Several observations have implicated oxidative stress and aggregation of the presynaptic protein ␣-synuclein in the pathogenesis of Parkinson disease. ␣-Synuclein has been shown to have affinity for unsaturated fatty acids and membranes enriched in polyunsaturated fatty acids, which are especially sensitive to oxidation under conditions of oxidative stress. One of the most important products of lipid oxidation is 4-hydroxy-2-nonenal (HNE), which has been implicated in the pathogenesis of Parkinson disease. Consequently, we investigated the effects of the interaction of HNE with ␣-synuclein. Incubation of HNE with ␣-synuclein at pH 7.4 and 37°C resulted in covalent modification of the protein, with up to six HNE molecules incorporated as Michael addition products. Fourier transform infrared and CD spectra indicated that HNE modification of ␣-synuclein resulted in a major conformational change involving increased ␤-sheet. HNE modification of ␣-synuclein led to inhibition of fibrillation in an HNE concentration-dependent manner. This inhibition of fibrillation was shown to be due to the formation of soluble oligomers based on size exclusion high pressure liquid chromatography and atomic force microscope data. Small angle x-ray scattering analysis indicated that the HNE-induced oligomers were compact and tightly packed. Treatment with guanidinium chloride demonstrated that the HNE-induced oligomers were very stable with an extremely slow rate of dissociation. Addition of 5 M HNE-modified oligomers to primary mesencephalic cultures caused marked neurotoxicity because the integrity of dopaminergic and GABAergic neurons was reduced by 95 and 85%, respectively. Our observations indicate that HNE modification of ␣-synuclein prevents fibrillation but may result in toxic oligomers, which could therefore contribute to the demise of neurons subjected to oxidative damage.

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Oxidative stress and aggregation of the presynaptic protein α-synuclein (α-Syn) are implied in the pathogenesis of Parkinson disease and several other neurodegenerative diseases. Various posttranslational modifications, such as oxidation, nitration and truncation, have significant effects on the kinetics of α-Syn fibrillation in vitro. α-Syn is a typical natively unfolded protein, which possesses some residual structure. The existence of long-range intra-molecular interactions between the C-terminal tail (residues 120–140) and the central part of α-Syn (residues 30–100) was recently established (Bertoncini et al. (2005) Proc Natl Acad Sci U S A 102, 1430–1435). Since α-Syn has four methionines, two of which (Met 1 and 5) are at the N-terminus and the other two (Met 116, 127) are in the hydrophobic cluster at the C-terminus of protein, the perturbation of these residues via their oxidation represents a good model for studying the effect of long-range interaction on α-Syn fibril formation. In this paper we show that Met 1, 116, and 127 are more protected from the oxidation than Met 5 likely due to the residual structure in the natively unfolded α-Syn. In addition to the hydrophobic interactions between the C-terminal hydrophobic cluster and hydrophobic central region of α-Syn, there are some long-range electrostatic interactions in this protein. Both of these interactions likely serve as auto-inhibitors of α-Syn fibrillation. Methionine oxidation affects both electrostatic and hydrophobic long-range interactions in α-Syn. Finally, oxidation of methionines by H2O2 greatly inhibited α-Syn fibrillation in vitro, leading to the formation of relatively stable oligomers, which are not toxic to dopaminergic and GABAergic neurons.

Manganese (Mn) is ubiquitous in mammalian systems and is essential for proper development and function, though it can also be toxic at elevated exposures. While essential biologic functions of Mn depend on its oxidation state [e.g., Mn(II), Mn(III)], little is known about how the oxidation state of elevated Mn exposures affect cellular uptake, and function/toxicity. Here we report the dynamics of EPR measurable Mn(II) in fresh human plasma and cultured PC12 cell lysates as a function of exposure to either manganese(II) chloride or manganese(III) pyrophosphate, and the effects of exposure to Mn(II) versus Mn(III) on total cellular aconitase activity and cellular Mn uptake. The results indicate that Mn(II) or Mn(III) added in vitro to fresh human plasma or cell lysates yielded similar amounts of EPR measurable Mn(II). In contrast, Mn added as Mn(III) was significantly more effective in inhibiting total cellular aconitase activity, and intact PC12 cells accumulated significantly more Mn when exposures occurred as Mn(III). Collectively, these data reflect the dynamic nature of Mn speciation in simple biological systems, and the importance of Mn oxidation/speciation state in mediating potential cellular toxicity. This study supports concern over increased environmental exposures to Mn in different oxidation states [Mn(II), Mn(III), and Mn(IV)] that may arise from combustion products of the gasoline antiknock additive methycyclopentadienyl manganese tricarbonyl (MMT).