Neurobiology of Disease 54 (2013) 139–149

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Neurobiology of Disease

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Blockage of CR1 prevents activation of rodent microglia

Helen Crehan a,b, John Hardy b, Jennifer Pocock a,⁎a Department of Neuroinflammation, University College London, Institute of Neurology, 1 Wakefield Street, London WC1 N1PJ, UKb Reta Lila Weston Laboratories and Department of Molecular Neuroscience, University College London, Institute of Neurology, Queen Square, London WC1N 3BG, UK

Abbreviations: Aβ, amyloid beta; AD, Alzheimer's diseaprecursor protein; CGC, cerebellar granule cells; CR1, compvitro; DHE, dihydroethidium; D-MEM, Dulbecco's modifieacid; ECL, enhanced chemiluminescence; ELISA, enzymeGWAS, genome-wide association study; IL-1β, interleukioxide synthase; LOAD, late-onset Alzheimer's disease;monochlorobimane; MGCM, microglial conditioned medNO, nitric oxide; O•−, superoxide; PBS, phosphate bufferedPI, propidium iodide; PMA, phorbol myristate acetate; PRAGE, receptor for advanced glycation end product;SDS-PAGE, sodium sulphate polyacrylamide gel electrophfactor-alpha.⁎ Corresponding author.

E-mail address: j.pocock@ucl.ac.uk (J. Pocock).Available online on ScienceDirect (www.scienced

0969-9961/$ – see front matter © 2013 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.nbd.2013.02.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 24 July 2012Revised 31 January 2013Accepted 19 February 2013Available online 27 February 2013

Keywords:Complement receptor 1MicrogliaAlzheimer's diseaseCytokinePhagocytosisAβ1–42

Lipopolysaccharide

The importance of the complement system in Alzheimer's disease (AD) pathogenesis has been emphasizedthrough recent genome wide association studies. However, the cellular and molecular role of these comple-ment proteins is not fully understood. Microglia express complement receptors and the activation of specificreceptors may increase Aβ clearance and reduce neurodegeneration. Here, we investigated the contributionof complement receptor 1 (CR1), the second most significant hit in GWAS studies, on microglia to neuronaldamage. We show that microglia displaying an activated phenotype demonstrate an increase in CR1 expres-sion. We also provide evidence that activation of microglial CR1 was detrimental to neurons and this corre-lated with an increase in microglial intracellular superoxide generation, and tumour necrosis factor-α(TNFα) and interleukin-1 β (IL-1β) secretion. Amyloid-β 42 (Aβ1–42)-treated microglia displayed an in-creased ability to phagocytose dextran beads following antibody blockage of CR1 but a decreased capacityto phagocytose fluorescent-tagged Aβ1–42. Together, these results indicate that microglial CR1 plays a rolein the neuronal death observed in AD and investigating this further may provide a possible strategy to controlneurotoxicity in the AD brain.

© 2013 Elsevier Inc. All rights reserved.

Introduction

Recent genome wide association studies (GWAS) in Alzheimer'sdisease have highlighted the importance of the complement cascade inthe pathogenesis of Alzheimer's disease (Brouwers et al., 2011; Crehanet al., 2012; Jones et al., 2010; Lambert et al., 2009). This work confirmsthe pathogenic relevance of earlier work implicating this cascade in thepathology of the disease (McGeer and McGeer., 2002; McGeer et al.,1989) and work showing that Aβ/amyloid deposits activate the comple-ment cascade (Guerreiro et al., 2012; Rogers et al., 1992; Velazquez et al.,1997).

The secondmost significant hit in these GWAS, after apolipoprotein E,was the CR1 gene which encodes complement receptor 1, a complement

se; Apo, apocynin; APP, amyloidlement receptor 1; DIV, days ind eagle medium; EA, ethacrynic-linked immunosorbent assay;n-1 beta; iNOS, inducible nitricLPS, lipopolysaccharide; MCB,ium; NGS, normal goat serum;saline; PFA, paraformaldehyde;VDF, polyvinylidene difluoride;ROS, reactive oxygen species;oresis; TNFα, tumour necrosis

irect.com).

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regulator and receptor for C3b (Hollingworth et al., 2011;Naj et al., 2011).Based on this analysis, we investigated the relationship between Aβ andCR1, by using blocking CR1 antibody (Anti-CD35) and small interferingRNA (siRNA) to assess whether this relationship could underpin the asso-ciation with disease.

The association observed between the CR1 loci and age-relatedcognitive decline and plaque burden has been implicated to have connec-tions to impaired clearance of Aβ plaques in the brain of AD patients(Chibnik et al., 2011). Although the recruitment of phagocytes andinflammatorymediators is intended to be beneficial, under certain condi-tions, these processes can prove harmful instead. Classical markers ofimmune-mediated damage have been identified in Alzheimer's disease(AD) brains includingmajor histocompatibility complex class I and II pos-itive microglia (McGeer et al., 1987; Perlmutter et al., 1992; Tooyama etal., 1990).

