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RNA Binds to Tau Fibrils and Sustains Template-Assisted Growth.

Dinkel PD, Holden MR, Matin N, Margittai M - Biochemistry (2015)

Bottom Line: These structural features are similar to those previously observed for heparin-induced fibrils, indicating that basic conformational properties are conserved, despite their being molecular differences of the nucleating agents.Furthermore, RNA sustains template-assisted growth and binds to the fibril surface and can be exchanged by heparin.These findings suggest that, in addition to mediating fibrillization, cofactors decorating the surface of Tau fibrils may modulate biological interactions and thereby influence the spreading of Tau pathology in the human brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado 80208, United States.

ABSTRACT
Tau fibrils are the main proteinacious components of neurofibrillary lesions in Alzheimer disease. Although RNA molecules are sequestered into these lesions, their relationship to Tau fibrils is only poorly understood. Such understanding, however, is important, as short fibrils can transfer between neurons and nonproteinacious factors including RNA could play a defining role in modulating the latter process. Here, we used sedimentation assays combined with electron paramagnetic resonance (EPR), fluorescence, and absorbance spectroscopy to determine the effects of RNA on Tau fibril structure and growth. We observe that, in the presence of RNA, three-repeat (3R) and four-repeat (4R) Tau form fibrils with parallel, in-register arrangement of β-strands and exhibit an asymmetric seeding barrier in which 4R Tau grows onto 3R Tau seeds but not vice versa. These structural features are similar to those previously observed for heparin-induced fibrils, indicating that basic conformational properties are conserved, despite their being molecular differences of the nucleating agents. Furthermore, RNA sustains template-assisted growth and binds to the fibril surface and can be exchanged by heparin. These findings suggest that, in addition to mediating fibrillization, cofactors decorating the surface of Tau fibrils may modulate biological interactions and thereby influence the spreading of Tau pathology in the human brain.

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Heparin replaces RNA on Tau fibrils. K18and K19 fibrils (25 μMmonomer added to 10% seeds) were grown in the presence of RNA (50μg/mL) and subsequently incubated for 24 h with fluorescein-conjugatedheparin (2.5 μM). (A, B) Normalized absorbance at 260 nm forsolubilized K18 and K19 pellets and supernatants. (C, D) Normalizedabsorbance at 490 nm for solubilized K18 and K19 pellets and supernatants.(E, F) Fluorescence anisotropy changes upon association of heparinwith K18/RNA and K19/RNA fibrils. (G, H) Fluorescence anisotropy changesupon association of heparin with K18/RNA and K19/RNA monomers. Tauconcentration, 6 μM; heparin concentration, 40 nM; excitation,480 nm; emission, 516 nm. Arrows mark additions of Tau fibrils orTau monomers to fluorescein-conjugated heparin. The large changesin fluorescence anisotropy observed in E and F imply that heparinbinds to Tau fibrils.
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fig6: Heparin replaces RNA on Tau fibrils. K18and K19 fibrils (25 μMmonomer added to 10% seeds) were grown in the presence of RNA (50μg/mL) and subsequently incubated for 24 h with fluorescein-conjugatedheparin (2.5 μM). (A, B) Normalized absorbance at 260 nm forsolubilized K18 and K19 pellets and supernatants. (C, D) Normalizedabsorbance at 490 nm for solubilized K18 and K19 pellets and supernatants.(E, F) Fluorescence anisotropy changes upon association of heparinwith K18/RNA and K19/RNA fibrils. (G, H) Fluorescence anisotropy changesupon association of heparin with K18/RNA and K19/RNA monomers. Tauconcentration, 6 μM; heparin concentration, 40 nM; excitation,480 nm; emission, 516 nm. Arrows mark additions of Tau fibrils orTau monomers to fluorescein-conjugated heparin. The large changesin fluorescence anisotropy observed in E and F imply that heparinbinds to Tau fibrils.

Mentions: Since heparin does notabsorb in the visible spectrum, it remainedunclear whether heparin had replaced RNA on the fibril. To addressthis question, we added fluorescein-conjugated heparin to Tau/RNAfibrils and incubated the samples for 24 h at 37 °C. As before,pellets and supernatants were adjusted to the same volume containing2% SDS (which solubilizes the fibrils). The absorbance at 260 nm revealedthat the majority of RNA was released from K18 and K19 fibrils (Figure 6A,B). The resultsare similar to those obtained for nonconjugated heparin (Figure 5A,D), suggestingthat the fluorescein modification and the change in average molecularweight (see Materials and Methods) did notaffect the ability of heparin to release RNA from the Tau fibril cores.Notice that, again, the release of RNA from K19 fibrils was less efficientthan that from K18 fibrils. This could reflect differences in RNAbinding between the conformationally distinct fibrils. Specifically,some of the RNA binding sites in K19 fibrils might be in the interiorrather than on the surface. Conjugated heparin was detected by measuringthe absorbance at 490 nm. Importantly, the majority of heparin (∼80%)associated with the K18 and K19 fibrils (Figure 6C,D). In both cases, the molar ratio of heparinto Tau was approximately 1:13.


RNA Binds to Tau Fibrils and Sustains Template-Assisted Growth.

