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Synergistic influence of phosphorylation and metal ions on tau oligomer formation and coaggregation with α-synuclein at the single molecule level.

Nübling G, Bader B, Levin J, Hildebrandt J, Kretzschmar H, Giese A - Mol Neurodegener (2012)

Bottom Line: While tau fibril formation is well-characterized, factors influencing tau oligomerization and molecular interactions of tau and α-synuclein are not well understood.We used a novel approach applying confocal single-particle fluorescence to investigate the influence of tau phosphorylation and metal ions on tau oligomer formation and its coaggregation with α-synuclein at the level of individual oligomers.Moreover, tau phosphorylation and Al3+ as well as Fe3+ enhanced both formation of mixed oligomers and recruitment of α-synuclein in pre-formed tau oligomers.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Neuropathology and Prion Research, Ludwig-Maximilians-Universität, Feodor-Lynen-Str, 23, 81377, Munich, Germany.

ABSTRACT

Background: Fibrillar amyloid-like deposits and co-deposits of tau and α-synuclein are found in several common neurodegenerative diseases. Recent evidence indicates that small oligomers are the most relevant toxic aggregate species. While tau fibril formation is well-characterized, factors influencing tau oligomerization and molecular interactions of tau and α-synuclein are not well understood.

Results: We used a novel approach applying confocal single-particle fluorescence to investigate the influence of tau phosphorylation and metal ions on tau oligomer formation and its coaggregation with α-synuclein at the level of individual oligomers. We show that Al3+ at physiologically relevant concentrations and tau phosphorylation by GSK-3β exert synergistic effects on the formation of a distinct SDS-resistant tau oligomer species even at nanomolar protein concentration. Moreover, tau phosphorylation and Al3+ as well as Fe3+ enhanced both formation of mixed oligomers and recruitment of α-synuclein in pre-formed tau oligomers.

Conclusions: Our findings provide a new perspective on interactions of tau phosphorylation, metal ions, and the formation of potentially toxic oligomer species, and elucidate molecular crosstalks between different aggregation pathways involved in neurodegeneration.

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Al3+promotes stronger formation of SDS stable tau and α-syn coaggregates than Fe3+, preferably after tau phosphorylation.A. Cross-correlation amplitudes (G (0)) show mild proaggregatory activity of DMSO and Fe3+, while Al3+ promotes intense protein oligomerization. Combinations of DMSO and metal ions show a synergistic effect especially for mTau. 2D histograms show the formation of smaller oligomers induced by DMSO compared to Al3+. B. Cross-correlation amplitude before (T0) and 100 min after (T100) addition of SDS to the aggregation assay. Significant reductions of aggregation levels at T100 are indicated by the symbols over the error bars, while significant differences between pTau and mTau are indicated by the symbols over the wide bars. C. Cross correlation amplitude at T100 normalized against T0 shows increased SDS-resistance of metal ion induced oligomers compared to DMSO. D. Scheme illustrating the appearance of tau and α-syn aggregates and coaggregates of the two proteins. Higher fluorescence intensities of detected oligomers indicate increased oligomer size. Measurements were taken from 16 independent samples; each sample was measured four times. Levels of significance are displayed as * = p < 0.05; ** = p < 0.01; ‡ = p < 0.001.
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Figure 3: Al3+promotes stronger formation of SDS stable tau and α-syn coaggregates than Fe3+, preferably after tau phosphorylation.A. Cross-correlation amplitudes (G (0)) show mild proaggregatory activity of DMSO and Fe3+, while Al3+ promotes intense protein oligomerization. Combinations of DMSO and metal ions show a synergistic effect especially for mTau. 2D histograms show the formation of smaller oligomers induced by DMSO compared to Al3+. B. Cross-correlation amplitude before (T0) and 100 min after (T100) addition of SDS to the aggregation assay. Significant reductions of aggregation levels at T100 are indicated by the symbols over the error bars, while significant differences between pTau and mTau are indicated by the symbols over the wide bars. C. Cross correlation amplitude at T100 normalized against T0 shows increased SDS-resistance of metal ion induced oligomers compared to DMSO. D. Scheme illustrating the appearance of tau and α-syn aggregates and coaggregates of the two proteins. Higher fluorescence intensities of detected oligomers indicate increased oligomer size. Measurements were taken from 16 independent samples; each sample was measured four times. Levels of significance are displayed as * = p < 0.05; ** = p < 0.01; ‡ = p < 0.001.

Mentions: In these experiments, 1% DMSO promoted the coaggregation of tau and α-syn in a similar manner for pTau and mTau (Figure 3A). On average, coaggregates were composed of 11 pTau + 8 α-syn monomers or 15 mTau + 12 α-syn monomers, respectively, as determined by FIDA analysis. Fe3+ also had a promotional influence on coaggregation, which was higher for pTau (p < 0.01, on average 53 pTau + 10 α-syn and 39 mTau + 3 α-syn monomers per oligomer). Upon combining DMSO and Fe3+, an additional effect exceeding the influence of one inducer alone can be seen (also see Additional files 4 and 5), while no significant difference in aggregation levels of pTau and mTau was detectable. Similar to our tau oligomerization experiments, Al3+ was the strongest inducer of tau and α-syn coaggregation, with pTau showing higher coaggregation activity than mTau (p < 0.001, Figure 3A; on average 131 pTau + 115 α-syn and 137 mTau + 80 α-syn monomers per oligomer). This difference was still detectable when combining Al3+ with DMSO, though to a lesser extent (p < 0.05). Mixed oligomers comprising both tau and α-syn appear scattered around the bisectrix of these histograms, while homogeneous aggregates are located close to the axes, as illustrated in Figure 3D.


