When doctors perform autopsies to look at the brain tissue of people with Alzheimer’s disease, they find toxic buildups of certain proteins – in particular, a protein called tau – in structures called ‘aggregates’. People with more severe dementia have more tau aggregates in their brain. Aggregates form when individual proteins stick together in repetitive patterns, much like the way a single Lego block might attach to another identical one. Like all proteins, tau is built from a string of amino acids that folds into a specific shape. Normally folded tau proteins do not form aggregates. It was not clear that an individual tau protein had two distinct forms—structures associated with health (“good”) or disease (“bad”).

Mirbaha et al. have now studied the folding pattern of purified tau proteins with a sophisticated technology called mass spectrometry. This technique can measure changes in tiny amounts of protein. Some of the purified proteins had been extracted from human brains (from people with and without Alzheimer’s). To detect which of the proteins were toxic, Mirbaha et al. also grew simple human cells in a dish that were designed to react specifically to the bad forms of tau. This allowed the good and bad forms of tau to be isolated.

Mirbaha et al. discovered that in the good form of tau the parts of the protein that allow it to stick to itself are hidden, folded inside. By contrast, the bad form of tau exposes the parts that allow it to aggregate, enabling the protein to build upon itself to form a large, toxic assembly.

The shape-shifting concept established by Mirbaha et al. might apply to other proteins that form toxic aggregates. This could help us to better understand how many other neurodegenerative diseases develop and progress. Recognizing that the shapes that tau forms can be categorized as either ‘good’ or ‘bad’ may also help to develop new treatments for Alzheimer’s disease. Drugs could be designed to stabilize the good form of tau, or to help remove the bad form from the brain. Furthermore, if the shape-shift described by Mirbaha et al. can be measured early enough in patients, it may allow treatments for Alzheimer’s before people have developed any detectable symptoms.

Amyloids are ordered protein assemblies, typically rich in beta sheet, that underlie multiple disorders such as Alzheimer’s disease (AD). Amyloid-forming proteins include tau, synuclein, and expanded polyglutamine proteins such as huntingtin, among many others. It is unknown how or why intracellular proteins such as tau transition from a relatively inert form to one that efficiently self-assembles into ordered structures in vivo. This process begins with the formation of a pathogenic ‘seed,’ a structure that serves as a template for homotypic fibril growth. This structural transition could be a critical event in the pathogenesis of neurodegeneration. Under defined conditions and relatively high concentrations (typically micromolar), recombinant tau monomer will form amyloid fibrils in vitro. However the basis of spontaneous assembly in cells is unknown. The conversion of a protein from a monomer to a large, ordered multimer could occur by several mechanisms, but the first step probably involves the formation of a seed. This event, and indeed the actual conformation or assembly state of the protein that constitutes the ‘minimal’ seed, has remained obscure. This has led to the idea that a seed is potentially transitory, arising from an equilibrium between two states: one relatively aggregation-resistant, and another that is short-lived. A seed could be a single molecule, or several. Based on extrapolation from kinetic aggregation studies, it has been suggested that a critical seed for tau and polyglutamine peptide amyloid formation is a single molecule (Chirita et al., 2005; Bhattacharyya et al., 2005; Kar et al., 2011), while an earlier study (among others [Ramachandran and Udgaonkar, 2013]) has proposed a tau multimer (Friedhoff et al., 1998). Isolation of the seed-competent form of tau could be critical to understanding the initiation of disease and the design of more effective diagnostics and therapeutics.

Tau forms amyloids that underlie neurodegeneration in a variety of neuropathological syndromes, collectively termed tauopathies (Lee et al., 2001). These include AD and frontotemporal dementias, among many others. Multiple groups, including ours, have now observed that tau will propagate an aggregated state from the outside to the inside of a cell, between cells, across synapses, and within brain networks (Sanders et al., 2016). In prior work, we used size exclusion chromatography (SEC) to define tau trimers as the minimal unit of spontaneous cellular uptake and intracellular amyloid formation, and proposed this as the smallest particle capable of propagating aggregates between cells (Mirbaha et al., 2015). This work involved application of ‘naked’ protein assemblies derived from recombinant protein or human brain onto cultured ‘biosensor’ HEK293 cells or primary neurons that express a tau aggregation reporter (Frost et al., 2009a; Holmes et al., 2014). Biosensor cells and primary neurons alike take up tau aggregates via macropinocytosis (Holmes et al., 2013). The aggregates subsequently serve as highly specific templates to trigger intracellular amyloid formation (Holmes et al., 2014; Sanders et al., 2014). We have also determined that preincubation of cationic lipids such as lipofectamine with tau seeds facilitates their direct transduction into a cell, bypassing the physiologic uptake mechanism (Holmes et al., 2014; Furman et al., 2015). Lipofectamine-mediated delivery into biosensor cells allows direct quantitation of seed titer for both tau and α-synuclein (Holmes et al., 2013).

Tau is intrinsically disordered upon isolation from bacteria or mammalian cells and is relatively inert in terms of spontaneous self-assembly. However under various conditions, including exposure to polyanions such as heparin, tau will form aggregates via nucleated self-assembly (Goedert et al., 1996; Pérez et al., 1996). It is unknown how these experimental conditions relate to the initiation of aggregation in human brain. We have now purified various stable forms of full-length tau monomer from recombinant sources and human brain. One is relatively inert and is stable for long periods. Another is ‘seed-competent,’ triggers amyloid formation in cells and in vitro, and exhibits intrinsic properties of self-assembly. We have used crosslinking with mass spectrometry (XL-MS) to probe the structures of these molecules. Models of discrete regions within the RD predict that differential exposure of hexapeptide motifs previously known to be important for amyloid formation distinguishes the two forms of tau. These models are supported by limited proteolysis studies. The identification of distinct and stable forms of tau monomer, including some that are uniquely seed-competent, bears directly on how we understand the initiation of protein aggregation in the tauopathies.

We propose that tau monomer occupies two distinct and stable conformational ensembles. One set of structures (collectively termed M_i) is relatively inert, while another has intrinsic ability to self-assemble, and acts as a template, or seed, for fibril growth in vitro and in cells (collectively termed M_s). Multiple controls indicated that our original preparation of fibril-derived M_s is in fact a monomer, uncontaminated by larger assemblies. Tau monomer purified from AD brain also had intrinsic seeding activity, and self-associated to produce larger seed-competent assemblies. A model restrained by the XL-MS data, and consistent with biochemical studies, predicts that VQIVYK and VQIINK sequences assume an open configuration in all types of M_s (fibril-derived, heparin-induced, and AD-derived). By contrast, the model predicts lack of VQIINK/VQIVYK exposure in M_i. Limited proteolysis studies are consistent with this idea, although clearly more detailed biochemical, biophysical, and structural analyses will be needed to test its validity. Taken together, these data establish a new concept for tau: this intrinsically disordered protein has multiple, stable monomeric states, functionally distinguished by the presence or absence of seeding activity.

