The Tumor Suppressor p53 Fine-Tunes Reactive Oxygen Species Levels and Neurogenesis via PI3 Kinase Signaling

A, Immunohistochemistry for p53 on E11 telencephalons. p53 immunoreactivity is ubiquitous in highly proliferating cells of E11 telencephalons, yet decreasing in the preplate that is at the outmost layer of the telencephalon. Scale bar, 50 μm. B, p53 immunoreactivity is detectable in the germinal zones of E13 mouse telencephalons, where proliferative neural progenitors reside. Notably, the intensity of p53 signal decreases considerably toward the cortical plate. C, Immunohistochemistry for nestin, a marker for proliferative NPCs on E13 telencephalons. The same regions that are strongly immunoreactive for nestin are positive also for p53 (B). D, Co-immunocytochemistry in E13 neurospheres co-stained for the pan-proliferative marker Ki67 and for p53. The great majority of p53+ NPCs are also Ki67+. No detectable p53 immunoreactivity is found in p53−/− neurospheres. Scale bars (C, D): 100 μm. E, The bar graph shows mean percentage (normalized to total number of Hoechst+ cells) ± SEM of Ki67+, p53+, and Ki67+/p53+ double-positive cells in neurospheres. ^★★p < 0.01 (Student's two-tail t test). F, The bar graph shows quantification of p53 mRNA during embryonic telencephalic development by quantitative real-time RT-PCR. p53 mRNA is most abundant at E13–E16, that is, during the neurogenic period of telencephalic development, decreasing remarkably thereafter. Mean p53 mRNA expression was normalized to β-actin. Three embryos per developmental stage and genotype were examined. Each experiment was conducted as triplicates. Error bars indicate ±SD. G, p53 in vitro mRNA levels decrease upon differentiation the highest amount of p53 mRNA being expressed in proliferative neurospheres when assessed by quantitative RT-PCR. Data represent mean p53 mRNA expression normalized to β-actin ± SD from two unrelated experiments, each triplicates (^★★p < 0.01; ^★★★p < 0.001, two-tail t test). wt, Wild type.

A, Immunostaining of E13 neurospheres for the neuroblast marker DCX (red). Nuclear staining with Hoechst is in blue. B, Quantification of DCX+ NPCs in neurospheres. A greater proportion of p53−/− NPCs are DCX+ compared to wild type (wt). Data are presented as percent mean ± SD of DCX+ cells in neurospheres. C–E, Quantification of mRNA levels for neurogenic genes in cultured neurospheres by qRT-PCR. p53−/− NPCs exhibit increased expression of several genes involved in neurogenesis. Mean mRNA expression normalized to β-actin ± SD is shown. Three independent experiments were conducted, each in triplicate. F, Immunocytochemistry for Tuj1 or GFAP in in vitro differentiating wild-type and p53−/− NPCs (DIV 7). Differentiating cells were stained for the neuronal marker Tuj1 (green) and the astrocyte marker GFAP (red) and with the nuclear dye Hoechst (blue). G, H, p53−/− NPCs generate more Tuj1+ neurons (G) and longer neurites (H) upon differentiation (DIV 7) compared to wild type. Values represent the mean percentage of Tuj1+ neurons (G) or of mean neurite length (H) ± SEM from three independent experiments. I, Quantification of the number of GFAP-positive cells in wild-type and p53−/− differentiating NPC cultures (DIV 7). No difference in astroglial differentiation can be detected between genotypes. Values represent mean percentage of GFAP+ cells when normalized to the total number of cells ± SD from three independent experiments. J, Immunoblotting of differentiating wild-type and p53−/− NPCs (DIV 7) display increased expression of neuronal markers Tuj1 and MAP2, but not of the astroglial marker GFAP. Similar results were obtained in independent experiments. Representative blots are shown. K, Immunoblotting shows that GABAergic (GAD65/GAD67) and glutamatergic (VGLUT2) differentiation is elevated in p53−/− differentiating NPC cultures (DIV 7). Similar results were obtained in independent experiments. Representative blots are shown. L, Immunoblotting of prenatal and postnatal telencephalic lysates for marker proteins as indicated. Three telencephalons were used per developmental stage and genotype. Representative blots and bar graphs with quantitation are shown. ^★p < 0.05; ^★★p < 0.01; ^★★★p < 0.001 (two-tail Student's t test). Scale bars: A, 100 μm; F, 50 μm.

A, FACS of DCFDA/DCX double-stained E13 neurospheres. p53−/− NPCs display an increase both in the total number of DCX++ (strongly positive) cells and in the number of DCX/DCFDA double-positive cells. Background (nonstained NPCs and nonspecific IgG) signal (gray bars) was used to set the threshold for signal detection. Three independent experiments with similar results were conducted. A representative experiment is shown. B, C, FACS after Ad-GFP-p53 or control Ad-GFP transduction of E13 p53−/− cultured NPCs shows a decrease in GFP/DCX+ NPCs after Ad-GFP-p53 transduction. The bar graph shows percentage of DCX+/GFP+ double-positive E13 NPCs by FACS (C). Data are the mean percentage of DCX+/GFP+ (strongly positive) double-positive NPCs (DCX+/GFP+ vs GFP+) ± SEM (N = 3). D, Immunocytochemistry for the neuronal marker Tuj1 (red) of Ad-GFP- and Ad-GFP-p53-transduced p53−/− differentiating NPCs (DIV 3). Proliferative NPCs were transduced with adenovirus before being plated for differentiation. Nuclei were stained with Hoechst (blue). E, Immunocytochemistry for the astrocyte marker GFAP (red) and Hoechst (blue) on Ad-GFP- and Ad-GFP-p53-transduced differentiating p53−/− NPCs as in D. Scale bars: 50 μm. F, Quantification of Tuj1+/GFP+ double-positive differentiating neurons (Tuj1+/GFP+ vs GFP+) after Ad-GFP and Ad-GFP-p53 transduction in NPCs. The bar graph shows that overexpression of p53 decreases the number of Tuj1+ neurons generated by differentiating p53−/− NPCs. The data are the mean percentage of Tuj1+/GFP+ double-positive neurons ± SEM from three independent experiments. G, The bar graph shows that Ad-GFP-p53 transduction decreases mean neurite length of Tuj1+/GFP+ double-positive differentiating p53−/− neurons compared to GFP expressing control neurons. Values represent mean neurite length (in micrometers) ± SD from two independent experiments. H, Quantification of GFAP+ astrocytes in Ad-GFP- and Ad-GFP-p53-transduced differentiating E13 NPCs. p53 overexpression does not affect astrogliogenesis. Mean percentage of GFAP+/GFP+ double-positive cells (GFAP+/GFP+ vs GFP+) ± SEM from two unrelated experiments is shown. ^★p < 0.05; ^★★p < 0.01 (Student's two-tail t test). wt, Wild type.

A, p53−/− NPCs do not exhibit increased proliferation compared to wild type (wt) according to P-H3 immunocytochemistry. E13 neurospheres were immunostained for the mitosis marker P-H3 (green). Nuclei were stained with Hoechst (blue). B, The bar graph shows quantification of P-H3+ cells in wild-type and p53−/− neurospheres (A). Data are presented as mean percentage of P-H3+ cells in neurospheres ± SD from three unrelated experiments. C, Immunostaining of neurospheres for the pan-proliferative marker Ki67 (red) does not reveal differences in proliferation between wild-type and p53−/− NPCs. Nuclei were stained with Hoechst (blue). D, The bar graph shows quantification of Ki67+ cells in wild-type and p53−/− neurospheres (C) in successive passages. Data are presented as mean percentage of Ki67+ cells in neurospheres ± SD from three independent experiments. E, The bar graph shows that proliferating P-H3+/DCX+ p53−/− neurospheres do not display differential proliferation compared to wild type. The percentage of DCX+/P-H3+ double-positive NPCs in neurospheres was normalized to the total number of DCX+ cells. Values represent mean percentage ± SD (N = 3). F, Immunohistochemistry for BrdU (red) on E16 sectioned wild-type and p53−/− telencephalons. Pregnant dams were killed after a 1 h BrdU pulse, and embryonic brains were dissected for IHC. G, Quantification of BrdU+ cells in the VZ–SVZ of E16 wild-type and p53−/− telencephalons does not reveal a difference in the number of S-phase (BrdU+) cells between genotypes. Data show mean percentage of BrdU+ cells ± SEM for VZ–SVZ (3 embryos per genotype and 3 sections per each embryo, successive sections, 40 μm apart). H, I, Immunohistochemistry for P-H3 (green) on E13 coronal sections of wild-type and p53−/− telencephalons. J, Quantification of P-H3+ cells in the VZ and SVZ of E13 wild-type and p53−/− telencephalons does not reveal a difference between genotypes in the percentage of mitotic cells. Data show the mean percentage of P-H3+ cells ± SEM for each germinal zone as indicated when counted from three embryos per genotype and three sections per each embryo (successive sections, 40 μm apart). Scale bars: 100 μm.

