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• W1971000103 abstract "Neuroinflammation is a hallmark of several neurodegenerative diseases, including Alzheimer’s disease (AD). Strong epidemiological and experimental evidence supports the use of nonsteroidal anti-inflammatory drugs to reduce AD risk. However, poor outcome in clinical trials and toxicity in a prevention trial have shifted focus away from these cyclooxygenase (COX) inhibitors to seek additional therapeutic targets in the prostaglandin pathway. Previously, the prostaglandin E2 receptor, EP2, was shown to regulate neuroinflammation and reduce Aβ plaque burden in transgenic mice. Unfortunately, widespread EP2 distribution and a direct effect on COX2 induction make EP2 a less desirable target. In this study, we link dedicator of cytokinesis 2 (DOCK2) to the prostaglandin pathway in the brain. Additionally, we show that DOCK2 regulates microglial innate immunity independent of COX2 induction and that DOCK2+ microglia are associated with human AD pathology. Together, these results suggest DOCK2 as a COX2 expression-independent therapeutic target for neurodegenerative diseases such as AD. Neuroinflammation is a hallmark of several neurodegenerative diseases, including Alzheimer’s disease (AD). Strong epidemiological and experimental evidence supports the use of nonsteroidal anti-inflammatory drugs to reduce AD risk. However, poor outcome in clinical trials and toxicity in a prevention trial have shifted focus away from these cyclooxygenase (COX) inhibitors to seek additional therapeutic targets in the prostaglandin pathway. Previously, the prostaglandin E2 receptor, EP2, was shown to regulate neuroinflammation and reduce Aβ plaque burden in transgenic mice. Unfortunately, widespread EP2 distribution and a direct effect on COX2 induction make EP2 a less desirable target. In this study, we link dedicator of cytokinesis 2 (DOCK2) to the prostaglandin pathway in the brain. Additionally, we show that DOCK2 regulates microglial innate immunity independent of COX2 induction and that DOCK2+ microglia are associated with human AD pathology. Together, these results suggest DOCK2 as a COX2 expression-independent therapeutic target for neurodegenerative diseases such as AD. Innate immune activation of the central nervous system is associated with several neurodegenerative diseases including Alzheimer’s disease (AD).1Lobsiger CS Cleveland DW Glial cells as intrinsic components of non-cell-autonomous neurodegenerative disease.Nat Neurosci. 2007; 10: 1355-1360Crossref PubMed Scopus (364) Google Scholar, 2Cimino PJ Keene CD Breyer RM Montine KS Montine TJ Therapeutic targets in prostaglandin E2 signaling for neurologic disease.Curr Med Chem. 2008; 15: 1863-1869Crossref PubMed Scopus (85) Google Scholar, 3Zipp F Aktas O The brain as a target of inflammation: common pathways link inflammatory and neurodegenerative diseases.Trends Neurosci. 2006; 29: 518-527Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 4Wyss-Coray T Inflammation in Alzheimer disease: driving force, bystander or beneficial response?.Nat Med. 2006; 12: 1005-1015Crossref PubMed Scopus (912) Google Scholar The major cellular component of this response, activated microglia, demonstrates both beneficial and deleterious effects on surrounding neurons.4Wyss-Coray T Inflammation in Alzheimer disease: driving force, bystander or beneficial response?.Nat Med. 2006; 12: 1005-1015Crossref PubMed Scopus (912) Google Scholar, 5Miller KR Streit WJ The effects of aging, injury and disease on microglial function: a case for cellular senescence.Neuron Glia Biol. 2007; 3: 245-253Crossref PubMed Scopus (108) Google Scholar, 6Shie FS Breyer RM Montine TJ Microglia lacking E prostanoid receptor subtype 2 have enhanced Abeta phagocytosis yet lack Abeta-activated neurotoxicity.Am J Pathol. 