Animals

Male 6–7 week old, WT C57BL/6J (stock 000664, denoted as B6) (H-2^b), B6.CB17-Prkdc^scid/SzJ (stock 001913, herein denoted as SCID) (H-2^b) and B10.BR-H2^k H2-T18^a/SgSnJ (stock 000465, herein denoted as B10.BR) (H-2^k) mice were purchased from Jackson Laboratories (Bar Harbor, ME). All animal procedures were in accordance with National Institutes of Health (NIH) guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Nebraska Medical Center (UNMC).

MPTP Intoxication

For chronic intoxication, B6 mice received 5 intraperitoneal (i.p.) injections at 24 hr intervals for 5 days of either vehicle (PBS, 10 ml/kg) or MPTP-HCl (30 mg/kg of free base in PBS) (Sigma-Aldrich, St. Louis, MO). For acute intoxication, mice received 4 i.p. injections, one every 2 hr, of either vehicle (PBS, 10 ml/kg) or MPTP-HCl (18 mg/kg of free base in PBS for B10.BR mice, 14 or 18 mg/kg for B6 mice). At selected time points following MPTP intoxication, mice were sacrificed and brains processed for subsequent analyses. MPTP handling and safety measures were in accordance with published guidelines [35].

Immunohistochemistry

At the time points indicated following MPTP intoxication, mice were transcardially perfused with 4% paraformaldehyde (PFA) in 0.1 M PBS using 0.9% saline as vascular rinse. Brains were post-fixed in 4% PFA overnight, kept in 30% sucrose for 2 days, snap frozen, embedded in OCT compound, and 30 µm sections cut on a cryostat (CM1900, Leica, Bannockburn, IL). The sections were collected in PBS and processed free-floating. Primary antibodies used for immunohistochemistry includes rabbit anti-TH antibody (1∶2000; Calbiochem/EMD Biosciences, Inc., San Diego, CA), rat anti-mouse CD11b or Mac-1 (1∶1,000; Serotec, Raleigh, NC), rat anti-CD3 (1∶800; BD Pharmingen, San Diego, CA,), rat anti-CD4 (BD Pharmingen), and rat anti-CD8 (BD Pharmingen). Immunostaining was visualized using diaminobenzidine (Sigma-Aldrich) as the chromogen and mounted on slides. TH, CD3-, CD4- and CD8-immunostained brain sections were counterstained with thionin (Sigma-Aldrich) as previously described [33], [36]. Fluoro-Jade C (Chemicon International, Inc., Temecula, CA) was used to stain degenerating neurons [37] and was detected as green fluorescence by fluorescence microscopy with FITC filter (Eclipse E800, Nikon, Inc., Melville, NY).

Stereology of TH-Positive Neurons

Total numbers of Nissl- and TH-stained neurons throughout the entire SNpc were counted stereologically in a blinded fashion with Stereo Investigator software (MicroBrightfield, Williston, VT) using the Optical Fractionator probe module as previously described [33].

Cloning α-Syn and 4YSyn

Total RNA from adult C57BL/6 mouse brain was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The full-length mouse α-Syn gene (504 bp) and 120 bp length encoding the C-terminal portion (4YSyn) was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using Platinum Taq DNA Polymerase High Fidelity (Invitrogen). The 5′ primer was designed to introduce a Nde1 site at position 1. This fragment was blunt cloned into the pZero-1 (Invitrogen) Eco RV site using standard cloning procedures. Transformed cells were plated on low salt agar containing Zeocyn and 3 mM IPTG. Colonies were screened using colony PCR with α-Syn primers. Colonies containing the full-length mouse α-Syn gene or the 3′ fragment encoding 4YSyn were grown overnight and plasmid DNA was isolated using standard mini-prep (Invitrogen). The gene was digested out of pZero with Nde1 and Xho1 and subcloned into the Nde1 and Xho1 sites in the pET-28a prokaryotic expression vector using DH5-α cells. Colonies were screened using colony PCR. Purified plasmids were submitted to the UNMC core facility for sequence confirmation. Plasmids containing the complete sequence were transformed into BL-21 E. coli cells for expression. Frozen glycerol stocks were maintained at −80°C.

4YSyn Expression

Glycerol stocks were streaked on Luria-Bertani (LB) agar plate containing 30 µg/ml kanamycin. A single colony was inoculated into LB broth containing 30 µg/ml kanamycin, grown for 8 hrs, and stored at 4°C until the following day. The starter culture was diluted 1∶100 into fresh liquid medium containing 30 µg/ml kanamycin and allowed to grow to an OD₆₀₀ = 0.6. Expression of recombinant protein was induced by the addition of 3 mM IPTG with continued incubation for 3 hrs at 37°C. Following induction, cells were centrifuged, weighed, and stored at −80°C until purification protocol was resumed. >90% of detectable 4YSyn was found in the soluble fraction.

Protein Purification and Nitration

Cell lysis was performed with Bug Buster reagent (Novagen/EMD Biosciences, Inc., San Diego, CA) at 5 ml/g cells with addition of EDTA-free protease inhibitor cocktail (Calbiochem). Benzonase nuclease (Novagen) was added to reduce viscosity during lysis following manufacturer's instructions. Insoluble cell debris was removed by centrifugation at 16,000×g for 20 min at 4°C. The soluble fraction was directly subjected to column affinity chromatography and was carried out in the following steps: His-tagged protein was bound to Ni-NTA His Bind Resin (Novagen) in Bug Buster reagent with the addition of imidazole (10 mM). The column was washed first with 50 mM NaH₂PO₄/300 mM NaCl/20mM imidazole, pH 8.0 and then with 50 mM NaH₂PO₄/300 mM NaCl/35mM imidazole, pH 8.0. Elution was carried out 50 mM NaH₂PO₄/300 mM NaCl/250 mM imidazole pH 8.0. Samples were separated by SDS-PAGE and stained with Brilliant Blue G-Colloidal Coomassie stain (Invitrogen) to confirm purity of the eluted fraction. Full-length α-Syn and 4YSyn were visualized by silver stain (Silver Xpress, Invitrogen). Purified full-length α-Syn was dialyzed in 50 mM NaH₂PO₄ buffer. Thrombin cleavage was carried out using biotinylated thrombin cleavage capture kit (Novagen) following manufacturer's instructions. Cleaved His-tags were removed with Ni-NTA resin. His-tag free full-length α-Syn and His-tagged 4YSyn (unable to remove the His-tag) were dialyzed against water for 24–48 hrs with multiple water changes, lyophilized, and weighed. Endotoxin was removed by polymyxin B agarose beads following manufacturer's instructions (Sigma-Aldrich) and tested for residual endotoxin by Limulus amebocyte lysate (LAL) assay (E-Toxate, Sigma-Aldrich). Recombinant α-Syn-derived proteins were endotoxin-free as all batches of purified proteins utilized tested below the limit of detection for endotoxin by LAL (<0.05 endotoxin units, EU).

Lyophilized protein was resuspended (2 mg protein/ml) in 50 mM NaH₂PO₄ buffer containing 5 mM FeCl₃ as a Lewis acid. Peroxynitrite (Upstate Biotechnology, Inc. Lake Placid, NY) was added dropwise to protein to achieve a 5 M excess while vigorously mixing the reaction mixture. Nitrated protein was dialyzed against water for 48 hrs using multiple water exchanges, lyophilized, and stored at −80°C.

