Fractalkine Attenuates Excito-neurotoxicity via Microglial Clearance of Damaged Neurons and Antioxidant Enzyme Heme Oxygenase-1 Expression
2010; Elsevier BV; Volume: 286; Issue: 3 Linguagem: Inglês
10.1074/jbc.m110.169839
ISSN1083-351X
AutoresMariko Noda, Yukiko Doi, Jianfeng Liang, Jun Kawanokuchi, Yoshifumi Sonobe, Hideyuki Takeuchi, Tetsuya Mizuno, Akio Suzumura,
Tópico(s)Heme Oxygenase-1 and Carbon Monoxide
ResumoGlutamate-induced excito-neurotoxicity likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases. Microglial clearance of dying neurons and associated debris is essential to maintain healthy neural networks in the central nervous system. In fact, the functions of microglia are regulated by various signaling molecules that are produced as neurons degenerate. Here, we show that the soluble CX3C chemokine fractalkine (sFKN), which is secreted from neurons that have been damaged by glutamate, promotes microglial phagocytosis of neuronal debris through release of milk fat globule-EGF factor 8, a mediator of apoptotic cell clearance. In addition, sFKN induces the expression of the antioxidant enzyme heme oxygenase-1 (HO-1) in microglia in the absence of neurotoxic molecule production, including NO, TNF, and glutamate. sFKN treatment of primary neuron-microglia co-cultures significantly attenuated glutamate-induced neuronal cell death. Using several specific MAPK inhibitors, we found that sFKN-induced heme oxygenase-1 expression was primarily mediated by activation of JNK and nuclear factor erythroid 2-related factor 2. These results suggest that sFKN secreted from glutamate-damaged neurons provides both phagocytotic and neuroprotective signals. Glutamate-induced excito-neurotoxicity likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases. Microglial clearance of dying neurons and associated debris is essential to maintain healthy neural networks in the central nervous system. In fact, the functions of microglia are regulated by various signaling molecules that are produced as neurons degenerate. Here, we show that the soluble CX3C chemokine fractalkine (sFKN), which is secreted from neurons that have been damaged by glutamate, promotes microglial phagocytosis of neuronal debris through release of milk fat globule-EGF factor 8, a mediator of apoptotic cell clearance. In addition, sFKN induces the expression of the antioxidant enzyme heme oxygenase-1 (HO-1) in microglia in the absence of neurotoxic molecule production, including NO, TNF, and glutamate. sFKN treatment of primary neuron-microglia co-cultures significantly attenuated glutamate-induced neuronal cell death. Using several specific MAPK inhibitors, we found that sFKN-induced heme oxygenase-1 expression was primarily mediated by activation of JNK and nuclear factor erythroid 2-related factor 2. These results suggest that sFKN secreted from glutamate-damaged neurons provides both phagocytotic and neuroprotective signals. IntroductionGlutamate toxicity is a major cause of neuronal cell death in various neurologic disorders, including ischemia, inflammation, epilepsy, and neurodegenerative diseases. Microglia, macrophage-like resident immune cells in the central nervous system, accumulate in the lesions observed in such neurodegenerative disorders as Alzheimer disease (AD) 2The abbreviations used are: ADAlzheimer diseaseDIVdays in vitroFKNfractalkineHO-1heme oxygenase-1Aβamyloid βPSphosphatidylserineTREM2triggering receptor expressed on myeloid cellsMFG-E8milk fat globule-EGF factor 8sFKNsoluble fractalkinePIpropidium iodideSnMPstannus