Amyloid proteins form progressively larger assemblies over time, and it has been difficult to define the composition of the minimal seed. Mandelkow and colleagues studied tau aggregation in vitro and concluded that a seed of 8–12 molecules existed in their experimental system (Friedhoff et al., 1998). By contrast, Kuret and colleagues posited an ‘intermediate’ of tau that could subsequently initiate self-assembly, and their data, based on extrapolation of tau concentrations needed to enable development of thioflavin fluorescence in vitro, were consistent with a monomeric seed (Chirita et al., 2005). Wetzel and colleagues also proposed that a monomer is the basis of a ‘thermodynamic nucleus’ that templates the aggregation of synthetic polyglutamine peptides (Bhattacharyya et al., 2005). However, no prior study has previously identified stable forms of tau monomer that seed amyloid formation.

The actual cause of tau aggregation in tauopathies is unknown. It has been proposed that dissociation of tau monomer from microtubules, possibly due to phosphorylation, allows high concentration and self-association to form pathogenic assemblies (Mandelkow and Mandelkow, 2012). In this study, using a single source of recombinant protein, we define distinctly structured seed-competent and inert forms of tau. We have similarly identified seed-competent species in human brain. In reality ‘seed-competent’ and ‘inert’ forms of tau almost certainly represent multiple structural ensembles separated by defined energy and/or kinetic barriers. The barrier for conversion of an inert to a seed-competent form of tau can apparently be overcome by incubation with heparin and/or incorporation into a fibril. In neurons, other factors such as post-translational modifications and heterologous binding events likely play a role. Identification of the factors that trigger conversion from inert to seed-competent forms will thus have obvious implications for understanding disease mechanisms.

Isolation of seed-competent monomer from AD brain, with a very mild purification that explicitly excludes sonication or vigorous tissue homogenization, strongly suggests that this form of tau exists in vivo. Furthermore, we observed that both recombinant M_s and AD-derived M_s build multimeric assemblies in vitro far more efficiently than M_i or control-derived monomer. Thus, we hypothesize that a uniquely structured form of tau may be required for efficient assembly growth in cells. This contrasts with the idea that multimeric assemblies uniquely stabilize the conformation of otherwise unstructured proteins as they incorporate into the growing fibril, or that liquid-liquid phase separation with extremely high local concentration underlies tau aggregation (Wegmann et al., 2018). Instead, we imagine that the initiation of aggregation in human brain might begin with a stable transition of tau monomer from an inert to a seed-competent form. To fully study this process will require more extensive biochemical purification of tau M_s from the earliest stages of disease.

M_s has a remarkably stable structure, as it resists heat denaturation at 95°C for up to 3 hr. This suggests a heretofore unrecognized conformation of tau that, to account for its slow denaturation, likely involves multiple intra-molecular interactions involving short and long range amino acid contacts. XL-MS provides some indication of what these might be, and crosslinks between aa150 and R1/R2 appear to mark a seed-competent conformation. In agreement with the XL-MS results, we observed that heat inactivation of M_s seeding activity occurs with a lag phase, rather than first order time-dependent decay. This implies a complex tertiary structure in which M_s has multiple seed-competent intermediates. Future XL-MS studies performed at different temperatures could reveal these structures. With more advanced methods to interrogate the structure of monomeric tau in patient material, we imagine that ‘seed-competent monomer’ will in fact represent myriad structures, depending on the underlying disease. This could provide an explanation for how a single tau protein might self-assemble into diverse amyloid strains. We note with excitement a recent study of the yeast prion Sup35 from the Tanaka laboratory. Like tau, Sup35 is intrinsically disordered, yet they have observed local structure that influences the conformations of fibrils it can form (Ohhashi et al., 2018).

Without further studies to identify structures of tau at higher resolution, we cannot know for certain why one form acts as a seed and another does not. However, we gained important insights when we modeled the configurations of R1R2 and R2R3 using Rosetta, with crosslinks as restraints. With obvious caveats, our models predicted that the local environment surrounding two hexapeptide motifs, VQIINK and VQIVYK, which are required for tau to form amyloid structures, may explain the differences between seed-competent and inert forms. In the models of M_i, and control brain-derived tau, these motifs lie buried in hairpin structures. By contrast, in M_s and AD-derived tau, both are exposed. VQIINK and VQIVYK thus might mediate intermolecular interaction in a growing assembly. In support of our structural model, the proteolysis experiments corroborate differences in exposure of the VQIINK and VQIVYK sequences in the R1R2 and R2R3 regions between M_i and M_s. We note with great enthusiasm the recent study of Fitzpatrick et al. (Fitzpatrick et al., 2017), which defined critical sequences of tau within the amyloid core that are based on VQIVYK and adjacent amino acids. Indeed, it has been recently observed that heparin binding involves residues spanning 270–290, and promotes expansion of the remainder of the molecule (Zhao et al., 2017). This is consistent with our predictions of relative exposure of VQIINK/VQIVYK. The diversity of exposed core elements (almost certainly beyond VQIINK/VQIVYK) could specify the formation of assemblies that give rise to distinct strains, as suggested by work from the Tanaka laboratory (Ohhashi et al., 2018). Consistent with this idea, the Fitzpatrick et al. study indicates that in AD-derived tau fibrils the VQIVYK sequence plays a key role in the core amyloid structure (along with adjacent amino acids), but the VQIINK sequence does not (Fitzpatrick et al., 2017). We also note that multiple disease-associated mutations in tau affect residues in close proximity to VQIINK/VQIVYK. For example, our models predict that serine or leucine substitutions at P301 (which cause dominantly inherited tauopathy) would uniquely destabilize the local structure and promote exposure of the VQIINK/VQIVYK sequences. Future experiments will test these ideas more definitively.

All data generated or analysed during this study are included in the manuscript and supporting files.

1. 1. Acker CM
   2. Forest SK
   3. Zinkowski R
   4. Davies P
   5. d'Abramo C

   (2013) Sensitive quantitative assays for tau and phospho-tau in transgenic mouse models

   Neurobiology of Aging 34:338–350.

   https://doi.org/10.1016/j.neurobiolaging.2012.05.010

   • PubMed
   • Google Scholar
2. 1. Bhattacharyya AM
   2. Thakur AK
   3. Wetzel R

   (2005) Polyglutamine aggregation nucleation: thermodynamics of a highly unfavorable protein folding reaction

   PNAS 102:15400–15405.

   https://doi.org/10.1073/pnas.0501651102

   • PubMed
   • Google Scholar
3. 1. Buschmann V
   2. Weston KD
   3. Sauer M

   (2003) Spectroscopic study and evaluation of red-absorbing fluorescent dyes

   Bioconjugate Chemistry 14:195–204.

   https://doi.org/10.1021/bc025600x

   • PubMed
   • Google Scholar
4. 1. Chattopadhyay K
   2. Saffarian S
   3. Elson EL
   4. Frieden C

   (2002) Measurement of microsecond dynamic motion in the intestinal fatty acid binding protein by using fluorescence correlation spectroscopy

   PNAS 99:14171–14176.

   https://doi.org/10.1073/pnas.172524899

   • PubMed
   • Google Scholar
5. 1. Chirita CN
   2. Congdon EE
   3. Yin H
   4. Kuret J

