Development of new fusion proteins for visualizing amyloid-β oligomers in vivo

Ochiishi, Tomoyo; Doi, Motomichi; Yamasaki, Kazuhiko; Hirose, Keiko; Kitamura, Akira; Urabe, Takao; Ebihara, Tatsuhiko; Shimura, Hideki

doi:10.1038/srep22712

Cited by 36 publications

(38 citation statements)

References 51 publications

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“…This situation is similar to that of other cases where an amyloidogenic protein fused to a compact reporter molecule loses its ability to aggregate into amyloid bers (e.g. PABPN1-CspB, Aβ-GFP) (Ochiishi et al, 2016, Buttstedt et al, 2010).…”

Section: Discussionsupporting

confidence: 79%

FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau filaments

Kaniyappan

Tepper

Biernat

et al. 2020

Preprint

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Tau aggregation into amyloid fibers based on the cross-beta structure is a hallmark of several Tauopathies, including Alzheimer Disease (AD). Trans-cellular propagation of Tau with pathological conformation has been suggested as a key disease mechanism. This is thought to cause the spreading of Tau pathology in AD by templated conversion of naive Tau in recipient cells into a pathological state, followed by assembly of pathological Tau fibers, similar to the mechanism of nucleated polymerization proposed for prion pathogenesis. In cell cultures, the process is often monitored by a FRET assay where the recipient cell expresses the Tau repeat domain (TauRD) with a pro-aggregant mutation, fused to GFP-based FRET pairs. Since the size of the reporter GFP (barrel of ~3nm x 4nm) is ~7 times larger than the β-strand distance (0.47nm), this points to a potential steric clash. Hence, we investigated the influence of the GFP tag on Tau or TauRD aggregation. Using biophysical methods (light scattering, atomic force microscopy (AFM), and scanning-transmission electron microscopy (STEM)), we found that the assembly of TauRD-GFP was severely inhibited and incompatible with that of Alzheimer filaments. These observations argue against the hypothesis that the propagation of Tau pathology in AD is caused by the prion-like templated aggregation of Tau protein, transmitted via cell-to-cell spreading of Tau. Thus, even though the observed local increase of FRET in recipient cells may be a valid hallmark of a pathological reaction, our data argue that it is caused a process distinct from assembly of TauRD filaments.

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Section: Discussionsupporting

confidence: 79%

FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau filaments

Kaniyappan

Tepper

Biernat

et al. 2020

Preprint

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“…This situation is similar to other cases where an amyloidogenic protein fused to a compact reporter molecule lost its ability to aggregate into amyloid fibers (e.g. PABPN1-CspB, Aβ-GFP) (Ochiishi et al, 2016, Buttstedt et al, 2010.…”

Section: Discussionsupporting

confidence: 75%

FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau fibers

Kaniyappan

Tepper

Biernat

et al. 2020

Preprint

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Abbreviations: AD Alzheimer disease AFM atomic force microscopy CNS central nervous system FRET fluorescence resonance energy transfer GFP green fluorescent protein PHF paired helical filament Tau FL full-length Tau protein, largest isoform in human CNS (Uniprot P10636-F, htau40) Tau FLΔK full-length Tau protein with pro-aggregant mutation ΔK280 Tau RD Tau repeat domain (amyloidogenic domain) Tau RDΔK Tau repeat domain with pro-aggregant mutation ΔK280 Sf9 cell line from Spodoptera frugiperda STEM scanning transmission electron microscopy EM electron microscopy MPL mass per length Wt wildtype AbstractTau aggregation into amyloid fibers based on the cross-beta structure is a hallmark of several Tauopathies, including Alzheimer Disease (AD). Trans-cellular propagation of Tau with pathological conformation has been suggested as a key disease mechanism. This is thought to cause the spreading of Tau pathology in AD by templated conversion of naive Tau in recipient cells into a pathological state, followed by assembly of pathological Tau fibers, similar to the mechanism proposed for prion pathogenesis. In cell cultures, the process is usually monitored by a FRET assay where the recipient cell expresses the Tau repeat domain (Tau RD , with pro-aggregant mutation, e.g., ΔK280 or P301L, ~13.5 kDa) fused to GFP-based FRET pairs (YFP or CFP, ~28 kD). Since the diameter of the reporter GFP (~3 nm) is ~6.5 times larger than the β-strand distance (0.47nm), this points to a potential steric clash. Hence, we investigated the influence of GFP tagged (N-or C-terminally) Tau RD and Tau FL (full-length Tau) on their aggregation behavior in vitro. Using biophysical methods (light scattering, atomic force microscopy (AFM), and scanningtransmission electron microscopy (STEM)), we found that the assembly of Tau RDΔK -GFP was severely inhibited, even in the presence of nucleation enhancers (heparin and/or pre-formed PHFs from Tau RDΔK ). Some rare fiber-like particles had a very different subunit packing from proper PHFs, as judged by STEM. The mass per length (MPL) values of Tau RDΔK fibrils are equivalent to 4.45 molecules/nm, close to the expected value for a paired-helical fiber with 2 protofilaments and cross-β structure. By contrast, the elongated particles formed by Tau RDΔK -GFP have MPL values around ~2, less than half of the values expected for PHFs, indicating that the subunit packing is distinct. Thus, both kinetic and structural observations are incompatible with a model whereby external Tau can form a template for PHF assembly of Tau-GFP in recipient cells. As a consequence, the observed local increase of FRET in recipient cells must be caused by other signalling processes.

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“…Fox and colleagues have used the similar approach to study the amyloid formation of amylin (also known as islet amyloid polypeptide) [38]. Recently, GFP molecule has also been used as a reporting protein in fusion constructs with A peptides for visualizing A oligomers in vivo [39]. However, in these and other studies [40][41][42][43], the impact of linker length on oligomerization or fibril formation was not evaluated.…”

Section: Choice Of a Modelmentioning

confidence: 99%

Establishment of Constraints on Amyloid Formation Imposed by Steric Exclusion of Globular Domains

Azizyan

Garro

Radkova

et al. 2018

Journal of Molecular Biology

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In many disease-related and functional amyloids, the amyloid-forming regions of proteins are flanked by globular domains. When located in close vicinity of the amyloid regions along the chain, the globular domains can prevent the formation of amyloids because of the steric repulsion. Experimental tests of this effect are few in number and non-systematic, and their interpretation is hampered by polymorphism of amyloid structures. In this situation, modeling approaches that use such a clear-cut criterion as the steric tension can give us highly trustworthy results. In this work, we evaluated this steric effect by using molecular modeling and dynamics. As an example, we tested hybrid proteins containing an amyloid-forming fragment of Aβ peptide (17-42) linked to one or two globular domains of GFP. Searching for the shortest possible linker, we constructed models with pseudo-helical arrangements of the densely packed GFPs around the Aβ amyloid core. The molecular modeling showed that linkers of 7 and more residues allow fibrillogenesis of the Aβ-peptide flanked by GFP on one side and 18 and more residues when Aβ-peptide is flanked by GFPs on both sides. Furthermore, we were able to establish a more general relationship between the size of the globular domains and the length of the linkers by using analytical expressions and rigid body simulations. Our results will find use in planning and interpretation of experiments, improvement of the prediction of amyloidogenic regions in proteins, and design of new functional amyloids carrying globular domains.

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Development of new fusion proteins for visualizing amyloid-β oligomers in vivo

Cited by 36 publications

References 51 publications

FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau filaments

FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau filaments

FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau fibers

Establishment of Constraints on Amyloid Formation Imposed by Steric Exclusion of Globular Domains

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