In normal brain, microglia become reactive, surround damaged ordead cells and clear cellular waste from the area to promote regenera-tion and repair (Fetler and Amigorena, 2005). Microglia generallyhave beneficial effects, but their overstimulation can promote neuro-toxicity due to pathogenic signals, includingAβ, resulting in the produc-tion of excess free radicals, pro-inflammatory cytokines, complementproteins and glutamate (Morales et al., 2010). In AD, microglia displayan early reactive phenotype (Lautner et al., 2011), and changes in theimmune response are another risk marker for the development of AD(Jones et al., 2010).

The expression and distribution of CR1 in humans and rodents con-trast because human CR1 is encoded by a separate gene to human CR2,

140 H. Crehan et al. / Neurobiology of Disease 54 (2013) 139–149

but murine CR2 encodes both CR1 and CR2 (Kurtz et al., 1990; Molina etal., 1990). However the protein sequence and function of the murinespecific gene, CR1-related protein Y (Crry), display more likeness tohuman CR1 thanmouse CR1 (Killick et al., 2012). In view of this relation-ship, we confirmed our findings using a CR1 blocking antibody withsiRNA to downregulate Crry.

Exposure of microglia to extrinsic C1q complement protein dem-onstrated a shift towards a pro-inflammatory phenotype, similar tothat seen after the exposure of microglia to LPS, with a release ininterleukin-6, tumour necrosis factor-α (TNFα), nitric oxide and anoxidative burst (Färber et al., 2009). Here we investigated howmodula-tion of CR1 signalling influenced themicroglial phenotype during expo-sure of activators complicit in microglial responses in AD. In this workwe show that blocking microglial CR1 has a positive effect on neuronalsurvival. We provide evidence that microglial CR1 can elicit a neuro-toxic effect which correlated with enhanced cytokine and superoxideproduction, but that blockage of CR1 does not contribute a protectiveresponse by triggering glutathione production or inhibiting iNOS induc-tion. We also demonstrated an increased ability of Aβ1–42 treatedmicroglia to phagocytose dextran beads following antibody blockageof CR1 but a reduced ability to phagocytosis fluorescent Aβ1–42.

Materials and methods

Animals and materials

Sprague Dawley rats were bred and reared in house from stockanimals from Charles River UK Ltd. (Kent, UK). Tissue culture compo-nents were from Invitrogen (Paisley, UK). Anti-CD35 functional blockingantibody, C3b receptor was from antibodies-online.com (Atlanta, USA),anti-iNOS was from BD Biosciences (Oxford, UK), anti-CD35 antibodieswere from Hycult biotech, and antibodies were raised against purifiedhuman C3bR (Uden, theNetherlands, clone 31R) andAbcam (Cambridge,UK, clone E11), and details of the immunogen for this antibody are notavailable. Anti-NeuN antibody was from Merck Millipore (Oxfordshire,UK), cleaved caspase-3 antibody was from Cell Signalling Technology(MA, USA), dextran beads were from Molecular Probes, Invitrogen(Paisley, UK), β-amyloid (1–42) HiLyte FluorTM 488-labeled was fromAnaSpec (Fremont, CA, USA), Vectashield mountant for fluorescencewas from Vector (Peterborough, UK), and enhanced chemiluminescencereagent (ECL) was from Amersham Pharmacia (Buckinghamshire, UK).ON-TARGETplus Crry siRNA and Cyclophilin B control siRNA waspurchased from Thermo-scientific (CO, USA). Quantikine Rat TNFα andIL-1β Immunoassay kits were from R&D Systems (Abingdon, UK).Anti-β-actin and all other reagents were from Sigma (Dorset, UK).

Cell culture preparation and treatment

Animals (5 day old Sprague Dawley rat pups) were sacrificedthrough cervical dislocation and decapitation in accordance withthe Animals (Scientific Procedures) Act of 1986 (United Kingdom).Cerebellar granular cell (CGC) neuronal–glial cultures were preparedas described (Piers et al., 2011). These cultures contain approximately 5–10% of microglia per 100 neurons as determined by Isolectin B4 stainingand NeuN staining (Pocock, unpublished observations), a ratio represen-tative of in vivo data. Mixed cultures of hippocampal neurons and glialcells were prepared as described previously with modifications, fromSprague–Dawley rat pups 2–4 days post-partum (Vaarmann et al.,2010). Hippocampi and cortex were removed into ice-cold phosphatebuffered saline (PBS). The tissue was minced and trypsinised (0.25% for5 min at 37 °C), triturated and plated on poly-D-lysine-coated coverslipsand cultured in Dulbecco's modified eagle medium (D-MEM) (GIBCO,Carlsbad, CA) supplementedwith 10% foetal bovine serum(FBS), penicil-lin and streptomycin (GIBCO, Carlsbad, CA).

Cultures were maintained at 37 °C at 5% CO2 and were viable forexperimentation at 7 days in vivo (DIV).

Microglial cells were isolated from the brains of 5 day old SpragueDawley rat pups using Percoll gradients as previously described(Hooper et al., 2009a). The BV2 microglial cell line was a kind giftfrom Dr Claudie Hooper (MRC Centre for Neurodegenerative Research,Institute of Psychiatry, Kings College London, UK) and was originallyobtained from Dr FS Tzeng (Department of Life Sciences, NationalCheng Kung University, Taiwan). Where indicated in the figure legend,CGCs, hippocampal neuronal–glial cultures, microglia or BV2s weretreated directly with LPS (1 μg/mL) or Aβ1–42 (20 nM) for 24 h or48 h with or without the pre-treatment with CR1 blocking antibody(2 μg/mL).

