Limits...
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

Schematic outline of the self-assembly mechanisms of αS in the absence and in the presence of ARA.(a) Without ARA, monomeric protein assembles according to the aberrant aggregation mechanism, as reported previously38. In this mechanism, αS forms disordered low-FRET oligomers, which convert to toxic and beta-sheet-rich high-FRET oligomers, which in turn convert into fibrils. (b) Upon the addition of ARA, the alpha-helical ARA-induced multimers are formed, comprising both αS and ARA. Equilibrium with monomer is consistent with the immediate ARA-induced oligomer formation, and equilibrium with low-FRET oligomers can account for the observed time-progression during first 6 hours of the reaction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5037366&req=5

f6: Schematic outline of the self-assembly mechanisms of αS in the absence and in the presence of ARA.(a) Without ARA, monomeric protein assembles according to the aberrant aggregation mechanism, as reported previously38. In this mechanism, αS forms disordered low-FRET oligomers, which convert to toxic and beta-sheet-rich high-FRET oligomers, which in turn convert into fibrils. (b) Upon the addition of ARA, the alpha-helical ARA-induced multimers are formed, comprising both αS and ARA. Equilibrium with monomer is consistent with the immediate ARA-induced oligomer formation, and equilibrium with low-FRET oligomers can account for the observed time-progression during first 6 hours of the reaction.

Mentions: The ARA-induced oligomers in our experiments were evidently distinct from the beta-sheet-rich oligomers formed during the aberrant aggregation of αS. Moreover, they were resistant to fibril formation, as judged from TEM imaging, and required ARA for their stabilization, as concluded from the ARA washing experiments. These species may therefore represent the products of an alternative reaction involving both αS and ARA, as is schematically illustrated in Fig. 6. Because their formation is fast and recruits monomeric αS, it can compete with the slow formation of high-FRET beta-sheet oligomers particularly when the total monomer concentration is low. We previously showed that the beta-sheet-rich oligomers of αS were the most cytotoxic species35 and caused damage to neuronal cells when present even at picomolar concentrations38, and reconfirmed their toxicity in the present study. Therefore, any competing mechanisms that inhibit their formation may be highly neuroprotective. This is consistent with the finding that disease-associated mutants of αS had a lower tendency to assemble into the FA-induced species compared to the wildtype αS, which leaves them in a free state and may ultimately lead to the generation of higher concentrations of toxic beta-sheet-rich oligomers via the aberrant aggregation mechanism. To note, this in vitro result is in striking agreement with what was shown for PD mutants of αS in intact neurons by both cell-penetrant crosslinking and fluorescent protein complementation48.


Arachidonic acid mediates the formation of abundant alpha-helical multimers of alpha-synuclein
Schematic outline of the self-assembly mechanisms of αS in the absence and in the presence of ARA.(a) Without ARA, monomeric protein assembles according to the aberrant aggregation mechanism, as reported previously38. In this mechanism, αS forms disordered low-FRET oligomers, which convert to toxic and beta-sheet-rich high-FRET oligomers, which in turn convert into fibrils. (b) Upon the addition of ARA, the alpha-helical ARA-induced multimers are formed, comprising both αS and ARA. Equilibrium with monomer is consistent with the immediate ARA-induced oligomer formation, and equilibrium with low-FRET oligomers can account for the observed time-progression during first 6 hours of the reaction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037366&req=5

f6: Schematic outline of the self-assembly mechanisms of αS in the absence and in the presence of ARA.(a) Without ARA, monomeric protein assembles according to the aberrant aggregation mechanism, as reported previously38. In this mechanism, αS forms disordered low-FRET oligomers, which convert to toxic and beta-sheet-rich high-FRET oligomers, which in turn convert into fibrils. (b) Upon the addition of ARA, the alpha-helical ARA-induced multimers are formed, comprising both αS and ARA. Equilibrium with monomer is consistent with the immediate ARA-induced oligomer formation, and equilibrium with low-FRET oligomers can account for the observed time-progression during first 6 hours of the reaction.
Mentions: The ARA-induced oligomers in our experiments were evidently distinct from the beta-sheet-rich oligomers formed during the aberrant aggregation of αS. Moreover, they were resistant to fibril formation, as judged from TEM imaging, and required ARA for their stabilization, as concluded from the ARA washing experiments. These species may therefore represent the products of an alternative reaction involving both αS and ARA, as is schematically illustrated in Fig. 6. Because their formation is fast and recruits monomeric αS, it can compete with the slow formation of high-FRET beta-sheet oligomers particularly when the total monomer concentration is low. We previously showed that the beta-sheet-rich oligomers of αS were the most cytotoxic species35 and caused damage to neuronal cells when present even at picomolar concentrations38, and reconfirmed their toxicity in the present study. Therefore, any competing mechanisms that inhibit their formation may be highly neuroprotective. This is consistent with the finding that disease-associated mutants of αS had a lower tendency to assemble into the FA-induced species compared to the wildtype αS, which leaves them in a free state and may ultimately lead to the generation of higher concentrations of toxic beta-sheet-rich oligomers via the aberrant aggregation mechanism. To note, this in vitro result is in striking agreement with what was shown for PD mutants of αS in intact neurons by both cell-penetrant crosslinking and fluorescent protein complementation48.

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