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.
Research article
FRET-based Tau seeding assay does not represent prion-like templated assembly of Tau filaments
https://doi.org/10.21203/rs.3.rs-20919/v2
This work is licensed under a CC BY 4.0 License
Journal Publication
published 16 Jul, 2020
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Tau, a microtubule-associated protein (MAPT, Uniprot P10636), has an important role in microtubule assembly and stabilization. Tau has a hydrophilic, mostly basic composition, is natively unfolded and is highly soluble. Nevertheless, Tau amyloidogenic aggregates characterize a wide range of neurodegenerative diseases known as Tauopathies (Wang and Mandelkow, 2016, Lee et al., 1988, Holtzman et al., 2016) including Alzheimer Disease (AD). Mutations in the Tau gene alone are sufficient to cause neurodegeneration (Hutton et al., 1998). Moreover Tau deposits in the brain correlate well with the memory decline, confirming the importance of Tau pathology in AD (Braak stages) (Nelson et al., 2012, Braak and Braak, 1991). Biophysical and structural studies show that soluble monomeric Tau, upon nucleation by polyanionic cofactors like heparin or RNA, can form insoluble paired helical filaments (PHFs) in vitro (Kampers et al., 1996, Goedert et al., 1996). However, the pathways causing Tau aggregation in neurons and of Tau-induced neurodegeneration are not well understood.
In AD, Tau pathology spreads from the entorhinal cortex to anatomically connected regions such as hippocampus, subiculum and cortex. The spatio-temporal progression of cognitive impairment correlates well with the Tau pathology, as assessed by hallmarks such as aggregation or hyperphosphorylation (Braak and Braak, 1991, Braak et al., 2006). This has led to the hypothesis that the disease progression in AD is caused by the cell-to-cell spreading of Tau protein itself in a pathological state (Holmes et al., 2014, Kfoury et al., 2012), rather than by some other signal. Consistent with this hypothesis, Tau can be secreted from neurons (concentration in ISF ~1 nM (Yamada et al., 2011)), secretion is enhanced by neuronal activation (Pooler et al., 2013, Wu et al., 2016), by exosomes (Wang et al., 2017) and by neuronal death (Arriagada et al., 1992). Extracellular Tau can be taken up by neighboring cells by several mechanisms including receptor mediated endocytosis, phagocytosis, muscarinic receptor-mediated or HSPG mediated uptake (Michel et al., 2014, Gomez-Ramos et al., 2008, Holmes et al., 2013, Guo and Lee, 2011). The internalized Tau is thought to induce the fibrous aggregation of endogenous Tau by templated self-assembly. This would promote further aggregation and propagation Tau pathology to other cells, by analogy to the mechanism proposed for prion pathogenesis (Colby and Prusiner, 2011, Prusiner, 2012) which is based on the concept of nucleated protein polymerization (Pollard and Borisy, 2003). Following the assumption that spreading of Tau protein is responsible for the spreading of neuronal pathology, current therapeutic approaches include the prevention of the pathological conformation of Tau, scavenging extracellular Tau by antibodies, blocking of Tau uptake by neurons, reducing Tau concentrations, and others (Tai et al., 2020, DeVos et al., 2017, Yanamandra et al., 2013).
The key methods for investigating the reactions of cellular Tau protein in response to external Tau are based on expressing aggregation-prone forms of Tau repeat domains (TauRD) labeled with fluorescent sensors, e.g. CFP, YFP. Their accumulation can be observed by local increases of fluorescence, FRET, or FLIM which indicate proximity within several nm (Kfoury et al., 2012, Gorlovoy et al., 2009). The repeat domain (RD) of Tau represents the assembly-competent core of Tau filaments, as it contains the hexapeptide motifs with a high propensity to generate β-structure (von Bergen et al., 2000, Wischik et al., 1988). Mutations such as ΔK280, P301L, P301S in the repeat domain enhance β-propensity of Tau and hence show stronger toxic effects (Mocanu et al., 2008, Santacruz et al., 2005). Although a FRET signal in a recipient cell requires only proximity of the XFP labels (<10nm) it is usually taken as a sign of pathological PHF-like Tau filaments, induced by the pathological conformation of the Tau subunits (termed seeds) penetrating into the cells. This would amount to a prion-like propagation of Tau pathology from cell to cell (Holmes et al., 2014).
From a structural perspective, the templated assembly of Tau into bona fide PHFs can only be concluded if the Tau aggregates are filaments with cross-β structure (axial repeat between strands ~0.47nm), similar to PHFs from AD brain (Fitzpatrick et al., 2017). We hypothesized that a large GFP reporter molecule (~28 kD) tagged onto TauRD (~13 kD) could inhibit the aggregation because of a steric clash: The size of the reporter GFP ( a barrel-shaped molecule ~3 x 4 nm) (Yang et al., 1996) is ~7 times larger than the β-strand distance (0.47nm) between the Tau molecules. To test this, we investigated the assembly forms of TauRD tagged with GFP either at their C-terminus or N-terminus, using several biophysical and microscopic methods. For comparison we also studied the assembly forms of GFP-tagged full-length Tau, and the same Tau proteins without GFP tags. The results showed that the self-assembly of TauRD is severely inhibited when tagged with GFP. In particular, even when fiber-like particles occurred they had a very different structure and mass distribution, distinct from that of PHF-like Tau fibers. We conclude that cell inclusions with enhanced FRET intensity do not result from a templated assembly of PHF-like Tau filaments, and do not result from the transfer of a pathological conformation.
Materials – All chemicals were obtained from Sigma-Aldrich (Taufkirchen, Germany), Fluka (Seelze, Germany), and Roth (Karlsruhe, Germany) in highest purity if not stated otherwise. Heparin 5000 or 16000 was purchased from Fisher Scientific (#BP2524-10). MSNL-10 probe for AFM measurements were from Bruker, USA.
Cells and viruses
Sf9 cells were obtained from Invitrogen (Life Technologies, DE) and grown at 27°C in monolayer culture with Grace's medium (Sigma /Life Technologies TM) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin (PS).
Sapphire Baculovirus DNA was obtained from Orbigen/Biozol (Eching, DE) and pVL1392 from Invitrogen (Life Technologies, EM). MultiBacTurbo system contains the acceptor plasmid pACEBac1 and the E.coli strain DH10MultiBac /Turbo contains the acceptor bacmid. MultiBacTurbo baculoviral DNA were obtained from EMBL Grenoble Outstation(Bieniossek et al., 2012).
Plasmids and baculovirus construction
The hTau40 cDNA, the longest Tau isoform in human CNS (2N4R), was tagged with 6xHis at the C-terminus using PCR amplification and the modified Tau-His cassette was introduced into pET3a plasmid (Novagen, DE) linearized with NdeI enzyme succeeding in pET3a/htau40/6xHis plasmid for the protein expression in E.coli. The cDNA cassette of GFP-hTau40 (consisting of M1 – L239 of GFP sequence bridged by one aa: His to htau40 (M1 – L441) sequence elongated with 6x His at the C-terminus was inserted into pET3a plasmid succeeding in E.coli expression vector pET3a GFP/htau40/6xHis.
The cDNA cassette of htau40-GFP (consisting of htau40 (M1-L441) bridged by 13 aa AH linker sequence: GAPGSAGSAAGSG to M1- L239 GFP terminated with 6xHis Tag was also inserted into pET3a plasmid leading to the pET3a htau40/GFP/6xHis expression vector for E.coli. Similarly, the cDNA cassette of GFP-TauRDΔK-6xHis consisting of GFP, pro-aggregant Tau repeat domain (4-repeat construct K18, M-Q244-E372, with the deletion mutation ΔK280 terminated with 6xHis Tag (GFP-TauRDΔK) was also inserted into pET3a plasmid, yielding the pET3a GFP-TauRDΔK-6xHis expression vector. This mutation strongly accelerates aggregation due to pronounced β propensity (von Bergen et al., 2001).
A second group of proteins: TauRDΔK –GFP and GFP/6xHis proteins was expressed in the baculovirus expression system. A cDNA cassette containing TauRDΔK tagged with GFP at its C-terminus (TauRDΔK–GFP) followed by a 6xHis tail was inserted into pVL1392 vector resulting in the plasmid pVL1392 TauRDΔK/GFP/6xHis. After mixing with Saphire TM baculovirus DNA, this plasmid was used for the generation of baculovirus and protein expression in Sf9 insect cells as described before (Biernat et al., 1993).
The GFP cDNA sequence fused to a 6xHis tail at its C-terminus was inserted into the pACEBac-1 acceptor vector to generate the pACEBac/GFP/6xHis plasmid. This plasmid was subsequently subjected for the Tn7- dependent integration into the baculoviral genome of DH10 MultiBacTurbo E.coli cells for the generation of baculovirus encoding GFP-His-tagged protein.
The purified MultiBacTurbo bacmid encoding GFP protein was transfected into Sf9 cells for the generation of baculoviruses and subsequent protein expression (Bieniossek et al., 2012).
An overview of the Tau constructs is shown in Fig. 2 (for sequence see Supplement Table S1).
Protein preparation and purification
Proteins were expressed either in E.coli or in insect Sf9-cells using the baculovirus expression system. The Tau proteins hTau40wt (2N4R) and TauRDΔK (K18 construct, (M) Q244-E372 with deletion of K280) were expressed in E.coli and purified as described earlier (Barghorn et al., 2005).
The proteins Tau-His (hTau40/6xHis; 2N4R), GFP-TauRDΔK (GFP- (M)Q244-E372 with deleted K280/6xHis) and GFP-Tau (GFP- hTau40/6xHis; 2N4R) and Tau-GFP (hTau40/-GFP/6xHis; 2N4R) were expressed as fusion proteins with 6x polyHis tail at the C-terminus in E.coli strain BL21(DE3) (Merck-Novagen, Darmstadt). After harvesting, the E.coli bacteria cell pellet was directly re-suspended in lysis buffer [50 mM Tris HCl pH 7.3, 300 mM NaCl, 10% glycerol, 0.5 mM TCEP, 10 mM Imidazol, 1 mM Benzamidin, 1 mM PMSF and 10µg/ml each of protease inhibitors leupeptin, aprotinin, and pepstatin], in the ratio 1g E.coli pellet to 10 ml lysis buffer and disrupted by French press.
Protein TauRDΔK-GFP (M) Q244-E372 with deletion of K280-GFP/6xHis), GFP/6xHis and also GFP-Tau (GFP- hTau40/6xHis; 2N4R) and (hTau40/-GFP/6xHis; 2N4R) were expressed in Sf9 insect cells from Invitrogen. Sf9 cells were infected with recombinant virus at a MOI of > 1 typically in six T150 cell culture flasks containing 75% confluent Sf9 cells. Cells were incubated for three days at 27°C, collected and re-suspended for preparation in the lysis buffer [50 mM Tris HCl pH 7.3, 300 mM NaCl, 10% glycerol, 0.5 mM TCEP, 10 mM Imidazol, 1 mM Benzamidin, 1 mM PMSF and 10 µg/ml each of protease inhibitors leupeptin, aprotinin, and pepstatin], in the ratio 1g Sf9 pellet to 10 ml lysis buffer and disrupted by French press.
The lysates of the E.coli bacteria and Sf9 cells were cleared by centrifugation in a Beckman Optima L- 80 XP Ultracentrifuge with a Ti45 rotor (40,000 rpm. for 60 min at 4 °C), applied to Ni2+ ion affinity chromatography His Trap FF column (GE Healthcare), and purified using an Äkta pure chromatography system (GE Healthcare). Following extensive wash with 12 column volumes (CV) of wash buffer (50mM Na phosphate buffer pH 7.2, 300 mM NaCl, and 25 mM imidazole) the protein was eluted with elution buffer (50mM Na Phosphate buffer pH 7.2, 300 mM and 1 M imidazol).
If necessary the protein breakdown products were separated in the second chromatography step using gel filtration on a Superdex G200 column (GE Healthcare, Freiburg). PBS buffer was used for gel filtration column (137 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7,4) with 1 mM DTT (freshly added).
Polymerization assays
Light scattering: Tau aggregation was monitored by 90° angle light scattering at 350nm in a FluoroMax spectrophotometer (HORIBA). 50 µM Tau protein in suspended in BES buffer, pH 7.0
(20 mM BES, 25 mM NaCl) and supplemented with 12.5µM heparin 16000 or heparin 5000. Heparin 5000 and 16000 induce the aggregates at the same level and there is no difference between the aggregates formed under these conditions. This mixture was incubated at 37°C. At different time points 20µl of the samples were analyzed by light scattering at 350 nm and then the samples were brought back to original tube for further aggregation.
Sedimentation assay and western blotting: The aggregated Tau samples (t=48 h) were sedimented at 61,000 rpm (TLA 100.3 rotor) for 1h at 4°C. The supernatant was collected and the pellet was resuspended to the same volume as supernatant. 5µl of the samples were resolved on 10% SDS gel and immunoblotted as described in (Kaniyappan et al., 2017). Later the blot was probed with K9JA (1:5000 dilution) and GFP (1:1000 dilution) antibodies and the signal was detected by chemiluminescence method.
