Background
Alzheimer’s disease (AD) is a neurodegenerative disorder characterised by senile plaques and neurofibrillary tangles. An important component of the plaques in the human brain is amyloid-β 1–42 (Aβ1-42), a 42 amino acid peptide fragment derived from sequential proteolytic cleavage of the amyloid precursor protein by beta- and gamma-secretases [
1]. Aβ1-42 plays a central role in mediating neurotoxicity and activates glial cells (astrocytes as well as microglia). Elevated levels of non-fibrillar [
2,
3] and fibrillar Aβ1–42 [
4,
5] lead to the release of proinflammatory cytokines by activated glial cells. This may subsequently lead to gliosis and cytotoxicity in neurons [
6‐
8]. However, the role of glial cells in the formation of amyloid plaques in Alzheimer’s disease remains unknown [
9,
10]. The underlying pathogenic mechanisms are not well understood, especially regarding the initial steps of cellular Aβ1-42 uptake and the induction of signal transduction and the consequence for the development of the disease. Recent studies suggest that the chemotactic G-protein-coupled receptor, formyl-peptide-receptor-like 1 (FPRL1), is involved in Aβ1-42 and PrP
106-126-induced activation and the internalisation in glial cells [
11‐
13]. Furthermore it is indicated that the FPRL1 is expressed on astrocytes and microglia and plays an essential role in the inflammatory response [
14].
However, it should be noted that a variety of further receptors were discussed to participate in Aβ1-42-induced glial cell activation and internalisation. In fact, previous results suggest an involvement of the scavenger receptor MARCO (macrophage receptor with collagenous structure)[
15,
16] and the receptor for advanced glycation endproducts (RAGE)[
17]. MARCO is a membrane glycoprotein that can bind to chemically modified low-density lipoproteins or Gram-positive and Gram negative bacteria [
18,
19]. For MARCO, our recent work shows no involvement in Aβ1-42-induced glial cell activation [
11]. However, we are able to show a physical and functional interaction between FPRL1 and MARCO in MARCO ligand fucoidan-induced signaling and in the host defense against brain infections [
11,
14]. RAGE is a multiligand receptor belonging to the immunoglobulin superfamily [
20].
In this study we analysed the expression of formyl peptide receptors and RAGE and their glial localisation using fluorescence microscopy and real-time RT-PCR in an APP/PS1 transgenic mouse model. The murine FPR gene family has at least six members in contrast to only three in humans.
Fpr1 encodes for the murine FPR1 (mFPR1), which is considered to be the murine orthologue of human FPR1, whereas
Fpr-rs2 (mFPR2) encodes for receptors that are similar to the human formyl peptide receptor like 1 (FPRL1) [
21]. Furthermore, we examined the involvement of FPRL1, FPR1 and RAGE in Aβ1–42-induced signalling by measured the extracellular-signal regulated kinase 1/2 (ERK 1/2) phosphorylation and cAMP levels in rat glial and transfected HEK293 cells. Also, the involvements of the RAGE receptor ligands S100B as well as AGE-induced signalling were examined. In addition, a functional and physical interaction between FPR1, FPRL1 and RAGE using co-immunoprecipitation and ERK1/2 phosphorylation and cAMP level measurement in rat glial and transfected HEK293 cells was determined. Furthermore, we analysed and quantified the co-localisation between different receptors and S100B or Aβ1–42 in transfected HEK293 cells using fluorescence microscopy. The results suggest that FPRL1 as well as FPR1 play an essential role in Aβ1–42-induced signal transduction in glial cells, and also show the capability of formyl peptide receptors to expand its ligand spectrum by interacting with the RAGE receptor.