MALDI-TOF Mass Spectrometry

MALDI-TOF mass spectrometric analysis was performed using a Voyager DE Pro mass analyzer (Applied Biosystems, Framingham, MA), which was externally calibrated prior to each assay. Data acquisition was performed using 500 laser shots. The MS scan range was set from 500 to 20,000 m/z. Saturated α-cyanohydroxycinnamic acid (Sigma-Aldrich) was used as matrix in these assays and samples were manually spotted onto MALDI targets.

Enzyme-Linked Immunosorbent Assay (ELISA)

Individual wells of Immunolon II ELISA plates (Thermo Electron Corp., Waltham, MA) were coated with 100 µl/well of native 4YSyn or N-4YSyn at 1 µg/ml PBS, pH 8.5. Plates were incubated for 2 hrs at 37°C and washed with 0.5% Tween20/PBS, pH 7.2 (PBS-T). Nonspecific binding was blocked by the addition of 1% bovine serum albumin in PBS, pH 7.2 (PBS-BSA) and incubation at 37°C for 1 hr. Plates were washed with PBS-T, 100 µl of 2-fold serial dilutions (from an initial 1∶50 dilution in PBS-BSA) of serum samples from MPTP- or PBS-treated mice were added to each well, and incubated at 37°C for 1 hr. Plates were washed with PBS-T and 100 µl/well of a 1∶5000 dilution of horseradish peroxidase (HRP)-conjugated anti-mouse IgG (SouthernBiotech, Birmingham, AL) was added. Plates were incubated at 37°C for 1 hr, washed with PBS-T, and reacted with 0.012% H₂O₂ and 2.2 mM o-phenylenediamine dihydrochloride (Sigma-Aldrich) in 100 µl of 0.1 M phosphate-citrate buffer, pH 5.0. The reaction was stopped with the addition of 2 N H₂SO₄, read at 490 nm on a microplate reader (Vmax Kinetic Microplate Reader, Molecular Devices Corporation, Sunnyvale, CA), and acquired data analyzed with interfacing SoftMax Pro software (Molecular Devices). Serum IgG concentrations were quantified from a standard curve prepared from known concentrations of mouse IgG (SouthernBiotech).

Immunization and Immune Cell Adoptive Transfers

B10.BR (H-2^K) mice were immunized with PBS, 50 µg of 4YSyn or N-4YSyn emulsified in an equal volume of CFA containing 1 mg/ml Mycobacterium tuberculosis (Sigma-Aldrich). B6 (H-2^b) mice were immunized with PBS, 10 µg of 4YSyn or N-4YSyn with or without CFA. While immunization with adjuvant were administered subcutaneous (s.c.) on either side of the tail base, s.c. injections without adjuvant were given at 5 different sites. Fourteen days after primary immunization, mice were boosted with their respective antigens. CFA recipient mice were boosted with their respective antigens emulsified in IFA (Sigma-Aldrich). Five days following their final immunizations, donor mice were sacrificed and single cell suspensions were prepared from the spleen and draining lymph nodes after lysing red blood cells with ammonium chloride-potassium (ACK) lysis buffer (0.15M NH₄Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA, pH7.2). T cells were enriched by using the PAN T cell isolation kit (Miltenyi Biotec, Auburn, CA) and by depletion of magnetically labeled cells employing AutoMACS (Miltenyi Biotec). Twelve hrs post-MPTP intoxication, both B10.BR and B6 mice received intravenous (i.v.) injections of 5×10⁷ spleen cells (SPC) in 0.25 ml of Hanks' balanced salt solution (HBSS). B10.BR mice also received 2.5×10⁷ purified T cells. SCID mice were reconstituted with i.v. injections of 8×10⁷ unfractionated SPC populations from WT B6 mice. RCS-SCID mice were rested for 4 wks prior to MPTP intoxication.

³H-Thymidine in vitro Proliferation Assays

Samples of pooled immunized donor cells used for adoptive transfer were tested for their proliferative capacity by ³H-thymidine incorporation after stimulation with either immunizing or irrelevant antigen. Donor SPC were plated at a density of 2×10⁶ cells/ml complete RPMI tissue culture media [RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 1× nonessential aa, 55 µM 2-mercaptoethanol, 100 U/ml penicillin, and 100 µg/ml streptomycin (Mediatech Inc., Herndon, VA)]. SPC from PBS, 4YSyn, and N-4YSyn immunized mice were stimulated with 0, 1, 10, 50 µg/ml of immunizing antigen, 4YSyn or N-4YSyn, and cultured at 37°C for 5 days. Cells were pulsed with 1 µCi ³H-thymidine/well for the final 18 hrs of culture, harvested onto glass fiber plates, and counted by β-scintillation spectrometry (TopCount, Packard-PerkinElmer Instruments, Wellesley, MA).

Western Blot Analysis

Ventral midbrain (VMB) and lymphoid organ protein extracts (80 µg/lane) were separated by 16% SDS-PAGE (Invitrogen) and transferred for 45 min onto 0.2 µm PVDF membranes (Millipore, Bedford, MA). Membranes were probed with rabbit antibodies to NT (1∶2000; Chemicon) or monoclonal rat antibodies to myelin basic protein (MBP, 1∶1000, Chemicon) or guinea pig antibodies to α-Syn (1∶1000; Ab-1, Oncogene/EMD Biosciences). Appropriate HRP-conjugated secondary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) were used to visualize blots using SuperSignal West Pico Chemiluminescent substrate and CCL-XPosure film (Pierce Biotechnology, Inc., Rockford, IL). Immunoblots were stripped and reprobed with antibodies to α-actin (1∶5000; Chemicon,) as an internal control.

Identification of α-Syn in MPTP-CLN

Anti-N-α/β-syn (clone nSyn12, mouse ascites, Upstate) that specifically recognizes N-α-Syn (14.5 kD) and N-β-Syn (17 kD) but not non-nitrated α/β-Syn was used for immunoprecipitation (IP). VMB and CLN from PBS or MPTP treated mice were dissected out, homogenized in ice-cold RIPA buffer, pH 7.4 and centrifuged at 10,000×g for 10 min at 4°C to remove cellular debris. Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology) were added to 2 mg total cellular protein, incubated for 1 hr at 4°C. Beads were centrifuged at 1,000×g for 5 min at 4°C. The supernatant was incubated with 40 µl anti-N-α-Syn overnight at 4°C on a rotating device, and then with Protein A/G PLUS-Agarose beads for 1 hr on a rotating device at 4°C. Immunoprecipitates were collected after centrifugation at 1,000×g for 5 min at 4°C, washed once with RIPA buffer and twice with PBS, resuspended in 40 µl of 1× electrophoresis sample buffer.

N-α-Syn IP samples were fractionated by large format 16% Tricine SDS-PAGE (Jule Inc., Milford, CT; BIORAD Laboratories, Inc, Los Angeles, CA) at constant voltage for 8–10 hrs. The gel was stained with highly sensitive SYPRO Ruby stain (Invitrogen) and scanned at excitation (400 nm) and emission (630 nm) wavelengths using Typhoon scanner (Amersham Biosciences, Piscataway, NJ) to visualize the protein bands. Small gel fragments (3–4 mm) corresponding to molecular weight (12–18 kD) were excised from each lane of the same gel stained with Coomassie. In brief, gel pieces were destained for 1 hr at room temperature using 100 µl of 50% ACN/50 mM NH₄CO₃. Gel pieces were dried and incubated with trypsin in 10 mM NH₄CO₃ (Promega, Madison, WI) overnight at 37°C. Peptides were extracted by washing gel pieces twice with 0.1% TFA and 60% ACN. Dried samples were resuspended in 12 µl of 0.1% formic acid in water for automated injection. All samples were purified using ZipTip (Millipore) prior to MS analysis. In-gel trypsin digested proteins were fractionated on microcapillary RP-C18 (Ciborowski et al., 2004). The resulting peptides were sequenced using Electrospray Ionization (ESI)-LC MS/MS (Proteome X System with LCQDecaPlus mass spectrometer, thermoElectron, Inc., San Jose, CA) with a nanospray configuration. The spectra obtained from LC-MS/MS analysis were searched against the NCBI.fasta rodent protein database using SEQUEST search engine (BioWorks 3.2 SR software from ThermoElectron, Inc, San Jose, CA.). Criteria for high confidence protein identification were used as previously published [38], [39], [40], [41], [42], [43].

Flow Cytometry

Single cell suspensions were prepared from deep CLN from C57BL/6 mice 20–24 hrs post PBS or MPTP (18 mg/kg) intoxication. Cell suspensions were analyzed for cell surface expression of CD11c, CD11b, and MHC class II (I-A^b). Also, prior to adoptive transfers, cell populations from immunized donors were stained for T cells using PE conjugated anti-mouse CD3ε (BD Pharmingen) and B cells with FITC conjugated anti-mouse CD19 (BD Pharmingen). Analysis was performed with a FACSCalibur flow cytometer interfaced with CellQuest software (BD-Biosciences, Immunocytometry Systems, San Jose, CA).

Determination of N-4YSyn-mediated Toxicity In Vitro

For proliferation analyses, purified T cells from naïve B6 mice were plated with SPC irradiated at 3000 rad (1:3) at 2×10⁶ cells/ml in complete RPMI tissue culture media and activated with anti-CD3 (0.5 µg/ml, 145-2C11; BD Pharmingen) in U-bottom 96-well tissue culture plates. Graded concentrations of 4YSyn or N-4YSyn were added to quadruplicate wells. After activation for 3 days, ³H-thymidine incorporation was performed as described previously.

To assess α-Syn-mediated cytotoxicity, purified T cells were stimulated with anti-CD3 and cultured at a density of 1×10⁶ cells/ml for 24 hrs in media alone or in the presence of 4YSyn or N-4YSyn at concentrations of 1, 3, 10, or 30 µg/ml. Cells were stained with PI, washed and the percentages of PI⁺ dead cells and MFI were analyzed by flow cytometry.

Macrophage and MES 23.5 Cultures

BMM were prepared from C57BL/6 adult male (6–12 weeks old) mice. The animals were sacrificed by CO₂ asphyxiation. Single cell suspensions of bone marrow cells were obtained from femur bone marrow cavities after flushing with HBSS, and red blood cells lysed with ACK buffer. The bone marrow cells were cultured in complete DMEM medium (Dulbecco's Modified Eagles Media supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin) containing 2 µg/ml macrophage colony stimulating factor (MCSF), a generous gift from Wyeth Pharmaceuticals (Cambridge, MA) in a 5% CO₂/37°C incubator. Non-adherent cells were removed from flasks at 1, 4, and 7 days by successive DMEM washes. Adherent BMM were harvested and replated for experiments following 7–14 days of culture. Cells from the MES 23.5 dopaminergic cell line kindly provided by Dr. Stanley Appel, were cultured in 75-cm² flasks in DMEM/F12 with 15 mM HEPES (Invitrogen) containing N2 supplement (Invitrogen), 100 U/ml of penicillin, 100 µg/ml streptomycin, and 5% FBS. Cells were grown to 80% confluence then co-cultured with BMM in serum free MEM/F12 at a density of 1×10⁵ cells (1:1) on sterile glass coverslips.

N-α-Syn SPC-Induced Microglia Cytotoxicity

SPC isolated from N-4YSyn (10 µg) immunized B6 mice were cultured in RPMI media and activated in vitro for 4 days with N-4YSyn (1 µg/ml). MES 23.5 cells or macrophages alone or MES 23.5 and macrophage co-cultures were stimulated with aggregated N-α-Syn (1.45 µg/ml) alone and in combination with either activated SPC or supernatants obtained from activated SPC for 24 hrs. Unstimulated cultures served as controls. Assays for viable and dead cells were performed with Live/Dead Viability/Cytotoxicity kit (Invitrogen) according to the manufacturer's protocol and viewed under a fluorescence microscope (Nikon Eclipse E800, Buffalo Grove, IL). Images were captured at 100× magnification and quantification of live (green) and dead (red) counts was performed from 4–8 different fields.

Cytokine array

Triplicate co-cultures of antigen presenting cells (APC) and T cells from PBS-, 4YSyn- and N-4YSyn-immunized mice were stimulated with 4YSyn or N-4YSyn. After 24 hours of culture, 50 µl samples were collected, centrifuged, and supernatants frozen at −80°C until utilized. Frozen supernatants were thawed only once and analyzed using the BD Cytometric Bead Array Mouse Th1/Th2 Kit (BD Biosciences, San Jose, CA) according to the manufacturer's instructions.

Statistical Analysis

All values are expressed as mean±SEM. Differences among normally distributed means were evaluated by Student's t test for two group comparisons or one-way ANOVA followed by Bonferroni post-hoc tests for pairwise comparisons amongst multiple data sets (Statistica v7, Statsoft, Tulsa, OK, and SPSS v13, SPSS, Inc., Chicago, IL) and were considered significant at p ≤ 0.05 unless otherwise indicated. Kolmogorov-Smirnov (K-S) analysis was performed for flow cytometric data analysis.

