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

Kaniyappan, Senthilvelrajan, Tepper, Katharina, Biernat, Jacek, Chandupatla, Ram Reddy, Hübschmann, Sabrina, Irsen, Stephan, Bicher, Sandra, Klatt, Christoph, Mandelkow, Eva-Maria, Mandelkow, Eckhard·2020·Molecular Neurodegeneration
DOI 10.1186/s13024-020-00389-1PMC PMC7364478

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Abstract

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 (Tau RD ) with a pro-aggregant mutation, fused to GFP-based FRET pairs. Since the size of the reporter GFP (barrel of ~ 3 nm × 4 nm) is ~ 7 times larger than the β-strand distance (0.47 nm), this points to a potential steric clash. Hence, we investigated the influence of the GFP tag on Tau FL or Tau RD aggregation. Using biophysical methods (light scattering, atomic force microscopy (AFM), and scanning-transmission electron microscopy (STEM)), we found that the assembly of Tau RD -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 by a process distinct from assembly of Tau RD filaments.

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Figures

Fig. 1 Schematic representation of effects of GFP label in preventing templated assembly of TauRD. (a) Self-assembly of Tau: Tau protein is highly hydrophilic and natively unfolded. In disease conditions, Tau becomes misfolded, attains β-structure and assembles into paired helical filaments. (b) Seeded (templated) assembly: Tau protein subunits elongate into long filaments using external Tau seeds as template (usually prepared by sonication of preformed fibrils). Note: In Tau assembly experiments in vitro, polyanions such as heparin must be added to initiate nucleation. (c)Pre-dominant hypothesis: In cell culture, Tau seeds (made from TauFL or TauRD) are internalized into acceptor cells. Endogenous TauRD is thought to attain the amyloid structure of the seeds and elongate the filaments. (d) Experimental observation: In cell culture studies TauRD-GFP is often used as endogenous Tau fusion protein, intended to elongate templating seed structures. However, steric hindrance by the large GFP molecule prevents fiber formation
Fig. 1 Schematic representation of effects of GFP label in preventing templated assembly of TauRD. (a) Self-assembly of Tau: Tau protein is highly hydrophilic and natively unfolded. In disease conditions, Tau becomes misfolded, attains β-structure and assembles into paired helical filaments. (b) Seeded (templated) assembly: Tau protein subunits elongate into long filaments using external Tau seeds as template (usually prepared by sonication of preformed fibrils). Note: In Tau assembly experiments in vitro, polyanions such as heparin must be added to initiate nucleation. (c)Pre-dominant hypothesis: In cell culture, Tau seeds (made from TauFL or TauRD) are internalized into acceptor cells. Endogenous TauRD is thought to attain the amyloid structure of the seeds and elongate the filaments. (d) Experimental observation: In cell culture studies TauRD-GFP is often used as endogenous Tau fusion protein, intended to elongate templating seed structures. However, steric hindrance by the large GFP molecule prevents fiber formationCC BY · PubMed Central
Fig. 2 Tau proteins and variants used in this study. (a) Bar diagram of longest isoform of Tau in human CNS comprising 441 amino acids (Uniprot P10636-F, alias hTau40, Tau-2N4R). Domains are depicted with 2 near-N-terminal inserts (N1, N2, pink), the proline-rich domains (P1, P2; blue), 4 pseudo-repeats (R1-R4, ~ 31 residues each, ochre, plus one less-conserved repeat R5), and the C-terminal tail. The amyloidogenic hexapeptide motifs are indicated at the beginning of R2, R3 (thick black line). The C-terminal half (MT assembly domain P2-R5) promotes both microtubule assembly and pathological aggregation of Tau, the N-terminal half (projection domain) projects from the surface of microtubules or from the core of Tau fibers, respectively. The “pro-aggregant” deletion mutation ΔK280 (red dot) lies near the beginning of R2, it increases the β-propensity of Tau and hence increases pathological aggregation. (b) Tau and GFP fusion proteins are schematically presented with their name listed on the left. The GFP-tag is positioned either at the N-terminus or C-terminus. Some constructs contain a C-terminal 6xHis-tag to aid in the purification (blue ellipse). A sequence of 13–14 amino acids was used as a linker (black line) in the two short Tau constructs GFP-TauRDΔK and TauRDΔK-GFP to increase the distance between the GFP and the repeat domain (see Table S1)