Methods
Reagents
Human Aβ1–42 and formyl-peptide-receptor antagonist WRW4 [
22] were purchased from Dr. P. Henklein (Charité, Berlin, Germany). Peptides were dissolved at 1 and 10 mM concentration in dimethylsulfoxide (DMSO), and Aβ1–42 is present in the soluble form. DMSO used as vehicle in a concentration of 0.1% showed no significant effects in the experiments. The RAGE agonists Advanced Glycation Endproduct-Bovine Serum Albumine (AGE-BSA) and S100 calcium binding protein B (S100B) were purchased from BioCat (Heidelberg, Germany) and Merck (Darmstadt, Germany). Forskolin and formyl-methionyl-leucyl-proline (fMLF) were obtained from Sigma-Aldrich, Munich, Germany.
APP/PS1 transgenic mouse model
The APP/PS1 transgenic mouse model used in this study (APPswe/PS1dE9-Line 85) co-expresses a chimeric mouse/human amyloid precursor protein (APP) 695 harboring the Swedish K670M/N671L mutations (Mo/HuAPPswe) and human presenilin 1 (PS1) with the exon-9 deletion mutation (PS1dE9) under control of the mouse prion protein promoter [
23]. The mouse line was obtained from Jackson Laboratory (B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/J; Stock-Number: 005864; Promoter:
Prnp, prion protein; created by David Borchelt 2006, University of California, referring to Jackson Laboratory). Wildtype littermates were used as controls. Mice were used at 12 months of age. Mice were fed standard lab chow and water
ad libitum and kept under a 12 h light/dark cycle.
Cloning of cDNA and plasmids
The pcDNA3.1-hFPRL1 plasmid containing a neomycin resistance gene was kindly provided by Dr. U. Rescher (Münster, Germany). The pcDNA3.1-hFPR1 containing a neomycin resistance gene was obtained from UMS cDNA Resource Center (Rolla, Missouri, USA). The pcDNA3.1-hRAGE and –hRAGEΔcyto plasmids, containing a neomycin resistance gene, were kindly provided by Prof. R. Donato (Perugia, Italy). RAGEΔcyto (ΔRAGE) is a RAGE mutant lacking the cytoplasmic domain [
24]. The inserts were subcloned into a pcDNA3.1 expression vector containing a Zeocin™ resistance gene (Invitrogen, Karlsruhe, Germany).
Cell culture
HEK293 cells (American Type Culture Collection, Rockville, MD, USA) were subcultivated in Dulbecco’s modified Eagle’s medium (DMEM; PAA Laboratories, Pasching, Austria) supplemented with 10% fetal calf serum (FCS) and 1% penicillin/streptomycin (Carl Roth, Karlsruhe, Germany). The transfection and selection of HEK293 cells expressing hFPRL1 was described previously [
11]. Using Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s protocol, HEK293 cells (American Type Culture Collection, Rockville, MD, USA) were first transfected with either pcDNA3.1-hFPR1 or –hFPRL1 plasmid. Stable transfectants were selected in the presence of 500 μg/ml G418 (Carl Roth, Karlsruhe, Germany). To generate hFPR1/hFPRL1 cell lines co-expressing hRAGE or hRAGEΔcyto, cells were subjected to a second round of transfection with pcDNA3.1-hRAGE or -hRAGEΔcyto and selected in the presence of 100 μg/ml Zeocin™ (Invitrogen, Karlsruhe, Germany).
Isolated cerebral cortices and rostral mesencephali from wistar rats (P2) were stripped of the meninges, minced and dissociated enzymatically with trypsin from bovine pancreas (Sigma-Aldrich, Taufkirchen, Germany) in phosphate-buffered saline and 50 μg/ml DNase I (Roche Molecular Biochemicals, Mannheim, Germany) for 30 min at 37°C and crushed mechanically with Pasteur pipettes. Astrocytes were prepared according to the protocol of McCarthy and DeVellis [
25], which allows the preparation of nearly pure cultures of astrocytes (> 97%) and cultivated in Dulbecco’s modified Eagle’s medium (DMEM; PAA Laboratories, Pasching, Austria) supplemented with 10% FCS. Suspended microglial cells were plated in 75 cm
2 cell culture flasks (Sarstedt, Nümbrecht, Germany) in microglial cell growth medium and harvested as described previously [
13]. The microglial cell growth medium (DMEM) containing 10% FCS (heat inactivating from 44-53°C) and antibiotics (penicillin and streptomycin). After about ten days, the cells begin to move away from the cell layer and swim in the supernatant. The cells are collected and then seeded in normal medium (DMEM, 10% FCS heat inactivated at 56°C, penicillin and streptomycin). Prior to replating microglial cells for different assays, cell number and viability were estimated by trypan blue exclusion. This procedure increased the purity of the microglial preparation to > 98% with only very few remaining astrocytes.
