Article pubs.acs.org/jpr
Amyloid-Beta-Activated Human Microglial Cells Through ERResident Proteins YongCheol Yoo,†,‡,§,▽ Kyunghee Byun,∥,⊥,▽ Taewook Kang,§ Delger Bayarsaikhan,∥ Jin Young Kim,§ Seyeoun Oh,∥ Young Hye Kim,§ Se-Young Kim,§ Won-Il Chung,† Seung U. Kim,# Bonghee Lee,*,∥,⊥ and Young Mok Park*,‡,§ †
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Daejeon 305-701, Republic of Korea ‡ Center for Cognition and Sociality, Institute for Basic Science (IBS), 5, Hwarang-ro 14-gil, Daejeon 305-811, Republic of Korea § Mass Spectrometer Research Center, Korea Basic Science Institute, 52 Eoeun-dong, Ochang, Chungcheongbuk-do 363-883, Republic of Korea ∥ Center for Genomics and Proteomics, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, 7-45, Songdo-dong, Yeonsu-ku, Incheon 406-840, Republic of Korea ⊥ Department of Anatomy and Cell Biology, Gachon University Graduate School of Medicine, 7-45 Songdo-dong, Yeonsu-gu, Incheon 406-840, Republic of Korea # Department of Medicine, University of British Columbia, 2775 Laurel Street, Vancouver, British Columbia V5Z 1M9, Canada S Supporting Information *
ABSTRACT: Microglial activation in the central nervous system is a key event in the neuroinflammation that accompanies neurodegenerative diseases such as Alzheimer’s disease (AD). Among cytokines involved in microglial activation, amyloid β (Aβ) peptide is known to be a key molecule in the induction of diverse inflammatory products, which may lead to chronic inflammation in AD. However, proteomic studies of microglia in AD are limited due to lack of proper cell or animal model systems. In this study, we performed a proteomic analysis of Aβ-stimulated human microglial cells using SILAC (stable isotope labeling with amino acids in cell culture) combined with LC−MS/MS. Results showed that 60 proteins increased or decreased their abundance by 1.5 fold or greater. Among these, ER-resident proteins such as SERPINH1, PDIA6, PDIA3, and PPIB were revealed to be key molecular biomarkers of human microglial activation by validation of the proteomic results by immunostaining, PCR, ELISA, and Western blot. Taken together, our data suggest that ER proteins play an essential role in human microglial activation by Aβ and may be important molecular therapeutic targets for treatment of AD. KEYWORDS: amyloid β, human microglia, tandem mass tags, LC−MS/MS
1. INTRODUCTION
Activated microglia are numerous in human and animal AD brain tissues, and many of them are found to accumulate in Aβ plaques in human AD brain tissues. This implies an important role for activated microglial cells in the progression of AD. Activated microglial cells also produce various proinflammatory factors, which may lead to chronic inflammation, and these neuroinflammations may accelerate the progression of AD.7−10 The changes in Aβ-stimulated microglia have been reported only in a murine model with a 2D gel electrophoresis approach.11 Even though microglial activation has been recognized as a therapeutic target in AD, promising microglial therapies have not yet been developed.10,12−15 However, there are no reports on the proteomic changes accompanying
Alzheimer’s disease (AD) is the most common neurodegenerative disorder in humans. The most prominent pathological features of AD are formation and accumulation in the brain of amyloid beta (Aβ) plaques and neurofibrillary tangles (NFTs). Insoluble extracellular aggregates, Aβ plaques, are mainly composed of Aβ1−40 and Aβ1−42 peptides, which are produced by proteolytic cleavage of the amyloid precursor protein and have been regarded as one of the main contributors to AD development.1−3 Microglial cells (brain-resident macrophages) are activated in response to many pathologic changes in central nervous system (CNS) homeostasis. Activation of microglia leads to several inflammatory responses such as scavenging, phagocytosis, cytotoxicity, antigen presentation, synaptic stripping, promotion of repair, and extracellular signaling to restore CNS homeostasis.4−6 © XXXX American Chemical Society
Special Issue: Environmental Impact on Health Received: September 11, 2014
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2. MATERIALS AND METHODS
distilled water, the gel pieces were rehydrated with 12.5 μg/μL trypsin (Promega) solution (in 50 mM NH4HCO3) and incubated overnight at 37 °C. Following digestion, peptides were extracted with 50% ACN/5% TFA (trifluoroacetic acid) solution at room temperature for 40 min. The supernatants were dried by vacuum centrifugation. The dried peptides were stored at −20 °C until MS analysis.
2.1. Human Microglial HMO6 Cell Culture and SILAC
2.5. Nanoflow Liquid Chromatography and Tandem MS/MS Analysis
activation of human microglial cells by Aβ and functional validations of these. Here we report for the first time the proteomic analysis of Aβ-stimulated human microglia using SILAC combined with LC−MS/MS and comprehensive analysis as well as validation of the proteomic results in brain tissues of an AD mouse model.
Our study organism was HMO6, an immortalized human microglial cell line. HMO6 cells were grown in Dulbecco’s modified Eagle’s medium-high glucose (DMEM-HG, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 20 mg/mL gentamicin (Sigma), in an incubator at 37 °C under 5% CO2/95% air as previously reported.16−18 For SILAC labeling, SILAC amino acids (MS50011, Invitrogen), SILAC Phosphoprotein ID and Quantitation Kit with Lysine (SP10005, Invitrogen), and SILAC medium (DMEM, Invitrogen) were used. Cells were grown in light medium containing [U−12C6]-L-lysine and [U−12C6]-L-arginine and heavy medium containing [U−13C6]-L-lysine and [U−13C6]-Larginine for 2 weeks for at least five generations. Aβ1−42 (Sigma) was added to the HMO6 cells at a concentration of 500 nM, and the cells were then incubated for up to 12 h before protein extraction.
The dried peptides were resuspended in 0.1% formic acid (FA) and analyzed online by nanoelectrospray ionization using a 7T LTQ-FT mass spectrometer (Thermofinnigan, USA) coupled to a UPLC system (Waters, USA). The peptide solution was trapped on a C18 trap column (180 μm i.d. × 2 cm, particle size 5 μm; Waters) at a flow rate of 5 μL/min. The trapped peptides were separated on a 20 cm homemade microcapillary column consisting of C18 (Aqua; particle size 3 μm) packed into 100 μm silica tubing with an orifice i.d. of 360 μm. The samples were loaded onto the column and then eluted with a 12 min gradient covering 10 to 50% of buffer B (buffer A: 0.1% FA in water; buffer B: 0.1% FA in ACN). Eluted peptides were electrosprayed directly off the column into the mass spectrometer at a voltage of 2.2 kV. A cycle of MS/MS spectra consisted of one full-scan mass spectrum (scan range of 400− 2000 m/z, resolution 100 000, isolation window ±1 Da) and up to five ion-trap data-dependent scans with the linear ion trap analyzer. All MS/MS spectra were recorded using a normalized collision energy of 35%. Target ions selected for MS/MS were dynamically excluded for 60 s. An activation q of 0.25 and an activation time of 30 ms were applied in MS/MS acquisitions.
2.2. Animals and Brain Tissue Preparation
Ten 5XFAD (AD model) mice were provided and used as previously described.19 For tissue preparation, all mice were perfused trans-cardially with 100 mL of heparinized saline at 18 °C, followed by 100 mL of 4% paraformaldehyde−lysine periodate in 0.1 M sodium phosphate buffer (pH 7.4). The brains were removed, placed in the same fixative for 4 h at 4 °C, and transferred into ice-cold 0.1 M phosphate-buffered saline (PBS) containing 20% sucrose. The brains were cut in a transverse plane at 10 μm thickness with a freezing microtome and were stored at −80 °C until use.