   (2005) Triggers of full-length tau aggregation: a role for partially folded intermediates

   Biochemistry 44:5862–5872.

   https://doi.org/10.1021/bi0500123

   • PubMed
   • Google Scholar
6. 1. Elias JE
   2. Gygi SP

   (2010) Target-decoy search strategy for mass spectrometry-based proteomics

   Methods in Molecular Biology 604:55–71.

   https://doi.org/10.1007/978-1-60761-444-9_5

   • PubMed
   • Google Scholar
7. 1. Fitzpatrick AWP
   2. Falcon B
   3. He S
   4. Murzin AG
   5. Murshudov G
   6. Garringer HJ
   7. Crowther RA
   8. Ghetti B
   9. Goedert M
   10. Scheres SHW

   (2017) Cryo-EM structures of tau filaments from Alzheimer's disease

   Nature 547:185–190.

   https://doi.org/10.1038/nature23002

   • PubMed
   • Google Scholar
8. 1. Friedhoff P
   2. von Bergen M
   3. Mandelkow EM
   4. Davies P
   5. Mandelkow E

   (1998) A nucleated assembly mechanism of alzheimer paired helical filaments

   PNAS 95:15712–15717.

   https://doi.org/10.1073/pnas.95.26.15712

   • PubMed
   • Google Scholar
9. 1. Frost B
   2. Jacks RL
   3. Diamond MI

   (2009a) Propagation of tau misfolding from the outside to the inside of a cell

   Journal of Biological Chemistry 284:12845–12852.

   https://doi.org/10.1074/jbc.M808759200

   • PubMed
   • Google Scholar
10. 1. Frost B
    2. Ollesch J
    3. Wille H
    4. Diamond MI

    (2009b) Conformational diversity of Wild-type tau fibrils specified by templated conformation change

    Journal of Biological Chemistry 284:3546–3551.

    https://doi.org/10.1074/jbc.M805627200

    • Google Scholar
11. 1. Furman JL
    2. Holmes BB
    3. Diamond MI

    (2015) Sensitive detection of proteopathic seeding activity with FRET flow cytometry

    Journal of Visualized Experiments 106:e53205–5.

    https://doi.org/10.3791/53205

    • PubMed
    • Google Scholar
12. 1. Goedert M
    2. Jakes R
    3. Spillantini MG
    4. Hasegawa M
    5. Smith MJ
    6. Crowther RA

    (1996) Assembly of microtubule-associated protein tau into Alzheimer-like filaments induced by sulphated glycosaminoglycans

    Nature 383:550–553.

    https://doi.org/10.1038/383550a0

    • PubMed
    • Google Scholar
13. 1. Grimm M
    2. Zimniak T
    3. Kahraman A
    4. Herzog F

    (2015) xVis: a web server for the schematic visualization and interpretation of crosslink-derived spatial restraints

    Nucleic Acids Research 43:W362–W369.

    https://doi.org/10.1093/nar/gkv463

    • PubMed
    • Google Scholar
14. 1. Holmes BB
    2. DeVos SL
    3. Kfoury N
    4. Li M
    5. Jacks R
    6. Yanamandra K
    7. Ouidja MO
    8. Brodsky FM
    9. Marasa J
    10. Bagchi DP
    11. Kotzbauer PT
    12. Miller TM
    13. Papy-Garcia D
    14. Diamond MI

    (2013) Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds

    PNAS 110:E3138–E3147.

    https://doi.org/10.1073/pnas.1301440110

    • PubMed
    • Google Scholar
15. 1. Holmes BB
    2. Furman JL
    3. Mahan TE
    4. Yamasaki TR
    5. Mirbaha H
    6. Eades WC
    7. Belaygorod L
    8. Cairns NJ
    9. Holtzman DM
    10. Diamond MI

    (2014) Proteopathic tau seeding predicts tauopathy in vivo

    PNAS 111:E4376–E4385.

    https://doi.org/10.1073/pnas.1411649111

    • PubMed
    • Google Scholar
16. 1. Jeganathan S
    2. von Bergen M
    3. Brutlach H
    4. Steinhoff HJ
    5. Mandelkow E

    (2006) Global hairpin folding of tau in solution

    Biochemistry 45:2283–2293.

    https://doi.org/10.1021/bi0521543

    • PubMed
    • Google Scholar
17. 1. Joachimiak LA
    2. Walzthoeni T
    3. Liu CW
    4. Aebersold R
    5. Frydman J

    (2014) The structural basis of substrate recognition by the eukaryotic chaperonin TRiC/CCT

    Cell 159:1042–1055.

    https://doi.org/10.1016/j.cell.2014.10.042

    • PubMed
    • Google Scholar
18. 1. Kadavath H
    2. Jaremko M
    3. Jaremko Ł
    4. Biernat J
    5. Mandelkow E
    6. Zweckstetter M

    (2015) Folding of the tau protein on microtubules

    Angewandte Chemie International Edition 54:10347–10351.

    https://doi.org/10.1002/anie.201501714

    • PubMed
    • Google Scholar
19. 1. Kahraman A
    2. Herzog F
    3. Leitner A
    4. Rosenberger G
    5. Aebersold R
    6. Malmström L

    (2013) Cross-link guided molecular modeling with ROSETTA

    PLoS ONE 8:e73411.

    https://doi.org/10.1371/journal.pone.0073411

    • PubMed
    • Google Scholar
20. 1. Kar K
    2. Jayaraman M
    3. Sahoo B
    4. Kodali R
    5. Wetzel R

    (2011) Critical nucleus size for disease-related polyglutamine aggregation is repeat-length dependent

    Nature Structural & Molecular Biology 18:328–336.

    https://doi.org/10.1038/nsmb.1992

    • PubMed
    • Google Scholar
21. 1. Laidler KJ

    (1984) The development of the Arrhenius equation

    Journal of Chemical Education 61:494.

    https://doi.org/10.1021/ed061p494

    • Google Scholar
22. 1. Lange OF
    2. Rossi P
    3. Sgourakis NG
    4. Song Y
    5. Lee HW
    6. Aramini JM
    7. Ertekin A
    8. Xiao R
    9. Acton TB
    10. Montelione GT
    11. Baker D

    (2012) Determination of solution structures of proteins up to 40 kDa using CS-Rosetta with sparse NMR data from deuterated samples

    PNAS 109:10873–10878.

    https://doi.org/10.1073/pnas.1203013109

    • PubMed
    • Google Scholar
23. 1. Lasker K
    2. Förster F
    3. Bohn S
    4. Walzthoeni T
    5. Villa E
    6. Unverdorben P
    7. Beck F
    8. Aebersold R
    9. Sali A
    10. Baumeister W

    (2012) Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach

    PNAS 109:1380–1387.

    https://doi.org/10.1073/pnas.1120559109

    • PubMed
    • Google Scholar
24. 1. Lee VM
    2. Goedert M
    3. Trojanowski JQ

    (2001) Neurodegenerative tauopathies

    Annual Review of Neuroscience 24:1121–1159.

