The cells of the olfactory bulb originate from the subventricular zone (SVZ) neuronal progenitors that differentiate into peri-glomerular and granule cells of the bulb.^1,2 These cells are different form the other central nervous system neurons because of their ability to divide after the expression of adult neuronal markers.^3,4 Several studies have reported the unusual cell cycle and mitotic behavior of these cells during the development of the nervous system, although the molecular mechanism responsible for the control of the prolonged cell cycle and adult neurogenesis remains elusive.^1,5,6

Tumor suppressor protein (p53) and cell cycle exit protein (Bax) largely participates in control of cell proliferation in the SVZ neuronal stream.^7,8 In vitro experiments involving knock out mice models have shown that over expression of p53 or Bax reduces the rate of cell proliferation and renewal in the developing olfactory bulb.^8,9 However, in the adult system, the repression of cell proliferation in the olfactory bulb by p53 is linked to the p53-mitochondria signaling in apoptosis.^8–10 The p53-miotchondria pathway is driven by oxidative stress induced through inhibition of the energy coupling process involving the cytochrome C oxidase (CcOX)- complex V of the electron transport chain- and transient release of reactive oxygen species (ROS).^11–13

Chemical agents (such as cyanide and other heavy metals) inhibit the oxygen carrying capacity of the CcOX (Heme a3-Cuβ binuclear center) leading to generation of ROS in the mitochondria matrix (oxidative stress).^14–16 The product of this stress pattern is the release of cytochrome C (Cyto C) into the cytoplasm which fixes Bax into the membrane of the mitochondria through tBID to create calcium surge in the cytoplasm.^17–20 Factors which facilitate the up regulation of Bax are usually associated with senescence, reduction of Bcl-2 and over expression of p53.^21–23 However, other non-Bax related mechanisms are known to induce increased expression of p53 in neurons during oxidative stress. Nitric oxide (NO), formed through the reaction of ROS with nitrogen containing compounds trigger apoptosis by increasing the expression of p53 in the nucleus and subsequently DNA cleavage.^10,24 These mechanisms are also important in tumor protection and self-renewal in the olfactory bulb through regulation of senescence, tumorgenesis and apoptosis.²⁵

The relationship between Bax and p53 expression in the cell cycle of granule and periglomerular cells of the adult olfactory bulb is still relatively unknown.²⁶ Furthermore the functional role of p53 in the prolonged cell cycle, oxidative stress, neuronal survival and apoptosis in the adult olfactory bulb in vivo is yet to be elucidated.^26,27 Previous studies have demonstrated dose-dependent mechanisms in oxidative stress cytotoxic response in different parts of the brain (also dependent on the cell cycle specific for each cell type).²⁸ A common evidence for the regional cytotoxic pathways is how the plastic brain regions respond differently when compared with the non-plastic cortex to the same assault. Also, neurons in these regions follow varying patterns of cell death for the same assault and threshold of assault.²⁹ The role of p53 and Bax in regulating proliferation and apoptosis in oxidative stress is a major switch that determines selective vulnerability of adult neurons in the olfactory bulb to varying thresholds of induced oxidative stress. In this study, we have elucidated the interplay of p53 and Bax expression in peri-glomerular and granule cells of the olfactory bulb. We investigated pharmacologically, whether the induction of oxidative stress through inhibition of CcOX at varying doses will generate a difference in p53/Bax expression in the neuroplasticolfactory bulb. Furthermore, we sought to identify the relationship between p53/Bax expression and cell viability in the adult olfactory bulb following induced oxidative stress.

In order to determine the p53/Bax relationship in proliferation or cell death of neuron in the adult olfactory bulb, we employed the use of cyanide-CcOX toxicity dose-dependent assault followed by histology and p53/Bax protein mapping of the bulb. Using stereological methods, we counted the granule and periglomerular cells of the bulb (n = 7) for n = 5 animals in each group (n = 35 fields per group). The counts were done in general histology (H&E), p53 and Bax immunohistochemistry to determine the overall cell count and protein specific expressions in these layers. In general histology (Figure 1A) the cell count for 10 mg/Kg treatment increased when compared to the control (***P<0.001) while no significant change in cell count was seen for the 20 mg/Kg treatment versus the control (Figure 1B). However, the increase in cell number in the 10 mg/kg treatment was not associated with any change in p53 expression in the olfactory bulb (Figure 2A).

This is intriguing as the p53 expression level in the control and 20 mg/Kg also showed no significant change (Figure 2A and 2B) similar to the observations in the total cell count (Figure 1A and 1B). Thus, for varying thresholds of induced oxidative stress, no change in p53 expression despite a change in cell count at 10 mg/kg BW treatment (Figure 2B). This suggests that p53 might play little or no role in the regulation proliferation or apoptosis in the adult olfactory bulb cells in induced oxidative stress.

We then examined the expression of Bax in the granule and periglomerular cell layers to determine the involvement of Bax versus p53 in oxidative stress driven cell proliferation or cell death (Figure 3A and 3B). We observed a significant decrease in the Bax expression at 10 mg/Kg treatment versus the control (P<0.01) and the 20 mg/Kg treatment (P<0.01) (Figure 3B). This also correlated with an increase in cell proliferation in total cell count (10 mg/Kg; Figure 1A and 1B). In all instances, the p53 and Bax levels did not change significantly for the control and 20 mg/Kg treatment (Figure 4). These findings suggest that p53 is relatively constant irrespective of the oxidative stress threshold and Bax acts independently in a dose-specific manner to regulate neuronal survival in the olfactory bulb. A lower dose (10 mg/Kg BW) induced proliferative changes and caused a reduction in the expression of Bax and with no corresponding change in p53 expression while 20 mg/Kg BW caused no significant change in Bax, p53 and cell count when compared to the control (Figure 4).