Fig. 2 Tau proteins and variants used in this study. (a) Bar diagram of longest isoform of Tau in human CNS comprising 441 amino acids (Uniprot P10636-F, alias hTau40, Tau-2N4R). Domains are depicted with 2 near-N-terminal inserts (N1, N2, pink), the proline-rich domains (P1, P2; blue), 4 pseudo-repeats (R1-R4, ~ 31 residues each, ochre, plus one less-conserved repeat R5), and the C-terminal tail. The amyloidogenic hexapeptide motifs are indicated at the beginning of R2, R3 (thick black line). The C-terminal half (MT assembly domain P2-R5) promotes both microtubule assembly and pathological aggregation of Tau, the N-terminal half (projection domain) projects from the surface of microtubules or from the core of Tau fibers, respectively. The “pro-aggregant” deletion mutation ΔK280 (red dot) lies near the beginning of R2, it increases the β-propensity of Tau and hence increases pathological aggregation. (b) Tau and GFP fusion proteins are schematically presented with their name listed on the left. The GFP-tag is positioned either at the N-terminus or C-terminus. Some constructs contain a C-terminal 6xHis-tag to aid in the purification (blue ellipse). A sequence of 13–14 amino acids was used as a linker (black line) in the two short Tau constructs GFP-TauRDΔK and TauRDΔK-GFP to increase the distance between the GFP and the repeat domain (see Table S1)CC BY · PubMed Central
Fig. 3 Full length wildtype Tau proteins (with or without GFP) induce microtubule assembly whereas the repeat domain does not. (a) Assembly of microtubules (10 μM tubulin) is supported by TauFL-wt (5 μM) without GFP or with GFP attached to either N- or C-terminus (top, green, blue, magenta curves). MT assembly levels and rates (t1/2 ~ min range) are comparable. Note that GFP alone shows no increase in light scattering (red curve). (b) Microtubule assembly is not supported by the repeat domain TauRDΔK, regardless of GFP (bottom curves: TauRDΔK (light green); GFP-TauRDΔK (blue); TauRDΔK–GFP (magenta); GFP alone (red)). As a control, TauFL-wt induces robust microtubule assembly (as in a, green). Note that the repeat domain R1-R4 is often denoted as “MTBD” domain in the literature, even though it binds and assembles microtubules poorly
Fig. 3 Full length wildtype Tau proteins (with or without GFP) induce microtubule assembly whereas the repeat domain does not. (a) Assembly of microtubules (10 μM tubulin) is supported by TauFL-wt (5 μM) without GFP or with GFP attached to either N- or C-terminus (top, green, blue, magenta curves). MT assembly levels and rates (t1/2 ~ min range) are comparable. Note that GFP alone shows no increase in light scattering (red curve). (b) Microtubule assembly is not supported by the repeat domain TauRDΔK, regardless of GFP (bottom curves: TauRDΔK (light green); GFP-TauRDΔK (blue); TauRDΔK–GFP (magenta); GFP alone (red)). As a control, TauFL-wt induces robust microtubule assembly (as in a, green). Note that the repeat domain R1-R4 is often denoted as “MTBD” domain in the literature, even though it binds and assembles microtubules poorlyCC BY · PubMed Central