2005; 166: 1163-1172Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 7Halle A Hornung V Petzold GC Stewart CR Monks BG Reinheckel T Fitzgerald KA Latz E Moore KJ Golenbock DT The NALP3 inflammasome is involved in the innate immune response to amyloid-beta.Nat Immunol. 2008; 9: 857-865Crossref PubMed Scopus (1716) Google Scholar, 8Hickman SE Allison EK El Khoury J Microglial dysfunction and defective beta-amyloid clearance pathways in aging Alzheimer's disease mice.J Neurosci. 2008; 28: 8354-8360Crossref PubMed Scopus (906) Google Scholar, 9Liang X Wang Q Hand T Wu L Breyer RM Montine TJ Andreasson K Deletion of the prostaglandin E2 EP2 receptor reduces oxidative damage and amyloid burden in a model of Alzheimer's disease.J Neurosci. 2005; 25: 10180-10187Crossref PubMed Scopus (195) Google Scholar The deleterious effects include microglial secretion of a variety of molecules including prostaglandins (PGs) that can mediate paracrine neurotoxicity. Indeed, activation of the PG pathway has been linked with neurotoxicity in a number of cell culture and in vivo models.2Cimino PJ Keene CD Breyer RM Montine KS Montine TJ Therapeutic targets in prostaglandin E2 signaling for neurologic disease.Curr Med Chem. 2008; 15: 1863-1869Crossref PubMed Scopus (85) Google Scholar, 6Shie FS Breyer RM Montine TJ Microglia lacking E prostanoid receptor subtype 2 have enhanced Abeta phagocytosis yet lack Abeta-activated neurotoxicity.Am J Pathol. 2005; 166: 1163-1172Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 10Ahmad AS Yun YT Ahmad M Maruyama T Dore S Selective blockade of PGE2 EP1 receptor protects brain against experimental ischemia and excitotoxicity, and hippocampal slice cultures against oxygen-glucose deprivation.Neurotox Res. 2008; 14: 343-351Crossref PubMed Scopus (44) Google Scholar, 11Jin J Shie FS Liu J Wang Y Davis J Schantz AM Montine KS Montine TJ Zhang J Prostaglandin E2 receptor subtype 2 (EP2) regulates microglial activation and associated neurotoxicity induced by aggregated alpha-synuclein.J Neuroinflammation. 2007; 4: 2Crossref PubMed Scopus (93) Google Scholar, 12Manabe Y Anrather J Kawano T Niwa K Zhou P Ross ME Iadecola C Prostanoids, not reactive oxygen species, mediate COX-2-dependent neurotoxicity.Ann Neurol. 2004; 55: 668-675Crossref PubMed Scopus (131) Google Scholar, 13Wu L Wang Q Liang X Andreasson K Divergent effects of prostaglandin receptor signaling on neuronal survival.Neurosci Lett. 2007; 421: 253-258Crossref PubMed Scopus (39) Google Scholar This is especially compelling because there are existing drugs that target the PG pathway, such as the cyclooxygenase (COX) inhibitors that inhibit PG production.Strong epidemiological evidence supports the efficacy of COX isozyme suppression in PG signaling by nonsteroidal anti-inflammatory drugs (NSAIDs) for AD therapy (reviewed in14McGeer PL McGeer EG NSAIDs and Alzheimer disease: epidemiological, animal model and clinical studies.Neurobiol Aging. 2007; 28: 639-647Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar). Recently, hard-gained knowledge about COX2 toxicity associated with NSAIDs has led academic and industry laboratories to pursue more specific targets.15Group AR Lyketsos CG Breitner JC Green RC Martin BK Meinert C Piantadosi S Sabbagh M Naproxen and celecoxib do not prevent AD in early results from a randomized controlled trial.Neurology. 2007; 68: 1800-1808Crossref PubMed Scopus (275) Google Scholar, 16Konstantinopoulos PA Lehmann DF The cardiovascular toxicity of selective and nonselective cyclooxygenase inhibitors: comparisons, contrasts, and aspirin confounding.J Clin Pharmacol. 