Inert and seed-competent tau monomers suggest structural origins of aggregation

  1. Hilda Mirbaha
  2. Dailu Chen
  3. Olga A Morazova
  4. Kiersten M Ruff
  5. Apurwa M Sharma
  6. Xiaohua Liu
  7. Mohammad Goodarzi
  8. Rohit V Pappu
  9. David W Colby
  10. Hamid Mirzaei
  11. Lukasz A Joachimiak  Is a corresponding author
  12. Marc I Diamond  Is a corresponding author
  1. University of Texas Southwestern Medical Center, United States
  2. University of Delaware, United States
  3. Washington University in St. Louis, United States
10 figures, 1 table and 1 additional file

Figures

Seeding activity of tau monomer in cells and in vitro.

(A, B) FL Cys-Tau(2A) was labeled with Alexa488 and resolved by SEC (A), or was fibrillized in the presence of heparin, labeled with Alexa488, sonicated, and the assemblies resolved by SEC (B). The …

https://doi.org/10.7554/eLife.36584.003
Figure 1—source data 1

Data for Mi and Ms seeding activity in cells and in vitro.

https://doi.org/10.7554/eLife.36584.004
Analyses of Mi and Ms by CD and FCS.

(A) CD spectra of Mi and Ms were similar. (B) FCS Diffusion times for Mi, MS, dimer, trimer, and ~10 mer, and the cross-correlation for Mi, Ms, dimer, trimer, and ≥10 mer were determined after …

https://doi.org/10.7554/eLife.36584.005
Figure 2—source data 1

Data for CD and FCS of Mi and Ms.

https://doi.org/10.7554/eLife.36584.006
Fidelity of SEC purification of assemblies.

SEC fidelity was tested by isolating Ms from fractions after fibril sonication. Remaining fractions were combined with Mi, and the mix was re-isolated by SEC. In Group 1, after the first isolation, …

https://doi.org/10.7554/eLife.36584.007
Figure 3—source data 1

Data for fidelity of SEC purification of assemblies.

https://doi.org/10.7554/eLife.36584.008
Heat denaturation of assemblies.

(A–C) Heat-induced dissociation of assemblies. (A) The SEC fraction containing Ms (B5) was heated to 95°C for 3 hr and re-isolated by SEC prior to testing the FRET biosensor assay. No loss in …

https://doi.org/10.7554/eLife.36584.009
Figure 4—source data 1

Data for heat denaturation of assemblies.

https://doi.org/10.7554/eLife.36584.010
Ms self-assembles.

Mi and Ms were incubated at 500 nM or with equivalent amounts (monomer equivalent) of dimer and trimer for various times prior to resolution by SEC. Assemblies were monitored by reading the …

https://doi.org/10.7554/eLife.36584.011
Figure 6 with 1 supplement
Heparin induces transition from Mi to Ms.

(A) Heparin treatment of FL WT tau was carried out for 15 min, 1 hr, or 4 hr. Samples were resolved by SEC, and fractions of various sizes were compared using the biosensor seeding assay. ‘Pre-SEC’ …

https://doi.org/10.7554/eLife.36584.013
Figure 6—source data 1

Data for heparin induced transition from Mi to Ms.

https://doi.org/10.7554/eLife.36584.015
Figure 6—figure supplement 1
SDS-PAGE of tau after sonication or heparin treatment.

(A) Two different FL WT tau preparations were sonicated or not, and 1.5 µg protein was then resolved by SDS-PAGE and coomassie stain. Sonication induced a small degree of protein fragmentation. (B) …

https://doi.org/10.7554/eLife.36584.014
Figure 7 with 1 supplement
Unique XL-MS patterns for different forms of Mi and Ms.

Tau monomers were prepared as described, heated at 95°C for 0, 3 or 24 hr, reacted with DSS, proteolyzed and analyzed by mass spectrometry to define intramolecular crosslinks. Diagrams represent …

https://doi.org/10.7554/eLife.36584.016
Figure 7—source data 1

Summary of triplicate XL-MS datasets.

https://doi.org/10.7554/eLife.36584.018
Figure 7—source data 2

Summary of consensus XL-MS datasets.

https://doi.org/10.7554/eLife.36584.019
Figure 7—figure supplement 1
Frequency of crosslinks decreases with heat incubation.