2.6. Protein Identification, Quantification, and Proteomic Data Analysis
The MS raw data from each gel fraction were processed for protein identification using the MaxQuant software (v.1.2.2.5) with a peptide mass tolerance of 20 ppm and a fragment mass tolerance of ±0.5 Da at a false discovery rate (FDR) of 1% for both the protein and peptide.21 All peak lists were identified against the human International Protein Index (IPI) database with decoy (IPI version 3.68, 87 061 entries) using the following parameters: enzyme, trypsin; missed cleavages ≤1; fixed modification, carbamidomethyl (C); variable modifications, oxidation (M) and phosphorylation (S,T,Y). Protein identifications were accepted only if they contained at least two peptides. Also, only the proteins with at least one unique peptide were accepted for quantification. Triple biological replicates were used to analyze proteomic data for this application. For relative protein quantification, the evidence.txt output file from MaxQuant was imported into Microsoft Excel and then for each IPI name, filtered as follows: MaxQuant score ≥100, not a contaminant or reversed sequence. An intensity from the respective IPI name was calculated by summing all normalized intensity using only unique peptides. The averaged ratio of normalized intensities was then calculated between Aβ nontreatment (light) and Aβ treatment (heavy). Proteins were accepted as a difference in their abundance with a ratio of 1.5 or greater between heavy and light. Also, a ratio was reserved for proteins having a equivalent ratio at least two of trials. Finally, Student’s t test was calculated for each IPI name (P < 0.05).
2.3. Protein Preparation
For protein extraction, HMO6 cells were harvested and washed three times with ice-cold PBS. They were then homogenized in lysis buffer (7 M urea, 2 M thiourea, and 4% CHAPS), sonicated, and centrifuged at 15 000 rpm for 15 min at 4 °C. The supernatant was collected and protein concentrations were measured using the Bradford assay (Pierce Biotechnology, Rockford, IL). 2.4. SDS-PAGE and In-Gel Digestion
For SDS-PAGE analyses, both heavy and light protein extracts were mixed in equal amounts, and 100 μg of the combined proteins was separated on a 10% SDS-polyacrylamide gel (18 × 16 cm). The SDS-PAGE (Bio-Rad) was run at 80 V for 30 min and then at 120 V until the end of the run. After separation, the gel was stained with Coomassie Brilliant Blue (CBB) R-250 and was cut into 10 slices. The excised bands were digested according to the in-gel digestion protocol.20 The band pieces were washed twice with 30% methanol for 5 min and destained with 50% acetonitrile (ACN) containing 10 mM NH4HCO3. They were then incubated with 10 mM DTT (dithiothreitol) in 100 mM NH4HCO3 for 60 min at 56 °C to cleave the reductively disulfide bonds in the proteins and were then incubated with 55 mM IAA (2-iodoacetamide) in 100 mM NH4HCO3 for 40 min at room temperature in the dark to block reformation of disulfide bonds. After washing with B
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Figure 1. MS and MS/MS spectra of two representative peptides. Among 41 up-regulated proteins, two significantly increased peptides were selected. Each peak of the MS reveals a strong increase in peptide abundance in Aβ-treated microglia compared with controls, and the MS/MS spectra show strong matches with each peptide sequence. (A) Tandem mass spectrum of peptide DTQSGSLLFIGR from SERPINH1. (B) Tandem mass spectrum of peptide LAAVDATVNQVLASR from PDIA6.
2.7. Gene Annotation, Upstream Regulator Analysis, and Gene-Enrichment Analysis
gene names of 60 identified proteins were used for analyzing protein−protein interactions.
Gene Ontology annotation enrichment analysis was performed using the DAVID Bioinformatics Resource (v6.7) developed by NIAID, at the National Institutes of Health. DAVID analysis enabled the detection of enrichment of our set of experimentally responsive genes with gene groups related to specific functions and cellular compartments. Ingenuity Pathway Analysis (IPA; Ingenuity Systems, http://www.ingenuity. com) was used to functionally annotate genes implicated in our observed glial response to Aβ in a search for common upstream regulators. The EASE score was adopted for measuring the degree of enrichment in genes with specific annotation terms based on the DAVID annotation system.
2.9. Immunofluorescence
Cells were grown on Lab-Tek II slide chambers (Nunc), rinsed with PBS, fixed in methanol for 15 min, and rinsed again with PBS. The fixed cells on slide chambers and brain slices were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-SERPINH1 antibody (1:1000, Abcam), rabbit anti-PDIA6 antibody (1:200, Abcam), rabbit anti-PDIA3 antibody (1:100, Abcam), rabbit anti-PPIB antibody (1:50, Abcam), mouse anti-PDI antibody (1:200, Abcam), and goat anti-Iba1 antibody (1:100, Abcam). After overnight incubation, the primary antibodies were washed with PBS three times and the slides were then incubated for 1 h at room temperature with one of the following secondary antibodies: Alexa Fluor 633 antimouse IgG (1:500, Invitrogen) or Alexa Fluor 488 antirabbit IgG (1:500, Invitrogen). After washing the secondary antibodies with PBS three times at 10 min intervals, slides were incubated with DAPI (4′,6-diamidino-2-phenylindole; 1 μg/
2.8. Protein−Protein Interaction Data from STRING
The initial weighted protein−protein interaction network was retrieved from the STRING (Search Tool for the Retrieval of Interacting Genes, version 9.5, http://string-db.org/). The C
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Figure 2. Gene Ontology analysis of increased and decreased proteins in microglia stimulated by Aβ. Increased and decreased proteins were submitted to the Gene Ontology database (Supplemental Table S4 in the Supporting Information). Categories were ranked by EASE score, a modified Fisher exact P value. The EASE score represents gene “enrichment”: the prevalence of genes from a given functional category in our list of Aβ-regulated genes compared with the prevalence of such genes in the entire human genome. The dashed line indicates an EASE score of 1.3, above which results are significant. The number of genes indicates the sum of proteins among increased and decreased. (A) Enrichment analysis for biological process. (B) Enrichment analysis for cellular component. (C) Enrichment analysis for molecular function.
three times at 10 min intervals. After washing, HRP-conjugated antirabbit secondary antibody (1:1000, Vector Laboratories) was added to the wells, which were then incubated for 1 h at room temperature. After washing out unbound HRPconjugated secondary antibody, color was developed by incubation with 3,3′,5,5′-tetramethylbenzidine (TMB) for 15 min, and the reaction then stopped with an equal volume of 2 M H2SO4. Absorbance in each well was measured at 450 nm using an ELISA plate reader (VERSA Max, Molecular Devices).
mL; Sigma-Aldrich) for 10 s for counterstaining of nuclei. After washing with PBS, coverslips were mounted onto glass slides using Vectashield mounting medium (Vector Laboratories) and examined under a laser confocal fluorescence microscope (LSM-710, Carl Zeiss). 2.10. Immunoblot Analysis
Whole cell lysates were prepared in RIPA buffer containing 4% CHAPS. Proteins from each group were separated on 4−12% polyacrylamide gels (Life Technology) and transferred to nitrocellulose membranes. The primary antibodies used were: rabbit anti-SERPINH1 antibody (1:1000, Abcam), rabbit antiPDIA6 antibody (1:3000, Abcam), rabbit anti-PDIA3 antibody (1:1000, Abcam), rabbit anti-PPIB antibody (1:500, Abcam), and rabbit anti-β-actin antibody (1:5000, Sigma-Aldrich).
2.12. Real-Time PCR
RNA was isolated from six biological replicates of each group using Qiagen RNeasy MiniKit (Qiagen), pooled, and subjected to first-strand cDNA synthesis using Reverse Transcription System (Promega) according to the manufacturer’s protocol. Quantitative real-time PCR was performed using Rotor-Gene 6000 (Corbett Lifescience). The threshold cycle number and reaction efficiency were determined using Rotor-Gene 6000 series software (version 2.7), and the 22 DDCT method was used for relative quantitation. The primers used are listed in Supplemental Table S1 in the Supporting Information.
2.11. Enzyme-Linked Immunosorbent Assay
The amounts of SERPINH1, PDIA6, PDIA3, and PPIB in cell lysates were determined by ELISA. We coated 96 wells by incubation with rabbit anti-SERPINH1 antibody (1:2000, Abcam), rabbit anti-PDIA6 antibody (1:2000, Abcam), rabbit anti-PDIA3 antibody (1:2000, Abcam), and rabbit anti-PPIB antibody (1:2000, Abcam) overnight at 4 °C. Cell lysates and supernatant were added to each well and incubated for 1 h for capture of analyzands by primary antibody. Unbound proteins were then eliminated from each well by washing with 1 × PBS D
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Table 1. Known Upstream Regulators of the Microglial Proteins Shown in This Study to Be More Abundant by Aβa upstream regulator NFE2L2 CD38 SYVN1 HRAS IL5 a
molecule type
predicted activation state
activation z score
p value of overlap
activated
2.234
5.21 × 10−9
activated activated activated activated
2.236 2.000 2.190 2.000
6.85 4.88 3.95 5.64
transcriptional regulator enzyme transporter enzyme cytokine
× × × ×
10−5 10−4 10−3 10−3
target molecules in data set PSMD12, PSMC3, PSMC1, PPIB, PDIA6, PDIA3, MOGS, FN1, ALDOA PDIA6, CKAP4, CD44, ASNS, ALDOA CD44, TARS, SLC3A2, PSMC3 RPS6, PRDX6, FN1, CD44, ASNS PDIA6, CKAP4, ASNS, ALDOA
Upstream regulator analysis was performed using the IPA. Bolded proteins are ER-resident.
