Alpha-Synuclein Oligomers Interact with Metal Ions to Induce Oxidative Stress and Neuronal Death in Parkinson's Disease

Abstract

Aims: Protein aggregation and oxidative stress are both key pathogenic processes in Parkinson's disease, although the mechanism by which misfolded proteins induce oxidative stress and neuronal death remains unknown. In this study, we describe how aggregation of alpha-synuclein (α-S) from its monomeric form to its soluble oligomeric state results in aberrant free radical production and neuronal toxicity.

Results: We first demonstrate excessive free radical production in a human induced pluripotent stem-derived α-S triplication model at basal levels and on application of picomolar doses of β-sheet-rich α-S oligomers. We probed the effects of different structural species of α-S in wild-type rat neuronal cultures and show that both oligomeric and fibrillar forms of α-S are capable of generating free radical production, but that only the oligomeric form results in reduction of endogenous glutathione and subsequent neuronal toxicity. We dissected the mechanism of oligomer-induced free radical production and found that it was interestingly independent of several known cellular enzymatic sources.

Innovation: The oligomer-induced reactive oxygen species (ROS) production was entirely dependent on the presence of free metal ions as addition of metal chelators was able to block oligomer-induced ROS production and prevent oligomer-induced neuronal death.

Conclusion: Our findings further support the causative role of soluble amyloid oligomers in triggering neurodegeneration and shed light into the mechanisms by which these species cause neuronal damage, which, we show here, can be amenable to modulation through the use of metal chelation.

FIG. 1.

Oligomeric α-S induces ROS production in control and SNCA triplication neurons. (A) Basal and oligomeric induced levels of ROS were assessed in young (50 days) and old (80 days) control and SNCA triplication differentiated neurons using dihydroethidium. Old neurons were exposed to oligomers and the rate of ROS production was recorded. (B) Young control and triplication neurons were exposed to 300 nM monomers, 300 nM oligomers (3 nM total oligomers), or 300 nM fibrils, and ROS production was recorded and compared with basal ROS production levels. (C) Aged neurons (80 days) were transfected with the cellular Hyper-3 construct, and H₂O₂ production in the presence of oligomers was recorded. Representative trace of H₂O₂ production rate in control aged neurons (black trace). A control pH construct (Hyper-CS) reported no effect on intracellular pH (gray trace). (D) Representative trace of H₂O₂ production in triplication neurons exposed to oligomers. (E) Quantification of basal and oligomer-induced H₂O₂ production in control and triplication neurons. (F) Immunocytochemistry for iPSC derived neurons for βIII-tubulin (green) and Hoechst (blue). (G) Cytosolic calcium response to glutamate and ATP in iPSC derived neurons (representative traces). Bars indicate the incubation period of either oligomers or H₂O₂. n ≥ 4 experiments. Error bars indicate SEM; *p < 0.05; **p < 0.01; ***p < 0.001. α-S, alpha-synuclein (protein); H₂O₂, hydrogen peroxide; ROS, reactive oxygen species; SNCA alpha-synuclein (gene). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 2.

The ability of α-S to induce ROS is dependent on its structural conformation. The ability of each different α-S conformation to induce ROS production in WT rat neurons was assessed using both labeled and unlabeled preparations of monomers (A), oligomers (B), and fibrils (C, D) Quantification of ROS production in WT rat midbrain neurons upon exposure to α-S species. WT rat midbrain neurons were transfected with the cellular Hyper-3 construct and H₂O₂ production in the presence of monomers (E), oligomers (F), and fibrils (G) and recorded (representative traces). (H) Quantification of α-S-induced H₂O₂ production rate in WT rat neurons. Bars indicate the incubation period of either α-S species or H₂O₂. n = 3 experiments. Error bars indicate SEM; *p < 0.05; ***p < 0.001. α-S, α-synuclein; WT, wild-type.

FIG. 3.

