The essential role of p53-up-regulated modulator of apoptosis (Puma) and its regulation by FoxO3a transcription factor in β-amyloid-induced neuron death

Abstract

Neurodegeneration underlies the pathology of Alzheimer disease (AD). The molecules responsible for such neurodegeneration in AD brain are mostly unknown. Recent findings indicate that the BH3-only proteins of the Bcl-2 family play an essential role in various cell death paradigms, including neurodegeneration. Here we report that Puma (p53-up-regulated modulator of apoptosis), an important member of the BH3-only protein family, is up-regulated in neurons upon toxic β-amyloid 1-42 (Aβ(1-42)) exposure both in vitro and in vivo. Down-regulation of Puma by specific siRNA provides significant protection against neuron death induced by Aβ(1-42). We further demonstrate that the activation of p53 and inhibition of PI3K/Akt pathways induce Puma. The transcription factor FoxO3a, which is activated when PI3K/Akt signaling is inhibited, directly binds with the Puma gene and induces its expression upon exposure of neurons to oligomeric Aβ(1-42). Moreover, Puma cooperates with another BH3-only protein, Bim, which is already implicated in AD. Our results thus suggest that Puma is activated by both p53 and PI3K/Akt/FoxO3a pathways and cooperates with Bim to induce neuron death in response to Aβ(1-42).

Keywords: Alzheimer Disease; Amyloid; Bcl-2 Family Proteins; Bim; Cell Death; Foxo; Neurodegeneration; Neurons; Puma; siRNA.

FIGURE 1.

Puma is induced by Aβ in cortical neurons. A, rat cortical neurons (7 days in vitro) were subjected to oligomeric Aβ (1.5 μm) for the indicated times, and total RNA was isolated, reverse-transcribed, and analyzed by semiquantitative PCR for Puma transcripts. α-Tubulin was used as a loading control. B, graphical representation of changes in Puma transcript level upon Aβ (1.5 μm) treatment on primary cultured rat cortical neurons at the indicated times by real-time PCR. 18 S was used as a loading control. Data are presented as -fold increase relative to untreated control (C) and represent mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01. C, cortical neurons were subjected to Aβ (1.5 μm) for the indicated times, and total tissue lysates were analyzed by Western blot for Puma level. A representative immunoblot shows Puma protein level at the indicated time points. ERK2 was used as a loading control. D, graphical representation of -fold increase of Puma protein level after Aβ treatment at different time points expressed relative to untreated control. Data represent mean ± S.E. (error bars) of three experiments. *, statistically significant differences from 0 h control; p < 0.05.

FIGURE 2.

Puma is induced following Aβ(1–42) infusion in vivo. Right hemispheres of brains of adult rats were infused with either Aβ(1–42) or PBS, and after 21 days, the animals were sacrificed, and brains were taken out following cardiac perfusion. The brains were cryosectioned and co-immunostained with Puma and NeuN antibodies; nuclei were stained with Hoechst dye. Representative images of five sections from three animals of each group with similar results are shown here. Scale bar, 100 μm. Images were taken for each case by using an inverted fluorescence microscope and camera set to the same exposure.

FIGURE 3.

Puma is elevated in AD transgenic mice brain. The brain sections of AβPPswe-PS1de9 transgenic mice and wild-type mice were analyzed for level of Puma expression. A, transgenic and wild-type brain slices were stained with Congo red to see the deposition of Aβ plaques. These plaques (arrows) were seen in transgenic brains, whereas they were absent in wild-type brain tissue. B, the brain sections of transgenic mice were co-immunostained with Puma and NeuN antibodies. Hoechst was used to stain nuclei. Representative images from six sections from three animals of each group with similar results are shown here. Scale bar, 100 μm. Inset, magnified version of the same section in each case. Images were taken for each case using an inverted fluorescence microscope and a camera set to the same exposure.

FIGURE 4.

Down-regulation of Puma by shRNA protects cortical neurons from death. A, primary cultured rat cortical neurons (5 days in vitro) were transfected with pSIREN-shPuma-zsgreen or control pSIREN-shRand-zsgreen (scrambled shRNA) and maintained for 48 h and then subjected to Aβ (1.5 μm) treatment for 72 h. Representative pictures of transfected neurons that were maintained in the presence or absence of Aβ for the indicated time periods are shown. Images were taken under a ×20 objective. B, graphical representation of the percentage of viable green cells after each time point. The numbers of surviving transfected (green) cells were counted under a fluorescence microscope just before Aβ treatment (C) and after 24, 48, and 72 h of the same treatment. Data are from three independent experiments, each with comparable results, and are shown as mean ± S.E., performed in triplicates. The asterisks denote statistically significant differences from control (shRand) at corresponding time points: *, p < 0.05; **, p < 0.001. C, Puma knockdown prevents neuronal degeneration. Sholl analysis of single imaged neurons by using ImageJ was done as described under “Experimental Procedures.” Data represent the mean ± S.E. (error bars) of six different neurons from three independent cultures for each class. *, statistically significant differences from shRand (control); p < 0.001.

FIGURE 5.