Fig. 4 Aggregation of Tau proteins with GFP tags monitored by light scattering and electron microscopy. (a-b) Attachment of GFP to Tau repeat domain severely inhibits aggregation, but full length Tau+GFP remains aggregation competent. Aggregation of Tau to PHF-like fibers was monitored by light scattering at 350 nm. 50 μM of TauFL-wt or TauRDΔK with or without GFP on either end was incubated in the presence of heparin 16,000 (12.5 μM) in BES buffer, pH 7.0, at 37 °C at different time intervals. (a) Unlabeled TauFL-wt (green) aggregates mostly into PHF-like fibers with t1/2 ~ 3 h. GFP–TauFL-wt (blue, t1/2 ~ 2 h) and TauFL-wt–GFP (magenta, t1/2 ~ 12 h) aggregate with distinct rates and reach higher final levels, consistent with the higher mass of the subunits and variations in elongation. (b) Unlabeled TauRDΔK (green curve) shows aggregation with t1/2 ~ 6 h into PHF-like fibers. By contrast, GFP-TauRDΔK (blue) shows no increase in light scattering, and TauRDΔK–GFP (magenta) reaches only a low level of light scattering saturating at ~ 2 h. Thus in both cases the aggregation of GFP-labeled protein is severely inhibited. (c-h) Electron microscopy reveals heterogeneous aggregation forms of Tau + GFP Samples of TauFL-wt, TauRDΔK with and without GFP aggregated for 24 h at 37 °C were placed on carbon grids and imaged by negative stain electron microscopy. Top row, TauFL-wt proteins with or without GFP forms typical long filaments (c-e), whereas repeat domain TauRDΔK does not (f-h, bottom row). (c) TauFL-wt aggregates into typical PHF-like twisted filaments (diameter ~ 28 nm). (d) GFP-TauFL-wt forms long filaments with increased diameter (~ 40 nm). (e) TauFL-wt-GFP forms long filaments and bundles (~ 28 nm). (f) Repeat domain TauRDΔK without GFP forms filaments of typical PHF-like morphology but smaller diameter (~ 21 nm). (g) GFP-TauRDΔK does not form filaments but only amorphous small aggregates. (h) TauRDΔK-GFP forms short filaments and oligomers with the length < 100 nm; average diameter ~ 37 nm
Fig. 4 Aggregation of Tau proteins with GFP tags monitored by light scattering and electron microscopy. (a-b) Attachment of GFP to Tau repeat domain severely inhibits aggregation, but full length Tau+GFP remains aggregation competent. Aggregation of Tau to PHF-like fibers was monitored by light scattering at 350 nm. 50 μM of TauFL-wt or TauRDΔK with or without GFP on either end was incubated in the presence of heparin 16,000 (12.5 μM) in BES buffer, pH 7.0, at 37 °C at different time intervals. (a) Unlabeled TauFL-wt (green) aggregates mostly into PHF-like fibers with t1/2 ~ 3 h. GFP–TauFL-wt (blue, t1/2 ~ 2 h) and TauFL-wt–GFP (magenta, t1/2 ~ 12 h) aggregate with distinct rates and reach higher final levels, consistent with the higher mass of the subunits and variations in elongation. (b) Unlabeled TauRDΔK (green curve) shows aggregation with t1/2 ~ 6 h into PHF-like fibers. By contrast, GFP-TauRDΔK (blue) shows no increase in light scattering, and TauRDΔK–GFP (magenta) reaches only a low level of light scattering saturating at ~ 2 h. Thus in both cases the aggregation of GFP-labeled protein is severely inhibited. (c-h) Electron microscopy reveals heterogeneous aggregation forms of Tau + GFP Samples of TauFL-wt, TauRDΔK with and without GFP aggregated for 24 h at 37 °C were placed on carbon grids and imaged by negative stain electron microscopy. Top row, TauFL-wt proteins with or without GFP forms typical long filaments (c-e), whereas repeat domain TauRDΔK does not (f-h, bottom row). (c) TauFL-wt aggregates into typical PHF-like twisted filaments (diameter ~ 28 nm). (d) GFP-TauFL-wt forms long filaments with increased diameter (~ 40 nm). (e) TauFL-wt-GFP forms long filaments and bundles (~ 28 nm). (f) Repeat domain TauRDΔK without GFP forms filaments of typical PHF-like morphology but smaller diameter (~ 21 nm). (g) GFP-TauRDΔK does not form filaments but only amorphous small aggregates. (h) TauRDΔK-GFP forms short filaments and oligomers with the length < 100 nm; average diameter ~ 37 nmCC BY · PubMed Central
Fig. 5 Atomic force microscopy reveals the changes in Tau filament assembly and decoration with GFP. AFM analysis of Tau fibrils formed in the presence of heparin for 24 h at 37 °C was imaged in tapping mode using an MSNL cantilever. Left row, overviews; center row, magnified details; right row, line scans showing distribution of height. TauFL-wt(a1) and GFP-TauFL-wt(b1) both form long fibrils (straight or twisted), but the height distribution shows characteristic differences because the attachment of GFP generates a halo on both sides of the filament core (compare a3 and b3, core and side bands indicated by dashed lines). The average height (thickness) of the fibril core is similar for TauFL-wt (7.7 nm ± 0.7 nm; n = 36) and GFP-TauFL-wt (7.0 nm ± 1.2 nm; n = 36). However, the overall thickness increases from TauFL-wt alone (38.1 nm ± 5.45 nm; n = 50) to GFP-TauFL-wt (96.01 nm ± 8.98 nm; n = 50). The two side peaks (b3) are ~ 3 nm wide, in good agreement with the shape of GFP. (c-d) The repeat domain constructs TauRDΔK forms well twisted filaments (c1) whereas TauRDΔK-GFP forms only few short filaments and more globular shaped aggregates (d1). The enlarged images (c2, d2) show that the short filaments of TauRDΔK-GFP are thicker than those of TauRDΔK. (c3 and d3) The widths and heights of TauRDΔK-GFP (width - 42.95 nm ± 8.32 nm; n = 50; height- 18.5 nm ± 3.1 nm; n = 36) fibrils are larger than those of TauRDΔK fibrils (width - 28.17 nm ± 3.35 nm; n = 50; height - 7.0 nm ± 1.1 nm; n = 36). The height scale for a1, b1, c1 and d1 is 0 to 15 nm
Fig. 5 Atomic force microscopy reveals the changes in Tau filament assembly and decoration with GFP. AFM analysis of Tau fibrils formed in the presence of heparin for 24 h at 37 °C was imaged in tapping mode using an MSNL cantilever. Left row, overviews; center row, magnified details; right row, line scans showing distribution of height. TauFL-wt(a1) and GFP-TauFL-wt(b1) both form long fibrils (straight or twisted), but the height distribution shows characteristic differences because the attachment of GFP generates a halo on both sides of the filament core (compare a3 and b3, core and side bands indicated by dashed lines). The average height (thickness) of the fibril core is similar for TauFL-wt (7.7 nm ± 0.7 nm; n = 36) and GFP-TauFL-wt (7.0 nm ± 1.2 nm; n = 36). However, the overall thickness increases from TauFL-wt alone (38.1 nm ± 5.45 nm; n = 50) to GFP-TauFL-wt (96.01 nm ± 8.98 nm; n = 50). The two side peaks (b3) are ~ 3 nm wide, in good agreement with the shape of GFP. (c-d) The repeat domain constructs TauRDΔK forms well twisted filaments (c1) whereas TauRDΔK-GFP forms only few short filaments and more globular shaped aggregates (d1). The enlarged images (c2, d2) show that the short filaments of TauRDΔK-GFP are thicker than those of TauRDΔK. (c3 and d3) The widths and heights of TauRDΔK-GFP (width - 42.95 nm ± 8.32 nm; n = 50; height- 18.5 nm ± 3.1 nm; n = 36) fibrils are larger than those of TauRDΔK fibrils (width - 28.17 nm ± 3.35 nm; n = 50; height - 7.0 nm ± 1.1 nm; n = 36). The height scale for a1, b1, c1 and d1 is 0 to 15 nmCC BY · PubMed Central
Fig. 6 STEM imaging reveals that packing of Tau in assembly conditions is severely altered by GFP tags. (a) Mass per length histograms (MPL) of fibrils aggregated for 24 h at 37 °C, with fitted peak values (in kDa/nm) listed for each protein. Examples of STEM images are shown for TauFL-wt(b), GFP-TauFL-wt(c), TauRDΔK(d); TauRDΔK-GFP fibrils (long) (e) and TauRDΔK-GFP fibrils (short) (f). MPL measurements were calibrated with tobacco mosaic virus (TMV, shown in the bottom histogram in a and in image g). The estimated molecules per nm are presented in Table 1. Note that TauRDΔK-GFP fibrils have been observed mostly as short and stubby fibrils, but that the long fibrils show a packing, less dense than the short fibrils
Fig. 6 STEM imaging reveals that packing of Tau in assembly conditions is severely altered by GFP tags. (a) Mass per length histograms (MPL) of fibrils aggregated for 24 h at 37 °C, with fitted peak values (in kDa/nm) listed for each protein. Examples of STEM images are shown for TauFL-wt(b), GFP-TauFL-wt(c), TauRDΔK(d); TauRDΔK-GFP fibrils (long) (e) and TauRDΔK-GFP fibrils (short) (f). MPL measurements were calibrated with tobacco mosaic virus (TMV, shown in the bottom histogram in a and in image g). The estimated molecules per nm are presented in Table 1. Note that TauRDΔK-GFP fibrils have been observed mostly as short and stubby fibrils, but that the long fibrils show a packing, less dense than the short fibrilsCC BY · PubMed Central