Heat denaturation of Mi and Ms (fibril-derived and heparin treated for 0.25 hr, 1 hr) decreases the abundance of consensus crosslink pairs (A). Columns represent data after exposure to 95°C for 0 …

https://doi.org/10.7554/eLife.36584.017
Figure 8 with 1 supplement
AD brain contains seed-competent monomer.

Tau from control and AD brains was immunoprecipitated and subjected to SEC. (A) SEC from control brain contained predominantly tau monomer. (B) SEC from AD brain contained a range of tau assembly …

https://doi.org/10.7554/eLife.36584.020
Figure 8—source data 1

Data characterization of patient samples.

https://doi.org/10.7554/eLife.36584.022
Figure 8—source data 2

Summary of patient-derived XL-MS datasets.

https://doi.org/10.7554/eLife.36584.023
Figure 8—figure supplement 1
Different brain homogenization methods yield similar crosslink patterns.

A single AD brain sample was homogenized using four different methods: (A) Dounce homogenization; (B) Pulse sonication; (C) Mechanical homogenization; (D) Mechanical homogenization followed by pulse …

https://doi.org/10.7554/eLife.36584.021
Figure 9 with 1 supplement
Models of Mi and Ms suggest differences in the R1R2 and R2R3 regions.

XL-MS identified pairs were used as restraints in Rosetta to create structural models of discrete tau domains. (A) Schematic highlighting the region of the RD encoding structural differences between …

https://doi.org/10.7554/eLife.36584.024
Figure 9—source data 1

Models of Mi, Ms, control and AD conformations of tau.

https://doi.org/10.7554/eLife.36584.026
Figure 9—source data 2

Rosetta energy and radius of gyration for structural ensembles.

https://doi.org/10.7554/eLife.36584.027
Figure 9—figure supplement 1
Energetics of Rosetta structural ensembles.

The ensembles are shown as a distribution of total energy of each model and radius of gyration for recombinant Mi (A), recombinant Ms (B), control brain-derived Mi (C) and AD-derived Ms (D). Figure …

https://doi.org/10.7554/eLife.36584.025
Figure 10 with 1 supplement
Proteolysis of Mi and Ms reveals distinct patterns.

(A) Mi and Ms were prepared as technical triplicates (N = 3), isolated by SEC, and passed through a 100kD filter immediately prior to exposure to trypsin for 1, 5, 10, 30, 60 and 120 min. Samples …

https://doi.org/10.7554/eLife.36584.028
Figure 10—source data 1

Summary of peptides and their abundance identified in the triplicate Mi and Ms proteolysis experiment.

https://doi.org/10.7554/eLife.36584.030
Figure 10—source data 2

Summary of ratiometric analysis of Mi and Ms peptide abundance.

https://doi.org/10.7554/eLife.36584.031
Figure 10—figure supplement 1
Proteolysis reveals localized differences between Mi and Ms.

The medians of the averaged (N = 3) kinetic profiles were compared as ratios for Mi and Ms. The data were compared to the mean of all ratios (red line) and standard deviation (dotted grey line). …