mesoporphyrinNrf2nuclear factor erythroid 2-related factor 2pre-Glupretreated with 10 μm glutamateANOVAanalysis of variance. and Parkinson disease, where this cell type is thought to have both neurotoxic and neuroprotective properties (1Farfara D. Lifshitz V. Frenkel D. J. Cell Mol. Med. 2008; 12: 762-780Crossref PubMed Scopus (178) Google Scholar). When activated by LPS, neurotoxic microglia release large amounts of glutamate through gap junction hemichannels (2Takeuchi H. Jin S. Wang J. Zhang G. Kawanokuchi J. Kuno R. Sonobe Y. Mizuno T. Suzumura A. J. Biol. Chem. 2006; 281: 21362-21368Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar, 3Takeuchi H. Clin. Exp. Neuroimmun. 2010; 1: 12-21Crossref Scopus (54) Google Scholar), resulting in neuronal damage. On the other hand, neuroprotective microglia release neurotrophic factors and anti-inflammatory cytokines, and remove unwanted debris via phagocytosis. We have shown that microglia activated by the Toll-like receptor 9 ligand CpG attenuate oligomeric amyloid β (Aβ) neurotoxicity by producing the antioxidant enzyme heme oxygenase-1 (HO-1) and phagocytosing Aβ (4Doi Y. Mizuno T. Maki Y. Jin S. Mizoguchi H. Ikeyama M. Doi M. Michikawa M. Takeuchi H. Suzumura A. Am. J. Pathol. 2009; 175: 2121-2132Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). HO-1 expression is up-regulated by various stressors, resulting in antioxidant effects to counteract neurodegenerative disease pathophysiology (5Elbirt K.K. Bonkovsky H.L. Proc. Assoc. Am. Physicians. 1999; 111: 438-447Crossref PubMed Scopus (276) Google Scholar, 6Pappolla M.A. Chyan Y.J. Omar R.A. Hsiao K. Perry G. Smith M.A. Bozner P. Am. J. Pathol. 1998; 152: 871-877PubMed Google Scholar). Furthermore, the antioxidative effects of HO-1 are derived from induction of various anti-inflammatory responses and other cytoprotective processes (7Syapin P.J. Br. J. Pharmacol. 2008; 155: 623-640Crossref PubMed Scopus (130) Google Scholar).Microglial phagocytosis maintains neural networks by clearing neurotoxic molecules, such as Aβ and cellular debris. Microglia express cell surface receptors that regulate phagocytosis, including phosphatidylserine (PS) receptor (8Fuller A.D. Van Eldik L.J. J. Neuroimmune. Pharmacol. 2008; 3: 246-256Crossref PubMed Scopus (98) Google Scholar), triggering receptor expressed on myeloid cells 2 (TREM2) (9Takahashi K. Rochford C.D. Neumann H. J. Exp. Med. 2005; 201: 647-657Crossref PubMed Scopus (736) Google Scholar), the scavenger receptor CD36 (10Stolzing A. Grune T. FASEB J. 2004; 18: 743-745Crossref PubMed Scopus (85) Google Scholar), and the purine receptor P2Y6 (11Koizumi S. Shigemoto-Mogami Y. Nasu-Tada K. Shinozaki Y. Ohsawa K. Tsuda M. Joshi B.V. Jacobson K.A. Kohsaka S. Inoue K. Nature. 2007; 446: 1091-1095Crossref PubMed Scopus (577) Google Scholar). Microglia also produce an opsonin, milk fat globule-EGF factor 8 (MFG-E8), which mediates signaling between microglia and apoptotic cells expressing such cell surface molecules as PS (12Hanayama R. Tanaka M. Miwa K. Shinohara A. Iwamatsu A. Nagata S. Nature. 2002; 417: 182-187Crossref PubMed Scopus (1030) Google Scholar). MFG-E8 may also be involved in Aβ phagocytosis, and its expression is reportedly reduced in AD (13Boddaert J. Kinugawa K. Lambert J.C. Boukhtouche F. Zoll J. Merval R. Blanc-Brude O. Mann D. Berr C. Vilar J. Garabedian B. Journiac N. Charue D. Silvestre J.S. Duyckaerts C. Amouyel P. Mariani J. Tedgui A. Mallat Z. Am. J. Pathol. 