2005; 45: 742-750Crossref PubMed Scopus (69) Google Scholar, 17Martin BK Szekely C Brandt J Piantadosi S Breitner JC Craft S Evans D Green R Mullan M Cognitive function over time in the Alzheimer's Disease Anti-inflammatory Prevention Trial (ADAPT): results of a randomized, controlled trial of naproxen and celecoxib.Arch Neurol. 2008; 65: 896-905Crossref PubMed Scopus (123) Google Scholar Through a series of studies we and others have demonstrated the pro-inflammatory, pro-oxidative, and pro-amyloidogenic nature of the prostaglandin E2 receptor (EP2) in mouse brain or primary cultures from mouse brain, suggesting it as a potentially beneficial therapeutic target for AD.4Wyss-Coray T Inflammation in Alzheimer disease: driving force, bystander or beneficial response?.Nat Med. 2006; 12: 1005-1015Crossref PubMed Scopus (912) Google Scholar, 9Liang X Wang Q Hand T Wu L Breyer RM Montine TJ Andreasson K Deletion of the prostaglandin E2 EP2 receptor reduces oxidative damage and amyloid burden in a model of Alzheimer's disease.J Neurosci. 2005; 25: 10180-10187Crossref PubMed Scopus (195) Google Scholar, 18Echeverria V Clerman A Dore S Stimulation of PGE receptors EP2 and EP4 protects cultured neurons against oxidative stress and cell death following beta-amyloid exposure.Eur J Neurosci. 2005; 22: 2199-2206Crossref PubMed Scopus (108) Google Scholar, 19Montine TJ Milatovic D Gupta RC Valyi-Nagy T Morrow JD Breyer RM Neuronal oxidative damage from activated innate immunity is EP2 receptor-dependent.J Neurochem. 2002; 83: 463-470Crossref PubMed Scopus (115) Google Scholar, 20Shie FS Montine KS Breyer RM Montine TJ Microglial EP2 is critical to neurotoxicity from activated cerebral innate immunity.Glia. 2005; 52: 70-77Crossref PubMed Scopus (100) Google Scholar While work to date with EP2 highlights a promising approach to PG-related therapeutics in neurodegenerative diseases, widespread organ and cellular distribution of EP make it a nonspecific therapeutic target. Moreover, EP2 activation regulates COX2 expression, at least in microglia, and so EP2 targeting may lead to a similar toxicity profile as relatively COX2-selective NSAIDs.20Shie FS Montine KS Breyer RM Montine TJ Microglial EP2 is critical to neurotoxicity from activated cerebral innate immunity.Glia. 2005; 52: 70-77Crossref PubMed Scopus (100) Google Scholar, 21Funk CD FitzGerald GA COX-2 inhibitors and cardiovascular risk.J Cardiovasc Pharmacol. 2007; 50: 470-479Crossref PubMed Scopus (243) Google Scholar, 22Yu Y Fan J Chen XS Wang D Klein-Szanto AJ Campbell RL FitzGerald GA Funk CD Genetic model of selective COX2 inhibition reveals novel heterodimer signaling.Nat Med. 2006; 12: 699-704Crossref PubMed Scopus (62) Google Scholar, 23Yu Y Fan J Hui Y Rouzer CA Marnett LJ Klein-Szanto AJ FitzGerald GA Funk CD Targeted cyclooxygenase gene (ptgs) exchange reveals discriminant isoform functionality.J Biol Chem. 2007; 282: 1498-1506Crossref PubMed Scopus (47) Google ScholarThe aims of this study were to discover and evaluate EP2-dependent regulators of microglial innate immune response that did not regulate COX2 expression. Indeed, we identified dedicator of cytokinesis 2 (DOCK2) expression as being nearly completely dependent on EP2 expression in microglia. DOCK2 was identified in 1999 as a member of the CDM family of proteins, which includes Caenorhabditis Elegans CED-5, human DOCK180, and Drosophila Melanogaster Myoblast City.24Nishihara H Kobayashi S Hashimoto Y Ohba F Mochizuki N Kurata T Nagashima K Matsuda M Non-adherent cell-specific expression of DOCK2, a member of the human CDM-family proteins.Biochim Biophys Acta. 1999; 1452: 179-187Crossref PubMed Scopus (76) Google Scholar To date, the majority of studies concerning DOCK2 have shown it to act as a guanyl-nucleotide exchange factor (GEF), which positively regulates Rac- (a Rho family small GTPase) mediated cellular processes such as lymphocyte migration.24Nishihara H Kobayashi S Hashimoto Y Ohba F Mochizuki N Kurata T Nagashima K Matsuda M Non-adherent cell-specific expression of DOCK2, a member of the human CDM-family proteins.Biochim Biophys Acta. 1999; 1452: 179-187Crossref PubMed Scopus (76) Google Scholar, 25Fukui Y Hashimoto O Sanui T Oono T Koga H Abe M Inayoshi A Noda M Oike M Shirai T Sasazuki T Haematopoietic cell-specific CDM family protein DOCK2 is essential for lymphocyte migration.Nature. 