Tau monomer encodes strains
- University of Texas Southwestern Medical Center, United States
- Washington University in St Louis, United States
Figures
Figure 1
Tau Ms from DS9 retains strain identity.
(A) Clones isolated from DS9 monomer (9.1–9.6) show morphological characteristics similar to DS9. (B) Limited proteolysis digests all the monomer from DS1, but reveals similar protease resistant band patterns for DS9 and DS9.1–9.6. Both DS9 and its sub-strains exhibited a band around 10 kD, and a second band between 20 and 25 kD. (C) Sedimentation analysis was performed on DS1, 9, and its substrains DS9.1–9.6. Total (T) lysate was resolved into supernatant (S) and pellet (P) fractions by ultracentrifugation. Supernatant to pellet ratio loaded on the gel was 1:1 for all samples. DS1 had tau in the supernatant, whereas DS9 and its substrains had tau predominantly in the pellet. The band represents RD-YFP at ~45 kD. (D) DS9 and sub-strains had similar seeding activities upon transduction into P301S FRET biosensor cells. Images are representative of thousands similar cells. Western blots are representative of at least three replicates. Seeding assays represent an individual experiment in which each data point represents a sample analyzed in triplicate. Error bars represent the standard deviation.
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Figure 1—source data 1
Source data for Figure 1D.
- https://doi.org/10.7554/eLife.37813.003
Figure 2
Tau Ms from DS10 creates multiple sub-strains.
(A) Clones isolated from DS10 monomer give rise to cells with multiple morphologies. Four sub-strains were discriminated based on multiple tests. (B) Limited proteolysis of RD-YFP using pronase differentiated the protease resistant cores in the sub-strains. Lane 1 represents DS1, which is comprised of RD-YFP monomer that is completely digested. (C) Sedimentation analysis of RD-YFP was performed on DS1, 10, and DS10.1–4. Total (T) lysate was resolved into supernatant (S) and pellet (P) fractions by ultracentrifugation. Supernatant to pellet ratio loaded on the gel was 1:1 for all samples. DS1 had RD-YFP in the supernatant; DS10, 10.1 and 10.2 had most RD-YFP in the pellet; DS10.3 and DS10.4 had mixed RD-YFP solubility. (D) DS10 sub-strains had distinct seeding activities upon transduction into P301S FRET biosensor cells. Images are representative of thousands similar cells. Western blots are representative of at least three replicates. Seeding assays represent an individual experiment in which each data point represents a sample analyzed in triplicate. Error bars represent the standard deviation.
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Figure 2—source data 1
Source data for Figure 2D.
- https://doi.org/10.7554/eLife.37813.005
Figure 3
Sub-strains trigger unique tau pathology in P301S mice.
10 μg of clarified lysate was injected into the left hippocampi of 3 mo P301S mice, followed by AT8 staining after 4 weeks. Coronal images are oriented with the injection site on the left. DS9 and DS9.1 induced similar pathology in the CA1 and CA3 regions. DS1 induced no pathology. DS10 and DS10.1 induced similar pathology in CA1 and CA3. In both cases, we observed AT8 staining in the cell body throughout CA1 and AT8-positive puncta throughout CA3. DS10.2 induced AT8 signal in both the cell body and along the axons in CA1. There was very little pathology in CA3. DS10.3 induced very little AT8 signal in CA1 and none in CA3. DS10.4 likewise induced little pathology in CA1 region and none in CA3. Each image represents an example from five mice (3 males/2 females or 2 males/3 females per group) treated identically. We noted no differences in induced pathology between males and females. The scale bars represent 200 μm for the whole brain and 50 μm for the CA1 and CA3 regions.
Figure 4
Ms derived from an AD patient produces a single strain.
