The c-Abl inhibitor, nilotinib, protects dopaminergic neurons in a preclinical animal model of Parkinson's disease

Abstract

c-Abl is activated in the brain of Parkinson's disease (PD) patients and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice where it inhibits parkin through tyrosine phosphorylation leading to the accumulation of parkin substrates, and neuronal cell death. In the present study, we evaluated the in vivo efficacy of nilotinib, a brain penetrant c-Abl inhibitor, in the acute MPTP-induced model of PD. Our results show that administration of nilotinib reduces c-Abl activation and the levels of the parkin substrate, PARIS, resulting in prevention of dopamine (DA) neuron loss and behavioral deficits following MPTP intoxication. On the other hand, we observe no reduction in the tyrosine phosphorylation of parkin and the parkin substrate, AIMP2 suggesting that the protective effect of nilotinib may, in part, be parkin-independent or to the pharmacodynamics properties of nilotinib. This study provides a strong rationale for testing other brain permeable c-Abl inhibitors as potential therapeutic agents for the treatment of PD.

Figure 1. The schematic diagram depicts the time schedule of intervention and analyses performed.

A schematic diagram depicts the experimental design of present study. Numerals represent the days experiments were conducted. Mice were pretreated with vehicle or nilotinib a week before MPTP injections and continued to be treated until experiments. On 7th day we injected saline or MPTP (2 h interval, 4 times, 20 mg/kg free base) in respective treatment groups. On 13th day the pole test was performed. On 14th day, mice were sacrificed for indicated studies. Following are animal numbers used for this studies, behavioural (n = 10), neurochemical (n = 5), immunohistochemistry (n = 5) and biochemical studies (n = 4) per each treatment group.

Figure 2. In vivo assessments of nilotinib in mouse brain.

(a) In vivo measurement of nilotinib levels in the brain: Nilotinib was dissolved in vehicle (10% NMP and 90% PEG 300) and administrated by oral gavage to mice. At 2 h the concentration of nilotinib was measured by using HPLC-UV analysis. Nilotinib crosses the blood brain barrier and shows pharmacological effective levels in brain. Oral gavage of vehicle treated mice brain showed no peaks corresponding to reference to nilotinib standards. (b) Nilotinib has no effect on the levels of MPP+: Levels of MPP+ in the striatum of vehicle and nilotinib treated mice injected with saline and MPTP (2 h interval, 4 times, 20 mg/kg free base). 90 min after the final injection of MPTP, MPP+ was measured by using HPLC-UV analysis. (c) Effect of nilotinib on DAT levels following MPTP-intoxication in the striatum: Immunoblots of striatal lysates from C57BL/6 mice treated with saline, MPTP, MPTP with nilotinib (25 mg/kg body wt, p.o.), or nilotinib alone. The striatal lysates were immunoblotted with anti-DAT and anti-β-actin antibodies. β-actin serves as a loading control. (d) Relative DAT levels normalized to total β-actin are indicated. (e) Nilotinib inhibits c-Abl activation following MPTP-intoxication in the ventral midbrain: Immunoblots of ventral midbrain lysates from C57BL/6 mice treated with saline, MPTP, MPTP with nilotinib (25 mg/kg body wt, p.o.), or nilotinib alone. The ventral midbrain lysates were immunoblotted with anti-phospho-c-Abl, anti-c-Abl, and anti-β-actin antibodies. β-actin serves as a loading control. (f) Relative phospho-c-Abl levels normalized to total c-Abl are indicated. All experiments were repeated three times and representative images of the immunoblots are shown. The Error bars represent the mean ± SEM. (n = 3–4 mice per group). Statistical significance was determined by performing two-way ANOVA followed by Bonferroni post test. ** p < 0.01, ***p < 0.001 for MPTP compared with Saline group, ###p<0.001 for Nil + MPTP compared with MPTP group. Full length blots are presented in the supplementary Figure S1. (ns: not significant).

Figure 3. c-Abl inhibitor, nilotinib protects against MPTP-induced dopamine depletion.

