Elevation of casein kinase 1ε associated with TDP-43 and tau pathologies in Alzheimer's disease

Abstract

Alzheimer's disease (AD) is characterized by the presence of extracellular amyloid β plaques and intraneuronal neurofibrillary tangles of hyperphosphorylated microtubule-associated protein tau in the brain. Aggregation of transactive response DNA-binding protein of 43 kDa (TDP-43) in the neuronal cytoplasm is another feature of AD. However, how TDP-43 is associated with AD pathogenesis is unknown. Here, we found that casein kinase 1ε (CK1ε) phosphorylated TDP-43 at Ser403/404 and Ser409/410. In AD brains, the level of CK1ε was dramatically increased and positively correlated with the phosphorylation of TDP-43 at Ser403/404 and Ser409/410. Overexpression of CK1ε promoted its cytoplasmic aggregation and suppressed TDP-43-promoted tau mRNA instability and tau exon 10 inclusion, leading to an increase of tau and 3R-tau expressions. Levels of CK1ε and TDP-43 phosphorylation were positively correlated with the levels of total tau and 3R-tau in human brains. Furthermore, we observed, in pilot immunohistochemical studies, that the severe tau pathology was accompanied by robust TDP-43 pathology and a high level of CK1ε. Taken together, our findings suggest that the elevation of CK1ε in AD brain may phosphorylate TDP-43, promote its cytoplasmic aggregation and suppress its function in tau mRNA processing, leading to acceleration/exacerbation of tau pathology. Thus, the elevation of CK1ε may link TDP-43 to tau pathogenesis in AD brain.

Keywords: CK1ε; TDP-43; TDP-43 proteinopathy; phosphorylation; tau; tau pathology.

Figure 1

Morphological varieties of TDP‐43 pathology in AD brain. Free‐floating sections of frontal cortex of AD cases were immunostained with anti‐TDP‐43 (A) or anti‐pS409/410‐TDP‐43 (B). The high magnified insert in (A) shows a presentative nuclear localization of TDP‐43. The morphology of TDP‐43 pathology in AD brain can be divided into four categories: I) somatodendritic inclusion, which could be amorphous (i), restricted (ii), skein‐like (iii) or granular (viii); II) dystrophic neurites (iv); III) compact round inclusion in the absence (v) or presence (vi) of apparent cellular profiles in proximity; and IV) highly condensed cobblestone‐like spots (vii). Clusters of compact round inclusions (ix) were also observed. Scale bars: 20 μm for low magnification, 10 μm for high magnification.

Figure 2

CK1ε interacts with and phosphorylates TDP‐43. A. GST‐TDP‐43 or GST coupled with glutathione–sepharose was incubated with rat brain extract. After washing, bound proteins were subjected to Western blots with indicated antibodies. B,C. Recombinant GST‐TDP‐43 was phosphorylated by CK1ε for various times as indicated. The phosphorylation products of TDP‐43 were analyzed by Western blots developed with site‐specific and phosphorylation‐dependent TDP‐43 antibodies (B). Levels of phosphorylated TDP‐43 at individual sites were plotted against the phosphorylation time (C). D. TDP‐43 or together with CK1ε was expressed in HEK‐293FT cells. The phosphorylation products of TDP‐43 were analyzed by Western blots developed with site‐specific and phosphorylation‐dependent TDP‐43 antibodies. E,F. TDP‐43 was overexpressed in HEK‐293FT cells for 40 h, then the cells were treated with 20 and 50 μM PF4800567, a CK1ε‐specific inhibitor, for 8 h. TDP‐43 was immunoprecipitated with anti‐HA and analyzed by Western blots with indicated antibodies (E). Average levels of phosphorylated TDP‐43 at each site were plotted against the concentration of PF4800567 (F).

Figure 3

CK1ε level is elevated in AD brain and is associated with an increase in TDP‐43 phosphorylation at Ser403/404 and Ser409/410. A. Brain frontal cortex homogenates from nine controls and ten AD cases were analyzed by Western blots. B–E. The levels of CK1ε (B), TDP‐43 phosphorylated at Ser379 (C), Ser403/404 (D) and Ser409/410 (E) are represented as scattered dots with mean ± SD. F–H. The level of CK1ε was plotted against the level of TDP‐43 phosphorylated at Ser379 (F), Ser403/404 (G) or Ser409/410 (H). The correlation between CK1ε and TDP‐43 was analyzed by Pearson (G and H) and Spearman (F) correlation. *P < 0.05; **P < 0.01; ****P < 0.0001.