Turbidity assays for MT assembly: Tau-induced microtubule assembly was monitored by 90° angle light scattering at 350nm in a FluoroMax spectrophotometer (HORIBA). 10 µM PC-purified Tubulin were mixed with 5 µM Tau protein in RB-Buffer (100 mM PIPES pH 6.9, 1 mM DTT, 1mM MgSO4, 1mM EGTA, 1 mM GTP). The polymerization was started by transferring the ice-cold Tubulin-Tau-solution to the 37°C warm cuvette-holder and the reaction started once the temperature was reached. Tubulin assembly was monitored for 15 min.
Electron microscopy and mass per length analysis
Sample preparation: Holey carbon grids (Quantifoil R2/1) covered with either 2 nm amorphous carbon (Quantifoil, R2/1+2 nm C) or graphene were used for sample preparation. The preparation of graphene grids was done according to a method described earlier (Pantelic et al., 2011).The sample concentration was adjusted for MPL measurements to a final concentration of 5–10 molecules / grid hole. The protein solution was mixed with Tobacco mosaic virus (TMV) prior to application to the grids. The TMV was used to calibrate the mass measurements. 10 µl sample was applied to the grids for 2 min. Excess liquid was blotted away using filter paper. Samples were washed 3 times with doubled distilled water to remove buffer salts and afterwards air dried or frozen and vacuum dried at – 80 °C for 12 h.
Scanning-transmission electron microscopy (STEM): For the MPL experiments, a Zeiss Libra200 MC Cs-STEM CRISP (Corrected Illumination Scanning Probe) was used. The instrument was operated at 200 kV. The CRISP is equipped with a monochromated Schottky-type field emission cathode and a double hexapole-design corrector for spherical aberrations of the illumination system (Cs-corrector). A high-angle annular dark field (HAADF) detector (Fischione Instruments, USA) was used for imaging. The images were recorded at a convergence angle of 16 mrad and an acceptance angle of 20 mrad. Images were recorded at a pixel size of 0.6 nm with a dwell time of 70 µs/px. The total dose per image was 400 e/nm2.
Mass determination was done using the software PCMASS32 (Schutz et al., 2015). In a first step images were calibrated using tobacco mosaic virus as calibration sample. Filament regions were manually selected and masked. The full datasets were plotted as histograms and fitted to Gaussian curves.
Transmission electron microscopy (TEM): Samples for negative stain transmission electron microscopy were placed on 200 mesh formvar-carbonated copper grids with (Plano, Quantifoil #S162). Grids were glow-discharged for 30 s (Baltec, MED010) prior to sample addition. The protein solution was removed after 3 min incubation time by filter-paper and the grids washed 3 times with ddH2O (grid on top of each drop) to remove buffer salts, then followed by staining with 2% uranyl acetate for 60 seconds and a finally removing the stain slowly with a wet-filter paper and rapid air drying. To prepare grids with microtubules for TEM, all steps were carried at out at 37°C and with pre-warmed solutions. All specimens were analyzed at 200kV using a JEOL JEM-2200FS TEM.
Atomic force microscopy (AFM)
AFM measurements were performed as described earlier (Kaniyappan et al., 2018). Briefly, Tau fibril samples were diluted in PBS buffer for a final concentration of 0.5 – 1µM. 30µl of the sample was placed onto freshly cleaved mica and allowed to adsorb for 10 minutes at room temperature. Unattached and excess proteins were removed by rinsing the sample with PBS for 4-5 times. Finally, the sample on the mica was filled with imaging buffer (10 mM Tris-HCl, pH 7.4, 50 mM KCl). AFM imaging was performed in oscillation mode using JPK NanoWizard® ULTRA Speed AFM system and MSNL-10 probe with “F” cantilever. The amplitude set point and the gains were adjusted manually to control the thermal drift and to achieve the minimal force between the cantilever and the sample. AFM images were processed by JPK data processing software. Fibril heights/widths were measured by the inbuilt cross-sections method of JPK data processing software. For the analysis of fibril width 50 fibrils per condition were measured .
Generation of Tau constructs to study the effect of fusion protein (GFP) on Tau
For in vitro studies, GFP-tags were fused to either full length Tau protein (2N4R) or the shorter repeat domain containing the “pro-aggregant” deletion mutation ΔK280 (TauRDΔK) at their N-terminus or C-terminus using a flexible linker (Fig. 2A, B). The proteins were expressed in E. coli, except TauRDΔK-GFP which could only be expressed in the baculovirus-Sf9 cell system. His-tags were added at the C-termini to aid in purification.
