FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau filaments
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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Posted 03 Jun, 2020
On 16 Jul, 2020
On 05 Jun, 2020
On 03 Jun, 2020
Received 03 Jun, 2020
On 28 May, 2020
Received 28 May, 2020
Invitations sent on 27 May, 2020
On 27 May, 2020
On 26 May, 2020
On 26 May, 2020
On 15 May, 2020
Received 21 Apr, 2020
Received 21 Apr, 2020
Invitations sent on 16 Apr, 2020
On 16 Apr, 2020
On 16 Apr, 2020
On 16 Apr, 2020
On 08 Apr, 2020
On 07 Apr, 2020
On 07 Apr, 2020
On 07 Apr, 2020
FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau filaments
Posted 03 Jun, 2020
On 16 Jul, 2020
On 05 Jun, 2020
On 03 Jun, 2020
Received 03 Jun, 2020
On 28 May, 2020
Received 28 May, 2020
Invitations sent on 27 May, 2020
On 27 May, 2020
On 26 May, 2020
On 26 May, 2020
On 15 May, 2020
Received 21 Apr, 2020
Received 21 Apr, 2020
Invitations sent on 16 Apr, 2020
On 16 Apr, 2020
On 16 Apr, 2020
On 16 Apr, 2020
On 08 Apr, 2020
On 07 Apr, 2020
On 07 Apr, 2020
On 07 Apr, 2020
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.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7