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

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.


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Experiments using physiological concentrations of αS and ARA.(a) Kinetic profile of oligomer formation at 2 μM αS in the presence of 10 μM ARA (dashed line), and in the absence of ARA under the same conditions (black line) (n = 3, std). (b) Comparison of apparent size distributions at 35 μM αS with 1 mM ARA (red), or 2 μM αS with 10 μM ARA (black). (c) Representative FRET efficiency histograms, resulting from sm-FRET analysis of the oligomers formed with either 35 μM αS with 1 mM ARA, or 2 μM αS with 10 μM ARA after 6 h. After this and later times, there was no difference in the appearance of the histograms, apart from lower total numbers detected in the lower-concentration samples, when the same protein concentration was used for the detection. (d) CD spectra of the solutions of 2 μM αS with 10 μM ARA after 24 h and enrichment using 100 kDa spinfilter. (e) Overlaid oligomer disaggregation profiles upon dilution into aqueous buffer to 280 pM of 35 μM αS samples with 1 mM ARA (red), and 2 μM αS with 10 μM ARA (grey) (n = 3, std). (f) Numbers of oligomers detected after >30 h using a range of αS isoforms, either A90C or pathological mutants, at 2 μM with 10 μM ARA (grey) or in the absence of ARA (red) (n = 6, sem).
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f5: Experiments using physiological concentrations of αS and ARA.(a) Kinetic profile of oligomer formation at 2 μM αS in the presence of 10 μM ARA (dashed line), and in the absence of ARA under the same conditions (black line) (n = 3, std). (b) Comparison of apparent size distributions at 35 μM αS with 1 mM ARA (red), or 2 μM αS with 10 μM ARA (black). (c) Representative FRET efficiency histograms, resulting from sm-FRET analysis of the oligomers formed with either 35 μM αS with 1 mM ARA, or 2 μM αS with 10 μM ARA after 6 h. After this and later times, there was no difference in the appearance of the histograms, apart from lower total numbers detected in the lower-concentration samples, when the same protein concentration was used for the detection. (d) CD spectra of the solutions of 2 μM αS with 10 μM ARA after 24 h and enrichment using 100 kDa spinfilter. (e) Overlaid oligomer disaggregation profiles upon dilution into aqueous buffer to 280 pM of 35 μM αS samples with 1 mM ARA (red), and 2 μM αS with 10 μM ARA (grey) (n = 3, std). (f) Numbers of oligomers detected after >30 h using a range of αS isoforms, either A90C or pathological mutants, at 2 μM with 10 μM ARA (grey) or in the absence of ARA (red) (n = 6, sem).

Mentions: In the above experiments ARA has been used at high concentration above its CMC value2639. Because ARA is a biologically relevant molecule and occurs in vivo at concentrations significantly below its CMC, we extended our study to more physiologically-relevant concentrations of 2–10 μM47 (Supplementary Fig. 6a). In addition, the concentrations of αS protein at the synapse were reported to be in the range 2–5 μM11. Therefore, in order to mimic the concentrations of both αS and ARA found in vivo, we combined 2 μM αS and 10 μM ARA. Under these conditions, a rapid multimerization was still observed shortly after the addition of ARA, as shown in Fig. 5a, and an increase in their numbers was present during the first 6 hours, resembling the timescales of the process at higher concentrations (Fig. 1a). The numbers of the detected oligomers, and their estimated concentrations (Supplementary Fig. 6b) were lower in comparison to the results in the high-concentration experiments, which highlights the challenge of monitoring this low-concentration process using more conventional bulk methods. Despite the lower overall numbers of oligomers, the growth of these species was again observed, with similar apparent size distributions compared to the higher-concentration reaction (Fig. 5b), and very similar FRET efficiency histograms (Fig. 5c). Further, we used CD to determine the conformation of these multimers. Preliminary attempts to measure the solutions containing 2 μM αS and 10 μM ARA resulted in the spectra indicating the presence of intrinsically disordered protein. This was consistent with the single-molecule observations that even though the multimers were present in the solutions, the majority of αS was still in its monomeric form, as is indicated by the low estimated concentrations of the multimers (Supplementary Fig. 6b). We therefore enriched the multimers using 100 kDa spin-filters, as described in Methods, and their CD spectrum indicated that these species were alpha-helically-folded, similarly to the species generated at 35 μM αS with 1 mM ARA (Fig. 5d). To note, the retention by the 100 kDa cutoff filter is consistent with the oligomers being the size of a tetramer and larger.


Arachidonic acid mediates the formation of abundant alpha-helical multimers of alpha-synuclein
Experiments using physiological concentrations of αS and ARA.(a) Kinetic profile of oligomer formation at 2 μM αS in the presence of 10 μM ARA (dashed line), and in the absence of ARA under the same conditions (black line) (n = 3, std). (b) Comparison of apparent size distributions at 35 μM αS with 1 mM ARA (red), or 2 μM αS with 10 μM ARA (black). (c) Representative FRET efficiency histograms, resulting from sm-FRET analysis of the oligomers formed with either 35 μM αS with 1 mM ARA, or 2 μM αS with 10 μM ARA after 6 h. After this and later times, there was no difference in the appearance of the histograms, apart from lower total numbers detected in the lower-concentration samples, when the same protein concentration was used for the detection. (d) CD spectra of the solutions of 2 μM αS with 10 μM ARA after 24 h and enrichment using 100 kDa spinfilter. (e) Overlaid oligomer disaggregation profiles upon dilution into aqueous buffer to 280 pM of 35 μM αS samples with 1 mM ARA (red), and 2 μM αS with 10 μM ARA (grey) (n = 3, std). (f) Numbers of oligomers detected after >30 h using a range of αS isoforms, either A90C or pathological mutants, at 2 μM with 10 μM ARA (grey) or in the absence of ARA (red) (n = 6, sem).
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f5: Experiments using physiological concentrations of αS and ARA.(a) Kinetic profile of oligomer formation at 2 μM αS in the presence of 10 μM ARA (dashed line), and in the absence of ARA under the same conditions (black line) (n = 3, std). (b) Comparison of apparent size distributions at 35 μM αS with 1 mM ARA (red), or 2 μM αS with 10 μM ARA (black). (c) Representative FRET efficiency histograms, resulting from sm-FRET analysis of the oligomers formed with either 35 μM αS with 1 mM ARA, or 2 μM αS with 10 μM ARA after 6 h. After this and later times, there was no difference in the appearance of the histograms, apart from lower total numbers detected in the lower-concentration samples, when the same protein concentration was used for the detection. (d) CD spectra of the solutions of 2 μM αS with 10 μM ARA after 24 h and enrichment using 100 kDa spinfilter. (e) Overlaid oligomer disaggregation profiles upon dilution into aqueous buffer to 280 pM of 35 μM αS samples with 1 mM ARA (red), and 2 μM αS with 10 μM ARA (grey) (n = 3, std). (f) Numbers of oligomers detected after >30 h using a range of αS isoforms, either A90C or pathological mutants, at 2 μM with 10 μM ARA (grey) or in the absence of ARA (red) (n = 6, sem).
Mentions: In the above experiments ARA has been used at high concentration above its CMC value2639. Because ARA is a biologically relevant molecule and occurs in vivo at concentrations significantly below its CMC, we extended our study to more physiologically-relevant concentrations of 2–10 μM47 (Supplementary Fig. 6a). In addition, the concentrations of αS protein at the synapse were reported to be in the range 2–5 μM11. Therefore, in order to mimic the concentrations of both αS and ARA found in vivo, we combined 2 μM αS and 10 μM ARA. Under these conditions, a rapid multimerization was still observed shortly after the addition of ARA, as shown in Fig. 5a, and an increase in their numbers was present during the first 6 hours, resembling the timescales of the process at higher concentrations (Fig. 1a). The numbers of the detected oligomers, and their estimated concentrations (Supplementary Fig. 6b) were lower in comparison to the results in the high-concentration experiments, which highlights the challenge of monitoring this low-concentration process using more conventional bulk methods. Despite the lower overall numbers of oligomers, the growth of these species was again observed, with similar apparent size distributions compared to the higher-concentration reaction (Fig. 5b), and very similar FRET efficiency histograms (Fig. 5c). Further, we used CD to determine the conformation of these multimers. Preliminary attempts to measure the solutions containing 2 μM αS and 10 μM ARA resulted in the spectra indicating the presence of intrinsically disordered protein. This was consistent with the single-molecule observations that even though the multimers were present in the solutions, the majority of αS was still in its monomeric form, as is indicated by the low estimated concentrations of the multimers (Supplementary Fig. 6b). We therefore enriched the multimers using 100 kDa spin-filters, as described in Methods, and their CD spectrum indicated that these species were alpha-helically-folded, similarly to the species generated at 35 μM αS with 1 mM ARA (Fig. 5d). To note, the retention by the 100 kDa cutoff filter is consistent with the oligomers being the size of a tetramer and larger.

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