    https://doi.org/10.1146/annurev.neuro.24.1.1121

    • PubMed
    • Google Scholar
25. 1. Leitner A
    2. Joachimiak LA
    3. Bracher A
    4. Mönkemeyer L
    5. Walzthoeni T
    6. Chen B
    7. Pechmann S
    8. Holmes S
    9. Cong Y
    10. Ma B
    11. Ludtke S
    12. Chiu W
    13. Hartl FU
    14. Aebersold R
    15. Frydman J

    (2012) The molecular architecture of the eukaryotic chaperonin TRiC/CCT

    Structure 20:814–825.

    https://doi.org/10.1016/j.str.2012.03.007

    • PubMed
    • Google Scholar
26. 1. Mandelkow EM
    2. Mandelkow E

    (2012) Biochemistry and cell biology of tau protein in neurofibrillary degeneration

    Cold Spring Harbor Perspectives in Medicine 2:a006247–7.

    https://doi.org/10.1101/cshperspect.a006247

    • PubMed
    • Google Scholar
27. 1. Mirbaha H
    2. Holmes BB
    3. Sanders DW
    4. Bieschke J
    5. Diamond MI

    (2015) Tau trimers are the minimal propagation unit spontaneously internalized to seed intracellular aggregation

    Journal of Biological Chemistry 290:14893–14903.

    https://doi.org/10.1074/jbc.M115.652693

    • PubMed
    • Google Scholar
28. 1. Morozova OA
    2. March ZM
    3. Robinson AS
    4. Colby DW

    (2013) Conformational features of tau fibrils from Alzheimer's disease brain are faithfully propagated by unmodified recombinant protein

    Biochemistry 52:6960–6967.

    https://doi.org/10.1021/bi400866w

    • PubMed
    • Google Scholar
29. 1. Ohhashi Y
    2. Yamaguchi Y
    3. Kurahashi H
    4. Kamatari YO
    5. Sugiyama S
    6. Uluca B
    7. Piechatzek T
    8. Komi Y
    9. Shida T
    10. Müller H
    11. Hanashima S
    12. Heise H
    13. Kuwata K
    14. Tanaka M

    (2018) Molecular basis for diversification of yeast prion strain conformation

    PNAS 115:2389–2394.

    https://doi.org/10.1073/pnas.1715483115

    • PubMed
    • Google Scholar
30. 1. Pérez M
    2. Valpuesta JM
    3. Medina M
    4. Montejo de Garcini E
    5. Avila J

    (1996) Polymerization of tau into filaments in the presence of heparin: the minimal sequence required for tau-tau interaction

    Journal of Neurochemistry 67:1183–1190.

    https://doi.org/10.1046/j.1471-4159.1996.67031183.x

    • PubMed
    • Google Scholar
31. 1. Ramachandran G
    2. Udgaonkar JB

    (2013) Mechanistic studies unravel the complexity inherent in tau aggregation leading to Alzheimer's disease and the tauopathies

    Biochemistry 52:4107–4126.

    https://doi.org/10.1021/bi400209z

    • PubMed
    • Google Scholar
32. 1. Rinner O
    2. Seebacher J
    3. Walzthoeni T
    4. Mueller LN
    5. Beck M
    6. Schmidt A
    7. Mueller M
    8. Aebersold R

    (2008) Identification of cross-linked peptides from large sequence databases

    Nature Methods 5:315–318.

    https://doi.org/10.1038/nmeth.1192

    • PubMed
    • Google Scholar
33. 1. Sanders DW
    2. Kaufman SK
    3. DeVos SL
    4. Sharma AM
    5. Mirbaha H
    6. Li A
    7. Barker SJ
    8. Foley AC
    9. Thorpe JR
    10. Serpell LC
    11. Miller TM
    12. Grinberg LT
    13. Seeley WW
    14. Diamond MI

    (2014) Distinct tau prion strains propagate in cells and mice and define different tauopathies

    Neuron 82:1271–1288.

    https://doi.org/10.1016/j.neuron.2014.04.047

    • PubMed
    • Google Scholar
34. 1. Sanders DW
    2. Kaufman SK
    3. Holmes BB
    4. Diamond MI

    (2016) Prions and protein assemblies that convey biological information in health and disease

    Neuron 89:433–448.

    https://doi.org/10.1016/j.neuron.2016.01.026

    • PubMed
    • Google Scholar
35. 1. von Bergen M
    2. Barghorn S
    3. Li L
    4. Marx A
    5. Biernat J
    6. Mandelkow EM
    7. Mandelkow E

    (2001) Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local beta-structure

    Journal of Biological Chemistry 276:48165–48174.

    https://doi.org/10.1074/jbc.M105196200

    • PubMed
    • Google Scholar
36. 1. von Bergen M
    2. Friedhoff P
    3. Biernat J
    4. Heberle J
    5. Mandelkow EM
    6. Mandelkow E

    (2000) Assembly of tau protein into alzheimer paired helical filaments depends on a local sequence motif ((306)VQIVYK(311)) forming beta structure

    PNAS 97:5129–5134.

    https://doi.org/10.1073/pnas.97.10.5129

    • PubMed
    • Google Scholar
37. 1. Wegmann S
    2. Eftekharzadeh B
    3. Tepper K
    4. Zoltowska KM
    5. Bennett RE
    6. Dujardin S
    7. Laskowski PR
    8. MacKenzie D
    9. Kamath T
    10. Commins C
    11. Vanderburg C
    12. Roe AD
    13. Fan Z
    14. Molliex AM
    15. Hernandez-Vega A
    16. Muller D
    17. Hyman AA
    18. Mandelkow E
    19. Taylor JP
    20. Hyman BT

    (2018) Tau protein liquid-liquid phase separation can initiate tau aggregation

    The EMBO Journal 37:e98049.

    https://doi.org/10.15252/embj.201798049

    • PubMed
    • Google Scholar
38. 1. Yanamandra K
    2. Kfoury N
    3. Jiang H
    4. Mahan TE
    5. Ma S
    6. Maloney SE
    7. Wozniak DF
    8. Diamond MI
    9. Holtzman DM

    (2013) Anti-tau antibodies that block tau aggregate seeding in vitro markedly decrease pathology and improve cognition in vivo

    Neuron 80:402–414.

    https://doi.org/10.1016/j.neuron.2013.07.046

    • PubMed
    • Google Scholar
39. 1. Zhao J
    2. Huvent I
    3. Lippens G
    4. Eliezer D
    5. Zhang A
    6. Li Q
    7. Tessier P
    8. Linhardt RJ
    9. Zhang F
    10. Wang C

    (2017) Glycan determinants of Heparin-Tau interaction

    Biophysical Journal 112:921–932.

    https://doi.org/10.1016/j.bpj.2017.01.024

    • PubMed
    • Google Scholar

[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for submitting your work entitled "Inert and seed-competent tau monomers elucidate the structural origins of aggregation" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Senior/Reviewing Editor. The reviewers have opted to remain anonymous.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

The reviewers raised major concerns over the principal finding that a form of heat-stable tau monomer possesses seeding activity. In this case, it seems that generating adequate controls could take longer than 2-3 months, and the outcome of such experiments seems uncertain. Furthermore, the secondary finding of constraints for monomer seed structure also requires major revisions or might even need to be removed as pointed out by reviewer 1. For these reasons, we cannot proceed with your paper. However, if you were able to gather the data needed to fully support your hypothesis, we will be prepared to give this study another round of consideration and to send it to the same reviewers.

Reviewer #1:

1) In this paper, the authors claim to have identified two distinct monomeric forms of tau, where one form is seed-competent and the other is inert. Somehow, the seed-competent monomer can be heated to 95°C for 3 hours and still be conformationally distinct from the inert monomer. This is an extraordinary claim that will need extraordinary evidence to support it. What kind of intra-molecular interactions would resist this type of treatment? Especially, given that tau is disordered to start with. What if there were contaminating filamentous tau species (for example, formed from self-assembly due to prolonged storage and/or freeze-thaw)? Such filaments may have a far greater seed-competency than the monomer, dimer and trimer fractions tested and, therefore, even a very small amount of contamination could have a big effect. This might form a much simpler explanation of the presented results. For this reason, it would be important to know how the seeding activities of the M_s fraction and AD-derived tau monomer compared to the total pool of molecular tau species and to tau filaments alone. The majority of published work on seeded tau aggregation has been conducted using filamentous tau seeds and it would, therefore, be very useful to have this direct comparison presented here. At present, the authors only test the effects of contamination by dimers and trimers of tau.

2) Also, the in vitro experiments all start with heparin-induced filament formation. These are then sonicated, centrifuged and put over a SEC column to obtain the seed-competent monomer. Yet, the authors in multiple places (including the title) state that they have elucidated the structural 'origins' of tau aggregation. This claim is not supported by the data: the data work backwards from filaments to monomers, which is not necessarily the same as starting from some 'aggregation origin' to form filaments. In this context, the authors' claim that the use of a dounce homogenizer would prevent the liberation of tau monomers from filaments during brain homogenization is also important. I am not convinced this is actually the case. Neurofibrillary tangles are very large, filling most of the somato-dendritic compartment, and are likely broken up by a dounce homogenizer. The authors should directly test this before concluding that AD brain-derived seed-competent tau monomers are not actually derived from tau filaments. The centrifugation steps used prior to size exclusion chromatography (10,000 xg, 10 min, for recombinant protein and 21,000 xg, 15 min, for brain homogenates) would pellet the majority of filamentous tau aggregates and tangles, thereby excluding them from comparisons.

3) The authors present cross-link mass-spectrometry data to study the conformation of the seed-competent and inert monomers. These experiments lead to the identification of a few distance restraints between different regions of the protein (Figure 7), which are then used to model the conformations using ROSETTA (Figure 8). It is outrageous to assume one can get anywhere near reliable 3D atomic models from such little data (the authors even describe differences in the accessibility of 2 hexapeptides inside this ~400-residue long protein!). The XL-MS data may be valuable, but Figure 8 and the corresponding section "Models of…" in the Results should be removed in a revised version of the paper. One prediction from Figure 4 would be that the red cross-link in Figure 7D and F would persist if samples were first incubated for 3 hours at 95^oC. In the context of necessary extraordinary evidence, the authors should include this experiment in a revised version of the manuscript.

Reviewer #2:

This paper provides evidence for a provocative hypothesis, that tau monomer with the VQIINK and VQIVYK sequences exposed in a consistent conformational ensemble is one of the seeds for initiating Tau aggregation.

While the data in this paper are generally supportive of this hypothesis, this paper was very hard to read/review critically as the authors forced the reviewers to go back to already published papers to get all the details-this paper has to be self-contained if I am to review the revised version.

Since M_s self-assembles efficiently under conditions where seeding is being assessed in the cell reporter line (heparin and Lipofectamine), how can the authors be confident that the monomer is doing the seeding and not a self-assembled M_s? This needs to be explicitly addressed and the conditions for the cell reported seeding experiments need to be discussed in detail.

Has the heparin + Lipofectamine only control been done in the cell reporter line? I am sorry if I missed this.

It seems important to have heavy and light Tau 50:50 in the crosslinking experiment to be certain that oligomeric crosslinks are not contributing.

This paper seems to be an important contribution, and I hope the authors can revise the paper not assuming the readers have read all their papers, while integrating the control results or discussing them if they have been done.

Reviewer #3:

This article characterizes the structural requirements for tau seeding activity. The approach is innovative, in that the identification of seeding activity follows physical characterization rather than kinetic analysis. The results are novel, in that they identify a monomeric form of full-length tau as having seeding activity. The work also is relevant for tauopathic disease, as it integrates analysis of brain tissue derived tau in the analysis. Despite these strengths, a weakness of the work is that it relies solely on hydrodynamic analysis to deduce tau structure, which depends on molecular shape as well as size. As a result, conclusions regarding the size of tau seeds seem premature and incomplete. Specifically, there is risk that the monomeric seeding activity identified by authors reflects break down of tau into aggregation-prone fragments.

1) Figure 1 shows size exclusion chromatograms for globular protein standards and for various tau species. Leaving aside for a moment that SEC should be calibrated in terms of hydrodynamic radius rather than MW, it is worrying that M_i tau elutes with ovalbumin, a globular protein of similar MW but a much smaller hydrodynamic radius (half!) than 2N4R tau protein (PMID: 14769047, 15823045, 19226187, et al.). Since M_i is a gel-filtered form of M_r, this result could be a sign of proteolysis, which can generate small monomeric fragments having greater aggregation propensity and possibly seeding activity. Proteolysis is always a concern when working with tau protein, but especially for M_s because it is generated by extensively incubation/sonication at elevated temperatures. Authors should provide SDS-PAGE or M_S data on M_h, M_i, M_r, and M_s so as to disclose to what extent these preparations are proteolyzed. Because the seeding assays are sensitive, generation of even minor amounts of aggregation-competent fragments could yield a positive and confounding seeding result.

2) Since SEC is sensitive to shape as well as size, it is inappropriate to interpret eluted tau species only in terms of size. FCS depends on shape and size too, so it does not provide a "second opinion". The seed depletion and spiking experiments (Figure 3) don't control for this problem either.

3) It seems that M_r should be treated the same as M_s, i.e. incubated and then sonicated, if it is to yield an M_i that can act as a control for M_s.

4) The legend to Figure 1D states that "Seeding activity of each fraction was evaluated by SEC". The meaning of this statement is not clear.

5) The seeding assay used 0N4R tau, whereas M_i and M_s seeds were prepared from 2N4R tau. Why was this done?

6) In the recently reported structure of tau fibrils (PMID: 28678775), the segment VQIVYK interacts with 373THKLTF378. Can authors comment on solvent exposure of THKLTF in their models?

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Thank you for resubmitting your work entitled "Inert and seed-competent tau monomers suggest structural origins of aggregation" for further consideration at eLife. Your revised article has been favorably evaluated by Anna Akhmanova as the Senior/Reviewing Editor, and three reviewers.

