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Mapping interactions with the chaperone network reveals factors that protect against tau aggregation

Nature Structural & Molecular Biology volume 25, pages 384–393 (2018)Cite this article

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Abstract

A network of molecular chaperones is known to bind proteins (‘clients’) and balance their folding, function and turnover. However, it is often unclear which chaperones are critical for selective recognition of individual clients. It is also not clear why these key chaperones might fail in protein-aggregation diseases. Here, we utilized human microtubule-associated protein tau (MAPT or tau) as a model client to survey interactions between ~30 purified chaperones and ~20 disease-associated tau variants (~600 combinations). From this large-scale analysis, we identified human DnaJA2 as an unexpected, but potent, inhibitor of tau aggregation. DnaJA2 levels were correlated with tau pathology in human brains, supporting the idea that it is an important regulator of tau homeostasis. Of note, we found that some disease-associated tau variants were relatively immune to interactions with chaperones, suggesting a model in which avoiding physical recognition by chaperone networks may contribute to disease.

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Fig. 1: In vitro tau aggregation screen identifies chaperones that inhibit or promote tau aggregation.
Fig. 2: Unique chaperone profiles with respect to tau aggregation suggest distinct mechanisms of anti-aggregation activity.
Fig. 3: Anti-aggregation chaperones bind to regions spanning the tau aggregation motifs.
Fig. 4: Tau fibrils formed in the presence of DnaJA2 have a reduced capacity to seed tau aggregation in cells.
Fig. 5: DnaJA2 levels are selectively increased in neurons positive for tau pathology in samples from patients with MCI and late-stage AD.

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Acknowledgements

We thank E. Poss and K. Widjaja for technical support, S. Ambadipudi for NMR analysis and J. Stoehr for advice on ThT assays. We are grateful to the Bardwell group (University of Michigan) for supplying chaperone proteins and M. Diamond (Tau consortium) for providing Clone 1 cells. Human autopsied brain tissue was provided by W. W. Seeley and the UCSF Neurodegenerative Disease Brain Bank, which is supported by the NIH (AG023501 and AG19724 to W. W. Seeley), the Tau Consortium, and the Consortium for Frontotemporal Dementia Research. This work was funded by grants to J.E.G. from the Tau Consortium, BrightFocus Foundation and the NIH (NS059690) and to F.T.F.T. from the NIH (GM104980 and GM111084).

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Author notes

  1. Deceased: Chad A. Dickey.

Authors and Affiliations

  1. Department of Neurology, University of California at San Francisco, San Francisco, CA, USA

    Sue-Ann Mok, Carlo Condello & Jason E. Gestwicki

  2. Department of Pharmaceutical Chemistry, University of California at San Francisco, San Francisco, CA, USA

    Rebecca Freilich, Anne Gillies, Taylor Arhar, Olivier Julien, Victoria A. Assimon, Jennifer N. Rauch, Bryan M. Dunyak & Jason E. Gestwicki

  3. Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Göttingen, Germany

    Javier Oroz, Harindranath Kadavath & Markus Zweckstetter

  4. Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA

    Jungsoon Lee & Francis T. F. Tsai

  5. llawarra Health and Medical Research Institute, School of Biological Sciences, University of Wollongong, Wollongong, New South Wales, Australia

    Mark R. Wilson

  6. Max-Planck-Institut für Biophysikalische Chemie, Goettingen, Germany

    Markus Zweckstetter

  7. Department of Neurology, University Medical Center Göttingen, University of Göttingen, Göttingen, Germany

    Markus Zweckstetter

  8. Department of Molecular Medicine and Byrd Alzheimer’s Research Institute, University of South Florida, Tampa, FL, USA

    Chad A. Dickey

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Contributions

S.-A.M., C.A.D. and J.E.G. conceived and designed the study. S.-A.M., R.F., C.C., J.O., H.K., A.G. and T.A. acquired data. S.-A.M., R.F., C.C., J.O., H.K. and O.J. analyzed and interpreted data. S.-A.M., F.T.F.T., M.R.W., M.Z., J.E.G. drafted and/or revised the manuscript. N.J., V.A.A., B.M.D., J.N.R., J.L., F.T.F.T., M.R.W. and M.Z. contributed reagents.

Corresponding author

Correspondence to Jason E. Gestwicki.

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The authors declare no competing interests.

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Integrated supplementary information

Supplementary Figure 1 Supporting data for in vitro tau aggregation screen.

a) Addition of Hsc70 to tau prior to aggregation induction leads to dose-dependent effects on aggregation. 0N4R tauWT incubated with indicated concentrations of Hsc70 or Hsc70NBD was induced to aggregate with the addition of heparin. The aggregation kinetic parameters (lag time, elongation rate constant, and amplitude) calculated from the corresponding ThT aggregation curves are plotted (mean ± SD, triplicates, representative of 3 independent experiments). b) Calculated elongation rate constant for tau variants tested in the in vitro aggregation screen (mean ± SD, triplicates). c and d) Replicate experiments demonstrating reproducibility of lag time effects. c) 18 chaperones were assayed for effects on 0N4R tauWT aggregation for two independent experiments. The fold change in lag time due to addition of chaperone was plotted for each experiment on the x and y axes, respectively (mean ± SD, triplicates for each experiment). The calculated values for the Spearman’s rank correlation are shown. d) Hsc70 was assayed against the entire panel of 0N4R tau variants in triplicate within the same plate for 3 independent experiments. The fold change in lag time due to addition of full-length Hsc70 was plotted for each replicate paradigm on the x and y axes, respectively (mean ± SD). The calculated values for the Spearman’s rank correlation are shown.