2001; 412: 826-831Crossref PubMed Scopus (363) Google Scholar, 26Janardhan A Swigut T Hill B Myers MP Skowronski J HIV-1 Nef binds the DOCK2-ELMO1 complex to activate rac and inhibit lymphocyte chemotaxis.PLoS Biol. 2004; 2: E6Crossref PubMed Scopus (99) Google Scholar, 27Nishihara H Maeda M Oda A Tsuda M Sawa H Nagashima K Tanaka S DOCK2 associates with CrkL and regulates Rac1 in human leukemia cell lines.Blood. 2002; 100: 3968-3974Crossref PubMed Scopus (63) Google Scholar, 28Nishihara H Maeda M Tsuda M Makino Y Sawa H Nagashima K Tanaka S DOCK2 mediates T cell receptor-induced activation of Rac2 and IL-2 transcription.Biochem Biophys Res Commun. 2002; 296: 716-720Crossref PubMed Scopus (23) Google Scholar, 29Sanui T Inayoshi A Noda M Iwata E Oike M Sasazuki T Fukui Y DOCK2 is essential for antigen-induced translocation of TCR and lipid rafts, but not PKC-theta and LFA-1, in T cells.Immunity. 2003; 19: 119-129Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 30Sanui T Inayoshi A Noda M Iwata E Stein JV Sasazuki T Fukui Y DOCK2 regulates Rac activation and cytoskeletal reorganization through interaction with ELMO1.Blood. 2003; 102: 2948-2950Crossref PubMed Scopus (90) Google Scholar, 31Shulman Z Pasvolsky R Woolf E Grabovsky V Feigelson SW Erez N Fukui Y Alon R DOCK2 regulates chemokine-triggered lateral lymphocyte motility but not transendothelial migration.Blood. 2006; 108: 2150-2158Crossref PubMed Scopus (65) Google Scholar The Rho family of small GTPases, including Rac, is known to be intimately associated with actin cytoskeleton processes as well as oxidative processes in phagocytic cells. In primary and immortalized microglial cell cultures, Rac1 activation has been shown to promote phagocytosis, including Aβ1-42 clearance.32Kitamura Y Shibagaki K Takata K Tsuchiya D Taniguchi T Gebicke-Haerter PJ Miki H Takenawa T Shimohama S Involvement of Wiskott-Aldrich syndrome protein family verprolin-homologous protein (WAVE) and Rac1 in the phagocytosis of amyloid-beta(1-42) in rat microglia.J Pharmacol Sci. 2003; 92: 115-123Crossref PubMed Scopus (37) Google Scholar, 33Ohsawa K Imai Y Kanazawa H Sasaki Y Kohsaka S Involvement of Iba1 in membrane ruffling and phagocytosis of macrophages/microglia.J Cell Sci. 2000; 113: 3073-3084PubMed Google Scholar With the exception of DOCK2’s role in neutrophil chemotaxis, there has been no literature describing its function in phagocytic cells even though it has been implicated in macrophage phagocytosis and NADPH oxidation.24Nishihara H Kobayashi S Hashimoto Y Ohba F Mochizuki N Kurata T Nagashima K Matsuda M Non-adherent cell-specific expression of DOCK2, a member of the human CDM-family proteins.Biochim Biophys Acta. 1999; 1452: 179-187Crossref PubMed Scopus (76) Google Scholar, 34Kunisaki Y Nishikimi A Tanaka Y Takii R Noda M Inayoshi A Watanabe K Sanematsu F Sasazuki T Sasaki T Fukui Y DOCK2 is a Rac activator that regulates motility and polarity during neutrophil chemotaxis.J Cell Biol. 2006; 174: 647-652Crossref PubMed Scopus (160) Google Scholar, 35Kunisaki Y Tanaka Y Sanui T Inayoshi A Noda M Nakayama T Harada M Taniguchi M Sasazuki T Fukui Y DOCK2 is required in T cell precursors for development of Valpha14 NK T cells.J Immunol. 