The major issues raised by the reviewers have been addressed in revision. Although there was some remaining skepticism about monomeric Tau as the seed, this paper merits publication. Undoubtedly, this hypothesis will generate a lot of interest and catalyze further experimentation to scrutinize this concept. This is good for the field, because if this paper ultimately stands the test of time, it will change how we think about tauopathies.

[Editors’ note: the author responses to the first round of peer review follow.]

Reviewer #1:

1) In this paper, the authors claim to have identified two distinct monomeric forms of tau, where one form is seed-competent and the other is inert. Somehow, the seed-competent monomer can be heated to 95°C for 3 hours and still be conformationally distinct from the inert monomer. This is an extraordinary claim that will need extraordinary evidence to support it. What kind of intra-molecular interactions would resist this type of treatment? Especially, given that tau is disordered to start with.

We were indeed initially as surprised as the reviewer to see the results of these tests, which imply strong, but not unheard-of, protein stability. Indeed, prion infectivity is well known to be resistant to boiling and autoclaving. We thus used XL-MS to test for structural changes over time at 95°C. In Figure 7 we have now studied two forms of seed-competent monomer (fibril-derived, and heparin-derived), tracking the persistence of the critical crosslinks over time. We observe that this crosslink tracks with seeding activity, being detectable at 3h, but gone by 24h of heating at 95°C. We also note (in unpublished data) that 8M urea treatment completely abolishes secondary structure detected by XL-MS, as would be predicted.

One of the major points of this paper is to challenge the notion that tau being “disordered” is synonymous with “unstructured.” Our data are consistent with it being a protein that adopts multiple, stable conformational ensembles, even as a monomer in solution. In support of this general notion, we were excited to read a recent publication from the Tanaka laboratory which reports that, as we have observed for tau, Sup35 monomer (another “intrinsically disordered” prion protein) exhibits signs of discrete local structures that trigger it to form distinct strains (Ohhashi et al., 2018).

What if there were contaminating filamentous tau species (for example, formed from self-assembly due to prolonged storage and/or freeze-thaw)? Such filaments may have a far greater seed-competency than the monomer, dimer and trimer fractions tested and, therefore, even a very small amount of contamination could have a big effect. This might form a much simpler explanation of the presented results.

This is an excellent hypothesis that we have excluded by multiple controls:

a) In all cases except those specified to involve a freeze-thaw, we immediately took monomer preps from the SEC column to transduce cells. There was no prolonged storage of frozen material.

b) In Figure 1 we now pass putative monomer through a 100kD filter prior to seeding, and find that the activity passes efficiently through the filter. By contrast, dimer is largely blocked, and trimer is almost completely blocked.

c) In Figures 1 and 6, we directly compare seeding activity of monomer, dimer, trimer, and fibrillar species in dose responses. Seeding activity per nM of material is actually very similar across assembly sizes, which argues for a proportional increase in seeding activity for fibrils per molecule (i.e. assemblies are more efficient than monomer). But for dimers and trimers (most likely to contaminate the SEC preps) to see artifactual seeding activity would require enormous contamination.

d) In Figure 3, within the limits of detection, the SEC column perfectly excludes larger species from the monomer fraction.

e)The SEC column is loaded with forms of tau that are soluble after a 10,000 x g spin, excluding large fibrils.

For this reason, it would be important to know how the seeding activities of the M_s fraction and AD-derived tau monomer compared to the total pool of molecular tau species and to tau filaments alone.

The seeding activity of recombinant monomer and fibrils are remarkably similar. Please see experiments cited in Figure 6. We did not directly compare recombinant M_s prepared in vitro with AD-derived M_s, however we have noted that they have relatively similar seeding efficiencies in multiple experiments.

The majority of published work on seeded tau aggregation has been conducted using filamentous tau seeds and it would, therefore, be very useful to have this direct comparison presented here. At present, the authors only test the effects of contamination by dimers and trimers of tau.

Based on our second response to reviewer #1, we contend that it is essentially impossible that filamentous tau is significantly contaminating our preparations. In Figure 6C we have now directly compared the seeding efficiency between monomer and fibrils (sonicated and unsonicated). We also refer the reviewer to Figure 8D. In this experiment we spiked tau KO mouse brain with recombinant fibrils or M_s, and carried out our standard purification with SEC. In this case, we isolated minimal signal from fibril-spiked brain in the ~20-mer fraction, but no signal in the monomer fraction. By contrast, after spiking the brain preparation with M_s we recovered robust monomer signal. It is thus highly unlikely that fibrils are contaminating our preparations.

2) Also, the in vitro experiments all start with heparin-induced filament formation. These are then sonicated, centrifuged and put over a SEC column to obtain the seed-competent monomer. Yet, the authors in multiple places (including the title) state that they have elucidated the structural 'origins' of tau aggregation. This claim is not supported by the data: the data work backwards from filaments to monomers, which is not necessarily the same as starting from some 'aggregation origin' to form filaments.

The reviewer is correct to point out that inferring “origins” of aggregation solely based on species fragmented from fibrils would be fraught. However, in addition to this approach (which is only how we first approached the question), we have also studied tau monomer that is converted by heparin exposure from an inert to seed-competent form, and have also isolated and studied brain-derived tau monomer. In Figure 6A we use brief exposure to heparin (prior to any larger assemblies forming) to create seed-competent monomer. Please note in Figure 6A that at 15 minutes the seeding activity purifies predominantly in the monomer fraction, and not larger assemblies. Thus, there is essentially no chance that the heparin-induced monomer we isolate was previously part of a larger assembly. Second, in Figure 8 we purify tau monomer from AD brain (controlling for possible liberation of tau monomer from fibrils (Figure 8D). This AD-derived monomer (but not control brain monomer) has intrinsic properties of self-assembly and seeding. Since (in the absence of heparin) we do not have “catalytic” control of tau monomer’s conformational change in vitro, to the best of our ability we have attempted to isolate a seed-competent monomer that was not previously part of a fibril. We thus feel justified in referring to structural “origins” of aggregation as we study this monomer in comparison to an inert form. However, we defer to the editor with regard to this disagreement in terminology.

In this context, the authors' claim that the use of a dounce homogenizer would prevent the liberation of tau monomers from filaments during brain homogenization is also important. I am not convinced this is actually the case.

We took very seriously this critique, and thus included a new control, described in Figure 8D of spiking mouse tau KO brain either with fibrils or M_s prior to dounce homogenization. There is no detectable liberation of tau monomer from fibrils following dounce homogenization.

Neurofibrillary tangles are very large, filling most of the somato-dendritic compartment, and are likely broken up by a dounce homogenizer. The authors should directly test this before concluding that AD brain-derived seed-competent tau monomers are not actually derived from tau filaments. The centrifugation steps used prior to size exclusion chromatography (10,000 xg, 10 min, for recombinant protein and 21,000 xg, 15 min, for brain homogenates) would pellet the majority of filamentous tau aggregates and tangles, thereby excluding them from comparisons.

We agree and are perhaps misunderstanding the point of this comment. As we are attempting here to define the smallest unit of seeding activity, this seems to be an appropriate step in the preparation of material.