(A) Clonal cell lines derived from AD-derived total lysate, AD(t), and Ms, AD(m) exhibited identical inclusion morphologies. (B) RD-YFP derived from AD(t) and AD(m) had similar solubility profiles. RD-YFP in DS1 was only present in the supernatant fraction. Total (T) lysate was resolved into supernatant (S) and pellet (P) fractions by ultracentrifugation. Supernatant to pellet ratio loaded on the gel was 1:1 for all samples. (C) Limited proteolysis of RD-YFP aggregates from AD(t) and AD(m) clonal lines produced identical band patterns. Lane 1 represents DS1, which is comprised of RD-YFP monomer that is completely digested. (D) Lysates of AD(t) and AD(m) clonal lines had similar seeding profiles. Images are representative of thousands similar cells. Western blots are representative of at least three replicates. Seeding assays represent an individual experiment in which each data point represents a sample analyzed in triplicate. Error bars represent the standard deviation.
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Figure 4—source data 1
Source data for Figure 4D.
- https://doi.org/10.7554/eLife.37813.012
Figure 5
Ms derived from a CBD patient produces three distinct strains.
(A) Clonal lines derived from CBD total lysate had two distinct inclusion patterns, CBD1(t), CBD2(t). Clonal lines derived from CBD Ms had two patterns identical to those from the total lysate, CBD1(m), CBD2(m), and a third, CBD3(m). (B) RD-YFP from CBD1(t) and CBD1(m) had mixed solubility. In CBD2(t) and CBD2(m), most RD-YFP was present in the supernatant. CBD3(m) had mixed solubility with most RD-YFP in the insoluble fraction. Total (T) lysate was resolved into supernatant (S) and pellet (P) fractions by ultracentrifugation. Supernatant to pellet ratio loaded on the gel was 1:1 for all samples. Lane 1 represents DS1, which is comprised of RD-YFP monomer that is completely digested. (C) RD-YFP aggregates from CBD1(t) and CBD1(m) exhibited similar patterns of proteolysis, with protease-resistant bands around 10–15 kD and a strong band around 15 kD. Proteolysis of RD-YFP from CBD2(t) and CBD2(m) only produced a strong band around 15 kD. CBD3(m) had a unique proteolysis pattern with a band around 10–15 kD. (D) Lysates from clones CBD1(t), CBD1(m), CBD2(t) and CBD2(m) had similar seeding profiles, while lysate from CBD3(m) had more potent seeding activity. Images are representative of thousands similar cells. Western blots are representative of at least three replicates. Seeding assays represent an individual experiment in which each data point represents a sample analyzed in triplicate. Error bars represent the standard deviation.
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Figure 5—source data 1
Source data for Figure 5D.
- https://doi.org/10.7554/eLife.37813.015
Figure 6
Model for families of monomer conformations.
We propose a model that discriminates two general conformational ensembles: Mi and Ms. Mi is inert, whereas Ms has seeding activity. Within Ms, multiple conformations exist that can encode individual or multiple strains. Once an assembly forms, a strain will be faithfully replicated; however, if Ms is isolated from the strain, it can assemble to form a defined set of sub-strains.
Author response image 1
Immunoprecipitation of seeding activity from DS10.2.
Lysates from DS10.2 cells expressing tau RD-YFP in an aggregated form were exposed to HJ9.3 antibody, which recognizes tau aa306-321, followed by immunoprecipitation. Total, supernatant, and pellet fractions were transduced via Lipofectamine into tau RD-CFP/YFP FRET biosensor cells, and the percentage of cells with aggregates was quantified by flow cytometry. After exposure to anti-tau antibody, no seeding activity remained in the supernatant, and all had been removed to the pellet fraction.
Author response image 2
Tau monomer from DS10.2 is predominantly full-length.
Diagram indicates tau RD-YFP and the epitope of HJ9.3, which was used for initial
Tables
Table 1
Sub-strains generated from DS9 monomer isolated by SEC or cutoff filter.
Tau RD-YFP monomer (Ms) was isolated from DS9 either by SEC or 100kD cutoff filter and inoculated into DS1 to create sub-strains. Multiple clones were isolated and characterized by morphology. Columns indicate the number of clones identified (n) and the percentage this represents of the total (%). A single sub-strain was observed regardless of purification method. Classification of cell morphology was performed using blinded analysis.
| Ms | SEC | 100kD filter | ||
|---|---|---|---|---|
| N | % | N | % | |
| 9.1 | 52 | 100 | 31 | 100 |
Table 2
Sub-strains generated from DS10 monomer isolated by SEC or cutoff filter.