Neurons were also exposed to microglial conditioned medium(MGCM) collected from primary microglia following 24 h treatmentand added to CGCs in a ratio of 1:1 with the original CGC medium andthe neurons incubated for a further 48 h, as previously described(Davenport et al., 2010).

Phagocytic assay

Treatedmicrogliawere incubatedwith 10 kDa TRITC-conjugated dex-tran beads (20 μg/mL) for 3 h or with β-Amyloid (1–42) HiLyte FluorTM

488-labeled (5 μg/mL) for 2 h at 37 °C. Cells were then incubatedwith 5 μg/mL of the nuclear stain 2′-[epoxyphenyl]-5-[4-methyl-1-piperazinyl]-2,5′-bi-1H-benzimidazol Hoechst 33342 (Hoechst33342) for 10 min and then washed in PBS to remove loose beadsand bead uptake was visualized by fluorescence microscopy. Fluores-cence images were captured using TRITC and FITC filter sets on a Zeissfluorescence microscope plus 40× Neofluor objective (Zeiss Axioskop2, Oberkochen, Germany). Data were collected from at least 3 fields ofview per coverslipwith each condition repeated on 3 coverslips in 3 sep-arate experiments. Labelled cells were counted as a percentage of totalcells per field. Control cells were incubated with beads alone at 37 °Cfor all conditions or at 4 °C, the latter as a binding control.

Assessment of apoptosis and cell death

Treated CGCs were incubated with 5 μg/mL Hoechst 33342 for20 min and 5 μg/mL propidium iodide (PI) for 30 min as previouslydescribed (Pinteaux-Jones et al., 2008). Apoptotic cells, which showbright blue pyknotic nuclei, and necrotic cells, which display red nuclei,were observed using DAPI (blue fluorochrome 364 nm) and TRITC (redfluorochrome, 543 nm) filters, respectively, on a Zeiss fluorescencemicroscopeplus 40×Neofluor objective (Zeiss Axioskop 2, Oberkochen,Germany). Cell counts were performed on a minimum of 3 fields percoverslip, 3 coverslips per treatment from 3 independent experiments.Treated hippocampal cells were fixed and stained for NeuN (see below)to assess neuronal loss as NeuN does not stain dead cells.

Immunocytochemistry

Treated microglia or hippocampal neuronal–glial cultures were fixedwith 4% paraformaldehyde (PFA), permeabilised with 100% ice-coldmethanol or 0.1% Triton-X100 and non-specific binding blocked with4% NGS. Cultures were incubated at 4 °C overnight with primary anti-body, NeuN (1:500) or cleaved caspase-3 (1:500) followed by incubationat room temperature with the appropriate secondary antibody for 2 h.Finally cells were counterstained with DAPI, mounted with Vectashieldand visualized by fluorescence microscopy.

Superoxide and glutathione live cell imaging

Treated primarymicroglia or BV2microgliawere assessed for super-oxide (O2

•−) production by incubation at 37 °C for 40 min withdihydroethidium (DHE) (5 μM) and were counterstained with Hoechst33342 as previously described (Mead et al., 2012). Duplicate conditionswere treated with apocynin (10 mM) to verify O2

•− production was via

141H. Crehan et al. / Neurobiology of Disease 54 (2013) 139–149

NADPH oxidase activity (Mead et al., 2012). Treated primary microgliaand BV2microglia were assessed for glutathione production by incuba-tion at 37 °C for 45 min withmonochlorobimane (MCB) (5 μg/mL) andcounterstained with PI. To confirm glutathione production, cells wereincubated with 1 mM ethacrynic acid (EA), a potent inhibitor of gluta-thione S-transferases (Ploemen et al., 1993) for 10 min before and dur-ing incubationwithMCB as previously described (Hooper et al., 2009b).Coverslips for superoxide or glutathione imaging were mounted insaline solution consisting of 153 mM NaCl, 3.5 mM KCl, 0.4 mMKH2PO4, 20 mM TES, 5 mM NaHCO3, 1.2 mM Na2SO4, 1.2 mM MgCl2,1.3 mM CaCl2, and 5 mM glucose, and observed immediately byfluorescence microscopy using the TRITC (red fluorochrome, 543 nm),FITC (green fluorochrome 488 nm) and DAPI (blue fluorochrome364 nm) filter sets on a Zeiss fluorescence microscope (Zeiss Axioskop2, Oberkochen, Germany). The mean cellular fluorescence of approxi-mately 50 cells/field, from three fields per coverslip, three coverslipsper condition and from three independent experiments was deter-mined using ImageJ software.

