p53 upregulated mediator of apoptosis (Puma) deficiency increases survival of adult neural stem cells generated physiologically in the hippocampus, but does not protect stem cells generated in surplus after an excitotoxic lesion

Introduction

A remarkable number of new cells are generated every day by neurogenesis in the adult hippocampus of rodents, but their fate seems to be limited by apoptotic cell death. Greater than 60% of newly formed neurons die by apoptosis [1], [, 2]. Members of the Bcl-2 protein family and the mitochondrial apoptosis pathways play a critical role in cell death of adult generated cells within the dentate gyrus (DG) [3], [, 4]. Apoptosis is triggered through the release of caspase-activating factors from mitochondria, a process initiated through Bax oligomerization. bax-deficient mice display not only reduced apoptosis in the hippocampal neurogenic region but also an increased number of mature DG cells [1]. Functional deficiencies [3], but also cognitive improvements resulted from these aberrantly surviving neurons [5]. Constitutively-activated Bax plays an important role for apoptosis in neural progenitors [6]. Conversely, overexpression of the anti-apoptotic protein Bcl-2, an inhibitor of Bax, significantly decreased apoptotic cell death in the granule cell layer (GCL) of the DG and also led to an increased total number of granule cells [7].

However, the exact requirements, mechanisms and signalling cascades in Bax involvement and apoptosis activation in neural stem cells (NSCs) are largely unknown. Bax activation necessitates the transcriptional and posttranslational activation of Bcl-2-homolgy domain 3 (BH3)-only proteins that are integrated into several stress-activated signalling pathways. p53-regulated mediator of apoptosis (Puma) is a transcriptional target of p53 and one of the most potent BH3-only proteins [8]. Puma directly activates Bax and neutralizes the activity of all known anti-apoptotic Bcl-2 family proteins. Our previous work showed that under physiological conditions the lack of Puma rescued neural precursor cells (NPC) in the hippocampus from apoptotic cell death suggesting these proteins contribute to controlling the fate of newly generated cells within this neurogenic region of the adult brain [9].

Enhanced or reduced rates of neurogenesis occur under certain physiological or pathophysiological conditions. Enriched environment [10], physical activity [11] or anti-depressant medication [12] positively regulate hippocampal neurogenesis. Excitotoxic injury [9], [13], [14], global and focal brain ischaemia [15], [, 16], and traumatic brain injury [17], [, 18], all characterized by increased release of the N-methyl-d-aspartate (NMDA) receptor agonist glutamate, stimulate neurogenesis in a process believed to facilitate repair. Conversely, stress [19] and aging [20] reduce the generation of new neurons.

The role of Puma in controlling pathophysiological alterations in adult neurogenesis is largely unknown. Puma was previously shown to rescue neural stem cells from physiological cell death [9]. Furthermore, Liu et al. (2010) showed that stem cell depletion within the DG of p53 knock-in mice, a model of genotoxic stress during aging, could be rescued by the deficiency of puma [21]. However, despite this, the role of Puma in precursor cell survival in vivo following excitotoxic injury remains largely uncharacterized. Hippocampal injection of NMDA provides a model of excitotoxic injury and causes pronounced degeneration of CA1 neurons, a model we previously established [22]. We therefore set out to explore the role of Puma in the regulation of neurogenesis following NMDA-induced excitotoxic injury to the mouse hippocampus.

Materials and methods

Stereotaxic injection of NMDA and BrdU administration

Surgical procedures were completed as previously published [14]. NMDA (10 mg/ml) was dissolved in phosphate-buffered saline (PBS) for the injection. Two groups of 10 puma^−/− and wt mice each underwent surgery. Six mice per group received intrahippocampal NMDA, and four mice per group received vehicle. Mice were anaesthetized via intraperitoneal (i.p.) injection of 20 mg/kg Avertin (100 g 2,2,2-tri-bromoethanol in 62 mL tert-amyl alcohol) and placed into a stereotaxic frame. Craniectomy was completed according to coordinates measured from Bregma (anterior–posterior=−1.9 mm, medial–lateral=+/−1.5 mm), following a midline incision. Prior to and after injection the needle was tested to exclude a potential block of the cannula. The injection needle was placed into the brain using a dorsal–ventral coordinate of −1.2 mm from the brain surface. NMDA (or vehicle) was administered at a rate of 0.1 μL/min. Following withdrawal of the needle, the incision was sutured and animals monitored until recovered from the anaesthetic.