CNS Antigens Drain to CLN Following MPTP Intoxication

To determine if CNS antigens drain to CLN during established neurodegeneration of the nigrostriatal pathway, their presence in ventral midbrain (VMB), cervical, axillary, inguinal, mesenteric lymph nodes and spleens were determined 24 hrs after MPTP-intoxication in C57BL6 mice. The presence of unmodified α-Syn was demonstrated in VMB and CLN (Figure 1A) as well as other lymph nodes and in the spleen from phosphate-buffered (PBS)- or MPTP-treated mice in anti-α-Syn-probed immunoblots (data not shown). These findings also confirmed the expression of unmodified α-Syn amongst cells of hematopoietic lineage [44]. N-α-Syn IP showed that N-α-Syn was present in the CLN of MPTP-treated, but not PBS-treated mice as similar molecular weight bands were observed from gels probed with SYPRO Red and Western blots performed with α-Syn antibodies (Figure 1B). To validate the presence of NT-modified α-Syn after MPTP treatment, N-α-Syn immunoprecipitates were obtained after in-gel tryptic digestion of 12–18 kD fragments acquired from the VMB and CLN and sequenced by LC-MS/MS. This regions was chosen as it represents the molecular mass ranges of oxidized α-Syn [6], [14], [45], [46], [47]. Sequence analysis demonstrated α-Syn peptides (yellow highlighted sequences, Figure 1C) in the VMB from both PBS- and MPTP-treated mice but exclusively in the CLN of MPTP-intoxicated mice (Table 1). α-Syn peptides were identified at >99.999% confidence (Table 1). Western blot analysis of lymphoid tissue homogenates using rabbit NT antibodies detected a single band with a molecular mass of ∼16–18 kD–which is comparable to that of α-Syn–in CLN from MPTP-intoxicated animals, but not in other lymph nodes or spleen (Figure 1D, blots of mesenteric lymph nodes and spleen are not shown). These results were confirmatory for the presence of N-α-Syn in the draining CLN. Another CNS antigen, MBP was also detected only in the CLN of MPTP intoxicated animals (Figure 1D). NT-modified proteins and MBP were absent in lymph nodes and spleens of control (PBS-injected) mice. These data suggest that brain proteins released as a consequence of nigrostriatal injury, drain to the deep CLN, placing them in organs associated with efficient presentation of antigen. To demonstrate the functional significance of these observations, single cell suspensions were prepared from CLN isolated from MPTP animals and controls, and analyzed by flow cytometry for MHC class II expression on CD11b⁺ APC (Figure 1E). Increased frequencies of CD11b⁺/MHC class II⁺ in MPTP-treated mice compared to PBS controls was taken as evidence of leukocyte activation in the deep CLN following MPTP-induced nigrostriatal injury. Supporting the induction of a α-Syn specific immune response, sera from WT B6 mice 21 days after chronic MPTP intoxication were analyzed for anti-α-Syn IgG and compared to animals that received PBS. Serum levels of α-Syn antibodies in mice exposed to MPTP were significantly increased (Figure 1F). Together, these results demonstrate that NT-modified α-Syn draining into the deep CLN is capable of eliciting a peripheral immune response.

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Figure 1. Drainage of N-α-Syn and MBP to CLN with macrophage activation and production of α-Syn serum antibodies after MPTP intoxication.

(A) Western blot of tissue homogenates from VMB and CLN of mice 20 hrs following treatment with PBS or MPTP, were probed with antibodies to α-Syn. (B) N-α/β Syn IP with (clone nSyn12 antibodies) against CLN homogenates from PBS or MPTP-treated mice. Immunoprecipitates were fractionated on a 16% polyacrylamide gel and the gel stained with SYPRO Red or blotted. The Western blot was probed with anti-α-Syn. (C) Proteins recovered from in-gel digestion of 12–18 kD fragments from anti-N-α/β Syn of CLN immunoprecipitates were were identified by LC-MS/MS. The sequence coverage by peptides identified by LC-MS/MS from the CLN of MPTP-treated mice is highlighted in yellow within the primary aa sequence of full-length mouse α-Syn. (D) Western blots of lymph node homogenates (Cervical, Axillary, and Inquinal) from mice treated with PBS or MPTP. Blots were probed with antibodies to nitrotyrosine (NT) or anti-myelin basic protein (MBP). (E) Flow cytometric analysis of CD11b and I-A^b expression in cells from CLN, show an increased number of CD11b⁺I-A⁺ cells 24 hrs after MPTP treatment compared to PBS administered animals (n = 3 mice/group). (F) Antibodies against α-Syn and N-α-Syn in sera of B6 WT mice on day 21 following MPTP intoxication (n = 8) or PBS control treatment (n = 5) as determined by anti-α-Syn specific ELISA. Sera from MPTP treated group contained significantly higher IgG antibodies directed against 4YSyn (p = 0.021) and N-4YSyn (p = 0.016) compared to PBS treated control sera. Comparisons of mean IgG concentrations ± SEM was by Student's t test.

https://doi.org/10.1371/journal.pone.0001376.g001

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Table 1. Probabilities (p values) of protein sequence matches within 12–18 kD bands from anti-N-α/β-synuclein immunoprecipitation and LC MS-MS analyses of VMB and CLN from PBS- or MPTP-treated mice.

https://doi.org/10.1371/journal.pone.0001376.t001

Adaptive Immunity Participates in MPTP-Nigral Degeneration

The presence of NT modifications of α-Syn in draining lymphatic tissue following MPTP-induced nigrostriatal injury, along with evidence of lymphoid-associated APC activation provided support for antigen presentation to T cells and subsequent immune responsiveness. To substantiate this idea, we explored whether an endogenous adaptive immune system was required for MPTP-induced nigrostriatal degeneration. B6 WT mice, B6 SCID mice, and B6 SCID mice reconstituted with 10⁸ B6 WT splenocytes (SPC) (RCS-SCID) were treated with PBS or a chronic MPTP regimen. Mice were sacrificed at 21 days after the last MPTP injection, and VMB sections immunostained for tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis (Figure 2A, left panels). The numbers of TH⁺ neurons in the SN showed a 33% reduction in WT B6 animals that received MPTP compared to those that received PBS (Figure 2B). No significant difference in the numbers of TH⁺ neurons was observed in MPTP-treated SCID mice compared to SCID mice that received PBS (Figure 2B). In contrast, immune reconstituted SCID mice (RCS-SCID) treated with MPTP showed significantly fewer TH⁺ neurons compared to the SCID MPTP group (Figure 2A and 2B). To validate the reconstitution of RCS-SCID mice, spleens were immunostained for CD3⁺ T cell distribution (Figure 2A, right panels). T cell repopulation was confirmed by the presence of CD3⁺ T cells in the periarteriolar lymphoid sheath of RCS-SCID mouse spleens (Figure 2A),

VMB and cerebellum control sections of WT, SCID, and RCS-SCID mice treated with MPTP were immunostained for T cells using antibodies against CD3, CD4, and CD8. CD3 immunostaining of MPTP-treated B6 mice demonstrated CD3⁺ cells in the VMB beginning at day 0, present at day 4 after MPTP intoxication (Figure 2C) that persisted to day 14. Both CD4⁺ and CD8⁺ subpopulations were also present in VMB of only MPTP-treated animals at 4 and 14 days (Figure 2C). No T cell accumulation was observed in PBS or MPTP-treated SCID mice at any time point (data not shown), whereas CD3⁺ T cell accumulations in VMB of RCS-SCID mice after MPTP-treatment were identified. Cerebellar tissue of MPTP animals had ≤1 CD3⁺ T cell per high power field examined present suggesting specific cell entry into affected regions. Taken together, these data support the occurrence of an adaptive immune response triggered by modified CNS antigens that modulates the vulnerability of the dopaminergic neurons to MPTP through the migration of T cells into the CNS.

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Figure 2. Nigral degeneration following MPTP-intoxication in B6 SCID mice before and after lymphoid cell reconstitution.