α-S-induced ROS is independent of the major cellular ROS production systems. (A) Unlabeled enriched oligomeric α-S was utilized to assess the dose-dependent effect of oligomers on ROS production. (B) Representative trace of mitochondrial ROS production (MitoSOX) in WT rat neurons upon oligomeric α-S exposure; 1 μM rotenone induced a significant increase of MitoSOX fluorescence. (C) Equivalent levels of labeled and unlabeled enriched α-S species were assessed for their ability to induce mitochondrial ROS production in WT neurons using the mitochondrial ROS sensing dye, MitoSOX. (D) Representative image of iPSC-derived neurons labeled with MitoTracker red CM-H(2)X ROS. (Di) Representative traces of mitochondrial ROS production rate in the presence of monomeric, oligomeric, or fibrillar α-S in iPSC-derived neurons. (E) Representative traces showing the marginal decrease in ROS levels by AEBSF pretreatment of iPSC-derived neuron, (F) preincubation of WT neurons with inhibitors (DPI; Selegiline; Oxypurinol; NDGA) of the major cellular sources of ROS failed to block ROS production induced by the unlabeled enriched oligomeric species. (G) Quantification of ROS levels in control, α-S triplication, and AEBSF-treated α-S triplication neurons. Bars indicate the incubation period of either α-S species or rotenone. n = 3 experiments. Scale bar = 10 μm; error bars indicate SEM. **p < 0.01. AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride; DPI, dibenziodolium chloride. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 4.

α-S oligomers induce ROS via a redox metal ion-driven mechanism. (A) Preincubation of the oligomers with different chelators, clioquinol, DFO, and D-PEN, for 10 min before addition to neurons resulted in a significant decrease in the oligomer-induced ROS signal. (B) Preincubation of cells with the same chelators for 15 min, followed by application of oligomers, also resulted in a significant reduction in the oligomer-induced ROS production. (C) Preincubation (15 min) of α-S triplication iPSC neurons cells with the chelators, followed by application of oligomers, resulted in significant decrease in oligomer-induced ROS production. Note that inhibitor of NADPH oxidase 20 μM AEBSF had no effect on ROS production induced by oligomers in iPSC neurons. (D) Basal ROS production in cells preincubated with different chelators (clioquinol, DFO, and D-PEN) alone before the application of oligomers. n = 3 experiments. Error bars indicate SEM. *p < 0.05; **p < 0.05; ***p < 0.001. D-PEN, 1,2-diphenyl-1,2-ethylenediamine; DFO, desferrioxamine; iPSC, induced pluripotent stem cell.

FIG. 5.

Exposure of neuronal cultures to oligomeric α-S reduces endogenous GSH levels and increases lipid peroxidation. (A) Reduced GSH levels of WT rat neurons after incubation with the different α-S species for 48 h; the levels of reduced GSH were assayed using MCB. (B) Treatment of neurons and astrocytes with the metal chelators, TPEN, DFO, or clioquinol, prevents the decrease in endogenous GSH levels caused by the oligomer-induced ROS production. (C) Control and α-S triplication cells were loaded with C11-Bodipy and lipid oxidation was recorded. Representative trace of oligomeric-α-S-induced lipid peroxidation in control iPSC neurons. (D) Representative traces of lipid peroxidation in α-S triplication cells preincubated with D-PEN, DFO, clioquinol, or solvent. (E) Quantification of lipid peroxidation rate in α-S triplication iPSC neurons preincubated with chelators or solvent. Bar indicates the incubation period of oligomers. n = 3 experiments. Error bars indicate SEM. **p < 0.01; ***p < 0.001. GSH, glutathione; MCB, monochlorobimane; TPEN, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine

FIG. 6.

α-S oligomers trigger oxidative stress-induced caspase-3 activation in neurons. WT rat midbrain neurons were loaded with NucView488 caspase-3 substrate, which fluoresces green upon caspase-3/7 activation. Neurons were exposed to (A) monomeric, (B) oligomeric, and (D) fibrillar α-S, respectively. (C) Representative image of basal and oligomeric α-S-induced caspase-3 activation. (E) Representative traces demonstrating the inhibition of oligomeric-induced caspase-3 activation in cells preincubated with 100 μM Trolox. (F) Quantification of apoptosis (caspase-3 activation) in untreated, DFO, or clioquinol-treated WT rat neurons exposed to PBS or oligomeric α-S. Bars indicate the incubation period of either α-S species or Trolox. Scale bar = 50 μm; n = 4 experiments; error bars indicate SEM. *p < 0.05; **p < 0.01. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars