The main findings of the present study were: 1) before irradiation, the numbers of newly born neurons generated in the dentate SGZ were lower in SOD KO mice compared to WT mice; 2) irradiation did not reduce the numbers of newly generated neurons in SOD KO mice; 3) both before and after irradiation the numbers of newly born astrocytes in SOD KO mice were significantly elevated relative to that seen in WT mice; and 4) changes in neuroinflammation, at least in the context of newly born activated microglia, are genotype dependent. These findings confirmed our earlier observations in EC-SOD KO mice, in that after irradiation an environment lacking SOD was much more permissive in the context of hippocampal neurogenesis [32
]. The mechanism(s) behind this observation are not yet known, but given that all SOD KO mice should have variable levels of increased oxidative stress, it likely will involve persistent and elevated levels of ROS.
SODs, which are critical elements of the cellular antioxidant defense mechanism [56
], are oxidoreductases that remove superoxide by catalyzing the dismutation of the superoxide radical to hydrogen peroxide. Hydrogen peroxide is then metabolized to molecular oxygen and water by catalase or peroxidases. The 3 different SOD isoforms catalyze the same chemical reaction, but have different enzymatic properties and distinct subcellular localizations. Therefore, deficiency in SOD, regardless of location, should result in relatively higher levels of ROS and altered redox state, which will induce a state of persistent oxidative stress. While ROS have often been considered to be hostile or destructive entities, data also exist showing that ROS can have beneficial effects [47
], and in the brain, they are critically involved in a number of important processes, particularly those involved in learning and memory formation [46
]. This information documents the paradoxical effects associated with ROS, where differing levels can either be good or bad, depending upon the circumstances [46
Here we found that partial depletion of the SOD1 and SOD2 isoforms was associated with reduced baseline levels of neurogenesis, but was also associated with a ‘protective’ effect after irradiation. These effects were not coupled with any obvious compensatory changes in other anti-oxidant enzymes (), at least in the context of western blot analyses of brain extracts. Furthermore, there were no compensatory changes in SOD1 or SOD2 activities (). It is possible that circulating antioxidants, including ascorbic acid, tocopherol, uric acid, bilirubin, proteins and other compounds, could be increased as a compensatory mechanism, but we did not address that in the current study. The fact that a partial deficiency in the SOD1 and SOD2 isoforms as seen here, as well as a full deficiency in SOD3 reported earlier [32
], all imparted a common protective effect in the hippocampus, suggests that there are common effectors throughout the cell capable of engaging prosurvival or differentiation pathways. While the precise mechanism(s) responsible for this type of response is not yet known, in a general sense this effect resembles a preconditioning [62
], adaptive (reviewed in [63
]), or inducible-like radioprotective response [64
], where a sublethal or potentially injurious stimulus (i.e. oxidative stress) induces tolerance to a subsequent and potentially more damaging insult (irradiation). Lower levels of SOD should lead to higher levels of oxidative stress [65
] which should in turn result in an increase in reactive species that are derived from superoxide, e.g. H2
, and data exist showing that the presence of such compounds can reduce the effects of a subsequent insult [66
]. In the CNS, this could involve specific trophic factors and signaling molecules that favor differentiation and long-term survival of newly generated neurons and which are up-regulated or activated in the brains of irradiated SOD KO mice. Such factors could include brain derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), and nitric oxide (NO), all of which have been shown to favor differentiation and survival of neurons [68
]. Regardless of the mechanism involved, our findings clearly suggest that SOD deficient mice have developed a resistance to radiation-induced inhibition of neurogenesis that may involve some type of adaptation within the microenvironment, without, necessarily, any compensatory changes in other major intracellular antioxidants.
