alpha-synuclein and 5-5-dimethyl-1-pyrroline-1-oxide

Parkinson's disease (PD) is a debilitating, progressive, neurodegenerative disorder characterized by progressive loss of dopaminergic neurons and motor deficits. Alpha-synuclein-containing aggregates represent a feature of a variety of neurodegenerative disorders, including PD; however, the mechanism that initiates and promotes intraneuronal alpha-synuclein aggregation remains unknown. We hypothesized protein radical formation as an initiating mechanism for alpha-synuclein aggregation. Therefore, we used the highly sensitive immuno-spin trapping technique to investigate protein radical formation as a possible mechanism of alpha-synuclein aggregation as well as to investigate the source of protein radical formation in the midbrains of Maneb- and paraquat-coexposed mice. Coexposure to Maneb and paraquat for 6 weeks resulted in active microgliosis, NADPH oxidase activation, and inducible nitric oxide synthase (iNOS) induction, which culminated in protein radical formation in the midbrains of mice. Results obtained with immuno-spin trapping and immunoprecipitation experiments confirmed formation of alpha-synuclein radicals in dopaminergic neurons of exposed mice. Free radical formation requires NADPH oxidase and iNOS, as indicated by decreased protein radical formation in knockout mice (P47phox(-/-) and iNOS(-/-)) and in mice treated with inhibitors such as FeTPPS (a peroxynitrite decomposition catalyst), 1400 W (an iNOS inhibitor), or apocynin (a NADPH oxidase inhibitor). Concurrence of protein radical formation with dopaminergic neuronal death indicated a link between protein radicals and disease progression. Taken together, these results show for the first time the formation and detection of the alpha-synuclein radical and suggest that NADPH oxidase and iNOS play roles in peroxynitrite-mediated protein radical formation and subsequent neuronal death in the midbrains of Maneb- and paraquat-coexposed mice.

Topics: alpha-Synuclein; Animals; Cyclic N-Oxides; Disease Models, Animal; Dopaminergic Neurons; Injections, Intraperitoneal; Male; Maneb; Mesencephalon; Mice, Inbred C57BL; Microglia; Models, Biological; NADPH Oxidases; Nitric Oxide Synthase Type II; Paraquat; Parkinson Disease; Peroxynitrous Acid; Spin Labels; Substantia Nigra; Tyrosine 3-Monooxygenase

The molecular mechanism underlying the flavonoid-induced inhibition of alpha-synuclein fibrillation was thoroughly examined by various biochemical and biophysical approaches. The noncovalent binding of the inhibitory flavonoids to alpha-synuclein and the covalent modification by the flavonoid quinone led to the restriction of the conformational changes in this natively unfolded protein and to the stabilization of soluble flavonoid-modified species of alpha-synuclein (monomers and oligomers). All of these factors rather than a single one contribute to the inhibition of WT alpha-synuclein fibrillation induced by the flavonoid. The structural requirements that appear necessary to provide a flavonoid the ability to inhibit alpha-synuclein fibrillation were determined to be vicinal dihydroxyphenyl moieties, irrespective of the ring position where they are located. Flavonoids with three vicinal hydroxyl groups exhibited enhanced inhibitory effects on alpha-synuclein fibrillation. The antioxidant activities of flavonoids were generally correlated with their in vitro inhibitory effects on alpha-synuclein fibrillation. The flavonoids inhibiting alpha-synuclein fibrillation and stabilizing the protein monomeric conformation can serve as a model for the development of therapeutic drugs in combating Parkinson's disease.

Topics: alpha-Synuclein; Animals; Binding Sites; Catalase; Cattle; Cyclic N-Oxides; Flavanones; Flavonoids; Free Radicals; Humans; Hydrogen Peroxide; Isoelectric Focusing; Mass Spectrometry; Mutation; Oxidation-Reduction; Protein Binding; Protein Conformation; Protein Stability; Time Factors; Tyrosine