c-Abl Inhibition Delays Motor Neuron Degeneration in the G93A Mouse, an Animal Model of Amyotrophic Lateral Sclerosis

<div><h3>Background</h3><p>Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive death of motor neurons. Although the pathogenesis of ALS remains unclear, several cellular processes are known to be involved, including apoptosis. A previous study revealed the apoptosis-related gene c-Abl to be upregulated in sporadic ALS motor neurons.</p> <h3>Methodology/Findings</h3><p>We investigated the possibility that c-Abl activation is involved in the progression of ALS and that c-Abl inhibition is potentially a therapeutic strategy for ALS. Using a mouse motor neuron cell line, we found that mutation of Cu/Zn-superoxide dismutase-1 (SOD1), which is one of the causative genes of familial ALS, induced the upregulation of c-Abl and decreased cell viability, and that the c-Abl inhibitor dasatinib inhibited cytotoxicity. Activation of c-Abl with a concomitant increase in activated caspase-3 was observed in the lumbar spine of G93A-SOD1 transgenic mice (G93A mice), a widely used model of ALS. The survival of G93A mice was improved by oral administration of dasatinib, which also decreased c-Abl phosphorylation, inactivated caspase-3, and improved the innervation status of neuromuscular junctions. In addition, c-Abl expression in postmortem spinal cord tissues from sporadic ALS patients was increased by 3-fold compared with non-ALS patients.</p> <h3>Conclusions/Significance</h3><p>The present results suggest that c-Abl is a potential therapeutic target for ALS and that the c-Abl inhibitor dasatinib has neuroprotective properties <em>in vitro</em> and <em>in vivo</em>.</p> </div

Dasatinib inhibits c-Abl phosphorylation in G93A mice.

<p>Protein levels of phosphorylated c-Abl (Tyr245), c-Abl, and activated caspase-3 were measured by western blot analysis using spinal cords from dasatinib- and vehicle-treated G93A mice (120 days old). GAPDH is shown as the loading control. hSOD1 and mSOD1 indicate human SOD1 and mouse endogenous SOD1, respectively. Western blot analysis is shown in duplicate. The animal number refers to individual animals. Parallel declines in c-Abl phosphorylation and activated caspase 3 were observed.</p

The effect of dasatinib on motor neuron survival in G93A mice.

<p>A: Spinal cord (L1-3) specimens from 120-day-old mice were immunostained with anti-ChAT antibody. The mice were administered the indicated amounts of dasatinib daily from postnatal day 56 to day 120 (n = 8 mice per group). Scale bar: 250 µm. B: The number of ChAT-positive neurons in the sections described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046185#pone-0046185-g006" target="_blank">Fig. 6A</a> was counted using Image J software. Dasatinib prevented the loss of ChAT-positive motor neurons in the ventral horn of G93A mice at doses of 15 mg/(kg·day) (<i>P</i><0.05) and 25 mg/(kg·day) (<i>P</i><0.01). Statistics were evaluated using 1-way ANOVA with Dunnett's post-hoc test. *<i>P</i><0.05, **<i>P</i><0.01. C: The area of ChAT-positive neurons in the sections described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046185#pone-0046185-g006" target="_blank">Fig. 6A</a> was determined using Image J software. Dasatinib increased the size of motor neuron cell bodies at doses of 15 and 25 mg/(kg·day) (<i>P</i><0.05). Statistics were evaluated using 1-way ANOVA with Dunnett's post-hoc test. *<i>P</i><0.05. D: To investigate the innervation status of NMJs, frozen quadriceps femoris specimens from 120-day-old mice were stained with alpha-BuTX (red) and anti-synaptophysin (green) or anti-SMI31 (green) antibodies. Representative NMJs visualized with the confocal laser scanning microscopy are shown. The mice were administered the indicated amounts of dasatinib daily from postnatal day 56 to day 120. Scale bar: 10 µm. E: The ratio of double-immunostained innervated NMJs to total NMJs is summarized. One hundred immunostained NMJs were investigated in each dasatinib-treated mouse (n = 3 mice per group). Dasatinib significantly ameliorated the destruction of NMJ innervation in G93A mice at doses of 5 (<i>P</i><0.05), 15, and 25 mg/(kg·day) (<i>P</i><0.01). Statistics were evaluated using 1-way ANOVA with Dunnett's post-hoc test. *<i>P</i><0.05, **<i>P</i><0.01.</p

c-Abl upregulation and activation in affected motor neurons of sporadic ALS patients.

<p>A: The protein expression of total c-Abl was measured by western blot analysis using an anti-c-Abl antibody and the lumbar spinal cord tissue from sporadic ALS (sALS) cases and controls. GAPDH is shown as an internal control. The patient number refers to individual patients. B: Densitometric analysis using Image J software revealed a significant difference in the amount of total c-Abl protein in the lumbar spinal cords of sALS patients and controls (<i>P</i><0.01). Data are presented as mean ± SEM. Statistical analysis was performed using Student's <i>t</i> test. **<i>P</i><0.01. C: Immunohistochemical analysis using paraffin-embedded spinal cords from control and sALS patients was carried out by staining with anti-c-Abl, anti-phosphorylated c-Abl (Tyr412), and anti-phosphorylated c-Abl (Tyr245) antibodies. Scale bar: 50 µm.</p

The effect of dasatinib on survival and disease progression in G93A mice.

