Article pubs.acs.org/jpr
Secretome Analyses of Aβ1−42 Stimulated Hippocampal Astrocytes Reveal that CXCL10 is Involved in Astrocyte Migration Wenjia Lai,† Jing Wu,† Xiao Zou,† Jian Xie,‡ Liwei Zhang,‡ Xuyang Zhao,† Minzhi Zhao,† Qingsong Wang,† and Jianguo Ji*,† †
The National Laboratory of Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, P. R. China ‡ Department of Neurosurgery, Beijing TianTan Hospital Affiliated to Capital Medical University, Beijing 100875, P. R. China S Supporting Information *
ABSTRACT: Amyloid-beta (Aβ) aggregation plays an important role in the development of Alzheimer’s disease (AD). In the AD brain, amyloid plaques are surrounded by reactive astrocytes, and many essential functions of astrocytes have been reported to be mediated by protein secretion. However, the roles of activated astrocytes in AD progression are under intense debate. To provide an in-depth view of the secretomes of activated astrocytes, we present in this study a quantitative profile of rat hippocampal astrocyte secretomes at multiple time points after both brief and sustained Aβ1−42 stimulation. Using SILAC labeling and LC−MS/MS analyses, we identified 19 up-regulated secreted proteins after Aβ1−42 treatment. These differentially expressed proteins have been suggested to be involved in key aspects of biological processes, such as cell recruitment, Aβ clearance, and regulation of neurogenesis. Particularly, we validated the role played by CXCL10 in promoting astrocyte aggregation around amyloid plagues through in vitro cell migration analysis. This research provides global, quantitative profiling of astrocyte secretomes produced on Aβ stimulation and hence provides a detailed molecular basis for the relationship between amyloid plaques and astrocyte aggregation; the findings thus have important implications for further investigations into AD development and therapy. KEYWORDS: cell migration, secreted protein, reactive astrocytes, Alzheimer’s disease, amyloid-beta, LC−MS/MS
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INTRODUCTION Neurodegenerative diseases greatly affect the health and quality of life of elderly people. Alzheimer’s disease (AD) is a neurodegenerative disease that can cause loss of learning and memory abilities in patients.1 It is characterized by amyloid plaque deposition, neurofibrillary tangles, loss of cholinergic neurons, and gliosis in the patient’s brain.2−4 The pathogenesis of AD is very complex, and several hypotheses have been proposed to explain it,5−7 e.g., mutation and polymorphism of associated genes, amyloid-beta (Aβ) deposition, tau protein hyperphosphorylation, calcium homeostasis and free radical metabolic disorders, neuronal apoptosis, and chronic inflammation. Astrocyte aggregation in amyloid plaques has been reported; this aggregation can be induced by Lib8 or MCP-1.9 The presence of reactive astrocytes along with microglia around amyloid plaques can lead to chronic inflammation.10 Some evidence also suggests that astrocytes can take up and degrade Aβ fibers9,11 which are the major constituent of amyloid plaques. This indicates that astrocytes play a role in AD pathogenesis. Astrocytes are widely distributed in the mammalian brain. Apart from supporting and separating neurons, astrocytes are © 2012 American Chemical Society
also involved in the regulation of neuronal development, axon guidance, synapse formation, synaptic signaling, neuronal survival, and immune response, as well as maintaining the environment of the neuronal network.12 In certain conditions, such as brain injury and disease, astrocytes are stimulated into the “reactive” state, in which they can secrete many proteins, including neurotrophins, cytokines, and chemokines, to realize their functions.13 Therefore, to better understand the impact of certain astrocyte functions on the physiology and pathology of central nervous system (CNS) diseases, it is important to study the levels of these secreted proteins and changes in these levels in different situations. Secretomics, a branch of proteomics, focuses on understanding the profile of all proteins secreted from cells or tissues. The use of mass spectrometry (MS) allows high-throughput identification of secreted proteins, and in vivo or in vitro quantitative methods can help to identify differentially expressed proteins. Secretomes of primary astrocytes isolated from mice,14−17 rats,18 and humans19 under certain conditions Received: September 23, 2012 Published: December 28, 2012 832
dx.doi.org/10.1021/pr300895r | J. Proteome Res. 2013, 12, 832−843
Journal of Proteome Research
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collected at 1 day, 4 days, and 9 days and replaced with new media at 1 day and 4 days.
