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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.


Related in: MedlinePlus

ARA depletion experiments and comparative cell assays.(a) Numbers of oligomers, detected by sm-FRET before and after decreasing the concentration of ARA by washing with excess buffer (see Methods) (n = 3, std). (b) The increase in the smallest oligomers (2-5-mers) is observed, whereas the fraction of larger species drops, indicating that oligomers undergo a partial dissociation upon separation from the acid. (c) CD spectrum acquired after washing the protein sample, showing that the alpha-helical conformation is preserved. The detection of intact small multimers and alpha-helical conformation indicate that ARA is still present in solution and bound to αS in these multimers. Thus, it is very difficult to fully separate the FA from αS under these conditions. (d) Cytoplasmic ROS production by monitoring the rate of the ratio of the oxidised to reduced form of dihydroethidium (n = 50–90 cells, sem). Application of αS oligomers (500 nM of total αS) lead to a significant increase in ROS production (222 ± 12.95% compared to 100% basal, n =  88 cells, P < 0.01 relative to basal level). Application of ARA-induced oligomers (500 nM of total αS) showed small increase in ROS generation (134 ± 7.78%, n = 73 cells, P < 0.05 relative to basal level). Application of washed ARA-induced oligomers after ARA depletion by centrifugation (500 nM of total αS) or application of ARA alone (14.2 μM ARA) produced close to basal levels of ROS. (e) Percentage of cell-death as measured by Hoechst/propidium iodide staining after overnight incubation with the αS-only or ARA-induced oligomers, or ARA (n = 6–9 fields of view, sem). αS-only oligomers caused an increased cell death (7.54 ± 1.57%, n(cells)  =  693, P < 0.05 relative to untreated group), while ARA-induced oligomers washed from excess ARA lead to basal levels of cell death (1.3 ± 0.52%, n  =  7 fields of view, n(cells) = 509). (f) Pro-inflammatory response measured by the production of THF-α in BV2 microglia after a 24-h incubation of the cells after treatment with αS-only oligomers, ARA-induced oligomers and ARA alone, added at a range of concentrations between 0.05–200 μM (n = 4, sem).
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f4: ARA depletion experiments and comparative cell assays.(a) Numbers of oligomers, detected by sm-FRET before and after decreasing the concentration of ARA by washing with excess buffer (see Methods) (n = 3, std). (b) The increase in the smallest oligomers (2-5-mers) is observed, whereas the fraction of larger species drops, indicating that oligomers undergo a partial dissociation upon separation from the acid. (c) CD spectrum acquired after washing the protein sample, showing that the alpha-helical conformation is preserved. The detection of intact small multimers and alpha-helical conformation indicate that ARA is still present in solution and bound to αS in these multimers. Thus, it is very difficult to fully separate the FA from αS under these conditions. (d) Cytoplasmic ROS production by monitoring the rate of the ratio of the oxidised to reduced form of dihydroethidium (n = 50–90 cells, sem). Application of αS oligomers (500 nM of total αS) lead to a significant increase in ROS production (222 ± 12.95% compared to 100% basal, n =  88 cells, P < 0.01 relative to basal level). Application of ARA-induced oligomers (500 nM of total αS) showed small increase in ROS generation (134 ± 7.78%, n = 73 cells, P < 0.05 relative to basal level). Application of washed ARA-induced oligomers after ARA depletion by centrifugation (500 nM of total αS) or application of ARA alone (14.2 μM ARA) produced close to basal levels of ROS. (e) Percentage of cell-death as measured by Hoechst/propidium iodide staining after overnight incubation with the αS-only or ARA-induced oligomers, or ARA (n = 6–9 fields of view, sem). αS-only oligomers caused an increased cell death (7.54 ± 1.57%, n(cells)  =  693, P < 0.05 relative to untreated group), while ARA-induced oligomers washed from excess ARA lead to basal levels of cell death (1.3 ± 0.52%, n  =  7 fields of view, n(cells) = 509). (f) Pro-inflammatory response measured by the production of THF-α in BV2 microglia after a 24-h incubation of the cells after treatment with αS-only oligomers, ARA-induced oligomers and ARA alone, added at a range of concentrations between 0.05–200 μM (n = 4, sem).