(A) Photomicrographs of TH-immunostained SN (left panels) and CD3-immunostained spleen sections (right panels) from B6 (WT), SCID, and reconstituted SCID (RCS-SCID) mice treated with PBS or MPTP and obtained on day 21 post-MPTP intoxication. Immunostaining for expression of CD3 in spleens show normal distributions of CD3⁺ T cells in B6 WT and RCS-SCID mice treated with PBS or MPTP. Note the absence of CD3⁺ T cells in spleens from SCID MPTP mice. (B) Quantification of TH+ neurons in the SN of B6 WT, SCID, or reconstituted (RCS) SCID mice treated with PBS or MPTP. Values represent mean number of TH+ neurons ± SEM for 5-9 mice per group. ^abcdefgPair-wise comparisons by Bonferroni post-hoc test: ^acdp<0.0001, ^befp<0.001, ^gp<0.05. (C) Coronal VMB sections of MPTP intoxicated B6 mice reacted with antibodies against CD3, CD4 and CD8 show positive immunostaining of cells with small, round lymphocytic morphology (magnification = 400X).

https://doi.org/10.1371/journal.pone.0001376.g002

Prediction of Mouse N-α-Syn Specific T Cell Epitopes

To test the probability of N-α-Syn induced adaptive immune responses, we compared the numbers of predicted α-Syn specific T cell epitopes with the propensity to bind class I MHC grooves [48] for the murine MHC haplotypes, H-2^k and H-2^b (see Table 2). However, since the last 40 aa of the mouse α-Syn contains 4 Tyr residues available for nitration, our analysis focused on this C-terminal α-Syn fragment. Table 2 shows that H-2K^k epitopes have a superior ability to bind with high and intermediate affinity α-Syn-derived 8-11-meric peptide fragments derived from the whole α-Syn molecule or from its C-terminal 38-meric fragment. These data demonstrate significant T cell induction potential. In fact, α-Syn has 100 potential T cell epitopes, 73 of which contain Tyr that were predicted to bind H-2K^k molecules, while only 8 and 15 potential epitopes may bind H-2D^b and H-2K^b molecules, respectively. This was also for nitrated epitopes containing Tyr residue including those with a Tyr residue within the central region of the epitope that presents prominently to the T cell receptor. These epitopes do not contain anchor aa that mediate binding to the MHC groove. Dramatic difference in the number of Tyr-containing T cell epitopes from C-terminal segment of α-Syn predicted to bind H-2K^k but not any of H-2^b molecules (Table 2, data in brackets) suggests a potential preference for H-2K^k versus H-2^b mice to induce T cell responses to nitrated C-terminal fragments of α-Syn. These fragments, if facing T cell receptors, have greater chances of inducing MHC class I-restricted CD8⁺ T cells due to lack of negative selection against N-α-Syn epitopes in the embryonic thymus. Further epitope prediction analysis revealed a number of 15-meric epitopes, including Tyr-containing peptides from C-terminal, that can bind with increased affinity class II MHC groove, thus increasing the propensity of inducing MHC class II-restricted CD4⁺ T cells specific for Tyr-containing α-Syn C-terminal fragments. Therefore, mice expressing MHC class I and II molecules of H-2^k haplotypes are capable of generating immune responses to NT-modified α-Syn. Interestingly, that α-Syn C-terminal fragment contains several Tyr-containing peptides with predicted significant binding affinity for IA^k and IA^b MHC molecules (18 and 14 epitopes, respectively) suggests a significant potential for CD4⁺ T cells of mice expressing IA^k or IA^b to respond to nitrated epitopes from α-Syn C-terminal.

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Table 2. Numbers of putative α-Syn epitopes for presentation to T cell receptors predicted from the binding potential of MHC class I and II molecules for the aa sequence of α-Syn.

https://doi.org/10.1371/journal.pone.0001376.t002

Purification and Nitration of Recombinant α-Syn

Based on our findings, we hypothesized that in PD, NT modifications of α-Syn could be a key step converting the endogenous protein to an immunogen. Here, we used the C-terminal 40 aa α-Syn fragment (4YSyn) as it contains all four tyrosine residues that are nitrated, thus limiting the possible specificities of epitopes capable of generating an immune response. For this, the mouse cDNA encoding the final 40 aa was cloned into the bacterial pET-28a His-tag expression vector and recombinant protein expressed in BL21 E. coli following isopropyl-β-D-thiogalactopyranoside (IPTG) induction. Expression of the recombinant protein exhibited no apparent toxicity to the bacterial expression system. Affinity-purified 4YSyn peptide from E. coli lysates was detected as a prominent single band using silver staining on 12% polyacrylamide gel (Figure 3B) and by Western blot using a polyclonal antibody raised against aa 120–140 of α-Syn (Figure 3C). Reverse-phase high performance liquid chromatography (RP-HPLC) analysis of isolated 4YSyn products demonstrated purities equal to or in excess of 97%. NT modifications of 4YSyn peptide (N-4YSyn) after peroxynitrite nitration was confirmed by Western blot using mouse monoclonal anti-NT antibody (Figure 3C).

Homogeneity of purified 4YSyn and its modified forms (aggregated and nitrated) was assessed based on: 1D SDS-PAGE (Figure 3B), Western blot (Figure 3C), and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (Figure 3D). The predominant peak for 4YSyn by MALDI-TOF analysis was 6592 m/z, which corresponded to the 6718 expected mass of purified recombinant α-Syn within <2% mass accuracy (Figure 3D and Table 3). To provide proof that the mass discrepancy originated from recombination errors within the His-tag region obtained during protein purification, but not within the biologically active portion of the molecule, we digested the recombinant 4YSyn protein with trypsin and measured masses of resulting fragments using MALDI-TOF. The observed masses of the generated fragments were Arg-cleaved 4YSyn and Lys-cleaved 4YSyn. These corresponded to the expected masses with 0.07% of mass accuracy. Next, we compared the mass of native 4YSyn to N-4YSyn. The oxidized peptide or its trypsin cleaved fragments revealed a 184 D mass increase that is analogous to the expected mass of 4 nitro groups corresponding to 4 NT- residues (Figure 3A and 3D). Based on these observations, we concluded that reaction of 4YSyn with peroxynitrite, under the conditions used in this study, efficiently nitrated all four available Tyr residues in 4YSyn.

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Figure 3. Characterization of purified and nitrated recombinant 4YSyn.

(A) Primary aa sequence of His-tagged 4YSyn peptide. The His-Tag sequence is highlighted in yellow. The sequence of 4YSyn (Syn_100–140) is shown underlined with 4 Tyr residues (magenta) as potent sites for nitration. Trypsin cleavage sites at Arg (arrowhead) and Lys (arrow) are shown. (B) Purified 4YSyn (lane 1) and N-4YSyn following nitration with peroxynitrite (lane 2) fractionated on a 10–20% polyacrylamide gel and visualized using silver stain. Covalently cross-linked oligomers are indicated by arrowheads. (C) Western blot confirmation of purified 4YSyn and its associated NT modifications following peroxynitrite treatment. (D) MALDI-TOF spectra of purified 4YSyn (top panel), N-4YSyn (middle panel), and 4YSyn after tryptic digest (lower panel).

https://doi.org/10.1371/journal.pone.0001376.g003

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Table 3. Theoretical and Observed Masses of 4YSyn, N-4YSyn and Tryptic Digest Fragments.

https://doi.org/10.1371/journal.pone.0001376.t003

N-4YSyn Induces Specific Immune Responses in B10.BR Mice

To test our predictions of immune responses to N-α-Syn, B10.BR (H-2^k) mice were immunized with N-4YSyn, 4YSyn, or PBS each emulsified in complete Freund's adjuvant (CFA) (Figure 4A). Fourteen days following the initial immunization, mice were boosted with their respective immunogens emulsified in incomplete Freund's adjuvant (IFA). Five days later, mice were sacrificed and SPC were tested for antigen-specific T cell proliferative responses to N-4YSyn or 4YSyn. Stimulation with 4YSyn yielded no significant immune responses regardless of whether mice were immunized with adjuvant containing PBS, 4YSyn or N-4YSyn (Figure 4B). In contrast, significant proliferative responses were afforded from SPC of mice immunized with N-4YSyn and challenged in vitro with N-4YSyn, but not 4YSyn. Moreover, N-4YSyn stimulated SPC from mice immunized with adjuvant containing 4YSyn or PBS failed to induce significant proliferative responses. These data indicate that immunization with N-4YSyn, but not 4YSyn is capable of inducing antigen specific immune responses to NT-modified CNS antigens.