Fig. 7 Models of organization of Tau fibrils fused to GFP. (a) Models of arrangement of GFP + Tau fusion constructs under aggregating conditions. The diagrams represent filaments lying on a support (grey line), viewed down the filament axis (black oval = core). (1, 2) TauFL-wt and GFP-TauFL-wt have a similar packing of their fibril cores (cross-β structure) with attached N- or C-terminal domains (1), plus GFP on either end (2). The PHF-like packing is possible because Tau is disordered and flexible, so that GFP molecules can be accommodated around the perimeter as a fuzzy “halo”. (3) TauRDΔK can form a PHF-like fibril core, but (4) GFP- TauRDΔK does not form any fibrils. (5) TauRDΔK-GFP may assemble into short aberrant fibrils, but the arrangement is not compatible with that of AD-like filaments and results in perturbed core (red distorted structure). The GFP creates an outside layer (analogous to the fuzzy coat of PHFs), but the packing is less dense and the elongation is strongly disturbed. (b) Diagram of a side view of a PHF-like filament, made up of two parallel β-sheets (red and blue) with antiparallel orientations (arrows). The axial separation of β-strands is 0.47 nm. Size comparison of β-strand and attached GFP. If the GFP were attached sufficiently far away from repeat domain (as in FLTau), the amyloid core could be formed, with GFP molecules accommodated around the perimeter. If the GFP is too close to the β-strand core (as in TauRD) this leads to steric hindrance which causes improper folding and disruption of the amyloid core
Fig. 7 Models of organization of Tau fibrils fused to GFP. (a) Models of arrangement of GFP + Tau fusion constructs under aggregating conditions. The diagrams represent filaments lying on a support (grey line), viewed down the filament axis (black oval = core). (1, 2) TauFL-wt and GFP-TauFL-wt have a similar packing of their fibril cores (cross-β structure) with attached N- or C-terminal domains (1), plus GFP on either end (2). The PHF-like packing is possible because Tau is disordered and flexible, so that GFP molecules can be accommodated around the perimeter as a fuzzy “halo”. (3) TauRDΔK can form a PHF-like fibril core, but (4) GFP- TauRDΔK does not form any fibrils. (5) TauRDΔK-GFP may assemble into short aberrant fibrils, but the arrangement is not compatible with that of AD-like filaments and results in perturbed core (red distorted structure). The GFP creates an outside layer (analogous to the fuzzy coat of PHFs), but the packing is less dense and the elongation is strongly disturbed. (b) Diagram of a side view of a PHF-like filament, made up of two parallel β-sheets (red and blue) with antiparallel orientations (arrows). The axial separation of β-strands is 0.47 nm. Size comparison of β-strand and attached GFP. If the GFP were attached sufficiently far away from repeat domain (as in FLTau), the amyloid core could be formed, with GFP molecules accommodated around the perimeter. If the GFP is too close to the β-strand core (as in TauRD) this leads to steric hindrance which causes improper folding and disruption of the amyloid coreCC BY · PubMed Central

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Metadata

Authors
Kaniyappan, S., Tepper, K., Biernat, J., Chandupatla, R. R., Hübschmann, S., Irsen, S., Bicher, S., Klatt, C., Mandelkow, E.-M., & Mandelkow, E.
Venue
Molecular Neurodegeneration
Publisher
Springer Science and Business Media LLC
Published
2020-07-16
Volume
15
Issue
1
ISSN
1750-1326
DOI
10.1186/s13024-020-00389-1
PMC
PMC7364478
Cited by
58
References
30
Language
en
Licence
Funders
  • DZNE
  • MPG
  • Cure Alzheimer's Fund
  • Katharina Hardt Foundation

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Bibliographic data: Semantic Scholar, OpenAlex, Crossref.

Contains information from the Semantic Scholar Academic Graph Datasets, made available under the ODC Attribution License (ODC-BY 1.0).

Semantic Scholar’s data is described in: Kinney, R., et al. (2023). The Semantic Scholar Open Data Platform. arXiv:2301.10140.