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Arachidonic acid mediates the formation of abundant alpha-helical multimers of alpha-synuclein

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ABSTRACT

The protein alpha-synuclein (αS) self-assembles into toxic beta-sheet aggregates in Parkinson’s disease, while it is proposed that αS forms soluble alpha-helical multimers in healthy neurons. Here, we have made αS multimers in vitro using arachidonic acid (ARA), one of the most abundant fatty acids in the brain, and characterized them by a combination of bulk experiments and single-molecule Fӧrster resonance energy transfer (sm-FRET) measurements. The data suggest that ARA-induced oligomers are alpha-helical, resistant to fibril formation, more prone to disaggregation, enzymatic digestion and degradation by the 26S proteasome, and lead to lower neuronal damage and reduced activation of microglia compared to the oligomers formed in the absence of ARA. These multimers can be formed at physiologically-relevant concentrations, and pathological mutants of αS form less multimers than wild-type αS. Our work provides strong biophysical evidence for the formation of alpha-helical multimers of αS in the presence of a biologically relevant fatty acid, which may have a protective role with respect to the generation of beta-sheet toxic structures during αS fibrillation.

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TEM images of aggregates.(a) αS aggregates in buffer (35 μM, 24 h, shaking). From left to right: before centrifugation (15 min at 14.2 r.p.m.), a mixture of oligomers and fibrils is observed; soluble oligomers are present in the supernatant after centrifugation; insoluble pellet after centrifugation contains fibrillar aggregates. Scale bars (left to right) 200 nm, 200 nm and 100 nm. (b) ARA-induced oligomers of αS (35 μM, 1 mM ARA, 24 h, non-shaking). No fibrils observed after centrifugation. Abundant soluble oligomers and oligomer agglomerates are present. Scale bars 100 nm (left) and 50 nm (right). (c) ARA acid micelles in buffer (1 mM ARA) in the absence of αS. Scale bars 1 μm (left) and 100 nm (right).
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f2: TEM images of aggregates.(a) αS aggregates in buffer (35 μM, 24 h, shaking). From left to right: before centrifugation (15 min at 14.2 r.p.m.), a mixture of oligomers and fibrils is observed; soluble oligomers are present in the supernatant after centrifugation; insoluble pellet after centrifugation contains fibrillar aggregates. Scale bars (left to right) 200 nm, 200 nm and 100 nm. (b) ARA-induced oligomers of αS (35 μM, 1 mM ARA, 24 h, non-shaking). No fibrils observed after centrifugation. Abundant soluble oligomers and oligomer agglomerates are present. Scale bars 100 nm (left) and 50 nm (right). (c) ARA acid micelles in buffer (1 mM ARA) in the absence of αS. Scale bars 1 μm (left) and 100 nm (right).

Mentions: Firstly, we found using transmission electron microscopy (TEM) that the ARA-induced oligomers had markedly different morphologies compared to the oligomers of αS formed in aqueous buffer (Fig. 2). In the case of αS samples in buffer solution, after 24 hours of incubation the solutions contained a mixture of monomers, soluble oligomeric species as well as insoluble fibrils (Fig. 2a). The majority of the insoluble fibrils could be removed by centrifugation, as judged by TEM and confirmed in our previous work38, therefore this step was introduced for the preparation of the αS-only oligomers. In the case of the samples with ARA-induced oligomers, abundant populations of oligomers were observed, frequently associated into higher-order assemblies (Fig. 2b). Interestingly, these aggregates were soluble, judged from the absence of fibrillar aggregates in TEM images and no precipitate upon centrifugation of the samples. In the absence of αS, at 1 mM ARA, micelles were formed (Fig. 2c). In the presence of the protein, however, the aggregates visualized using TEM were smaller compared to the micellar structures, which is consistent with the previously reported observation that αS is able to disrupt the micelles of ARA26. Overall, ARA-induced oligomers were larger and had less regular shapes than the αS-only oligomers, probably due to the association with the FA, and had a tendency to assemble into higher-order soluble agglomerates. They looked similar in morphology to the previously reported oligomers of αS in the excess of docosahexaenoic acid29.