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Figure 4. Experimental protocol for adoptive transfer and lymphocyte proliferation assessment of donor SPC in B10.BR mice.

(A) B10.BR (H-2^K) mice were immunized with PBS, 50 µg 4YSyn, or 50 µg N-4YSyn emulsified in CFA. Mice were boosted 14 days later with PBS or their respective antigens in IFA. After 5 days, donor mice were sacrificed and single cell suspensions were prepared from the draining inguinal lymph nodes and spleen, and T cells were enriched by negative selection. Twelve hours after the final MPTP injection, 5×10⁷ donor immune SPC or 2.5×10⁷ T cells were adoptively transferred to MPTP-treated recipient mice. SPC were evaluated for antigen specificity prior to adoptive transfer by lymphocyte proliferation assays. SN of recipients were evaluated after 28 days of MPTP treatment for migration of T cells, survival of dopaminergic neurons, and reactive microglia. (B). SPC were tested for antigen specific proliferation by culturing in the presence of media alone or media containing 3 µg/ml of immunizing antigens for 5 days and using standard ³H-thymidine incorporation assays.

https://doi.org/10.1371/journal.pone.0001376.g004

Adoptive Transfer of N-4YSyn SPC and T Cells Exacerbates MPTP-induced Microglial Activation and Dopaminergic Neuronal Death

In light of the fact that modified α-Syn is capable of evading tolerance and inducing reactive T cells, we next tested whether modified α-Syn-activated T cells could exacerbate MPTP-induced dopaminergic neurodegeneration. The experimental scheme for adoptive transfer of SPC or purified T cells from immunized animals is outlined in Figure 4A. For these studies, B10.BR (H-2^k) donor mice were immunized and boosted with N-4YSyn or 4YSyn, and SPC were adoptively transferred to MPTP-treated syngeneic recipients. To delineate effects due specifically to T cells, CD3⁺ T cells were enriched by negative selection and transferred to an additional group of MPTP-treated animals. Flow cytometric analysis showed that the enriched population from N-4YSyn mice was 94% CD3⁺ T cells (Figure 5A). Adoptive transfer of purified T cells from N-4YSyn immunized donors to MPTP intoxicated mice revealed CD3⁺ T cell infiltrates in the SNpc on day 2 after MPTP treatment (Figure 5B).

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Figure 5. Adoptive transfer of SPC and purified T cells from N-4YSyn vaccinated B10.BR donors leads to infiltration of T cells in the SNpc of MPTP mice on day 2.

(A) Frequency of CD3⁺ T cells and CD19⁺ B cells before and after enrichment of T cells. Population of enriched T-cells was 94% CD3⁺ prior to adoptive transfer to B10.BR mice. (B) Sections throughout the SNpc were immunostained for CD3 and counterstained with thionin. Clusters of CD3⁺ cells are observed within the SNpc (arrowheads) as seen at 100× magnification (left). Magnification (600X) of boxed area (left panel) is shown (right panel). CD3⁺ cells are small and round exhibiting lymphocyte morphology.

https://doi.org/10.1371/journal.pone.0001376.g005

MPTP treated mice showed fluorescent neurons within the SN using the degenerating cell marker Fluoro-Jade C by day 2, but not by day 7 (Figure 6, left and middle panels) confirming previous kinetic data regarding MPTP-induced nigral neuronal death obtained by silver staining techniques [49]. MPTP treated mice that received immune cells from N-4YSyn immune mice showed more Fluoro-Jade C stained neurons within the SN by day 2 than MPTP-intoxication alone, and, in contrast to the latter, Fluoro-Jade C stained neurons within the SN by day 7 as well. In the PBS control group, no Fluoro-Jade C stained neurons were observed at any time point. Following MPTP administration, microgliosis is striking and immediate. Our initial time course studies are in line with these findings and show that the microgliosis and dopaminergic neurodegeneration in B10.BR mice are virtually resolved respectively by days 4 and 7 post-MPTP injection (Figure 6, right and middle panels, respectively). However, adoptive transfer of SPC from B10.BR mice, regardless of immunization protocol, was associated with a persistent microglial response, as evidenced by quantitative morphology with Mac-1 immunostaining (Figure 6, right panel). Counts of Mac-1⁺ microglia were greatest (p<0.0001) in MPTP mice treated with SPC from N-4YSyn immunized mice [84.1±7.0/mm² (mean±SEM)] compared to those from mice treated with MPTP and SPC from 4YSyn immunized mice (26.9±3.5/mm²), MPTP alone (27.7±3.2/mm²), or PBS (0.7±0.3/mm²). These data suggest that the adaptive immune components of H-2^k mice following MPTP administration contribute to the neuroinflammatory phenotype seen in these animals.

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Figure 6. SPC from N-4YSyn immunized B10.BR mice exacerbate MPTP-induced dopaminergic neurodegeneration and induce microglial responses in the SNpc.

Photomicrographs from VMB sections stained with Fluoro-Jade C (left and middle panels) and Mac-1 antibody (right panels). PBS controls (PBS/none) exhibit an absence of Fluoro-Jade C stained dead neurons on days 2 and 7, and only faint Mac-1 immunoreactivity on day 4 post-treatment. In MPTP-treated mice that received SPC from PBS/adjuvant treated donors (MPTP/PBS), Fluoro-Jade C stained neurons are evident at day 2, but not detectable by day 7. MPTP-treated mice that received SPC from 4YSyn immunized donors (MPTP/4YSyn), also exhibits dead fluorescent neurons by day 2 comparable to the MPTP/PBS control group, and only rare degenerating neurons are visible by day 7. Mac-1 immunoreactivity in those mice is comparably resolved to levels seen in MPTP/PBS control group. SPC transfers from N-4YSyn immunized donors to MPTP-treated mice (MPTP/N-4YSyn) induced a robust and prolonged microglial response, conspicuously enhanced when compared to MPTP/PBS-treated controls, with concomitant neuronal death still evident by Fluoro-Jade C staining at day 7.