2006; 176: 4640-4645Crossref PubMed Scopus (27) Google ScholarFollowing our discovery, we further investigated the role of DOCK2 in microglial function relating to phagocytosis and neurotoxicity as well as regulation of COX2 expression. We also sought to establish relevance of DOCK2 expression to AD pathogenesis by evaluating its expression pattern in human brain. To date, there has been no literature providing evidence for DOCK2 expression or function in the brain. Our identification and subsequent characterization of DOCK2 in brain may highlight its potential as a microglial-specific, COX2-expression independent therapeutic target for neurodegenerative diseases, such as AD.Materials and MethodsAnimal and Human UseWild-type C57Bl/6 mice (Jackson, Bar Harbor, Maine), Dock2−/− mice (Dr. Yoshinori Fukui, Kyushu University, Japan), and EP2−/− mice (Dr. Richard Breyer, Vanderbilt University) were used with approval by the University of Washington Institutional Animal Care and Use Committee. Well-characterized human tissue was obtained from the University of Washington’s Alzheimer’s Disease Research Center in complete compliance with Institutional Review Board-approved protocols (Postmortem intervals <8 hours).Primary Cell CulturePrimary mouse microglia and neurons were cultured as previously described.6Shie FS Breyer RM Montine TJ Microglia lacking E prostanoid receptor subtype 2 have enhanced Abeta phagocytosis yet lack Abeta-activated neurotoxicity.Am J Pathol. 2005; 166: 1163-1172Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 20Shie FS Montine KS Breyer RM Montine TJ Microglial EP2 is critical to neurotoxicity from activated cerebral innate immunity.Glia. 2005; 52: 70-77Crossref PubMed Scopus (100) Google Scholar Microglia at DIV 7–15 and neurons at DIV 8 were used for experiments.Pharmacological Cell TreatmentSoluble Aβ1-42 (Bachem, Torrance, CA) was freshly prepared as previously described.6Shie FS Breyer RM Montine TJ Microglia lacking E prostanoid receptor subtype 2 have enhanced Abeta phagocytosis yet lack Abeta-activated neurotoxicity.Am J Pathol. 2005; 166: 1163-1172Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 20Shie FS Montine KS Breyer RM Montine TJ Microglial EP2 is critical to neurotoxicity from activated cerebral innate immunity.Glia. 2005; 52: 70-77Crossref PubMed Scopus (100) Google Scholar Primary microglia in 6-well plates (1 well = 1 × 106 cells) were treated with Dulbecco’s modified Eagle’s medium (DMEM) containing Aβ1-42 (10 μmol/L)/interferon-γ (10 pg/ml) for 6 hours. DMEM containing 10% fetal bovine serum and 100 ng/ml of lipopolysaccharide (EMD Chemicals, Gibbstown, NJ) was added to microglia in 96-well plates (1 well = 5 × 104 cells) for 6- or 24-hour treatment.Gene ExpressionTotal Microglial RNA was extracted using Trizol (Invitrogen, Carlsbad, CA) and purified using an RNeasy Mini column (Qiagen, Valencia, CA). RNA quality was verified using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA). Total RNA was labeled and hybridized to Mouse Genome 430 2.0 Arrays (Affymetrix, Santa Clara, CA). Four independent gene chips were hybridized. The intensity value for each probe was calculated using GeneChip operating software (GCOS) version 1.4 (Affymetrix, Santa Clara, CA), which was followed by data quality validation. Raw microarray data were processed and analyzed using GeneTraffic (Iobion Informatics, La Jolla, CA) along with Statistical Analysis of Microarray methodology to produce pair comparison lists containing Affymetrix gene probe identifications, their relative fold-change, and the statistical indicator False Determining Rate. From these data, the False Determining Rate of 5% was used as the cutoff for gene probe significance. Validation by quantitative real-time PCR was done using validated