Nitrated α–Synuclein Immunity Accelerates Degeneration of Nigral Dopaminergic Neurons

The neuropathology of Parkinson's disease (PD) includes loss of dopaminergic neurons in the substantia nigra, nitrated alpha-synuclein (N-alpha-Syn) enriched intraneuronal inclusions or Lewy bodies and neuroinflammation. While the contribution of innate microglial inflammatory activities to disease are known, evidence for how adaptive immune mechanisms may affect the course of PD remains obscure. We reasoned that PD-associated oxidative protein modifications create novel antigenic epitopes capable of peripheral adaptive T cell responses that could affect nigrostriatal degeneration.Nitrotyrosine (NT)-modified alpha-Syn was detected readily in cervical lymph nodes (CLN) from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxicated mice. Antigen-presenting cells within the CLN showed increased surface expression of major histocompatibility complex class II, initiating the molecular machinery necessary for efficient antigen presentation. MPTP-treated mice produced antibodies to native and nitrated alpha-Syn. Mice immunized with the NT-modified C-terminal tail fragment of alpha-Syn, but not native protein, generated robust T cell proliferative and pro-inflammatory secretory responses specific only for the modified antigen. T cells generated against the nitrated epitope do not respond to the unmodified protein. Mice deficient in T and B lymphocytes were resistant to MPTP-induced neurodegeneration. Transfer of T cells from mice immunized with N-alpha-Syn led to a robust neuroinflammatory response with accelerated dopaminergic cell loss.These data show that NT modifications within alpha-Syn, can bypass or break immunological tolerance and activate peripheral leukocytes in draining lymphoid tissue. A novel mechanism for disease is made in that NT modifications in alpha-Syn induce adaptive immune responses that exacerbate PD pathobiology. These results have implications for both the pathogenesis and treatment of this disabling neurodegenerative disease

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

<p>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 <i>in vitro</i> 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 <i>post-hoc</i> tests. p<0.01 compared to cultures treated with ^aMedia, ^bN-4YSyn, or ^cN-4YSyn+SPC.</p

Characterization of purified and nitrated recombinant 4YSyn.

<p>(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).</p

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

<p>(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 <i>post-hoc</i> 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.</p

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

<p>Theoretical and Observed Masses of 4YSyn, N-4YSyn and Tryptic Digest Fragments.</p

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

a<p>Anti-CD3 stimulated T cells were cultured for 24 hrs in media alone or in the presence of different concentrations of 4YSyn or N-4YSyn, stained with 2 µg/ml of the vital dye, PI, washed, and assessed by flow cytometric analysis for uptake of PI.</p>b<p>Percentage of T cells susceptible to PI permeation as determined by flow cytometric analysis.</p>c<p>Mean fluorescence intensity of PI-stained T cells.</p>d<p>D statistic at α = 0.001 of Kolmogorov-Smirnov (K-S) analysis for fluorescence intensities of PI-stained T cells as the sigmoidal function of the accumulated cell frequency curve and fluorescence intensity (channel number) compared to the curve of PI-stained T cells from the medium control (asterisk) as computed by Cellquest software (BD Biosciences) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001376#pone.0001376-Young1" target="_blank">[88]</a>.</p>e<p>Index of similarity = D/[(n_c+n_t)/(n_c·n_t)]^1/2, where n_c and n_t are the number of events in cell frequency curves for medium control (c) and test substance (t), respectively (BD Biosciences) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001376#pone.0001376-Young1" target="_blank">[88]</a>. An index of similarity = 0 indicates the curves are identical.</p

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

<p>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 <i>post-hoc</i> tests. ^ap<0.01 compared with T cells stimulated with anti-CD3 and cultured in media alone.</p

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.

<p>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.</p

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

<p>(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.</p