https://doi.org/10.7554/eLife.36584.029

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Cell line
(HEK293)
Tau RD P301S FRET
Biosensor
Produced by Diamond
lab, also available from
ATCC
ATCC CRL-3275
biological
sample (mouse)
Tau KO mouseJackson Laboratories7251
biological
sample (human)
Alzheimer's Disease
brain, stage VI, frozen
Washington University
in St. Louis
62579, 62732, 61473
biological sample
(human)
Human normal brainWashington University
in St. Louis
60974, 607, 64238
antibodyHJ8.5Produced by Diamond labN/AMonoclonal antibody
against human tau
antibodyPolyclonal Rabbit
Anti-Human Tau
Dako, DenmarkA0024
antibodyECL Anti-rabbit IgG
Horseradish Peroxidase F(ab)
fragment
GE HealthcareNA9340V
recombinant proteinHuman tau 2N4R
(Full Length WT-tau)
Produced by Diamond labN/AMAEPRQEFEV
MEDHAGTYGL
GDRKDQGGYT
MHQDQEGDTD
AGLKESPLQT
PTEDGSEEPG
SETSDAKSTP
TAEDVTAPLV
DEGAPGKQAA
AQPHTEIPEG
TTAEEAGIGD
TPSLEDEAAG
HVTQARMVSK
SKDGTGSDDK
KAKGADGKTK
IATPRGAAPP
GQKGQANATR
IPAKTPPAPK
TPPSSGEPPK
SGDRSGYSSP
GSPGTPGSRS
RTPSLPTPPT
REPKKVAVVR
TPPKSPSSAK
SRLQTAPVPM
PDLKNVKSKI
GSTENLKHQP
GGGKVQIINK
KLDLSNVQSK
CGSKDNIKHV
PGGGSVQIVY
KPVDLSKVTS
KCGSLGNIHH
KPGGGQVEVK
SEKLDFKDRV
QSKIGSLDNI
THVPGGGNKK
IETHKLTFRE
NAKAKTDHGA
EIVYKSPVVS
GDTSPRHLSN
VSSTGSIDMV
DSPQLATLAD
EVSASLAKQG L
recombinant proteinTau (2A)Produced by Diamond labN/AMAEPRQEFEV
MEDHAGTYGL
GDRKDQGGYT
MHQDQEGDTD
AGLKESPLQT
PTEDGSEEPG
SETSDAKSTP
TAEDVTAPLV
DEGAPGKQAA
AQPHTEIPEG
TTAEEAGIGD
TPSLEDEAAG
HVTQARMVSK
SKDGTGSDDK
KAKGADGKTK
IATPRGAAPP
GQKGQANATR
IPAKTPPAPK
TPPSSGEPPK
SGDRSGYSSP
GSPGTPGSRS
RTPSLPTPPT
REPKKVAVVR
TPPKSPSSAK
SRLQTAPVPM
PDLKNVKSKI
GSTENLKHQP
GGGKVQIINK
KLDLSNVQSK
AGSKDNIKHV
PGGGSVQIVY
KPVDLSKVTS
KAGSLGNIHH
KPGGGQVEVK
SEKLDFKDRV
QSKIGSLDNI
THVPGGGNKK
IETHKLTFRE
NAKAKTDHGA
EIVYKSPVVS
GDTSPRHLSN
VSSTGSIDMV
DSPQLATLAD
EVSASLAKQG L
recombinant proteinCys-Tau (2A)Produced by Diamond labN/AMAEPRQEFEV
MEDHACGTYGL
GDRKDQGGYT
MHQDQEGDTD
AGLKESPLQT
PTEDGSEEPG
SETSDAKSTP
TAEDVTAPLV
DEGAPGKQAA
AQPHTEIPEG
TTAEEAGIGD
TPSLEDEAAG
HVTQARMVSK
SKDGTGSDDK
KAKGADGKTK
IATPRGAAPP
GQKGQANATR
IPAKTPPAPK
TPPSSGEPPK
SGDRSGYSSP
GSPGTPGSRS
RTPSLPTPPT
REPKKVAVVR
TPPKSPSSAK
SRLQTAPVPM
PDLKNVKSKI
GSTENLKHQP
GGGKVQIINK
KLDLSNVQSK
AGSKDNIKHV
PGGGSVQIVY
KPVDLSKVTS
KAGSLGNIHH
KPGGGQVEVK
SEKLDFKDRV
QSKIGSLDNI
THVPGGGNKK
IETHKLTFRE
NAKAKTDHGA
EIVYKSPVVS
GDTSPRHLSN
VSSTGSIDMV
DSPQLATLAD
EVSASLAKQG L
commercial assay or kitmicro BCA Protein Assay KitThermo Scientific23235
chemical compoundHeparin sodium salt from
porcine intestinal mucosa
SigmaH4784

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