3. RESULTS AND DISCUSSION
(PDIA3, PPIB, PRDX6, SLC3A2, PDIA6, ACTN1, PDIA4, HSPD1, and FN1), and adherens junction (CD44, ITGA2, ACTN1, MYH9, and CTNNA1). Molecular function analysis showed strong changes in the following categories: aminoacyltRNA ligase activity (IARS, TARS, DARS, LARS, KARS, and MARS), collagen binding (CD44, ITGA2, SERPINH1, and FN1), intramolecular oxidoreductase (including protein disulfide isomerase) activity (PDIA3, PDIA6, PDIA4, and TPI1P1), nucleotide binding (DARS, HK1, ASNS, ACLY, MYH9, KARS, IARS, TARS, PSMC3, PSMC1, LARS, HSPD1, and MARS), and cofactor binding (GOT2, SHMT2, UGDH, ASNS, and HADHA). According to these Gene Ontology analysis, microglia stimulated by Aβ seem to change the level of proteins involved in protein synthesis, correct folding of proteins, and clearance of aberrant proteins, mainly in the ER. All ER proteins increased or decreased in our data set are ER chaperone and foldase involved in protein folding.22 Aberrant proteins such as Aβ are known to cause ER stress, which may regulate ER chaperone and foldase expression for correct folding of protein. Cell adhesion and cell shape changes are also implicated, which are known to be involved in activation of microglia.23
3.1. Proteomic Analysis
To investigate the protein changes in human microglial activation by Aβ, we used quantitative proteomic techniques. SILAC combined with LC−MS/MS revealed more than 600 proteins, and among these proteins, 60 proteins showed significant changes in their abundance by 1.5 fold or greater in microglia treated with Aβ as compared with controls and well shown in Tables (Supplemental Tables S2 and S3 in the Supporting Information). These selected proteins were required to show significant Aβ responses in at least two out of three independent experiments. The MS/MS spectra for two representative proteins, SERPINH1 and PDIA6, are shown in Figure 1. In each case, each peak of the MS spectrum showed a significant increase in the peptide abundance in microglia stimulated by Aβ compared with controls (Figure 1). 3.2. Enrichment Analysis of Increased and Decreased Proteins in Microglia Stimulated by Aβ
For functional analysis of regulated proteins in microglia stimulated by Aβ, 41 increased proteins and 19 decreased proteins were submitted to the Gene Ontology database to check for commonalities in their biological process, cellular component, and molecular function (Supplemental Table S4 in the Supporting Information). Among these results, the main functional categories are shown in Figure 2 ranked according to their EASE score, which is a modified Fisher’s exact P value. The EASE score represents the gene enrichment: the prevalence of genes from a given functional category in our list of Aβ-regulated genes compared with the prevalence of such genes in the entire human genome. When the EASE score is higher than 1.3, the functional category is considered to be enriched above the average proportion, thereby implicating that function in the cells’ response to our experimental intervention. Biological process analysis demonstrated strong changes in the following categories: tRNA aminoacylation for protein translation (IARS, TARS, DARS, LARS, KARS, and MARS), cell redox homeostasis (PDIA3, PRDX6, PDIA6, and PDIA4), cellmatrix adhesion (CD44, ITGA2, ACTN1, and FN1), response to organic substrate (GOT2, CD44, VN1R5, ITGA2, ASNS, HSPD1, HDAC9, and SERPINH1), regulation of cell shape (ALDOA, MYH9, and FN1), negative regulation of ubiquitin− protein ligase activity (PSMD12, PSMC3, and PSMC1), and cell adhesion-related proteins (CD44, COL6A1, ITGA2, ACTN1, MYH9, CTNNA1, and FN1). Cellular component analysis displayed strong changes in the following categories: mitochondria (GOT2, SHMT2, ALDH1B1, LRRC59, ATP5C1, STOML2, HK1, HSPD1, HADHA, and KARS), ER (PDIA3, PPIB, PDIA6, PDIA4, and SERPINH1) and ERGolgi intermediate compartment (PDIA6, ANPEP, SERPINH1, and FN1), cytoplasmic membrane-bounded vesicle
3.3. Upstream Regulator Analysis of Up-Regulated Proteins in Microglia Stimulated by Aβ
To search for upstream regulators that may increase the expression of proteins in microglia stimulated by Aβ, IPA was used. The upstream regulators found were as follows: one transcriptional regulator (NFE2L2), two enzymes (CD38, HRAS), one transporter (SYVN1), and one cytokine (IL5) (Table 1). NFE2L2, called nuclear factor (erythroid-derived 2)like 2 and also known as Nrf2, is a transcription factor that regulates the expression changes involved in the oxidative stress response, such as upregulation of NAD(P)H:quinone oxidoreductase 1 (NQO1),24 heme oxygenase-1,25 the glutathione Stransferase (GST) family,26 and so on. Oxidative stress is one of the leading causes of neurodegenerative disease, including AD, and thus NFE2L2 has recently been highlighted as a therapeutic target for neurodegenerative disease.27 Most of the target molecules regulated by NFE2L2 in our data set are related to ER stress. As the result of recent studies, NFE2L2 is also known to be involved in ER stress clearance mechanisms.28 PPIB (peptidyl-prolyl cis−trans isomerase B), PDIA6 (protein disulfide isomerase 6), PDIA3 (protein disulfide isomerase 3), and MOGS (mannosyl-oligosaccharide glucosidase) are ER proteins in our data set involved in protein-folding regulation by ER.22 3.4. Protein−Protein Interaction Analysis
Protein−protein interaction analysis of the 60 regulated proteins was performed using STRING, which is a large database of known and predicted protein interactions.29 The confidence score of a predicted interaction indicates high E
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Figure 3. Protein−protein interaction network of regulated proteins in microglia stimulated by Aβ. Protein−protein interaction analysis was performed using STRING, with high-confidence scores being those over 0.7.
Table 2. Resident ER Proteins Involved in Protein Folding IPI accession
gene symbol
protein name
coverage (%)
sequence length
number unique peptides
IPI00299571 IPI00032140 IPI00328170 IPI00646304 IPI00025252 IPI00009904
PDIA6 SERPINH1 MOGS PPIB PDIA3 PDIA4
protein disulfide-isomerase A6 serpin H1 mannosyl-oligosaccharide glucosidase peptidyl-prolyl cis−trans isomerase B protein disulfide-isomerase A3 protein disulfide-isomerase A4
20.7 40.7 3.6 28.2 24.2 13.3
492 418 837 216 505 645
4 14 1 3 8 10
ratio (H/L) 3.27 2.68 1.58 1.57 1.56 0.66
± ± ± ± ± ±
1.14 0.18 0.13 0.15 0.25 0.12
four ER proteins in microglial cells in the AD model mouse strain 5XFAD. Immunocytochemistry showed that the expression levels of four ER proteins identified proteomically were increased in HMO6 cells after Aβ1−42 treatment and that all were located in the ER (Figure 4). For quantitative analysis, Western blot, ELISA, and real-time PCR were used. Protein levels of PDIA6, PDIA3, and PPIB were increased in both Western blots and ELISAs of extracts of HMO6 cells after Aβ1−42 treatment. Levels of each protein increased in a time-dependent manner during Aβ1−42 treatment and were maximal after 12 h. RNA levels of PDIA6, PDIA3, and PPIB by real-time PCR were also dramatically increased and were also maximal after Aβ1−42 treatment for 12 h. The protein and RNA levels of SERPINH1 were also increased after Aβ1−42 treatment for 12 h by ELISA and real-time PCR, but no changes were observed for this protein by Western blot (Figure 5). Also, the real-time PCR data of additional genes showed
confidence if it is above 0.7. The results predicted interactions of proteins in our data set with several resident ER proteins related to regulation of correct protein folding in the ER (Figure 3). 3.5. ER Proteins
In this study, we detected several ER proteins involved in protein folding. Two PDIs (protein disulfide isomerase: PDIA3 and PDIA6), one foldase (PPIB), one carbohydrate processing enzyme (MOGS), and one heat shock protein (SERPINH1: serpin peptidase inhibitor, clade H) were up-regulated and one PDI (PDIA4) was slightly down-regulated by Aβ in microglia (Table 2). To validate the proteomic analysis results from HMO6 cells, we examined four of these proteins, SERPINH1, PDIA6, PDIA3, and PPIB, by immunostaining, Western blot, ELISA, and real-time PCR. Additionally, immunostaining of brain slices was used to verify the presence of increased expression levels of F
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Figure 4. Expression levels of SERPINH1, PDIA6, PDIA3, and PPIB in the ER of human microglial cells after Aβ1−42 treatment. Triplelabeled confocal microscopic image analyses were used to study the expression of (A) SERPINH1, (B) PDIA6, (C) PDIA3, and (D) PPIB. Also imaged are PDI (an ER marker, red) and DAPI (a nucleus marker, blue). Shown are HMO6 cells before and after treatment with 500 nM Aβ1−42 for 12 h. Scale bar = 100 μm.