Small interfering RNA transfection

BV2 microglia were grown on a 24-well plate at a density of25,000 per well in D-MEM supplemented with 10% FBS, penicillinand streptomycin. ON-TARGETplus Crry DNA (0.6 μM) (Dharmacon,CO, USA) and Polymag beads (Oz biosciences, Marseille, France)were added in a 1:1 ratio to 200 μL serum-free D-MEM per welland incubated at room temperature for 30 min with vigorous shaking.BV2 microglia were transfected with ON-TARGETplus cyclophilin Bcontrol siRNA (Dharmacon, CO, USA) as controls. The DNA/bead mixwas then added to the BV2 microglia and placed on a Magnefect nanoII system (nanoTherics, Staffordshire, UK) at 2 Hz for 1 h at 0.5 mmdisplacement in a tissue culture incubator (5% CO2, 37 °C). Then BV2microglia were incubated at 37 °C for 12 h prior to treatment with LPS(1 μg/mL) and Aβ1–42 (20 nM) for a further 24 h.

Western blot analysis

Primary cultured microglia, CGCs and BV2 microglia were harvestedinto standard lysis buffer (Kingham and Pocock., 2000) incubated onice for 10 min, before being centrifuged for 10 min at 14,000 g to pelletthe nuclear and membrane fractions. A Bradford assay was carried outto determine the protein concentration of the cell lysate supernatantsusing bovine serum albumin as a standard. Protein was separated by10% SDS-PAGE and transferred to PVDF membranes (Immobilon P,Millipore). The membranes were washed in Tween-20 Tris buffer saline(TTBS: 10 mM Tris HCL, 150 mM NaCl, 0.5% Tween-20, pH 7.4) for10 min, and then blocked in 5% non-fat milk solution in TTBS for 1 h atroom temperature. Primary antibodies, incubated overnight at 4 °C,were anti-iNOS (1:2000), anti-CD35 (31R) Hycult biotech (1:100),anti-CD35 (E11) Abcam plc, Cambridge, UK (1:500) or anti-β-actin(1:2000), followed by incubationwith appropriate secondary antibodiesat room temperature for 1 h and visualization by ECL. In addition, 45 μgof protein fromhuman lymphoblastswas also run as positive controls forCR1 expression (Barth et al., 1978). Immuno-blots shown are represen-tative of three independent experiments.

ELISA analysis of secreted cytokines

TNFα and Interleukin 1β (IL-1β) concentrations in cell culturesupernatants were quantified using Quantikine Rat TNFα/IL-1β Immu-noassay kit according to the manufacturer's instructions (R&D Systems,Abingdon, UK). Cytokine concentrations in cell supernatants weredetermined against a standard curve of TNFα/IL-1β. For each condition,the cytokine content in supernatants was analysed from three cover-slips of microglia in three independent experiments with each sampleassayed in duplicate.

Results

CR1 expression is increased following microglial activation and antibodyblockage of CR1 modulated microglial phagocytosis

Since there is controversy about the ability of microglia to expressCR1, we tested two different antibodies on lysates from primary culturesof rat microglia. These cultures were ascertained to be 100% pure (byIsolectin B4 fluorescence staining, data not shown). Fig. 1a shows theresultant western blot with antibody CD35 (E11) from Abcam andFig. 1b shows the resultant western blot with antibody CD35 (31R)from Hycult biotech. In each case a standard of human lymphoblastswas also analysed for each antibody. In order to assess how CR1 signal-ling influences the pro-inflammatory phenotype associated withmicroglial responses in AD, we investigated the influence of activatorssuch as LPS and Aβ1–42 on microglial CR1 expression (Fig. 1). Therewas an increase in CR1 protein expression following activation for 24 hof primary rat microglial activation with either LPS (1 μg/mL) or Aβ1–42

(20 nM).Exposure of primary rat microglia to either LPS (1 μg/mL) or Aβ1–42

(20 nM) decreased dextran bead uptake (Figs. 2a and c). In the case ofLPS, phagocytosis of beads was not reversed by co incubation with CR1blocking antibody. However, CR1 block reversed Aβ1–42 suppression ofdextran bead phagocytosis back to control levels. Phagocytosis is adynamic process requiring energy and phagocytosis of dextran beadswas significantly reduced when the assay was carried out at 4 °C, thuseliminating binding or a superficial cell-association as an explanationfor the results observed (Kvarstein, 1969; Peterson et al., 1977; Prattenand Lloyd, 1986) (Fig. 2b). The experiment was repeated measuringphagocytosis of HiLyte Fluor™ 488-labeled Aβ1–42 (Figs. 2d and e) andthe results exhibited a differential response comparedwith phagocytosisof dextran beads. Following antibody blockage of CR1 in microglia treat-ed with LPS (1 μg/mL) or Aβ1–42 (20 nM), there was a significant reduc-tion in fluorescent Aβ1–42 phagocytosis compared with LPS or Aβ1–42

treatment alone. Again, conduction of the experiments at 4 °C revealedlittle phagocytosis, therefore confirming that the fluorescence observedwas due to phagocytosis.