Arachidonic acid mediates the formation of abundant alpha-helical multimers of alpha-synuclein
TEM images of aggregates.(a) αS aggregates in buffer (35 μM, 24 h, shaking). From left to right: before centrifugation (15 min at 14.2 r.p.m.), a mixture of oligomers and fibrils is observed; soluble oligomers are present in the supernatant after centrifugation; insoluble pellet after centrifugation contains fibrillar aggregates. Scale bars (left to right) 200 nm, 200 nm and 100 nm. (b) ARA-induced oligomers of αS (35 μM, 1 mM ARA, 24 h, non-shaking). No fibrils observed after centrifugation. Abundant soluble oligomers and oligomer agglomerates are present. Scale bars 100 nm (left) and 50 nm (right). (c) ARA acid micelles in buffer (1 mM ARA) in the absence of αS. Scale bars 1 μm (left) and 100 nm (right).
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Related In: Results  -  Collection

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f2: TEM images of aggregates.(a) αS aggregates in buffer (35 μM, 24 h, shaking). From left to right: before centrifugation (15 min at 14.2 r.p.m.), a mixture of oligomers and fibrils is observed; soluble oligomers are present in the supernatant after centrifugation; insoluble pellet after centrifugation contains fibrillar aggregates. Scale bars (left to right) 200 nm, 200 nm and 100 nm. (b) ARA-induced oligomers of αS (35 μM, 1 mM ARA, 24 h, non-shaking). No fibrils observed after centrifugation. Abundant soluble oligomers and oligomer agglomerates are present. Scale bars 100 nm (left) and 50 nm (right). (c) ARA acid micelles in buffer (1 mM ARA) in the absence of αS. Scale bars 1 μm (left) and 100 nm (right).
Mentions: Firstly, we found using transmission electron microscopy (TEM) that the ARA-induced oligomers had markedly different morphologies compared to the oligomers of αS formed in aqueous buffer (Fig. 2). In the case of αS samples in buffer solution, after 24 hours of incubation the solutions contained a mixture of monomers, soluble oligomeric species as well as insoluble fibrils (Fig. 2a). The majority of the insoluble fibrils could be removed by centrifugation, as judged by TEM and confirmed in our previous work38, therefore this step was introduced for the preparation of the αS-only oligomers. In the case of the samples with ARA-induced oligomers, abundant populations of oligomers were observed, frequently associated into higher-order assemblies (Fig. 2b). Interestingly, these aggregates were soluble, judged from the absence of fibrillar aggregates in TEM images and no precipitate upon centrifugation of the samples. In the absence of αS, at 1 mM ARA, micelles were formed (Fig. 2c). In the presence of the protein, however, the aggregates visualized using TEM were smaller compared to the micellar structures, which is consistent with the previously reported observation that αS is able to disrupt the micelles of ARA26. Overall, ARA-induced oligomers were larger and had less regular shapes than the αS-only oligomers, probably due to the association with the FA, and had a tendency to assemble into higher-order soluble agglomerates. They looked similar in morphology to the previously reported oligomers of αS in the excess of docosahexaenoic acid29.

View Article: PubMed Central - PubMed

ABSTRACT

The protein alpha-synuclein (αS) self-assembles into toxic beta-sheet aggregates in Parkinson’s disease, while it is proposed that αS forms soluble alpha-helical multimers in healthy neurons. Here, we have made αS multimers in vitro using arachidonic acid (ARA), one of the most abundant fatty acids in the brain, and characterized them by a combination of bulk experiments and single-molecule Fӧrster resonance energy transfer (sm-FRET) measurements. The data suggest that ARA-induced oligomers are alpha-helical, resistant to fibril formation, more prone to disaggregation, enzymatic digestion and degradation by the 26S proteasome, and lead to lower neuronal damage and reduced activation of microglia compared to the oligomers formed in the absence of ARA. These multimers can be formed at physiologically-relevant concentrations, and pathological mutants of αS form less multimers than wild-type αS. Our work provides strong biophysical evidence for the formation of alpha-helical multimers of αS in the presence of a biologically relevant fatty acid, which may have a protective role with respect to the generation of beta-sheet toxic structures during αS fibrillation.

No MeSH data available.


Related in: MedlinePlus