Dinkel PD, Holden MR, Matin N, Margittai M - Biochemistry (2015)

Heparin replaces RNA on Tau fibrils. K18and K19 fibrils (25 μMmonomer added to 10% seeds) were grown in the presence of RNA (50μg/mL) and subsequently incubated for 24 h with fluorescein-conjugatedheparin (2.5 μM). (A, B) Normalized absorbance at 260 nm forsolubilized K18 and K19 pellets and supernatants. (C, D) Normalizedabsorbance at 490 nm for solubilized K18 and K19 pellets and supernatants.(E, F) Fluorescence anisotropy changes upon association of heparinwith K18/RNA and K19/RNA fibrils. (G, H) Fluorescence anisotropy changesupon association of heparin with K18/RNA and K19/RNA monomers. Tauconcentration, 6 μM; heparin concentration, 40 nM; excitation,480 nm; emission, 516 nm. Arrows mark additions of Tau fibrils orTau monomers to fluorescein-conjugated heparin. The large changesin fluorescence anisotropy observed in E and F imply that heparinbinds to Tau fibrils.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4526887&req=5

fig6: Heparin replaces RNA on Tau fibrils. K18and K19 fibrils (25 μMmonomer added to 10% seeds) were grown in the presence of RNA (50μg/mL) and subsequently incubated for 24 h with fluorescein-conjugatedheparin (2.5 μM). (A, B) Normalized absorbance at 260 nm forsolubilized K18 and K19 pellets and supernatants. (C, D) Normalizedabsorbance at 490 nm for solubilized K18 and K19 pellets and supernatants.(E, F) Fluorescence anisotropy changes upon association of heparinwith K18/RNA and K19/RNA fibrils. (G, H) Fluorescence anisotropy changesupon association of heparin with K18/RNA and K19/RNA monomers. Tauconcentration, 6 μM; heparin concentration, 40 nM; excitation,480 nm; emission, 516 nm. Arrows mark additions of Tau fibrils orTau monomers to fluorescein-conjugated heparin. The large changesin fluorescence anisotropy observed in E and F imply that heparinbinds to Tau fibrils.
Mentions: Since heparin does notabsorb in the visible spectrum, it remainedunclear whether heparin had replaced RNA on the fibril. To addressthis question, we added fluorescein-conjugated heparin to Tau/RNAfibrils and incubated the samples for 24 h at 37 °C. As before,pellets and supernatants were adjusted to the same volume containing2% SDS (which solubilizes the fibrils). The absorbance at 260 nm revealedthat the majority of RNA was released from K18 and K19 fibrils (Figure 6A,B). The resultsare similar to those obtained for nonconjugated heparin (Figure 5A,D), suggestingthat the fluorescein modification and the change in average molecularweight (see Materials and Methods) did notaffect the ability of heparin to release RNA from the Tau fibril cores.Notice that, again, the release of RNA from K19 fibrils was less efficientthan that from K18 fibrils. This could reflect differences in RNAbinding between the conformationally distinct fibrils. Specifically,some of the RNA binding sites in K19 fibrils might be in the interiorrather than on the surface. Conjugated heparin was detected by measuringthe absorbance at 490 nm. Importantly, the majority of heparin (∼80%)associated with the K18 and K19 fibrils (Figure 6C,D). In both cases, the molar ratio of heparinto Tau was approximately 1:13.

Bottom Line: These structural features are similar to those previously observed for heparin-induced fibrils, indicating that basic conformational properties are conserved, despite their being molecular differences of the nucleating agents.Furthermore, RNA sustains template-assisted growth and binds to the fibril surface and can be exchanged by heparin.These findings suggest that, in addition to mediating fibrillization, cofactors decorating the surface of Tau fibrils may modulate biological interactions and thereby influence the spreading of Tau pathology in the human brain.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of Denver, Denver, Colorado 80208, United States.

ABSTRACT
Tau fibrils are the main proteinacious components of neurofibrillary lesions in Alzheimer disease. Although RNA molecules are sequestered into these lesions, their relationship to Tau fibrils is only poorly understood. Such understanding, however, is important, as short fibrils can transfer between neurons and nonproteinacious factors including RNA could play a defining role in modulating the latter process. Here, we used sedimentation assays combined with electron paramagnetic resonance (EPR), fluorescence, and absorbance spectroscopy to determine the effects of RNA on Tau fibril structure and growth. We observe that, in the presence of RNA, three-repeat (3R) and four-repeat (4R) Tau form fibrils with parallel, in-register arrangement of β-strands and exhibit an asymmetric seeding barrier in which 4R Tau grows onto 3R Tau seeds but not vice versa. These structural features are similar to those previously observed for heparin-induced fibrils, indicating that basic conformational properties are conserved, despite their being molecular differences of the nucleating agents. Furthermore, RNA sustains template-assisted growth and binds to the fibril surface and can be exchanged by heparin. These findings suggest that, in addition to mediating fibrillization, cofactors decorating the surface of Tau fibrils may modulate biological interactions and thereby influence the spreading of Tau pathology in the human brain.

Show MeSH
Related in: MedlinePlus