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Nitrated alpha-synuclein immunity accelerates degeneration of nigral dopaminergic neurons.

Benner EJ, Banerjee R, Reynolds AD, Sherman S, Pisarev VM, Tsiperson V, Nemachek C, Ciborowski P, Przedborski S, Mosley RL, Gendelman HE - PLoS ONE (2008)

Bottom Line: We reasoned that PD-associated oxidative protein modifications create novel antigenic epitopes capable of peripheral adaptive T cell responses that could affect nigrostriatal degeneration.T cells generated against the nitrated epitope do not respond to the unmodified protein.These results have implications for both the pathogenesis and treatment of this disabling neurodegenerative disease.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurovirology and Neurodegenerative Disorders, Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska, USA.

ABSTRACT

Background: 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.

Methods and findings: 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.

Conclusions: 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.

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Scheme for immunization, lymphocyte proliferation assessment, and adoptive transfer of donor SPC in B6 mice.(A) B6 (H-2b) 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-2b) 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.
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pone-0001376-g008: Scheme for immunization, lymphocyte proliferation assessment, and adoptive transfer of donor SPC in B6 mice.(A) B6 (H-2b) 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-2b) 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.

Mentions: Based on MHC binding affinity algorithms, mice expressing H-2b were predicted to respond poorly to α-Syn epitopes; yet 21 days after chronic MPTP-intoxication, B6 mice that express the H-2b haplotype yielded significant antibody responses to N-α-Syn. This suggested that mice expressing H-2b 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-2b) 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.


Nitrated alpha-synuclein immunity accelerates degeneration of nigral dopaminergic neurons.

Benner EJ, Banerjee R, Reynolds AD, Sherman S, Pisarev VM, Tsiperson V, Nemachek C, Ciborowski P, Przedborski S, Mosley RL, Gendelman HE - PLoS ONE (2008)

Scheme for immunization, lymphocyte proliferation assessment, and adoptive transfer of donor SPC in B6 mice.(A) B6 (H-2b) 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-2b) 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2147051&req=5

pone-0001376-g008: Scheme for immunization, lymphocyte proliferation assessment, and adoptive transfer of donor SPC in B6 mice.(A) B6 (H-2b) 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-2b) 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.
Mentions: Based on MHC binding affinity algorithms, mice expressing H-2b were predicted to respond poorly to α-Syn epitopes; yet 21 days after chronic MPTP-intoxication, B6 mice that express the H-2b haplotype yielded significant antibody responses to N-α-Syn. This suggested that mice expressing H-2b 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-2b) 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.

Bottom Line: We reasoned that PD-associated oxidative protein modifications create novel antigenic epitopes capable of peripheral adaptive T cell responses that could affect nigrostriatal degeneration.T cells generated against the nitrated epitope do not respond to the unmodified protein.These results have implications for both the pathogenesis and treatment of this disabling neurodegenerative disease.

View Article: PubMed Central - PubMed

Affiliation: Center for Neurovirology and Neurodegenerative Disorders, Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska, USA.

ABSTRACT

Background: 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.

Methods and findings: 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus