Phosphorylation of TAR DNA-binding Protein of 43 kDa (TDP-43) by Truncated Casein Kinase 1δ Triggers Mislocalization and Accumulation of TDP-43

Abstract

Intracellular aggregates of phosphorylated TDP-43 are a major component of ubiquitin-positive inclusions in the brains of patients with frontotemporal lobar degeneration and ALS and are considered a pathological hallmark. Here, to gain insight into the mechanism of intracellular TDP-43 accumulation, we examined the relationship between phosphorylation and aggregation of TDP-43. We found that expression of a hyperactive form of casein kinase 1 δ (CK1δ1-317, a C-terminally truncated form) promotes mislocalization and cytoplasmic accumulation of phosphorylated TDP-43 (ubiquitin- and p62-positive) in cultured neuroblastoma SH-SY5Y cells. Insoluble phosphorylated TDP-43 prepared from cells co-expressing TDP-43 and CK1δ1-317 functioned as seeds for TDP-43 aggregation in cultured cells, indicating that CK1δ1-317-induced aggregated TDP-43 has prion-like properties. A striking toxicity and alterations of TDP-43 were also observed in yeast expressing TDP-43 and CK1δ1-317. Therefore, abnormal activation of CK1δ causes phosphorylation of TDP-43, leading to the formation of cytoplasmic TDP-43 aggregates, which, in turn, may trigger neurodegeneration.

Keywords: ALS (Lou Gehrig disease); TAR DNA-binding protein of 43 kDa (TDP-43); casein kinase 1δ; frontotemporal lobar degeneration (FTLD); phosphorylation; protein aggregation; protein kinase.

FIGURE 1.

Biochemical evidence of phosphorylated TDP-43 aggregation by CK1δ1-317. Shown are immunoblot analyses of proteins extracted from SH-SY5Y cells co-expressing both TDP-43 and either empty vector, Myc-tagged CK1α1, Myc-tagged CK1α2, Myc-tagged CK1δ, Myc-tagged CK1ϵ, FLAG-tagged CK1δ1-317, HA-tagged CK2, HA-tagged Cdc-7, or FLAG-tagged ASK. Proteins were extracted from cells with 1% Sarkosyl, and the Sar-sup and Sar-ppt were subjected to immunoblot analyses. The blots were probed using anti-phosphorylated TDP-43 (anti-pS409/410) polyclonal and anti-TDP-43 monoclonal (anti-TDP mono) antibodies, a mixture of anti-Myc monoclonal and anti-FLAG monoclonal antibodies, a mixture of anti-HA monoclonal and anti-FLAG monoclonal antibodies, and anti-tubulin α antibody. Because FLAG-CK1δ1-317 is predicted to be ∼35 kDa, the band of ∼43 kDa may correspond to its ubiquitinated form. Note that the band of phosphorylated TDP-43 is observed in the Sar-sup and Sar-ppt of cells expressing CK1δ1-317 (arrowheads).

FIGURE 2.

Microscopic analyses of phosphorylated TDP-43 inclusions by CK1δ1-317. Shown are confocal microscopic analyses of cells expressing TDP-43 alone, FLAG-CK1δ1-317 alone, or both. These cells were immunostained with anti-phosphorylated TDP-43 (pS409/410) polyclonal, anti-FLAG monoclonal, anti-ubiquitin (Ub) monoclonal, and anti-p62 monoclonal antibodies and counterstained with Hoechst 33342. Scale bars = 20 nm.

FIGURE 3.

Time course characterization of cells expressing TDP-43 and CK1δ1-317. Shown are immunoblot analyses of cells expressing TDP-43 alone, FLAG-CK1δ1-317 alone, or both. Sar-sup and Sar-ppt fractions were prepared from cells and subjected to immunoblot analyses. The blots were probed using anti-phosphorylated TDP-43 (anti-pS409/410) monoclonal, anti-TDP-43 monoclonal (anti-TDP mono), anti-FLAG monoclonal, and anti-tubulin α antibodies. Note that endogenous TDP-43 is phosphorylated and aggregated in cells expressing FLAG-CK1δ1-317 alone. The arrowheads show phosphorylated TDP-43.

FIGURE 4.