have already been identified using quantitative proteomics methods. However, little is known about secretory alterations in reactive astrocytes present in amyloid plaques. Here, we used the LC−MS/MS approach along with stable isotope labeling with amino acids (SILAC) to investigate alterations in secretomes of astrocytes during Aβ treatment and identified the secreted proteins that were involved in reactive astrocyte function. Nineteen proteins were identified based on their significantly increased levels in Aβ-treated astrocyte media, and their potential functions in astrocyte, microglia, neurons, and amyloid plaques were illustrated with the help of a protein network. Moreover, higher levels of the C−X−C motif chemokine 10 (CXCL10) was detected in Aβ-stimulated cells, which mediated in vitro hippocampal astrocyte migration. CXCL10, also known as interferon-γ inducible protein of 10 kDa (IP-10), is a small cytokine that is associated with the inflammatory process20 and mediates leukocyte migration to the lesion.21 Our results suggest that CXCL10 also participated in astrocyte recruitment in AD.
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Aβ1−42 Uptake and Degradation Assay
To determine Aβ uptake by astrocytes, cells were treated with 4 μM Aβ 488 in serum-free DMEM for 1 day, 4 days, and 9 days and washed three times with HBSS before they were examined under an Olympus IX-71 research inverted system microscope (OLYMPUS, Tokyo, Japan). For degradation tests, 4 μM Aβ 488 was added to the media and incubated for 24 h; then, the cells were washed with HBSS, and new media without Aβ 488 were provided. Aβ in the astrocytes was viewed microscopically at 12, 24, 36, and 48 h. Cell Viability and Apoptosis Assay
Astrocytes grown in 96-well plates were treated with Aβ1−42 or not at the designated times, and cell viability was analyzed as described previously.24 Briefly, 0.5 mg/mL of MTT (Sigma, MO, USA) was added to each well and incubated at 37 °C for 2−3 h. After the plates were centrifuged and media were removed, dimethyl sulfoxide (DMSO) was added at 150 μL/ well to dissolve the formazan, and absorbance was measured at 570 nm using a model 680 Microplate Reader (Bio-Rad, CA, USA). The percentage of viable cells was normalized by comparison with the 1 day controls. Astrocytes were cultured in 12-well plates and incubated with or without Aβ1−42 for 1 day, 4 days, and 9 days. Cells were collected, and cell apoptosis/death was analyzed using annexin V/FITC Kits (Beyotime, Jiangsu, China) under an Olympus microscope.
MATERIALS AND METHODS
Primary Culture of Astrocytes
Mixed glia culture was derived from the hippocampus of newborn Sprague−Dawley (SD) rats (Vitalriver, Beijing, China) as described previously22 and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco BRL, NE, USA), 50 IU/ml penicillin, and 50 mg/mL streptomycin (Invitrogen, NM, USA) at 37 °C in an atmosphere containing 95% humidified air and 5% CO2, with the medium changed every 3 days. After 9 days of culture, astrocytes were purified by shaking the flasks at 37 °C overnight, and then the cells were digested with trypsin (Gibco BRL, NE, USA) and transplanted into plates coated with poly lysine (Sigma, MO, USA) for subsequent experiments.