RNA isolation and real time RT-PCR
Total RNA was isolated using the peqGold Trifast reagent (Peqlab, Erlangen, Germany) according to the manufacturer’s instructions. RNA samples were reverse-transcribed by moloney murine leukemia virus (MMLV) reverse transcriptase (Fermentas, Burlington, Canada) and random hexamer primers (Invitrogen, Darmstadt, Germany). The cDNA products were used immediately for SYBR green (Applied Biosystems, Darmstadt, Germany) real-time RT-PCR for mus(m)FPR1, mFPR2 and RAGE. Gene expression was monitored using the StepOne Plus apparatus (Applied Biosystems, Darmstadt, Germany) according to manufacturer’s protocol [
26]. Relative quantification was performed using the ΔCt method which results in ratios between target genes and a housekeeping reference gene (18 s). cDNA was amplified using gene-specific primers described in Table
1. The specificity of the amplification reaction was determined by a melting curve analysis. We performed relative quantification of the signals normalising to the Geomean of the gene signal from m18s, ribosomal protein L13a (RPL13a) and TATA box binding protein (TBP; all primers Eurofins MWG Operon, Ebersberg, Germany, for primer sequences please see Table
1) for SYBR Green real time RT-PCR.
Table 1
Primer sequences for real-time RT-PCR gene analysis
mFPR1 | for | 5’-CACAATCCAAGTCCGTGAACG-3’ | 57 |
| rev | 5’-CAGCTGTTGAAGAAAGCCAAGG-3’ | |
mFPR2 | for | 5’-CTGAATGGATCAGAAGTGGTGG-3’ | 56 |
| rev | 5’-CCCAAATCACTAGTCCATTGCC-3’ | |
mRAGE | for | 5’-TGACCGCAGTGTAAAGAGTCCC-3’ | 59 |
| rev | 5’-CCCTTAGCTGGCACTTAGATGG-3’ | |
m18s | for | 5’-GAATAATGGAATAGGACCGCGG-3’ | 57 |
| rev | 5’-AAGAATTTCACCTCTAGCGGCG-3’ | |
mTBP | for | 5’-AGAACAATCCAGACTAGCAGCA-3’ | 58 |
| rev | 5’-GGGAACTTCACATCACAGCTC-3’ | |
mRPL13a | for | 5’-GAATAATGGAATAGGACCGCGG-3’ | 60 |
| rev | 5’-GGCTCGGAATTGGTAGGGG-3’ | |
Western blotting
For Western blot analysis of MAP kinase phosphorylation, rat glial or HEK293 cells were seeded in DMEM containing 10% FCS. Cells were harvested in a lysis buffer (50 mM Tris pH 7.5, 100 mM NaCl, 5 mM EDTA, 1% Triton, 2 mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1 mM glycerol 2-phosphate, 1 mM phenylmethylsulfonylfluoride). Proteins (5 μg for pERK and ERK2) were resolved in SDS sample buffer, and a Western blotting procedure was performed as previously described in detail [
12]. Membranes were incubated with polyclonal primary antibodies against pERK1/2 (1:500; sc-7383; Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C and subsequent detection was performed with peroxidase-labeled secondary antibodies (Sigma-Aldrich, Munich, Germany). Antibody binding was detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Essex, UK). The membranes were then stripped and re-probed with anti-ERK2 (1:500; sc-1647; Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibody as a loading control. The Western blot bands were densitometrically evaluated with the program Quantity One (Bio-Rad, Munich, Germany), the pERK-bands were adjusted with their respective ERK-bands and subsequently, the values were referred to control (=100%).
Co-immunoprecipitation
Co-immunoprecipitation was performed as previously described in detail in Brandenburg et al. 2010 [
10]. Cells (1.5 x 10
6/plates for astrocytes and transfected HEK293 cells, 10 x 10
6/plates for microglia) were plated onto 100 mm dishes and grown to 80% confluence. Cells were washed twice with phosphate-buffered saline and harvested into ice-cold lysis buffer (10 mM Tris–HCl, pH 7.6, 5 mM EDTA, 3 mM EGTA, 250 mM sucrose, 10 μM iodoacetamide, and a mixture of proteinase inhibitors: 0.2 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 1 μg/ml pepstatin A, 1 μg/ml aprotinin, and 10 μg/ml bacitracin). Subsequently, the cell suspensions were incubated for 30 min on ice and homogenised. The homogenates were then centrifuged at 500 g for 5 min at 4°C to remove not disrupted cells and nuclei. Membranes were pelleted at 20.000 g for 30 min at 4°C, and pellets were lysed in detergent buffer (20 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM EDTA, 3 mM EGTA, 4 mg/ml β-dodecylmaltoside, and the proteinase inhibitors listed above) for 1 h on ice. Lysates were centrifuged at 20.000 g for 30 min at 4°C, and the protein content of the resulting supernatant was determined using a BCA protein assay (Pierce, Rockford, IL, USA). Receptor proteins were immunoprecipitated with 50 μl protein G agarose beads preloaded with 5 μg anti-FPR1 or anti-FPRL1 antibodies (for rat glial cells from Santa Cruz; for HEK cells transfected with hFPR1 or hFPRL1, from MBL, Woburn, MA, USA or Abcam (FPRL1, ab13177)) overnight at 4°C. Beads were washed five times with detergent buffer and eluted into 200 μl of SDS-sample buffer (62.5 mM Tris–HCl, pH 6.8, 2% SDS, 20% glycerol, 100 mM DL-dithiotreitol, and 0.005% bromphenol blue) at 60°C for 20 min. After SDS–polyacrylamide gel electrophoresis and electroblotting, membranes were incubated with rabbit anti-FPR1, FPRL1, RAGE (Abcam, Cambridge, UK; ab3611) antibodies overnight at 4°C. Immunoreactive bands were visualized using the enhanced chemiluminescence detection system mentioned above.
Determination of receptor activity by measuring cyclic AMP accumulation
5 x 104 astrocytes/well, or 7.5 x 104 microglia/well or 1.5 x 104 transfected HEK cells were seeded in a 96 well culture plate with DMEM containing 10% FCS and incubated for 48 h. The medium was removed and replaced with 100 μl of serum-free Opti-MEM medium containing 10 μM forskolin (for astrocytes, Sigma) or 25 μM forskolin (for microglial or HEK cells) plus agonist. Different forskolin concentrations were used because there is a difference in cell sensitivities to forskolin-stimulated adenylate cyclase activity. For the formyl peptide receptors antagonist WRW4, glial cells were pre-incubated in Opti-MEM medium containing 10 μM WRW4 for 30 min. The cells were incubated at 37°C for 15 min, and the reaction was terminated by the removal of the culture medium and addition of 70 μl lysis buffer (for HEK cells) or 100 μl lysis buffer (for glial cells) followed by 10 min incubation at room temperature. cAMP content was determined using a commercial available colorimetric kit (Millipore, Schwalbach, Germany).
