Identification of Proteins Involved in Microglial Endocytosis of r-Synuclein Jun Liu,†,‡,§ Yong Zhou,† Yan Wang,‡ Hanson Fong,| Thomas M. Murray,| and Jing Zhang*,‡ Departments of Pathology and Materials Science & Engineering, University of Washington School of Medicine, Seattle, Washington 98104 Received March 17, 2007
Aggregated R-synuclein, a protein playing pivotal roles in the pathogenesis of Parkinson disease (PD) and related synucleinopathy, has been shown to activate microglia, the key cells in neuroinflammation. However, the mechanisms by which aggregated R-synuclein enters microglia remain uncharacterized. In this study, we first replicated our previous results with a modified protocol that generated aggregated R-synuclein more efficiently. Next, using two recently developed proteomic techniques, SILAC (Stable Isotope Labeling of Amino acid in Cell cultures) and PROCEED (PROteome of Cell Exposed Extracellular Domains), we studied the plasma membrane proteins of primary cultured microglia that might be interacting with aggregated R-synuclein and mediating its internalization. The results demonstrated that 250 nM R-synuclein, aged for 6 h with a magnetic stir bar, was just as potent in activating microglia as the aggregated R-synuclein produced by aging without constant agitation for 7 days. The proteomic analysis identified 111 membrane proteins; of these, 46 proteins were altered in relative abundance in the membrane compartment after treatment with aggregated R-synuclein for 3 h. Two of these proteins, clathrin and calnexin, were further evaluated with Western blotting, demonstrating good agreement with quantitative proteomics. Finally, immunocytochemical as well as co-immunoprecipitation studies indicated that clathrin was indeed co-localized with internalized R-synuclein in microglia. These results suggest for the first time that microglial activation secondary to internalization of aggregated R-synuclein likely requires participation of clathrin, which is an essential protein of the polyhedral coat of coated pits and vesicles that play major roles in endocytosis and vesicular trafficking. Keywords: R-synuclein • microglia • SILAC • PROCEED • clathrin • Parkinson disease
Introduction Parkinson disease (PD) is a progressive movement disorder. Its clinical features are largely due to the relatively selective loss of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc) and a few other brainstem structures in the early stage of the disease. Formation of cytoplasmic inclusions called Lewy bodies in the remaining neurons has been considered a pathological hallmark of PD.1 The underlying pathogenesis for PD development remains elusive, although more recent studies have indicated that an inflammatory process in the SNpc, characterized by activation of resident innate cell microglia, likely either initiates or aggravates nigral neurodegeneration in PD.2-4 This is because microglia, when activated overtly, produce a variety of proinflammatory and * To whom correspondence should be addressed. Jing Zhang, MD, PhD, Division of Neuropathology, University of Washington School of Medicine, Box 359660 Harborview Medical Center, Seattle, Washington 98104; Phone. 206-341-5245; Fax, (206) 341-5249; E-mail,
[email protected]. † Authors contributed equally. ‡ Department of Pathology. § Permanent Address: Department of Neurology & Institute of Neurology, Ruijin Hospital, Shanghai Jiao-Tong University School of Medicine, Shanghai 200025, China. | Department of Materials Science & Engineering.
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neurotoxic factors, e.g., reactive oxygen species (ROS), tumor necrosis factor-R (TNFR), interleukin-1β (IL-1β), and nitric oxide (NO) that result in collateral damage to brain.5,6 Several mechanisms have been proposed to activate microglia in the setting of PD, including exposure to environmental parkinsonian toxicants4,7 and endogenous molecules like neuromelanin8 and aggregated R-synuclein.9 The role of R-synuclein in microglial activation is appealing because it is a relatively abundant protein in the brain, comprising ∼1% of total brain protein, and is a major component of Lewy bodies in PD or dementia with Lewy body disease (DLB).10 The precise mechanism leading to R-synuclein aggregation and subsequent neurodegeneration is still controversial. Most investigators believe that R-synuclein aggregates via partially folded intermediates, which form a critical nucleus, followed by formation of protofibrils (7 nm wide) and then mature fibrils (10 nm wide). Protofibrils are also known as soluble oligomers and assume a unique morphology under electron microscopy.11-13 The role of R-synuclein in microglial activation is relatively new. Following our initial description that aggregated R-synuclein, presumably released from dying neurons, can activate microglia, a more recent study has suggested that cells actively secrete R-synuclein, both monomeric and aggregated forms, 10.1021/pr0701512 CCC: $37.00
2007 American Chemical Society
r-Synuclein Endocytosis by Microglia
to extracellular space, a process that can be greatly enhanced when mitochondrial and proteasomal functions are impaired.14 This further indicates that extracellular aggregated R-synuclein may indeed provide a mechanism underlying relentless nigral neurodegeneration in PD. As a part of our ongoing effort in defining the mechanisms related to microglial activation secondary to exposure to aggregated R-synuclein,9 we focused on discovering novel cell surface and plasma membrane proteins via proteomics that might be interacting with R-synuclein and mediating its internalization. In this study, we began by modifying our production of R-synuclein aggregates with a more recently published method that produced R-synuclein aggregation much more efficiently.13 We then employed two unique proteomic techniques to identify membrane proteins that were altered by R-synuclein aggregates. One of these proteomic methods is called SILAC (Stable Isotope Labeling of Amino acid in Cell cultures), which offers the advantage of removing most artifacts owing to sample processing, as cells are labeled even before experimental manipulation starts.15 The other proteomic platform is PROCEED (PROteome of Cell Exposed Extracellular Domains), which enriches plasma membrane proteins before proteomic analysis.16 With these methods, we identified 111 proteins that appeared to be associated with the plasma membrane or membrane related functions; of these, 46 proteins displayed significant changes in relative abundance in extracellular compartment of microglia treated with aggregated R-synuclein. We further validated two of these proteins, clathrin and calnexin, with Western blotting and confirmed their relative changes as determined by quantitative proteomics. Finally, we demonstrated that clathrin was indeed co-localized with R-synuclein by confocal and co-immunoprecipitation analysis.
