MAPK-activated Protein Kinase 2 Deficiency in Microglia Inhibits Pro-inflammatory Mediator Release and Resultant Neurotoxicity
2006; Elsevier BV; Volume: 281; Issue: 33 Linguagem: Inglês
10.1074/jbc.m513646200
ISSN1083-351X
AutoresAinsley A. Culbert, Stephen D. Skaper, David Howlett, Nicholas A. Evans, Laura Facci, Peter E. Soden, Z Seymour, Florence Guillot, Matthias Gaestel, Jill Richardson,
Tópico(s)Melanoma and MAPK Pathways
ResumoMAPK-activated protein kinase 2 (MAPKAP K2 or MK2) is one of several kinases directly regulated by p38 MAPK. A role for p38 MAPK in the pathology of Alzheimer disease (AD) has previously been suggested. Here, we provide evidence to suggest that MK2 also plays a role in neuroinflammatory and neurodegenerative pathology of relevance to AD. MK2 activation and expression were increased in lipopolysaccharide (LPS) + interferon γ-stimulated microglial cells, implicating a role for MK2 in eliciting a pro-inflammatory response. Microglia cultured ex vivo from MK2-deficient (MK2–/–) mice demonstrated significant inhibition in release of tumor necrosis factor α, KC (mouse chemokine with highest sequence identity to human GROs and interleukin-8), and macrophage inflammatory protein 1α on stimulation with LPS + interferon γ or amyloid-β peptide (1–42) compared with MK2+/+ wild-type microglia. Consistent with an inhibition in pro-inflammatory mediator release, cortical neurons co-cultured with LPS + interferon γ-stimulated or amyloid-β peptide (1–42)-stimulated MK2–/– microglia were protected from microglial-mediated neuronal cell toxicity. In a transgenic mouse model of AD in which amyloid precursor protein and presenilin-1 harboring familial AD mutations are overexpressed in specific regions of the brain, elevated activation and expression of MK2 correlated with β-amyloid deposition, microglial activation, and up-regulation of tumor necrosis factor α, macrophage inflammatory protein 1α, and KC gene expression in the same brain regions. Our data propose a role for MK2 in AD brain pathology, for which neuroinflammation involving cytokines and chemokines and overt neuronal loss have been documented. MAPK-activated protein kinase 2 (MAPKAP K2 or MK2) is one of several kinases directly regulated by p38 MAPK. A role for p38 MAPK in the pathology of Alzheimer disease (AD) has previously been suggested. Here, we provide evidence to suggest that MK2 also plays a role in neuroinflammatory and neurodegenerative pathology of relevance to AD. MK2 activation and expression were increased in lipopolysaccharide (LPS) + interferon γ-stimulated microglial cells, implicating a role for MK2 in eliciting a pro-inflammatory response. Microglia cultured ex vivo from MK2-deficient (MK2–/–) mice demonstrated significant inhibition in release of tumor necrosis factor α, KC (mouse chemokine with highest sequence identity to human GROs and interleukin-8), and macrophage inflammatory protein 1α on stimulation with LPS + interferon γ or amyloid-β peptide (1–42) compared with MK2+/+ wild-type microglia. Consistent with an inhibition in pro-inflammatory mediator release, cortical neurons co-cultured with LPS + interferon γ-stimulated or amyloid-β peptide (1–42)-stimulated MK2–/– microglia were protected from microglial-mediated neuronal cell toxicity. In a transgenic mouse model of AD in which amyloid precursor protein and presenilin-1 harboring familial AD mutations are overexpressed in specific regions of the brain, elevated activation and expression of MK2 correlated with β-amyloid deposition, microglial activation, and up-regulation of tumor necrosis factor α, macrophage inflammatory protein 1α, and KC gene expression in the same brain regions. Our data propose a role for MK2 in AD brain pathology, for which neuroinflammation involving cytokines and chemokines and overt neuronal loss have been documented. The role of p38 mitogen-activated protein kinase (MAPK) 2The abbreviations used are: MAPK, mitogen-activated protein kinase; MK2, MAPK-activated protein kinase 2; AD, Alzheimer disease; CNS, central nervous system; LPS, lipopolysaccharide; IFNγ, interferon γ; TNF-α, tumor necrosis factor-α; MIP-1α, macrophage inflammatory protein 1α; WT, wild type; Aβ, β-amyloid protein; FCS, fetal calf serum; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; RT, reverse transcriptase; IL, interleukin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 2The abbreviations used are: MAPK, mitogen-activated protein kinase; MK2, MAPK-activated protein kinase 2; AD, Alzheimer disease; CNS, central nervous system; LPS, lipopolysaccharide; IFNγ, interferon γ; TNF-α, tumor necrosis factor-α; MIP-1α, macrophage inflammatory protein 1α; WT, wild type; Aβ, β-amyloid protein; FCS, fetal calf serum; LDH, lactate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; RT, reverse transcriptase; IL, interleukin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. in the regulation of cytokine biosynthesis is now well estab lished (1Han J. Lee J.D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2390) Google Scholar, 2Lee J.C. Laydon J.T. McDonnell P.C. Gallagher T.F. Kumar S. Green D. McNulty D. Blumenthal M.J. Heys J.R. Landvatter S.W. Strickler J.E. McLaughlin M.M. Siemens I.R. Fisher S.M. Livi G.P. White J.R. Adams J.L. Young P.R. Nature. 1994; 372: 739-746Crossref PubMed Scopus (3113) Google Scholar). MAPK-activated protein kinase 2 (MK2) is one of several kinases that are regulated through direct phosphorylation by p38 MAPK, and MK2 has therefore been a candidate for an effector role in p38 action in the inflammatory response. Studies using MK2–/– mice support this hypothesis, because mice lacking MK2 are resistant to endotoxic shock, largely due to a reduction in serum tumor necrosis factor-α (TNF-α) levels (3Kotlyarov A. Neininger A. Schubert C. Eckert R. Birchmeier C. Volk H.D. Gaestel M. Nat. Cell Biol. 1999; 1: 94-97Crossref PubMed Scopus (675) Google Scholar). Furthermore, macrophages and spleen cells taken from MK2-deficient animals show inhibition in the release of a range of pro-inflammatory mediators (3Kotlyarov A. Neininger A. Schubert C. Eckert R. Birchmeier C. Volk H.D. Gaestel M. Nat. Cell Biol. 1999; 1: 94-97Crossref PubMed Scopus (675) Google Scholar, 4Kotlyarov A. Gaestel M. Biochem. Soc. Trans. 2002; 30: 959-963Crossref PubMed Scopus (76) Google Scholar). Alzheimer disease (AD) is a neurodegenerative disorder that affects primarily hippocampal and neocortical brain regions resulting in a progressive loss of cognitive and memory function and ultimately dementia. Post-mortem diagnosis of AD is facilitated by the presence of extracellular plaques comprising β-amyloid protein (Aβ), and neuroinflammation is a persistent pathological hallmark. Microglia, the resident macrophages of the brain, are responsible for eliciting such an immune response in the CNS. These immune cells of monocytic lineage are activated by a wide range of stimuli and release pro-inflammatory mediators that, in turn, induce microglial autoactivation, thereby amplifying the inflammatory response. It has been proposed that elevated levels of β-amyloid in AD brain induces microglial activation and consequent release of pro-inflammatory cytokines, chemokines, and other potentially neurotoxic substances (5McGeer P.L. McGeer E.G. Prog. Neuropsychopharmacol. Biol. Psychiatry. 2003; 5: 741-749Crossref Scopus (460) Google Scholar, 6McGeer P.L. McGeer E.G. Neurobiol. Aging. 