Motor neuron preservation and decrease of in vivo TDP-43 phosphorylation by protein CK-1δ kinase inhibitor treatment

Abstract

Pathogenesis of amyotrophic lateral sclerosis (ALS), a devastating disease where no treatment exists, involves the compartmentalization of the nuclear protein TDP-43 (TAR DNA-binding protein 43) in the cytoplasm which is promoted by its aberrant phosphorylation and others posttranslational modifications. Recently, it was reported that CK-1δ (protein casein kinase-1δ) is able to phosphorylate TDP-43. Here, the preclinical efficacy of a benzothiazole-based CK-1δ inhibitor IGS-2.7, both in a TDP-43 (A315T) transgenic mouse and in a human cell-based model of ALS, is shown. Treatment with IGS-2.7 produces a significant preservation of motor neurons in the anterior horn at lumbar level, a decrease in both astroglial and microglial reactivity in this area, and in TDP-43 phosphorylation in spinal cord samples. Furthermore, the recovery of TDP-43 homeostasis (phosphorylation and localization) in a human-based cell model from ALS patients after treatment with IGS-2.7 is also reported. Moreover, we have shown a trend to increase in CK-1δ mRNA in spinal cord and significantly in frontal cortex of sALS cases. All these data show for the first time the in vivo modulation of TDP-43 toxicity by CK-1δ inhibition with IGS-2.7, which may explain the benefits in the preservation of spinal motor neurons and point to the relevance of CK-1δ inhibitors in a future disease-modifying treatment for ALS.

Figure 1

In vivo experiment with IGS-2.7. (a) Chemical structure of IGS-2.7 and its IC₅₀ values on CK-1. (b) Experimental design of i.p. administration of the compound IGS-2.7 (c) Effects of IGS-2.7 treatment on body weight gain in TDP-43 and wild-type mice. Values are expressed as means ± SEM; N ≥ 8 animals in each group. Data were assessed by repeat measures two-way ANOVA followed by the Bonferroni test (*p < 0.05, **p < 0.01, ***p < 0.001 vs. WT-Veh group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. TDP-43-Veh).

Figure 2

Photomicrographs of histological samples in the anterior horn of the spinal cord of TDP-43 mice and wild-type controls. (a) Representative images of Nissl staining of motor neurons (black arrows), ChAT, Iba-1 and GFAP immunostained sections (b) Quantification of the different markers. Total average number of motor neurons (Nissl, ChAT+), microglia (Iba-1+) and astrocytes (GFAP+) is shown. Data are expressed as a percentage over the WT-Veh group. Values are expressed as means ± SEM, N ≥ 6 in each group. Data were assessed by one-way ANOVA followed by the Bonferroni test (*p < 0.05, **p < 0.01, ***p < 0.001 vs. WT-Veh group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001 vs. TDP-43-Veh). Scale bars = 200 μm.

Figure 3

Effects of IGS-2.7 on phosphorylation of TDP-43 in the spinal cord of wild-type and transgenic TDP-43 mice. (a) Spinal cord lysates from wild-type and transgenic TDP-43 mice were used to determine the levels of pTDP-43 and TDP-43. (b) Representative immunoblots are shown. Densitometric analyses represent the ratio of pTDP-43/total TDP-43 and are the mean ± SEM of 8 observations per group. Data were assessed by one-way ANOVA followed by the Bonferroni test (*p < 0.05 WT-Veh group; ^#p < 0.05 vs. TDP-43-Veh).

Figure 4

Effect of IGS-2.7 on TDP-43 phosphorylation in immortalized lymphocytes from control and sporadic ALS individuals. Immortalized lymphocytes were seeded at an initial density of 1 × 10⁶ × ml⁻¹ in absence or presence of IGS-2.7 (2.5 and 5 μM). 24 h after drugs addition, cells were harvested and processed for Western blotting analysis. (a) Densitometric measurements were performed on individual immunoblots and values indicate the mean of p-TDP-43 versus the corresponding total TDP-43 levels ± SEM for experiments carried out with 4 different cell lines for each group. (b) Representative immunoblot is shown. Data were assessed by one-way ANOVA and post hoc Fisher’s analysis (***p < 0.001 significantly different from control cells; ^##p < 0.01 and significantly different from untreated ALS cells).

Figure 5

Subcellular localization of TDP-43 after IGS-2.7 treatment of lymphoblasts from control and ALS subjects. Lymphoblasts were seeded at 10⁶ cells × ml⁻¹ and incubated in presence or absence of IGS-2.7 (5 μM) for 24 h. (a) Cells were stained with anti-TDP-43 antibody followed by a secondary antibody labeled with Alexa Fluor 488. DAPI was included in the mounting media to stain the nucleus. TDP-43 protein localization was assessed by confocal laser scanning microscopy. Merged images show that treatment with IGS-2.7 prevented the higher cytosolic accumulation (red arrows) of TDP-43 protein in ALS patients. Scale bars = 11 μM. Magnified cells from images are shown on the right panels for better visualization (Scale bars = 3 μm) (b) Quantification of TDP-43 cytosolic localization in lymphoblasts from ALS patients compared to controls. Data are expressed as mean ± SEM for experiments carried out with 4 different cell lines for each group. Data were assessed by one-way ANOVA and post hoc Fisher’s analysis (**p < 0.01 from control cells; ^###p < 0.001 from untreated ALS cells).

Figure 6

Relative expression of CSNK1D and CSNK1E mRNA levels in post-mortem samples from control and ALS subjects and immunohistochemical analysis. (a) CSNK1D and CSNK1E mRNA levels in the anterior horn of the spinal cord and (b) frontal cortex area 8 of sALS cases and controls, *p < 0.05. (c) CK-1δ immunoreactivity in the anterior horn of the spinal cord at lumbar level in control and ALS cases showing CK-1δ immunoreactivity in the cytoplasm of neurons and neuronal processes in control. Similar reaction is found in the remaining motor neurons in devastated anterior horn in the ALS case together with increased CK-1δ immunoreactivity in reactive glial cells (arrows). Paraffin sections, slightly counterstained with haematoxylin; scale bar = 25 µm.