Mentions: Lastly, we investigated whether ARA itself was a constituent of the ARA-induced oligomers. Due to the observed tendency to assemble into large agglomerates (Fig. 2b), it was presumed that the association of ARA-induced oligomers into larger aggregates could occur via the free fatty acid molecules, and we set out to test whether these species could remain stable upon decreasing the concentration of ARA in solution. To address this, ARA- and αS-containing samples were prepared as described above, and the concentration of the acid was decreased by washing with the excess of aqueous buffer and by subsequently concentrating the protein solutions, as described in Methods. This resulted in the increase in the numbers of recovered oligomers (Fig. 4a), as well as an 11 ± 5% increase in the population of small species consisting of less than 6 apparent monomer units, and a drop in the sub-population of larger oligomers (Fig. 4b). This indicated that the multimers had undergone a partial dissociation during the process (Fig. 4b), suggesting that the excess of fatty acid molecules acts to stabilize the larger multimers. Nevertheless, the finding that the majority of the aggregates could be recovered and remained sufficiently stable to be detected at picomolar concentrations of the protein in the sm-FRET experiments implies a degree of stability, and indicates a strong binding of ARA to αS in these aggregates. These observations are compatible with the previous reports of the stability of the FA-induced multimers of αS upon chromatographic procedures2329, and highlight a challenge of removing FAs from αS under these conditions. The result that the oligomers partially dissociated upon the decrease of FA suggests that ARA is a stabilizing constituent of these aggregates, which is consistent with previous related findings that αS co-aggregated with anionic lipids43. Consistent with this, the CD spectrum of αS solution, recorded after decreasing the concentration of ARA, indicated that the alpha-helical conformation was preserved in the samples (Fig. 4c).


