Inert and seed-competent tau monomers suggest structural origins of aggregation
- University of Texas Southwestern Medical Center, United States
- University of Delaware, United States
- Washington University in St. Louis, United States
Figures
Figure 1
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 …
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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
Figure 2
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 …
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Figure 2—source data 1
Data for CD and FCS of Mi and Ms.
- https://doi.org/10.7554/eLife.36584.006
Figure 3
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, …
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Figure 3—source data 1
Data for fidelity of SEC purification of assemblies.
- https://doi.org/10.7554/eLife.36584.008
Figure 4
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 …
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Figure 4—source data 1
Data for heat denaturation of assemblies.
- https://doi.org/10.7554/eLife.36584.010
Figure 5
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 …
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Figure 5—source data 1
Data for Ms self-assembly.
- https://doi.org/10.7554/eLife.36584.012
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’ …
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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) …
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 …
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Figure 7—source data 1
Summary of triplicate XL-MS datasets.
- https://doi.org/10.7554/eLife.36584.018
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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 …
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 …
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Figure 8—source data 1
Data characterization of patient samples.
- https://doi.org/10.7554/eLife.36584.022
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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 …
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 …
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Figure 9—source data 1
Models of Mi, Ms, control and AD conformations of tau.
- https://doi.org/10.7554/eLife.36584.026
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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 …
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 …
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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
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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). …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional 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 mouse | Jackson Laboratories | 7251 | |
| biological sample (human) | Alzheimer's Disease brain, stage VI, frozen | Washington University in St. Louis | 62579, 62732, 61473 | |
| biological sample (human) | Human normal brain | Washington University in St. Louis | 60974, 607, 64238 | |
| antibody | HJ8.5 | Produced by Diamond lab | N/A | Monoclonal antibody against human tau |
| antibody | Polyclonal Rabbit Anti-Human Tau | Dako, Denmark | A0024 | |
| antibody | ECL Anti-rabbit IgG Horseradish Peroxidase F(ab) fragment | GE Healthcare | NA9340V | |
| recombinant protein | Human tau 2N4R (Full Length WT-tau) | Produced by Diamond lab | N/A | MAEPRQEFEV 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 protein | Tau (2A) | Produced by Diamond lab | N/A | MAEPRQEFEV 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 protein | Cys-Tau (2A) | Produced by Diamond lab | N/A | MAEPRQEFEV 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 kit | micro BCA Protein Assay Kit | Thermo Scientific | 23235 | |
| chemical compound | Heparin sodium salt from porcine intestinal mucosa | Sigma | H4784 |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.36584.032
