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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 dissociates from Tau fibrils. K18 and K19 fibrils(25 μMmonomer added to 10% seeds) were grown in the presence of RNA (50μg/mL) and subsequently incubated for 24 h with heparin (50μM). (A) Normalized absorbance at 260 nm for solubilized K18pellet and supernatant. (B) K18/RNA fibrils. (C) K18/heparin fibrilsafter heparin/RNA exchange. (D) Normalized absorbance at 260 nm forsolubilized K19 pellet and supernatant. (E) K19/RNA fibrils. (F) K19/heparinfibrils after heparin/RNA exchange. Intensities represent mean ±SD (n = 3 experiments). Bar, 400 nm. The data suggestthat heparin replaces the majority of RNA that is bound to K18 andK19 fibrils.
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fig5: RNA dissociates from Tau fibrils. K18 and K19 fibrils(25 μMmonomer added to 10% seeds) were grown in the presence of RNA (50μg/mL) and subsequently incubated for 24 h with heparin (50μM). (A) Normalized absorbance at 260 nm for solubilized K18pellet and supernatant. (B) K18/RNA fibrils. (C) K18/heparin fibrilsafter heparin/RNA exchange. (D) Normalized absorbance at 260 nm forsolubilized K19 pellet and supernatant. (E) K19/RNA fibrils. (F) K19/heparinfibrils after heparin/RNA exchange. Intensities represent mean ±SD (n = 3 experiments). Bar, 400 nm. The data suggestthat heparin replaces the majority of RNA that is bound to K18 andK19 fibrils.

Mentions: The question arose as to whetherRNA/Tau fibril interactions could be perturbed by polyanions. We hypothesizedthat heparin, a polysulfonated glycosaminoglycan, might affect theinteractions by competing for binding sites. In order to test thishypothesis, we added a 2-fold molar excess of heparin (based on theconcentration of Tau monomers) to preformed K18/RNA fibrils. Afterincubation for 24 h at 37 °C, RNA had almost completely transferredinto the supernatant (Figure 5A). The filamentous nature of K18 fibrils before and afterthe addition of heparin was verified by negative stain transmissionelectron microscopy (Figure 5B,C). Next, an equivalent set of experiments was carried outwith K19 fibrils. Again, addition of heparin caused transfer of RNAinto the supernatant (Figure 5D), although, in this case, transfer was less efficient. Importantly,in the absence of heparin, RNA was almost exclusively found in thepellet (Figure 4).The addition of heparin to K19/RNA fibrils did not affect the filamentousnature of the fibrils (compare Figure 5E with Figure 5F). The data indicate that heparin causes the release of RNAfrom Tau fibrils.


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

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

RNA dissociates from Tau fibrils. K18 and K19 fibrils(25 μMmonomer added to 10% seeds) were grown in the presence of RNA (50μg/mL) and subsequently incubated for 24 h with heparin (50μM). (A) Normalized absorbance at 260 nm for solubilized K18pellet and supernatant. (B) K18/RNA fibrils. (C) K18/heparin fibrilsafter heparin/RNA exchange. (D) Normalized absorbance at 260 nm forsolubilized K19 pellet and supernatant. (E) K19/RNA fibrils. (F) K19/heparinfibrils after heparin/RNA exchange. Intensities represent mean ±SD (n = 3 experiments). Bar, 400 nm. The data suggestthat heparin replaces the majority of RNA that is bound to K18 andK19 fibrils.
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fig5: RNA dissociates from Tau fibrils. K18 and K19 fibrils(25 μMmonomer added to 10% seeds) were grown in the presence of RNA (50μg/mL) and subsequently incubated for 24 h with heparin (50μM). (A) Normalized absorbance at 260 nm for solubilized K18pellet and supernatant. (B) K18/RNA fibrils. (C) K18/heparin fibrilsafter heparin/RNA exchange. (D) Normalized absorbance at 260 nm forsolubilized K19 pellet and supernatant. (E) K19/RNA fibrils. (F) K19/heparinfibrils after heparin/RNA exchange. Intensities represent mean ±SD (n = 3 experiments). Bar, 400 nm. The data suggestthat heparin replaces the majority of RNA that is bound to K18 andK19 fibrils.
Mentions: The question arose as to whetherRNA/Tau fibril interactions could be perturbed by polyanions. We hypothesizedthat heparin, a polysulfonated glycosaminoglycan, might affect theinteractions by competing for binding sites. In order to test thishypothesis, we added a 2-fold molar excess of heparin (based on theconcentration of Tau monomers) to preformed K18/RNA fibrils. Afterincubation for 24 h at 37 °C, RNA had almost completely transferredinto the supernatant (Figure 5A). The filamentous nature of K18 fibrils before and afterthe addition of heparin was verified by negative stain transmissionelectron microscopy (Figure 5B,C). Next, an equivalent set of experiments was carried outwith K19 fibrils. Again, addition of heparin caused transfer of RNAinto the supernatant (Figure 5D), although, in this case, transfer was less efficient. Importantly,in the absence of heparin, RNA was almost exclusively found in thepellet (Figure 4).The addition of heparin to K19/RNA fibrils did not affect the filamentousnature of the fibrils (compare Figure 5E with Figure 5F). The data indicate that heparin causes the release of RNAfrom 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