2001; 22: 799-809Crossref PubMed Scopus (435) Google Scholar, 7Akiyama H. Barger S. Barnum S. Bradt B. Bauer J. Cole G.M. Cooper N.R. Eikelenboom P. Emmerling M. Fiebich B. Finch C.E. Frautschy S. Griffin W.S.T. Hampel H. Hull M. Landreth G. Lue L.F. Mrak R. Mackenzie I.R. McGeer P.L. O'Banion M.K. Pachter J. Pasinetti G. Plata-Salaman C. Rogers J. Rydel R. Shen Y. Streit W. Strohmeyer R. Tooyama I. Van Muiswinkel F.L. Veerhuis R. Walker D. Webster S. Wegrzyniak B. Wenk G. Wyss-Coray T. Neurobiol. Aging. 2000; 21: 383-421Crossref PubMed Scopus (3585) Google Scholar, 8Eikelenboom P. Bate C. Van Gool W.A. Hoozemans J.J. Rozemuller J.M. Veerhuis R. Williams A. Glia. 2002; 40: 232-239Crossref PubMed Scopus (366) Google Scholar, 9Rogers J. Strohmeyer R. Kovelowski C.J. Li R. Glia. 2002; 40: 260-269Crossref PubMed Scopus (331) Google Scholar). Indeed, activated microglia can cause neuronal cell death in vitro (10Chao C.C. Hu S. Molitor T.W. Shaskan E.G. Peterson P.K. J. Immunol. 1992; 149: 2736-2741PubMed Google Scholar, 11Flavin M.P. Zhao G. Ho L.T. Glia. 2000; 29: 347-354Crossref PubMed Scopus (96) Google Scholar, 12Golde S. Chandran S. Brown G.C. Compston A. J. Neurochem. 2002; 82: 269-282Crossref PubMed Scopus (74) Google Scholar, 13Parvathenani L.K. Tertyshnikova S. Greco C.R. Roberts S.B. Robertson B. Posmantur R. J. Biol. Chem. 2003; 278: 13309-13317Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). The neuroinflammatory changes and microglial activation observed in AD pathology are therefore hypothesized to contribute to neuronal cell loss and associated dementia in this disease, as well as neuronal injury and neurodegeneration in other CNS disorders resulting from trauma, ischemia, or inflammation (14Diemel L.T. Copelman C.A. Cuznre M.L. Neurochem. Res. 1998; 23: 341-347Crossref PubMed Scopus (72) Google Scholar, 15Giulian D. Am. J. Hum. Genet. 1999; 65: 13-18Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar, 16Kaul M. Garden G.A. Lipson S.A. Nature. 2001; 401: 988-994Crossref Scopus (1050) Google Scholar). Modulation of the inflammatory response may retard the progression of AD through a reduction in neurodegeneration caused by chronic activation of microglia (17McGeer P.L. Schulzer M. McGeer E.G. Neurology. 1996; 47: 425-432Crossref PubMed Scopus (1280) Google Scholar, 18Stewart W.F. Kawas C. Corrada M. Metter E.J. Neurology. 1997; 48: 626-632Crossref PubMed Scopus (1036) Google Scholar, 19Zandi P.P. Anthony J.C. Hayden K.M. Mehta K. Mayer L. Breitner J.C.S. Neurology. 2002; 59: 880-886Crossref PubMed Scopus (301) Google Scholar). Identification of regulators of inflammation relevant to AD pathology is therefore of great therapeutic importance. Activation of the p38 MAPK pathway has been linked to inflammatory pathology both in AD and in mouse models of the disease (20Hensley K. Floyd R.A. Zheng N.-Y. Nael R. Robinson X.N. Pye Q.N. Stewart C.A. Geddes J. Markesbery W.R. Patel E. Johnson G.V.W. Bing G. J. Neurochem. 1999; 72: 2053-2058Crossref PubMed Scopus (311) Google Scholar, 21Giovannini M.G. Scali C. Prosperi C. Bellucci A. Vannucchi M.G. Rosi S. Pepeu G. Casamenti F. Neurobiol. Dis. 2002; 11: 257-274Crossref PubMed Scopus (198) Google Scholar, 22Koistinaho M. Kettunen M.I. Goldsteins G. Keinanen R. Salminen A. Ort M. Bures J. Liu D. Kauppinen R.A. Higgins L.S. Koistinaho J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 1610-1615Crossref PubMed Scopus (152) Google Scholar, 23Hwang D.Y. Cho J.S. Lee S.H. Chae K.R. Lim H.J. Min S.H. Seo S.J. Song Y.S. Song C.W. Paik S.G. Sheen Y.Y. Kim Y.K. Exp. Neurol. 2004; 186: 20-32Crossref PubMed Scopus (60) Google Scholar). In addition, Aβ activates p38 MAPK in cultured microglial cells (24Atzori C. Ghetti B. Piva R. Srinivasan A.N. Zolo P. Delisle M.B. Mirra S.S. Migheli A. J. Neuropathol. Exp. Neurol. 