https://doi.org/10.1371/journal.pone.0001376.g006

Based on these findings, we next investigated whether the immune response mediated by N-α-Syn affects degeneration of dopaminergic cell bodies in the SNpc. To test this, MPTP-intoxicated B10.BR mice received 5×10⁷ donor SPC from mice immunized with PBS, 4YSyn, or N-4YSyn, or 2.5×10⁷ enriched T cells from 4YSyn-immunized donors. PBS- and MPTP-treated mice that did not receive cells served as controls for no neuronal loss and loss attributable to MPTP treatment alone, respectively. PBS-treated mice that received N-4YSyn immunized donor SPC served as additional controls. VMB sections were obtained from mice at 2, 7, and 28 days following MPTP treatment and immunostained for TH (Figure 7A). Stereological analysis showed that MPTP induced a 45% reduction of SN TH⁺ neurons compared to PBS controls (Figure 7B). Similar results were observed in MPTP-injected mice that received SPC from PBS or 4YSyn immune donors (MPTP/PBS/SPC and MPTP/4YSyn/SPC, respectively). In contrast, recipients that received immune N-4YSyn SPC (MPTP/N-4YSyn/SPC) or N-4YSyn T cells (MPTP/N-4YSyn/T Cells) exhibited significantly greater reductions of SNpc TH⁺ neurons (64 and 63%, respectively) compared to all other MPTP-treated animals (Figure 7B). PBS-treated mice that received immune SPC from N-4YSyn immunized donors showed no change in TH⁺ neuron numbers, demonstrating the necessity for an initiating neuronal insult. Significant effects from any treatment were not observed among the numbers of non-dopaminergic neurons (Nissl+TH-). Correlation analysis of total Nissl+ neurons compared to TH+ and TH- neurons demonstrated that the number of total neurons strongly correlated with numbers of TH+ neurons (r = 0.981, p<0.0001) compared to numbers of TH- neurons (r = 0.522, p = 0.004). This confirmed that differences in TH+ neuron counts are due to differences in numbers of structurally intact neurons and eliminates the possibility that differences resulted from the down-regulation of TH itself. Thus, these data demonstrate that adaptive immune responses against the nitrated form of α-Syn exacerbated MPTP-induced nigrostriatal degeneration.

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Figure 7. N-4YSyn immunization with adjuvant exacerbates dopaminergic neuronal cell loss in B10.BR mice.

(A) All panels show TH-positive neurons in the SN from mice treated with: [top row, L to R] PBS or MPTP alone, MPTP and SPC from N-4YSyn immunized donors (MPTP+N-4YSyn), [bottom row, L to R] PBS and SPC from N-4YSyn immunized donors (PBS+N-4YSyn), MPTP and SPC from 4YSyn immunized donors (MPTP+4YSyn), and MPTP and T cells from N-4YSyn immunized donors (MPTP+N-4YSyn T Cells). Tissues collected 28 days post-MPTP treatment (B) Counts of nigral Nissl+ (blue bars), TH+ (red bars), and TH- (green bars) neurons on day 28 after MPTP treatment as determined by stereological analysis. Control groups included mice treated with PBS alone (n = 4), MPTP alone (n = 8), and PBS animals that received immune effector SPC from N-4YSyn immunized donor mice PBS/N-4YSyn/SPC (n = 6). Experimental groups included MPTP/PBS/SPC (n = 6), MPTP/4YSyn/SPC (n = 8), MPTP/N-4YSyn/SPC (n = 9), and MPTP mice which received purified T cells from N-4YSyn vaccinated donors. Values are means ± SEM. p<0.01 compared to the following treatment groups: ^aPBS, ^bMPTP, ^cMPTP/4YSyn/SPC.

https://doi.org/10.1371/journal.pone.0001376.g007

Based on MHC binding affinity algorithms, mice expressing H-2^b were predicted to respond poorly to α-Syn epitopes; yet 21 days after chronic MPTP-intoxication, B6 mice that express the H-2^b haplotype yielded significant antibody responses to N-α-Syn. This suggested that mice expressing H-2^b have the potential to develop immune responses to N-α-Syn that may affect disease progression. To assess that possibility, we immunized and boosted B6 (H-2^b) mice with 4YSyn or N-4YSyn either in PBS or emulsified in adjuvant (Figure 8A). Five days after the final boost SPC were harvested, assessed for antigen specific lymphocyte proliferation, and adoptively transferred to MPTP-intoxicated recipients. Stimulation of SPC from PBS-treated controls indicated that neither 4YSyn nor N-4YSyn induced significant proliferation above medium background levels (Figure 8B). In contrast, stimulation of SPC from N-4YSyn/PBS immunized mice with N-4YSyn induced a significant lymphocyte proliferative response indicating that immunization with N-4YSyn/PBS in the absence of adjuvant is capable of inducing an antigen specific adaptive immunity.

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Figure 8. Scheme for immunization, lymphocyte proliferation assessment, and adoptive transfer of donor SPC in B6 mice.

(A) B6 (H-2^b) mice were immunized with 10 µg 4YSyn in PBS, 50 µg N-4YSyn in CFA, 10 µg N-4YSyn in PBS or PBS in CFA. Mice were boosted 14 days later with their respective antigens as formulated previously with or without IFA. After 5 days, single lymphoid cell suspensions were prepared and assessed for antigen-specific responses in standard lymphocyte proliferation assays. Single cell suspensions were pooled and adoptively transferred to MPTP-treated syngeneic recipients 12 hrs after the final MPTP injection. 5×10⁷ donor immune SPC were adoptively transferred to MPTP-treated recipient mice. Survival of dopaminergic neurons in the SN of recipient mice were evaluated after 7 days. (B) Antigen specific proliferation of SPC from B6 (H-2^b) mice (n = 5/group) immunized with PBS/CFA or N-4YSyn/CFA, and cultured for 5 days in media alone (green bars) or in the presence 1 µg/ml of 4YSyn (blue bars) or N-4YSyn (red bars). Cultures were pulsed for 18 hrs, cells harvested and ³H-thymidine incorporation counted by β-scintillation spectrometry. Values represent mean stimulation indices±SEM and analyzed by ANOVA and Bonferroni post-hoc tests. ^ap = 0.0478.

https://doi.org/10.1371/journal.pone.0001376.g008

TH stained sections of VMB from MPTP-intoxicated mice that received SPC from donors immunized with N-4YSyn either in PBS excipient or adjuvant showed significant dopaminergic neuronal losses compared to MPTP-treated mice or those that received SPC from 4YSyn immunized mice (Figure 9A), suggesting 4YSyn immunization increased the MPTP-induced lesion. Next, we assessed the sections by stereological analysis to obtain estimates of dopaminergic neuronal survival and loss after treatment compared to PBS-treated mice. MPTP-intoxication of B6 mice induced 43% loss of TH+ nigral neurons compared to PBS-treated controls (Figure 9B). Numbers of dopaminergic neurons from MPTP mice treated with SPC from 4YSyn immunized mice were not significantly different compared to MPTP-treated mice and showed a similar 42% loss of neurons. However, adoptive transfer of SPC from N-4YSyn/Adjuvant immune donors significantly increased MPTP-induced dopaminergic neuron loss to 58%. Interestingly, SPC from mice immunized with N-4YSyn without adjuvant induced a 69% loss of nigral TH+ neurons after MPTP intoxication, which was significantly greater than losses due to SPC from 4YSyn- or N-4YSyn/Adjuvant immunized donors. Neither MPTP treatment nor adoptive transfer of immune SPC significantly affected numbers of non-dopaminergic neurons. Thus, taken together these data demonstrate that N-α-Syn, but not unmodified α-Syn, has the capacity to induce specific immune responses by which exacerbates neuronal loss in the context of dopaminergic neurodegeneration. Moreover, these results in H-2^b mice, predicted to provide poor immune response, suggests that epitopes modified by inflammatory processes may function unlike their tolerated unmodified self-analogues to induce immune responses to levels sufficient to alter disease progression.

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Figure 9. Lymphocytes from N-4YSyn immunization exacerbate nigral dopaminergic neuronal loss in B6 mice.