Materials and Methods Reagents. All tissue culture media and supplements were obtained from Invitrogen (Carlsbad, CA). All experiments related to rodents were conducted in compliance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Washington. Purified human wildtype recombinant R-synuclein (MW: 14 kDa; endotoxin 98% pure as determined by OX-42-IR and GFAP-IR. Immunocytochemistry and Quantitative Analysis. Immunostaining was performed as described previously.18,19 The 4% paraformaldehyde-fixed cells were blocked and incubated overnight at 4 °C with primary antibodies diluted in antibody diluents (anti-R-synuclein, 1:180; anti-clathrin, 1:50; anti-OX42, 2 µg/mL), followed by fluorescent labeled secondary antibodies, i.e., cross-absorbed goat anti-mouse (Alexa Fluor 488, Eugene, OR) and donkey anti-goat (Alexa Fluor 568, Eugene, OR) antisera. Nuclei from all the cells were stained by TO-PRO-3, a monomeric cyanine nucleic acid stain from Invitrogen (Carlsbad, CA). Images were recorded using a laser scanning confocal microscope (Bio-Rad LS2000). For quantitative analysis of OX-42 positive microglia, cells were counted in four randomly chosen fields in each well, and a total of three wells were counted for each experimental condition, with the data expressed as % of TO-PRO-3 stained cell nuclei. Western Blotting. For analysis of the extent of internalization of human R-synuclein, microglia were harvested, washed 3 times with ice-cold PBS, homogenized in 2% SDS lysis buffer, sonicated, and centrifuged at 14 000× g for 15 min at 4 °C. The supernatant was collected, and protein concentrations were measured using the BCA assay (Pierce Biotechnology, Inc., Rockford, IL). Proteins (20 µg) from each sample were loaded onto 8-16% sodium dodecyl sulfate polyacrylamide gels (SDSPAGE). Following separation, the proteins were transferred to PVDF membranes (Bio-Rad Laboratories, Hercules, CA) and probed overnight at 4 °C with primary antibodies. After washing with TBS-T (0.1% Tween 20 in TBS), HRP-conjugated secondJournal of Proteome Research • Vol. 6, No. 9, 2007 3615
research articles ary antibodies were added for 1 h at room temperature, and detections were carried out by enhanced chemiluminescence (ECL). Membrane fractions of microglial cells were collected on the basis of our previous protocol 20. Immunoprecipitation. Following 3 h exposure to aggregated R-synuclein, microglia were washed in ice-cold PBS, scraped off the plates, and underwent lysis in RIPA buffer (1% NP40, 0.5% SDS, 150 mM NaCl, 10 mM Tris-HCl, 1 mM PMSF, pH 7.4, supplemented with a protease inhibitor mixture (Sigma)). Total cell lysate was centrifuged at 14 000× g for 15 min before the supernatant was collected. Tosylated Dynabeads (2 × 107 of Dynabeads; Dynal Biotech, Invitrogen) were covalently conjugated to either anti-R-synuclein or anti-clathrin according to the manufacturer instruction. The beads were incubated with the supernatant overnight at 4 °C and then magnetically concentrated, leaving processed supernatant behind. The beads were washed three times with RIPA buffer and the immunoprecipitate was eluted using 2% Laemmli sample buffer before Western blot analysis with antibodies against clathrin and R-synuclein respectively. Prostaglandin E2 (PGE2) ELISA Assay. The prostaglandin E2 (PGE2) monoclonal ELISA kit was obtained from Cayman Chemical (Ann Arbor, MI). Assays were performed following the manual from the manufacturer. SILAC Microglial Proteome Labeling. SILAC labeling of microglial cells was carried out using a method described by us previously with minor modifications.15,18 Microglia were cultured as described above with the exception of using arginine-depleted media. For cultures exposed to aggregated R-synuclein and vehicle treatment, 12C6 L-arginine (light) and 13C L-arginine (heavy) were added respectively. Cells were 6 incubated at 37 °C for at least 2 weeks, after which microglial cells were shaken off, counted, and seeded into 24 well plates (0.5 × 106 cells per well). Aggregated R-synuclein was added to the culture media with a final concentration of 250 nM, and incubated for 3 h before harvest. Separation and Digestion of Extracellular Domains of Membrane Proteins - PROCEED. After