Figure 5. Quantitation of SERPINH1, PDIA6, PDIA3, and PPIB in human microglial cells after Aβ1−42 treatment. (A,B) Time-dependent changes in intracellular (cell lysate) SERPINH1, PDIA6, PDIA3, and PPIB in HMO6 cells, treated with Aβ1−42 for 3, 6, 9, and 12 h, were determined by immunoblotting (A) and ELISA (B). β-actin was used as an internal control for equal protein loading in each lane. (C) mRNA levels of SERPINH1, PDIA6, PDIA3, and PPIB were measured in HMO6 cells after Aβ1−42 treatment, by real-time PCR. *, P < 0.05; **, P < 0.01.
similar tendency with proteomic data (Supplemental Figure S1 in the Supporting Information). For validation in the AD model mouse 5XFAD, immunohistochemical staining was used. Each mouse brain was triplelabeled for one of four ER-resident proteins (green), an activated microglial cell marker (IbaI, red), and DAPI (blue). Activated microglial cells (red) were seen only in 5XFAD mouse brains. Each ER protein (green) was increased in the 5XFAD mouse brain and was colocalized with activated microglial cells (Figure 6). The ER has diverse and important functions, such as posttranslational modification of proteins, transport of proteins to
their target sites, protein quality control, maintenance of Ca2+ homeostasis, and synthesis of steroids and lipids. Disorders of these ER functions cause ER stress, which is known to be one of the leading causes of neurodegenerative disease, including AD.30 Aβ1−42 induces ER stress through the regulation of ER Ca2+ homeostasis involving phopholipase C activation31 and glycogen synthase kinase-3β activation and Tau phosphorylation.32 ER stress induced by Aβ is involved in mitochondrial G
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amyloid load. Interestingly, PGRN is expressed in neurons and microglia but not in astrocytes or oligodendrocytes,47 and mouse PGRN was up-regulated more than 60-fold in activated microglia.48 PGRN also plays a role as a chemoattractant for microglia and induces endocytotic activity in these cells.49 In our data, PDIA3 levels increased in microglia stimulated by Aβ. Because the well-known function of PDIA3 is to correct the folding of newly synthesized glycoproteins in the ER, we predicted that the increased PDIA3 levels in activated microglia may be associated with upregulation of numerous glycoproteins such as secretory and membrane proteins, including PGRN. Figure 6. Distribution of SERPINH1, PDIA6, PDIA3, and PPIB in normal and 5XFAD mouse brain. Triple-labeled confocal microscopic image analyses were used to study the expression of (A) SERPINH1, (B) PDIA6, (C) PDIA3, and (D) PPIB. Also imaged are Iba1 (an activated-microglia marker, red) and DAPI (a nucleus marker, blue). Shown are normal and 5XFAD mouse brain slices. Scale bar = 50 μm.
4. CONCLUSIONS In this study, we provide a proteomic analysis of Aβ stimulation of human microglia using SILAC labeling followed by LC− MS/MS analysis. Among the implicated proteins, 60 proteins showed significant differences in relative abundance after incubation of microglia with Aβ compared with controls. Our data identified the ER-resident proteins as potential biomarkers of human AD development based on microglia-mediated inflammation. These markers including SERPINH1, PDIA6, PDIA3, and PPIB can be represented as potential molecular targets for human AD treatment.
dysfunction, which is also known to be the one of main cause of AD.33,34 Most studies of ER stress indicate that the cause is disruption of protein folding. Many proteins, such as molecular chaperones, foldases, and glycosylation-related proteins, are involved in protein folding regulation in the ER.28 In light of these observations, our results may be evidence that Aβinduced microglial activation is accompanied by Aβ-induced ER stress. For example, SERPINH1 is a collagen-binding stress protein and molecular chaperone involved in the maturation of collagen residing in the endoplasmic reticulum.35−37 A recent study found physical interaction and colocalization of SERPINH1 and amyloid precursor protein in neurons. SERPINH1 was also reported to be enriched in amyloid plaques in mouse brain and to modulate the levels of extracellular Aβ peptides in neuron cultures.38 However, in contrast with the results of previous studies of some ER chaperones such as BiP/GRP78 (glucose-regulated protein 78), calnexin, and calreticulin,39−41 overexpression of SERPINH1 increased the level of secreted Aβ peptides, and reducing SERPINH1 expression with siRNA or chemically inhibiting its activity decreased the levels of secreted Aβ peptides in neuron cultures. Taken together, these results predict that SERPINH1 affects the degradation of Aβ peptides in the extracellular environment by inhibiting proteases.38 Another example of an ER-resident protein from our data set is PDIA3. PDIA3 is a protein disulfide isomerase 3 and is known as ERP57 (57-kD ER protein), GRP58 (58-kD glucoseregulated protein), and 1,25D3-MARRS (membrane-associated rapid response steroid-binding receptor). This protein is present mainly in ER and other subcellular locations such as the plasma membrane, cytosol, and nucleus.42,43 Erickson and his colleagues (2005) reported that PDIA3 and calreticulin were bound to Aβ in cerebrospinal fluid (CSF).44,45 They suggested that PDIA3 and calreticulin that became bound to normally N-glycosylated Aβ could prevent plaque formation, resulting from the precipitation of Aβ. These results suggest one reason why Aβ plaque is not formed in young people. Very little information is published on the roles of PDIA3 in microglia. In one report, progranulin (PGRN) was found to bind to ER molecular chaperones including some PDIs.46 PGRN is up-regulated in an AD model mouse compared with control, and its expression level is significantly correlated with
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ASSOCIATED CONTENT
S Supporting Information *
Supplemental Figure S1. Real-time PCR data confirming differential expression of the six selected up- and downregulated genes. Supplemental Table S1. Primer sequences used for real-time PCR. Supplemental Table S2. Increased proteins in microglia stimulated by Aβ. Supplemental Table S3. Decreased proteins in microglia stimulated by Aβ. Supplemental Table S4. Gene Ontology annotation analysis using DAVID Bioinformatics Resource (v6.7). This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*Y.M.P.: E-mail: [email protected]. Tel: +82-43-240-5160. Fax: +82-43-240-5059. *B.L.: E-mail: [email protected]. Tel: +82-32-899-6582. Fax: +82-32-899-6519. Author Contributions ▽
Y.Y. and K.B. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by IBS-R001-D1, the Gachon University research fund of 2014 (GCU 2014-5102, 5105) and HI13C2098.
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REFERENCES
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Protein Disulfide Isomerase Family Proteins. PLoS One 2011, 6 (10), e26454. (47) Pereson, S.; Wils, H.; Kleinberger, G.; McGowan, E.; Vandewoestyne, M.; et al. Progranulin expression correlates with dense-core amyloid plaque burden in Alzheimer disease mouse models. J. Pathol. 2009, 219 (2), 173−181. (48) Petkau, T. L.; Neal, S. J.; Orban, P. C.; MacDonald, J. L.; Hill, A. M.; et al. Progranulin expression in the developing and adult murine brain. J. Comp. Neurol. 2010, 518 (19), 3931−3947. (49) Pickford, F.; Marcus, J.; Camargo, L. M.; Xiao, Q.; Graham, D.; et al. Progranulin Is a chemoattractant for microglia and stimulates their endocytic activity. Am. J. Pathol. 2011, 178 (1), 284−295.