Supplementary Figure 2 Data analysis of additional calculated kinetic parameters from tau aggregation screen.

a) Heatmap representing the effect of individual chaperones on the elongation rate constant parameter of aggregation for each tau variant. For each chaperone-tau combination, the log2 fold change in elongation rate of tau when aggregated in the absence or presence of an equimolar concentration of chaperone was plotted. Red and blue represent chaperone–dependent decreases or increases in aggregation elongation rate constant, respectively. Grey = not tested. b) Dose-dependent effects of chaperone screen hits on tau aggregation lag time. Fold change in lag time of 0N4R tauWT (10 μM) in the presence of multiple chaperone concentrations are plotted (mean ± SD, triplicates). c) Comparison of DnaJA1 and DnaJA2 effects on tau aggregation kinetics. 0N4R tauWT (7.5 µM) was aggregated in the presence of an equimolar concentration of DnaJA1 or DnaJA2. The ThT fluorescence for each reaction is plotted (mean ± SD from triplicate wells is plotted, representative of 2 independent experiments). d) Heatmap of absolute values for lag time kinetic parameter for 0N4R tauWT and ΔK280 in the presence of chaperones. The calculated lag time (h) is displayed according to the indicated color scale for each chaperone-tau combination (1:1 stoichiometry). The lag time value (h) for each tau variant without chaperone is included.

Supplementary Figure 3 DnaJA2 and Hsc70 present similar anti-aggregation behaviors when heparin concentrations are varied or the alternate tau accelerant arachadonic acid is employed.

a) 0N4R tauWT (10 μM) was induced with arachadonic acid in the absence or presence of 5 μM Hsc70 or DnaJA2. Thioflavin T fluorescence was used to monitor the aggregation process (mean ± SD from triplicate wells is plotted). b) ITC traces of heparin (200 μM/1 mM) titrated into cells containing 100 μM Hsc70 or DnaJA2. The average calculated Kd from duplicate experiments is indicated. c) Example aggregation curves of 0N4R tauWT (10 μM) induced with a range of concentrations of heparin in the presence of 5 μM Hsc70 or DnaJA2 (mean ± SD, triplicates).

Supplementary Figure 4 Representative data of Hsc70, Hsp72 and Hsp90 binding to the tau peptide array from three independent experiments.

Raw signal intensity (rfu) of chaperone binding as assayed via fluorescently-tagged His-antibody is plotted for each peptide. The N-terminal amino acid is used to mark the position of each peptide within the 2N4R tau sequence. Dotted red line for each plot represents the mean of the corresponding array dataset. False positive peptide values in the array are not graphed for clarity. Bound peptide regions as defined in materials and methods are indicated by (*).

Supplementary Figure 5 Chaperone screen hits present during the aggregation of tau reduce the amount of pelletable tau material.

a) SDS-PAGE of pellet and supernatant fractions from tau aggregation samples. 10 μM tau samples aggregated for 24 h in the presence of indicated chaperones (20 μM) were centrifuged at 100,000g for 1 h and equal fractions of reactions were subject to SDS-PAGE. b) Quantification of bands corresponding to tau (bars) and indicated chaperone (circles) for each aggregated tau sample is plotted as the fraction of the total amount present in the supernatant plus pellet (mean ± SEM from 3 independent experiments). c) Chaperone addition to pre-formed tau fibrils does not alter their ability to seed aggregation in clone 1 cells. Samples of 0N4R tauWT (10 μM) aggregated in the presence of indicated chaperones (20 μM) were compared to samples of 0N4R tauWT fibrils formed without chaperones followed by incubation with 20 μM of chaperone for 1 h. Equal amounts of tau from each sample (blue, chaperone added prior to aggregation, red, chaperone added after aggregation) were transfected into clone 1 cells and the percentage of cells that formed punctae are plotted (mean ± SD, triplicates).

Supplementary Figure 6 Immunostaining of DnaJA2 and other chaperones in samples from patients with MCI and AD.

Fixed brain sections from MCI, late-stage AD, or non-demented control (NDC) samples were co-stained for chaperone (red), phospho-tau (AT8, green) antibodies as well as the amyloid-binding small molecule, FSB (white). Nuclei were visualized with propidium iodide (PI) stain. a and b) Low magnification images show that MCI samples have a low incidence of AT8 positive neurons compared to late-stage AD samples. Scale bar = 100 microns. c) In an MCI sample, 3D rendering of one face of a neuron showing DnaJA2 is interspersed between or surrounds areas of AT8 staining. Colocalization between DnaJA2 and AT8 is not evident. The neuron shown is the same as presented in Fig. 5b (main text). d) Representative image from late-stage AD samples showing increased DnaJA2 staining adjacent (arrows), but not within, an AT8 and FSB positive neuron. Scale bar = 10 microns. e) DnaJA2 antibody does not detect other J-proteins such as the closely related family member, DnaJA1. Dot blot of recombinant chaperones with the same DnaJA2 antibody used for immunostaining. Ponceau stain of the same blot to verify protein transfer to membrane (representative of 2 independent experiments). f-h) Representative images of chaperone staining patterns in MCI and non-demented control samples. Scale bar = 10 microns for all images. Samples probed with antibodies against f) Hsp72, g) Hsp27 and h) Hsc70. Arrowheads mark neurons in h). Note that sections stained for Hsp27 could not be co-stained for phospho-tau (AT8).

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Mok, SA., Condello, C., Freilich, R. et al. Mapping interactions with the chaperone network reveals factors that protect against tau aggregation. Nat Struct Mol Biol 25, 384–393 (2018). https://doi.org/10.1038/s41594-018-0057-1

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