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Increased α-synuclein phosphorylation and nitration in the aging primate substantia nigra.

ABSTRACT: Post-translational modifications of α-synuclein occur in the brain of patients affected by Parkinson's disease and other α-synucleinopathies, as indicated by the accumulation of Lewy inclusions containing phosphorylated (at serine 129) and nitrated α-synuclein. Here we found that phospho-Ser 129 and nitrated α-synuclein are also formed within dopaminergic neurons of the monkey substantia nigra as a result of normal aging. Dopaminergic cell bodies immunoreactive for phospho-Ser 129 and nitrated α-synuclein were rarely seen in adult mature animals but became significantly more frequent in the substantia nigra of old primates. Dual labeling with antibodies against phospho-Ser 129 and nitrated α-synuclein revealed only limited colocalization and mostly stained distinct sub-populations of dopaminergic neurons. Age-related elevations of modified protein paralleled an increase in the number of neurons immunoreactive for unmodified α-synuclein, supporting a relationship between higher levels of normal protein and enhanced phosphorylation/nitration. Other mechanisms were also identified that likely contribute to α-synuclein modifications. In particular, increased expression of Polo-like kinase 2 within neurons of older animals could contribute to phospho-Ser 129 α-synuclein production. Data also indicate that a pro-oxidant environment characterizes older neurons and favors α-synuclein nitration. Aging is an unequivocal risk factor for human α-synucleinopathies. These findings are consistent with a mechanistic link between aging, α-synuclein abnormalities and enhanced vulnerability to neurodegenerative processes.

SUBMITTER: McCormack AL

PROVIDER: S-EPMC3366084 | biostudies-other | 2012

REPOSITORIES: biostudies-other

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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.

Project description:α-Synuclein (α-syn) is a small presynaptic protein distributed ubiquitously in the central and peripheral nervous system. In normal conditions, α-syn is found in soluble form, while in Parkinson's disease (PD) it may phosphorylate, aggregate, and combine with other proteins to form Lewy bodies. The purpose of this study was to evaluate, in nonhuman primates, whether α-syn expression is affected by age and neurotoxin challenge. Young adult (n = 5, 5-10 years old) and aged (n = 4, 23-25 years old) rhesus monkeys received a single unilateral carotid artery injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Three months post-MPTP the animals were necropsied by transcardiac perfusion, and their brains extracted and processed with immunohistochemical methods. Quantification of tyrosine hydroxylase (TH)-positive substantia nigra (SN) neurons showed a significant 80-89% decrease in the side ipsilateral to MPTP administration in young and old animals. Optical density of TH- immunoreactivity (-ir) in the caudate and putamen presented a 60-70% loss compared with the contralateral side. α-Syn-ir was present in both ipsi- and contra- lateral MPTP-treated nigra, caudate, and putamen, mostly in fibers; its intracellular distribution was not affected by age. Comparison of α-syn-ir between MPTP-treated young and aged monkeys revealed significantly higher optical density for both the ipsi- and contralateral caudate and SN in the aged animals. TH and α-syn immunofluorescence confirmed the loss of nigral TH-ir dopaminergic neurons in the MPTP-treated side of intoxicated animals, but bilateral α-syn expression. Colabeling of GAD67 and α-syn immunofluorescence showed that α-syn expression was present mainly in GABAergic fibers. Our results demonstrate that, 3 months post unilateral intracarotid artery infusion of MPTP, α-syn expression in the SN is largely present in GABAergic fibers, regardless of age. Bilateral increase of α-syn expression in SN fibers of aged, compared with young rhesus monkeys, suggests that α-syn-ir may increase with age, but not after neurotoxin-induced dopaminergic nigral cell loss.

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Increased α-synuclein phosphorylation and nitration in the aging primate substantia nigra.

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