Fluorescence microscopy
Formalin-fixed and paraffin-embedded 5 μm whole coronary brain sections were examined. For immunofluorescence staining, sections were deparaffinized, pretreated for 3 x 7 min with microwaving in citric acid buffer, permeabilized with 0.1% Triton X in PBS for 10 min at room temperature and after blocking with 1.5% bovine serum albumine (BSA; Sigma-Aldrich, Taufkirchen, Germany) in TRIS incubated with either polyclonal rabbit anti-mFPR1 (1:100; ab101701; Abcam, Cambridge, UK), anti-mFPR2 (1:100; sc-18191; Santa Cruz Biotechnology), anti-RAGE (1:100; ab3611; Abcam, Cambridge, UK) and monoclonal mouse anti-GFAP (1:250; ab10062; Abcam, Cambridge, UK) or polyclonal goat anti-Iba1 (1:100; ab5076; Abcam, Cambridge, UK) overnight at 4°C. Finally, the slices were incubated with donkey anti-rabbit AlexaFluor 488 (Molecular Probes, Darmstadt, Germany) and goat anti mouse Cy3 (Sigma-Aldrich, Taufkirchen, Germany) or rabbit anti-goat AlexaFluor 555 (all 1:250; Molecular Probes, Darmstadt, Germany) for 1 h at room temperature.
Fluorescence staining of primary and cell cultures
Glial or HEK293 cells were grown on glass coverslips. Coverslips were previously coated with poly-L-lysine according the instruction (Sigma-Aldrich, Munich, Germany). Transfected HEK293 cells were exposed to RAGE agonist S100B (5 μg/ml = 2.4 μM) for 2 h at 37°C. After fixation with 4% paraformaldehyde and 0.2% picric acid in a phosphate buffer at pH 6.9 [
11] for 30 min and permeabilisation with 0.1% TritonX in PBS, cells were blocked in 0.1 M Tris–HCl pH 7.5 containing 1.5% BSA for 10 min. Coverslips were incubated at 4°C overnight with primary antibodies for FPR, FPRL1 or RAGE (for rat glial cells: FPR/FPRL1 from Santa Cruz; RAGE from AbD Serotec, Düsseldorf, Germany (goat) or Abcam (rabbit); for transfected HEK cells: hFPR1 from MBL (rabbit) or Santa Cruz (goat); hFPRL1 from Abcam (rabbit) or Everest Biotech, Oxfordshire, UK (goat); RAGE from AbD Serotec and S100B from Abcam (ab868, rabbit); Aβ1–42 from Santa Cruz (sc-58495, mouse) and diluted in TRIS containing 1.5% BSA. Finally, the coverslips were incubated with donkey anti-rabbit or anti-goat AlexaFluor 488 (Molecular Probes, Darmstadt, Germany) and goat anti rabbit Cy3 (Millipore) or rabbit anti-goat AlexaFluor 555 (all 1:250; Molecular Probes, Darmstadt, Germany) for 1 h at room temperature. Nuclear staining was performed with bisbenzimide (Sigma-Aldrich, Taufkirchen, Germany). Cells were digitally photographed using a LSM7 DUO laser confocal microscope (Zeiss, Göttingen, Germany).
Determination of co-localisation
The Pearson coefficient is a measure of the strength of linear relationship between two signals. Instead, Spearman coefficient is a measure of how well any monomeric function between the variables can describe the relationship. The Pearson-Spearman correlation (PSC) co-localisation plugin for ImageJ was used to calculate co-localisation between target receptors RAGE, FPR1 or FPRL1 and S100B or Aβ1-42 [
27,
28]. A subselection as a region of interest (ROI) was set up around the plasma membrane using the Selection Brush with a width of 25 pixels. The value for the background intensity noise threshold was set up to 40 to calculate the coefficients.
Statistical analysis
All in vitro experiments were performed at least in triplicate and the values are expressed as mean ± SEM. For statistical comparison, ANOVA test was used followed by Bonferroni’s correction. A value of p < 0.05 was considered statistically significant. The GraphPad Prism 5.0 software was used for statistical calculation (Graph Pad Software, San Diego, CA, USA).