Immunofluorescence Staining and Western Blot Analysis
The cultured cells were washed twice with HBSS and fixed with 4% polyoxymethylene for 20 min at RT, washed three times with 0.1 M PBS, permeabilized with 1% Triton X-100 for 20 min at RT, washed three times with PBST (0.05% Tween 20 in 0.1 M PBS), blocked with 10% BSA in PBST for 1 h at 37 °C, and then incubated with primary antibody rabbit antirat GFAP (Abcam, Cambridge, U.K.) in 1% BSA overnight at 4 °C. The cells were washed three times with PBST, after which secondary antibody TRITC-conjugated goat antirabbit IgG (Southern Biotech, AL, USA) in 1% BSA was added for 1 h at RT in the dark; the cells were washed twice with PBS, stained with 0.1 μg/mL of 4′,6-diamidino-2-phenylindole (DAPI) for 5 min, and washed again with PBS. Finally, 50% glycerin (w/v, in PBS) was added, and cells were observed for fluorescence under the Olympus microscope. Astrocytes and their culture media after Aβ treatment were collected. Cells were lysed directly using loading buffer (0.1 M Tris-HCl, 20% glycerol, 5% sodium dodecyl sulfate (SDS), and 2% β-mercaptoethanol), and proteins in the media were isolated using the same procedure as in the LC−MS/MS sample preparation mentioned later on and then mixed with loading buffer. The protein samples were separated on 13% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, CA, USA). Then, the membrane was blocked with 5% milk and incubated with rabbit antirat CXCL10 (LifeSpan BioSciences, WA, USA), rabbit antirat SPARC-like protein 1 (SPARCL1) (Bioss, Beijing, China), rabbit antirat pancreatic triglyceride lipase (PTL) (Biorbyt, CA, USA), and mouse antirat β-actin (Southern Biotech, AL, USA) overnight at 4 °C separately, after washing three times with PBST. Horseradish peroxide
SILAC Labeling
SILAC Protein ID & Quantitation Media Kits (Invitrogen, NM, USA) were used to label astrocytes. For secreted proteins analysis, astrocytes were cultured under the same conditions mentioned above but with the media changed to DMEM containing L -arginine and L-lysine (light medium) or 13 15 13 L- C6 N4-arginine and L- C6-lysine (heavy medium) supplemented with 10% dialyzed FBS, 50 IU/ml penicillin, and 50 mg/mL streptomycin. After purification, astrocytes were transplanted to 25 cm2 flasks and kept in SILAC media for 1 month before Aβ treatment.23 Aβ1−42 Treatment of Astrocytes
Human Aβ1−42 (Sigma, MO, USA) and fluoresced human Aβ1−42 (Aβ 488) (Otwobiotech, Guangzhou, China) were dissolved in Hanks’ balanced salt solution (HBSS) (M&C Gene Technology, Beijing, China) at 2 and 1 mM, respectively, and incubated at 37 °C for 3 days before use. Astrocytes cultured for about 1 month were washed twice with HBSS and then cultured in serum-free DMEM containing 4 μM Aβ1−42 or not at 37 °C for 1 day, 4 days, and 9 days. For SILAC-labeled astrocytes, forward and reverse experiments were conducted. In the forward experiments, 4 μM Aβ1−42 was added to serum-free heavy medium, and HBSS-containing light medium was used as the control; in the reverse experiments, 4 μM Aβ1−42 was added to serum-free light medium, and HBSS-containing heavy medium was used as the control. The SILAC media were 833
dx.doi.org/10.1021/pr300895r | J. Proteome Res. 2013, 12, 832−843
Journal of Proteome Research
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protein interactions and analyze GO biological process enrichment.
(HRP)-conjugated goat antirabbit IgG and HRP-conjugated goat antimouse IgG (Southern Biotech, AL, USA) were added, and ECL (Millipore, CA, USA) was used to detect the bands.
Real-Time PCR
Extraction and Digestion of Secreted Proteins
Total RNA was extracted using Trizol (Invitrogen, NM, USA) from Aβ1−42-treated and control astrocytes every 1 day, 4 days, and 9 days. The HiFi-MMLV cDNA kit (BeiJing Cowin Biotech Co. Ltd., Beijing, China) was used for mRNA reverse transcription on the S1000 Thermol Cycler (BioRad, CA, USA). Real-time PCR was carried out with the CFX96 realtime system (BioRad, CA, USA) using the GoTaq qPCR Master Mix (Promega, WI, USA) according to its manuscript. Relative mRNA expression levels were normalized to actin mRNA levels, and the results were analyzed by the 2−ΔΔCT method.35 The primer sequences of the genes are listed in Table S1 (Supporting Information).
Heavy/light media collected from treated astrocytes at 1 day, 4 days, and 9 days were mixed together with the corresponding control media. Mixed media were centrifuged at 4000g for 15 min at 4 °C, filtered through a 0.22 μm filter, and concentrated using Amicon Ultra Centrifugal Filter Devices 3000 Da (Millipore, MA, USA); then, the media were washed using 25 mM NH4HCO3, reduced with 10 mM DTT (Sigma, MO, USA) for 40 min at RT, alkylated with 55 mM iodoacetamide (Sigma, MO, USA) for 30 min at RT, and washed twice with 25 mM NH4HCO3. Every step was followed by centrifugation at 7000g for 30 min at 4 °C. Finally, the mixtures were transferred to 0.6 mL tubes, digested with trypsin at 37 °C overnight, and dried using a vacuum drier (Bio-Rad, CA, USA). Before MS/ MS analysis, the peptides were desalted and dried again.
Cell Migration Assay
Astrocyte-conditioned medium (ACM) was obtained after incubation of astrocytes for 2 days in medium with or without 4 μM Aβ1−42. After treatment, astrocytes were washed twice with HBSS and incubated with serum-free DMEM at 37 °C for 48 h. Then, the ACM was collected, centrifuged, and stored at −80 °C before use. Cell migration was analyzed using the transwell assay. The transwell assay was performed using 24-well Nunc cell culture inserts (pore size, 8 μm) (NUNC, Denmark). Astrocytes were harvested and resuspended in DMEM with 1% FBS and then transplanted into the upper chamber at 1 × 105 cells/well. ACM, recombinant rat CXCL 10 protein (Peprotech, NJ, USA), CXCL 10 antibody (LifeSpan BioSciences, WA, USA), and normal rabbit IgG (Santa Cruz, CA, USA) were added into the base wells and incubated at 37 °C for 24 h. Nonmigrated cells were removed by cotton swab, and migrated cells on the other side of the membrane were fixed using 10% methanol for 10 min and stained with 0.1% crystal violet solution (Beyotime, Jiangsu, China) for 20 min. Photomicrographs of migrated cells were taken, after which the crystal violet stain was dissolved with 33% acetic acid and the absorbance was detected at 590 nm.
LC−MS/MS
The peptide mixtures dissolved in 10 μL of 0.2% formic acid were centrifuged at 13 000g for 10 min before analysis with LC−MS/MS using the LTQ-Orbitrap mass spectrometer (Thermo Fisher Scientific Inc., USA). Peptide separation was conducted using the nanoflow liquid chromatography system (Micro-Tech Scientific, USA) with a C18 reverse-phase column at a flow rate of 500 nL/min. A 240 min gradient of 5% to 32% acetonitrile in 0.1% formic acid was used. The instrument was operated in the positive ion mode, with the ESI spray voltage set at 2.3 kV and the collision energy set at 35%. Raw data were acquired in the profile mode using the Xcalibur software (version 2.2). Each survey full-scan MS spectrum (m/z, 300− 1600) was followed by MS/MS acquisition. Survey spectra with a resolution of 60 000 at 400 m/z after accumulation to a target value of 1 000 000 were acquired for CID fragmentation on the five most intense ions from the MS spectrum. Protein Quantification and Data Analysis
All MS raw data were identified and quantified with the MaxQuant software (version 1.0.12.31).25 The Rat International Protein Index (IPI) decoy database containing both forward and reversed protein sequences, which was generated using the Sequence Reverser tool in MaxQuant software against the International Protein Index (IPI) Rat database (version 3.87, 39 925 entries) from European Bioinformatics Institute (EBI) as described,26 was used for database searching by Mascot Daemon. The search parameters were enzyme, trypsin; fixed modification, carbamidomethyl (Cys); variable modification, oxidation (Met), N-acetylation, L-13C615N4-arginine, and 13 L- C6-lysine; maximum number of missing cleavages, 1; MS tolerance, 5 ppm; MS/MS tolerance, 0.5 Da; and false discovery rate (FDR),