Taken together the findings of this study suggest the central role of Bax in determining oxidative stress induced proliferation or degeneration in the adult olfactory bulb. We have also shown that Bax-expression is dependent on the threshold on the induced stress while p53 remained unchanged irrespective of the stress threshold. The expression of p53 and Bax in the olfactory bulb also correlated to the outcomes of the total cell count. Cell proliferation was observed in the 10 mg/Kg treatment (Figure 1A and 1B), an effect linked with Bax repression (Figure 3A and 3B) and no change in p53 expression (Figure 2A–B and Figure 4). No significance was recorded in the cell count for the 20 mg/Kg treatment. Also, Bax and p53 expression was not changed significantly when compared to the control. An important inference is the expression pattern of Bax in relation to the induced-stress threshold and total cell count in the olfactory bulb. We observed that a decrease in Bax without a corresponding change in p53 expression at 10 mg/Kg treatment caused an increase in the olfactory bulb cell count (Figure 4 and 1B), while for the 20 mg/Kg treatment no significant change in p53/Bax expression and cell count (Figure 4). Also, the expression of p53 showed no significant change for the varying stress threshold (0, 10 and 20 mg/Kg BW) (Figure 2A and B). These findings confirms that the expression of Bax is dependent on the threshold of the induced stress which modulates an increase in Bax during apoptosis or its decrease in proliferation. This indicates that threshold of induced oxidative stress and the associated change in the expression of Bax are responsible for neuronal survival in oxidative stress rather than p53.

The interplay between p53 and Bax has long been described in the cell viability and apoptosis.^28,32,33 Recent studies have shown that an increase in p53 or Bax often corresponds to reduce cell viability through the modulation of calcium signaling in mitochondria-linked apoptosis.^34,35 Bax is central to the “suggested” p53-motochondria apoptotic pathway by directing ROS-induced calcium-shift through its ability to increase mitochondria membrane permeability.^34–36 A major comparison is the role of Bax as a calcium transport regulator in oxidative stress³⁶ versus its role as a cell cycle exit protein in death of olfactory bulb neurons (apoptosis).³⁷ First, an increased level of Bax in the cell cycle implies an increase in number of cells exiting the cell cycle (apoptosis) and reduction of Bax expression will promote cell viability and proliferation (retaining more cells in the cell cycle). This has been shown in vitro as cell proliferation increased following the use of pharmacological inhibitors of Bax³⁸, and in vivo in genetic deletion rodent models (Bax-/-).³⁹ Second, following the release of Cytochrome C in cyanide induced oxidative stress Bax activates Bid signaling (tBid) to facilitate the release of mitochondria calcium into the cytoplasm causing autophagy.^19,20 This is achieved through the kiss and run mechanism of both Bax and tBid in increasing mitochondria membrane permeability (ψ_m). Studies have shown that tBid runs faster if it has been kissed by Bax, thus an elevated Bax in oxidative stress indicates calcium based excitotoxicity through a mitochondria pathway.^40,41 Thus, either as a cycle exit protein or calcium-shift inductor, Bax modulates the survival of neurons; in the former through apoptosis and the later through calcium toxicity/autophagy.

The actual relationship between p53 and calcium signaling is vague. Although high levels of p53 have been reported to correspond to an increase in cerebral calcium, the central role of Bax in Ca^2-P53 cross talk remains elusive.^42,43 In oxidative stress, it is known that ROS formation facilitates the formation of NO which can raise the nuclear level of P53 and induce apoptosis. Further, the ROS also facilitates “ROS-dependent calcium” release from the mitochondria through the Cytochrome C-Bax-tBid signaling pathway.³⁸ We can deduce form these points that ROS-induced NO production and Cytochrome C release are inductors of P53 and Bax increase respectively. Thus, associated calcium-shift in ROS-linked elevated P53 can be said to be Bax-dependent rather than being induced through an increased expression of P53.^34,38

Our findings suggest that Bax is not dependent on P53 in modulating oxidative stress and neuronal cell viability in the adult olfactory bulb (Figure 4). Furthermore, the extent of Bax inhibition was dependent on the threshold of the oxidative stress (Figure 3A, 3B and Figure 4) while P53 showed no significant change when compared to the control. A decrease in Bax also caused an increased cell proliferation in the olfactory bulb raising the question as to role of P53 in cell viability in the adult olfactory bulb. Other studies have reported the role of P53 in cell proliferation in the developing olfactory bulb such that genetic deletion of P53 increased number of the cells of the SVZ.^7,34 However, in the adult bulb, genetic deletion P53 caused no significant increase in cell proliferation²⁶ while genetic deletion or up regulation of Bax altered the olfactory bulb density significantly;³⁸ further supporting the importance of Bax in neuronal viability in oxidative stress.

Although P53 play an important role in development of the olfactory bulb, our findings suggest that it has little contribution in neuronal cell viability and proliferation in the adult olfactory bulb. No significant change in P53 was observed irrespective of treatment dose and cell count while Bax expression was reduced at 10 mg/Kg treatment and was associated with an increased cell count. We conclude that regulation of survival of neurons in the adult olfactory bulb, following induced-oxidative stress was more dependent of the expression of Bax and the treatment concentration rather than P53 expression.