TaqMan Gene Expression Assays (Applied Biosystems, Foster City, CA) and analyzed through relative quantitation in an ABI 7500 Real-Time PCR instrument (SDS v1.3.1). The comparative threshold cycle method (ΔΔCT), with glyceraldehyde-3-phosphate dehydrogenase as the endogenous reference, was used to determine relative message abundance.Western BlottingStandardized protein concentrations from cellular lysate were determined by BCA protein assay (Pierce, Rockford, IL) and subjected to SDS-polyacrylamide gel electrophoresis. Anti-DOCK2 (Millipore, Temecula, CA) and anti-COX2 (Calbiochem, Gibbstown, NJ) antibodies were used at 1:1000. Detection was done using HRP-based enhanced chemiluminescence.ImmunohistochemistryBrains were dissected out from PBS-perfused adult mice frozen in cryopreservative. Postmortem human brain tissue was placed directly into cryopreservative and stored at −80°C. For all tissue, 10 μm sections were cut using a cryostat. Sections were placed in cold acetone for 1 to 2 minutes followed by a 1-hour room temperature block. Primary antibodies (anti-DOCK2 [Millipore, Temecula, CA], anti-NeuN [Millipore, Temecula, CA], anti-CD68 [Serotec, Raleigh, NC], anti-glial fibrillary acidic protein [Dako, Capinteria, CA], anti-paired helical filament-Tau AT100 [Pierce, Rockford, IL], anti-Aβ 6E10 [Covance, Berkeley, CA]) were diluted 1:100 and incubated for 1 hour at room temperature. Sections were rinsed with Tris-saline. Secondary antibodies (Invitrogen, Carlsbad, CA) (1:200), tomato lectin (1:100), or Ricinus communis agglutinin 1(RCA1) lectin (Vector Laboratories, Burlingame, CA) (1:100) were added for 1 hour at room temperature. Sections were rinsed followed by 1 to 2 minutes in 70% ethanol. Saturated sudan black was added for 3 minutes. Sections were then rinsed with 70% ethanol and ddH2O. Tissue was mounted in ProLong Gold Antifade with 4,6-diamidino-2-phenylindole (Invitrogen, Carlsbad, CA). Imaging was done using an Olympus FV-1000 confocal microscope.Cytokine InductionMultiplex analysis of mouse cytokines (tumor necrosis factor [TNF]-α, monocyte chemoattractant protein [MCP-1]) from culture medium was done using immunobead-based multiplex assays (Millipore, Billerica, MA) according to manufacturer instructions and data were collected using the LiquiChip Workstation from Qiagen. Standard curves were constructed from authentic kit standards. Data are represented as a percent induction after standardizing the amount of cytokine containing media by dividing this by the total amount of protein from its respective cellular lysate.PhagocytosisMicroglia were plated overnight in 96-well plates (3 × 104 cells/well). Medium was replaced with DMEM containing 2 μm fluorescent microspheres (Invitrogen, Carlsbad, CA) (1:1700). Following a 6-hour incubation, cells were rinsed with cold PBS and collected after brief trypsinization. Cells were re-suspended in 4% paraformaldehyde-PBS and fixed for 1 hour at 4°C. Cells were subjected to flow cytometry (FACScan, BD Biosciences, San Jose, CA) with threshold values set at FSC 50, SSC 52, and FL1–3 52. Data were collected as the mean fluorescence intensity for each measure.Co-Culture NeurotoxicityPrimary microglia were seeded in 24-well collagen-treated transwell inserts (Fisher Scientific, Hanover Park, IL) at 1 × 105 cells/well in DMEM. From primary neurons, all but 500 μl conditioned media was removed and replaced with fresh serum-free Neurobasal Medium (0.5 mmol/L glutamine, 1× B27) with or without 1 μg/ml LPS. Butaprost or vehicle was added at 10 μmol/L. Co-cultures were incubated for 24 hours. Medium was collected and neurotoxicity was assessed using a lactate dehydrogenase (LDH) cytotoxicity assay (Sigma, St. Louis, MO) according to manufacturer’s protocol.StatisticsStatistical