Following surgery, we assessed the incorporation of BrdU into newly synthesized DNA. 5-Bromo-2-deoxyuridine (BrdU, 50 mg/kg; Sigma-Aldrich, Ireland) was administered, via the intraperitoneal route, at day 6–9 after NMDA or vehicle administration [9]. which represents the time of peak neurogenesis after excitotoxic insult [14]. To quantify the BrdU-positive cells generated and their survival, animals were sacrificed either one day after the last BrdU treatment (short-term survival group) or seven days after BrdU administration (long-term survival group) (Schematic Figure 1).

Figure 1:

Schematic of experimental regimen. Mice were injected intrahippocampally with NMDA (10 mg/mL) or saline. Following a recovery period, the mice received 50 mg/kg of BrdU (days 6–9 following excitotoxic injury). Following an additional 24 h (day 10 after surgery) or an additional week (day 16 after surgery) mice were sacrificed for subsequent procedures.

Results

Following surgery and perfusion, we quantified the generation of new cells in the DG in sections at the respective time points. Notably, in wt animals the NMDA-induced insult resulted in an expected and significantly increased generation of new cells only one day after the last BrdU injection when compared to control animals that did not receive NMDA [Figures 2A, B, short term, control 14.8±0.8 (n=4 animals) vs. NMDA 29.2±0.8 (n=6 animals) BrdU positive cells/10 µm brain section, p=0.02] After long-term survival, a decreased number of BrdU-positive cells in both groups (NMDA and control) was noted, and no significance was reached between NMDA and control [control 9.6±0.7 (n=4 animals) vs. NMDA 16.2±3 (n=6 animals) BrdU-positive cells/10 µm brain section; Figures 2B, D]. In puma knock-out mice, like in the wild-type, treatment with NMDA resulted in significantly increased precursor cell numbers one day after BrdU-injection [control 22.8±1.9 (n=4 animals) vs. NMDA 39.5±5.3 (n=6 animals) BrdU-positive cells/10 µm brain section, p=0.01] (Figures 2C, D). Interestingly, in the NMDA treated groups BrdU-positive cell numbers decreased to control levels seven days after BrdU injections [NMDA one day 39.5±5.3 (n=6 animals) vs. NMDA seven days 22.7±1.2 (n=6 animals) BrdU-positive cells/10 µm brain section, p=0.02] suggesting that puma-deficiency did not protect the surplus of newly generated cells from dying (Figures 2B, D).

Figure 2:

Proliferation and survival of newly generated cells in the DG after NMDA-induced injury. Wt animals and puma^−/− were subjected to intrahippocampal NMDA or saline administration. Daily BrdU injections (50 mg/kg, i.p.) at day 6–9 after NMDA treatment and transcardial perfusion one day (short-term survival group) or seven days (long-term survival group) after the last BrdU injection were performed. Following immunolabelling, 10 µm brain sections were assessed for BrdU-incorporation into newly generated cells in the DG (A, C, white lines demarcate the blades of the DG). The number of BrdU+cells were counted and compared between the different groups (B, D). Note the significant increase in BrdU+cells in wt and puma^−/− mice following intrahippocampal NMDA injections in the short-term survival groups compared to control treated animals of both genotypes. After long-term survival the number of BrdU+cells had decreased in NMDA and control treated wt animals with no significant difference between the two groups. In the puma^−/− animals no difference in the number of BrdU+cells comparing control animals from the short-term survival group to the long-term survival group was found. BrdU cell numbers after NMDA treatment had decreased in the long-term survival group to the level of cell numbers from control animals. n=4 animals for all control groups, n=6 for all NMDA treatment groups. Scale bar in (A, C) 200 µm. GCL, granule cell layer. Error bars represent SEM. *p≤0.05.

