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First published online 2 November 2004
doi: 10.1242/jcs.01505

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GSK-3ß inhibition/ß-catenin stabilization in ventral midbrain precursors increases differentiation into dopamine neurons

Gonçalo Castelo-Branco^*, Nina Rawal^* and Ernest Arenas

Laboratory of Molecular Neurobiology, Medical Biochemistry and Biophysics, Karolinska Institute, Scheeles väg 1, A1, plan 2, 17177 Stockholm, Sweden

View larger version (61K): [in a new window]   Fig. 1. Indirubin-3-monoxime (I3M) and kenpaullone (KP) increase the number of tyrosine hydroxylase positive (TH+) neurons in ventral mesencephalon (VM) precursor cultures. (A) Dose-response experiments in E14.5 VM precursors indicate that 15 µM KP and 3-5 µM I3M are optimal doses to increase TH+ cell numbers. Statistical analysis was performed using one-way ANOVA with Fisher's post-hoc test. *P<0.05; **0.01<P<0.001; ***P<0.001. (B) Treatment of E14.5 VM precursors with 15 µM KP or 5 µM I3M for 24 hours decreases GSK-3ß protein levels as assessed by immunoblotting. ß-actin was used as a loading control. (C) TH/Hoechst 33258 immunostaining shows an increase in the number of TH immunoreactive cells 3 days after treatment with 15 µM KP or 5 µM I3M. Bar, 50 µm.

View larger version (55K): [in a new window]   Fig. 2. Treatment with KP or I3M increases neuronal differentiation of VM precursors, particularly into DA neurons. (A,B) Immunostaining with antibodies against nestin/Hoechst 33258 (A) and vimentin/Hoechst 33258 (B), showed a reduction in the number of neuronal precursors (nestin+) after treatment with I3M, with no effect on glial precursors (vimentin+). (C-F) 15 µM KP or 5 µM I3M increased the number of immature Tuj1+ (C,F) and mature MAP2+ (D,F) neurons and the proportion of TH+ DA neurons (E,F) after 3 days in vitro. Statistical analysis was performed using one-way ANOVA with Boneferroni's multiple comparison test (A,B) and two-tailed unpaired t-tests (C-E). Significance levels as for Fig. 1. Bar, 25 µm.

View larger version (62K): [in a new window]   Fig. 3. GSK-3ß inhibition does not regulate cell survival or proliferation of VM precursors or MN9D cells but induces their morphological differentiation. (A) Treatment with 15 µM KP or 5 µM I3M did not change the total number of VM cells after 3 days in culture, as assessed by Hoechst 33258 staining. (B) Proliferation, as assessed by BrdU incorporation, was not affected by 15 µM KP, but was significantly decreased with 5 µM I3M. (C,D) Immunostaining with -Active Caspase 3 showed a decreased number of positive cells 3 days after treatment with 5 µM I3M, but not with KP. (E,F) Treatment of MN9D cells with 15 µM KP for 4 days did not alter the total number of cells (Hoechst 33258) or their proliferation (BrdU incorporation). (G) Treatment with 15 µM KP for 4 days induced morphological differentiation of MN9D cells. Statistical analysis was performed using two-tailed unpaired t-test (A, E and F), Kruskal-Wallis test (B) and one-way ANOVA with Boneferroni's multiple comparison test (C). Significance levels as for Fig. 1. Bar, 50 µm.

View larger version (34K): [in a new window]   Fig. 4. KP upregulates c-ret mRNA and increases conversion of Nurr1+ precursors into TH+ neurons. (A) Real time RT-PCR analysis revealed that c-ret mRNA levels were upregulated upon treatment with 15 µM KP. (B) 15 µM KP increased the conversion of VM Nurr1+ precursors into TH+ neurons. (C) TH/Nurr1 double immunostaining showed an increase in the number of immunoreactive cells 3 days after treatment with 15 µM KP. Statistical analysis was performed using a two-tailed Wilcoxon's signed rank test (A) and a two-tailed unpaired t-test (B). Significance levels as for Fig. 1.

View larger version (81K): [in a new window]   Fig. 5. GSK-3ß inhibitors stabilize ß-catenin and overexpression of ß-catenin increases the number of TH+ neurons. (A) Treatment of VM precursors with 15 µM KP or 5 µM I3M for 24 hours increased both ß-catenin and TH protein levels, as assessed by immunoblotting. ß-actin was used as a loading control. (B,C) TH immunostaining shows a twofold increase in the number of positive cells out of the total number of cells (Hoechst 33258) two days after transfection with ß-catenin, compared to transfection with an empty control vector. Statistical analysis was performed using a two-tailed unpaired t-test. Significance levels as for Fig. 1. Bar, 50 µm.