Arachidonic acid mediates the formation of abundant alpha-helical multimers of alpha-synuclein
ARA depletion experiments and comparative cell assays.(a) Numbers of oligomers, detected by sm-FRET before and after decreasing the concentration of ARA by washing with excess buffer (see Methods) (n = 3, std). (b) The increase in the smallest oligomers (2-5-mers) is observed, whereas the fraction of larger species drops, indicating that oligomers undergo a partial dissociation upon separation from the acid. (c) CD spectrum acquired after washing the protein sample, showing that the alpha-helical conformation is preserved. The detection of intact small multimers and alpha-helical conformation indicate that ARA is still present in solution and bound to αS in these multimers. Thus, it is very difficult to fully separate the FA from αS under these conditions. (d) Cytoplasmic ROS production by monitoring the rate of the ratio of the oxidised to reduced form of dihydroethidium (n = 50–90 cells, sem). Application of αS oligomers (500 nM of total αS) lead to a significant increase in ROS production (222 ± 12.95% compared to 100% basal, n =  88 cells, P < 0.01 relative to basal level). Application of ARA-induced oligomers (500 nM of total αS) showed small increase in ROS generation (134 ± 7.78%, n = 73 cells, P < 0.05 relative to basal level). Application of washed ARA-induced oligomers after ARA depletion by centrifugation (500 nM of total αS) or application of ARA alone (14.2 μM ARA) produced close to basal levels of ROS. (e) Percentage of cell-death as measured by Hoechst/propidium iodide staining after overnight incubation with the αS-only or ARA-induced oligomers, or ARA (n = 6–9 fields of view, sem). αS-only oligomers caused an increased cell death (7.54 ± 1.57%, n(cells)  =  693, P < 0.05 relative to untreated group), while ARA-induced oligomers washed from excess ARA lead to basal levels of cell death (1.3 ± 0.52%, n  =  7 fields of view, n(cells) = 509). (f) Pro-inflammatory response measured by the production of THF-α in BV2 microglia after a 24-h incubation of the cells after treatment with αS-only oligomers, ARA-induced oligomers and ARA alone, added at a range of concentrations between 0.05–200 μM (n = 4, sem).
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f4: ARA depletion experiments and comparative cell assays.(a) Numbers of oligomers, detected by sm-FRET before and after decreasing the concentration of ARA by washing with excess buffer (see Methods) (n = 3, std). (b) The increase in the smallest oligomers (2-5-mers) is observed, whereas the fraction of larger species drops, indicating that oligomers undergo a partial dissociation upon separation from the acid. (c) CD spectrum acquired after washing the protein sample, showing that the alpha-helical conformation is preserved. The detection of intact small multimers and alpha-helical conformation indicate that ARA is still present in solution and bound to αS in these multimers. Thus, it is very difficult to fully separate the FA from αS under these conditions. (d) Cytoplasmic ROS production by monitoring the rate of the ratio of the oxidised to reduced form of dihydroethidium (n = 50–90 cells, sem). Application of αS oligomers (500 nM of total αS) lead to a significant increase in ROS production (222 ± 12.95% compared to 100% basal, n =  88 cells, P < 0.01 relative to basal level). Application of ARA-induced oligomers (500 nM of total αS) showed small increase in ROS generation (134 ± 7.78%, n = 73 cells, P < 0.05 relative to basal level). Application of washed ARA-induced oligomers after ARA depletion by centrifugation (500 nM of total αS) or application of ARA alone (14.2 μM ARA) produced close to basal levels of ROS. (e) Percentage of cell-death as measured by Hoechst/propidium iodide staining after overnight incubation with the αS-only or ARA-induced oligomers, or ARA (n = 6–9 fields of view, sem). αS-only oligomers caused an increased cell death (7.54 ± 1.57%, n(cells)  =  693, P < 0.05 relative to untreated group), while ARA-induced oligomers washed from excess ARA lead to basal levels of cell death (1.3 ± 0.52%, n  =  7 fields of view, n(cells) = 509). (f) Pro-inflammatory response measured by the production of THF-α in BV2 microglia after a 24-h incubation of the cells after treatment with αS-only oligomers, ARA-induced oligomers and ARA alone, added at a range of concentrations between 0.05–200 μM (n = 4, sem).
Mentions: Lastly, we investigated whether ARA itself was a constituent of the ARA-induced oligomers. Due to the observed tendency to assemble into large agglomerates (Fig. 2b), it was presumed that the association of ARA-induced oligomers into larger aggregates could occur via the free fatty acid molecules, and we set out to test whether these species could remain stable upon decreasing the concentration of ARA in solution. To address this, ARA- and αS-containing samples were prepared as described above, and the concentration of the acid was decreased by washing with the excess of aqueous buffer and by subsequently concentrating the protein solutions, as described in Methods. This resulted in the increase in the numbers of recovered oligomers (Fig. 4a), as well as an 11 ± 5% increase in the population of small species consisting of less than 6 apparent monomer units, and a drop in the sub-population of larger oligomers (Fig. 4b). This indicated that the multimers had undergone a partial dissociation during the process (Fig. 4b), suggesting that the excess of fatty acid molecules acts to stabilize the larger multimers. Nevertheless, the finding that the majority of the aggregates could be recovered and remained sufficiently stable to be detected at picomolar concentrations of the protein in the sm-FRET experiments implies a degree of stability, and indicates a strong binding of ARA to αS in these aggregates. These observations are compatible with the previous reports of the stability of the FA-induced multimers of αS upon chromatographic procedures2329, and highlight a challenge of removing FAs from αS under these conditions. The result that the oligomers partially dissociated upon the decrease of FA suggests that ARA is a stabilizing constituent of these aggregates, which is consistent with previous related findings that αS co-aggregated with anionic lipids43. Consistent with this, the CD spectrum of αS solution, recorded after decreasing the concentration of ARA, indicated that the alpha-helical conformation was preserved in the samples (Fig. 4c).

View Article: PubMed Central - PubMed

ABSTRACT

The protein alpha-synuclein (&alpha;S) self-assembles into toxic beta-sheet aggregates in Parkinson&rsquo;s disease, while it is proposed that &alpha;S forms soluble alpha-helical multimers in healthy neurons. Here, we have made &alpha;S 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 F&#1255;rster 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 &alpha;S form less multimers than wild-type &alpha;S. Our work provides strong biophysical evidence for the formation of alpha-helical multimers of &alpha;S 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 &alpha;S fibrillation.

No MeSH data available.


Related in: MedlinePlus