To correlate the increased proliferation to a potential increase in neurogenesis, we used the neuronal progenitor cell marker doublecortin (DCX) and determined DCX-positive cells in the dentate gyrus. Within the short-term group, significantly increased numbers of DCX-positive cells were counted following NMDA treatment in wt animals (control 73.7±2.6 vs. NMDA 139.4±1.9 DCX-positive cells/10 µm brain section, p=0.05; Figure 3A, B). Our results suggested that NMDA-induced excitotoxicity increased neurogenesis. Following long-term survival, wt NMDA-treated animals showed an elevated level of DCX-positive cell numbers compared to control (control 78.9±5.4 vs. NMDA 98.1±3.5 DCX-positive cells/10 µm brain section, p=0.05; Figure 3B). Their DCX cell number however was already decreased when contrasted to the NMDA-treated/short-term survival group (NMDA short-term survival 139.4±2.6 vs. NMDA long-term survival 98.1±3.5 DCX-positive cells/10 µm brain section, p=0.05; Figure 3B). Even though neurogenesis in the injury group was still heightened long-term after injury, neurogenesis was decreased compared to the earlier NMDA treatment time point, suggesting that the increased level of early neurons could not be maintained. Next, we examined DCX-positive cells in the puma knockouts. Within the short-term survival group the number of DCX-positive cells was significantly increased after NMDA treatment compared to control-treated animals (control 121.6±8.2 vs. NMDA 201.1±12.5 DCX-positive cells/10 µm brain section, p=0.01) suggesting that neurogenesis was not impaired by puma ablation (Figure 3C, D). However, the number of DCX-positive cells had decreased to control levels during the long-term survival, similarly to the effect observed by BrdU cell counts (NMDA/10 days 201.1±12.5 vs. NMDA/16days 120.7±8.8 DCX-positive cells/10 µm brain section, p=0.01; Figure 3D). It appeared from these results that the deficiency in puma could not protect early neurons generated as a consequence of NMDA-induced injury.

Figure 3:

Neurogenesis and survival of newly generated cells in the DG after NMDA-induced injury. Wt animals and puma^−/− animals were subjected to intrahippocampal NMDA or saline administration followed by transcardial perfusion at short-term survival and long-term survival time points. Ten micrometre brain sections were assessed by anti-DCX immunohistochemistry (A, C, white lines demarcate the blades of the DG). DCX+cells were counted and compared between the different groups (B, D). DCX+cells were significantly more following NMDA treatment in both groups (short and long-term survival). Cell numbers decreased significantly from day 10–16 after NMDA treatment (wt, D). In puma^−/− animals the number of DCX+cells was significantly increased short-term after NMDA administration but the numbers were decreased to control levels at the long-term survival time point (D). n=4 animals for all control groups, n=6 for all NMDA treatment groups. Scale bar in (A, B) 200 µm. GCL, granule cell layer. Error bars represent SEM. *p≤0.05.

Finally, we analysed whether neuronal development was influenced in the newly generated cells after NMDA-induced excitotoxicity, using BrdU and DCX co-labelling (Figure 4A, B). One day after the last BrdU injection, 83–89% of BrdU-positive cells expressed the neuronal progenitor cell marker DCX in both control as well as NMDA-treated animals, suggesting that BrdU was incorporated within the same percentage of progenitor cells (Figure 4C). These numbers were approximately halved in the long-term control survival group, in sham-treated animals (wt and puma^−/−) 39% of the BrdU positive cells still expressed DCX (Figure 4C). In contrast following NMDA treatment, between 54% (wt) and 65% (puma^−/−) of the BrdU cells were positive for DCX (Figure 4C). Interestingly, a trend towards significance was observed only for the puma^−/− mice (control 39% vs. NMDA65% BrdU to BrdU/DCX cells, p=0.072).