treatments, microglia labeled with either heavy or light arginine were rinsed 3× with dye-free HBSS buffer to remove bovine serum proteins from culture media. HBSS buffer (250 µL) with 1 µg of proteinase K (Promega, Madison, WI) was introduced to each well and incubated for 3 h at 37 °C to cleave those exposed extracellular proteins from cell surface. The “shaved” peptides were collected, and the two samples were mixed at a 1:1 ratio for R-synuclein (light): control (heavy) based on microglial cell amount. The cell membrane integrity after protease incubation was then evaluated by Trypain Blue staining. To achieve efficient cleavage, the mixture of peptides and protein fragments was fully denatured in the presence of 8 M urea combined with 0.1% SDS before digestion by MS grade trypsin following a standard in-solution digestion protocol.21 The trypsin digested sample was then run on C-18 desalting column (Millipore, Bedford, MA) for further purification and stored at -80 °C until ran on µLC-MS/MS. Peptide Analysis by µLC-MS/MS. The purified stable isotope labeled peptides from each sample were separated by an online two-dimensional microcapillary high performance liquid chromatography system, which integrated a strong cation-exchange (SCX) column (100 mm in length × 0.32 mm inner diameter; particle size: 5 µm) with two alternating RP µC18 columns (100 mm in length × 0.18 mm inner diameter), followed by analysis directly with tandem mass spectrometry (MS/MS) using an LCQ 3616
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DECA XP PLUS quadruple ion trap mass spectrometer (ThermoElectron, San Jose, CA). Settings for the µLC-MS/MS were as previously described.18,22 Briefly, six fractions were eluted from SCX using a binary gradient of 2-90% solvent D (1.0 M ammonium chloride and 0.1% formic acid in 5% acetonitrile) versus solvent C (0.1% formic acid in 5% acetonitrile). Each fraction was injected onto a reverse phase (RP) µC18 column automatically with the peptides being resolved using a 120 min binary gradient of 5-80% solvent B (0.1% formic acid in acetonitrile) versus solvent A (0.1% formic acid in water). A flow rate of 160 µL/min with a split ratio of 1/80 was used. Peptides were eluted directly into the electrospray ionization (ESI) ion trap mass spectrometer. To determine the amino acid sequence, the mass spectrometer operated in a data-dependent MS/MS acquisition mode (a full-scan mass spectrum followed by MS/MS scans of the three most intense ions detected in the previous MS scan), using 35% of the normalized collision energy for obtaining MS/MS spectra. The dynamic exclusion function was enabled for 3 min. Database Search and Peptide/Protein Analysis. Original RAW files obtained from 6 fractions of each sample (a total of five samples) were converted to mzXML files separately, and MS/MS spectra were analyzed automatically using the computer program SEQUEST, which searched tandem mass spectra against International Protein Index (IPI) database (ipi.rat.fasta, V3.24). Search parameters for SEQUEST used in this study were the following: tryptic, maximum one miss cleavage; +6 Da for 13 C6 isotopic-labeled arginine, +16 Da for oxidized methionine as differential options, and +57 Da for carbamidomethyl cysteine as static option; mass tolerance ( 3Da; original forward database. Database search results from 6 fractions were combined and statistically analyzed using PeptideProphet, which effectively computes a probability for the likelihood of each identification being correct (on a scale of 0 to 1) in a data-dependent fashion.23 Considering the low sequence coverage of our sample set, a minimum PeptideProphet probability score (P) filter of 0.9 was used to remove low probability peptides, which equaled to the false positive rate about 0.7%. The identified peptides (PeptideProphet probability score (P) g 0.9) were further grouped into proteins using software ProteinProphet to enhance confidence of protein identification based on statistical models.24 ProteinProphet allowed filtering of large-scale data sets with assessment of predictable sensitivity and false-positive identification error rates. In our study, only proteins with a high probability of accuracy (
