Age-dependent changes in TDP-43 levels in a mouse model of Alzheimer disease are linked to Aβ oligomers accumulation

Abstract

Background: Transactive response DNA-binding protein 43 (TDP-43) is the pathological protein found in frontotemporal lobar degeneration with ubiquitin positive inclusions and in amyotrophic lateral sclerosis. In diseased tissue, TDP-43 translocates from its physiological nuclear location into the cytoplasm, where it accumulates. Additionally, C-terminal fragments of TDP-43 accumulate in affected brain regions and are sufficient to cause TDP-43 mislocalization and cytoplasmic accumulation in vitro. TDP-43 also accumulates in 30% of Alzheimer disease (AD) cases, a finding that has been highly reproducible. The role of TDP-43 in AD and its relation with Aβ and tau pathology, the two neuropathological hallmarks of AD, remains to be elucidated.

Results: Here we show that levels of TDP-43 and its ~35 kDa C-terminal fragment are significantly increased in the 3×Tg-AD mice, an animal model of AD that develops an age-dependent cognitive decline linked to the accumulation of Aβ and tau. We also report that the levels of TDP-43 and its C-terminal fragment correlate with the levels of soluble Aβ oligomers, which play a key role in AD pathogenesis. Notably, genetically reducing Aβ42 production restores the levels of TDP-43 and its ~35 kDa C-terminal fragment to control levels.

Conclusions: These data suggest a possible relation between Aβ oligomers and TDP-43.

Figure 1

3×Tg-AD mice have higher levels of TDP-43 and TDP-35. (A) Representative Western blots of proteins from the low salt fraction extracted from 2-month-old 3×Tg-AD and NonTg mice and probed with the indicated antibodies. A longer exposure time was necessary to see the less abundant low molecular weight fragments. (B) Quantitative analysis of the blots shows that at 2 months of age the levels of TDP-43 and TDP-35 were similar between 3×Tg-AD and NonTg mice (n = 6/genotype). (C) Representative Western blots of proteins from the low salt fraction extracted from 6-month-old 3×Tg-AD and NonTg mice. (D) Quantitative analysis of the blots shows that at 6 months of age the levels of TDP-43 and TDP-35 were significantly increased (p = 0.04 and p = 0.01, respectively) in the brains of the 3×Tg-AD mice compared to age- and gender-matched NonTg mice (n = 6/genotype). (E) Representative Western blots of proteins from the low salt fraction extracted from 12-month-old 3×Tg-AD and NonTg mice (n = 6/genotype). (F) Quantitative analysis of the blots shows that at 12 months of age the levels of TDP-43 and TDP-35 were similar between 3×Tg-AD and NonTg mice (n = 6/genotype). Abbreviation: exp = exposure. Data are presented as ± SEM and analyzed by t-test analysis.

Figure 2

Age-dependent changes in TDP-43 and TDP-35 levels. (A) Representative Western blots of proteins from the low salt fraction extracted from 2-, 6- and 12-month-old NonTg mice and probed with a polyclonal antibody raised against TDP-43 antibody. A longer exposure time was necessary to see the less abundant low molecular weight fragments. (B) Quantitative analysis of the blots shows that the levels of TDP-43 and TDP-35 were similar across the three different ages analyzed (n = 6/time-point). (C) Representative Western blots of proteins from the low salt fraction extracted from 2-, 6- and 12-month-old 3×Tg-AD mice and probed with a polyclonal antibody raised against TDP-43 antibody. (B) Quantitative analysis of the blots shows that the levels of TDP-43 and TDP-35 were significantly higher at 6 months of age (n = 6/time-point). Abbreviation: exp = exposure. Data are presented as ± SEM and analyzed by one way ANOVA.

Figure 3

Age-dependent changes in TDP-43 and TDP-35 cellular localization. (A) Representative Western blots of proteins from the cytosolic and nuclear fractions extracted from 2-month-old NonTg and 3×Tg-AD mice and probed with the a polyclonal anti-TDP antibody. A longer exposure was necessary to see the less abundant low molecular weight fragments. (B-C) Quantitative analysis of the blots shows that at 2 months of age the nuclear and cytosolic levels of TDP-43 and TDP-35 were not statistically significant between 3×Tg-AD and NonTg mice (n = 6/genotype). (D) Representative Western blots of proteins from the cytosolic and nuclear fractions extracted from 6-month-old NonTg and 3×Tg-AD mice and probed with a polyclonal anti-TDP antibody. (E-F) Quantitative analysis of the blots shows that at 6 months of age the nuclear levels of TDP-43 and TDP-35 were not statistically significant between 3×Tg-AD and NonTg mice (n = 6/genotype). In contrast, the cytosolic levels of TDP-43 and TDP-35 were significantly higher in the 3×Tg-AD mice compared to NonTg mice (p = 0.03 and p = 0.01, respectively). (G) Representative Western blots of proteins from the cytosolic and nuclear fractions extracted from 12-month-old NonTg and 3×Tg-AD mice and probed with a polyclonal anti-TDP antibody. (H-I) Quantitative analysis of the blots shows that at 12 months of age the nuclear and cytosolic levels of TDP-43 and TDP-35 were not statistically significant between 3×Tg-AD and NonTg mice (n = 6/genotype). Abbreviation: exp = exposure. Data are presented as ± SEM and analyzed by t-test analysis.