3) The authors present cross-link mass-spectrometry data to study the conformation of the seed-competent and inert monomers. These experiments lead to the identification of a few distance restraints between different regions of the protein (Figure 7), which are then used to model the conformations using ROSETTA (Figure 8). It is outrageous to assume one can get anywhere near reliable 3D atomic models from such little data (the authors even describe differences in the accessibility of 2 hexapeptides inside this ~400-residue long protein!).

First, we thank the reviewer for pointing out a certain absurdity in attributing any structure to the extreme N- and C-termini of tau. Although we never attempted to imply 3D structural knowledge of tau based on XL-MS, we have now studiously avoided any reference to “structure” as it would be implied by NMR, cryo-EM, or crystallography. Instead, we have attempted to make very clear that XL-MS provides restraints for a “structural model.” Furthermore, we have now conscientiously avoided any reference to global tau structure, and have focused only on putative hairpin regions in the repeat domain. Based on the fragment libraries employed in our ROSETTA simulation there is a propensity to form secondary structure surrounding the two amyloid cores, VQIVYK and VQIINK, which leads to a diversity of models that contain hairpin-like structures within the RD1/RD2 or RD2/DR3 interface. When the seed-competent crosslinks to the RD2 region are used in the simulation we shift the equilibrium away from hairpin conformations at these sites, more so in RD1/RD2 than in RD2/RD3. Furthermore, we have limited any interpretation of XL-MS to regions in which multiple consensus crosslinks to RD2 exist. This allows us to build a model that is in fact fairly testable (and which we are pursuing in ongoing work). To this end, in Figure 10 we have now added limited proteolysis data that is consistent with our model. We hope we have made clear in the Results and Discussion that actual atomic-level resolution will require different types of studies. XL-MS and the model it supports provide a starting point with which to investigate actual structures in tau that allow it to self-assemble and act as a seed. In our reference to a “structural model” we are only intending to leave the reader with an intriguing and plausible hypothesis for our findings.

The XL-MS data may be valuable, but Figure 8 and the corresponding section "Models of…" in the Results should be removed in a revised version of the paper. One prediction from Figure 4 would be that the red cross-link in Figure 7D and F would persist if samples were first incubated for 3 hours at 95^oC. In the context of necessary extraordinary evidence, the authors should include this experiment in a revised version of the manuscript.

Based on this excellent suggestion we have now included this data in a new Figure 7. We hope the reviewer is satisfied with the qualifications we have put on the interpretations of our XL-MS results, and don’t believe that proposing a very limited, local structural model based on ROSETTA is too bold.

Reviewer #2:

This paper provides evidence for a provocative hypothesis, that tau monomer with the VQIINK and VQIVYK sequences exposed in a consistent conformational ensemble is one of the seeds for initiating Tau aggregation.

While the data in this paper are generally supportive of this hypothesis, this paper was very hard to read/review critically as the authors forced the reviewers to go back to already published papers to get all the details-this paper has to be self-contained if I am to review the revised version.

We have attempted to elucidate all methods used in this version.

Since M_s self-assembles efficiently under conditions where seeding is being assessed in the cell reporter line (heparin and Lipofectamine), how can the authors be confident that the monomer is doing the seeding and not a self-assembled M_s? This needs to be explicitly addressed and the conditions for the cell reported seeding experiments need to be discussed in detail.

This is indeed a difficult question. The reviewer correctly points out that it is impossible to know what exactly happens within cells. We believe the data in Figure 5 and 8, however, are important. In these cases, we observe that monomer assembles quickly to form larger seed-competent assemblies (especially in the case of AD-derived monomer), while the larger assemblies themselves (dimer, trimer) will not self-associate. We are not certain how further to discuss the cell seeding experiments, other than to describe our methods, which we hope now are quite clear, and have been extensively reported in prior publications.

Has the heparin + Lipofectamine only control been done in the cell reporter line? I am sorry if I missed this.

Every experiment includes Lipofectamine-treated buffer alone as a control. In experiments summarized in Figure 6 we also “transfected” cells with heparin alone and measured seeding. There was no effect, and we have alluded to this in the figure legend. We didn’t see the need to include completely negative data for this, but can include it if desired. Please note also that we see seeding activity from AD-derived monomer that has never had exposure to heparin.

It seems important to have heavy and light Tau 50:50 in the crosslinking experiment to be certain that oligomeric crosslinks are not contributing.

In the XL-MS experiments we used multiple controls to rule out multimers. Please note Figure 6—figure supplement 1, in which we display an example of a gel in which we have studied cross-linked heparin-induced M_s, and find no evidence of multimerization. Critically, as part of our experimental protocol in every case when crosslinking monomer we run a control gel to rule out higher-order species (dimers, etc.). Additionally, when we probed the structures of M_i and M_s using limited proteolysis we passed the two forms of tau through a 100kD filter immediately prior to trypsin exposure, thus excluding multimers.

Reviewer #3:

[…] A weakness of the work is that it relies solely on hydrodynamic analysis to deduce tau structure, which depends on molecular shape as well as size. As a result, conclusions regarding the size of tau seeds seem premature and incomplete. Specifically, there is risk that the monomeric seeding activity identified by authors reflects break down of tau into aggregation-prone fragments.

In this manuscript we have indeed used hydrodynamic analyses to infer sizes of assemblies, resolving those that are monomer from dimers, trimers, etc. We make no claims about the precise hydrodynamic radii. Additionally, in the current resubmission, we use other methods to infer monomer structure (e.g. double-label fluorescence correlation spectroscopy, 100kD filter cutoffs, and molecular crosslinking to confirm monomer structure). In responses below we address the question of seed-competent tau fragments in more detail.

1) Figure 1 shows size exclusion chromatograms for globular protein standards and for various tau species. Leaving aside for a moment that SEC should be calibrated in terms of hydrodynamic radius rather than MW, it is worrying that M_i tau elutes with ovalbumin, a globular protein of similar MW but a much smaller hydrodynamic radius (half!) than 2N4R tau protein (PMID: 14769047, 15823045, 19226187, et al.).

We thank the reviewer for educating us as to an appropriate indicator for standards on SEC, and have altered our figures accordingly. We do not intend to use SEC on the Superdex 200 column as a precise marker of hydrodynamic radius or molecular size. Instead we are simply seeking to pick fractions where tau monomer is likely to be separated from larger assemblies. In choosing the fraction B5 as the source of monomer, we do so recognizing that tau monomer appears to reflect an ensemble of structures that are slightly polydisperse as they come off of the column (especially in comparison to ovalbumin, which is more monodisperse). In multiple other experiments described especially in response to reviewer #1, we have attempted to test whether the minimal tau seed could be a multimer, and we find no evidence for this. As a final point, in our prior manuscript (Mirbaha et al., 2015), we carried out crosslinking of assemblies derived from putative monomer, dimer, and trimer fractions, and ran out the samples on a gel (Figure 1E, F, G). While we were studying tau RD, please note the fidelity of our SEC, in which we observed crosslinked assemblies that correlated with our predicted assembly sizes.