CR1 modulation has an effect on neuronal death

Evidence suggests that apoptosis can stimulate the complementsystem, and such activation contributes to the phagocytosis of subse-quent apoptotic cells (Köhl, 2006). Direct treatment of CGC cultureswith LPS or addition of MGCM from LPS-treated microglia both inducedsignificant cell death (Figs. 3a and c), suggesting the neurotoxin(s)responsible was soluble and stable. No significant death was inducedwhen CGC cultures were directly treatedwith Aβ1–42 (Fig. 3a) but signif-icant death was observed when CGC cultures were treated with condi-tioned medium from microglia treated with Aβ1–42 (Fig. 3c), suggestingthat Aβ1–42-MGCMcontains stable neurotoxinswhichmaynot be potentin mixed neuronal–glial cultures. An alternative possibility is that Aβ1–42

takes longer to act than LPS. A significant reduction in death wasobserved in CGCs directly pre-treated with CR1 functional blocking anti-body compared with CGCs treated with LPS alone (Fig. 3a). Also, Fig. 3bindicates that the total cell number did not vary between treatmentsreducing the possibility of the decrease in apoptosis being due to an in-crease in phagocytosis of the apoptotic cells or floating off of dead cells.No difference in CGC death levels was seen following direct treatmentwith Aβ1–42 with or without the presence of CR1 blocking antibody(Fig. 3a). Fig. 3c indicated that neuronal death was enhanced after treat-ment of CGC cultures with microglial conditioned medium (MGCM)from microglia treated with LPS or Aβ1–42. This neuronal death wasreduced when the CGC cultures were treated with MGCM from primarymicroglia treated with Aβ1–42 and CR1 blocking antibody. Similar find-ings were observed with hippocampal neurons (Fig. 3e).

Fig. 1. CR1 expression is increased in primary rat microglia treated with microglial activators LPS (1 μg/mL) and Aβ1–42 (20 nM). Western blot with (a) antibody CD35 (E11) fromAbcam and (b) with antibody CD35 (31R) from Hycult biotech. In each case a standard of human lymphoblastswas also analysed for each antibody as a positive control. Densitometry ofexpression relative to the loading control, β-actin and against control CR1 expression was performed on three separate blots. Data were analysed by Student's t-test of expression versuscontrol, ***ρ b 0.001, **ρ b 0.01 and *ρ b 0.05.

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Release of pro-inflammatory cytokines frommicroglia is detrimentalto neurons (Smith et al., 2012; Taylor et al., 2005;Wang et al., 2003) andgenetic studies have reported an association between TNFα and IL-1βpolymorphisms and AD (Di Bona et al., 2008, 2009). Therefore in orderto elucidate the reason for the neurotoxic effect observed in Fig. 3, wemeasured levels of soluble neurotoxins such as TNFα and IL-1β presentin MGCM following 24 h treatment with microglial activators and CR1blocking antibody (Fig. 4). There was an increase in TNFα in MGCMfrom microglia treated with LPS (1 μg/mL) compared with controlMGCM (Fig. 4a). TNFα levels were significantly reduced in MGCM frommicroglia co-treated with CR1 blocking antibody and LPS. No differencewas seen in MGCM from microglia treated with Aβ1–42 (20 nM) orwith MGCM from microglia treated with Aβ1–42 and CR1 blockingantibody compared with control. Fig. 4b indicates a decrease in IL-1βlevels in MGCM from the microglia co-treated with CR1 blockingantibody plus Aβ1-42 compared with Aβ1-42 activator alone. No signif-icant release of IL-1β or regulation by CR1 block was observed followingLPS stimulation (Fig. 4b).We alsomeasured IL-6 and IL-10 release but nosignificant modulation with CR1 block was observed (data not shown).

CR1 block inhibits O2•− production in microglia

The immune system can battle against invading pathogens through arespiratory burst which is a rapid release of reactive oxygen species(ROS) from, in this case microglia. Previously, there has been a positivecorrelation between an increase in respiratory burst and expression ofC3b, the binding of which to its corresponding receptor, CR1, promotessignalling to NADPH oxidase to produce superoxide (Gessler andDahinden, 2003; Hoogerwerf et al., 1990). Here, primarymicroglia treat-ed with LPS showed a significantly higher intracellular O2

•− productioncompared with control and this was attenuated by functional antibodyCR1 block (Figs. 5a and b). A similar significant reduction in O2

•− produc-tion was observed in primary microglia treated with Aβ1–42 and CR1block compared with Aβ1–42 treatment alone. Primary microglia werealso pre-treated with apocynin, an inhibitor of NADPH oxidasewhich prevents superoxide production through inhibiting thetranslocation and assembly of the active NADPH oxidase complex(Mead et al., 2012; Stefanska and Pawliczak., 2008) and O2

•− pro-duction was inhibited on these cells. Similar O2

•− production wasobserved in BV2 microglia (Figs. 5c and d). Levels of O2

•− were

reduced in microglia co-treated with CR1 blocking antibody com-pared with LPS and Aβ1–42 alone. O2

•− productionwas also measuredin BV2 microglia transfected with Crry siRNA, the murine orthologof the human CR1 gene (Killick et al., 2012). LPS or Aβ1–42 treated BV2microglia displayed significantly increased O2

•− production comparedwith control and this was reduced in cells transfected with Crry siRNAprior to LPS and Aβ1–42 treatment (Figs. 5e and f). BV2 cells transfectedwith cyclophilin B control siRNAprior to treatmentwith LPS demonstrat-ed similar O2

•− production to control. O2•− positive fluorescence was also

inhibited in BV2microglia pre-treatedwith theNADPHoxidase inhibitor,apocynin, prior to LPS or Aβ1–42 treatment.