2001; 60: 1190-1197Crossref PubMed Scopus (147) Google Scholar). Because MK2 is an immediate downstream kinase of p38 MAPK, we hypothesized that MK2 itself might play a role in neuroinflammation and neurodegeneration relevant to AD. Here we have investigated the role of MK2 in microglial cell activation, and the resultant effects on microglial-mediated neuronal cell toxicity. In addition, we have correlated our findings in vitro with a potential role for MK2 in a transgenic mouse model of AD. Materials and Reagents—Tissue culture media, B27 supplements, antibiotics, fetal calf serum (FCS), and recombinant mouse and rat interferon-γ (IFNγ) were purchased from Invitrogen; lipopolysaccharide (LPS; Escherichia coli 026:B6) and 1,1,1,3,3,3-hexafluoro-2-propanol were from Sigma; tissue culture plasticware was from Nunc (Roskilde, Denmark). TRIzol reagent was from Invitrogen. Reagents for reverse transcription were from Invitrogen and mouse and rat genomic DNA were from Clontech (Palo Alto, CA). TaqMan PCR universal master mixture was from Applied Biosystems (Warrington, UK). Antibodies for immunohistochemistry were to phosphotyrosine (mouse monoclonal sc-7020, Santa Cruz) and Aβ40 (G30 rabbit polyclonal raised against CMVG-GVV and showing C-terminal specificity for Aβ40). Antibodies for imaging by Cellomics Arrayscan technology were to βIII-tubulin (TUJI mAb, Covance Research Products, Princeton, NJ) and Alexa 488-labeled secondary antibody was from Molecular Probes (Leiden, Netherlands). Antibodies for Western blotting to p38 MAPK, phospho-p38 MAPK (Thr180/Tyr182), and MK2 were from Cell Signaling Technology. The MK2 immunoprecipitation kinase assay kit was purchased from Upstate Cell Signaling (Milton Keynes, UK) and the enhanced chemiluminescence (ECL) kit was from Amersham Biosciences. Chemokine and cytokine enzyme-linked immunosorbent assay (ELISA) duoset kits were from R & D Systems (Abingdon, UK). Aβ(1–42) peptide was purchased from California Peptide Research (Napa, CA). CellTiter 96 Non-Radioactive Cell Proliferation Assay (MTT) and CytoTox® non-radioactive cytotoxicity assay kit (lactate dehydrogenase, LDH assay) were from Promega (Southampton, UK). Transgenic Mice—TASTPM mice, which are transgenic mice overexpressing the 695-amino acid isoform of human amyloid precursor protein (APP695) harboring the Swedish double familial Alzheimer disease mutation (K670N,M671L) and human presenilin-1 harboring the familial mutation M146V were generated and maintained at GlaxoSmithKline as described in detail previously (25Richardson J.C. Kendal C.E. Anderson R. Priest F. Gower E. Soden P. Gray R. Topps S. Howlett D.R. Lavendar D. Clarke N.J. Barnes J.C. Haworth R. Stewart M.G. Rupniak H.T.R. Neurosci. 2003; 122: 213-228Crossref PubMed Scopus (82) Google Scholar, 26Howlett D.R. Richardson J.C. Austin A. Parsons A.A. Bate S.T. Davies D.C. Gonzalez M.I. Brain Res. 2004; 1017: 130-136Crossref PubMed Scopus (166) Google Scholar). In this double transgenic line, both transgenes are under the control of the Thy-1 regulatory cassette, which directs expression to the cerebral cortex, amygdala, dentate gyrus, CA1–CA3, and the cerebellum. Expression of transgenes results in progressive amyloid deposition and pathology in the brain. For ex vivo tissue analyses, animals were humanely sacrificed by intraperitoneal injection of a lethal dose of pentobarbital sodium (Euthatal, Rhone Merieux, Harlow, UK). MK2–/– mice were generated in the laboratory of Professor Matthias Gaestel (Germany) and provided to GSK on a mixed 129v × C57BL/6 background as described previously (3Kotlyarov A. Neininger A. Schubert C. Eckert R. Birchmeier C. Volk H.D. Gaestel M. Nat. Cell Biol. 1999; 1: 94-97Crossref PubMed Scopus (675) Google Scholar). Microglia were cultured ex vivo from wild-type (WT) and MK2–/– mice as described below. Culture of NTW8 Microglial Cells—Mouse microglial NTW8 cells were cultured as previously described (27Chessell I.P. Michel A.D. Humphrey P.P.A. Br. J. Pharmacol. 