Since M_i is a gel-filtered form of M_r, this result could be a sign of proteolysis, which can generate small monomeric fragments having greater aggregation propensity and possibly seeding activity. Proteolysis is always a concern when working with tau protein, but especially for M_s because it is generated by extensively incubation/sonication at elevated temperatures.

While we have been concerned about proteolysis, we have found no evidence of this in the absence of sonication (which indeed can produce fragments, as in Figure 6—figure supplement 1A). First of all, we emphasize that we find seed-competent tau monomer in conditions that do not involve sonication, and which do not change tau size. For example, in the new manuscript, please see Figure 6—figure supplement 1B, in which we have taken inert and heparin-induced seed-competent monomers, crosslinked them (or not) and resolved them by SDS-PAGE. While there are several species visible (it is very difficult to have a completely pure monomer preparation), even with the over-loaded gel the reviewer will observe that there is no evidence of significantly smaller fragments or larger assemblies associated with the seeding activity. We have also carried out multiple controls to rule out multimerization, even of small fragments, as referenced in prior responses. In particular, we refer the reviewer to double-label FCS experiments in Figure 2, in which we find no evidence that multiple labels are incorporated into putative monomers, while we easily detect them in putative dimers and trimers. Furthermore, the heat denaturation steps, which we show are sufficient to dissociate oligomers, do not change the seeding activity (or elution characteristics) of the seed-competent monomer. Finally, in Figure 7C we observe that sonication has no effect on the seeding activity of otherwise inert monomer, M_i, despite causing a minor degree of fragmentation (Figure 6—figure supplement 1A). We believe at this point the data do not support the hypothesis that we are somehow purifying tiny amounts of seed-competent, aggregated fragments that account for the activity of M_s.

Authors should provide SDS-PAGE or M_S data on M_h, M_i, M_r, and M_s so as to disclose to what extent these preparations are proteolyzed. Because the seeding assays are sensitive, generation of even minor amounts of aggregation-competent fragments could yield a positive and confounding seeding result.

This is an excellent caveat. In response to this reviewer we performed SDS-

PAGE on sonicated monomer and observed evidence of fragmentation (Figure 6—figure supplement 1A). In Figure 6C we observe that sonication does not produce seed-competent species from M_i. Nonetheless we additionally confirmed by other means that we were not generating aggregation-prone fragments by simply treating tau monomer (confirmed intact) with heparin, and also by isolating seed-competent monomer through gentle methods from AD brain. It seems to us to be incredibly unlikely that 15min of heparin exposure is sufficient to fragment tau to form cryptic seed-competent fragments than then spontaneously self-assemble.

2) Since SEC is sensitive to shape as well as size, it is inappropriate to interpret eluted tau species only in terms of size. FCS depends on shape and size too, so it does not provide a "second opinion". The seed depletion and spiking experiments (Figure 3) don't control for this problem either.

We take the reviewer’s point, and would emphasize only that control proteins elute from the SEC as rough markers of tau size. We make no specific reference to the “molecular weight” of the tau assemblies we elute from the SEC columns. Furthermore, the column we used (Superdex 200) is designed to resolve an enormous window of assembly sizes. It is not ideal to resolve ovalbumin and tau, and thus it is not surprising that these two proteins co-elute to some extent. We have predominantly been interested in resolving monomer, dimer, trimer, etc., and not attempting to determine a precise hydrodynamic radius. While FCS and SEC do use similar metrics to infer size, they are not identical (FCS does not involve interaction of protein with a column substrate), and the use of double-label FCS enables us to rule out multimeric assemblies of tau within the putative monomer fraction. Likewise, the seed depletion and spiking experiments are not done to determine size, per se, but to rule out the possibility of contamination of larger species (putative dimers and trimers) into the monomer fraction.

3) It seems that M_r should be treated the same as M_s, i.e. incubated and then sonicated, if it is to yield an M_i that can act as a control for M_s.

As part of our revision we have directly tested whether sonication of M_i produces seeding activity in the absence of heparin (Figure 6C). It does not. We would also point out that we can produce M_s without sonication, whether by brief (15min) exposure of recombinant monomer to heparin, or by purification of monomer from AD brain.

4) The legend to Figure 1D states that "Seeding activity of each fraction was evaluated by SEC". The meaning of this statement is not clear.

We have clarified this statement.

5) The seeding assay used 0N4R tau, whereas M_i and M_s seeds were prepared from 2N4R tau. Why was this done?

This was done purely for “historical” reasons because the Colby lab uses this form of tau for their seeding assays.

6) In the recently reported structure of tau fibrils (PMID: 28678775), the segment VQIVYK interacts with 373THKLTF378. Can authors comment on solvent exposure of THKLTF in their models?

The R4/R’ interface (which includes the THKLTF region) according to secondary structure and modeling analysis as well as previous work is predicted to be disordered. In our models, both M_i and M_s, the THKLTF region is solvent exposed and not engaged in any interactions. The published cryoEM structure of the PHFs is also not consistent with many of the previous studies using solid state NMR and other methods. The conditions under which the fibrils form and the presence of the PTMs could favor a specific conformational arrangement of a fibril. Notably, the core of the fibril mediated by RD3 and RD4 in cryoEM structure buries many charged residues, which is quite surprising as this should destabilize the contacts between VQIVYK and THKLTF. More experiments will reveal the importance of this regions role in fibrils, in particular in strains, beyond this structure of a fibril derived from a single AD patient as recently reported.

Author details

1. Hilda Mirbaha

   Center for Alzheimer's and Neurodegenerative Diseases, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Conceptualization, Resources, Supervision, Writing—original draft, Writing—review and editing

   Competing interests

   No competing interests declared

2. Dailu Chen

   Center for Alzheimer's and Neurodegenerative Diseases, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Investigation, Methodology, Writing—original draft

   Competing interests

   No competing interests declared

3. Olga A Morazova

   Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, United States

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

4. Kiersten M Ruff

   Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, United States

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3240-1856

5. Apurwa M Sharma

   Center for Alzheimer's and Neurodegenerative Diseases, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

6. Xiaohua Liu

   Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

7. Mohammad Goodarzi

   Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology

8. Rohit V Pappu

   Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, United States

   Contribution

   Investigation, Methodology, Writing—original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-2568-1378

9. David W Colby

   Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, United States

   Contribution

   Resources, Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

10. Hamid Mirzaei

    Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, United States

    Contribution

    Resources, Data curation, Formal analysis, Supervision, Investigation, Methodology, Writing—original draft, Project administration

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-5013-3432

11. Lukasz A Joachimiak

    Center for Alzheimer's and Neurodegenerative Diseases, University of Texas Southwestern Medical Center, Dallas, United States

    Contribution

    Data curation, Formal analysis, Supervision, Methodology, Writing—original draft, Project administration, Writing—review and editing

    For correspondence

    lukasz.joachimiak@utsouthwestern.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-3061-5850

12. Marc I Diamond

    Center for Alzheimer's and Neurodegenerative Diseases, University of Texas Southwestern Medical Center, Dallas, United States

    Contribution

    Formal Analysis

    For correspondence

    Marc.Diamond@UTSouthwestern.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-8085-7770

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