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Amyloid-Beta-Activated Human Microglial Cells Through ERResident Proteins YongCheol Yoo,†,‡,§,▽ Kyunghee Byun,∥,⊥,▽ Taewook Kang,§ Delger Bayarsaikhan,∥ Jin Young Kim,§ Seyeoun Oh,∥ Young Hye Kim,§ Se-Young Kim,§ Won-Il Chung,† Seung U. Kim,# Bonghee Lee,*,∥,⊥ and Young Mok Park*,‡,§ †
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Guseong-dong, Daejeon 305-701, Republic of Korea ‡ Center for Cognition and Sociality, Institute for Basic Science (IBS), 5, Hwarang-ro 14-gil, Daejeon 305-811, Republic of Korea § Mass Spectrometer Research Center, Korea Basic Science Institute, 52 Eoeun-dong, Ochang, Chungcheongbuk-do 363-883, Republic of Korea ∥ Center for Genomics and Proteomics, Lee Gil Ya Cancer and Diabetes Institute, Gachon University, 7-45, Songdo-dong, Yeonsu-ku, Incheon 406-840, Republic of Korea ⊥ Department of Anatomy and Cell Biology, Gachon University Graduate School of Medicine, 7-45 Songdo-dong, Yeonsu-gu, Incheon 406-840, Republic of Korea # Department of Medicine, University of British Columbia, 2775 Laurel Street, Vancouver, British Columbia V5Z 1M9, Canada S Supporting Information *
ABSTRACT: Microglial activation in the central nervous system is a key event in the neuroinflammation that accompanies neurodegenerative diseases such as Alzheimer’s disease (AD). Among cytokines involved in microglial activation, amyloid β (Aβ) peptide is known to be a key molecule in the induction of diverse inflammatory products, which may lead to chronic inflammation in AD. However, proteomic studies of microglia in AD are limited due to lack of proper cell or animal model systems. In this study, we performed a proteomic analysis of Aβ-stimulated human microglial cells using SILAC (stable isotope labeling with amino acids in cell culture) combined with LC−MS/MS. Results showed that 60 proteins increased or decreased their abundance by 1.5 fold or greater. Among these, ER-resident proteins such as SERPINH1, PDIA6, PDIA3, and PPIB were revealed to be key molecular biomarkers of human microglial activation by validation of the proteomic results by immunostaining, PCR, ELISA, and Western blot. Taken together, our data suggest that ER proteins play an essential role in human microglial activation by Aβ and may be important molecular therapeutic targets for treatment of AD. KEYWORDS: amyloid β, human microglia, tandem mass tags, LC−MS/MS
1. INTRODUCTION
Activated microglia are numerous in human and animal AD brain tissues, and many of them are found to accumulate in Aβ plaques in human AD brain tissues. This implies an important role for activated microglial cells in the progression of AD. Activated microglial cells also produce various proinflammatory factors, which may lead to chronic inflammation, and these neuroinflammations may accelerate the progression of AD.7−10 The changes in Aβ-stimulated microglia have been reported only in a murine model with a 2D gel electrophoresis approach.11 Even though microglial activation has been recognized as a therapeutic target in AD, promising microglial therapies have not yet been developed.10,12−15 However, there are no reports on the proteomic changes accompanying
Alzheimer’s disease (AD) is the most common neurodegenerative disorder in humans. The most prominent pathological features of AD are formation and accumulation in the brain of amyloid beta (Aβ) plaques and neurofibrillary tangles (NFTs). Insoluble extracellular aggregates, Aβ plaques, are mainly composed of Aβ1−40 and Aβ1−42 peptides, which are produced by proteolytic cleavage of the amyloid precursor protein and have been regarded as one of the main contributors to AD development.1−3 Microglial cells (brain-resident macrophages) are activated in response to many pathologic changes in central nervous system (CNS) homeostasis. Activation of microglia leads to several inflammatory responses such as scavenging, phagocytosis, cytotoxicity, antigen presentation, synaptic stripping, promotion of repair, and extracellular signaling to restore CNS homeostasis.4−6 © XXXX American Chemical Society
Special Issue: Environmental Impact on Health Received: September 11, 2014
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2. MATERIALS AND METHODS
distilled water, the gel pieces were rehydrated with 12.5 μg/μL trypsin (Promega) solution (in 50 mM NH4HCO3) and incubated overnight at 37 °C. Following digestion, peptides were extracted with 50% ACN/5% TFA (trifluoroacetic acid) solution at room temperature for 40 min. The supernatants were dried by vacuum centrifugation. The dried peptides were stored at −20 °C until MS analysis.
2.1. Human Microglial HMO6 Cell Culture and SILAC
2.5. Nanoflow Liquid Chromatography and Tandem MS/MS Analysis
activation of human microglial cells by Aβ and functional validations of these. Here we report for the first time the proteomic analysis of Aβ-stimulated human microglia using SILAC combined with LC−MS/MS and comprehensive analysis as well as validation of the proteomic results in brain tissues of an AD mouse model.
Our study organism was HMO6, an immortalized human microglial cell line. HMO6 cells were grown in Dulbecco’s modified Eagle’s medium-high glucose (DMEM-HG, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 20 mg/mL gentamicin (Sigma), in an incubator at 37 °C under 5% CO2/95% air as previously reported.16−18 For SILAC labeling, SILAC amino acids (MS50011, Invitrogen), SILAC Phosphoprotein ID and Quantitation Kit with Lysine (SP10005, Invitrogen), and SILAC medium (DMEM, Invitrogen) were used. Cells were grown in light medium containing [U−12C6]-L-lysine and [U−12C6]-L-arginine and heavy medium containing [U−13C6]-L-lysine and [U−13C6]-Larginine for 2 weeks for at least five generations. Aβ1−42 (Sigma) was added to the HMO6 cells at a concentration of 500 nM, and the cells were then incubated for up to 12 h before protein extraction.
The dried peptides were resuspended in 0.1% formic acid (FA) and analyzed online by nanoelectrospray ionization using a 7T LTQ-FT mass spectrometer (Thermofinnigan, USA) coupled to a UPLC system (Waters, USA). The peptide solution was trapped on a C18 trap column (180 μm i.d. × 2 cm, particle size 5 μm; Waters) at a flow rate of 5 μL/min. The trapped peptides were separated on a 20 cm homemade microcapillary column consisting of C18 (Aqua; particle size 3 μm) packed into 100 μm silica tubing with an orifice i.d. of 360 μm. The samples were loaded onto the column and then eluted with a 12 min gradient covering 10 to 50% of buffer B (buffer A: 0.1% FA in water; buffer B: 0.1% FA in ACN). Eluted peptides were electrosprayed directly off the column into the mass spectrometer at a voltage of 2.2 kV. A cycle of MS/MS spectra consisted of one full-scan mass spectrum (scan range of 400− 2000 m/z, resolution 100 000, isolation window ±1 Da) and up to five ion-trap data-dependent scans with the linear ion trap analyzer. All MS/MS spectra were recorded using a normalized collision energy of 35%. Target ions selected for MS/MS were dynamically excluded for 60 s. An activation q of 0.25 and an activation time of 30 ms were applied in MS/MS acquisitions.
2.2. Animals and Brain Tissue Preparation
Ten 5XFAD (AD model) mice were provided and used as previously described.19 For tissue preparation, all mice were perfused trans-cardially with 100 mL of heparinized saline at 18 °C, followed by 100 mL of 4% paraformaldehyde−lysine periodate in 0.1 M sodium phosphate buffer (pH 7.4). The brains were removed, placed in the same fixative for 4 h at 4 °C, and transferred into ice-cold 0.1 M phosphate-buffered saline (PBS) containing 20% sucrose. The brains were cut in a transverse plane at 10 μm thickness with a freezing microtome and were stored at −80 °C until use.