1997; 121: 1429-1437Crossref PubMed Scopus (113) Google Scholar). Cells were switched to serum-free medium 2 h prior to cell stimulation with LPS and IFNγ, or Aβ(1–42) peptide. In Vitro Kinase Assay—Lysates prepared either from NTW8 microglial cells or cortical CNS tissues from WT and TASTPM mice were assayed for MK2 activity using the MK2 immunoprecipitation kinase assay kit (Upstate Cell Signaling) according to the manufacturer's instructions. In brief, lysates were prepared from NTW8 microglial cells by washing cell monolayers in ice-cold PBS, followed by extraction in the lysis buffer supplied. For mouse cortical tissue, dissected tissue was powdered under liquid nitrogen and protein extracts were prepared as described previously (28Cross D.A.E. Methods Mol. Biol. 2001; 124: 147-159PubMed Google Scholar). All lysates were clarified by centrifugation, and MK2 was immunoprecipitated from samples containing 1 mg of total cellular protein. Kinase reactions were performed by incubating immunoprecipitated MK2 at 30 °C for 45 min with 10 ng of recombinant Hsp27 in the kinase assay buffer provided. Phosphorylation of Hsp27 was used as a measure of MK2 activity; phospho-Hsp27 and immunoprecipitated MK2 was resolved by SDS-PAGE followed by Western blot analysis. Blots were developed using ECL and quantified where shown by densitometry. Quantitative RT-PCR—Total RNA was isolated from CNS tissues from WT and TASTPM mice, rat primary CNS cells, and NTW8 microglial cells using TRIzol reagent according to the manufacturer's instructions. First strand cDNA syntheses from equal quantities of RNA and aliquoting of the resulting cDNA products for subsequent parallel TaqMan PCR were all performed as described in detail previously (29Ginham R. Harrison D.C. Facci L. Skaper S.D. Philpott K.L. Neurosci. Lett. 2001; 302: 113-116Crossref PubMed Scopus (26) Google Scholar). Additional reactions were performed using genomic DNA to produce a standard curve relating threshold cycle to template copy number. Primer (F and R) and probe (P) sets were designed from mouse or rat sequences in the GenBank™ data base using Primer Express software (PerkinElmer Life Sciences); mouse GAPDH (F) 5′-GAACATCATCCCTGCATCCA-3′, (R) 5′-CCAGTGAGCTTCCCGTTCA-3′, and (P) 5′-CTTGCCCACAGCCTTGGCAGC-3′; mouse MIP-1α (F) 5′-AGCTGACACCCCGACTGC-3′, (R) 5′-GTCAACGATGAATTGGCGTG-3′, and (P) 5′-TGCTGCTTCTCCTACAGCCGGAAGAT-3′; mouse TNFα (F) 5′-TCCAGGCGGTGCCTATGT-3′, (R) 5′-GAGCGTGGTGGCCCC-3′, and (P) 5′-TCAGCCTCTTCTCATTCCTGCTTGTGG-3′; mouse MK2 (F) 5′-CACCCCTGGATCATGCAATC-3′, (R) 5′-CCTTCAGGACACGGCTGGT-3′, and (P) 5′-CGAAGGTCCCTCAGACTCCACTGCA-3′; rat MK2 (F) 5′-CACCCGTGGATCATGCAA-3′, (R) 5′-TCAGGACGCGGCTGGT-3′, and (P) 5′-CGAAGGTCCCTCAGACTCCACTGCA-3′; mouse KC (F) 5′-GCGCCTATCGCCAATGAG-3′, (R) 5′-CTTGAGGTGAATCCCAGCCAT-3′, and (P) 5′-TGCGCTGTCAGTGCCTGCAGAC-3′. All TaqMan probes contained 6-FAM at the 5′ end and the quencher dye, 6-carboxytetramethylrhodamine at the 3′ end. Immunohistochemistry Staining for β-Amyloid Protein and Activated Microglia—Brains from TASTPM mice were hemisected in the sagittal plane and immersion-fixed in 4% paraformaldehyde in 0.1 m phosphate-buffered saline (PBS), pH 7.4, for 48 h. The brain was then processed into paraffin wax and 7-μm sections were cut and subjected to standard immunohistochemical techniques. Briefly, the sections were de-waxed, re-hydrated through a series of graded alcohols, and washed in distilled water followed by PBS. To enhance antigenicity for phosphotyrosine and Aβ, sections were treated