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. 2017 Jul;103:154-162.
doi: 10.1016/j.nbd.2017.04.012. Epub 2017 Apr 20.

TDP-43 Expression Influences Amyloidβ Plaque Deposition and Tau Aggregation

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Free PMC article

TDP-43 Expression Influences Amyloidβ Plaque Deposition and Tau Aggregation

Stephani A Davis et al. Neurobiol Dis. .
Free PMC article

Abstract

Although the main focus in Alzheimer's disease (AD) has been an investigation of mechanisms causing Aβ plaque deposition and tau tangle formation, recent studies have shown that phosphorylated TDP-43 pathology is present in up to 50% of sporadic cases. Furthermore, elevated phosphorylated TDP-43 has been associated with more severe AD pathology. Therefore, we hypothesized that TDP-43 may regulate amyloid-beta precursor protein (APP) trafficking and tau phosphorylation/aggregation. In order to examine the role of TDP-43 in AD, we developed a transgenic mouse that overexpresses hippocampal and cortical neuronal TDP-43 in a mouse expressing familial mutations (K595N and M596L) in APP and presenilin 1 (PSEN1ΔE9). In our model, increased TDP-43 was related to increased tau aggregation as evidenced by thioflavin S-positive phosphorylated tau, which may implicate TDP-43 expression in pre-tangle formation. In addition, there was increased endosomal/lysosomal localization of APP and reduced Aβ plaque formation with increased TDP-43. Furthermore, there was decreased calcineurin with elevated TDP-43 expression. Since calcineurin is a phosphatase for TDP-43, the decreased calcineurin expression may be one mechanism leading to an increase in accumulation of diffuse phosphorylated TDP-43 in the hippocampus and cortex. We further show that when TDP-43 is knocked down there is an increase in calcineurin. In our model of selective TDP-43 overexpression in an APP/PSEN1 background, we show that TDP-43 decreases Aβ plaque deposition while increasing abnormal tau aggregation. These observations indicate that TDP-43 may play a role in regulating APP trafficking and tau aggregation. Our data suggest that TDP-43 could be a putative target for therapeutic intervention in AD affecting both Aβ plaque formation and tauopathy.

Keywords: APP; APP/PS1; Alzheimer's disease; Calcineurin; TARDBP; TDP-43; Tau.

Conflict of interest statement

Conflict of interest: The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1. Generation of Camk2a-tTA/hTDP-43/APP/PS1 mice
A) Camk2a-tTA mice were crossed with APPswe/PS1dE9 (APP/PS1) mice. Camk2a-tTa or Camk2a-tTA/APP/PS1 mice were then crossed with TetO mice containing human TDP-43 (tetO-hTDP-43) and were fed Doxicycline (Dox) to keep hTDP-43 expression off during development. Mice were weaned 22 days after birth and Dox was removed from their diet. Upon removal of Dox, the tTA expressed from the Camk2a promoter was free to bind to the tetO promoter, thus allowing expression of the human TDP-43 transgene. B) Immunofluorescence staining probed for human-specific TDP-43 (555nm, red, Abnova) and total TDP-43 (488nm, green, ProteinTech Group). C) Western blot of 9-month old mice confirms expression of human TDP-43 (Abnova) and APP (6E10) in transgenic mice. There was a significant reduction in endogenous TDP-43 in Camk2a/hTDP-43 and Camk2a/hTDP-43/APP/PS1 mice detected with mouse-specific anti-TDP-43 antibody. D) Western blot quantitation of normalized endogenous TDP-43. E) Western blot quantification of normalized total TDP-43 expression. (*p<0.05, ***p<0.0001).
Figure 2
Figure 2. TDP-43 overexpression reduces calcineurin
A) Western blots for APP/PS1 and wildtype primary cortical neurons infected with human TDP-43 virus. B) Western blot of calcineurin expression in HEK293T cells transfected with either non-targeting siRNA (siNC) or siTDP-43. Knockdown of TDP-43 increased calcineurin expression. C and D) Quantitation of normalized calcineurin expression by Western blots in A and B, respectively. *p<0.05, **p<0.001, One-way ANOVA with Bonferroni multiple comparison correction was performed for A. Student t-tests was performed for B. E) Phosphorylated TDP-43(Ser409/410) was more prevalent in Camk2a/hTDP-43/APP/PS1 mice but also observed in APP/PS1 mice. Arrows indicate cytoplasmic accumulation of diffuse endogenous phosphorylated TDP-43(S409/410) (phospho-TDP, Cosmobio, green; TDP-43, Genscript, red; DAPI, blue) 1000X magnification.
Figure 3
Figure 3. Selective expression of TDP-43 in the hippocampus and cortex alters Aβ plaque deposition and localization of APP
A) Sagittal sections from APP/PS1 and Camk2a/hTDP-43/APP/PS1 (littermates) display a distinct change in plaque deposition with both 10D5 (A) and 6E10 (B). C) Total Aβ plaques were blindly counted throughout the cortex and hippocampus of 3 sections from 3 different mice per genotype and immunostained with 6E10 antibody. Camk2a/hTDP-43/APP/PS1 mice had a significant reduction in the number of Aβ plaques. D) Intracellular levels of APP (6E10, Covance, red, 555nm) co-localized with the early endosomal protein RAB5 (Cell Signaling, green, 488nm), 1000X magnification. E) Representative staining shows an increased co-localization of APP (6E10, red, 555nm) and the late endosomal/lysosomal marker LAMP2 (Cell Signaling, green, 488nm) in Camk2a-tta/hTDP-43/APP/PS1 mice, 1000X magnification. Nuclei, shown in blue, are labeled with DAPI.
Figure 4
Figure 4. Phosphorylated tau aggregation in mice expressing TDP-43
A) PHF1 (phospho-Tau Ser396/404) Western blot in 9-month-old mice. Asterisk indicates phospho-tau at a higher molecular weight of approximately 100kD. B) Quantification of PHF1 and higher molecular weight PHF1 (C) normalized to total Tau. D) Thioflavin S/pTAU(Ser396)-positive cortical cells in Camk2a/hTDP-43/APP/PS1 mice are similar to what is observed in those with AD. Arrows point to cells that are positive for both thioflavin S staining and pTau(Ser396), 400X, (thioflavin S green; pTAU Ser396, red; and TDP-43, blue). Graphs show mean and standard deviation, n=3, One-way ANOVA and Tukey’s correction for multiple comparison, *** indicate p-value<0.0001.
Figure 5
Figure 5. Total TDP-43 colocalizes with thioflavin S and phosphorylated Tau in AD
A, B, and C are hippocampal sections from three separate cases of neuropathologically confirmed AD (Braak NFT stage V). Immunofluorescence staining probed with total TDP-43 (N-terminal, rabbit polyclonal, 647nm, blue), phosphorylated Tau (Ser396) (555nm, red, Cell Signaling), and thioflavin S (488nm, green). Arrows indicate cells with cytoplasmic accumulation of TDP-43 staining positive for thioflavin S and negative for pTau (Ser396). Asterisks indicate thioflavin S positive tangles colocalized with TDP-43 and pTAU(Ser396), 400X magnification.

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