See the published version of this article at Molecular Neurodegeneration.
This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research article
FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau filaments
Senthilvelrajan Kaniyappan
German Center for Neurodegenerative Diseases (DZNE)
Corresponding Author
ORCiD: https://orcid.org/0000-0002-0606-2769
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published version at Molecular Neurodegeneration.
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.
Keywords
Tau protein, propagation, seeding, Alzheimer, amyloid
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
This is a list of supplementary files associated with this preprint. Click to download.
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
Version 2
Posted 03 Jun, 2020
Published
On 16 Jul, 2020
No community comments so far
Editorial decision: Accept
On 05 Jun, 2020
Reviewer #2 agreed
On 03 Jun, 2020
Review #2 received
Received 03 Jun, 2020
Reviewer #1 agreed
On 28 May, 2020
Review #1 received
Received 28 May, 2020
Reviewers invited
Invitations sent on 27 May, 2020
Editor assigned
On 27 May, 2020
Submission checks complete
On 26 May, 2020
Editor invited
On 26 May, 2020
No comments provided
Editorial decision: Minor revision
On 15 May, 2020
Review #1 received
Received 21 Apr, 2020
Review #2 received
Received 21 Apr, 2020
Reviewers invited
Invitations sent on 16 Apr, 2020
Reviewer #1 agreed
On 16 Apr, 2020
Reviewer #2 agreed
On 16 Apr, 2020
Reviewer #3 agreed
On 16 Apr, 2020
Editor assigned
On 08 Apr, 2020
First submitted
On 07 Apr, 2020
Submission checks complete
On 07 Apr, 2020
Editor invited
On 07 Apr, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Neurology
Learn more about our company.
This preprint is under consideration at Molecular Neurodegeneration. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research article
FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau filaments
Senthilvelrajan Kaniyappan
German Center for Neurodegenerative Diseases (DZNE)
Corresponding Author
ORCiD: https://orcid.org/0000-0002-0606-2769
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
Version 2
Posted 03 Jun, 2020
Published
On 16 Jul, 2020
No community comments so far
Editorial decision: Accept
On 05 Jun, 2020
Reviewer #2 agreed
On 03 Jun, 2020
Review #2 received
Received 03 Jun, 2020
Reviewer #1 agreed
On 28 May, 2020
Review #1 received
Received 28 May, 2020
Reviewers invited
Invitations sent on 27 May, 2020
Editor assigned
On 27 May, 2020
Submission checks complete
On 26 May, 2020
Editor invited
On 26 May, 2020
No comments provided
Editorial decision: Minor revision
On 15 May, 2020
Review #1 received
Received 21 Apr, 2020
Review #2 received
Received 21 Apr, 2020
Reviewers invited
Invitations sent on 16 Apr, 2020
Reviewer #1 agreed
On 16 Apr, 2020
Reviewer #2 agreed
On 16 Apr, 2020
Reviewer #3 agreed
On 16 Apr, 2020
Editor assigned
On 08 Apr, 2020
First submitted
On 07 Apr, 2020
Submission checks complete
On 07 Apr, 2020
Editor invited
On 07 Apr, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
Neurology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published version at Molecular Neurodegeneration.
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