GFP does not interfere with microtubule assembly induced by full-length Tau.
We first asked whether GFP fusion to Tau affects the physiological function of promoting microtubule assembly as monitored by light scattering at 350 nm (Gaskin et al., 1975) and verified by electron and fluorescence microscopy (Fig. S1A-C). Full length Tau, either untagged or labeled with GFP at either end, is competent to induce MT assembly with similar time courses (t1/2 ~ 1-2min, Fig. 3A, top curves). GFP alone does not support MT assembly (Fig. 3A, bottom curve). By contrast, the Tau repeat domain alone, either with or without ΔK280 mutation, and with or without GFP tag, is not competent to cause MT polymerization (Fig. 3B, bottom curves). This is consistent with the weak binding of TauRD to tubulin and microtubules, which becomes pronounced only when the domains flanking the repeats are present (Gustke et al., 1994). The results mean that GFP moieties attached to the either end of full-length Tau are sufficiently flexible and/or far from the MT binding domain to allow assembly without steric hindrance.
GFP tags create steric hindrance against aggregation of Tau repeat domain.
The central question of this paper is to see whether GFP fusion of the Tau repeat domain allows aggregation into bona fide PHF-like filaments. To test this, TauFL-wt (Fig. 4A) and TauRDΔK (Fig. 4B) and with and without GFP were incubated with the nucleating reagent heparin16000 at 37°C, and aggregation was monitored by UV light scattering at 350 nm. In the case of the repeat domain, the overall extent of aggregation of TauRDΔK (Fig. 4B, green) was 3-fold higher than that of
TauRDΔK-GFP (magenta) whereas GFP-TauRDΔK (blue) showed almost no response, indicating that GFP attached to either end of the Tau repeat domain was strongly inhibitory, even though it carried the pro-aggregant mutation ΔK280. A different picture emerges from full length Tau: Here the unlabeled and GFP-labeled proteins showed a robust increase of light scattering during assembly (Fig. 4A), with some variation in assembly rates and final levels. Note that GFP increases the Tau subunit mass by ~28kD/48kD~58%, roughly consistent with the increase in scattering.
GFP fused TauRDΔK can form amorphous small oligomers and aberrant filaments
The data from light scattering and sedimentation assays (data not shown) were correlated with structural investigations using electron and atomic force microscopy. Full length TauFL-wt aggregated into long (straight and twisted) fibrils (apparent diameter ~24 nm, Fig. 4C).
GFP-TauFL-wt also formed straight or twisted filaments (Fig. 4D) but appeared somewhat thicker in negative stain (~40 nm), consistent with the extra mass of GFP. TauFL-wt-GFP formed long, straight or twisted fibrils with a tendency to coalesce into bundles (Fig. 4E). As a control, the His-tag on TauFL-wt-His had no influence on assembly (similar to Fig 4C, data not shown).
More pertinent to the present study are the results on the shorter construct TauRDΔK which readily forms PHF-like fibers with β-structure (Fig. 4F, see (Barghorn et al., 2000)). However, in contrast to full-length Tau, filament formation was strongly inhibited by GFP-tags on TauRDΔK. Fusion of GFP to the N-terminus (GFP-TauRDΔK) completely abolished fiber aggregation, and only small amorphous assemblies were generated (Fig. 4G). GFP fusion to TauRDΔK at C-terminus (TauRDΔK‑GFP) made it difficult to purify in E.coli due to protein instability, and hence the protein was expressed in Sf9 cells. This protein formed predominantly oligomers and few elongated particles with thicker diameters, distinct from PHFs (~37 nm, Fig. 4H).
Atomic force microscopy of fibrils assembled from full-length Tau-GFP reveals GFP decoration around the core
As an alternative imaging approach we used AFM in tapping mode of unstained Tau aggregates. The spatial resolution in the x-y-plane is limited by the size of the tip, but the z-height is recorded accurately via the tip touching the protein surface. Fig. 5 shows overview images of Tau filaments (left), magnified views (middle), and plots of the height distribution across filaments. TauFL-wt shows long twisted or straight filaments (Fig. 5A) with heights of ~8 nm and apparent widths of ~42 nm, enlarged ~2-fold by the tip-broadening effect, compared with the actual width 10-25 nm (Wegmann et al., 2010). Filaments of GFP-TauFL-wt (Fig. 5B) show a similar core (height ~7nm), but in addition a surrounding halo of height ~3nm which matches the size of the GFP moiety. Unlike GFP tagged full length Tau, TauRDΔK-GFP aggregates are mostly small oligomers and some short thick filaments with height of ~16nm (Fig. 5D), about twice the value of unlabeled fibers (Fig. 5C).
