DDX3 binding with CK1ε was closely related to motor neuron degeneration of ALS by affecting neurite outgrowth

Abstract

Amyotrophic lateral sclerosis (ALS) is a chronic neurodegenerative disease characterized by progressive degeneration of motor neurons. The pathogenesis of ALS remains largely unknown. RNA helicase DDX3 is a multifunctional protein involved in several steps of gene expression. Casein kinase 1ε (CK1ε) is an important signal molecule of Wnt signaling pathway and is closely related to neurite growth. However, the roles of DDX3 and CK1ε in the pathogenesis of ALS remain unclear. In this study, we first investigated the expression of DDX3 and CK1ε in the spinal cord of SOD1-G93A ALS transgenic mice using RT-PCR, Western blot and immunohistochemical technique. Results showed that the altered expression of DDX3 and CK1ε was found in the spinal cord of ALS mice. DDX3 and CK1ε positive cells were mainly distributed in the anterior horn of spinal cord and co-localized with neurons not with glial cells, suggesting that the altered expression of DDX3 and CK1ε was closely related to motor neuron degeneration of ALS. Moreover, we selected NSC34 cell line and transfected pEGFP-G93A-SOD1 plasmid to further examine the mechanism. Knockdown of DDX3 that uses small interfering RNA (siRNA) decreased the mRNA and protein levels of CK1ε significantly and inhibited neurite outgrowth of SOD1 mutant NSC34 cells in vitro. Co-immunoprecipitation kit confirmed that DDX3 could band with CK1ε in vivo. Our data suggested that DDX3 binding with CK1ε was closely related to motor neuron degeneration of ALS by affecting neurite outgrowth. Thus, elucidating the underlying mechanisms of ALS is crucial for future development of ALS treatments.

Keywords: Amyotrophic lateral sclerosis; CK1ε; DDX3; NSC34 cells; SOD1-G93A transgenic mice.

Figure 1

The altered expression of DDX3 in the spinal cords of SOD1-G93A transgenic mice (ALS) and wide-type (WT) mice. A. Representative RT-PCR images of DDX3. β-actin was used as loading control. B. Representative western blot images of DDX3 protein. GAPDH was used as loading control. C. The level of DDX3 mRNA as analyzed by RT-PCR (n = 4). D. The level of DDX3 protein as analyzed by western blot (n = 4). E. Confocal microscopy images showing the co-localization of DDX3 with β-tubulin III or GFAP. Representative confocal images were taken of gray matter of 108-day-old mice. Scale bar = 50 μm. *P < 0.05, **P < 0.01, ***P < 0.001, vs. WT.

Figure 2

Immunohistochemical (IHC) staining showing the distribution of DDX3 and CK1ε in the spinal cords of ALS mice and wide-type (WT) mice. The representative images were taken of gray matter (GM) and white matter (WM) of the spinal cords at different stages. 95 d (A, B, G, H); 108 d (C, D, I, J); 122 d (E, F, K, L); GM (1); WM (2). WT (A, C, E, G, I, K); ALS (B, D, F, H, J, L). Scale bar = 50 μm.

Figure 3

The altered expression of CK1ε in the spinal cords of SOD1-G93A transgenic mice (ALS) and wide-type (WT) mice. A. Representative RT-PCR images of CK1ε. β-actin was used as loading control. B. Representative western blot images of CK1ε protein. GAPDH was used as loading control. C. The level of CK1ε mRNA as analyzed by RT-PCR (n = 4). D. The level of CK1ε protein as analyzed by western blot (n = 4). E. Confocal microscopy images showing the co-localization of CK1ε with β-tubulin III or GFAP. Representative confocal images were taken of gray matter of 108-day-old mice. Scale bar = 50 μm. ***P < 0.001, vs. WT.

Figure 4

DDX3 can band with CK1ε in the spinal cord of ALS mice but not in NSC34 cells transfected with pEGFP-G93A-SOD1. Knockdown of DDX3 significantly decreased the mRNA and protein levels of CK1ε in vitro. NSC34 cells were co-transfected with DDX3 siRNA (si-DDX3) and pEGFP-G93A-SOD1 or control siRNA (Con) and pEGFP-G93A-SOD1. A. Co-immunoprecipitation kit confirmed that DDX3 can band with CK1ε in the spinal cord of ALS mice. CK1ε antibody was used to co-immunoprecipitate DDX3. Cell lysate (input) and immunoprecipitated products (Co-IP) were blotted with the DDX3 and CK1ε antibody respectively. B. Co-immunoprecipitation kit confirmed that DDX3 can not band with CK1ε in the NSC34 cells transfected with pEGFP-G93A-SOD1 at 48 h after transfection. Cell lysate (input) and immunoprecipitated products (Co-IP) were blotted with the DDX3 and CK1ε antibody respectively. C. Representative RT-PCR images of DDX3 in the co-transfected NSC34 cells. β-actin was used as loading control. D. Representative western blot images of DDX3 protein in the co-transfected NSC34 cells. GAPDH was used as loading control. E. RT-PCR analysis illustrating the knockdown of DDX3 (n = 4). F. Western blot analysis illustrating the knockdown of DDX3 protein. The amount of DDX3 was quantified and normalized against GAPDH (n = 4). G. Representative RT-PCR images of CK1ε in the co-transfected NSC34 cells. β-actin was used as loading control. H. Representative western blot images of CK1ε protein in the co-transfected NSC34 cells. GAPDH was used as loading control. I. RT-PCR analysis illustrating the down-regulation of CK1ε (n = 4). J. Western blot analysis illustrating the down-regulation of CK1ε protein. The amount of CK1ε was quantified and normalized against GAPDH (n = 4).

Figure 5

Knockdown of DDX3 reduced the cell proliferation and inhibited the neurite outgrowth of SOD1 mutant NSC34 cells in vitro. A. Growth curve of NSC34 cells co-transfected with DDX3 siRNA (si-DDX3) and pEGFP-G93A-SOD1 or control siRNA (Con) and pEGFP-G93A-SOD1 as determined by MTS assay at different time points. Each data point is the mean ± SD of three independent experiments. B. Representative images showing the neurites of NSC34 cells co-transfected with si-DDX3 and pEGFP-G93A-SOD1 or Con and pEGFP-G93A-SOD1 at different time points. Bar = 200 µm. C. Bar chart showing the percentage of cells having one or more neurites in the NSC34 cells co-transfected with si-DDX3 and pEGFP-G93A-SOD1 or Con and pEGFP-G93A-SOD1. Each data point is the mean ± SD of three independent experiments. D. Bar chart showing the lengths of the cell’s longest neurite in the NSC34 cells co-transfected with si-DDX3 and pEGFP-G93A-SOD1 or Con and pEGFP-G93A-SOD1. Each data point is the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, vs. Con and pEGFP-G93A-SOD1.