analyses were performed as described in each results section using GraphPad Prism software (GraphPad Software Inc., San Diego CA). All experiments were performed with at least n = 4 unless otherwise specified.ResultsDOCK2 Expression Is EP2-DependentFirst, we sought observable associated transcriptional changes in activated EP2−/− mouse primary microglia. Using cDNA microarray analysis followed by qRT-PCR validation, we screened for transcriptional events related to the EP2 pathway that may lead to a candidate gene to study further. From this analysis, a total of 17 genes were identified as significantly differentially expressed between EP2−/− and wild-type microglia after exposure to soluble Aβ1-42(Table 1). One of these genes, DOCK2, was selected for further study because existing literature suggest that its expression might be microglia specific. Indeed, DOCK2 was validated as being significantly down-regulated by tenfold in EP2−/− microglia (Figure 1A). Next, we determined if the observed difference in Dock2 mRNA was due to Aβ1-42 exposure or a property of the EP2−/− genotype itself. We performed quantitative real-time PCR for wild-type and EP2−/− primary microglia either not treated (NT) or exposed to Aβ1-42(Figure 1A). Dock2 gene expression level from NT wild-type cells were set to a relative value of 1 for normalization. Dock2 transcriptional levels were about tenfold lower in EP2−/− microglia compared with wild-type, regardless of treatment conditions (***P < 0.001 determined by Two-way analysis of variance with Bonferroni posttest correction, n = 4). DOCK2 protein expression was evaluated by Western blot analysis to see if protein levels correlate with that of the transcript. Cells challenged by LPS were also observed for extended testing of possible treatment effects. Corresponding with transcript, DOCK2 protein was only observed in wild-type microglia, again regardless of treatment type (Figure 1B). These findings indicate that regulation of DOCK2 transcription and protein expression is a consequence of genetic ablation of EP2, and not a result of microglial activation.Table 1Discovery of EP2-Dependent Dock2 mRNA Expression in MicrogliaGene symbolGene titleGenBankTranscript ratio (WT:EP2−/−)BC020077cDNA sequence BC020077NM_1455492.11NntNicotinamide nucleotide transhydrogenaseNM_0087102.05Rnf13Ring finger protein 13NM_0118830.77Serinc1Serine incorporator 1NM_0197600.76Cyp51Cytochrome P450, family 51NM_0200100.74Idi1Isopentenyl-diphosphate delta isomeraseNM_1453600.71Phf20l1PHD finger protein 20-like 1XM_4844760.69HnrpabHeterogeneous nuclear ribonucleoprotein A/BNM_0104480.65ScocShort coiled-coil proteinNM_0010391370.649330182L06RikRIKEN cDNA 9330182L06 geneNM_1727060.63A830039H10RikRIKEN cDNA A830039H10 geneNM_1721530.53Peli2Pellino 2NM_0336020.42Zfp236Zinc finger protein 236XM_4847520.24GmfbGlia maturation factor, betaNM_0220230.195031439G07RikRIKEN cDNA 5031439G07 geneNM_0010332730.16Ang1Angiogenin, ribonuclease A family, member 1NM_0074470.11Dock2Dedicator of cytokinesis 2NM_0333740.11Microarray gene expression data shows that Dock2 mRNA is decreased by approximately 10-fold in EP2−/− primary microglia stimulated by Aβ in vitro when compared with wild-type (WT). Open table in a new tab One nonphysiological interpretation of the in vitro findings is that DOCK2 expression in wild-type microglia changes as a result of cell culture technique, possibly due to loss of inhibition by interacting neurons or astrocytes in the brain. To test if this difference in DOCK2 expression also exists in vivo, we harvested several organs from adult mice and determined DOCK2 expression (Figure 1C). Dock2−/− mice were used as a negative control for antibody specificity. Confirming the results of others, DOCK2 was