Taken together, the kinetic and structural data show that attachment of GFP to TauRDΔK on either side severely inhibits its aggregation, and the small fraction of fiber-like aggregates is distinct from PHFs.
Mass-per-length analysis by STEM discriminates PHF-like filaments from aberrant structures
PHFs from Alzheimer brains typically consist of two protofilaments with cross-β structure (Fitzpatrick et al., 2017). Given the axial spacing between β-strands of ~0.47 nm, the molecular density of protein subunits would be 1/0.47=2.13 units per nm in each protofilaments, or ~4.26 molecules per nm in a PHF, irrespective of the molecular weight of the subunits. Thus the mass-per-length (MPL) should be ~4.2 times the subunit MW. The MPL value can be determined by STEM which allows one to distinguish PHF-like subunit packing from aberrant structures. Tau filaments with and without GFP were prepared at 37°C for 24 h for STEM analysis and the MPL data were compared with standard MPL data of tobacco mosaic virus (TMV). The results (Fig. 6 and Table 1) show that fibers assembled from the unlabeled repeat domain TauRDΔK agree very well with the theoretical expectations (~4.4 molecules/nm). Fibers from unlabeled full-length
TauFL-wt have ~20% lower values, ~3.4 molecules/nm, even though their appearance by EM or AFM is similar to those of the repeat domain. Both observations are in excellent agreement with our earlier study using a different set of instruments and protein preparations (von Bergen et al., 2006). The apparent decrease by ~20% of TauFL-wt can be explained by the fact that the "fuzzy coat" of full-length Tau is spread out around the perimeter so that its contrast is partly buried in the background. On the other hand, in the case of GFP-labeled proteins, the small fraction of fiber-like structures had very different subunit packings, e.g. ~0.9 or 2.0 for the "long" and "short" fibrils of TauRDΔK-GFP, and ~2.2 for GFP-TauFL-wt fibrils. These values are clearly incompatible with a PHF-like packing of molecules, yet their GFP moieties are close enough to generate FRET signals. MPL data evidently show that GFP fusion changes the aggregation pattern and packing of Tau molecules in filaments (see Table 1 for MPL values).
The progression of Alzheimer Disease can be subdivided into several stages, as judged by abnormal changes in the neuronal protein Tau (notably hyperphosphorylation and aggregation) which spread in the brain with a predictable spatio-temporal sequence, following axonally connected pathways (Braak and Braak, 1991). This suggests that the signal of toxicity spreads via interconnected neurons and/or cells closely associated with them. Various modes of transmitting a toxic signal between cells can be envisaged (Brundin et al., 2008, Walsh and Selkoe, 2016). Currently one of the favored mechanisms is based on the "prion-like" spreading of Tau protein, whereby a misfolded and aggregation-prone form of Tau is transferred from a donor to an acceptor cell where it nucleates the conversion of native Tau to a misfolded state and thus causes aggregation (Vaquer-Alicea and Diamond, 2019, Holmes and Diamond, 2017). This is consistent with the fact that Tau is a neuron-specific protein, and that only neurons develop the abnormal changes of Tau that show up as neurofibrillary tangles or neuropil threads, consisting of bundles of paired helical filaments (PHF) or straight fibers of Tau (SF) (Fitzpatrick et al., 2017). A variant of the hypothesis is that the transfer of pathogenic Tau may occur not directly from donor to acceptor neuron, but indirectly via microglia and exosomes (Asai et al., 2015).
