The c-Abl/p73 pathway induces neurodegeneration in a Parkinson's disease model

Abstract

Parkinson's disease is the second most common neurodegenerative disorder. Although it is clear that dopaminergic neurons degenerate, the underlying molecular mechanisms are still unknown, and thus, successful treatment is still elusive. One pro-apoptotic pathway associated with several neurodegenerative diseases is the tyrosine kinase c-Abl and its target p73. Here, we evaluated the contribution of c-Abl and p73 in the degeneration of dopaminergic neurons induced by the neurotoxin 6-hydroxydopamine as a model for Parkinson's disease. First, we found that in SH-SY5Y cells treated with 6-hydroxydopamine, c-Abl and p73 phosphorylation levels were up-regulated. Also, we found that the pro-apoptotic p73 isoform TAp73 was up-regulated. Then, to evaluate whether c-Abl tyrosine kinase activity is necessary for 6-hydroxydopamine-induced apoptosis, we co-treated SH-SY5Y cells with 6-hydroxydopamine and Imatinib, a c-Abl specific inhibitor, observing that Imatinib prevented p73 phosphorylation, TAp73 up-regulation, and protected SH-SY5Y cells treated with 6-hydroxydopamine from apoptosis. Interestingly, this observation was confirmed in the c-Abl conditional null mice, where 6-hydroxydopamine stereotaxic injections induced a lesser reduction of dopaminergic neurons than in the wild-type mice significantly. Finally, we found that the intraperitoneal administration of Imatinib prevented the death of dopaminergic neurons induced by injecting 6-hydroxydopamine stereotaxically in the mice striatum. Thus, our findings support the idea that the c-Abl/p73 pathway is involved in 6-hydroxydopamine degeneration and suggest that inhibition of its kinase activity might be used as a therapeutical drug in Parkinson's disease.

Keywords: 6-hydroxydopamine; Neurodegeneration; Parkinson's disease; c-Abl; p73.

Fig. 1

6-OHDA treatments up-regulate phosphorylated c-Abl protein levels. (A) Immunofluorescence of phospho-c-Abl-Tyr412 in SH-SY5Y cells treated with 100 µM 6-OHDA. (B-G) Western blot of total lysates of SH-SY5Y (B) and PC12 (E) cell cultures treated with 100 µM 6-OHDA and their densitometry quantifications (C and D, and F and G, respectively). β-actin was used as a loading control. Western blot quantifications for phosphorylated tyr 412 and tyr 245 were normalized by total c-Abl. n = 4 (C and D) and 5 (F and G). Statistical analysis was done using one-way ANOVA and Tukey's post hoc test * p < 0.05; ** p < 0.001. Mean ± SEM.

Fig. 2

Phosphorylated and pro-apoptotic TAp73 specific isoform form of p73 are increased in cells treated with 6-OHDA. Western blot was performed from total lysates of SH-SY5Y cell culture treated with 100 µM 6-OHDA at 0.5, 2 or 4 h. The membranes were incubated with total p73 (A), phospho-p73 (C), TAp73 (E) or ΔNp73 (G) antibodies. Then, membranes were reprobed with GAPDH antibody as loading control. Densitometric quantifications were performed for total p73 (B), phospho-p73 (D), TAp73 (F) or ΔNp73 (H), normalized by GAPDH, its loading control. One-way ANOVA Tukey's post hoc test were performed. * p < 0.05; *** p < 0.001; ns = not significant. n = 3. Mean ± SEM.

Fig. 3

Imatinib prevents cell death induced by 6-OHDA. (A-B) Apoptotic nuclei assay was assessed by Hoetsch staining in SH-SY5Y cell culture co-treated with 100 µM (A) or 250 µM (B) 6-OHDA and 10 µM Imatinib. Representative control and apoptotic (6-OHDA) nuclei are shown in A. (C) MTT assay of SH-SY5Y cell culture treated with 100 µM or 250 µM 6-OHDA and 10 µM Imatinib. One-way ANOVA and Tukey's post hoc test were performed *** p < 0.001; ns = not significant. n = 4. Mean ± SEM.

Fig. 4

Imatinib reduces phosphorylated-p73 protein levels induced by 6-OHDA. (A) Total lysates of SH-SY5Y cell cultures treated with 100 µM 6-OHDA or 10 µM Imatinib for 0.5, 2, or 4 h were incubated with antibodies against total or phosphorylated p73. Then, membranes were reprobed with GAPDH antibodies as a loading control. (B) Total lysates of SH-SY5Y cell cultures treated with 100 µM 6-OHDA or 10 µM Imatinib for 0.5, 2, or 4 h were incubated with antibodies against total or phosphorylated c-Abl. Then, membranes were reprobed with β-actin antibodies as a loading control.

Fig. 5

Conditional ablation of c-Abl prevents reduction of tyrosine hydroxylase-positive cells induced by 6-OHDA stereotaxic injection. (A-B) Wild-type (c-Abl WT) and neuronal-specific c-Abl null mice (c-Abl cKO) were stereotaxically injected with 6-OHDA at the striatum, and 7 days after, immunohistochemistry for tyrosine hydroxylase was performed in coronal brain sections at the substantia nigra (A). Then TH+ neurons were quantified (B). Data are expressed as mean ± SEM. n = 5 (c-Abl WT) and n = 4 (c-Abl cKO). * p < 0.05. Scale bar: 100 µm.

Fig. 6

Pharmacological c-Abl inhibition reduced 6-OHDA-induced dopaminergic cell loss. (A-B) WT C57B adult mice were injected with 6-OHDA (2 µl of 5 mg/ml stock solution) in the striatum. Mice were divided into two groups; untreated and treated with Imatinib (25 mg/kg) for 7 days, one injection per day. Then, immunostaining for tyrosine hydroxylase was performed in coronal slices containing the substantia nigra (A). Then TH+ neurons were quantified (B). Data are expressed as mean ± SEM. n = 4 (control) and n = 5 (Imatinib). * p < 0.05. Scale bar: 100 µm.