by microwaving in 0.01 m citrate buffer, pH 6.0 (2 × 5 min at 300 watts), followed by immersion in 80% formic acid for 8 min. Sections were then incubated in 0.3% H2O2 in PBS for 30 min at room temperature to quench endogenous peroxidase activity. To enable labeling of both Aβ plaques and microglia, sections were incubated overnight at 4 °C with both the rabbit polyclonal G30 (3 μg/ml) and the mouse monoclonal to phosphotyrosine (1:1000) diluted in primary layer diluent (0.3% Triton X-100, 0.01% sodium azide, and 2% normal serum in PBS). Immunohistochemistry was completed with appropriate secondary biotinylated antibodies (Vector Laboratories Ltd., Peterborough, UK) diluted 1:500 in secondary layer diluent (0.3% Triton X-100 in PBS), followed by avidin-biotin complexation (Vector ABC, Vector Laboratories Ltd., Peterborough, UK) and visualized using diaminobenzidine (for phosphotyrosine) and VIP (for Aβ40) according to the manufacturer's data sheets (Vector Laboratories Ltd.). Isolation of Primary Microglia—Rat microglia were prepared from cerebral cortices of 1–2-day-old rat pups of either sex (strain: CD, Charles River, Margate, UK) (30Rosin C. Bates T.E. Skaper S.D. J. Neurochem. 2004; 90: 1173-1185Crossref PubMed Scopus (69) Google Scholar). Animals were euthanaized in accordance with the 1986 Animals (Scientific Procedures) Act. The final cell pellet from cortex was resuspended in Dulbecco's modified Eagle's medium (high glucose Dulbecco's modified Eagle's medium with l-glutamine) supplemented with 10% FCS and 100 units/ml penicillin + 50 μg/ml streptomycin, and plated in 75-cm2 poly-l-lysine-coated tissue culture flasks (Corning, Corning, NY) at a density of 1.5 brains per flask. Culture medium was changed after 24 h and then twice per week. Microglia were isolated on day 14 by shaking the flasks on an orbital shaker (New Brunswick Scientific) at 200 rpm for 2 h (37 °C). The attached cell monolayers were highly enriched in astrocytes. The culture supernatant was transferred to plastic Petri dishes (Sterilin) and incubated for 45 min at 37 °C (5% CO2, 95% air) to allow differential adhesion of microglia. The adherent microglial cells were maintained in growth medium until harvested for addition to neuronal cell cultures (3–5 days later). Microglial cell cultures from genotypically matched normal and homozygous MK2–/– mice were also prepared. Mouse microglia were isolated from mixed glial cell cultures as described above for rat, except that animals were used at postnatal days 7–8 (to obtain greater cell yields). Harvested mouse microglia were used both for cytokine release assays, and in co-cultures of rat cortical neurons and mouse microglia. Co-cultures utilizing mouse microglia were established in the same manner as microglia derived from rat cortex. Cytokine/Chemokine Release Assays from Primary Mouse Microglia and NTW8 Mouse Microglial Cells—Microglia were plated in wells of a 96-well plate (poly-d-lysine, BD Biosciences) at a density of 100,000 cells per well and allowed to adhere overnight. Cells were stimulated to release pro-inflammatory mediators in medium containing 100 ng/ml LPS and 20 units/ml mouse IFNγ, or 30 μm Aβ(1–42) peptide. Prior to cell stimulations, Aβ peptide was prepared for this purpose by dissolving the peptide in 1,1,1,3,3,3-hexafluoro-2-propanol to a concentration of 1 mm. The 1,1,1,3,3,3-hexafluoro-2-propanol solvent was then evaporated, and the peptide film was re-dissolved in Me2SO to a concentration of 5 mm. Serum-free medium was pre-warmed to 37 °C and added to dilute the peptide to 100 μm, before adding to cells to give a final concentration of 30 μm. Cell supernatants were harvested after 24 h and cytokine and chemokine release was assayed by ELISA according to the manufacturer's instructions. For experiments employing either LPS + IFNγ or Aβ(1–42) treatment, cell viability was measured by MTT or LDH assay, respectively, according to the manufacturer's instructions. Isolation of Cortical Neurons—Primary cerebral cortical cell cultures were prepared from gestational day 18 fetuses of time-mated CD rats (Charles River) (31Skaper S.D. Facci L. Milani D. Leon A. Toffano G. Conn P.M. Methods in Neurosciences. 2. Academic Press, San Diego1990: 17-33Google Scholar), with minor modifications. Briefly, after removal of meninges, cerebral cortices were dissected and dissociated with a papain tissue dissociation kit (Worthington, Lakewood, NJ) following the manufacturer's instructions. Cells were resuspended in Neurobasal medium containing 2% B27 supplements, 1 mm sodium pyruvate, 2 mm l-glutamine and penicillin (100 units/ml)/streptomycin (50 μg/ml), and plated in poly-d-lysine-coated 48-well plates, 1 × 105 cells/well in 0.35 ml. Poly-d-lysine-coated culture wells were exposed overnight to medium containing 10% FCS prior to cell seeding. After 2 days, cultures received 0.35 ml/well of plating medium, but containing B27 supplement without antioxidants. Cultures were maintained at 37 °C in a 5% CO2 humidified atmosphere. Neuronal cells were used on days 5–6 for experiments. Co-cultures of Microglia and Cortical Neurons—Microglia were collected by mechanically scraping into culture medium. After centrifugation (200 × g for 5 min) cells were resuspended in Neurobasal medium containing 2% B27 supplements (without antioxidants), 1 mm sodium pyruvate, 2 mm l-glutamine and penicillin/streptomycin. Co-cultures were prepared by plating a suspension of isolated microglia (rat or mouse) on neurons maintained for 5 days in vitro in a 1:1 ratio. Cells were incubated for 30 min and then washed once with plating medium to remove non-adherent contaminating macroglia and debris, and then refed with fresh plating medium. Injury Induction in LPS + IFNγ-stimulated Co-cultures—Neuron-microglia co-cultures were treated the day following addition of microglia to the neuronal cell monolayers. Culture medium was replaced with an equal volume of fresh medium containing LPS (0.5 μg/ml) and rat or mouse IFNγ (500 units/ml). Analogous treatments were carried out for cultures composed of only cortical neurons or microglia. All cultures were incubated at 37 °C for 3 days, after which time cell viability was determined by LDH release. Neurotoxicity Assay in LPS + IFNγ-stimulated Co-cultures—LDH release into the culture medium was used as a measure of cell death, and has been utilized previously to quantify neuronal cell injury in microglia-neuron co-cultures (12Golde S. Chandran S. Brown G.C. Compston A. J. Neurochem. 2002; 82: 269-282Crossref PubMed Scopus (74) Google Scholar, 13Parvathenani L.K. Tertyshnikova S. Greco C.R. Roberts S.B. Robertson B. Posmantur R. J. Biol. Chem. 2003; 278: 13309-13317Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). LDH activity in the cell culture supernatants was determined after 72 h, using the CytoTox® non-radioactive cytotoxicity assay kit following the manufacturer's instructions. Values of LDH release are expressed as milliunits/ml, unless indicated otherwise. Microglia and cortical neurons were also independently cultured for 72 h in the presence of stimuli, and LDH release from the microglia alone ± stimuli were subtracted out from the values obtained from the combination of microglia and cortical neurons. Quantitative Analysis of Neuronal Cell Injury by Arrayscan—All incubations were carried out at room temperature unless otherwise stated. Cells were fixed in PBS containing 4% (w/v) paraformaldehyde, 10% (w/v) sucrose, and 15 μg/ml Hoechst 33342. The