Synergistic influence of phosphorylation and metal ions on tau oligomer formation and coaggregation with α-synuclein at the single molecule level.

Nübling G, Bader B, Levin J, Hildebrandt J, Kretzschmar H, Giese A - Mol Neurodegener (2012)

Al3+promotes stronger formation of SDS stable tau and α-syn coaggregates than Fe3+, preferably after tau phosphorylation.A. Cross-correlation amplitudes (G (0)) show mild proaggregatory activity of DMSO and Fe3+, while Al3+ promotes intense protein oligomerization. Combinations of DMSO and metal ions show a synergistic effect especially for mTau. 2D histograms show the formation of smaller oligomers induced by DMSO compared to Al3+. B. Cross-correlation amplitude before (T0) and 100 min after (T100) addition of SDS to the aggregation assay. Significant reductions of aggregation levels at T100 are indicated by the symbols over the error bars, while significant differences between pTau and mTau are indicated by the symbols over the wide bars. C. Cross correlation amplitude at T100 normalized against T0 shows increased SDS-resistance of metal ion induced oligomers compared to DMSO. D. Scheme illustrating the appearance of tau and α-syn aggregates and coaggregates of the two proteins. Higher fluorescence intensities of detected oligomers indicate increased oligomer size. Measurements were taken from 16 independent samples; each sample was measured four times. Levels of significance are displayed as * = p < 0.05; ** = p < 0.01; ‡ = p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472288&req=5

Figure 3: Al3+promotes stronger formation of SDS stable tau and α-syn coaggregates than Fe3+, preferably after tau phosphorylation.A. Cross-correlation amplitudes (G (0)) show mild proaggregatory activity of DMSO and Fe3+, while Al3+ promotes intense protein oligomerization. Combinations of DMSO and metal ions show a synergistic effect especially for mTau. 2D histograms show the formation of smaller oligomers induced by DMSO compared to Al3+. B. Cross-correlation amplitude before (T0) and 100 min after (T100) addition of SDS to the aggregation assay. Significant reductions of aggregation levels at T100 are indicated by the symbols over the error bars, while significant differences between pTau and mTau are indicated by the symbols over the wide bars. C. Cross correlation amplitude at T100 normalized against T0 shows increased SDS-resistance of metal ion induced oligomers compared to DMSO. D. Scheme illustrating the appearance of tau and α-syn aggregates and coaggregates of the two proteins. Higher fluorescence intensities of detected oligomers indicate increased oligomer size. Measurements were taken from 16 independent samples; each sample was measured four times. Levels of significance are displayed as * = p < 0.05; ** = p < 0.01; ‡ = p < 0.001.
Mentions: In these experiments, 1% DMSO promoted the coaggregation of tau and α-syn in a similar manner for pTau and mTau (Figure 3A). On average, coaggregates were composed of 11 pTau + 8 α-syn monomers or 15 mTau + 12 α-syn monomers, respectively, as determined by FIDA analysis. Fe3+ also had a promotional influence on coaggregation, which was higher for pTau (p < 0.01, on average 53 pTau + 10 α-syn and 39 mTau + 3 α-syn monomers per oligomer). Upon combining DMSO and Fe3+, an additional effect exceeding the influence of one inducer alone can be seen (also see Additional files 4 and 5), while no significant difference in aggregation levels of pTau and mTau was detectable. Similar to our tau oligomerization experiments, Al3+ was the strongest inducer of tau and α-syn coaggregation, with pTau showing higher coaggregation activity than mTau (p < 0.001, Figure 3A; on average 131 pTau + 115 α-syn and 137 mTau + 80 α-syn monomers per oligomer). This difference was still detectable when combining Al3+ with DMSO, though to a lesser extent (p < 0.05). Mixed oligomers comprising both tau and α-syn appear scattered around the bisectrix of these histograms, while homogeneous aggregates are located close to the axes, as illustrated in Figure 3D.

Bottom Line: While tau fibril formation is well-characterized, factors influencing tau oligomerization and molecular interactions of tau and α-synuclein are not well understood.We used a novel approach applying confocal single-particle fluorescence to investigate the influence of tau phosphorylation and metal ions on tau oligomer formation and its coaggregation with α-synuclein at the level of individual oligomers.Moreover, tau phosphorylation and Al3+ as well as Fe3+ enhanced both formation of mixed oligomers and recruitment of α-synuclein in pre-formed tau oligomers.

View Article: PubMed Central - HTML - PubMed

Affiliation: Center for Neuropathology and Prion Research, Ludwig-Maximilians-Universität, Feodor-Lynen-Str, 23, 81377, Munich, Germany.

ABSTRACT

Background: Fibrillar amyloid-like deposits and co-deposits of tau and α-synuclein are found in several common neurodegenerative diseases. Recent evidence indicates that small oligomers are the most relevant toxic aggregate species. While tau fibril formation is well-characterized, factors influencing tau oligomerization and molecular interactions of tau and α-synuclein are not well understood.

Results: We used a novel approach applying confocal single-particle fluorescence to investigate the influence of tau phosphorylation and metal ions on tau oligomer formation and its coaggregation with α-synuclein at the level of individual oligomers. We show that Al3+ at physiologically relevant concentrations and tau phosphorylation by GSK-3β exert synergistic effects on the formation of a distinct SDS-resistant tau oligomer species even at nanomolar protein concentration. Moreover, tau phosphorylation and Al3+ as well as Fe3+ enhanced both formation of mixed oligomers and recruitment of α-synuclein in pre-formed tau oligomers.

Conclusions: Our findings provide a new perspective on interactions of tau phosphorylation, metal ions, and the formation of potentially toxic oligomer species, and elucidate molecular crosstalks between different aggregation pathways involved in neurodegeneration.

Show MeSH
Related in: MedlinePlus