2.6. Protein Identification, Quantification, and Proteomic Data Analysis
The MS raw data from each gel fraction were processed for protein identification using the MaxQuant software (v.1.2.2.5) with a peptide mass tolerance of 20 ppm and a fragment mass tolerance of ±0.5 Da at a false discovery rate (FDR) of 1% for both the protein and peptide.21 All peak lists were identified against the human International Protein Index (IPI) database with decoy (IPI version 3.68, 87 061 entries) using the following parameters: enzyme, trypsin; missed cleavages ≤1; fixed modification, carbamidomethyl (C); variable modifications, oxidation (M) and phosphorylation (S,T,Y). Protein identifications were accepted only if they contained at least two peptides. Also, only the proteins with at least one unique peptide were accepted for quantification. Triple biological replicates were used to analyze proteomic data for this application. For relative protein quantification, the evidence.txt output file from MaxQuant was imported into Microsoft Excel and then for each IPI name, filtered as follows: MaxQuant score ≥100, not a contaminant or reversed sequence. An intensity from the respective IPI name was calculated by summing all normalized intensity using only unique peptides. The averaged ratio of normalized intensities was then calculated between Aβ nontreatment (light) and Aβ treatment (heavy). Proteins were accepted as a difference in their abundance with a ratio of 1.5 or greater between heavy and light. Also, a ratio was reserved for proteins having a equivalent ratio at least two of trials. Finally, Student’s t test was calculated for each IPI name (P < 0.05).
2.3. Protein Preparation
For protein extraction, HMO6 cells were harvested and washed three times with ice-cold PBS. They were then homogenized in lysis buffer (7 M urea, 2 M thiourea, and 4% CHAPS), sonicated, and centrifuged at 15 000 rpm for 15 min at 4 °C. The supernatant was collected and protein concentrations were measured using the Bradford assay (Pierce Biotechnology, Rockford, IL). 2.4. SDS-PAGE and In-Gel Digestion
For SDS-PAGE analyses, both heavy and light protein extracts were mixed in equal amounts, and 100 μg of the combined proteins was separated on a 10% SDS-polyacrylamide gel (18 × 16 cm). The SDS-PAGE (Bio-Rad) was run at 80 V for 30 min and then at 120 V until the end of the run. After separation, the gel was stained with Coomassie Brilliant Blue (CBB) R-250 and was cut into 10 slices. The excised bands were digested according to the in-gel digestion protocol.20 The band pieces were washed twice with 30% methanol for 5 min and destained with 50% acetonitrile (ACN) containing 10 mM NH4HCO3. They were then incubated with 10 mM DTT (dithiothreitol) in 100 mM NH4HCO3 for 60 min at 56 °C to cleave the reductively disulfide bonds in the proteins and were then incubated with 55 mM IAA (2-iodoacetamide) in 100 mM NH4HCO3 for 40 min at room temperature in the dark to block reformation of disulfide bonds. After washing with B
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Figure 1. MS and MS/MS spectra of two representative peptides. Among 41 up-regulated proteins, two significantly increased peptides were selected. Each peak of the MS reveals a strong increase in peptide abundance in Aβ-treated microglia compared with controls, and the MS/MS spectra show strong matches with each peptide sequence. (A) Tandem mass spectrum of peptide DTQSGSLLFIGR from SERPINH1. (B) Tandem mass spectrum of peptide LAAVDATVNQVLASR from PDIA6.
2.7. Gene Annotation, Upstream Regulator Analysis, and Gene-Enrichment Analysis
gene names of 60 identified proteins were used for analyzing protein−protein interactions.
Gene Ontology annotation enrichment analysis was performed using the DAVID Bioinformatics Resource (v6.7) developed by NIAID, at the National Institutes of Health. DAVID analysis enabled the detection of enrichment of our set of experimentally responsive genes with gene groups related to specific functions and cellular compartments. Ingenuity Pathway Analysis (IPA; Ingenuity Systems, http://www.ingenuity. com) was used to functionally annotate genes implicated in our observed glial response to Aβ in a search for common upstream regulators. The EASE score was adopted for measuring the degree of enrichment in genes with specific annotation terms based on the DAVID annotation system.
2.9. Immunofluorescence
Cells were grown on Lab-Tek II slide chambers (Nunc), rinsed with PBS, fixed in methanol for 15 min, and rinsed again with PBS. The fixed cells on slide chambers and brain slices were incubated overnight at 4 °C with the following primary antibodies: rabbit anti-SERPINH1 antibody (1:1000, Abcam), rabbit anti-PDIA6 antibody (1:200, Abcam), rabbit anti-PDIA3 antibody (1:100, Abcam), rabbit anti-PPIB antibody (1:50, Abcam), mouse anti-PDI antibody (1:200, Abcam), and goat anti-Iba1 antibody (1:100, Abcam). After overnight incubation, the primary antibodies were washed with PBS three times and the slides were then incubated for 1 h at room temperature with one of the following secondary antibodies: Alexa Fluor 633 antimouse IgG (1:500, Invitrogen) or Alexa Fluor 488 antirabbit IgG (1:500, Invitrogen). After washing the secondary antibodies with PBS three times at 10 min intervals, slides were incubated with DAPI (4′,6-diamidino-2-phenylindole; 1 μg/
2.8. Protein−Protein Interaction Data from STRING
The initial weighted protein−protein interaction network was retrieved from the STRING (Search Tool for the Retrieval of Interacting Genes, version 9.5, http://string-db.org/). The C
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Figure 2. Gene Ontology analysis of increased and decreased proteins in microglia stimulated by Aβ. Increased and decreased proteins were submitted to the Gene Ontology database (Supplemental Table S4 in the Supporting Information). Categories were ranked by EASE score, a modified Fisher exact P value. The EASE score represents gene “enrichment”: the prevalence of genes from a given functional category in our list of Aβ-regulated genes compared with the prevalence of such genes in the entire human genome. The dashed line indicates an EASE score of 1.3, above which results are significant. The number of genes indicates the sum of proteins among increased and decreased. (A) Enrichment analysis for biological process. (B) Enrichment analysis for cellular component. (C) Enrichment analysis for molecular function.
three times at 10 min intervals. After washing, HRP-conjugated antirabbit secondary antibody (1:1000, Vector Laboratories) was added to the wells, which were then incubated for 1 h at room temperature. After washing out unbound HRPconjugated secondary antibody, color was developed by incubation with 3,3′,5,5′-tetramethylbenzidine (TMB) for 15 min, and the reaction then stopped with an equal volume of 2 M H2SO4. Absorbance in each well was measured at 450 nm using an ELISA plate reader (VERSA Max, Molecular Devices).
mL; Sigma-Aldrich) for 10 s for counterstaining of nuclei. After washing with PBS, coverslips were mounted onto glass slides using Vectashield mounting medium (Vector Laboratories) and examined under a laser confocal fluorescence microscope (LSM-710, Carl Zeiss). 2.10. Immunoblot Analysis
Whole cell lysates were prepared in RIPA buffer containing 4% CHAPS. Proteins from each group were separated on 4−12% polyacrylamide gels (Life Technology) and transferred to nitrocellulose membranes. The primary antibodies used were: rabbit anti-SERPINH1 antibody (1:1000, Abcam), rabbit antiPDIA6 antibody (1:3000, Abcam), rabbit anti-PDIA3 antibody (1:1000, Abcam), rabbit anti-PPIB antibody (1:500, Abcam), and rabbit anti-β-actin antibody (1:5000, Sigma-Aldrich).
2.12. Real-Time PCR
RNA was isolated from six biological replicates of each group using Qiagen RNeasy MiniKit (Qiagen), pooled, and subjected to first-strand cDNA synthesis using Reverse Transcription System (Promega) according to the manufacturer’s protocol. Quantitative real-time PCR was performed using Rotor-Gene 6000 (Corbett Lifescience). The threshold cycle number and reaction efficiency were determined using Rotor-Gene 6000 series software (version 2.7), and the 22 DDCT method was used for relative quantitation. The primers used are listed in Supplemental Table S1 in the Supporting Information.
2.11. Enzyme-Linked Immunosorbent Assay
The amounts of SERPINH1, PDIA6, PDIA3, and PPIB in cell lysates were determined by ELISA. We coated 96 wells by incubation with rabbit anti-SERPINH1 antibody (1:2000, Abcam), rabbit anti-PDIA6 antibody (1:2000, Abcam), rabbit anti-PDIA3 antibody (1:2000, Abcam), and rabbit anti-PPIB antibody (1:2000, Abcam) overnight at 4 °C. Cell lysates and supernatant were added to each well and incubated for 1 h for capture of analyzands by primary antibody. Unbound proteins were then eliminated from each well by washing with 1 × PBS D
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Table 1. Known Upstream Regulators of the Microglial Proteins Shown in This Study to Be More Abundant by Aβa upstream regulator NFE2L2 CD38 SYVN1 HRAS IL5 a
molecule type
predicted activation state
activation z score
p value of overlap
activated
2.234
5.21 × 10−9
activated activated activated activated
2.236 2.000 2.190 2.000
6.85 4.88 3.95 5.64
transcriptional regulator enzyme transporter enzyme cytokine
× × × ×
10−5 10−4 10−3 10−3
target molecules in data set PSMD12, PSMC3, PSMC1, PPIB, PDIA6, PDIA3, MOGS, FN1, ALDOA PDIA6, CKAP4, CD44, ASNS, ALDOA CD44, TARS, SLC3A2, PSMC3 RPS6, PRDX6, FN1, CD44, ASNS PDIA6, CKAP4, ASNS, ALDOA
Upstream regulator analysis was performed using the IPA. Bolded proteins are ER-resident.
3. RESULTS AND DISCUSSION
(PDIA3, PPIB, PRDX6, SLC3A2, PDIA6, ACTN1, PDIA4, HSPD1, and FN1), and adherens junction (CD44, ITGA2, ACTN1, MYH9, and CTNNA1). Molecular function analysis showed strong changes in the following categories: aminoacyltRNA ligase activity (IARS, TARS, DARS, LARS, KARS, and MARS), collagen binding (CD44, ITGA2, SERPINH1, and FN1), intramolecular oxidoreductase (including protein disulfide isomerase) activity (PDIA3, PDIA6, PDIA4, and TPI1P1), nucleotide binding (DARS, HK1, ASNS, ACLY, MYH9, KARS, IARS, TARS, PSMC3, PSMC1, LARS, HSPD1, and MARS), and cofactor binding (GOT2, SHMT2, UGDH, ASNS, and HADHA). According to these Gene Ontology analysis, microglia stimulated by Aβ seem to change the level of proteins involved in protein synthesis, correct folding of proteins, and clearance of aberrant proteins, mainly in the ER. All ER proteins increased or decreased in our data set are ER chaperone and foldase involved in protein folding.22 Aberrant proteins such as Aβ are known to cause ER stress, which may regulate ER chaperone and foldase expression for correct folding of protein. Cell adhesion and cell shape changes are also implicated, which are known to be involved in activation of microglia.23
3.1. Proteomic Analysis
To investigate the protein changes in human microglial activation by Aβ, we used quantitative proteomic techniques. SILAC combined with LC−MS/MS revealed more than 600 proteins, and among these proteins, 60 proteins showed significant changes in their abundance by 1.5 fold or greater in microglia treated with Aβ as compared with controls and well shown in Tables (Supplemental Tables S2 and S3 in the Supporting Information). These selected proteins were required to show significant Aβ responses in at least two out of three independent experiments. The MS/MS spectra for two representative proteins, SERPINH1 and PDIA6, are shown in Figure 1. In each case, each peak of the MS spectrum showed a significant increase in the peptide abundance in microglia stimulated by Aβ compared with controls (Figure 1). 3.2. Enrichment Analysis of Increased and Decreased Proteins in Microglia Stimulated by Aβ
For functional analysis of regulated proteins in microglia stimulated by Aβ, 41 increased proteins and 19 decreased proteins were submitted to the Gene Ontology database to check for commonalities in their biological process, cellular component, and molecular function (Supplemental Table S4 in the Supporting Information). Among these results, the main functional categories are shown in Figure 2 ranked according to their EASE score, which is a modified Fisher’s exact P value. The EASE score represents the gene enrichment: the prevalence of genes from a given functional category in our list of Aβ-regulated genes compared with the prevalence of such genes in the entire human genome. When the EASE score is higher than 1.3, the functional category is considered to be enriched above the average proportion, thereby implicating that function in the cells’ response to our experimental intervention. Biological process analysis demonstrated strong changes in the following categories: tRNA aminoacylation for protein translation (IARS, TARS, DARS, LARS, KARS, and MARS), cell redox homeostasis (PDIA3, PRDX6, PDIA6, and PDIA4), cellmatrix adhesion (CD44, ITGA2, ACTN1, and FN1), response to organic substrate (GOT2, CD44, VN1R5, ITGA2, ASNS, HSPD1, HDAC9, and SERPINH1), regulation of cell shape (ALDOA, MYH9, and FN1), negative regulation of ubiquitin− protein ligase activity (PSMD12, PSMC3, and PSMC1), and cell adhesion-related proteins (CD44, COL6A1, ITGA2, ACTN1, MYH9, CTNNA1, and FN1). Cellular component analysis displayed strong changes in the following categories: mitochondria (GOT2, SHMT2, ALDH1B1, LRRC59, ATP5C1, STOML2, HK1, HSPD1, HADHA, and KARS), ER (PDIA3, PPIB, PDIA6, PDIA4, and SERPINH1) and ERGolgi intermediate compartment (PDIA6, ANPEP, SERPINH1, and FN1), cytoplasmic membrane-bounded vesicle
3.3. Upstream Regulator Analysis of Up-Regulated Proteins in Microglia Stimulated by Aβ
To search for upstream regulators that may increase the expression of proteins in microglia stimulated by Aβ, IPA was used. The upstream regulators found were as follows: one transcriptional regulator (NFE2L2), two enzymes (CD38, HRAS), one transporter (SYVN1), and one cytokine (IL5) (Table 1). NFE2L2, called nuclear factor (erythroid-derived 2)like 2 and also known as Nrf2, is a transcription factor that regulates the expression changes involved in the oxidative stress response, such as upregulation of NAD(P)H:quinone oxidoreductase 1 (NQO1),24 heme oxygenase-1,25 the glutathione Stransferase (GST) family,26 and so on. Oxidative stress is one of the leading causes of neurodegenerative disease, including AD, and thus NFE2L2 has recently been highlighted as a therapeutic target for neurodegenerative disease.27 Most of the target molecules regulated by NFE2L2 in our data set are related to ER stress. As the result of recent studies, NFE2L2 is also known to be involved in ER stress clearance mechanisms.28 PPIB (peptidyl-prolyl cis−trans isomerase B), PDIA6 (protein disulfide isomerase 6), PDIA3 (protein disulfide isomerase 3), and MOGS (mannosyl-oligosaccharide glucosidase) are ER proteins in our data set involved in protein-folding regulation by ER.22 3.4. Protein−Protein Interaction Analysis
Protein−protein interaction analysis of the 60 regulated proteins was performed using STRING, which is a large database of known and predicted protein interactions.29 The confidence score of a predicted interaction indicates high E
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Figure 3. Protein−protein interaction network of regulated proteins in microglia stimulated by Aβ. Protein−protein interaction analysis was performed using STRING, with high-confidence scores being those over 0.7.