(A) All panels show TH⁺ neurons in the SN from mice treated with PBS or MPTP alone, MPTP and SPC from 4YSyn immunized donors (MPTP+4YSyn), MPTP and SPC from N-4YSyn immunized donors (MPTP+N-4YSyn) and lastly, MPTP and SPC from N-4YSyn+CFA immunized donors (MPTP+N-4YSyn CFA). (B) Counts of nigral TH⁺ and TH⁻ neurons on day 7 after MPTP treatment. Experimental groups included mice treated with PBS alone (n = 7), MPTP alone (n = 7), MPTP/4YSyn (n = 7), MPTP/N-4YSyn/CFA (n = 6) and MPTP/N-4YSyn (n = 6). Values are means ± SEM. Analysis by ANOVA with Bonferroni post-hoc tests indicated ^ap<0.0001 compared to PBS control: ^bp<0.001 compared to MPTP group; ^cp<0.001 compared to MPTP/4YSyn group; and ^dp<0.03 compared to MPTP/4YSynCFA SPC.

https://doi.org/10.1371/journal.pone.0001376.g009

Nitrated α-Syn Inhibits Proliferation of Anti-CD3 Activated T Cells

We observed that antigen specific proliferative responses were inhibited by N-4YSyn in a dose dependent manner (r² = 0.96, p = 0.002). Significant inhibition was not seen by 4YSyn (r² = 0.53, p = 0.273). To rule out an antigen specific suppressive effect, purified T cells obtained from naïve mice were stimulated with anti-CD3 for 24 hrs in media or in the presence of 4YSyn or N-4YSyn at concentrations of 1, 3, 10 and 30 µg/ml. N-4YSyn inhibited proliferation of anti-CD3 stimulated T cells in a dose dependent fashion (r^r = 0.6803, p<0.0001). A significant inhibition of 31 and 47% was observed when stimulated T cells were co-cultured at N-4YSyn concentrations of 10 and 30 µg/ml (Figure 10). In contrast, proliferation of anti-CD3 stimulated T cells was not inhibited by 4YSyn (p = 0.435) or by increasing 4YSyn concentration (r² = 0.1554, p = 0.0854). Moreover, the inverse effect on anti-CD3 activated T cells with increasing N-4YSyn concentration was greater (p = 0.016) compared to that induced by 4YSyn. Second, to assess whether this effect was due to a cytotoxic mechanism, anti-CD3 stimulated T cells were stained for the membrane impermeant DNA dye, propidium iodide (PI) that is excluded from intact, living cells, and analyzed by flow cytometry. T cells cultured in the presence of N-4YSyn exhibited a dose-dependent increase in the mean fluorescent intensity (MFI) of PI (r² = 0.9261, p = 0.0008) and in the percentage of PI⁺ dead T cells (r² = 0.9743, p = 0.0018) (Table 4). In comparison, percentages of PI stained dead T cells cultured with 4YSyn and the PI MFI were not different from the basal level in absence of either of the peptides and did not change with increasing 4YSyn concentration (r² = 0.5983, p = 0.1249, and r² = 0.5016, p = 0.1807, respectively). Taken together, increasing N-4YSyn concentrations strongly correlated with inhibition of T cell proliferation, percent PI⁺ T cells, and increased MFI for PI (r>0.945, p<0.0154 for all comparisons combined). In contrast, no significant correlations were associated with increasing 4YSyn concentrations. Thus, cytotoxic interference of immune response induction by N-α-Syn may affect disease progression as these processes are shared by both effector and regulatory T cells.

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Figure 10. N-4YSyn-mediated inhibition of T cell proliferation.

Proliferative responses of anti-CD3 stimulated T cells from naïve B6 mice in presence of graded concentrations of 4YSyn or N-4YSyn (1, 3, 10, 30 µg/ml) or in media alone (0 µg/ml). T cells were cultured for 72 hrs and pulsed with ³H-thymidine for the final 18 hrs of culture. Harvested cells were counted for ³H-thymidine uptake by β-scintillation spectrometry and proliferation was expressed as mean counts per min (CPM)±SEM for quadruplicate samples and evaluated by ANOVA with Bonferroni post-hoc tests. ^ap<0.01 compared with T cells stimulated with anti-CD3 and cultured in media alone.

https://doi.org/10.1371/journal.pone.0001376.g010

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Table 4. N-4YSyn-induced cytotoxicity of stimulated T cells.

https://doi.org/10.1371/journal.pone.0001376.t004

N-4YSyn-Stimulated Immune SPC Enhance Dopaminergic Cell Death

To directly test the neurotoxic capacity of the N-4YSyn immune response, proliferating T cells or supernatants from N-4YSyn stimulated SPC of N-4YSyn immunized mice were assessed in live/dead assays with co-cultures of N-α-Syn activated bone marrow-derived macrophages (BMM) and MES 23.5 cells. Minimal cytotoxicity was afforded from co-cultures of unstimulated BMM/MES 23.5 co-cultures with virtual 100% MES 23.5 cell survival, whereas activation with aggregated N-α-Syn resulted in 7% cell death after 24 hrs (Figure 11). Activated and proliferating T cells from 5 day cultures of antigen-specific stimulation of N-4YSyn immune SPC when added to activated BMM/MES 23.5 cultures induced 21% cell death (N-4YSyn+SPC), while supernatants (Sup) from those SPC cultures resulted in 44% cell death (N-4YSyn+SPC Sup), and no cytotoxicity to activated BMM cultured in the absence of MES 23.5 cells (Macrophages+SPC Sup). In transwell studies wherein N-4YSyn-stimulated BMM was separated from MES 23.5 targets, virtually all cytotoxicity was concentrated among the MES 23.5 cell population (MES 23.5 Transwell) with no cytotoxicity attributed to BMM. T cells isolated from N-4YSyn-immunized donors and re-stimulated in vitro with N-4Ysyn, but not 4YSyn, showed TNF-α levels (26.0±1.2 pg/ml) that were increased (p<0.008) compared to levels from T cells of either PBS or 4YSyn immunized mice which were below the limits of detection (data not shown).

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Figure 11. N-4YSyn activated immune SPC induces macrophage-mediated dopaminergic cell death.

Representative fluorescence photomicrographs are shown of live (green) and dead (red) cells from 24 hr macrophage/MES 23.5 co-cultures in the presence of media alone, N-4YSyn, N-4YSyn and antigen-stimulated SPC of N-4YSyn-immunized mice (N-4YSyn+SPC), or N-4YSyn and the supernatants from antigen-stimulated SPC of N-4YSyn-immunized mice (N-4YSyn+SPC Sup). Antigen-stimulated SPC from N-4YSyn immune mice were induced in vitro with N-4YSyn, and cells and supernatants for use in the assay were harvested after 5 days of culture. Controls included macrophages cultured in the presence of SPC supernatants from antigen-stimulated SPC of N-4YSyn-immunized mice (Macrophages+SPC Sup); macrophages cultured in the presence of N-4YSyn alone (Macrophages+N4YSyn); and transwell cultures of plated MES 23.5 cells and macrophages in the transwell stimulated with N-4YSyn. Frequencies (±SEM) of dead (red) cells for 4-8 fields/assay are provided in the lower right corner of each panel. Differences in the mean frequencies of dead cells were evaluated by ANOVA and Bonferroni post-hoc tests. p<0.01 compared to cultures treated with ^aMedia, ^bN-4YSyn, or ^cN-4YSyn+SPC.

https://doi.org/10.1371/journal.pone.0001376.g011