Figure 4:

BrdU and DCX co-labelling in the dentate gyrus after NMDA-induced excitotoxicity. Wt animals and mice deficient in puma were subjected to intrahippocampal NMDA or saline administration. Daily BrdU injections (50 mg/kg, i.p.) at day 6–9 after NMDA treatment and transcardial perfusion one day (short-term survival group) or seven days (long-term survival group) after the last BrdU injection were performed. Ten µm brain sections were assessed for BrdU and DCX expression. In the short-term survival group the majority of BrdU-incorporated cells expressed DCX in both wt and puma^−/− animals. Arrows point at double-labelled cells, open arrowheads at BrdU+/DCX- cells (A, B). In the long-term group only 39% of BrdU+cells still expressed DCX in control groups of wt and puma^−/− mice. NMDA treatment increased the proportion of BrdU+/DCX+cells to 54% in wt animals and 65% in puma^−/− mice, this effect did not reach significance (C). Scale bar in (A, B) 50 µm. GCL, granule cell layer.

In order to determine if neuronal maturation was influenced in the newly generated cells after NMDA-induced excitotoxicitywe performed co-labelling with BrdU and the post-mitotic marker NeuN (Figures 5A, B). Again, double-labelled cells were assessed within the DG. Following short-term survival (10 days after excitotoxic insult), only a small percentage of BrdU-positive cells already expressed NeuN (16–18%; Figure 5C), reflecting the short-time window. However, the majority of BrdU cells expressed NeuN a week later (77–88%; Figure 5D). There was no difference in the proportion of BrdU/NeuN-positive cells comparing the different genotypes and treatments within the two analysed time points. These results suggested that phenotypic fate of the cells was preserved regardless of genotype and treatment (control vs. NMDA).

Figure 5:

BrdU and NeuN co-labelling in the dentate gyrus after NMDA-induced excitotoxicity. Wt animals and puma^−/− mice received intrahippocampal NMDA or saline and daily BrdU injections (50 mg/kg, i.p.) at day 6–9 after NMDA. Transcardial perfusion one day (short-term survival group) or seven days (long-term survival group) after the last BrdU injection was performed. Ten micro metre brain sections were assessed for BrdU and NeuN (A, B). One day following the last BrdU injection only around 18% of BrdU+cells expressed NeuN. Seven days after the last BrdU injection (long-term survival group), the majority of BrdU-incorporated cells expressed NeuN in both wt and puma^−/− animals (C). There was no difference in the proportion of BrdU+/NeuN- to BrdU+/NeuN+cells (C lower row). White arrows in (A) and (B) point at BrdU+/NeuN+cells magnified below. There was no difference between genotypes and treatment groups (C upper row). Scale bar in (A, B) 50 µm. GCL, granule cell layer.

Discussion

We examined the role of the BH3-only protein Puma in proliferation and survival of newly generated cells within the DG following an excitotoxic insult to the hippocampal CA1 subfield. We explored whether puma-deficiency influenced the NMDA-induced up-regulation of proliferation and/or the subsequent death of these cells. While neurogenesis was up-regulated in wild-type and puma-deficient animals following NMDA-induced excitotoxicity to the hippocampal CA1 subfield, the gene knock-out could not rescue this surplus of newly generated cells from subsequent apoptotic cell death. These results are different from earlier work, whereby we demonstrated the lack of the BH3-only protein Puma significantly increased the survival of neuronal progenitor cells, though their proliferation was unaffected in naïve animals [9]. Similar results have been observed in hematopoietic multipotent progenitors [24]. These differences are likely due to the stimulating event, in this case an excitotoxic insult.

