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

Takashi Nonaka,Genjiro Suzuki,Yoshinori Tanaka,Fuyuki Kametani,Shinobu Hirai,Haruo Okado,Tomoyuki Miyashita,Minoru Saitoe,Haruhiko Akiyama,Hisao Masai,Masato Hasegawa

doi:10.1074/jbc.m115.695379

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.

Highlights

Introduction of Protein Aggregates asSeeds into Cultured Cells—Cells co-expressing TAR DNA-binding protein of 43 kDa (TDP-43) and CK1␦1-317 were incubated for 3 days and harvested
We found that expression of a hyperactive form of casein kinase 1 ␦ (CK1␦1-317, a C-terminally truncated form) promotes mislocalization and cytoplasmic accumulation of phosphorylated TDP-43 in cultured neuroblastoma SH-SY5Y cells
Our results clearly show that phosphorylation of TDP-43 by abnormally activated CK1␦ causes both cytoplasmic aggregation of TDP-43 and cytotoxicity in vitro and in vivo, establishing a novel mechanism of neurodegeneration that is likely to be relevant to the pathogenesis of diseases such as Frontotemporal lobar degeneration (FTLD) and ALS

Summary

Experimental Procedures

Antibodies—Monoclonal and polyclonal (anti-Ser(P)-409/ 410) antibodies against a synthetic phosphopeptide of TDP-43 have been reported previously [13, 25]. The cells were maintained at 37 °C under a humidified atmosphere of 5% (v/v) CO2 in air They were grown to 50% confluence in 6-well culture dishes for transient expression and transfected with each expression vector (usually 1 ␮g) using XtreamGENE9 (Roche) according to the instructions of the manufacturer. Fractionation of Cellular Proteins and Immunoblotting— SH-SY5Y cells grown in a 6-well plate were transfected with several expression vectors. Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Exon 9 Skipping Assay—SH-SY5Y cells grown in 6-well plates were transfected with 0.5 ␮g of reporter plasmid pSPL3CFTR exon 9, including the repeat sequence of TG11T7 [16], pcDNA3.1-TDP-43, and/or pcDNA3.1-CK1␦1-317 (total 1.5 ␮g of plasmids), using XtreamGENE9 (Roche). Real-time PCR—SH-SY5Y cells grown in 6-well plates were transfected with 1 ␮g of pcDNA3.1-TDP-43 and/or pcDNA3.1-CK1␦1-317 (total 2 ␮g of plasmids), using XtreamGENE9 (Roche). The obtained spectra were analyzed with Proteome Discoverer (Thermo Fisher Scientific) and Mascot software (Matrix Science)

JOURNAL OF BIOLOGICAL CHEMISTRY

Results

Discussion