Figure 4

TDP-43 and TDP-35 levels do not correlate with tau phosphorylated at Thr181. (A) Representative microphotographs of 3×Tg-AD brain sections depicting hippocampal CA1 pyramidal neurons stained with the anti-tau antibody AT270, which recognize tau phosphorylated at Thr181 (n = 6/age group). (B) Representative Western blots of proteins extracted from 6- and 12-month old 3×Tg-AD mice probed with the AT270 antibody. β-actin is used as a loading control. (C) Quantitative analysis of the blots shows that the steady-state levels of AT270 were significantly higher in the brains of 12-month-old 3×Tg-AD mice compared to 6-month-old mice (n = 6/age group; p < 0.05). Data are presented as ± SEM and analyzed by t-test analysis. (D-E) Linear regression analysis indicated that the levels of AT270 in the brains of 6-month-old 3×Tg-AD mice did not correlate with the levels of TDP-43 (r² = 0.09148, p = 0.56) and TDP-35 (r² = 0.1491, p = 0.45). (F-G) Similarly, no correlation between AT270 and TDP-43 (r² = 0.4644, p = 0.14) and TDP-35 (r² = 0.4469, p = 0.15) levels was observed at 12 months of age.

Figure 5

Aβ oligomers correlate with TDP-43 and TDP-35. (A-D) Representative microphotographs showing 3×Tg-AD hippocampal sections stained with the antibody M71/3, which is specific for Aβ oligomers. Panels C and D show higher magnification views of panels A and B, respectively (n = 6/age group). (E) Representative dot blots of protein extracted from 2-, 6-, and 12-month-old 3×Tg-AD mice and probed with the antibody A11, which is specific for Aβ oligomers. (F) Quantitative analysis of the dot blots shows that at 6 months of age, A11 levels were significantly higher compared to 2-month-old mice (p < 0.001) and 12-month-old mice (p < 0.05). (G-H) Linear regression analysis indicated that the levels of A11 in the brains of 6-month-old 3×Tg-AD mice significantly correlated with the levels of TDP-43 (r² = 0.9330, p = 0.001) and TDP-35 (r² = 0.6749, p = 0.045). (I) Similarly, the levels of A11 in the brains of 12-month-old 3×Tg-AD mice significantly correlated with TDP-43 (r² = 0.7259, p = 0.031). (J) A strong trend was observed when analyzing A11 and TDP-35 levels in 12-month-old 3×Tg-AD mice (r² = 0.6277, p = 0.06). The dotted lines in panels G-J represent 95% confidence intervals.

Figure 6

Reduction of Aβ₄₂ levels decreases TDP-43 and TDP-35 levels. (A-B) Representative microphotographs depicting CA1 pyramidal neurons from 6-month-old 3×Tg-AD and APP/tau mice stained with an anti-Aβ antibody (n = 6/group). (C) Representative microphotograph depicting CA1 pyramidal neurons from APP/tau mice stained with the anti-oligomeric antibody M17/3. (D) Representative Western blots of protein extracted from the brains of 6-month-old NonTg, APP/tau and 3×Tg-AD mice (low salt fraction), probed with a polyclonal anti-TDP-43 antibody. β-Actin is used as a loading control. A longer exposure time was necessary to see the less abundant low molecular weight fragments. (E-F) Quantitative analysis of the blots (n = 6/genotype) shows that in APP/tau mice, the steady-state levels of TDP-43 and TDP-35 are significantly lower in APP/tau mice compared to 3×Tg-AD mice (p < 0.05). The levels of TDP-43 and TDP-35 were not statistically significant different between APP/tau and NonTg mice. Data are presented as means ± SEM and were analyzed using one-way ANOVA following by post hoc Bonferroni test to determine individual differences in groups. Abbreviation: exp = exposure.