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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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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 (Syn100–140) 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).
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pone-0001376-g003: 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 (Syn100–140) 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).

Mentions: 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).


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)

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 (Syn100–140) 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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2147051&req=5

pone-0001376-g003: 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 (Syn100–140) 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).
Mentions: 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).

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