3R Tau Inhibits 4R Tau Assembly.

<p>Increasing molar concentrations (0, 1, 2, or 3 µM) of 3R or 4R tau were added to 8 µM 4R tau assembly reactions containing 0.04 mg/ml heparin and DTT, and Thioflavin S binding fluorescence was measured after 24 and 48 hours. 3R0N or 4R0N tau spiked into 4R0N tau reactions after 24 hours (A) and 48 hours (B). 3R1N and 4R1N tau spiked into 4R1N tau reactions after 24 hours (C) and 48 hours (D). 3R2N and 4R2N tau spiked into 4R2N tau reactions after 24 hours (E) and 48 hours (F). Fluorescence was normalized to 4R0N (A and B), 4R1N (C and D), and 4R2N (E and F) assembly and the relative fluorescence is shown. Statistical analysis was performed using a one way ANOVA and all pairwise multiple comparisons were done by the Student-Newman- Keuls Method, with p<0.05 considered significant. An * denotes significance relative to unspiked 4R tau assembly reactions. Addition of 3R tau isoforms to 4R tau assembly reactions showed a dose dependent inhibition of tau assembly, in spite of an overall increase in tau protein levels, relative to unspiked 4R tau assembly reactions (p = 0.018, p = 0.001, and p = 0.001 for 1 µM, 2 µM, and 3 µM 3R0N, respectively, for day 1 and p = 0.046 and p = 0.026 for addition of 2 µM and 3 µM 3R0N, respectively, for day 2). Additionally, 4R1N and 4R2N tau assembly reactions spiked with 3R1N and 3R2N tau isoforms, respectively, showed negative correlations between 4R tau assembly and additions of increasing concentrations of 3R tau isoforms (p<0.05 for both 4R1N and 4R2N) using the Spearman Rank Order Correlation test in Sigmaplot. Assembly of 4R tau reactions were either unchanged or showed a dose dependent increase in tau assembly following addition of more 4R tau isoforms.</p

Early degenerative phenotype in iTDP-43^14A mice at P5 in the absence of FTLD-like TDP-43 aggregation.

<p>(A) Monoclonal antibody to human TDP-43 showed expression at P5 remained restricted to previously characterized regions of hippocampus, cortex and striatum. (B) Western blotting of brain lysate of P5 non-transgenic (NT) and iTDP-43^14A demonstrated increased levels of activated caspase 3 in iTDP-43^14A mice. (C) Abundant caspase 3 immunoreactivity in the cortex of iTDP-43^14A mice that was virtually absent in NT mice, suggestive of elevated cell death in iTDP43^14A compared to NT mice. iTDP-43^14A mice were also characterized by increased ubiquitin staining in the upper layers of the cortex compared to NT mice, which upon higher magnification appeared to be completely diffuse and cytoplasmic. (D) Immunohistochemistry for hTDP-43 and p403/404 and immunofluorescence using antibodies to total TDP-43 and p409/410 TDP-43. Significant amounts of cytoplasmic hTDP-43 were observed in iTDP-43 mice (arrowheads). Note that this cytoplasmic staining was also observed in NT mice (arrowheads) with antibodies to total TDP-43 (tTDP-43 Ab1) and TDP-43 phosphorylated at 409/410 (p409/410). Scale bars in D = 50 µm.</p

Changes in tau filament morphology in mixed isoform reactions.

<p>Filaments from 48 hour single isoform or mixed isoform assembly reactions were examined by electron microscopy. Representative filaments from reactions containing only 3R0N tau were observed as A) loosely twisted paired helical filaments or B) straight filaments while those formed in single 4R0N reactions contained predominantly C–D) straight filaments. The filament ends in these pure reactions were mostly observed to be well defined and cleanly stained. Filaments from the mixed isoform reactions were also able to form E) loose paired helical filament and F) straight filaments though the filament ends G–J) were often not well defined and demonstrated splaying or amorphous aggregation. The scale bar is 100 nm.</p

Expression of human TDP-43 in iTDP-43^14A and iTDP-43^8A mice in the postnatal period.

<p>Immunohistochemical detection of hTDP-43 expression in cortex (CTX), hippocampus (HIP) and striatum (STR) in iTDP-43^14A (A) and iTDP-43^8A (B). Western analysis of organs demonstrated specificity of hTDP-43 expression to the brain in both iTDP-43^14A (C) and iTDP-43^8A (D) (SC = spinal cord, He = heart, Lu = lung, Li = liver, Ki = kidney, St = stomach, SM = skeletal muscle, Sp = spleen, Br = brain). (E) Brain weight measurement of non-transgenic (NT) and iTDP-43^14A mice at postnatal stages until 2 months of age (P60) (*<i>p</i><0.05, **<i>p</i><0.01, *** p<0.001, unpaired two tailed <i>T-test</i>). (F) Expression of hTDP-43 at indicated postnatal time points for iTDP-43^14A. (G) Expression of hTDP-43 at indicated postnatal time points for iTDP-43^14A (14) compared to iTDP-43^8A (8).</p

Ratio of 3R to 4R tau isoforms determines the extent of tau assembly.