2007; 170: 921-929Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). These observations suggest that microglia may have both neurotoxic and neuroprotective roles under physiologic or pathologic conditions.Recently, several lines of evidence have suggested that damaged neurons are not merely passive targets of microglia, but rather regulate microglial activity through cytokines, nucleotides, and chemokines (14Biber K. Neumann H. Inoue K. Boddeke H.W. Trends Neurosci. 2007; 30: 596-602Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar). Degenerating neurons also produce signaling molecules that regulate microglia-mediated phagocytosis and neuroprotection. Some of this signaling may be controlled by chemokines and chemokine receptors, which are widely expressed throughout the central nervous system (15Tran P.B. Miller R.J. Nat. Rev. Neurosci. 2003; 4: 444-455Crossref PubMed Scopus (257) Google Scholar). We hypothesized that the CX3C chemokine fractalkine (FKN; CX3CL1), which has been detected as both soluble and membrane-anchored forms, plays a pivotal role in signaling between degenerating neurons and microglia, because FKN and its receptor CX3CR1 are highly expressed in brain tissue (16Pan Y. Lloyd C. Zhou H. Dolich S. Deeds J. Gonzalo J.A. Vath J. Gosselin M. Ma J. Dussault B. Woolf E. Alperin G. Culpepper J. Gutierrez-Ramos J.C. Gearing D. Nature. 1997; 387: 611-617Crossref PubMed Scopus (570) Google Scholar), particularly in neurons and microglia (17Harrison J.K. Jiang Y. Chen S. Xia Y. Maciejewski D. McNamara R.K. Streit W.J. Salafranca M.N. Adhikari S. Thompson D.A. Botti P. Bacon K.B. Feng L. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 10896-10901Crossref PubMed Scopus (884) Google Scholar, 18Nishiyori A. Minami M. Ohtani Y. Takami S. Yamamoto J. Kawaguchi N. Kume T. Akaike A. Satoh M. FEBS Lett. 1998; 429: 167-172Crossref PubMed Scopus (293) Google Scholar, 19Maciejewski-Lenoir D. Chen S. Feng L. Maki R. Bacon K.B. J. Immunol. 1999; 163: 1628-1635PubMed Google Scholar). We previously demonstrated that FKN functions as an intrinsic inhibitor of microglial neurotoxicity (20Mizuno T. Kawanokuchi J. Numata K. Suzumura A. Brain Res. 2003; 979: 65-70Crossref PubMed Scopus (252) Google Scholar). In addition, FKN is neuroprotective in rat hippocampal neuronal cultures (21Limatola C. Lauro C. Catalano M. Ciotti M.T. Bertollini C. Di Angelantonio S. Ragozzino D. Eusebi F. J. Neuroimmunol. 2005; 166: 19-28Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Finally, FKN has been shown to enhance the clearance of apoptotic cells by macrophages (22Miksa M. Amin D. Wu R. Ravikumar T.S. Wang P. Mol. Med. 2007; 13: 553-560Crossref PubMed Scopus (89) Google Scholar).In the present study, we show that soluble FKN (sFKN) is released from glutamate-exposed neurons, resulting in MFG-E8-induced enhancement of microglial phagocytosis of neuronal debris. Moreover, this chemokine induces HO-1 expression, which suppresses glutamate neurotoxicity to promote neuronal survival.DISCUSSIONAppropriate clearance of neuronal debris by microglia is essential for the maintenance of healthy neuronal networks (36Neumann H. Kotter M.R. Franklin R.J. Brain. 2009; 132: 288-295Crossref PubMed Scopus (749) Google Scholar). Various signals from damaged neurons, including nucleotides and chemokines, must therefore recruit and activate microglia (11Koizumi S. Shigemoto-Mogami Y. Nasu-Tada K. Shinozaki Y. Ohsawa K. Tsuda M. Joshi B.V. Jacobson K.A. Kohsaka S. Inoue K. Nature. 2007; 446: 1091-1095Crossref PubMed Scopus (577) Google Scholar, 14Biber K. Neumann H. Inoue K. Boddeke H.W. Trends Neurosci. 2007; 30: 596-602Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar). Microglia respond to FKN through CX3CR1. In a previous study (20Mizuno T. Kawanokuchi J. Numata K. Suzumura A. Brain Res. 2003; 979: 65-70Crossref PubMed Scopus (252) Google Scholar), we found that FKN serves a neuroprotective function against activated microglia-induced neurotoxicity. A lack of CX3CR1 reportedly results in progressive neuronal cell death in an animal model of neurodegenerative disease (37Cardona A.E. Pioro E.P. Sasse M.E. Kostenko V. Cardona S.M. Dijkstra I.M. Huang D. Kidd G. Dombrowski S. Dutta R. Lee J.C. Cook D.N. Jung S. Lira S.A. Littman D.R. Ransohoff R.M. Nat. Neurosci. 2006; 9: 917-924Crossref PubMed Scopus (1126) Google Scholar). Despite a controversial report showing that knocking out CX3CR1 prevents neuronal loss in a mouse model of AD (38Fuhrmann M. Bittner T. Jung C.K. Burgold S. Page R.M. Mitteregger G. Haass C. LaFerla F.M. Kretzschmar H. Herms J. Nat. Neurosci. 2010; 13: 411-413Crossref PubMed Scopus (410) Google Scholar), many others have offered evidence supporting the neuroprotective roles of FKN. Plasma sFKN level are lower in patients with AD (39Kim T.S. Lim H.K. Lee J.Y. Kim D.J. Park S. Lee C. Lee C.U. Neurosci. Lett. 2008; 436: 196-200Crossref PubMed Scopus (85) Google Scholar). Thus, FKN that is released from damaged neurons may signal microglia for support, although the precise mechanisms have not yet been clarified. Interestingly, FKN cleavage is reportedly enhanced in neuropathic pain (40Milligan E.D. Sloane E.M. Watkins L.R. J. Neuroimmunol. 2008; 198: 113-120Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) and cerebral ischemia (41Dénes A. Ferenczi S. Halász J. Környei Z. Kovács K.J. J. Cereb. Blood Flow Metab. 2008; 28: 1707-1721Crossref PubMed Scopus (212) Google Scholar), and in response to apoptosis inducers (8Fuller A.D. Van Eldik L.J. J. Neuroimmune. Pharmacol. 2008; 3: 246-256Crossref PubMed Scopus (98) Google Scholar) and glutamate (21Limatola C. Lauro C. Catalano M. Ciotti M.T. Bertollini C. Di Angelantonio S. Ragozzino D. Eusebi F. J. Neuroimmunol. 2005; 166: 19-28Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 29Chapman G.A. Moores K. Harrison D. Campbell C.A. Stewart B.R. Strijbos P.J. J. Neurosci. 2000; 20: 87-91Crossref Google Scholar).In this study, we found that sFKN is released from mouse cortical neurons when they are excitotoxically damaged by glutamate. sFKN then enhances phagocytic uptake of neuronal debris by microglia. Microglial phagocytosis has been generally assessed by uptake of fluorescently labeled beads. In some reports, the SH-SY5Y and Neuro2a neuroblastoma cell lines and the BV-2 microglial cell line have been used in phagocytosis assays (8Fuller A.D. Van Eldik L.J. J. Neuroimmune. Pharmacol. 2008; 3: 246-256Crossref PubMed Scopus (98) Google Scholar, 42Hsieh C.L. Koike M. Spusta S.C. Niemi E.C. Yenari M. Nakamura M.C. Seaman W.E. J. Neurochem. 2009; 109: 1144-1156Crossref PubMed Scopus (292) Google Scholar). It is preferred, however, to use damaged neurons to assess phagocytosis of neuronal debris by microglia. Here, we developed a novel approach to assess microglial phagocytic uptake using DiI-labeled neurons. The reliability of the assay was validated by colocalization of DiI-labeled debris and the phagocytic marker Rab-7. In this assay, microglial phagocytosis of neuronal debris was markedly enhanced by 10 nm sFKN. This sFKN concentration is similar to that released from neurons after exposure to 10 μm glutamate (Fig. 1C).We then explored phagocytosis-related factors that were expressed in microglia in response to sFKN. sFKN enhanced expression of the PS receptor MFG-E8 in microglia, which is involved in clearing apoptotic cells (8Fuller A.D. Van Eldik L.J. J. Neuroimmune. Pharmacol. 