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  1. Author Response:

    Reviewer #3 (Public Review):

    This paper addresses important questions about how two chaperones, DNAJA2 and DNAJB1, interfere with tau aggregation. The description of their interaction mechanisms and their interaction regions is both novel and interesting to the field of chaperone and tau aggregation. The use of NMR and kinetic analysis is compelling to obtain useful information. This information will be useful to understand the importance and the mode of action of the chaperones in a biological context.

    In general, no attention is paid to how aggregation is triggered. There is no mention of heparin in the results and the quantity of heparin used to trigger aggregation is not written in the method. This is a crucial aspect that is relevant to what aggregation pathway is modeled in vitro, in particular in the light of recent results showing that heparin-induced fibrils are different from brain-extracted fibrils (Fichou et al. Chem Comm 2018, Zhang et al., elife 2019).

    My major concern in this paper is about the assumption that tau aggregation-prone conformers were generated. The conjecture that an excess of heparin increases the population of aggregation-prone conformers is not justified. On the contrary, excess of heparin was shown to form off-pathway oligomers (thus not aggregation-prone) (Ramachandra & Udgaonkar JBC 2011) that do not exhibit exposed PHF6(*) (Fichou et al, Frontiers in neurosciences 2019) expected to be the signature of aggregation-prone conformers (Eschmann et al. Scientific reports 2017; Chen et al. Nat. Comm 2019). If more aggregation conformations were present, they would aggregate more easily, and not be stable as it happens when heparin concentration is increased. Thus, I don't believe that the interpretation that the chaperone interfere with aggregation-prone conformers is justified by the data on heparin-tau complex. In general heparin-tau complex should not be use a proxy for aggregation-prone conformations in the different result sections and in the discussion.

    We thank the reviewer for their valuable comments.

    Following the reviewer’s suggestion, we have added a description to the method section indicating that the aggregation was triggered by the addition of heparin (at 4:1 tau:heparin ratio for the unseeded experiments) or with 1% tau seeds (for the seeded experiments).

    In addition we fully agree that the heparin generated tau fibers are different from brain-extracted fibrils and therefore tried to avoid making any conclusions regarding the mode of chaperone association with the mature fibrils themselves.

    We do however stand behind our statement that excess heparin increases the population of the aggregation-prone extended tau conformers. The seeding competent monomers (Ms) reported by Mirbaha al. eLife 2018 and Chen et al. Nat. Comm 2019 were generated by the incubation of tau with equimolar concentrations of heparin, similar to that used in our manuscript to generate the aggregation-prone species. Furthermore, Mirbaha al. eLife 2018 and Chen et al. Nat. Comm 2019 found the tau species to be similarly expanded both when purified from AD patients and generated in the presence of equimolar heparin concentration.

    However, in light of the reviewer’s comments and in order to confirm that in our NMR experiments the heparin-bound tau monomer indeed displays an expanded conformation, we recorded RDC measurements on our samples. Tau alone, in the absence of heparin, showed large RDC values (10-20 Hz) in the PHF6 regions. The increased RDCs in these regions, which were visible using two alignment media, indicate the presence of a compaction in these regions, in agreement with previous reports. Addition of heparin, however, drastically reduced the value of these RDCs, indicating that the compaction is no longer present in heparin bound tau species. These experiments are added as Figure 4 - figure supplement 3 to the revised manuscript.

    Lastly, while we fully agree with the statement that excess of heparin prevents tau aggregation, this does not necessarily imply that these tau species are not found in the expanded conformation. The non-monotonous change of the aggregation propensity of tau4R as a function of heparin is suggestive of a concentration-enhancement mechanism. At sub-stoichiometric concentration of heparin, multiple tau molecules can interact with a single heparin molecule (at least 2:1 tau:heparin complexes can form), thus increasing the local concentration of tau4R and therefore the likelihood for nucleus formation. An excess of heparin will naturally shift the stoichiometry of the complexes towards 1:1 tau:heparin assemblies. Under these conditions, the effective concentration of tau is rather low (essentially the bulk concentration), i.e., the bimolecular rate of two tau molecules forming a nucleus, and thus the rate of aggregation, will be drastically reduced.

    Furthermore, our NMR experiments do not only show an increase in the accessibility of the PHF6 motifs in the presence of heparin, but this change in the accessibility also correlates with the competence of DNAJB1 to bind tau. Importantly, since DNAJB1 does not bind to heparin alone, it is therefore most plausible to assume that a more expanded conformation of tau is formed in complex with heparin, and that this comprises a substantial contribution to the aggregation of tau.

    The data on P301L/S mutant is more convincing. However, the deduction that DNAJB1 binds P301L/S better because of the exposure of PHF6 is plausible but purely hypothetical (and it should be described as is). I'm in particular concerned with the facts that (i) the PHF6-exposed conformers are likely to represent a small population in the P301L/S mutants and (ii) PHF6* is not exposed in heparin-tau complexes (Fichou et al, Frontiers in neurosciences 2019) and yet they bind DNAJB1.