Discussion
Our study shows the involvement of formyl peptide receptors FPR1 and FPRL1 in Aβ1-42-induced signal transduction in glial and transfected HEK293 cells. This confirmed and extended our previous results for the FPRL1 [
11,
13]. Interestingly, our results also show an involvement of the high affinity receptor FPR1 in Aβ1-42-induced signal transduction in transfected HEK293 cells. A previous result from Le et al. [
29] reported that Aβ1–42 is able to activate FPR1 in transfected HEK293 and a rat basophilic leukemia cell line, but that the receptor’s efficacy in mediating cell migration and activation is much lower than that of FPRL1. In our transfected HEK293 cells, we did not observe a clear difference between FPR1 and FPRL1 expressing cells in Aβ1-42-induced ERK1/2 phosphorylation or a change of cAMP accumulation (Figures
8 and
9). Our previous results with small inferring RNA against FPR1 in primary astrocytes did not result in an inhibition of Aβ1-42-induced ERK1/2 phosphorylation. However, the results could be explained by the low FPR1 expression in the astrocytes as our previous results had shown [
11]. It should be noted that microglial cells show a higher endogen FPR1 expression. In addition, other receptors are discussed for Aβ1-42-induced glial cell activation. Other groups have reported that the scavenger receptor MARCO (macrophage receptor with collagenous structure), a cell surface glycoprotein, plays a role in the internalisation and Aβ1–42-mediated microglia activation [
16]. Our previous results in astrocytes and transfected HEK293 cells did not show an involvement of MARCO in Aβ1-42-induced ERK1/2 phosphorylation and change of cAMP accumulation [
11]. Recent works suggested that scavenger receptors mediate Aβ internalisation in microglial cells or activation of perivascular macrophages [
30,
31]. A further receptor, which is discussed in the context of the Aβ1-42-induced glial cell activation, is RAGE. Previous studies had shown that RAGE binds Aβ1–42 with high affinity in microglial cells and neurons [
17]. It was suggested that RAGE-dependent signaling in microglial cells contributes to neuroinflammation and Aβ accumulation as well as impaired learning/memory in an APP/PS1 transgenic mouse model [
32]. The crossing of these mice with an inactive RAGE mutant resulted in a decrease of Aβ levels and amyloid plaque load. It should be noted that other working groups were not able to determine an effect of RAGE in an APP/PS1 transgenic mouse model [
33]. However, our present results show a strong increase of mice formyl peptide receptors mFPR1 and 2, the mice homologon to human FPR1 and FPRL1, as well as RAGE expression in the hippocampus and for mFPR2 also in the cortex of the used APP/PS1 transgenic mice (Figure
3). Nevertheless, it must be noted that nothing is known about the increase of receptor expression in the human AD brain. The increase was co-localised to astrocytes and microglia cells (Figures
1 and
2). Also for MARCO, we were able to detect a strong increase of expression co-localised to glial cells (data not shown). This extended previous results for RAGE and MARCO [
34,
35]. Altogether, the increasing receptor expression during the course of Aβ1-42 deposition in APP/PS1 transgenic mice could be a sign of enhanced inflammation including glial cell activation. The receptor activation of different signal transduction pathways including NADPH oxidase or NFκB is increased. This may be associated with an increased production of proinflammatory cytokines and reactive oxygen species [
36‐
38]. However, a recent study showed that the mFPR2 acted as an anti-inflammatory receptor [
39]. It could also be a sign of the increased uptake and clearance of Aβ. Our previous work showed the involvement of FPRL1 in glial cells mediated Aβ1-42 internalisation [
13]. Possible, the increase of receptor expression represents a protective function against increased Aβ concentration in the brain. The receptor-mediated internalisation could be an interesting point to influence the plaque as well as AD development. In this context, previous results showed that the glial cells are able to internalise Aβ, although the uptake is dependant on Aβ forms and size [
13,
40‐
42]. Further studies must explore this topic.
