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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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Seeding properties of 3R and 4R Tau. Tau fibrilseeds (10% monomerequivalents) were mixed with monomers of K18 and K19 (98% cysteinefree, 2% acrylodan labeled at position 310). As the labeled Tau monomersincorporate into the fibrils, the emission maxima shift to the blue.Inverse emission maxima plotted against time depict fibril growth:(A) K18 seeded growth and (B) K19 seeded growth. Values representmean ± SD (n = 3 experiments). Tau concentration,10 μM; cofactor, 20 μg/mL polyA RNA; excitation, 360 nm.An asymmetric seeding barrier prevents 3R Tau from growing onto 4RTau seeds but not vice versa.
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fig2: Seeding properties of 3R and 4R Tau. Tau fibrilseeds (10% monomerequivalents) were mixed with monomers of K18 and K19 (98% cysteinefree, 2% acrylodan labeled at position 310). As the labeled Tau monomersincorporate into the fibrils, the emission maxima shift to the blue.Inverse emission maxima plotted against time depict fibril growth:(A) K18 seeded growth and (B) K19 seeded growth. Values representmean ± SD (n = 3 experiments). Tau concentration,10 μM; cofactor, 20 μg/mL polyA RNA; excitation, 360 nm.An asymmetric seeding barrier prevents 3R Tau from growing onto 4RTau seeds but not vice versa.

Mentions: Despite the structural commonalities,packing interactions between β-sheets could vary substantially,resulting in different overall conformations. Indeed, K18 and K19fibrils, both of which (in the presence of heparin) form parallel,in-register β-strands,35 are conformationallydistinct.45 In order to gain further insightsinto the structure of RNA-induced fibrils, we next investigated theirseeding properties. Since RNA interferes with the commonly used thioflavinassays of Tau fibril formation,46 we chosean acrylodan-based fluorescence assay to monitor seeded fibril growth.36 In this assay, acrylodan is attached to a singlecysteine of the protein and fibril assembly is observed through theblueshift in the fluorescence emission maximum that occurs when thelabel moves from an aqueous environment in the disordered monomerto a hydrophobic environment in the folded fibril. First, K18 andK19 monomers were individually labeled at position 310 and mixed withtheir respective cysteine-free counterparts (molar ratio of 1:50).These mixtures were then combined with 10 mol % seeds (monomer equivalents)of K18 and K19. Samples were excited at 360 nm, and inverse emissionmaxima were plotted as a function of time (Figure 2). While K18 monomers grow onto K18 seeds,K19 monomers do not (Figure 2A). K19 seeds, however, facilitate growth of both K19 andK18 monomers, although the latter monomers grow less efficiently (Figure 2B). The data indicatethat the previously observed asymmetric seeding barrier for heparin-mediatedfibril growth is also preserved for fibrils formed in the presenceof RNA. Furthermore, the data suggest that important structural propertiesof the fibrils are conserved.


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

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

Seeding properties of 3R and 4R Tau. Tau fibrilseeds (10% monomerequivalents) were mixed with monomers of K18 and K19 (98% cysteinefree, 2% acrylodan labeled at position 310). As the labeled Tau monomersincorporate into the fibrils, the emission maxima shift to the blue.Inverse emission maxima plotted against time depict fibril growth:(A) K18 seeded growth and (B) K19 seeded growth. Values representmean ± SD (n = 3 experiments). Tau concentration,10 μM; cofactor, 20 μg/mL polyA RNA; excitation, 360 nm.An asymmetric seeding barrier prevents 3R Tau from growing onto 4RTau seeds but not vice versa.
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Related In: Results  -  Collection

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

fig2: Seeding properties of 3R and 4R Tau. Tau fibrilseeds (10% monomerequivalents) were mixed with monomers of K18 and K19 (98% cysteinefree, 2% acrylodan labeled at position 310). As the labeled Tau monomersincorporate into the fibrils, the emission maxima shift to the blue.Inverse emission maxima plotted against time depict fibril growth:(A) K18 seeded growth and (B) K19 seeded growth. Values representmean ± SD (n = 3 experiments). Tau concentration,10 μM; cofactor, 20 μg/mL polyA RNA; excitation, 360 nm.An asymmetric seeding barrier prevents 3R Tau from growing onto 4RTau seeds but not vice versa.
Mentions: Despite the structural commonalities,packing interactions between β-sheets could vary substantially,resulting in different overall conformations. Indeed, K18 and K19fibrils, both of which (in the presence of heparin) form parallel,in-register β-strands,35 are conformationallydistinct.45 In order to gain further insightsinto the structure of RNA-induced fibrils, we next investigated theirseeding properties. Since RNA interferes with the commonly used thioflavinassays of Tau fibril formation,46 we chosean acrylodan-based fluorescence assay to monitor seeded fibril growth.36 In this assay, acrylodan is attached to a singlecysteine of the protein and fibril assembly is observed through theblueshift in the fluorescence emission maximum that occurs when thelabel moves from an aqueous environment in the disordered monomerto a hydrophobic environment in the folded fibril. First, K18 andK19 monomers were individually labeled at position 310 and mixed withtheir respective cysteine-free counterparts (molar ratio of 1:50).These mixtures were then combined with 10 mol % seeds (monomer equivalents)of K18 and K19. Samples were excited at 360 nm, and inverse emissionmaxima were plotted as a function of time (Figure 2). While K18 monomers grow onto K18 seeds,K19 monomers do not (Figure 2A). K19 seeds, however, facilitate growth of both K19 andK18 monomers, although the latter monomers grow less efficiently (Figure 2B). The data indicatethat the previously observed asymmetric seeding barrier for heparin-mediatedfibril growth is also preserved for fibrils formed in the presenceof RNA. Furthermore, the data suggest that important structural propertiesof the fibrils are conserved.

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