar cellular is present in as by not In with these and as shown for glial the of to the medium was of the neurons with supernatant from microglia with and was completely abolished in cultures treated with both TDZDs NP00111 and NP01138 from and the induced by other could also be by TDZD compounds. we treated cortical neurons with or glutamate and by shown in Fig. both an in the number of similar to found an inflammatory stimulus was The of NP00111 or NP01138 protected neuronal cultures from both and These indicate that the protective role of the TDZDs is not to a of TDZDs on PPARγ NP00111 and NP01138 and the thiazolidinediones, such as to a of the nuclear receptor PPARγ in the of these compounds. are known of PPARγ (16Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3469) Google Scholar), activation has been shown to play a role in cellular proliferation, differentiation, and G.W. Glass C.K. Opin. 2002; PubMed Scopus Google Scholar, J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). suggests the that PPARγ activation could be involved in the of the effects of these TDZDs in cells. We in by a containing PPARγ response a we used rosiglitazone, a known of In cells with and expression for PPARγ and a of was treatment with NP00111 and NP01138 was similar or the of to the for These results demonstrate that these compounds are to a containing PPARγ response It has been shown that the PPARγ receptor transcription as a (review in Ref. A. A. Rev. 2001; PubMed Scopus Google Scholar). of nuclear receptor be activated by of or receptor and are activated in the of both we the response of the to the or in with rosiglitazone, or Fig. that treatment of cells with in a in the cells were with with rosiglitazone, or a activation of the was These suggest that the effects of the TDZDs could be by activation of In of the shown we activation of PPARγ is involved in the neuroprotective and anti-inflammatory effects of the TDZDs have also been as inhibitors and it has been postulated that could be of potential therapeutic use for the treatment of Alzheimer disease, we also the effect of an inhibitor of on neuronal cell death induced by medium activated microglia and proinflammatory neuronal cultures were with the PPARγ antagonist GW9662 S. R. I. J. Immunol. 2004; PubMed Scopus Google Scholar, J. J. C. P.J. J. J. Cell 2004; PubMed Scopus Google Scholar, C. S. N. L. T.M. Y. R. G. J. Exp. Med. 2004; PubMed Scopus Google Scholar) to NP00111 and NP01138 and the cell-free supernatant from microglia. neuronal cultures were with LiCl to with the shown in Fig. and GW9662 the effect of TDZDs on and their neuroprotective effects in cultures of cortical of neuronal cultures with the inhibitor LiCl and did not have any effect the in cells by treatment with medium from microglia. The in was also not by of neuronal cultures with These results suggest an of the nuclear receptor PPARγ in the neuroprotective action of both compounds. we the action of NP00111 and NP01138 on glial cells was also dependent on PPARγ activation. and microglia cultures were treated with GW9662 or and production was be in Fig. the in the medium of astrocytes and microglia with were similar to found in the other of these cultures with the PPARγ antagonist GW9662 the effect of both TDZDs on production These suggest that NP00111 and NP01138 are through activation of the PPARγ receptor and not through their of In the present we have demonstrated that NP00111 and two thiadiazolidinone compounds, suppress inflammatory activation of astrocytes and microglia in vitro and cortical neurons from cell death and that these effects could be by a mechanism dependent on PPARγ activation. a central role in the pathogenesis of several brain and F.C. Feuerstein G.Z. 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Our results demonstrate that NP00111 and NP01138 inhibit production and TNF-α from and microglia In addition, these TDZDs completely of such as and TNF-α, and the expression of and the proinflammatory COX-2, in response to Hence, these results suggest that these compounds could be of therapeutic value in brain diseases and brain damage where processes are We have also shown that NP00111 and NP01138 not the inflammatory response by glial cells also have a neuroprotective in primary cortical These compounds cortical neurons from induced by cell-free medium from microglial In addition, TDZDs protected cortical neurons against and that these compounds are potent neuroprotective agents against a of neuronal by TDZDs was with a in COX-2, and expression and indicate in to glial neurons M. M. T. J. 1998; 71: PubMed Scopus Google Scholar, G. I. J.H. J. 1996; 66: PubMed Scopus Google Scholar, I. Y. T. S. Neurosci. 1995; PubMed Scopus Google Scholar), production of be neurotoxic J.A. 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Our results extend these studies and present potential of PPARγ as agents against results indicate that TDZDs be neuroprotective and anti-inflammatory compounds in neuronal cells in activation of the nuclear receptor Our study suggests therapeutic of TDZDs in brain disorders, such as multiple sclerosis, and Alzheimer where inflammatory responses play a role.

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