Figure 4

CK1ε suppresses the function of TDP‐43 in tau mRNA processing and promotes TDP‐43 aggregation. A,B. GFP‐tailed with tau 3′‐UTR was co‐expressed with TDP‐43 and/or CK1ε in HEK‐293FT cells or treated the cells with 50 μM PF4800567 for 8 h. The levels of GFP mRNA and protein were analyzed by qRT‐PCR and Western blots developed with indicated antibodies, respectively. C,D. Overexpression of CK1ε together with TDP‐43 or treated with 50 μM PF4800567 for 8 h in HEK‐293FT cells transfected with mini‐tau gene, pCI/SI9‐LI10. The splicing products were analyzed by RT‐PCR and expression of TDP‐43 and CK1ε were analyzed by Western blots. The ratio of tau exon 10 inclusion/exclusion were calculated. E. TDP‐43 was co‐expressed with CK1ε in HeLa cells and immunostained with anti‐TDP‐43 (Green) and anti‐CK1ε (Red). Scale bar, 20 µm. F. Cells with cytoplasmic TDP‐43 aggregates were quantitated. The data are presented as mean ± SD. *P < 0.05; **p < 0.01.

Figure 5

Phosphorylation of TDP‐43 is associated with the increase in the levels of total tau and 3R‐tau/total tau in AD brain. A,B. Levels of 3R‐tau (RD3) and total‐tau (R134d) in frontal cortices from nine AD and nine age‐matched control cases were determined by immuno‐dot blots (A) and quantified by densitometry (B). C,D. The levels of total tau (C) and the ratio of 3R‐tau/total tau (D) in frontal brain homogenates determined by immuno‐dot blots were plotted against the levels of CK1ε, pS379‐TDP‐43, pS403/404‐TDP‐43, and pS409/410‐TDP‐43, respectively. The Pearson (CK1ε, pS403/404‐TDP‐43 and pS409/410‐TDP‐43 to total tau or 3R‐tau/total tau) and Spearman (pS379‐TDP‐43 to total tau or 3R‐tau/total tau) correlation coefficient r were calculated. The data are presented as mean ± SD. ***P < 0.001.

Figure 6

High level of CK1ε is associated with profound tau and TDP‐43 pathologies in AD. A. Immunohistochemical staining of phosphorylated tau (pSer202/pThr205, AT8), TDP‐43 and phosphorylated TDP‐43 (pSer409/410). For tau staining, magnified views of the boxed areas in low magnification are shown below the low‐magnification images. High magnification as inserts shows the representative morphology of staining. AD case 1 showed much higher density than AD case 2 of AT8‐positive tangles (hollow arrows) and dystrophic neurites (hollow arrowheads) and TDP‐43‐positive and pSer409/410‐TDP‐43‐positive inclusions (filled dark arrows). Filled dark arrowheads indicate seemingly normal nuclear TDP‐43 staining. B. Double immunofluorescence staining showed the association of tau pathology with TDP‐43 pathology. Sections were counterstained with TOPRO 3 iodide for nuclei. Within numerous AT8‐positive neurofibrillary tangles (arrows a) and dystrophic neurites (arrows b), TDP‐43 inclusions were seen to colocalize with AT8 staining in neurofibrillary tangles (arrows c) or to separately locate in the somatodendritic compartment (arrows d) and dystrophic neurites (arrows e) or appear as cobblestone‐like condensed spots of varying sizes (arrows f). Defuse and weak staining of nuclei by TDP‐43 (arrows g) were also seen. C. Western blots showed the expression levels of CK1ε, TDP‐43, pSer409/410‐TDP‐43, total tau (R134d) and pSer202/pThr205‐tau (AT8). AD case 1 exhibited much higher levels of the proteins examined, except TDP‐43, than AD case 2 and the control case. Scale bars: (A) 500 μm for low magnification, 10 μm for inserts and 50 μm for the rest; (B) 20 μm.