    We thank the reviewer for this very important comment. In order to provide experimental proof for the increased expansion of the P301L/S mutants we performed RDC measurements, and compared the RDC values to those of wild-type tau monomer (which showed compaction in the PHF6 region). The measured RDCs showed a clear decrease in the PHF6 RDC values of the mutant tau compared to the monomer, despite the two samples being aligned to the same degree. We further recorded the RDC measurements in two separate alignment media (Pf1 bacteriophage or polyethylene glycol/hexanol), and the same ~15% decrease was observed for both, demonstrating that the mutants are slightly more expanded that the wt protein.

    We indeed find that the expanded, PHF6-exposed conformers represent only a small population (~15%) of the overall conformational ensemble of the P301S/L mutants. This small percentage of exposure is, however, entirely consistent with P301S/L tau mutant only interacting weakly with DNAJB1 chaperone, compared to binding of heparin-bound tau species (which present a fully expanded conformational ensemble).

    The new RDC experiments, as well as a better explanation of the differential binding of DNAJB1 to tau mutants and heparin-bound species, are added to the revised version of the manuscript.

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  2. Evaluation Summary:

    This paper addresses the intriguing hypothesis that different molecular chaperones may recognize and bind distinct tau species, and thus may use different mechanisms to prevent tau aggregation. The findings are very interesting and advance our understanding of how chaperones can counteract the deleterious effect of tau amyloidogenesis.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    The authors used NMR spectroscopy and kinetic aggregation assays to investigate how heat-shock protein HSPB1 and two J-proteins (Hsp40), DNAJB1 and DNAJA2, interact with tau and prevent its aggregation. All three chaperones prevent or reduce tau aggregation, but interestingly the authors convincingly demonstrate that they interact with distinct tau species. NMR data (Fig. 1) show that the same two regions within tau are being recognized by HSPB1 and DNAJA2, which seem to bind to the monomeric tau, but DNAJB1 does not appear to bind tau (or rather it binds very weakly). The authors followed up on this interesting observation to show that DNAJB1 does reduce tau aggregation by interacting with a soluble oligomeric tau species. Some interesting observations about distinct specificities of the substrate-binding domains in the two J-proteins are also reported. Overall, this is beautiful work.

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  4. Reviewer #2 (Public Review):

    This is a carefully performed study that combines a range of biophysical experiments to study molecular and mechanistic aspects of the roles of the tau aggregation suppressors HSPB1 and two Hsp40 family members, DNAJA2 and DNAJB1. The differential roles and mechanism involved are dissected and rationalized by the involvement of distinct substrate binding domains CTDI and CTDII in the two Hsp40 proteins. The findings provide novel insight into mechanistic features of the modulation of tau aggregation by heat shock proteins using a combination of in vitro experiments and will form the basis for further studies and validation in a cellular context.

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  5. Reviewer #3 (Public Review):

    This paper addresses important questions about how two chaperones, DNAJA2 and DNAJB1, interfere with tau aggregation. The description of their interaction mechanisms and their interaction regions is both novel and interesting to the field of chaperone and tau aggregation. The use of NMR and kinetic analysis is compelling to obtain useful information. This information will be useful to understand the importance and the mode of action of the chaperones in a biological context.

    In general, no attention is paid to how aggregation is triggered. There is no mention of heparin in the results and the quantity of heparin used to trigger aggregation is not written in the method. This is a crucial aspect that is relevant to what aggregation pathway is modeled in vitro, in particular in the light of recent results showing that heparin-induced fibrils are different from brain-extracted fibrils (Fichou et al. Chem Comm 2018, Zhang et al., elife 2019).

    My major concern in this paper is about the assumption that tau aggregation-prone conformers were generated. The conjecture that an excess of heparin increases the population of aggregation-prone conformers is not justified. On the contrary, excess of heparin was shown to form off-pathway oligomers (thus not aggregation-prone) (Ramachandra & Udgaonkar JBC 2011) that do not exhibit exposed PHF6(*) (Fichou et al, Frontiers in neurosciences 2019) expected to be the signature of aggregation-prone conformers (Eschmann et al. Scientific reports 2017; Chen et al. Nat. Comm 2019). If more aggregation conformations were present, they would aggregate more easily, and not be stable as it happens when heparin concentration is increased. Thus, I don't believe that the interpretation that the chaperone interfere with aggregation-prone conformers is justified by the data on heparin-tau complex. In general heparin-tau complex should not be use a proxy for aggregation-prone conformations in the different result sections and in the discussion.

    The data on P301L/S mutant is more convincing. However, the deduction that DNAJB1 binds P301L/S better because of the exposure of PHF6 is plausible but purely hypothetical (and it should be described as is). I'm in particular concerned with the facts that (i) the PHF6-exposed conformers are likely to represent a small population in the P301L/S mutants and (ii) PHF6* is not exposed in heparin-tau complexes (Fichou et al, Frontiers in neurosciences 2019) and yet they bind DNAJB1.

    Was this evaluation helpful?