Lipid peroxidation is essential for α-synuclein-induced cell death

Abstract

Parkinson's disease is the second most common neurodegenerative disease and its pathogenesis is closely associated with oxidative stress. Deposition of aggregated α-synuclein (α-Syn) occurs in familial and sporadic forms of Parkinson's disease. Here, we studied the effect of oligomeric α-Syn on one of the major markers of oxidative stress, lipid peroxidation, in primary co-cultures of neurons and astrocytes. We found that oligomeric but not monomeric α-Syn significantly increases the rate of production of reactive oxygen species, subsequently inducing lipid peroxidation in both neurons and astrocytes. Pre-incubation of cells with isotope-reinforced polyunsaturated fatty acids (D-PUFAs) completely prevented the effect of oligomeric α-Syn on lipid peroxidation. Inhibition of lipid peroxidation with D-PUFAs further protected cells from cell death induced by oligomeric α-Syn. Thus, lipid peroxidation induced by misfolding of α-Syn may play an important role in the cellular mechanism of neuronal cell loss in Parkinson's disease. We have found that aggregated α-synuclein-induced production of reactive oxygen species (ROS) that subsequently stimulates lipid peroxidation and cell death in neurons and astrocytes. Specific inhibition of lipid peroxidation by incubation with reinforced polyunsaturated fatty acids (D-PUFAs) completely prevented the effect of α-synuclein on lipid peroxidation and cell death.

Keywords: deuterated PUFA; lipid peroxidation; oxidative stress; α-synuclein.

Scheme 1

(a) Chain reaction of lipid peroxidation is initiated by a reactive oxygen species (ROS)‐mediated hydrogen abstraction from a bis‐allylic site within a polyunsaturated fatty acids (PUFA). The carbon‐based radical thus formed reacts with O₂ to yield an alkylperoxy radical, which abstracts hydrogen from a neighbouring PUFA in the membrane. This propagation of the chain process can be terminated by antioxidants or radical recombination. The chain process generates a large variety of toxic moieties including peroxides and carbonyls. (b) The essential PUFAs, n‐6 Lin and n‐3 Lnn, from which higher essential PUFAs can be built in vivo by enzymatic desaturation and extension. (c) Deuteration at the bis‐allylic sites protects D2‐Lin and D4‐Lnn from the rate‐limiting step of lipid peroxidation.

Figure 1

The ability of α‐synuclein to induce reactive oxygen species (ROS) is dependent on the form of its conformation. ROS production is significantly higher when primary glio‐neuronal co‐culture was treated with aggregated (oligomeric) form of alpha‐synuclein, compared to those treated with non‐aggregated (monomeric) form of alpha‐synuclein (a and b). (a) shows representative traces of dihydroethidium (HEt) measurements whereas (b) summarizes the effects of the two forms of alpha‐synuclein on the ROS production in percentage. ns: not significant. C – effect of pre‐incubation (20 min) of neurons and astrocytes with 100 μM Trolox on α‐synuclein‐induced ROS production.

Figure 2

Aggregated, but not non‐aggregated form of α‐synuclein produces lipid peroxidation. Equivalent amounts of aggregated and non‐aggregated α‐synuclein have been tested for their ability to evoke lipid peroxidation in mixed glio‐neuronal culture, using the C‐11 BODIPY 581/591 lipid peroxidation probe. The addition of the aggregated form of α‐synuclein only, induced lipid peroxidation. (a), representative traces showing the increase in basal rate of lipid peroxidation only after addition of oligomeric α‐synuclein, (b), summary of the results given in percentage and (c), effect of 100 μM Trolox on lipid peroxidation under exposure of aggregated α‐synuclein. (d) Effect of aggregated α‐synuclein on DAF‐FM fluorescence.

Figure 3

α‐synuclein‐induced lipid peroxidation could be prevented by deuterated polyunsaturated fatty acids (PUFAs). No changes in the basal rate of lipid peroxidation (representative traces) are evoked by aggregated α‐synuclein in D4‐Lnn (a and c) and D2‐Lin (b and c) – pre‐treated co‐cultures from rat neurons and astrocytes in C‐11 Bodipy assay in comparison to the non‐deuterated forms of PUFA (H‐Lin and H‐Lnn). (c), Histogram summarizing changes in the rate of lipid peroxidation (in percentage) in the presence of deuterated PUFAs. ***p < 0.0001; n.s. non‐significant.

Figure 4

Deuterated polyunsaturated fatty acids (PUFAs) prevent α‐synuclein‐induced rise in lipid peroxidation in both, cortical neurons and cortical astrocytes. Aggregated form of α‐Syn stimulates lipid peroxidation in both neurons (a1, a2) and astrocytes (b1, b2), however, statistically significant level of the protective D4‐Lnn effect is reached only in astrocytes (p < 0.0001). c1, c2 – experiments, using alternative lipid peroxidation indicator BODIPY 655/675.

Figure 5

Protective effect of deuterated polyunsaturated fatty acid (PUFA) against α‐synuclein‐induced cell death. About 100 nM aggregated (but not non‐aggregated) alpha‐synuclein significantly increased the number of dead cells in primary glio‐neuronal cultures (a). Pre‐incubation of the cells with deuterated PUFA‐containing medium (either D4‐Lnn or D2‐Lin) significantly reduced the number of dead cells (given in percent) in the culture (a and b). Cell death was assessed using PI/Hoechst assay to label dead cells and total number of cells, respectively.