fixative was removed and cells were washed once with 200 μl of PBS. Cells were then blocked in PBS, 1% (w/v) bovine serum albumin, 0.1% (w/v) Triton X-100 (1 h), followed by incubation for 1 h with anti-βIII-tubulin primary antibody (1:1000 in blocking buffer). Cells were next washed 3 times with blocking buffer and then incubated 1 h with Alexa 488-labeled secondary antibody (1:300 in blocking buffer). Cells were subsequently washed 3 times with PBS prior to imaging. Image acquisition within the 48-well plate and subsequent neurite outgrowth measurements were performed using Cellomics Arrayscan technology (Swallowfield, UK). Ultraviolet light was used to illuminate Hoechst 33342-labeled nuclei, allowing automated focusing upon the cells. A second excitation wavelength of 488 nm was used to obtain pictures of specifically labeled neurons. These images were then analyzed using Cellomics neurite outgrowth software to identify and measure neurites that fit defined criteria. Data are expressed as perimeter squared divided by 4 × π × area (“P2/A”). Exposure times for each wavelength were determined empirically by the user. Injury Induction in Aβ(1–42)-stimulated Cortical Neuron/Microglia Co-cultures—Microglia isolated from the cerebral cortex of normal and MK2–/– mice were added to cell culture inserts (PET membrane, 0.4 μm pore size, BD Biosciences) at 7.5 × 104 cells per insert in Dulbecco's modified Eagle's medium with 10% FCS (0.4 ml/insert), and placed in a 24-well plate (notched for inserts) in Dulbecco's modified Eagle's medium with 10% FCS (0.8 ml/well). The following day, inserts were washed once with Neurobasal medium containing 2% B27 supplements (without antioxidants), 1 mm sodium pyruvate, 2 mm l-glutamine and penicillin/streptomycin, and transferred to a 24-well plate of rat cortical neurons (6 days in vitro, 2 × 105 cells per well). The porous membrane allows free diffusion of molecules. The distance between the neuron monolayer and microglia on the insert membrane is 1 mm, according to the manufacturer's description. Aβ(1–42) was then added to the microglia-containing inserts (15 μm in the above Neurobasal medium); controls received an equal volume of medium with 0.2% Me2SO. The co-cultures were returned to the incubator for another 3 days. Neurotoxicity Assay in Aβ(1–42)-stimulated Co-cultures—At the end of the incubation period, microglia-containing inserts were removed and neuronal cell viability was evaluated by a colorimetric method based upon the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by mitochondrial dehydrogenases to a blue formazan product (30Rosin C. Bates T.E. Skaper S.D. J. Neurochem. 2004; 90: 1173-1185Crossref PubMed Scopus (69) Google Scholar, 31Skaper S.D. Facci L. Milani D. Leon A. Toffano G. Conn P.M. Methods in Neurosciences. 2. Academic Press, San Diego1990: 17-33Google Scholar). Statistics—Data are given as mean ± S.D. or S.E. Statistical analyses to determine group differences were performed either by two-sample equal variance Student's t test, or by one-way analysis of variance, followed by Dunnett's post-hoc test for comparisons involving more than two data groups. MK2 Expression Is Enriched in Primary Microglial Cells Compared with Primary Astrocytes and Neurons—To investigate the relative expression of MK2 in cell types within the CNS, we compared expression of MK2 in rat primary cultures of cortical neurons, microglia, and astrocytes by quantitative RT-PCR (Fig. 1). GFAP, CD11b, and MAP2 expression as markers of astrocytes, microglia, and neurons, respectively, confirmed culture purity (data not shown). MK2 expression was found to be highly enriched in microglial cells, supporting a role for MK2 function
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