<p>Rotarod activity, grip strength, body weight, and survival rate in G93A mice with or without dasatinib treatment (0, 5, 15, and 25 mg/(kg·day)). Survival of G93A mice was improved by dasatinib at a dose of 25 mg/(kg·day) compared with vehicle treatment (Log-rank test, <i>P</i><0.01, 25 mg/(kg·day) vs. vehicle), whereas a lower dose of dasatinib (5 mg/(kg·day)) had no significant effect on life span. Weight loss was also ameliorated by dasatinib at a dose of 25 mg/(kg·day) compared with vehicle treatment (2-way ANOVA, <i>P</i><0.01, 25 mg/(kg·day) vs. vehicle). The administration of dasatinib at 25 mg/(kg·day) similarly ameliorated grip strength (2-way ANOVA, <i>P</i><0.01, 25 mg/(kg·day) vs. vehicle). The difference in physical function between the groups as assessed by rotarod was not significant by 2-way ANOVA, although a beneficial tendency of dasatinib was observed.</p

Dasatinib reduces cytotoxicity of mutant SOD1s in NSC-34 cells.

<p>A: Protein levels of phosphorylated c-Abl (Tyr245), c-Abl, phosphorylated c-Src (Tyr416), c-Src, and GAPDH in NSC-34 cells overexpressing human wild-type or mutant SOD1s treated with various concentrations of dasatinib or SU6656 were measured by western blot. Cells were cultured in serum-free culture medium with doxycycline (Dox, 2 µg/ml), and western blot was performed at 24 h after dasatinib or SU6656 addition. B: Cells were grown in 96-well collagen-coated plates (3,500 cells per well) with doxycycline (Dox, 2 µg/ml) in culture medium containing 10% FBS for 16 h. Culture medium was then replaced with 1% FBS-containing medium including the indicated concentrations of dasatinib and 2 µg/ml doxycycline (Dox). MTS assays were performed at 24 h after addition of dasatinib or SU6656. Viability was measured as the level of absorbance at 490 nm. Absorbance at 490 nm was expressed as the mean ± SEM (n = 6). Ratios of relative cell viability based on the MTS assay were calculated to determine the beneficial effect of dasatinib in mutant cells overexpressing SOD1s. Absorbance at 490 nm was standardized relative to the absorbance at each corresponding time point for 0 nM dasatinib. Cell viability assay confirmed that dasatinib significantly reduced the cytotoxicity of mutant SOD1s, whereas SU6656 did not. Statistics were evaluated using 1-way ANOVA with Dunnett's post-hoc test. *<i>P</i><0.05, **<i>P</i><0.01. C: Cells were grown in 96-well collagen-coated plates (3,500 cells per well) with doxycycline (Dox, 2 µg/ml) in culture medium containing 10% FBS for 16 h. Culture medium was then replaced with 1% FBS-containing medium with the indicated concentrations of dasatinib and 2 µg/ml doxycycline (Dox). LDH assays were performed at 24 h after dasatinib or SU6656 addition. Cytotoxicity was measured as the level of absorbance at 490 nm. Ratios of relative LDH release were calculated to determine the beneficial effect of dasatinib in mutant cells overexpressing SOD1s. Absorbance at 490 nm was standardized relative to the absorbance at each corresponding time point for 0 nM dasatinib. LDH assay confirmed that dasatinib significantly reduced the cytotoxicity of mutant SOD1s, whereas SU6656 did not. Values represent the mean ± SEM of the ratio of LDH release (n = 4). Statistics were evaluated using 1-way ANOVA with Dunnett's post-hoc test. *<i>P</i><0.05, **<i>P</i><0.01.</p

Inducible expression system of wild-type and mutant SOD1s in NSC-34 cells.

<p>A: NSC-34 cells were stably transduced with an inducible lentivirus expressing human Myc-tagged wild-type or mutant SOD1 protein. Cells were cultured with or without doxycycline (Dox, 2 µg/ml) for 48 h to induce SOD1 protein. Tubulin is shown as a loading control. hSOD1 and mSOD1 indicate human SOD1 and mouse endogenous SOD1, respectively. B: Cell viability assay based on the MTS method showed that overexpression of both types of mutant SOD1, G93A and G85R, caused cytotoxicity in serum-free culture medium. Mock indicates mock-transfected NSC-34 cells. Data are presented as mean ± SEM. Statistics were evaluated using 1-way ANOVA with Dunnett's post-hoc test. *<i>P</i><0.05, **<i>P</i><0.01 C: Cytotoxicity detection assay using the LDH release method revealed that overexpression of both types of mutant SOD1, G93A and G85R, caused cytotoxicity in serum-free culture medium. Data are presented as mean ± SEM. Statistics were evaluated using 1-way ANOVA with Dunnett's post-hoc test. *<i>P</i><0.05, **<i>P</i><0.01.</p