We found higher numbers of BrdU and doublecortin-positive cells in Puma-deficient mice, concurrent with our previous results [9]. Interestingly, it has also been shown that telencephalic ventricular zone neuronal precursor cells (NPCs) were protected from AraC-induced cell death in puma-heterozygous animals [23]. Puma is a BH3-only protein and potent mediator of the intrinsic, mitochondrial pathway of apoptosis. It either activates Bax or Bak with mitochondrial outer membrane pore formation, or it sequesters the anti-apoptotic members of the Bcl-2 family [25]. Puma, potently activated in neurons following endoplasmic reticulum stress [26], is required for ER-stress induced apoptosis [27], but not for cell death following NMDA-mediated toxicity [22]. Puma also controls cell death induced by trophic factor withdrawal in neurons [28]. Importantly, suppression of neuronal electrical activity by NMDA-receptor blockers resulted in Puma-dependent apoptosis under trophic factor withdrawal [29]. Common to these cellular stress conditions is that pro-apoptotic Puma expression is induced by Foxo3a [30]. Foxo3a is expressed in neuronal precursor cells under basal conditions and positively regulates precursor cell numbers [31], [, 32], Foxo3a may be diverted to gene promoters unrelated to pro-apoptotic proteins in Puma knock-out mice. Transcription of puma is also activated by E2F1 [33]. E2F1^−/− mice, similarly to our previous study on bim and puma^−/− mice [9], show decreased levels of apoptosis [34] suggesting that E2F1-induced transcription of puma might be responsible for the death of newly generated cells within the hippocampus. Overall, as there currently is no evidence that Puma directly affects the cell cycle, it may be assumed that the protective effects of Puma-deficiency on neuronal precursor cells is due to their protection from apoptotic cell death [35], [, 9].

In this study the puma^−/− animals used represent a global knock-out model where puma is lacking in all cells, including NSPCs [23], [, 36]. puma^−/− mice were generated from ES cells that lacked puma exons 2 and 3, which encompass the start site and the BH3 region of puma. However, indirect effects of puma gene deletion in other cell types such as neurons, cannot be excluded and may have impacted on NPC survival. Apoptotic cells secrete factors that stimulate cell proliferation and may regulate survival in neighbouring cells, e.g. through the WNT/beta-catenin pathway (reviewed in [37]). However a deficiency in puma in non-NPCs would potentially limit such paracrine signalling. It is also possible that the lack of puma in non-NPCs switches their cell death to a more necrotic phenotype, which in turn may activate inflammatory processes.

NMDA injections up-regulated proliferating precursor cells and neurogenesis. Inhibitors of NMDA-receptor signalling increase the density of the granule cell layer [38], [, 39]. Physiological concentrations of glutamate were thus suggested to limit neurogenesis. Excitotoxic levels of glutamate-receptor agonists were previously shown to stimulate neurogenesis in the dentate gyrus [13], [, 40]. Glutamate-responsive NMDA-receptors are expressed on proliferating cells of the adult subgranular zone of the dentate gyrus [41] and their positive influence on neurogenesis seems to involve CaMKIV-dependent CREB signalling [42]. Importantly, the transcription factor CREB currently emerges centre-stage as a positive regulator of neurogenesis [43]. Synaptic activity is a potent activator of CREB while suppressing pro-apoptotic protein Puma expression [29]. Puma is mainly activated by transcriptional mechanisms. While CREB-responsive elements can be found in the Puma-promoter [44], these two proteins may be regulated contrariwise following an NMDA-stimulus to neural precursor cells.

Analysis of proliferation and survival of the newly generated cells within the DG of wt mice revealed that while proliferation was significantly increased short-term following NMDA treatment, those cells did not survive an additional week and apoptotic cell death most likely accounted for the reduced cell numbers. Thus, Puma-deficiency failed to protect the new precursor cells and newly formed neurons from cell death. In fact, when cell death was calculated for BrdU-positive cells and compared between control and NMDA treated animals, in control animals about 66% of the cells had survived an additional week, but only 53% of the BrdU-positive cells were still present at this time point in animals subjected to intrahippocampal NMDA injection. This is in agreement with a study by Takasawa and colleagues who showed that brain injury increased proliferation, but also decreased the survival to 20% of the newly generated cells compared to control animals where 90% of the cells survived [45].

