Environmental and Genetic Factors Support the Dissociation Between α-Synuclein Aggregation and Toxicity

Abstract

Synucleinopathies are a group of progressive disorders characterized by the abnormal aggregation and accumulation of α-synuclein (aSyn), an abundant neuronal protein that can adopt different conformations and biological properties. Recently, aSyn pathology was shown to spread between neurons in a prion-like manner. Proteins like aSyn that exhibit self-propagating capacity appear to be able to adopt different stable conformational states, known as protein strains, which can be modulated both by environmental and by protein-intrinsic factors. Here, we analyzed these factors and found that the unique combination of the neurodegeneration-related metal copper and the pathological H50Q aSyn mutation induces a significant alteration in the aggregation properties of aSyn. We compared the aggregation of WT and H50Q aSyn with and without copper, and assessed the effects of the resultant protein species when applied to primary neuronal cultures. The presence of copper induces the formation of structurally different and less-damaging aSyn aggregates. Interestingly, these aggregates exhibit a stronger capacity to induce aSyn inclusion formation in recipient cells, which demonstrates that the structural features of aSyn species determine their effect in neuronal cells and supports a lack of correlation between toxicity and inclusion formation. In total, our study provides strong support in favor of the hypothesis that protein aggregation is not a primary cause of cytotoxicity.

Keywords: H50Q mutation; copper; inclusions; protein aggregation; α-synuclein.

Fig. 1.

Copper ions promote the intracellular accumulation of H50Q aSyn inclusions. (A) The addition of Cu²⁺ to the medium of cells transfected with different aSyn mutations revealed the formation of intracellular inclusions only in the presence of the H50Q aSyn mutant (white arrows). (Scale bar, 5 μm.) (B) The effect observed was not associated with an increase in intracellular levels of expression of the H50Q mutant, as determined by immunoblot analysis. (C) The interaction between Cu²⁺ and H50Q promotes an increase in cytotoxicity compared with WT −Cu²⁺, WT +Cu²⁺, and H50Q −Cu²⁺. MW, molecular weight/molecular mass. *P < 0.05; **P < 0.01.

Fig. 2.

Biophysical characterization of the effect of copper ions on the aggregation of WT and H50Q aSyn. (A) Aggregation kinetics of WT and H50Q aSyn with and without Cu²⁺ evaluated by normalized Th-T binding at the indicated time points. Fluorescence intensity peak at 482 nm was used to probe for amyloid formation. (B) Fluorescence intensity at the end-time point of the Th-T binding assay. (C) Remaining amount of soluble aSyn determined by sedimentation assay at the end time point of aggregation. (D) TEM images of the resulting aggregates stained with uranium acetate. (Scale bar, 500 nm.) (E) Partial PK digestion of the aggregates visualized by Coomasie staining of SDS/PAGE gels. Each lane represents different digestion times: 0, 1, 2.5, 5, and 10 min.

Fig. 3.

Copper ions reduce the seeding capacity of aSyn aggregates. Time-dependence of 1H NMR spectra of monomeric aSyn upon addition of aggregation seeds [4% (vol/vol)]. The integrated intensity of ¹H signals in two regions (0.50–1.05 ppm, 6.50–8.00 ppm) was calculated and plotted as a function of time. The solution with 70-μM monomeric protein (WT or H50Q) contained either no Cu²⁺ or an equimolar concentration of Cu²⁺, as indicated.

Fig. 4.

Cellular effects as a result of the exogenous application of aSyn aggregates. (A) Cytotoxicity quantification in cultures exposed to aggregates compared with the vehicle control, measured as the presence of adenylate kinase in the medium. (B) Cell death quantification, measured as the reduction in the number of cells. (C) Increase in the percentage of condensed nuclei in the same experiment represented in B. (D) Quantification of neurite length of cells exposed to aggregates compared with the vehicle control (Left) and representative images of MAP2 stained cultures, used to obtain the former parameter (Right). (Scale bars, 20 μm.) (E) Quantification of MAP2 and Tau levels by Western blot. (F) Astrogliosis caused by the application of aSyn aggregates, measured by Western blot quantification of GFAP (Left), and representative images evidencing the presence of reactive astrocytes (Right). (Scale bars, 20 μm.) *P < 0.05; **P < 0.01; ***P < 0.001 for comparison between samples and their appropriate vehicle control. ^#P < 0.05; ^#P < 0.01 for comparison between different samples.

Fig. 5.

Formation of S129 p-aSyn⁺ inclusions upon exposure to aSyn aggregates. (A) Representative images of each culture showing the presence of p-aSyn (S129) inclusions in cells treated with exogenous aggregates. (Scale bars, 20 μm.) (B) Distinct types of p-aSyn inclusions classified as neuritic, perykarial and nuclear. (Scale bars, 20 μm.) (C) Western blot measurements of p-aSyn (S129) in cell lysates. (D) Soluble/insoluble distribution of the cellular aSyn detected with a rat-specific aSyn antibody. **P < 0.01; ***P < 0.001 for comparison between samples and their appropriate vehicle control. ^##P < 0.01 for comparison between different samples.

Fig. 6.

Distribution of exogenous and endogenous aSyn in the S129 p-aSyn inclusions. (A) Neuritic (Top), perykarial (Middle), and nuclear (Bottom) p-aSyn inclusions show partial or total colocalization with rat-specific aSyn. (B) Presence and absence of labeled aSyn aggregates in perykarial and nuclear p-aSyn inclusions. (C) Confocal live cell imaging of cultures exposed to labeled aSyn aggregates and infected with a GFP-expressing lentivirus demonstrating the cellular distribution of the exogenous species at different time points after exposure. (Scale bars, 10 μm.)