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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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RNA sustains template-assisted fibril growth of 3R and4R Tau.(A) Schematic diagram illustrating consecutive cycles of seeding andgrowth used in the experiment below. Beginning with cycle 2, RNA wasomitted from all reactions. Only control experiments contained RNAthroughout. After each cycle (24 h of growth at 22 °C), fibrilswere sedimented by ultracentrifugation (100 000g) and analyzed by SDS-PAGE and Coomassie staining. (B) Fibril growthin the presence (left panel) and absence (right panel) of polyA RNA:upper panels, K18; lower panels, K19. Each reaction contained 10%seeds and 25 μM Tau. PolyA RNA, 125 μg/mL. (C, D) Taufibril seeds (10% monomer equivalents) were mixed with K18 and K19monomers (98% cysteine free, 2% acrylodan-labeled at position 310).(C) K18 growth onto K18 seeds in the presence and absence of RNA.(D) K19 growth onto K19 seeds in the presence and absence of RNA.The data demonstrate that RNA sustains template-assisted fibril growth.
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fig3: RNA sustains template-assisted fibril growth of 3R and4R Tau.(A) Schematic diagram illustrating consecutive cycles of seeding andgrowth used in the experiment below. Beginning with cycle 2, RNA wasomitted from all reactions. Only control experiments contained RNAthroughout. After each cycle (24 h of growth at 22 °C), fibrilswere sedimented by ultracentrifugation (100 000g) and analyzed by SDS-PAGE and Coomassie staining. (B) Fibril growthin the presence (left panel) and absence (right panel) of polyA RNA:upper panels, K18; lower panels, K19. Each reaction contained 10%seeds and 25 μM Tau. PolyA RNA, 125 μg/mL. (C, D) Taufibril seeds (10% monomer equivalents) were mixed with K18 and K19monomers (98% cysteine free, 2% acrylodan-labeled at position 310).(C) K18 growth onto K18 seeds in the presence and absence of RNA.(D) K19 growth onto K19 seeds in the presence and absence of RNA.The data demonstrate that RNA sustains template-assisted fibril growth.

Mentions: Although it is clear thatRNA is needed for nucleation, its role in fibril growth is not established.Here, we asked whether Tau fibril propagation could be sustained ifRNA was omitted in consecutive cycles of seeding. The overall designof these experiments is outlined schematically in Figure 3A. Tau fibrils were first grownin a seeded reaction that included cofactor (cycle 1, Figure 3A). These fibrils were thensonicated and subjected to four consecutive cycles of seeding withcofactor either present or absent (cycle 2–5, Figure 3A). After each cycle, the sampleswere divided into two fractions: one for seed production via sonicationand another for fibril sedimentation and SDS-PAGE analysis. The Coomassie-stainedgels revealed that K18 and K19 fibrils propagated in the presenceof RNA with similar amounts of fibril mass produced in each cycle(Figure 3B, left panels).When RNA was omitted during the elongation steps, fibril formationceased (Figure 3B,right panels). The weak bands observed for cycles 2 and 3 (respectiveintensities of 24 and 2.0% for K18 and 24 and 2.1% for K19) are dueto the seeds that were added to the reactions, and in the case ofcycle 2, some residual aggregation as cofactor was carried over fromcycle 1. The results demonstrate that RNA drives the amplificationof Tau fibrils.


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

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

RNA sustains template-assisted fibril growth of 3R and4R Tau.(A) Schematic diagram illustrating consecutive cycles of seeding andgrowth used in the experiment below. Beginning with cycle 2, RNA wasomitted from all reactions. Only control experiments contained RNAthroughout. After each cycle (24 h of growth at 22 °C), fibrilswere sedimented by ultracentrifugation (100 000g) and analyzed by SDS-PAGE and Coomassie staining. (B) Fibril growthin the presence (left panel) and absence (right panel) of polyA RNA:upper panels, K18; lower panels, K19. Each reaction contained 10%seeds and 25 μM Tau. PolyA RNA, 125 μg/mL. (C, D) Taufibril seeds (10% monomer equivalents) were mixed with K18 and K19monomers (98% cysteine free, 2% acrylodan-labeled at position 310).(C) K18 growth onto K18 seeds in the presence and absence of RNA.(D) K19 growth onto K19 seeds in the presence and absence of RNA.The data demonstrate that RNA sustains template-assisted fibril growth.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: RNA sustains template-assisted fibril growth of 3R and4R Tau.(A) Schematic diagram illustrating consecutive cycles of seeding andgrowth used in the experiment below. Beginning with cycle 2, RNA wasomitted from all reactions. Only control experiments contained RNAthroughout. After each cycle (24 h of growth at 22 °C), fibrilswere sedimented by ultracentrifugation (100 000g) and analyzed by SDS-PAGE and Coomassie staining. (B) Fibril growthin the presence (left panel) and absence (right panel) of polyA RNA:upper panels, K18; lower panels, K19. Each reaction contained 10%seeds and 25 μM Tau. PolyA RNA, 125 μg/mL. (C, D) Taufibril seeds (10% monomer equivalents) were mixed with K18 and K19monomers (98% cysteine free, 2% acrylodan-labeled at position 310).(C) K18 growth onto K18 seeds in the presence and absence of RNA.(D) K19 growth onto K19 seeds in the presence and absence of RNA.The data demonstrate that RNA sustains template-assisted fibril growth.
Mentions: Although it is clear thatRNA is needed for nucleation, its role in fibril growth is not established.Here, we asked whether Tau fibril propagation could be sustained ifRNA was omitted in consecutive cycles of seeding. The overall designof these experiments is outlined schematically in Figure 3A. Tau fibrils were first grownin a seeded reaction that included cofactor (cycle 1, Figure 3A). These fibrils were thensonicated and subjected to four consecutive cycles of seeding withcofactor either present or absent (cycle 2–5, Figure 3A). After each cycle, the sampleswere divided into two fractions: one for seed production via sonicationand another for fibril sedimentation and SDS-PAGE analysis. The Coomassie-stainedgels revealed that K18 and K19 fibrils propagated in the presenceof RNA with similar amounts of fibril mass produced in each cycle(Figure 3B, left panels).When RNA was omitted during the elongation steps, fibril formationceased (Figure 3B,right panels). The weak bands observed for cycles 2 and 3 (respectiveintensities of 24 and 2.0% for K18 and 24 and 2.1% for K19) are dueto the seeds that were added to the reactions, and in the case ofcycle 2, some residual aggregation as cofactor was carried over fromcycle 1. The results demonstrate that RNA drives the amplificationof Tau fibrils.

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