CR1 block does not alter glutathione production or iNOS expression inmicroglia

The complement system can be responsible for tissue damage inautoimmune diseases (Mollnes et al., 2002) and glutathione plays arole in the defence system as an antioxidant compound (Perricone etal., 2011). Microglial cells produce glutathione for defence against ROSproduced by microglia or other cells such as astrocytes in the brain(Hirrlinger et al., 2000) and glial cells are the main source of glutathionefor neurons (Keelan et al., 2001). Therefore glutathione production wasinvestigated following treatment of microglia with CR1 blocking anti-body and microglial activators. There was no change in mean MCBfluorescence per cell following incubation with CR1 blocking antibody,LPS and Aβ1–42 (Fig. 6a). Grey bars (Fig. 6a) represent microglia pre-incubated with ethacrynic acid, a potent inhibitor of glutathioneS-transferases (Ploemen et al., 1993) to confirm that fluorescence wasdue to glutathione binding to MCB. Because reactive oxygen speciesand reactive nitrogen species produced by microglia can react to formthe highly toxic peroxynitrite in response to LPS or Aβ stimulation(Kim et al., 2009; Neher et al., 2011), we determined whether CR1modulated iNOS expression, as this may explain reduced neurotoxicityafter microglial CR1 block. However we did not see regulation ofLPS-induced iNOS expression by CR1 block (Fig. 6b) and no significantexpression of iNOS was observed following Aβ1–42-stimulation ofmicroglia. This suggests that modulation of O2

•− production by CR1block may be sufficient to reduce microglial neurotoxicity. Taken togeth-er, these data indicate that neurotoxicity is not driven by direct iNOS orglutathione modulation.

Fig. 2. CR1 functional blocking antibody modulates microglial phagocytosis following treatment with Aβ or LPS. Quantification and images of primary microglial phagocytosis ofdextran beads (a,c) or fluorescently tagged Aβ1–42 (d,e) after direct treatment of microglia with Aβ1–42 (20 nM) or LPS (1 μg/mL) in the presence or absence of CR1 blockingantibody (1 mg/mL) for 24 h. (b) Quantification of primary microglial phagocytosis carried out as in (a) at 4 °C. Phagocytosis was measured as a percentage of the number ofcells with a fluorescence signal to total microglial cell number/field of view from 3 fields per coverslip, 3 coverslips per condition, from 3 separate experiments. Data were analysedby ANOVA followed by Student's t-test with pair-wise analysis as indicated, ***ρ b 0.001, **ρ b 0.01 and *ρ b 0.05.

143H. Crehan et al. / Neurobiology of Disease 54 (2013) 139–149

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Fig. 3. CR1 functional blocking antibody protects against microglial-evoked neurotoxicity following direct and indirect treatment with Aβ1–42 (20 nM) or LPS (1 μg/mL) in the presence orabsence of CR1 blocking antibody (1 mg/mL). (a) Live–dead cell staining of CGC neuronal cultureswith Hoechst 33342 (white bars) and propidium iodide (grey bars) after 48 h direct treat-ment. (b) Total cell numberper condition. (c) Live–dead cell staining of CGC cultures treatedwithMGCM frommicroglia treatedwith theconditions in (a) for 48 hwith+veCtrl representingcells with no treatment of MGCM. (d) Total cell number per condition after MGCM treatment of CGCs. (e) Hippocampal neuronal protection under the same conditionsmonitored by NeuNstaining. In all of the experiments data are the mean of 3 fields per coverslip, 3 coverslips per condition from 3 separate experiments. Data were analysed by ANOVA followed by Student'st-test with pair-wise analysis as indicated, ***ρ b 0.001, **ρ b 0.01 and *ρ b 0.05.

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Discussion

It is hypothesized that in LOAD, it is not the rate at which Aβ is accu-mulated that is impaired but its clearance (Mawuenyega et al., 2010).CR1 is known to be involved in immune clearance of opsonised patho-gens on erythrocytes (Kavai, 2008; Li et al., 2010), and so it is thoughtthat this receptor, which we have shown now to be present on microg-lia, may provide a role in the clearance of Aβ or lack thereof in the ADbrain. In healthy conditions, C3b, the ligand for CR1, is spontaneouslyproduced and destroyed. However, in the presence of a pathogen, orin this instance Aβ peptides, C3b binds to the peptide and through theaction of cofactors B and D develops into C3 convertase (Tohyama andYamamura, 2006). C3b is then cleaved to C3bi, which surrounds Aβ

and results in the capture of Aβ by the phagocyte, which expressesCR3, the receptor for C3bi.When CR1 is blocked, it prevents the cleavageof C3b at two sites to produce C3bi, therefore preventing phagocytosis.

Microglia activated with LPS have been demonstrated to be a goodcellular model to study neuroinflammation and Toll-like receptorshave been identified as important mediators of neuroinflammation(Arroyo et al., 2011; Le et al., 2001; Sheng et al., 2003). In order to exam-ine the phagocytic capability of microglia in a proinflammatory envi-ronment, the cells were treated with LPS, of which the resultingactivation is mediated by Toll-like receptor 4 (TLR4) (Chow et al.,1999; Lien et al., 2000). A significant reduction in phagocytosis ofdextran beads was observed in microglia treated with LPS. This wasnot reversed by LPS and CR1 blocking antibody compared with control

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LPS LPS + CR1 block

Aβ1-42 Aβ1-42 +CR1 block

Fig. 4. ELISA analysis of TNFα and IL-1β secretion from primary cultured rat microglia following treatment with LPS (1 μg/mL), Aβ1–42 (20 nM) and CR1 blocking antibody (1 μg/mL),alone and in combination, for 24 h. Reduced TNFα secretion followed LPS treatment inmicroglia pre-treatedwith CR1 blocking antibody 4a. A decrease in IL-1β secretion 4bwas observedin microglia pre-treated with CR1 blocking antibody compared with Aβ1–42 treatment alone. Data are the mean of supernatant values performed in duplicate from 3 separate coverslipsper condition taken from 3 independent experiments and values were analysed by ANOVA followed by Student's t-test with pair-wise analysis as indicated. **ρ b 0.01 and *ρ b 0.05.

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at 37 °C (Figs. 2a and c), suggesting that CR1 does not play a role in ageneral phagocytosis promoted by activation of microglia through theTLR4 signalling pathway. Dextran bead phagocytosis in microglia previ-ously exposed to Aβ1–42 was also significantly lower than in controlcells (Fig. 2a). However, an increase in phagocytosis of dextran beadswas observed in microglia treated with Aβ1–42 and CR1 blocking anti-body compared with Aβ1–42 alone (Figs. 2a and c). Microglia exposedto Aβundergo phagocytosis through a different receptor-linked pathwayto that induced by LPS. Microglia interact with fAβ through an Aβ cellsurface receptor CD36, α6β integrin and CD47 (Koenigsknecht andLandreth., 2004) as well as to the Receptor for Advanced GlycationEnd-product (RAGE) (Fang et al., 2010; Yan et al., 1996). Furthermore,microglial phagocytosis of fluorescently tagged Aβ1–42 following LPS orcold Aβ1–42 stimulation was prevented by CR1 block, suggesting thatCR1 block may have distinct consequences for different phagocytoticpathways inmicroglia. Thismay reflect changes in ongoing phagocytosisversus pinocytosis (Pratten and Lloyd, 1984).

To explore the consequences of blocking CR1 onmicroglial–neuronalinteractions, CGC apoptosis and necrosis were measured following 48 htreatment with MGCM from microglia treated with CR1 blocking anti-body, LPS or Aβ1–42, and also after direct treatment of the neuronal-enriched cultureswith these activators. Therewas an increase in apopto-sis following direct treatment of CGCs with LPS compared with control(Fig. 3a), which could be possibly due to an increase in TNFα (Fig. 4a).The percentage of neuronal death through apoptosis was significantlylower in CGCs that were pre-treated with CR1 blocking antibody. Totalcell number was measured to eliminate the possibility that a reductionin neuronal death was a consequence of changes in cell number(Fig. 3b). It was considered that this may be due to CR1 affecting TNFαshedding (Xaus et al., 2000) (Fig. 4). An increase in TNFα can cause anupregulation of C3b (Ogle et al., 1990). CR1 blockmight result in an accu-mulation of C3b due to reduced binding and subsequent removal fromcirculation. Excess C3b deposition results in red cell deposition of whichCR1 binding can have a protective effect (Odhiambo et al., 2008). Thiswouldprovide a possible explanation for a decrease in apoptosis observedas there was less TNFα produced in the cells pre-treated with CR1blocking antibody and LPS, compared with LPS alone. A decrease ofIL-1β was observed with Aβ1–42 and CR1 blocking antibody comparedwith Aβ1–42 alone (Fig. 4b). The rescuing effect observed with directtreatment of CGCs with CR1 blocking antibody together with LPS wasnot replicated in neurons directly treated with Aβ1–42. This may be dueto a direct effect of Aβ on neurons, as we and others have reportedpreviously (Taylor et al., 2003; Yan et al., 1996). However the increasedapoptosis of CGCs treated with Aβ1–42 MGCM for 48 h (Fig. 3c) was

significantly decreased when the neurons were exposed to MGCM frommicroglia treated with CR1 blocking antibody together with Aβ1–42.Binding of Aβ1–42 to the “receptor for advanced glycation end products”(RAGE) can induce a microglial mediated inflammatory and neurotoxicresponse (Yan et al., 1996) and Aβ oligomers, fibrils and aggregates canall bind to this receptor (Sturchler et al., 2008). This behaviour creates aharmful cellular environment, in which the persistent activation of mi-croglia causes them to adopt a predominantly neurotoxic phenotype,resulting in neuronal cell death (Fang et al., 2010). The decrease in ap-optosis seen in CGCs treated with MGCM from microglia treated withAβ1–42 and CR1 blocking antibody suggests that manipulating CR1 hasa positive effect on neuronal survival (Fig. 3c).