2008; 3: 246-256Crossref PubMed Scopus (98) Google Scholar). We also found that MFG-E8 expression was up-regulated under excitotoxic conditions, such as those mediated by high levels of glutamate. Moreover, expression levels of the other PS receptor T cell immunoglobulin mucin domain 4, TREM2, and CD36 also dose dependently increased in response to sFKN treatment (data not shown). These molecules may also promote phagocytic activity in microglia. Thus, sFKN released from damaged neurons may provide an "eat me" signal through several different pathways. On the other hand, sFKN-treated microglia exhibited dose-dependent neuroprotective effects against glutamate-induced neurotoxicity, suggesting that sFKN functions as a "help me" signal from damaged neurons. We found that the enhanced phagocytosis and the clearance of damaged neurons by FKN-treated microglia promote survival of neurons via MFG-E8. The neuroprotection of 10 nm sFKN can be due to microglial phagocytic activity via MFG-E8.FKN is reported to activate various intracellular signaling pathways, including ERK1/2 (21Limatola C. Lauro C. Catalano M. Ciotti M.T. Bertollini C. Di Angelantonio S. Ragozzino D. Eusebi F. J. Neuroimmunol. 2005; 166: 19-28Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 32Cambien B. Pomeranz M. Schmid-Antomarchi H. Millet M.A. Breittmayer V. Rossi B. Schmid-Alliana A. Blood. 2001; 97: 2031-2037Crossref PubMed Scopus (79) Google Scholar, 33Meucci O. Fatatis A. Simen A.A. Miller R.J. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 8075-8080Crossref PubMed Scopus (320) Google Scholar), JNK (32Cambien B. Pomeranz M. Schmid-Antomarchi H. Millet M.A. Breittmayer V. Rossi B. Schmid-Alliana A. Blood. 2001; 97: 2031-2037Crossref PubMed Scopus (79) Google Scholar), p38 (32Cambien B. Pomeranz M. Schmid-Antomarchi H. Millet M.A. Breittmayer V. Rossi B. Schmid-Alliana A. Blood. 2001; 97: 2031-2037Crossref PubMed Scopus (79) Google Scholar, 43Clark A.K. D'Aquisto F. Gentry C. Marchand F. McMahon S.B. Malcangio M. J. Neurochem. 2006; 99: 868-880Crossref PubMed Scopus (86) Google Scholar), and PI3K (19Maciejewski-Lenoir D. Chen S. Feng L. Maki R. Bacon K.B. J. Immunol. 1999; 163: 1628-1635PubMed Google Scholar, 33Meucci O. Fatatis A. Simen A.A. Miller R.J. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 8075-8080Crossref PubMed Scopus (320) Google Scholar, 44Lyons A. Lynch A.M. Downer E.J. Hanley R. O'Sullivan J.B. Smith A. Lynch M.A. J. Neurochem. 2009; 110: 1547-1556Crossref PubMed Scopus (140) Google Scholar). Among these pathways, PI3K and p38 MAPK signaling mediates the toxic activities of microglia through production of certain proinflammatory cytokines. We examined signaling pathways that may have been involved in sFKN-induced microglial neuroprotection using the microglial cell line BV-2; with the aid of specific MAPK inhibitors, we demonstrated that sFKN acts through ERK and JNK MAPK, but not through p38 MAPK. Moreover, we showed that JNK MAPK signaling drives the expression of the antioxidant enzyme HO-1 via Nrf2 nuclear translocation. Nrf2 is an important transcription factor for the expression of genes encoding phase II detoxifying and antioxidant enzymes (45Alam J. Cook J.L. Am. J. Respir. Cell Mol. Biol. 2007; 36: 166-174Crossref PubMed Scopus (294) Google Scholar). The subcellular localization of Nrf2 is a key regulator of HO-1 expression in response to various stress stimuli (34Surh Y.J. Kundu J.K. Li M.H. Na H.K. Cha Y.N. Arch. Pharm. Res. 2009; 32: 1163-1176Crossref PubMed Scopus (120) Google Scholar, 46Naidu S. Vijayan V. Santoso S. Kietzmann T. Immenschuh S. J. Immunol. 