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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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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).
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pone-0001376-g002: 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).

Mentions: 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),


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)

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).
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pone-0001376-g002: 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).
Mentions: 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),

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