title: Characterization of a novel adult murine immortalized microglial cell line and its activation by amyloid-beta.
abstract: BACKGROUND
  Alzheimer's disease is associated with amyloid-beta (Aβ)-induced microglia activation. This pro-inflammatory response promotes neuronal damage, and therapies are sought to limit microglial activation. Screening efforts to develop new pharmacological inhibitors require a robust in vitro cell system. Current models lack significant responses to Aβ, and their use in examining age-related neurodegenerative diseases is questionable. For example, the commonly used BV-2 microglial line was derived from embryonic mononuclear cells and its activation by various stimuli is limited. To this end, we have established a new immortalized microglial (IMG) cell line from adult murine brain. The objective of this study was to characterize Aβ-induced activation of IMG cells, and here, we demonstrate the ability of cannabinoids to significantly reduce this inflammatory response.
  
  
  METHODS
  Microglial cells derived from adult murine brain were immortalized via infection with the v-raf/v-myc retrovirus under conditions that selectively promote microglia growth. The presence or absence of markers CD11b and F4/80 (microglial), NeuN (neuronal), and GFAP (astrocytic) was assessed by immunofluorescence microscopy and western blotting. Using IMG and BV-2 cells, levels of pro- and anti-inflammatory transcripts in response to extracellular stimuli were determined by quantitative PCR (qPCR). Phagocytosis of fluorescent beads and fluorescein isothiocyanate (FITC)-labeled Aβ oligomers was assessed using flow cytometry and fluorescence microscopy. FITC-Aβ uptake was quantified using a fluorescence plate reader. The ability of cannabinoids to mitigate Aβ-induced expression of inducible nitric oxide synthase (iNOS) was evaluated.
  
  
  RESULTS
  IMG cells express the microglial markers CD11b and F4/80 but not NeuN or GFAP. Relative to BV-2 cells, IMG cells increased iNOS (>200-fold) and Arg-1 (>100-fold) in response to pro- and anti-inflammatory stimuli. IMG cells phagocytose foreign particles and Aβ oligomers, with the latter trafficked to phagolysosomes. Aβ-induced activation of IMG cells was suppressed by delta-9-tetrahydrocannabinol and the CB2-selective agonist JWH-015 in a time- and concentration-dependent manner.
  