Inhibition of PI3K signaling induces Puma in cortical neurons. A, primary cultures of rat cortical neurons were treated with LY294002 (50 μm) or pifithrin-α (50 μm) for 8 h with or without Aβ (1.5 μm), and the proteins were analyzed by Western immunoblotting using enhanced chemiluminescence for the expression of Puma and actin (loading control). B, graphical representation of densitometric analysis of -fold change of Puma level in the indicated conditions. Data represent ± S.E. (error bars) of three experiments. *, significant differences from control; p < 0.03. C, cultured cortical neurons were treated with pifithrin-α with or without Aβ for 8 h, and the expression of phospho-p53^Ser-15 and total p53 was assessed by Western blot. D, graphical representation of densitometric analysis of fold change of phospho-p53^Ser-15 level in the indicated conditions. Data represent means ± S.E. of three experiments. *, p < 0.05. E, cultured cortical neurons were treated with or without Aβ in the presence and absence of pifithrin-α. Data are represented as mean ± S.E. of three independent experiments. *, p < 0.05. F, cortical neurons were subjected to Aβ (1.5 μm) for the indicated times, and total tissue lysates were analyzed by Western blot for BAX. Actin was used as loading control. G, graphical representation of -fold increase of BAX protein level after Aβ treatment at different time points expressed relative to untreated control. Data represent mean ± S.E. of three experiments with three replicate cultures. *, significant differences from control; p < 0.03.

FIGURE 6.

Knockdown of FoxO3a by shRNA represses up-regulation of endogenous Puma in neuronal cells subjected to Aβ treatment. A, cortical neurons were transfected with shFoxO3a or shRand and maintained for 48 h and then treated with Aβ (1.5 μm) for 8 h, after which they were immunostained with antibodies against Puma (red). Images were taken under a ×63 objective. B, percentage of stained cells pertains to the proportions of transfected cells (green) that show Puma staining either greater than that of non-treated control neurons (High) or equal to or less than that of non-treated neurons (Low). Data represent the mean ± S.E. (error bars) of three experiments. The number of cells evaluated per culture was ∼50. *, p < 0.01. C, graphical representation of corrected total cell fluorescence of Puma in neurons transfected with shRand or shFoxO3a following Aβ exposure. Difference in intensity of Puma staining was quantified by ImageJ as described under “Experimental Procedures.” Data represent mean ± S.E. of 60 different cells from three independent experiments. *, p < 0.03. D, PC12 cells were transfected with shFoxO3a or shRand and primed as described under “Experimental Procedures,” and then the down-regulation of endogenous FoxO3a was analyzed by Western blotting with anti-FoxO3a antibody. E, graphical representation of densitometric analysis of -fold change of FoxO3a level upon transfection with shFoxO3a or shRand in the presence or absence of Aβ (5 μm). Data represent mean ± S.D. (error bars) of two independent experiments. *, p < 0.05. F, PC12 cells were transfected with shFoxO3a or shRand and primed, and then the down-regulation of endogenous Puma was analyzed by Western blotting with anti-Puma antibody. G, graphical representation of densitometric analysis of -fold change of Puma level upon transfection with shFoxO3a or shRand in the presence or absence of Aβ (5 μm). Data represent mean ± S.D. of two independent experiments; *, p < 0.05.

FIGURE 7.

FoxO3a directly binds with intron 1 of the rat Puma gene and regulates its induction upon Aβ treatment. A, schematic representation of Puma-luc reporter consisting of intron-1 of the rat Puma gene. B, cortical neurons were co-transfected with 0.3 μg of Puma-luc reporter and 0.1 μg of Renilla luciferase expression construct pRL-CMV with 0.3 μg of either shRand (control) or shFoxO3a. The cultures were maintained for 48 h and then subjected to overnight Aβ treatment, after which luciferase activity was assayed and represented as -fold change of luciferase activity. Data represent mean ± S.E. (error bars) of four experiments. *, p < 0.05. C, cortical neurons were co-transfected with 0.4 μg of either wild type Puma-luc reporter or FoxO3a-mutated construct and 0.1 μg of Renilla luciferase expression construct pRL-CMV. The cultures were maintained for 48 h and then subjected to overnight Aβ treatment, after which luciferase activity was assayed and represented as -fold change of luciferase activity. Data represent mean ± S.E. of four experiments. *, p < 0.05. D, primary cultures of rat cortical neurons were treated with or without Aβ for 8 h. An equal number of cells were processed for ChIP assay using anti-FoxO3a antibody for immunoprecipitation. The immunoprecipitated materials were subjected to PCR using primers against the portion of the Puma promoter that flanks the FoxO3a-binding site. PCR products were verified by agarose gel electrophoresis. Templates were DNA from cells before ChIP (Input) or DNA from immunoprecipitated (IP) materials. PCR assays were conducted after ChIP, using samples from cells that were either left untreated (Control) or treated with Aβ. E, graphical representation of FoxO3a association with the Puma gene. Quantitative PCR was performed using material derived from cultured cortical neurons treated as in D. Association of FoxO3a with Puma Forkhead response element (Puma FHRE level) in the presence or absence of Aβ was determined by quantitative PCR after ChIP, using samples from cells that were either left untreated (Control) or treated with Aβ. Numbers on the y axis represent the levels of FoxO3a association with the Puma promoter region after normalizing to Ct values from input samples. Data shown are means ± S.E. *, p < 0.05.

FIGURE 8.

Knockdown of both Bim and Puma together provided better protection than knockdown of individual genes against Aβ-induced neuron death. Primary cultured rat cortical neurons (5 days in vitro) were transfected with pSIREN-shPuma-zsgreen or with pSIREN-shBim-zsgreen or co-transfected with both or with pSIREN-shRand-zsgreen (control); maintained for 48 h; and then subjected to Aβ (1.5 μm) treatment for the indicated times. Live cells were counted under a fluorescence microscope after each time point. Data represent three independent experiments, each with comparable results, and are shown as mean ± S.E. (error bars), performed in triplicates. The asterisks denote statistically significant differences from shPuma at corresponding time points: *, p < 0.05; **, p < 0.01.

FIGURE 9.

Schematic representation of Puma activation by FoxO3a and of neuron death in response to Aβ.