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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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Related in: MedlinePlus

Tau phosphorylation verified by western blot and SIFT analysis.A. Mass spectroscopy showed that recombinant human tau (isoform 5, 42967 Da) is of high purity. B. While mock phosphorylation (mTau) does not influence SDS-PAGE mobility of recombinant tau, a typical band shift is observed upon tau phosphorylation (pTau). C. SIFT analysis showed labeling of pTau oligomers with the phosphorylated tau specific AT-8 antibody in presence of 1% DMSO indicating antibody binding, while no coaggregation was observed with mTau. Data was normalized against the coaggregation level of mTau with the T46 antibody, which does not require tau phosphorylation. 2D histograms depicting antibody (x-axis) and protein (y-axis) interactions show coaggregates of T46 with both pTau and mTau, while AT-8 only coaggregates with pTau. A scheme describing the appearance of mixed aggregates in 2D histograms is included in Figure 3. Upon combining AT-8 and mTau, only DMSO induced tau aggregates are visible along the y-axis, similar to the control antibody 6E10. Measurements were taken from 12 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 1: Tau phosphorylation verified by western blot and SIFT analysis.A. Mass spectroscopy showed that recombinant human tau (isoform 5, 42967 Da) is of high purity. B. While mock phosphorylation (mTau) does not influence SDS-PAGE mobility of recombinant tau, a typical band shift is observed upon tau phosphorylation (pTau). C. SIFT analysis showed labeling of pTau oligomers with the phosphorylated tau specific AT-8 antibody in presence of 1% DMSO indicating antibody binding, while no coaggregation was observed with mTau. Data was normalized against the coaggregation level of mTau with the T46 antibody, which does not require tau phosphorylation. 2D histograms depicting antibody (x-axis) and protein (y-axis) interactions show coaggregates of T46 with both pTau and mTau, while AT-8 only coaggregates with pTau. A scheme describing the appearance of mixed aggregates in 2D histograms is included in Figure 3. Upon combining AT-8 and mTau, only DMSO induced tau aggregates are visible along the y-axis, similar to the control antibody 6E10. Measurements were taken from 12 independent samples; each sample was measured four times. Levels of significance are displayed as * = p < 0.05; ** = p < 0.01; ‡ = p < 0.001.

Mentions: Mass spectroscopy showed that recombinant protein tau is of high purity and does not contain significant amounts of cleavage products (Figure 1A). Notably, fluorescence labeling of tau with Alexa dyes did not alter the protein’s electrophoretic mobility, though mass spectroscopy showed an increase in tau’s molecular weight after labeling (data not shown). We confirmed in vitro phosphorylation of human recombinant tau by demonstrating a typical band shift of the protein in western blot (see Figure 1B) [17]. We further evaluated whether the SIFT method can be employed to detect phosphorylated protein tau by labeling with Alexa-488 tagged antibodies AT-8 and T46. While T46 detects both phosphorylated and unphosphorylated tau, the AT-8 antibody requires the protein to be phosphorylated at specific sites (Goedert et al, 1995). The Aβ specific antibody 6E10 was used as a negative control. SIFT analysis shows that both T46488 and AT-8488, but not 6E10488 bind to phosphorylated tau647 (pTau, Figure 1C), while only T46488 binds to mock phosphorylated tau647 (mTau, also see materials and methods section).


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)

Tau phosphorylation verified by western blot and SIFT analysis.A. Mass spectroscopy showed that recombinant human tau (isoform 5, 42967 Da) is of high purity. B. While mock phosphorylation (mTau) does not influence SDS-PAGE mobility of recombinant tau, a typical band shift is observed upon tau phosphorylation (pTau). C. SIFT analysis showed labeling of pTau oligomers with the phosphorylated tau specific AT-8 antibody in presence of 1% DMSO indicating antibody binding, while no coaggregation was observed with mTau. Data was normalized against the coaggregation level of mTau with the T46 antibody, which does not require tau phosphorylation. 2D histograms depicting antibody (x-axis) and protein (y-axis) interactions show coaggregates of T46 with both pTau and mTau, while AT-8 only coaggregates with pTau. A scheme describing the appearance of mixed aggregates in 2D histograms is included in Figure 3. Upon combining AT-8 and mTau, only DMSO induced tau aggregates are visible along the y-axis, similar to the control antibody 6E10. Measurements were taken from 12 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 1: Tau phosphorylation verified by western blot and SIFT analysis.A. Mass spectroscopy showed that recombinant human tau (isoform 5, 42967 Da) is of high purity. B. While mock phosphorylation (mTau) does not influence SDS-PAGE mobility of recombinant tau, a typical band shift is observed upon tau phosphorylation (pTau). C. SIFT analysis showed labeling of pTau oligomers with the phosphorylated tau specific AT-8 antibody in presence of 1% DMSO indicating antibody binding, while no coaggregation was observed with mTau. Data was normalized against the coaggregation level of mTau with the T46 antibody, which does not require tau phosphorylation. 2D histograms depicting antibody (x-axis) and protein (y-axis) interactions show coaggregates of T46 with both pTau and mTau, while AT-8 only coaggregates with pTau. A scheme describing the appearance of mixed aggregates in 2D histograms is included in Figure 3. Upon combining AT-8 and mTau, only DMSO induced tau aggregates are visible along the y-axis, similar to the control antibody 6E10. Measurements were taken from 12 independent samples; each sample was measured four times. Levels of significance are displayed as * = p < 0.05; ** = p < 0.01; ‡ = p < 0.001.
Mentions: Mass spectroscopy showed that recombinant protein tau is of high purity and does not contain significant amounts of cleavage products (Figure 1A). Notably, fluorescence labeling of tau with Alexa dyes did not alter the protein’s electrophoretic mobility, though mass spectroscopy showed an increase in tau’s molecular weight after labeling (data not shown). We confirmed in vitro phosphorylation of human recombinant tau by demonstrating a typical band shift of the protein in western blot (see Figure 1B) [17]. We further evaluated whether the SIFT method can be employed to detect phosphorylated protein tau by labeling with Alexa-488 tagged antibodies AT-8 and T46. While T46 detects both phosphorylated and unphosphorylated tau, the AT-8 antibody requires the protein to be phosphorylated at specific sites (Goedert et al, 1995). The Aβ specific antibody 6E10 was used as a negative control. SIFT analysis shows that both T46488 and AT-8488, but not 6E10488 bind to phosphorylated tau647 (pTau, Figure 1C), while only T46488 binds to mock phosphorylated tau647 (mTau, also see materials and methods section).

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