  
  CONCLUSIONS
  IMG cells recapitulate key features of microglial cell activation. As an example of their potential pharmacological use, cannabinoids were shown to reduce activation of Aβ-induced iNOS gene expression. IMG cells hold promising potential for drug screening, mechanistic studies, and functional investigations directed towards understanding how Aβ interacts with microglia.
authors.affiliation: Department of Genetics and Complex Diseases, Harvard School of Public Health, 655 Huntington Avenue, Boston, MA, 02115, USA. rmccarth@hsph.harvard.edu.
authors.forename: Ryan C
authors.initials: RC
authors.lastname: McCarthy
authors.affiliation: Department of Genetics and Complex Diseases, Harvard School of Public Health, 655 Huntington Avenue, Boston, MA, 02115, USA. dahyuu@mail.cmu.edu.tw.|Present Address: Graduate Institute of Neural and Cognitive Sciences, China Medical University, Taichung, Taiwan, Republic of China. dahyuu@mail.cmu.edu.tw.
authors.forename: Dah-Yuu
authors.initials: DY
authors.lastname: Lu
authors.affiliation: Department of Genetics and Complex Diseases, Harvard School of Public Health, 655 Huntington Avenue, Boston, MA, 02115, USA. aalkhateeb@mgh.harvard.edu.|Present Address: The Wellman Center for Photomedicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA. aalkhateeb@mgh.harvard.edu.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Alkhateeb
authors.affiliation: Department of Genetics and Complex Diseases, Harvard School of Public Health, 655 Huntington Avenue, Boston, MA, 02115, USA. garde046@umn.edu.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Gardeck
authors.affiliation: Department of Genetics and Complex Diseases, Harvard School of Public Health, 655 Huntington Avenue, Boston, MA, 02115, USA. clee@hsph.harvard.edu.
authors.forename: Chih-Hao
authors.initials: CH
authors.lastname: Lee
authors.affiliation: Department of Genetics and Complex Diseases, Harvard School of Public Health, 655 Huntington Avenue, Boston, MA, 02115, USA. wessling@hsph.harvard.edu.
authors.forename: Marianne
authors.initials: M
authors.lastname: Wessling-Resnick
mesh_terms: D016229:Amyloid beta-Peptides / Q000378:metabolism*; D000704:Analysis of Variance; D000818:Animals; D000943:Antigens, Differentiation / Q000378:metabolism; D039481:CD11b Antigen / Q000378:metabolism; D002478:Cells, Cultured; D005434:Flow Cytometry; D005786:Gene Expression Regulation / Q000502:physiology; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D066298:In Vitro Techniques; D053583:Interleukin-1beta / Q000378:metabolism; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D010586:Phagocytes / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: D016229:Amyloid beta-Peptides; D000943:Antigens, Differentiation; D039481:CD11b Antigen; D005904:Glial Fibrillary Acidic Protein; D053583:Interleukin-1beta; D012333:RNA, Messenger; C036763:monocyte-macrophage differentiation antigen
references.citation: Neuropharmacology. 2012 Sep;63(4):653-66
references.pmid: 22634229
references.citation: J Biol Chem. 2002 Jun 21;277(25):22131-9
references.pmid: 11940570
references.citation: Expert Opin Ther Targets. 2015 Apr;19(4):497-506
references.pmid: 25435348
references.citation: Trends Neurosci. 1996 Aug;19(8):312-8
references.pmid: 8843599
references.citation: Mol Pharmacol. 2011 Jun;79(6):964-73
references.pmid: 21350020
references.citation: Front Cell Neurosci. 2014 Apr 22;8:112
references.pmid: 24795567
references.citation: Nat Neurosci. 2014 Jan;17(1):131-43
references.pmid: 24316888
references.citation: Biomed Res Int. 2014;2014:437483
references.pmid: 25197646
references.citation: Int Immunopharmacol. 2014 Sep;22(1):73-83
references.pmid: 24975832
references.citation: J Biol Chem. 2011 Feb 4;286(5):3693-706
references.pmid: 20971854
references.citation: Neurobiol Aging. 2014 Dec;35(12):2746-2760
references.pmid: 25002035
references.citation: Mol Neurobiol. 2016 Mar;53(2):1181-1194
references.pmid: 25598354
references.citation: Methods Mol Biol. 2013;1041:17-23
references.pmid: 23813365
references.citation: J Neurochem. 2008 Oct;107(2):557-69
references.pmid: 18717813
references.citation: J Neurosci. 2005 Sep 7;25(36):8240-9
references.pmid: 16148231
references.citation: Nat Rev Neurol. 2015 Jan;11(1):56-64
references.pmid: 25385337
references.citation: J Neuroinflammation. 2012 Jan 16;9:8
references.pmid: 22248049
references.citation: J Immunol. 2000 May 1;164(9):4893-8
references.pmid: 10779799
references.citation: J Neuroinflammation. 2012 Sep 04;9:210
references.pmid: 22947253
references.citation: J Neurosci. 2014 Apr 23;34(17):5882-94
references.pmid: 24760848
references.citation: J Neuroimmunol. 1990 May;27(2-3):229-37
references.pmid: 2110186
references.citation: J Neurosci. 2008 Aug 13;28(33):8354-60
references.pmid: 18701698
references.citation: Neurobiol Dis. 2015 Jan;73:24-35
references.pmid: 25204716
references.citation: Science. 2010 Nov 5;330(6005):841-5
references.pmid: 20966214
references.citation: J Neuroinflammation. 2014 Jun 03;11:98
references.pmid: 24889886
references.citation: Nat Rev Neurol. 2014 Apr;10(4):217-24
references.pmid: 24638131
references.citation: Glia. 2007 Oct;55(13):1374-84
references.pmid: 17661344
references.citation: FEBS Lett. 2014 Aug 25;588(17):2952-6
references.pmid: 24911209
references.citation: Nat Rev Neurol. 2010 Apr;6(4):193-201
references.pmid: 20234358
references.citation: Brain Res Dev Brain Res. 1999 Nov 18;117(2):145-52
references.pmid: 10567732
references.citation: J Neurosci. 2005 Feb 23;25(8):1904-13
references.pmid: 15728830
references.citation: PLoS One. 2008;3(11):e3604
references.pmid: 18978948
references.citation: Nature. 1985 Dec 19-1986 Jan 1;318(6047):667-70
references.pmid: 4079980
references.citation: FEBS Lett. 2007 Feb 6;581(3):475-8
references.pmid: 17240371
references.citation: Glia. 2013 Jan;61(1):121-7
references.pmid: 22927325
references.citation: J Biol Chem. 2004 Apr 9;279(15):15441-7
references.pmid: 14754894
references.citation: J Neuroinflammation. 2012 May 31;9:115
references.pmid: 22651808
references.citation: Proc Natl Acad Sci U S A. 1991 Aug 15;88(16):7438-42
references.pmid: 1651506
references.citation: Mol Neurobiol. 2010 Jun;41(2-3):242-7
references.pmid: 20195798
references.citation: Cell Mol Neurobiol. 2015 Jan;35(1):71-83
references.pmid: 25149075
references.citation: Neuropathol Appl Neurobiol. 2013 Feb;39(1):3-18
references.pmid: 23252647
references.citation: Methods Mol Biol. 2013;1041:11-6
references.pmid: 23813364