Nilotinib or vehicle administered mice were subjected to acute MPTP injections (20 mg/kg, MPTP free base X 4, every 2 h). Striatal dopamine (DA) and metabolites levels were analysed 7 days after the last MPTP injection by HPLC-ECD analysis. (a) Nilotinib rescues DA loss and (b) DOPAC in the striatum of MPTP mice. (c) Striatal levels of HVA and (d) 3MT were restored in mice treated with nilotinib. (e) There is a significant decrease in DA turnover [(DOPAC+HVA/DA) and (f) (DOPAC+3MT/DA)] in the striatum of nilotinib treated mice. Error bars represent the mean ± SEM, n = 5 mice per group. Two-way ANOVA was used to test significant and followed with post-hoc Bonferroni test to compare with the multiple group. **p<0.0002, *** p<0.0001 for MPTP with compare control group, #p<0.05, ### p<0.001 for Nil+ MPTP compared MPTP group. (ns: not significant).

Figure 4. Nilotinib prevents MPTP-induced dopaminergic neurodegeneration.

Seven days following the last MPTP injection, the number of TH-positive neurons was analysed in the substantia nigra pars compacta (SNpc) of vehicle and nilotinib treated mice in the presence or absence of MPTP treatment by unbiased stereologic counting. (a) Representative photomicrographs from coronal mesencephalon sections containing tyrosine hydroxylase (TH)-positive neurons of (a and c) vehicle and (b and d) nilotinib mice treated with saline or MPTP. (b) Stereology counts of TH and (c) Nissl-positive neurons in the SNpc from mice injected with saline and MPTP with or without treatment of nilotinib. Error bars represent the mean ± SEM, n = 5 mice per group. Two-way ANOVA was used to test significance and followed with post-hoc Bonferroni test to compare with the multiple group. *** p<0.0001 for MPTP compared to control group, #p<0.01 for Nil+ MPTP compared to MPTP group.

Figure 5. Nilotinib prevents MPTP-induced dopaminergic terminal loss.

Seven days following the last MPTP injection, striatal TH-immunopositive fiber density was estimated. (a) Representative photomicrograph of striatal sections stained for TH immunoreactivity. From top to bottom panel, (a and c) vehicle and (b and d) nilotinib mice treated with saline or MPTP respectively. (b) Quantification of dopaminergic fiber densities in the striatum by using Image J software (NIH). Significant TH fiber density loss was observed in striatum after MPTP intoxication. Administration of nilotinib significantly decreased the loss of striatal TH-positive fibers after MPTP treatment. Error bars represent the mean +/−SEM, n = 5 mice per group for Striatal TH-fiber density. Two-way ANOVA was used to test significance and followed with Bonferrioni post hoc test to compare with the multiple groups. ***p < 0.0001 for MPTP compared with control group. ###p < 0.001 for Nil plus MPTP compared to the MPTP group.

Figure 6. Nilotinib protects against MPTP-induced behavioral deficits.

The sixth day after the last MPTP injection, the pole test was performed in vehicle or nilotinib treated mice. Behavioral abnormalities were improved in mice administered with nilotinib. Error bars represent the mean ± SEM, n = 10 mice per group for behavioural studies. Two-way ANOVA was used to test significance and followed with post-hoc Bonferroni test to compare with the multiple groups. ** p<0.001 for MPTP with compared to the control group, ###p<0.001 for Nil plus MPTP compared to the MPTP group.

Figure 7. Nilotinib is unable to prevent MPTP-induced parkin phosphorylation and accumulation of AIMP2, but reduces PARIS in the ventral midbrain of MPTP-treated mice.

(a) Immunoblots of parkin immunoprecipitation samples from C57/BL6 mice treated with saline, MPTP only, MPTP with nilotinib (25 mg/kg body wt), or nilotinib alone (25 mg/kg body wt). Immunoblotting with an anti-phosphotyrosine antibody shows tyrosine-phosphorylated parkin and an anti-parkin antibody shows immunoprecipitated parkin. Brain lysates were immunoblotted with anti-AIMP2 and anti-PARIS antibodies to monitor their levels with β-actin serving as a loading control. (b) Normalized levels of tyrosine-phosphorylated parkin (p-parkin), AIMP2, and PARIS are indicated. The Error bars represent the mean ± SEM (n = 4 mice per group). Two-way ANOVA was used to test significance and followed with post-hoc Bonferroni test to compare with the multiple groups. ** p<0.001 for MPTP with compared to the control group, ##p<0.001 for Nil plus MPTP compared to the MPTP group. All experiments were repeated three times and representative images of the immunoblots are shown. Full length blots are presented in the supplementary Figure S2.