Tau RD-YFP monomer (Ms) was isolated from DS10 by either by SEC or 100kD cutoff filter and inoculated into DS1 to create sub-strains. Multiple clones were isolated and characterized by morphology. Columns indicate the number of clones identified (n) and the percentage this represents of the total (%). Isolation of Ms from DS10 by SEC or 100 kD cutoff filter each enabled a similar proportion of sub-strains to form. Classification of cell morphology was performed using blinded analysis.
| Ms | SEC | 100kD filter | ||
|---|---|---|---|---|
| N | % | N | % | |
| 10.1 | 19 | 36 | 12 | 43 |
| 10.2 | 11 | 21 | 3 | 11 |
| 10.3 | 7 | 13 | 5 | 20 |
| 10.4 | 5 | 9 | 3 | 11 |
| 10.5 (sectored) | 11 | 21 | 4 | 15 |
| Total | 53 | 100 | 27 | 100 |
Table 3
Quantification of second generation of sub-strains obtained from DS10.
Ms from each sub-strain of DS10 (10.1–10.5) was inoculated into DS1, and clones of the induced strains were characterized. DS10.1 largely produced a single predominant strain identical to DS10.1 (92%) and another strain DS10.5 that rapidly sectored (8%). DS10.2–10.4 each recreated all other strains. Columns indicate the number (n) of clones characterized and the percentage of the total (%) in each case. Classification of cell morphology was performed using blinded analysis.
| Induced clone | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 10.1 | 10.2 | 10.3 | 10.4 | 10.5 (sectored) | Total | ||||||||
| Ms | n | % | n | % | n | % | n | % | n | % | n | % | |
| 10.1 | 45 | 92 | 0 | 0 | 0 | 0 | 0 | 0 | 4 | 8 | 49 | 100 | |
| 10.2 | 3 | 6 | 37 | 79 | 2 | 4 | 2 | 4 | 3 | 7 | 47 | 100 | |
| 10.3 | 11 | 21 | 3 | 6 | 21 | 42 | 4 | 8 | 12 | 23 | 51 | 100 | |
| 10.4 | 17 | 35 | 3 | 7 | 13 | 27 | 12 | 24 | 3 | 7 | 48 | 100 | |
Table 4
Analysis of AT8 signal in hippocampi of injected mice.
PS19 mice were inoculated with sub-strains from DS9 and DS10 at 3 months into the hippocampus (n = 5 each). After 8 weeks, coronal sections of hippocampus were analyzed by a blinded reviewer educated on different sections as to the characteristics of each clone. One error occurred in analysis of 40 brains.
| Strain inoculated | Blinded analysis |
|---|---|
| DS1 | DS1 were correctly classified. |
| DS9 | DS9 and DS9.1 were indistinguishable. |
| DS9.1 | |
| DS10 | DS10 and DS10.1 were indistinguishable. |
| DS10.1 | |
| DS10.2 | DS10.2 were correctly classified |
| DS10.3 | DS10.3 were correctly classified. |
| DS10.4 | DS10.4 correctly classified 4/5 times, 1/5 incorrectly classified as DS10.3 |
Table 5
Sub-strains generated from AD monomer isolated by SEC or cutoff filter.
Tau monomer (Ms) from AD brain was purified by immunoprecipitation followed by SEC or passage through a 100kD cutoff filter, prior to inoculation into DS1 cells. Columns indicate the number of clones identified (n) and the percentage this represents of the total (%). A single AD sub-strain was identified regardless of purification method. Classification of cell morphology was performed using blinded analysis.
| Ms | SEC | 100kD filter | ||
|---|---|---|---|---|
| N | % | N | % | |
| AD(m) | 47 | 100 | 23 | 100 |
Table 6
Sub-strains generated from CBD monomer isolated by SEC or cutoff filter.