Table 2. Resident ER Proteins Involved in Protein Folding IPI accession
gene symbol
protein name
coverage (%)
sequence length
number unique peptides
IPI00299571 IPI00032140 IPI00328170 IPI00646304 IPI00025252 IPI00009904
PDIA6 SERPINH1 MOGS PPIB PDIA3 PDIA4
protein disulfide-isomerase A6 serpin H1 mannosyl-oligosaccharide glucosidase peptidyl-prolyl cis−trans isomerase B protein disulfide-isomerase A3 protein disulfide-isomerase A4
20.7 40.7 3.6 28.2 24.2 13.3
492 418 837 216 505 645
4 14 1 3 8 10
ratio (H/L) 3.27 2.68 1.58 1.57 1.56 0.66
± ± ± ± ± ±
1.14 0.18 0.13 0.15 0.25 0.12
four ER proteins in microglial cells in the AD model mouse strain 5XFAD. Immunocytochemistry showed that the expression levels of four ER proteins identified proteomically were increased in HMO6 cells after Aβ1−42 treatment and that all were located in the ER (Figure 4). For quantitative analysis, Western blot, ELISA, and real-time PCR were used. Protein levels of PDIA6, PDIA3, and PPIB were increased in both Western blots and ELISAs of extracts of HMO6 cells after Aβ1−42 treatment. Levels of each protein increased in a time-dependent manner during Aβ1−42 treatment and were maximal after 12 h. RNA levels of PDIA6, PDIA3, and PPIB by real-time PCR were also dramatically increased and were also maximal after Aβ1−42 treatment for 12 h. The protein and RNA levels of SERPINH1 were also increased after Aβ1−42 treatment for 12 h by ELISA and real-time PCR, but no changes were observed for this protein by Western blot (Figure 5). Also, the real-time PCR data of additional genes showed
confidence if it is above 0.7. The results predicted interactions of proteins in our data set with several resident ER proteins related to regulation of correct protein folding in the ER (Figure 3). 3.5. ER Proteins
In this study, we detected several ER proteins involved in protein folding. Two PDIs (protein disulfide isomerase: PDIA3 and PDIA6), one foldase (PPIB), one carbohydrate processing enzyme (MOGS), and one heat shock protein (SERPINH1: serpin peptidase inhibitor, clade H) were up-regulated and one PDI (PDIA4) was slightly down-regulated by Aβ in microglia (Table 2). To validate the proteomic analysis results from HMO6 cells, we examined four of these proteins, SERPINH1, PDIA6, PDIA3, and PPIB, by immunostaining, Western blot, ELISA, and real-time PCR. Additionally, immunostaining of brain slices was used to verify the presence of increased expression levels of F
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Figure 4. Expression levels of SERPINH1, PDIA6, PDIA3, and PPIB in the ER of human microglial cells after Aβ1−42 treatment. Triplelabeled confocal microscopic image analyses were used to study the expression of (A) SERPINH1, (B) PDIA6, (C) PDIA3, and (D) PPIB. Also imaged are PDI (an ER marker, red) and DAPI (a nucleus marker, blue). Shown are HMO6 cells before and after treatment with 500 nM Aβ1−42 for 12 h. Scale bar = 100 μm.
Figure 5. Quantitation of SERPINH1, PDIA6, PDIA3, and PPIB in human microglial cells after Aβ1−42 treatment. (A,B) Time-dependent changes in intracellular (cell lysate) SERPINH1, PDIA6, PDIA3, and PPIB in HMO6 cells, treated with Aβ1−42 for 3, 6, 9, and 12 h, were determined by immunoblotting (A) and ELISA (B). β-actin was used as an internal control for equal protein loading in each lane. (C) mRNA levels of SERPINH1, PDIA6, PDIA3, and PPIB were measured in HMO6 cells after Aβ1−42 treatment, by real-time PCR. *, P < 0.05; **, P < 0.01.
similar tendency with proteomic data (Supplemental Figure S1 in the Supporting Information). For validation in the AD model mouse 5XFAD, immunohistochemical staining was used. Each mouse brain was triplelabeled for one of four ER-resident proteins (green), an activated microglial cell marker (IbaI, red), and DAPI (blue). Activated microglial cells (red) were seen only in 5XFAD mouse brains. Each ER protein (green) was increased in the 5XFAD mouse brain and was colocalized with activated microglial cells (Figure 6). The ER has diverse and important functions, such as posttranslational modification of proteins, transport of proteins to
their target sites, protein quality control, maintenance of Ca2+ homeostasis, and synthesis of steroids and lipids. Disorders of these ER functions cause ER stress, which is known to be one of the leading causes of neurodegenerative disease, including AD.30 Aβ1−42 induces ER stress through the regulation of ER Ca2+ homeostasis involving phopholipase C activation31 and glycogen synthase kinase-3β activation and Tau phosphorylation.32 ER stress induced by Aβ is involved in mitochondrial G
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amyloid load. Interestingly, PGRN is expressed in neurons and microglia but not in astrocytes or oligodendrocytes,47 and mouse PGRN was up-regulated more than 60-fold in activated microglia.48 PGRN also plays a role as a chemoattractant for microglia and induces endocytotic activity in these cells.49 In our data, PDIA3 levels increased in microglia stimulated by Aβ. Because the well-known function of PDIA3 is to correct the folding of newly synthesized glycoproteins in the ER, we predicted that the increased PDIA3 levels in activated microglia may be associated with upregulation of numerous glycoproteins such as secretory and membrane proteins, including PGRN. Figure 6. Distribution of SERPINH1, PDIA6, PDIA3, and PPIB in normal and 5XFAD mouse brain. Triple-labeled confocal microscopic image analyses were used to study the expression of (A) SERPINH1, (B) PDIA6, (C) PDIA3, and (D) PPIB. Also imaged are Iba1 (an activated-microglia marker, red) and DAPI (a nucleus marker, blue). Shown are normal and 5XFAD mouse brain slices. Scale bar = 50 μm.
4. CONCLUSIONS In this study, we provide a proteomic analysis of Aβ stimulation of human microglia using SILAC labeling followed by LC− MS/MS analysis. Among the implicated proteins, 60 proteins showed significant differences in relative abundance after incubation of microglia with Aβ compared with controls. Our data identified the ER-resident proteins as potential biomarkers of human AD development based on microglia-mediated inflammation. These markers including SERPINH1, PDIA6, PDIA3, and PPIB can be represented as potential molecular targets for human AD treatment.
dysfunction, which is also known to be the one of main cause of AD.33,34 Most studies of ER stress indicate that the cause is disruption of protein folding. Many proteins, such as molecular chaperones, foldases, and glycosylation-related proteins, are involved in protein folding regulation in the ER.28 In light of these observations, our results may be evidence that Aβinduced microglial activation is accompanied by Aβ-induced ER stress. For example, SERPINH1 is a collagen-binding stress protein and molecular chaperone involved in the maturation of collagen residing in the endoplasmic reticulum.35−37 A recent study found physical interaction and colocalization of SERPINH1 and amyloid precursor protein in neurons. SERPINH1 was also reported to be enriched in amyloid plaques in mouse brain and to modulate the levels of extracellular Aβ peptides in neuron cultures.38 However, in contrast with the results of previous studies of some ER chaperones such as BiP/GRP78 (glucose-regulated protein 78), calnexin, and calreticulin,39−41 overexpression of SERPINH1 increased the level of secreted Aβ peptides, and reducing SERPINH1 expression with siRNA or chemically inhibiting its activity decreased the levels of secreted Aβ peptides in neuron cultures. Taken together, these results predict that SERPINH1 affects the degradation of Aβ peptides in the extracellular environment by inhibiting proteases.38 Another example of an ER-resident protein from our data set is PDIA3. PDIA3 is a protein disulfide isomerase 3 and is known as ERP57 (57-kD ER protein), GRP58 (58-kD glucoseregulated protein), and 1,25D3-MARRS (membrane-associated rapid response steroid-binding receptor). This protein is present mainly in ER and other subcellular locations such as the plasma membrane, cytosol, and nucleus.42,43 Erickson and his colleagues (2005) reported that PDIA3 and calreticulin were bound to Aβ in cerebrospinal fluid (CSF).44,45 They suggested that PDIA3 and calreticulin that became bound to normally N-glycosylated Aβ could prevent plaque formation, resulting from the precipitation of Aβ. These results suggest one reason why Aβ plaque is not formed in young people. Very little information is published on the roles of PDIA3 in microglia. In one report, progranulin (PGRN) was found to bind to ER molecular chaperones including some PDIs.46 PGRN is up-regulated in an AD model mouse compared with control, and its expression level is significantly correlated with
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ASSOCIATED CONTENT
S Supporting Information *
Supplemental Figure S1. Real-time PCR data confirming differential expression of the six selected up- and downregulated genes. Supplemental Table S1. Primer sequences used for real-time PCR. Supplemental Table S2. Increased proteins in microglia stimulated by Aβ. Supplemental Table S3. Decreased proteins in microglia stimulated by Aβ. Supplemental Table S4. Gene Ontology annotation analysis using DAVID Bioinformatics Resource (v6.7). This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*Y.M.P.: E-mail: [email protected]. Tel: +82-43-240-5160. Fax: +82-43-240-5059. *B.L.: E-mail: [email protected]. Tel: +82-32-899-6582. Fax: +82-32-899-6519. Author Contributions ▽
Y.Y. and K.B. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by IBS-R001-D1, the Gachon University research fund of 2014 (GCU 2014-5102, 5105) and HI13C2098.
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