<p>Thioflavin S binding fluorescence of different ratios of 3R and 4R tau isoforms in the presence of 0.04 mg/ml heparin with or without DTT was measured at 48 hours or steady state. 3R0N tau isoforms mixed with increasing molar fractions of 4R0N tau under non-reducing (A) and reducing (B) conditions. Ratios of 3R0N to 4R0N were as follows: 8 µM 3R0N (0% 4R0N), 6 µM 3R0N to 2 µM 4R0N (25% 4R0N), 4 µM 3R0N to 4 µM 4R0N (50% 4R0N), 2 µM 3R0N to 6 µM 4R0N (75% 4R0N) and 8 µM 4R0N (100% 4R0N). 3R2N tau isoforms mixed with increasing molar fractions of 4R2N tau under non-reducing (C) and reducing (D) conditions. Ratios of 3R2N to 4R2N were the same as for 3R0N to 4R0N. Statistical analysis was performed using a one way ANOVA (for 30/40 mixes with and without DTT and for 32/42 mixes with DTT) or by Kruskal-Wallis One Way Analysis of Variance on Ranks (for 32/42 without DTT) and all pairwise multiple comparisons were done by the Student-Newman- Keuls Method, with p<0.05 considered significant. An * denotes significance relative to 3R tau assembly, # denotes significance relative to 4R tau assembly, and ∧ denotes significance relative to the equimolar ratio of 4R:3R tau assembly.</p

3R tau isoforms inhibit 4R tau aggregation.

<p>Different 4R tau isoform assembly reactions alone or spiked with increasing molar concentrations of 3R tau isoforms, with or without N-terminal inserts, were centrifuged at 100,000×g to separate free and polymerized tau. Coomassie stained gels of the pellets containing polymerized tau from A) 8 µM 4R0N spiked with 3R0N, C) 8 µM 4R0N spiked with 4R0N, with the following samples for each gel: 4R (0 µM 3R), 4R spiked with 1 µM 3R, 4R spiked with 2 µM 3R, and 4R spiked with 3 µM 3R. Densitometric analysis of pelleting gels B) 4R0N spiked with 3R0N and D) 4R0N spiked with 4R0N, was performed using Image J software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010810#pone.0010810-Abramoff1" target="_blank">[19]</a>. Statistical analysis performed using the Spearman Rank Correlation test in Sigmaplot showed a negative correlation between 4R tau assembly and additions of increasing concentrations of 3R tau isoforms (p<0.05), but a positive correlation between 4R tau assembly and additions of increasing concentrations of 4R tau isoforms (p = 0.05).</p

Schematic representation of human tau and Coomassie blue-stained gel of recombinant tau.

<p>(A) Full-length 3-repeat tau (3R2N) and 4-repeat tau (4R2N) containing N-terminal inserts encoded by exons 2 and 3, and microtubule (MT)-binding repeats encoded by exons 9–12. The 6 different tau isoforms are generated by alternative splicing of exons 2, 3, and 10 shown in white. The tau isoforms 3R0N and 4R0N would not include exons 2 and 3. Exclusion of alternatively spliced exon 10 generates 3-repeat tau isoforms. Inclusion or exclusion of alternatively spliced exons 2 and 3 generates 3R1N, 4R1N, 3R2N, or 4R2N tau isoforms. Potential heparin binding sites (HEPBS) are indicated by bars. (B) Purified recombinant tau proteins used in the study were loaded heavy at 2 ug per well and separated on 10% SDS-PAGE gels before staining with Coomassie brilliant blue R-250 to demonstrate purity.</p

Tau Isoform Assembly Kinetics.

<p>The kinetics of 3R (A) and 4R (B) tau isoform assembly was measured using thioflavin S binding fluorescence in the presence of 0.04 mg/ml heparin with or without DTT. Each time point represents 2–5 experiments performed on separate days with background fluorescence (no tau present in reaction) subtracted from each experiment. (C) The thioflavin S data presented in A and B is the integrated value from thioflavin S binding curves as shown for 4R, 3R, and no tau reactions in the presence of DTT at day 1. (D–E) Electron micrographs of 4R0N tau assembly reactions containing 0.04 mg/ml heparin with DTT can be used to confirm the presence of tau filaments.</p

Tau aggregation decreased in mixed isoform reactions.

<p>Assembly reactions of single tau isoforms and different ratios of 3R0N/4R0N tau isoforms were centrifuged at 100,000×g to separate free from aggregated or polymerized tau. Coomassie stained gels of the pellets containing polymerized tau from A) 3R0N mixed with 4R0N with the following samples for each gel: 8 µM 3R0N (0% 4R0N), 6 µM 3R0N to 2 µM 4R0N (25% 4R0N), 4 µM 3R0N to 4 µM 4R0N (50% 4R0N), 2 µM 3R0N to 6 µM 4R0N (75% 4R0N) and 8 µM 4R0N (100% 4R0N). Densitometric analysis of pelleting gels B) 3R0N mixed with 4R0N was performed using Image J software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010810#pone.0010810-Abramoff1" target="_blank">[19]</a>. Results were normalized to 4R values and relative % tau aggregation is shown. Statistical analysis was performed using a one way ANOVA and all pairwise multiple comparisons were done by the Student-Newman-Keuls Method, with p<0.05 considered significant. An * denotes significance relative to 4R tau assembly and a ∧ denotes significance relative to the equimolar ratio of 4R:3R tau assembly.</p

Biochemistry of iTDP-43^14A brain lysates at P5.

<p>(A) Western blotting using two antibodies to total TDP-43 (tTDP-43 Ab1 and tTDP-43 Ab2) demonstrated increased levels of low molecular weight species at 35 kDa (arrow) and 25 kDa (arrowhead) in iTDP-43^14A mice relative to NT mice. These species were not observed using antibodies to the C-terminus (405–414) or N-terminus (3–12) of TDP-43. (B) Western blot analysis of high salt (HS), myelin floatation buffer (MFB), sarkosyl (SARK) and urea fractions using antibody to human TDP-43. Note that human TDP-35 (arrow) is present in the urea fraction but is absent from MFB and SARK fractions, N = non-transgenic, T = iTDP-43^14A. (C) Antibody to murine Tdp-43 demonstrated reduction of mTdp-43 in brain compared to NT mice. (D) Quantification of blot in (C), **<i>p</i><0.01, unpaired two tailed <i>t-</i>tes<i>t</i>.</p