To elucidate the mechanisms by which this neuroprotection mightoccur, we investigated how microglial CR1 manipulation influencedmicroglial superoxide generation. Treatingmicroglia with CR1 blockingantibody before the addition of LPS or Aβ1–42 resulted in decreased in-tracellular O2

•− production (Fig. 5). Binding of C3b to CR1was previouslyfound to stimulate a respiratory burst leading to the production of O2

•−

(Hoogerwerf et al., 1990). Therefore the reducedO2•− productionwe ob-

served may be influenced by the lower availability of CR1 present tobind any C3b. SimilarO2

•−productionwas observed following treatmentof primary microglia (Figs. 5a and b) and the BV2 microglial cell line(Figs. 5c and d). When glutathione levels were measured followingtreatment with CR1 blocking antibody, LPS, and Aβ1–42, there were nodifferences in production, suggesting that CR1 does not modulatepotential ROS toxicity through enhanced glutathione production(Fig. 6a). Since nitric oxide production via iNOS expression in mi-croglia could contribute to increased neurotoxicity via the produc-tion of neurotoxic peroxynitrite formed in combination with O2

•−,we investigatedwhether this wasmodulated by CR1 block. However,western blotting revealed that iNOS expression was not modulated(Fig. 6b) but since O2

•− is modulated by CR1 block, it is possible thatthis can modulate the generation of peroxynitrite.

These results demonstrate that CR1 plays a role in microglial phago-cytosis, particularlywhen triggered by Aβ. Thismodulation is interestingas it revealed CR1 block to enhance dextran phagocytosis but reduce Aβphagocytosis. Thus, taken together, GWAS studies on mutations in CR1may lead to loss of function and a decrease in Aβ clearance, which corre-lates with our data. What is also clear here is the proinflammatory influ-ence CR1 has on neurotoxicity, and our data suggest that this is viaactions of microglial O2

•− and cytokine production. Mice express theCr1-related protein Y (Crry) which is more similar to human Cr1 in itsprotein sequence and function than murine Cr1 and deletion of Crry hasbeen recently shown to influence tau phosphorylation and complement

Fig. 5. CR1 functional blocking antibody modulates intracellular superoxide (O2•−) production in primary rat microglia and in BV2 murine microglia. Live cell imaging of O2

•− gen-eration monitored with dihydroethidium (DHE, pink fluorescence) in cells producing intracellular O2

•− in (a) primary microglia and (c) BV2 cells, after treatment with Aβ1–42

(20 nM), phorbol myristate acetate (PMA, 10 ng/mL) or LPS (1 μg/mL) in the presence or absence of CR1 blocking antibody (1 mg/mL) for 24 h or apocynin (10 mM) duringDHE loading, as a control for the specificity of DHE fluorescence. (b, d) Quantification of the percentage of microglia positive for O2

•− after treatments given in (a) (black bars).(e,f) siRNA knockdown of Crry, the mouse homologue to CR1 results in a modulation of O2

•− production after treatment with Aβ1–42 (20 nM) or LPS (1 μg/mL) for 24 h, withapocynin as a control for DHE fluorescence (Apo, 10 mM). Data were analysed by ANOVA followed by Student's t-test with pair-wise analysis as indicated. ***ρ b 0.001,**ρ b 0.01 and *ρ b 0.05.

146 H. Crehan et al. / Neurobiology of Disease 54 (2013) 139–149

Fig. 6. CR1 functional blocking antibody does not alter glutathione production or iNOS expression in microglia. (a) CR1 functional blocking antibody does not alter glutathione pro-duction in primary rat microglia investigated by live cell imaging of monochlorobimane (MCB) fluorescence. Quantification of meanMCB fluorescence per microglial cell after treat-ment with Aβ1–42 (20 nM) or LPS (1 μg/mL) in the presence or absence of CR1 blocking antibody (1 mg/mL) for 24 h (white bars) and co-treatment with ethacrynic acid (1 mM)as a control for MCB fluorescence (grey bars). Data are the mean ± SEM of cellular fluorescence in approximately 50 cells per field, 3 fields per coverslip, 3 coverslips per conditionfrom 3 independent experiments analysed with ImageJ software. Data were analysed by ANOVA followed by pair-wise analysis with control using Student's t-test. (b) CR1 func-tional blocking antibody does not modulate iNOS expression. Western blot and densitometric analysis of iNOS expression in (bi) primary rat microglia or (bii) BV2 microgliaafter treatment with Aβ1–42 (20 nM) or LPS (1 μg/mL) in the presence or absence of CR1 blocking antibody (1 mg/mL) for 24 h. Blots are representative. Data analyses were car-ried out on three separate blots from three independent experiments and compared by ANOVA and with control using Student's t-test.

147H. Crehan et al. / Neurobiology of Disease 54 (2013) 139–149

production (Jaconson andWeis, 2008; Killick et al., 2012). Our results forCrry knockdown inmurine BV2microglia are the same as those using theCR1 blocking antibody in rat microglia, suggesting similar downstreampathways. Considering the recent data demonstrating a genetic link be-tween CR1 and LOAD, it is crucial to determine what impact this has onthe cellular and molecular consequences of microglial activation in AD.

Acknowledgments

This study was supported by the Corsan Family Foundation.

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