Ms from CBD brain was purified by immunoprecipitation followed by SEC or passage through a 100kD cutoff filter, prior to inoculation into DS1 cells. CBD sub-strains, CBD1-3(m), were quantified. Isolation of Ms from CBD brain by SEC or cutoff filter enabled a similar proportion of sub-strains to form. Columns indicate the number of clones identified (n) and the percentage this represents of the total (%). Ms created similar strain patterns regardless of filtration method. Classification of cell morphology was performed using blinded analysis.
| Ms | SEC | 100kD filter | ||
|---|---|---|---|---|
| N | % | N | % | |
| CBD1(m) | 20 | 36 | 4 | 22 |
| CBD2(m) | 18 | 33 | 7 | 39 |
| CBD3(m) | 17 | 31 | 7 | 39 |
| Total | 55 | 100 | 18 | 100 |
Table 7
Quantification of strains derived from CBD-derived sub-strains.
Monomeric RD-YFP derived from each CBD sub-strain was used to inoculate DS1. The resultant clones were then characterized by morphology. Ms from each sub-strain recreated all three, with a preference for the strain of origin. Columns indicate the number of clones identified (n) and the percentage this represents of the total (%). Classification of cell morphology was performed using blinded analysis.
| Induced clone | ||||||||
|---|---|---|---|---|---|---|---|---|
| CBD1 | CBD2 | CBD3 | Total | |||||
| Input Ms | n | % | n | % | n | % | n | % |
| CBD1(m) | 30 | 57 | 7 | 15 | 10 | 20 | 47 | 100 |
| CBD2(m) | 9 | 17 | 29 | 62 | 10 | 20 | 48 | 100 |
| CBD3(m) | 13 | 26 | 11 | 23 | 31 | 60 | 55 | 100 |
Key resources table
| Reagent | Designation | Source | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (human) | tau RD (LM)-YFP | PMID: 24857020 | ||
| Cell line (human) | DS1; DS9; DS10 | PMID: 24857020 | ||
| Cell line (human) | Tau RD P301S FRET Biosensor | PMID: 25261551, ATCC | ATCC CRL-3275, RRID:CVCL_DA04 | |
| Cell line (human) | DS9.1; DS10.1; DS10.2; DS10.3,DS10.4, AD(t); AD(m); CBD1(t); CBD2(t); CBD1(m); CBD2(m); CBD3(m) | This paper | These are cell lines created from tau seeds (total lysate and/or seed-competent monomer) that derived either from clonal cell lines DS9 and DS10, or Alzheimer’s or Corticobasal Degeneration disease brain samples that were inoculated into DS1 cell lines, as described in the Materials and Methods, and Results sections. | |
| Antibody (rabbit) | TauA (polyclonal against QTAP…KIGSTENL) | This paper | Antibodies used at dilution indicated in Materials and Methods section (1:1000). | |
| Antibody (rabbit) | Anti-GFP (polyclonal) | Rockland Inc. | Rockland antibodies: 600-401-215, RRID:AB_828167 | Antibodies used at dilution indicated in Materials and Methods section (1:1000) |
| Antibody (mouse) | HJ 9.3 (monoclonal against tau RD) | PMID:24075978, PMID:29566794 | RRID:AB_2721235 | Antibodies used at dilution indicated in Materials and Methods section (1:2000) |
| Antibody (donkey) | ECL Anti-Rabbit | GE Lifesciences | NA9340V, | Antibodies used at dilution indicated in Materials and Methods section (1:2000) |
| Antibody (sheep) | ECL Anti-Mouse | GE Lifesciences | NA931V | Antibodies used at dilution indicated in Materials and Methods section (1:2000) |
| Commercial assay | Amersham ECL Western Blotting reagent | GE Lifesciences | GE Lifesciences: RPN2236 | |
| Commercial assay | 100 kD Spin filter | Corning Spin-X UF | Corning: 431481 | |
| Commercial assay | Agarose beads | Pierce protein A/G Plus | Thermo fisher: 20423 | |
| Software | Graphpad Prism | Graphpad software LLC | ||
| Software | FlowJo | FlowJo LLC | ||
| Other | Lipofectamine 2000 | Thermofisher | Thermo fisher: 11668019 | transfection reagent |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.37813.019
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Tau monomer encodes strains
eLife 7:e37813.
https://doi.org/10.7554/eLife.37813