The physiological properties of TDP-43 are altered in cells co-expressing TDP-43 and CK1δ1-317. A, CFTR exon 9 skipping assay of transfected cells. Gel electrophoresis of the RT-PCR products of RNA from transfected cells was performed. The RNAs from SH-SY5Y cells transfected with the reporter plasmid pSPL3-CFTR exon 9 plus pcDNA3.1 expression vectors were used as templates for RT-PCR analysis. The products were analyzed by electrophoresis in 1.5% agarose gel. The band intensities (−exon 9) were quantified and the results are expressed as mean + S.E. (n = 3). ****, p < 0.0001 by Student's t test. a.u., arbitrary unit. B, quantification of endogenous HDAC6 mRNA levels in several transfected cells by real-time PCR. The mRNA ratio of HDAC6/hypoxanthine-guanine phosphoribosyltransferase (HPRT) is expressed as mean ± S.E. (n = 3). *, p < 0.05 by Student's t test.

FIGURE 5.

Kinase activity of CK1δ1-317 is required for the accumulation of phosphorylated TDP-43. Shown are immunoblot analyses of cells expressing FLAG-CK1δ1-317 WT, K38R (KR), or K38A (KA) with or without TDP-43. Sar-sup and Sar-ppt were prepared from cells and subjected to immunoblot analyses. The blots were probed using anti-phosphorylated TDP-43 (anti-pS409/410) monoclonal, anti-FLAG monoclonal, and anti-tubulin α antibodies. Note that endogenous TDP-43 is phosphorylated and aggregated in cells expressing the WT alone. The arrowhead shows phosphorylated TDP-43.

FIGURE 6.

Phosphorylation at Ser-393/395 of TDP-43 is important for its aggregation induced by CK1δ1-317. A, mass spectrometric identification of phosphorylation sites of aggregated TDP-43 induced by CK1δ1-317. Red Ser residues are phosphorylation sites of intracellular accumulated TDP-43 by CK1δ1-317. B, immunoblot analyses of non-transfected cells (lane 1), cells expressing FLAG-CK1δ1-317 alone (lane 2), and cells co-expressing FLAG-CK1δ1-317 and either TDP-43 wild-type (lane 3), S393A/S395A (lane 4), S403A/S404A (lane 5), or S393A/S395A/S403A/S404A (lane 6) mutant. Sar-sup and Sar-ppt were prepared from cells and subjected to immunoblot analyses. The blots were probed using anti-phosphorylated TDP-43 (anti-pS409/410) monoclonal, anti-TDP monoclonal (anti-TDP mono), and anti-tubulin α antibodies. The arrowheads shows phosphorylated TDP-43. C, the immunoreactivity of phosphorylated TDP-43 (arrowheads in B) of Sar-ppt positive for anti-pS409/410 (top panel) and anti-TDP monoclonal antibody (bottom panel) was quantified, and the results are expressed as mean ± S.E. (n = 3). **, p < 0.01 by Student's t test; n.s., not significant. a.u., arbitrary unit.

FIGURE 7.

Insoluble phosphorylated TDP-43 functions as seeds for intracellular TDP-43 aggregation. Confocal microscopic analyses of cells expressing TDP-43 ΔNLS alone, cells treated with the Sar-ppt prepared from cells expressing TDP-43 and FLAG-CK1δ1-317 (Sar-ppt seeds), and cells expressing TDP-43 ΔNLS and treated with Sar-ppt seeds. These cells were immunostained with anti-phosphorylated TDP-43 (pS409/410) polyclonal antibody and counterstained with Hoechst 33342. Scale bars = 20 nm.

FIGURE 8.

Cytotoxic effects of phosphorylated TDP-43 aggregates induced by CK1δ1-317 in yeast. A, spotting assay to compare cytotoxicity in yeast cells expressing TDP-43 alone, cells expressing CK1δ1-317 alone, and cells expressing both TDP-43 and CK1δ1-317. We used serial dilutions of yeast cells transformed with galactose-inducible constructs. Transformants were spotted on agar plates with (inducing) or without (non-inducing) galactose, and growth was assessed after 48 h. B, immunoblot analyses of yeast cells expressing TDP-43 alone, cells expressing CK1δ1-317 alone, and cells expressing both TDP-43 and CK1δ1-317. Sar-sup and Sar-ppt were prepared from these cells and subjected to immunoblot analyses. The blots were probed using anti-TDP-43 monoclonal (anti-TDP mono) and anti-phosphorylated TDP-43 (anti-pS409/410) monoclonal antibodies. C, immunofluorescence analyses of yeast cells expressing GFP-tagged TDP-43 (TDP-43-GFP) alone and cells expressing both TDP-43-GFP and CK1δ1-317. Cells were counterstained with Hoechst 33342. Note that GFP-positive aggregates (arrowheads) can be observed in cells expressing TDP-43-GFP and CK1δ1-317.