PT  - JOURNAL ARTICLE
AU  - Senthilvelrajan Kaniyappan
AU  - Katharina Tepper
AU  - Jacek Biernat
AU  - Ram Reddy Chandupatla
AU  - Sabrina Hübschmann
AU  - Stephan Irsen
AU  - Sandra Bicher
AU  - Christoph Klatt
AU  - Eva-Maria Mandelkow
AU  - Eckhard Mandelkow
TI  - FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau fibers
AID  - 10.1101/2020.03.25.998831
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.03.25.998831
4099  - http://biorxiv.org/content/early/2020/03/25/2020.03.25.998831.short
4100  - http://biorxiv.org/content/early/2020/03/25/2020.03.25.998831.full
AB  - 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 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 (TauRD, 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) TauRD and TauFL (full-length Tau) on their aggregation behavior in vitro. Using biophysical methods (light scattering, atomic force microscopy (AFM), and scanning-transmission electron microscopy (STEM)), we found that the assembly of TauRDΔK-GFP was severely inhibited, even in the presence of nucleation enhancers (heparin and/or pre-formed PHFs from TauRDΔK). 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 TauRDΔK 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 TauRDΔK-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.ADAlzheimer diseaseAFMatomic force microscopyCNScentral nervous systemFRETfluorescence resonance energy transferGFPgreen fluorescent proteinPHFpaired helical filamentTauFLfull-length Tau protein, largest isoform in human CNS (Uniprot P10636-F, htau40)TauFLΔKfull-length Tau protein with pro-aggregant mutation ΔK280TauRDTau repeat domain (amyloidogenic domain)TauRDΔKTau repeat domain with pro-aggregant mutation ΔK280Sf9cell line from Spodoptera frugiperdaSTEMscanning transmission electron microscopyEMelectron microscopyMPLmass per lengthWtwildtype