Interestingly, a similar effect was also observed in the DG of puma^−/− mice. Within the control treated animals no decrease in the number of BrdU-positive cells was detected following an additional week of survival, suggesting that the lack in Puma protected those cells from apoptotic cell death, as previously observed [9]. However, in puma^−/− animals with intrahippocampal NMDA injection, a decrease of BrdU-positive cells, to control levels, following long-term survival was detected, suggesting that deficiency in puma did not protect the newly generated cells following injury. A similar observation was found for doublecortin-positive precursors. It is also possible that other pro-apoptotic BH3-only proteins such as Bcl-rambo control the survival of NPCs, particularly in the absence of puma [46].

BrdU/DCX-double positive NPC’s were numerically higher in Puma-knockout DG following injury in the long-term survival, but this effect was not statistically significant. BrdU/NeuN-positive neuron numbers were also not significantly higher in Puma-deficient compared to wild-type animals in the long-term survival group following NMDA. It has been described that there is a transition of DCX to NeuN expression during neuronal maturation, during an intermediate state both markers are expressed. Our data cautiously suggest that more of the new cells generated under the influence of NMDA-induced injury were still in this intermediate state, whereas more cells of control animals had already matured into neurons. In a previous study the time course of neuronal maturation after ischemia was similar to that under normal conditions [47], [, 48]. However, Tanaka and co-workers (2004) conducted their study using gerbils, GFP-retrovirus injection and a transient global ischemia model, while in our study back-crossed C57BL/6 mice, BrdU injection and a model of excitotoxicity were used. These variables may account for the differences observed in neuronal maturation following brain injury. Supporting our study, similar decelerated maturation of newly generated cells within the hippocampus has been demonstrated in the aged brain [49], though the molecular mechanisms responsible are yet to be uncovered.

We previously showed Puma-deficient neurons are not protected from excitotoxic cell death and similar sizes of lesions are produced in wild type and Puma-deficient mice [22]. A delayed release of endogenous glutamate stores, on already differentiated neurons as a sequela to NMDA-injury, is expected to have a similarly excitotoxic effect in both genotypes. On the other hand trophic signalling following glutamate-receptor activation may underlie the minimal increase in BrdU/NeuN double-positive neurons in the long-term survival group across the genotypes and treatment. In newly-born cells, differential sets of growth and transcription factors may have been activated. We show that such pathways are Puma independent, with unfavourable outcomes for the survival of the newborn cells regardless of the genotype. On the contrary, decreased neuronal differentiation could also have affected the survival of the newly generated cells, since it has been suggested that forming functional connections and integrating into the existing network is necessary for the survival of the cells [50]. Therefore, decreased neuronal maturation could have contributed to the decrease of cell survival within the DG also under Puma-deficient conditions. As the same amount of BrdU-positive cells expressed the neuronal marker NeuN one week following the last BrdU administration the phenotypic fate seems to be preserved among different genotypes (puma^−/− and wt) which is consistent with observations by others [1].

One potential limitation of this study was the use of only male mice. While the impact of gender on physiology and behaviour is important, the intention of this study was to examine the effect of PUMA knockout on the survival regeneration and survival of cells following an acute excitotoxic injury. Oestradiol has been consistently shown to exhibit neuroprotective activities and there is evidence that this is mediated, at least partly, through the NMDA receptor [51]. Hence the use of female mice in this study would have introduced significant variability due to the stage of oestrus cycle and potentially reduced the chances of detecting an effect of PUMA knockout. Despite this, it would be important in the future to determine the impact of gender in this paradigm. One other limitation of our study is that we used DCX as a marker for neurogenesis. However it should be mentioned that DCX is also expressed in neural progenitor cells, which frequently undergo apoptosis during the first wave of selection [52]. While we did not perform DCX/NeuN double staining, even DCX/NeuN double positive cells may not survive the second wave of apoptosis when integration into neuronal circuits fails.

Conclusion

Taken together, we showed that while excitotoxic injury to the hippocampal CA1 subfield increased proliferation in the DG, those cells could not be protected from subsequent apoptosis in mice deficient in the pro-apoptotic protein Puma, suggesting that Puma is not involved in cell survival of the newly generated cells in response to excitotoxic injury.