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Preferentially increased nitration of alpha-synuclein at tyrosine-39 in a cellular oxidative model of Parkinson's disease.

ABSTRACT: Alpha-synuclein is a major component of Lewy bodies, proteinacious inclusions which are a major hallmark of Parkinson's disease (PD). Lewy bodies contain high levels of nitrated tyrosine residues as determined by antibodies specific for 3-nitrotyrosine (3NT) and via mass spectrometry (MS). We have developed a multiple reaction monitoring (MRM) mass spectrometry method to sensitively quantitate the 3NT levels of specific alpha-synuclein tyrosine residues. We found a 9-fold increase (relative to controls) in levels of 3NT at Tyr-39 of alpha-synuclein in an inducible transgenic cellular model of Parkinson's disease in which monoamine oxidase B (MAO-B) is overexpressed and which emulates several features of PD. Increased nitration of Tyr-39 on endogenous alpha-synuclein via elevations in MAO-B levels could be abrogated by the addition of deprenyl, a specific MAO-B inhibitor. The increased levels of 3NT was selective for Tyr-39 as no significant increases in 3NT levels were detected at other tyrosine residues present in the protein (Tyr-125, Tyr-133, and Tyr-136). This is the first report of increased 3NT levels of a specific tyrosine in a PD model and the first use of MRM mass spectrometry to quantify changes in 3NT modifications at specific sites within a target protein.

SUBMITTER: Danielson SR

PROVIDER: S-EPMC2748813 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Preferentially increased nitration of alpha-synuclein at tyrosine-39 in a cellular oxidative model of Parkinson's disease.

Danielson Steven R SR Held Jason M JM Schilling Birgit B Oo May M Gibson Bradford W BW Andersen Julie K JK

Analytical chemistry 20090901 18

Alpha-synuclein is a major component of Lewy bodies, proteinacious inclusions which are a major hallmark of Parkinson's disease (PD). Lewy bodies contain high levels of nitrated tyrosine residues as determined by antibodies specific for 3-nitrotyrosine (3NT) and via mass spectrometry (MS). We have developed a multiple reaction monitoring (MRM) mass spectrometry method to sensitively quantitate the 3NT levels of specific alpha-synuclein tyrosine residues. We found a 9-fold increase (relative to c ...[more]

PMID: 19697948

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Project description:Methamphetamine is an amphetamine-type psychostimulant that can damage dopaminergic neurons and cause characteristic pathological changes similar to neurodegenerative diseases such as Parkinson's disease. However, its specific mechanism of action is still unclear. In the present study, we established a Parkinson's disease pathology model by exposing SH-SY5Y cells and C57BL/6J mice to methamphetamine. In vitro experiments were performed with 0, 0.5, 1.0, 1.5, 2.0 or 2.5 mM methamphetamine for 24 hours or 2.0 mM methamphetamine for 0-, 2-, 4-, 8-, 16-, and 24-hour culture of SH-SY5Y cells. Additional experimental groups of SH-SY5Y cells were administered a nitric oxide inhibitor, 0.1 mM N-nitro-L-arginine, 1 hour before exposure to 2.0 mM methamphetamine for 24 hours. In vivo experiments: C57BL/6J mice were intraperitoneally injected with N-nitro-L-arginine (8 mg/kg), eight times, at intervals of 12 hours. Methamphetamine 15 mg/kg was intraperitoneally injected eight times, at intervals of 12 hours, but 0.5-hour after each N-nitro-L-arginine injection in the combined group. Western blot assay was used to determine the expression of nitric oxide synthase, α-synuclein (α-Syn), 5G4, nitrated α-synuclein at the residue Tyr39 (nT39 α-Syn), cleaved caspase-3, and cleaved poly ADP-ribose polymerase (PARP) in cells and mouse brain tissue. Immunofluorescence staining was conducted to measure the positive reaction of NeuN, nT39 α-Syn and 5G4. Enzyme linked immunosorbent assay was performed to determine the dopamine levels in the mouse brain. After methamphetamine exposure, α-Syn expression increased; the aggregation of α-Syn 5G4 increased; nT39 α-Syn, nitric oxide synthase, cleaved caspase-3, and cleaved PARP expression increased in the cultures of SH-SY5Y cells and in the brains of C57BL/6J mice; and dopamine levels were reduced in the mouse brain. These changes were markedly reduced when N-nitro-L-arginine was administered with methamphetamine in both SH-SY5Y cells and C57BL/6J mice. These results suggest that nT39 α-Syn aggregation is involved in methamphetamine neurotoxicity.

Project description:α-Synuclein (αSyn) is central to the neuropathology of Parkinson's disease (PD) due to its propensity for misfolding and aggregation into neurotoxic oligomers. Nitration/oxidation of αSyn leads to dityrosine crosslinking and aggregation. Myeloperoxidase (MPO) is an oxidant-generating enzyme implicated in neurodegenerative diseases. In the present work we have examined the impact of MPO in PD through analysis of postmortem PD brain and in a novel animal model in which we crossed a transgenic mouse expressing the human MPO (hMPO) gene to a mouse expressing human αSyn-A53T mutant (A53T) (hMPO-A53T). Surprisingly, our results show that in PD substantia nigra, the hMPO gene is expressed in neurons containing aggregates of nitrated αSyn as well as MPO-generated HOCl-modified epitopes. In our hMPO-A53T mouse model, we also saw hMPO expression in neurons but not mouse MPO. In the mouse model, hMPO was expressed in neurons colocalizing with nitrated αSyn, carbamylated lysine, nitrotyrosine, as well as HOCl-modified epitopes/proteins. RNAscope in situ hybridization confirmed hMPO mRNA expression in neurons. Interestingly, the hMPO protein expressed in hMPO-A53T brain is primarily the precursor proMPO, which enters the secretory pathway potentially resulting in interneuronal transmission of MPO and oxidative species. Importantly, the hMPO-A53T mouse model, when compared to the A53T model, exhibited significant exacerbation of motor impairment on rotating rods, balance beams, and wire hang tests. Further, hMPO expression in the A53T model resulted in earlier onset of end stage paralysis. Interestingly, there was a high concentration of αSyn aggregates in the stratum lacunosum moleculare of hippocampal CA2 region, which has been associated in humans with accumulation of αSyn pathology and neural atrophy in dementia with Lewy bodies. This accumulation of αSyn aggregates in CA2 was associated with markers of endoplasmic reticulum (ER) stress and the unfolded protein response with expression of activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), MPO, and cleaved caspase-3. Together these findings suggest that MPO plays an important role in nitrative and oxidative damage that contributes to αSyn pathology in synucleinopathies.

Dataset Information

Preferentially increased nitration of alpha-synuclein at tyrosine-39 in a cellular oxidative model of Parkinson's disease.

Publications

Preferentially increased nitration of alpha-synuclein at tyrosine-39 in a cellular oxidative model of Parkinson's disease.

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