2009; 182: 7048-7057Crossref PubMed Scopus (97) Google Scholar). Nuclear translocation and phosphorylation of Nrf2 are induced by ERK1/2, p38, and PI3K (34Surh Y.J. Kundu J.K. Li M.H. Na H.K. Cha Y.N. Arch. Pharm. Res. 2009; 32: 1163-1176Crossref PubMed Scopus (120) Google Scholar). Our study showed that sFKN enhances the nuclear translocation of Nrf2 via activation of JNK MAPK in microglia. HO-1, a member of the heat shock protein family, is a lysosomal enzyme that oxidatively cleaves heme to produce biliverdin, carbon monoxide, and iron. This enzyme also helps to reduce oxidative stress-induced factors, such as reactive oxygen species. Numerous studies have demonstrated that up-regulation of HO-1 expression in the central nervous system may be beneficial to counteract neuroinflammation and neurodegenerative diseases (7Syapin P.J. Br. J. Pharmacol. 2008; 155: 623-640Crossref PubMed Scopus (130) Google Scholar).The various MAPK pathways operate independently of each other (47Takekawa M. Tatebayashi K. Saito H. Mol. Cell. 2005; 18: 295-306Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). In this study, only JNK MAPK was involved in HO-1 expression through Nrf2 nuclear translocation. ERK signaling was also involved in the neuroprotective effects of sFKN. ERK contributes to cellular survival, proliferation, and differentiation through phosphorylation of a number of transcription factors, including cAMP response element-binding protein, activating transcription factor 1, and Myc (48Chang F. Steelman L.S. Lee J.T. Shelton J.G. Navolanic P.M. Blalock W.L. Franklin R.A. McCubrey J.A. Leukemia. 2003; 17: 1263-1293Crossref PubMed Scopus (593) Google Scholar). Further studies are needed to clarify the neuroprotective mechanism of the ERK pathway. Nevertheless, the neuroprotective effects of sFKN may be mediated by both the JNK-Nrf2-HO-1 and ERK signaling pathways.Our data are consistent with the scheme shown in Fig. 8. Mild to moderate neuronal damage induced by glutamate enhances sFKN release from cellular membranes, and sFKN induces the expression of MFG-E8, JNK-Nrf2-HO-1, and ERK in microglia through CX3CR1. MFG-E8 enhances the phagocytic uptake of neuronal debris and thereby promotes neuronal survival, and JNK-Nrf2-HO-1 exerts antioxidant effects. Expression of MFG-E8, which is involved in Aβ phagocytosis by macrophages, is reduced in AD brains (13Boddaert J. Kinugawa K. Lambert J.C. Boukhtouche F. Zoll J. Merval R. Blanc-Brude O. Mann D. Berr C. Vilar J. Garabedian B. Journiac N. Charue D. Silvestre J.S. Duyckaerts C. Amouyel P. Mariani J. Tedgui A. Mallat Z. Am. J. Pathol. 2007; 170: 921-929Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar).Furthermore, Nrf2 gene therapy has been shown to improve memory in the mouse model of AD (49Kanninen K. Heikkinen R. Malm T. Rolova T. Kuhmonen S. Leinonen H. Ylä-Herttuala S. Tanila H. Levonen A.L. Koistinaho M. Koistinaho J. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 16505-16510Crossref PubMed Scopus (242) Google Scholar). Therefore, FKN may prevent some of the pathogenic processes that underlie AD. Our study also suggests that the FKN-CX3CR1 signaling axis is crucial for healthy neuronal networks, and may provide a pharmacotherapeutic target in the treatment of neurodegenerative diseases. IntroductionGlutamate toxicity is a major cause of neuronal cell death in various neurologic disorders, including ischemia, inflammation, epilepsy, and neurodegenerative diseases. Microglia, macrophage-like resident immune cells in the central nervous system, accumulate in the lesions observed in such neurodegenerative disorders as Alzheimer disease (AD) 2The abbreviations used are: ADAlzheimer diseaseDIVdays in vitroFKNfractalkineHO-1heme oxygenase-1Aβamyloid βPSphosphatidylserineTREM2triggering receptor expressed on myeloid cellsMFG-E8milk fat globule-EGF factor 8sFKNsoluble fractalkinePIpropidium iodideSnMPstannus mesoporphyrinNrf2nuclear factor erythroid 2-related factor 2pre-Glupretreated with 10 μm glutamateANOVAanalysis of variance. and Parkinson disease, where this cell type is thought to have both neurotoxic and neuroprotective properties (1Farfara D. Lifshitz V. Frenkel D. J. Cell Mol. Med. 2008; 12: 762-780Crossref PubMed Scopus (178) Google Scholar). When activated by LPS, neurotoxic microglia release large amounts of glutamate through gap junction hemichannels (2Takeuchi H. Jin S. Wang J. Zhang G. Kawanokuchi J. Kuno R. Sonobe Y. Mizuno T. Suzumura A. J. Biol. Chem. 2006; 281: 21362-21368Abstract Full Text Full Text PDF PubMed Scopus (570) Google Scholar, 3Takeuchi H. Clin. Exp. Neuroimmun. 2010; 1: 12-21Crossref Scopus (54) Google Scholar), resulting in neuronal damage. On the other hand, neuroprotective microglia release neurotrophic factors and anti-inflammatory cytokines, and remove unwanted debris via phagocytosis. We have shown that microglia activated by the Toll-like receptor 9 ligand CpG attenuate oligomeric amyloid β (Aβ) neurotoxicity by producing the antioxidant enzyme heme oxygenase-1 (HO-1) and phagocytosing Aβ (4Doi Y. Mizuno T. Maki Y. Jin S. Mizoguchi H. Ikeyama M. Doi M. Michikawa M. Takeuchi H. Suzumura A. Am. J. Pathol. 2009; 175: 2121-2132Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). HO-1 expression is up-regulated by various stressors, resulting in antioxidant effects to counteract neurodegenerative disease pathophysiology (5Elbirt K.K. Bonkovsky H.L. Proc. Assoc. Am. Physicians. 1999; 111: 438-447Crossref PubMed Scopus (276) Google Scholar, 6Pappolla M.A. Chyan Y.J. Omar R.A. Hsiao K. Perry G. Smith M.A. Bozner P. Am. J. Pathol. 1998; 152: 871-877PubMed Google Scholar). Furthermore, the antioxidative effects of HO-1 are derived from induction of various anti-inflammatory responses and other cytoprotective processes (7Syapin P.J. Br. J. Pharmacol. 2008; 155: 623-640Crossref PubMed Scopus (130) Google Scholar).Microglial phagocytosis maintains neural networks by clearing neurotoxic molecules, such as Aβ and cellular debris. Microglia express cell surface receptors that regulate phagocytosis, including phosphatidylserine (PS) receptor (8Fuller A.D. Van Eldik L.J. J. Neuroimmune. Pharmacol. 2008; 3: 246-256Crossref PubMed Scopus (98) Google Scholar), triggering receptor expressed on myeloid cells 2 (TREM2) (9Takahashi K. Rochford C.D. Neumann H. J. Exp. Med. 2005; 201: 647-657Crossref PubMed Scopus (736) Google Scholar), the scavenger receptor CD36 (10Stolzing A. Grune T. FASEB J. 2004; 18: 743-745Crossref PubMed Scopus (85) Google Scholar), and the purine receptor P2Y6 (11Koizumi S. Shigemoto-Mogami Y. Nasu-Tada K. Shinozaki Y. Ohsawa K. Tsuda M. Joshi B.V. Jacobson K.A. Kohsaka S. Inoue K. Nature. 2007; 446: 1091-1095Crossref PubMed Scopus (577) Google Scholar). Microglia also produce an opsonin, milk fat globule-EGF factor 8 (MFG-E8), which mediates signaling between microglia and apoptotic cells expressing such cell surface molecules as PS (12Hanayama R. Tanaka M. Miwa K. Shinohara A. Iwamatsu A. Nagata S. Nature. 