1. Biochemistry and Chemical Biology
  2. Neuroscience
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Tau monomer encodes strains

  1. Apurwa M Sharma
  2. Talitha L Thomas
  3. DaNae R Woodard
  4. Omar M Kashmer
  5. Marc I Diamond  Is a corresponding author
  1. University of Texas Southwestern Medical Center, United States
  2. Washington University in St Louis, United States
Research Advance
Cite this article as: eLife 2018;7:e37813 doi: 10.7554/eLife.37813
8 figures, 8 tables and 1 additional file

Figures

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.

https://doi.org/10.7554/eLife.37813.002
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.

https://doi.org/10.7554/eLife.37813.004
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.

https://doi.org/10.7554/eLife.37813.009
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.

https://doi.org/10.7554/eLife.37813.011
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.

https://doi.org/10.7554/eLife.37813.014
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.

https://doi.org/10.7554/eLife.37813.018
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.

https://doi.org/10.7554/eLife.37813.006
MsSEC100kD filter
N%N%
9.15210031100
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.

https://doi.org/10.7554/eLife.37813.007
MsSEC100kD filter
N%N%
10.119361243
10.21121311
10.3713520
10.459311
10.5 (sectored)1121415
Total5310027100
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.

https://doi.org/10.7554/eLife.37813.008
Induced clone
10.110.210.310.410.5 (sectored)Total
Msn%n%n%n%n%n%
10.145920000004849100
10.236377924243747100
10.3112136214248122351100
10.4173537132712243748100
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.

https://doi.org/10.7554/eLife.37813.010
Strain inoculatedBlinded analysis
DS1DS1 were correctly classified.
DS9DS9 and DS9.1 were indistinguishable.
DS9.1
DS10DS10 and DS10.1 were indistinguishable.
DS10.1
DS10.2DS10.2 were correctly classified
DS10.3DS10.3 were correctly classified.
DS10.4DS10.4 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.

https://doi.org/10.7554/eLife.37813.013
MsSEC100kD filter
N%N%
AD(m)4710023100
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.

https://doi.org/10.7554/eLife.37813.016
MsSEC100kD filter
N%N%
CBD1(m)2036422
CBD2(m)1833739
CBD3(m)1731739
Total5510018100
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.

https://doi.org/10.7554/eLife.37813.017
Induced clone
CBD1CBD2CBD3Total
Input Msn%n%n%n%
CBD1(m)3057715102047100
CBD2(m)9172962102048100
CBD3(m)13261123316055100
Key resources table
ReagentDesignationSourceIdentifiersAdditional information
Gene
(human)
tau RD
(LM)-YFP
PMID: 24857020
Cell line
(human)
DS1; DS9; DS10PMID: 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 paperThese 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 paperAntibodies 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_2721235Antibodies used at
dilution indicated
in Materials and
Methods section
(1:2000)
Antibody (donkey)ECL
Anti-Rabbit
GE LifesciencesNA9340V,Antibodies
used at dilution
indicated in
Materials and
Methods
section
(1:2000)
Antibody (sheep)ECL
Anti-Mouse
GE
Lifesciences
NA931VAntibodies 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 filterCorning
Spin-X UF
Corning: 431481
Commercial
assay
Agarose
beads
Pierce protein
A/G Plus
Thermo
fisher: 20423
SoftwareGraphpad
Prism
Graphpad
software LLC
SoftwareFlowJoFlowJo LLC
OtherLipofectamine
2000
ThermofisherThermo
fisher: 11668019
transfection
reagent

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data have been provided for Figures 1, 2, 4 and 5.

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