Interestingly, in this study, we have demonstrated a physical interaction between FPR1 or FPRL1 and RAGE in glial and transfected HEK293 cells by co-immunoprecipitation and fluorescence microscopy for the first time (Figures
567). The densitometric quantification and quantitative statistical co-localisation show that the interaction was stronger between FPRL1 and RAGE compared to FPR1 and RAGE in glial cells. Differences between astrocytes, microglial cells and in transfected HEK293 cells were detectable. In FPR1 as well as FPRL1 and ΔRAGE, a RAGE mutant lacking the cytoplasmic and transducing domain, co-expressing HEK293 cells, showed that the interaction was significantly but not completely reduced (Figure
7). The intracellular domain is possibly involved in the binding between FPR1/FPRL1 and RAGE. The function of this interaction remains unclear. However, our previous results showed a physical and functional interaction between FPR1/FPRL1 and MARCO [
11]. The findings suggest that the receptors interaction influences the receptor activity by cross-phosphorylation and desensitisation of downstream signalling pathways. Such influence was also detected for other receptor classes. For example, studies have shown an extensive cross-talk between opioid- and somatostatin-receptors mediated analgesic responses and pain-processing pathways [
43,
44]. For the interaction between FPR1/FPRL1 and MARCO as well as RAGE, it is possible that the pattern recognition receptors complement, modulate and enhance each other of their effects. By this, they could enhance the response of the innate immune system or even the inflammatory response in Alzheimer's disease.
For the signal transduction pathways, our results for ERK1/2 phosphorylation and the change of cAMP accumulation show that the specific formyl peptide receptors antagonist WRW4 inhibited RAGE ligands S100B- as well as AGE-BSA-induced glial cell activation (Figure
4). Furthermore, the findings confirmed the importance of the FPRL1 in Aβ1–42-induced signal transduction. However, the results with transfected HEK293 cells showed that S100B- or AGE-BSA-induced ERK1/2 phosphorylation and inhibition of cAMP level is not mediated by FPRL1, whereas FPR1 is involved (Figures
8 and
9). Interestingly, FPR1 expressing HEK293 cells also mediate an Aβ1–42-induced ERK1/2 phosphorylation and a change of cAMP accumulation, whereas our results did not confirm an involvement of RAGE. In addition, a co-transfection of RAGE in FPR1 or FPRL1 expressing cells resulted in an amplification of the S100B- as well as AGE-BSA-induced signal transduction. For the involvement of the receptors in S100B or Aβ1–42-induced signalling, the immunofluorescence and quantitative statistical co-localisation confirmed that RAGE but also FPR1 binding S100B, whereas the co-localisation of Aβ1–42 and FPRL1 is strongest (Figure
10). Altogether, the findings suggest complementary and synergic action between the receptors. The elucidation of consequences for receptor activities and inflammation as well as the progression of the AD need further investigations. Nevertheless, previous kinetics studies showed a binding of Aβ and RAGE in endothelial cells and cortical neurons [
45]. I can be assumed that the binding depends on the Aβ forms (monomeric, oligomeric or fibrillary). For the present study, we used non-fibrillary Aβ1-42. In addition, it must be noted that in the primary cells, the mediation of the effect by other receptors cannot be excluded. Furthermore, it was shown that microglial cells behave differently depending upon age in interaction with fibrillary Aβ [
46]. Further studies with receptor-deficient cells should bring more clarity here.
In conclusion, there is a substantial interest to identify the cell surface receptors that bind and mediate the intracellular effects of Aβ1-42 in glial cells. Consequently, FPR1/FPRL1 and RAGE or MARCO interactions may explain how formyl peptide receptors interact with a menagerie of structurally diverse pro- and anti-inflammatory ligands associated with different diseases including amyloidosis, Alzheimer’s disease, prion disease and HIV, or with bacterial components [
14,
21]. Interactions with other receptors may support and modulate the cellular reaction to such structurally diverse ligands by the formyl peptide receptors. Altogether, we hypothesise that formyl peptide receptors play a central role in neurodegenerative mechanisms and physiological regulatory processes.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
AS, JM and LOB designed as well as performed experiments, and drafted the manuscript. AE and FM performed experiments. SJ helped to accomplish experiments and revised the manuscript. CJW and TP co-conceived of the study, participated in its design and coordination, and helped draft the manuscript. All authors have read and approved the final version of this manuscript.