In the "prion-like" hypothesis, the concept of "seeding" plays an important role. However, the term has acquired two distinct meanings which can lead to ambiguities. In the strict sense (and by analogy with other cytoskeletal fibers (Pollard and Borisy, 2003)), seeding it refers to the nucleated self-assembly of native tau into PHF-like filaments in the acceptor cell onto the template provided by the incoming tau, thereby forming tau polymers with a misfolded conformation (Fig. 1). More generally it denotes the transfer of a pathogenic species ("misfolded" Tau) from donor to acceptor cell, resulting in a pathological response (e.g. FRET, phosphorylation, loss of solubility). The tacit assumption is that some species in this transformation (e.g. Tau polymers or oligomers) is pathogenic for the acceptor cell. A widely-used experimental procedure to test the pathogenic potential of a Tau preparation (e.g. from AD brain or transgenic animals) is based on a reporter cell expressing the Tau repeat domain (~13 kDa) fused to XFP [GFP or variants] (Holmes et al., 2014). Exposing the FRET reporter cell to the extracellular Tau preparation may cause a local accumulation of fluorescence (or FRET, if TauRD is fused to FRET pairs like CFP/YFP). By analogy with the "prion-like" hypothesis, such inclusion with elevated fluorescence or FRET are interpreted as bona fide PHF-like aggregates, resulting from the internalization of the extracellular Tau and subsequent templated assembly of the TauRD-XFP molecules.
We argue that this is an over-interpretation of the fluorescence data on structural grounds. As shown in this paper (Fig. 6, 7), steric hindrance prevents TauRD-fusion proteins to assemble into PHF-like fibers. In a proper PHF the core has a tightly packed cross-β-structure, with a 0.47 nm distance between adjacent β-strands (Fitzpatrick et al., 2017). However, this is not compatible with the size of an attached GFP molecule (size ~3x4 nm). Even if a templating Tau assembly were to reach the cytosol of an acceptor cell, it could not combine with the reporter TauRD-GFP fusion molecules to propagate PHF-like structures. 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 fibers (e.g. PABPN1-CspB, Aβ-GFP) (Ochiishi et al., 2016, Buttstedt et al., 2010).
How is it then possible to explain local accumulations of GFP-labeled TauRD? One key factor is that the FRET effect extends up to distances of ~10nm, considerably further than the spacing of secondary structures and sizes of the involved protein molecules, and it allows variable relative orientations of molecules. While full-length Tau tends to associate with microtubules, this is not the case for TauRD which has only a weak affinity for MT (Fig. 3) and instead is rather uniformly distributed in the cytoplasm. However, TauRD has a natively unfolded structure and can undergo multiple weak interactions with other hydrophilic or charged molecules (Mukrasch et al., 2009). Examples are the interactions with ribosomes (Papasozomenos and Binder, 1987, Koren et al., 2019), stress granules (Apicco et al., 2018, Moschner et al., 2014), transport granules (Mercken et al., 1995, Konzack et al., 2007, Aronov et al., 2002). Moreover, several groups showed recently examples of how the low-complexity composition of Tau or TauRD enables it to become spontaneously enriched locally in phase-separated membrane-less compartments, particularly in combination with RNA or cytoskeletal proteins (Wegmann et al., 2018, Zhang et al., 2017, Hernandez-Vega et al., 2017, Ambadipudi et al., 2017).
Neurofibrilly changes in Tau were originally described as one of the cellular hallmarks of AD (Alzheimer, 1907). Their progressive distribution in the brain, correlating with clinical stages (Braak and Braak, 1991), and the discovery of disease-causing mutations in Tau (Goedert and Spillantini, 2001) led to the concept that pathological changes in Tau protein might be causative via the spreading of Tau between brain cells (Clavaguera et al., 2009). This led to a variety of approaches to test the hypothesis, notably cells expressing XFP-labeled full-length Tau or TauRD showing a sensitive FRET reaction (Gorlovoy et al., 2009, Kfoury et al., 2012). The appearance of FRET was described as a "seeding" reaction, however this term has acquired a dual meaning which can lead to ambiguity: On one hand it refers to nucleated assembly of a Tau filament, on the other it may mean the induction of some pathological property (e.g. accumulation of insoluble material, phosphorylation, changes in fluorescence). Several authors have applied bimolecular complementation assays in cells (e.g. split GFP or split luciferase), which by definition measure the direct binding of two proteins, usually in the ratio of two Tau+one complemented reporter molecule (Wang et al., 2017, Wegmann et al., 2016, Chun et al., 2007, Mirbaha et al., 2015, Kim et al., 2015). In these cases one obtains information on dimerization or at best oligomerization, so that conclusions about Tau aggregation cannot be made. An interesting exception is that of Chun et al., 2007, who tagged TauRD with a short fragment of GFP and expressed the remainder of GFP separately, leading to a ratio of 1 TauRD+1 GFP, the same ratio as we used in vitro. Their conclusion was that GFP prevents PHF assembly in cells, in agreement with our observation. Other authors devised seeding assays with readouts based mainly on the reduction of Tau solubility in cells, e.g. (Falcon et al., 2015, Strang et al., 2018). This increases the range and flexibility of biochemical investigation of triggers (e.g. preparations from AD tissue or transgenic animals) and responses (e.g. abnormal changes in Tau, dependence on mutations), but makes the interpretation in terms of PHF aggregation less specific. Thus, sensor cells are valuable tools in assessing pathogenic properties of extracellular triggers, but there is no need to link this to the templated assembly of Tau filaments. By analogy, in ALS research extracellular stress signals can be tested by their effect on cytoplasmic foci of FUS-GFP, without making assumptions on structures of FUS fibers (Marrone et al., 2019).