observed in the primary lymphoid tissue of wild-type mice24Nishihara H Kobayashi S Hashimoto Y Ohba F Mochizuki N Kurata T Nagashima K Matsuda M Non-adherent cell-specific expression of DOCK2, a member of the human CDM-family proteins.Biochim Biophys Acta. 1999; 1452: 179-187Crossref PubMed Scopus (76) Google Scholar; however, no DOCK2 was detected in lymphoid tissue harvested from EP2−/− mice. Much longer Western blot film exposure time demonstrated for the first time that DOCK2 is indeed present at low levels in wild-type brain (Figure 1C). DOCK2 was not detected in EP2−/− or Dock2−/− brain. To test if the loss of Dock2 leads to loss of EP2, we did a functional assay for the presence of EP2 in Dock2−/− microglia using the selective EP2 agonist butaprost. For Dock2−/− microglia, butaprost effectively decreases LPS-stimulated TNF-α induction, providing evidence that EP2 activity is not DOCK2-dependent (Figure 2A) (**P < 0.01 determined by one-way analysis of variance with Bonferroni corrected comparisons, n = 4). Together, these experiments confirm that DOCK2 is transcriptionally down-regulated in the absence of EP2.Figure 2DOCK2 is a COX2-independent regulator of microglial innate immune responses. A: The EP2 agonist butaprost shows that Dock2−/− microglia have EP2 functionally present (**P < 0.01 and ***P < 0.001). A–D: Quantification of in vitro primary mouse microglial response to LPS stimulation shows Dock2−/− microglia to have decreased TNF-α cytokine induction (A) (*P < 0.05), decreased MCP-1 chemokine induction (B) (*P < 0.05), decreased phagocytosis (C) (*P < 0.05), and decreased paracrine neurotoxicity (D) (*P < 0.05) when compared with wild-type (WT) (LDH=lactate dehydrogenase). (No significance = n.s.). Total significant difference between wild-type and Dock 2−/− micrological genotype (**P < 0.01, n = 4). E: Western blot images show COX2 induction following LPS stimulation of both wild-type and Dock2−/− primary microglia.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DOCK2 Localizes to Microglia in Mouse BrainHaving discovered that DOCK2 is expressed in wild-type microglia in vitro and weakly in wild-type brain in vivo, we used immunohistochemistry to determine whether DOCK2 is expressed by microglia in vivo. Double-labeling immunohistochemistry using cryopreserved wild-type mouse hippocampus revealed that DOCK2 cellular staining is present and that it co-localized exclusively with established microglial markers tomato lectin (Figure 1D) and CD68. DOCK2 did not co-localize with the neuronal marker NeuN or the astrocytic marker glial fibrillary acidic protein. Immunohistochemistry did not reveal cellular staining for DOCK2 in EP2−/− or Dock2−/− hippocampus. Therefore, DOCK2 expression in mouse brain is restricted to wild-type microglia.DOCK2 Regulates Microglial ResponseWe tested if DOCK2 down-regulation had physiological relevance to activated microglial innate immune response, namely cytokine induction, phagocytosis, paracrine neurotoxicity, and COX2 induction. Primary microglia derived from wild-type and Dock2−/− mice underwent 24-hour LPS exposure and cell culture media was collected for quantification of pro-inflammatory cytokines" @default.
• W1971000103 created "2016-06-24" @default.
• W1971000103 creator A5004929037 @default.
• W1971000103 creator A5030256643 @default.
• W1971000103 creator A5030952055 @default.
• W1971000103 creator A5033683673 @default.
• W1971000103 creator A5067728043 @default.
• W1971000103 date "2009-10-01" @default.
• W1971000103 modified "2023-10-16" @default.
• W1971000103 title "DOCK2 Is a Microglial Specific Regulator of Central Nervous System Innate Immunity Found in Normal and Alzheimer’s Disease Brain" @default.
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