A, Immunocytochemistry for the neuronal marker Tuj1 (green) and nuclear stain Hoechst (blue) of 1 μm H₂O₂ preprimed wild-type (wt) and p53−/− differentiating NPCs (DIV 5). Neurospheres were pretreated with H₂O₂ in proliferation conditions and plated subsequently for differentiation. B, The bar graph shows that pretreatment of neurospheres with H₂O₂ increases neurogenic differentiation in wild-type but not in p53-null NPCs. Data represent the mean percentage of Tuj1+ cells normalized to the total number of differentiating cells (Hoechst+ cells) ± SEM from three independent experiments. C, Immunocytochemistry for the neuronal marker Tuj1 (green) and nuclear stain Hoechst (blue) of antioxidant N-acetyl-cysteine (1 mm) pretreated wild-type and p53−/− differentiating NPCs (DIV 5). Neurospheres were pretreated with NAC in proliferation conditions and plated subsequently for differentiation. D, The bar graph shows that pretreatment of proliferative NPCs with antioxidant NAC decreases neurogenic differentiation in p53−/− cells only. Data represent the mean percentage of Tuj1+ cells normalized to the total number of differentiating cells (Hoechst+ cells) ± SEM from three independent experiments. ^★★p < 0.01; ^★★★p < 0.001 (two-tail t test). E, Immunoblotting of E16 p53−/− telencephalic lysates for neuroblast/immature neuron marker DCX, whose expression is reduced by NAC. Pregnant dams were injected daily from E10 onward, either with control saline or antioxidant NAC. Embryos were killed at E16. Antioxidant treatment decreased the amount of DCX (N = 3). Scale bars: 100 μm.

A–F, Expression of genes that promote neurogenesis is responsive to redox regulation when assessed by qRT-PCR (A–D). PdgfRα (E) was used as a ROS regulated positive control (cntr) transcript. Cdkn1a/p21 (F) served as a negative control. The expression of neurogenesis-related genes was measured in neurospheres treated either with 1 μm H₂O₂ or 1 mm NAC. Data represent mean mRNA expression normalized to β-actin ± SEM from three unrelated experiments, each as triplicates. ^★★p < 0.01; ^★★★p < 0.001 (two-tail Student's t test).

A, Expression of Sesn2 mRNA is regulated by ROS. Neurospheres were treated with 1 μm H₂O₂. H₂O₂ increased Sesn2 expression by qRT-PCR. Values represent mean Sesn2 mRNA expression normalized to β-actin ± SEM from three unrelated experiments, each as triplicate. B, Expression of Sesn2 mRNA is induced by exogenous p53 expression in p53−/− NPCs. RT-PCR quantification of Sesn2 mRNA from Ad-GFP- and Ad-GFP-p53-transduced proliferative wild-type and p53−/− NPCs. Shown is mean mRNA expression normalized to β-actin ± SD. Independent experiments were conducted in triplicate. C, Sesn2 mRNA is downregulated in E13–E16 p53−/− telencephalons compared to wild type (qRT-PCR). Data represent mean Sesn2 mRNA expression normalized to β-actin ± SD. Three embryos per developmental stage and genotype were examined (each sample in triplicate). D, Immunoblotting for Sesn2 after Ad-GFP and Ad-GFP-p53 infection in p53−/− E13 neurospheres shows that Ad-GFP-p53 induces expression of Sesn2 to the same level as in the wild type. A representative immunoblot is shown. E, Immunoblotting of prenatal telencephalic E11–E16 samples for Sesn2. Sesn2 protein is downregulated in p53−/− telencephalons. Three embryos per developmental stage and genotype were examined. Representative immunoblot is shown. F–H, Expression of exogenous Sesn2 by electroporation decreases ROS in ex vivo E13 telencephalons. ROS of either EF-Sesn2 (G) or EF-empty vector (H) ex vivo electroporated E13 telencephalons were measured by FACS for DCFDA. Plasmid DNA was injected into the lateral ventricle and after 20 h cortices were dissociated to single cells and stained for the ROS indicator DCFDA and FACS was performed. Non-injected hemispheres were used as controls. Data are presented as the mean percentage of DCFDA++ (strongly positive) cells of all cells ± SEM. Two embryos were used for EF-empty vector and five for EF-Sesn2 electroporation. ^★p < 0.05; ^★★★p < 0.001 (two-tail Student's t test).

A, FACS of E13 in vitro NPCs for the ROS indicator DCFDA and for P-Akt. p53−/− NPCs exhibit an increase in P-Akt/DCFDA double-positive cells and an increase in the total number of P-Akt+ NPCs. Background signal (nonspecific IgG-stained NPCs; gray bars) was used to set the threshold for signal detection. Percentages show P-AKT/DCFDA double-positive cells. Three independent experiments with similar results were performed. A representative experiment is shown. B, Double immunocytochemistry for Tuj1/Akt phospho-S473 on differentiating wild-type (wt) and p53−/− NPCs (DIV 5). Scale bar, 100 μm. C, The bar graph shows quantification of P-Akt- and Tuj1-positive cells (B). p53−/− differentiating NPCs display an increase in both P-Akt- and Tuj1-positive cells. Data represent the mean percentage of positive cells ± SD from three independent experiments. D, FACS for P-Akt after transduction of p53−/− NPCs with Ad-GFP-p53 or control Ad-GFP (18 h). E, The bar graph shows a decrease in the number of P-Akt+ NPCs after Ad-GFP-p53 transduction compared to control Ad-GFP. Values represent the mean percentage of GFP+/P-Akt+ strongly double-positive cells normalized to GFP+ cells ± SEM from three independent experiments. Gray bars indicate cells not positive for GFP+/P-Akt. ^★★p < 0.01; ^★★★p < 0.001 (two-tail Student's t test).