Our earlier studies on the problem of somatodendritic Tau "missorting" have shown that changes in Tau distribution can be triggered by extracellular stimuli independently of Tau, for example various stress signals, including oxidative stress or Aβ oligomers, or changes in protein degradation systems (Zempel et al., 2013, Balaji et al., 2018). In particular, accumulations of GFP-labeled Tau that mimic seeding can be triggered in neurons by cytokines such as TNFα released from activated microglia (Gorlovoy et al., 2009). It emphasizes the growing evidence that hallmarks of Tau pathology in neurons can be initiated by inflammatory signals from microglia (Ising et al., 2019). This can prompt both the local accumulation, aggregation of assembly-competent Tau as well as other abnormal changes like hyperphosphorylation (e.g. by reducing the activity of phosphatases) or loss of solubility. Noteworthy in this context, "seeding" is observed primarily with heterogeneous Tau preparations that are tissue- or cell-derived, but is inefficient with purified tau samples.
How do these arguments affect approaches to therapy? Earlier attempts to prevent tau pathology were directed at pathological changes of Tau within neurons, assuming they were the carriers of toxicity. Examples are the inhibition of kinases or activation of phosphatases to reduce hyperphosphorylation (Medina, 2018), or inhibitor compounds to block aggregation (Wilcock et al., 2018, Wischik et al., 2014, Pickhardt et al., 2015). Overall these attempts were not successful as treatments. The "prion-like" hypothesis shifted the emphasis to the transfer of tau between cells, which could conceptually be intercepted by Tau-specific antibodies (Yanamandra et al., 2013, Congdon et al., 2016, Gu et al., 2013, Novak et al., 2017). Despite some encouraging results in transgenic mice, success of this approach is still uncertain and would not be expected if tau protein is not the carrier of pathogenicity, as suggested here.
Assembly of Tau-GFP is severely inhibited and the aberrant structures formed are incompatible with that of Alzheimer filaments (Fig.1). Our 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. As a consequence, the observed local increase of FRET in recipient cells in Tau-FRET based assays must be caused by alternative processes such as stress granules or liquid liquid phase transition.
Ethical Approval and Consent to participate – Not applicable
Consent for publication: All authors have given their consent for publication.
Availability of supporting data: All data generated or analysed during this study are included in this published article [and its supplementary information files].
Competing interests: The authors declare that they have no competing interests.
Funding
This project was supported by funding from DZNE, MPG, Katharina Hardt Foundation and Cure Alzheimer's Fund.
Authors' contributions
SK and KT designed, performed and analyzed all invitro biophysical experiments. SK, KT and CK performed electron microscopy analysis. SK performed AFM experiments and analysis. JB and SH performed recombinant protein preparation. RRC performed invitro microtubule assembly experiment. SI and SB performed STEM experiment. SK, KT, EMM and EM wrote the manuscript. EMM and EM supervised the study. All authors read and approved the final manuscript.
Acknowledgements
We thank Ms. Carola Troger for her assistance with STEM experiments and the CAESAR electron microscope facility.
Authors' information
1 DZNE, German Center for Neurodegenerative Diseases, Bonn, Germany
2 CAESAR Research Center, Bonn, Germany
* SK and KT are joint first authors
- Corresponding authors
AD Alzheimer disease
AFM atomic force microscopy
CNS central nervous system
FRET fluorescence resonance energy transfer
GFP green fluorescent protein
PHF paired helical filament
TauFL full-length Tau protein, largest isoform in human CNS (Uniprot P10636-F, htau40)
TauFLΔK full-length Tau protein with pro-aggregant mutation ΔK280
TauRD Tau repeat domain (amyloidogenic domain)
TauRDΔK 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
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Table 1 Summary of the effects of Tau + GFP fusion proteins on aggregation, stimulation of microtubule assembly, and packing of subunits in Tau filaments