2002; 417: 182-187Crossref PubMed Scopus (1030) Google Scholar). MFG-E8 may also be involved in Aβ phagocytosis, and its expression is reportedly reduced in AD (13Boddaert J. Kinugawa K. Lambert J.C. Boukhtouche F. Zoll J. Merval R. Blanc-Brude O. Mann D. Berr C. Vilar J. Garabedian B. Journiac N. Charue D. Silvestre J.S. Duyckaerts C. Amouyel P. Mariani J. Tedgui A. Mallat Z. Am. J. Pathol. 2007; 170: 921-929Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). These observations suggest that microglia may have both neurotoxic and neuroprotective roles under physiologic or pathologic conditions.Recently, several lines of evidence have suggested that damaged neurons are not merely passive targets of microglia, but rather regulate microglial activity through cytokines, nucleotides, and chemokines (14Biber K. Neumann H. Inoue K. Boddeke H.W. Trends Neurosci. 2007; 30: 596-602Abstract Full Text Full Text PDF PubMed Scopus (572) Google Scholar). Degenerating neurons also produce signaling molecules that regulate microglia-mediated phagocytosis and neuroprotection. Some of this signaling may be controlled by chemokines and chemokine receptors, which are widely expressed throughout the central nervous system (15Tran P.B. Miller R.J. Nat. Rev. Neurosci. 2003; 4: 444-455Crossref PubMed Scopus (257) Google Scholar). We hypothesized that the CX3C chemokine fractalkine (FKN; CX3CL1), which has been detected as both soluble and membrane-anchored forms, plays a pivotal role in signaling between degenerating neurons and microglia, because FKN and its receptor CX3CR1 are highly expressed in brain tissue (16Pan Y. Lloyd C. Zhou H. Dolich S. Deeds J. Gonzalo J.A. Vath J. Gosselin M. Ma J. Dussault B. Woolf E. Alperin G. Culpepper J. Gutierrez-Ramos J.C. Gearing D. Nature. 1997; 387: 611-617Crossref PubMed Scopus (570) Google Scholar), particularly in neurons and microglia (17Harrison J.K. Jiang Y. Chen S. Xia Y. Maciejewski D. McNamara R.K. Streit W.J. Salafranca M.N. Adhikari S. Thompson D.A. Botti P. Bacon K.B. Feng L. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 10896-10901Crossref PubMed Scopus (884) Google Scholar, 18Nishiyori A. Minami M. Ohtani Y. Takami S. Yamamoto J. Kawaguchi N. Kume T. Akaike A. Satoh M. FEBS Lett. 1998; 429: 167-172Crossref PubMed Scopus (293) Google Scholar, 19Maciejewski-Lenoir D. Chen S. Feng L. Maki R. Bacon K.B. J. Immunol. 1999; 163: 1628-1635PubMed Google Scholar). We previously demonstrated that FKN functions as an intrinsic inhibitor of microglial neurotoxicity (20Mizuno T. Kawanokuchi J. Numata K. Suzumura A. Brain Res. 2003; 979: 65-70Crossref PubMed Scopus (252) Google Scholar). In addition, FKN is neuroprotective in rat hippocampal neuronal cultures (21Limatola C. Lauro C. Catalano M. Ciotti M.T. Bertollini C. Di Angelantonio S. Ragozzino D. Eusebi F. J. Neuroimmunol. 2005; 166: 19-28Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Finally, FKN has been shown to enhance the clearance of apoptotic cells by macrophages (22Miksa M. Amin D. Wu R. Ravikumar T.S. Wang P. Mol. Med. 2007; 13: 553-560Crossref PubMed Scopus (89) Google Scholar).In the present study, we show that soluble FKN (sFKN) is released from glutamate-exposed neurons, resulting in MFG-E8-induced enhancement of microglial phagocytosis of neuronal debris. Moreover, this chemokine induces HO-1 expression, which suppresses glutamate neurotoxicity to promote neuronal survival.
Referência(s)