Magnesium Inhibits Spontaneous and Iron-induced Aggregation of α-Synuclein

Abstract

Multiple studies implicate metals in the pathophysiology of neurodegenerative diseases. Disturbances in brain iron metabolism are linked with synucleinopathies. For example, in Parkinson's disease, iron levels are increased and magnesium levels are reduced in the brains of patients. To understand how changes in iron and magnesium might affect the pathophysiology of Parkinson's disease, we investigated binding of iron to ␣-synuclein, which accumulates in Lewy bodies. Using fluorescence of the four tyrosines in ␣-synuclein as indicators of metal-related conformational changes in ␣-synuclein, we show that iron and magnesium both interact with ␣-synuclein. ␣-Synuclein exhibits fluorescence peaks at 310 and 375 nm. Iron lowers both fluorescence peaks, while magnesium increases the fluorescence peak only at 375 nm, which suggests that magnesium affects the conformation of ␣-synuclein differently than iron. Consistent with this hypothesis, we also observe that magnesium inhibits ␣-synuclein aggregation, measured by immunoblot, cellulose acetate filtration, or thioflavine-T fluorescence. In each of these studies, iron increases ␣-synuclein aggregation, while magnesium at concentrations >0.75 mM inhibits the aggregation of ␣-synuclein induced either spontaneously or by incubation with iron. These data suggest that the conformation of ␣-synuclein can be modulated by metals, with iron promoting aggregation and magnesium inhibiting aggregation. Parkinson's disease (PD)1 is a common motor disorder that affects about 1% of population over the age of 65 (1). The disease is characterized by progressive neurodegeneration predominantly affecting dopaminergic neurons in the nigrostriatal system (2). The degenerating neurons develop intracellular inclusions, termed Lewy bodies, which are composed of a dense core of filamentous and granular material (3). Recent studies indicate that ␣-synuclein is a major filamentous component of Lewy bodies (3,4). Genetic studies suggest that ␣-synuclein plays a key role in the pathophysiology of PD, because mutations in ␣-synuclein, at A53T or A30P, are associated with early-onset familial PD (5, 6).The accumulation of aggregated protein underlies the pathophysiology of many neurodegenerative disorders, and increasing evidence suggests that aggregated ␣-synuclein plays a key role in the pathophysiology of PD. ␣-Synuclein has a strong tendency to aggregate and does so spontaneously in vitro at a slow rate (7-9). Both the A53T and the A30P mutations in PD increase the tendency of ␣-synuclein to aggregate. Many studies in cultured neurons, and some studies in transgenic animals, suggest that ␣-synuclein aggregation is linked to cellular toxicity and neurodegeneration (10 -12). In cell culture, formation of ␣-synuclein aggregates correlates with cell injury (10). Overexpressing ␣-synuclein in Drosophila leads to an age-dependent accumulation of aggregated ␣-synuclein and associated neurodegeneration (12). Masliah and colleagues also observed that aggregated ␣-synuclein is associated with loss of marke...

Dose- and time-dependent α-synuclein aggregation induced by ferric iron in SK-N-SH cells

“…Our studies further confirm that high concentrations of ferric iron can induce α-synuclein aggregation in dopaminergic cell lines, in a dose-and time-dependent way, which is toxic to the cells. This is consistent with other reports that ferrous [21] or ferric iron [22] could promote purified α-synuclein monomer folding, oligomerization, and aggregation in vitro. Co-treatment with iron and other free radical generators, such as dopamine and hydrogen peroxide, can also induce α-synuclein aggregation [20] .…”

“…The high concentration of ferric iron has been proved to promote the aggregation of A53T mutant α-synuclein in dopaminergic cells [20] . In vitro studies also show that when incubated with ferrous [21] or ferric iron [22] , α-synuclein folding, oligomerization, and aggregation would be promoted. …”

Oligomeric alpha‐synuclein and its role in neuronal death

“…However, further analysis suggested that a-Syn binds only two Cu(II) ions per monomer: the first of which binds with a dissociation constant in the 0.1-50 lM range and the second with much lower affinity (400-500 lM) (36). Although there is a consensus that a-Syn also binds iron, the reported binding affinities are very different (37)(38)(39). Other metals, such as Mn(II), Co(II), and Ni(II) also bind to a-Syn, however, the affinity for these metals is much lower (K d 1 mM) (37).…”

Inhibition of the Fe(III)-Catalyzed Dopamine Oxidation by ATP and Its Relevance to Oxidative Stress in Parkinson’s Disease

“…17 Since neuromelanin is believed to be produced from oxidation of excess DA, and the loss of dopaminergic neurons in SN is accompanied by an accumulation of ferric ions, the synergy between DA and iron in the etiology of PD has become increasingly evident. 18 Furthermore, iron-induced DA neurotoxicity has been confirmed by cell culture experiments, which revealed that cell viability decreases dramatically in the presence of DA and iron. 19,20 In vitro, iron has been shown to catalyze the oxidation of DA by oxygen to generate H 2 O 2 , neurotoxic intermediates, and neuromelanin (Scheme 1).…”