Astrocytes are now recognized as dynamic regulators of a variety of neuron-related functions, including neurogenesis [72
]. In fact, it has been suggested that the astrocytes within the neurogenic niche are highly specialized and contribute to the regulation of proliferation and fate specification of neural precursor cells [74
]. In the present study, before irradiation the relative abundance of newly born astrocytes was 5–9 fold higher in SOD KO mice compared to WT controls (), and the ratio of new neurons to new astrocytes was about 17 in WT and 1–2 in the SOD KO mice. The finding of increased numbers of newly generated astrocytes in SOD1 and SOD2 KO mice needs to be considered in the context of an increase in steady-state levels of superoxide due to a ~ 50% reduction in CuZnSOD or MnSOD. It is possible that increased steady-state levels of superoxide and downstream ROS derived from superoxide alter the differentiation pattern of neuronal progenitor cells in favor of astroglial lineage. Alternatively, it may be that astrocytes are more resistant to superoxide than neurons, leading to a better outcome in terms of long-term survival. The notion that astrocytes are more resistant to superoxide and other ROS is consistent with previous findings showing that primary astrocytes are more resistant to menadione induced apoptotic cell death than primary neuronal cultures [75
] and that astrocytes are more efficient in repairing oxidative mitochondrial DNA damage mediated by menadione [76
]. Astrocytes tend to produce lower steady-state levels of ROS during metabolism, and primary astrocytes derived from hippocampus have been shown to have lower steady-state level lipid peroxidation than primary neurons derived from the same region [77
]. It has been shown in different experimental systems that a more oxidized cellular environment facilitates progenitor cell differentiation as opposed to proliferation [33
], but whether the more oxidized redox state favors differentiation toward one cell lineage over another is less clear. In our experimental system, the data suggest that the SOD deficient environment favors differentiation toward an astrocytic lineage.
It is particularly interesting to note that prior to irradiation, the average number of newly born cells that differentiated into astrocytes in SOD1 mice (1112 ± 157) was higher than that seen in SOD2 (586 ± 102), which in turn was higher than the value for SOD3 (291 ± 98; [32
]). A critical redox-related factor associated with differentiation may be more predominant in the cytoplasm rather than in the mitochondria or extracellularly, but this conclusion is highly speculative. Additionally, when the number of newly born cells that differentiate into neurons prior to irradiation was analyzed between the different SOD isoforms the opposite trend was found, where SOD1, SOD2 and SOD3 deficient mice have an average of 1048 ±40, 1355 ± 290, and 1793 ± 226 [32
] new neurons, respectively. These trends are provocative and it is tempting to speculate that the different cellular compartments from which the SOD isoforms evolved may contain redox-sensitive factors that play a deciding role in the lineage commitment during precursor cell differentiation [78
Irradiation had little or no effect on newly born cells that become astrocytes, regardless of genotype, but the new neuron to new astrocyte ratio after irradiation fell to about 5 in WT mice but remained around 1–1.5 in the KO animals. Given the supportive role of astrocytes in neurogenesis [72
], the relatively higher numbers of the newly generated astrocytes in the KO mice may have promoted the survival of newly born neurons after irradiation, although our data do no provide definitive proof of this thesis. Alternatively, the higher numbers of newly born astrocytes seen after irradiation in the SOD KO mice might simply be associated with gliosis, which has been shown to be mediated in part by reactive species [79
]. Finally, there are data suggesting that the putative stem cell in the adult mammalian brain is astrocytic [80
], so it may be that the SOD deficient background favors the selection/survival of stem like cells in the dentate SGZ. This latter idea is supported, in part, by data suggesting that oxidative stress and redox regulation play important roles in self-renewal and differentiation in specific precursor cell populations [33
]. Whether or not the differences in the astrocytic cell population is responsible for ‘protecting’ neurogenesis after irradiation, or is merely a sign of some sort of non-specific activation or process due to a persistent oxidative stress needs to be further investigated.
Previous studies have suggested that neuroinflammation, and in particular elevated numbers of activated microglia, may negatively impact neurogenesis after irradiation [12
]. However, with the wide diversity of cell types involved, including astrocytes, and differences in activation state, it is now being recognized that neuroinflammation may be supportive as well as detrimental to neurogenesis (reviewed in [81
]). In fact, in our previous EC-SOD study we saw that there were more activated microglia in EC-SOD KO mice than in WT mice, and concluded that in an SOD deficient background, increased numbers of activated microglia had no apparent association with neurogenesis [32
]. In general, this agrees with work from others showing that microglial phenotype critically influences the ability of these cells to support or impair cell renewal processes in the adult brain [83
], and suggests that microglia may respond differently to a given stimulus (irradiation) depending upon the presence of another and preceding stimulus (oxidative stress). The present data further complicate this issue inasmuch as irradiation induced a very substantial increase in numbers of newly born activated microglia in the irradiated SOD2 background, but had no apparent effect in the SOD1 background, while both showed a protective effect in the context of neurogenesis. This could mean that the numbers of newly born activated microglia per se
play a limited role in the observed changes in neurogenesis, or that the deficiency of mitochondrial SOD impacts specific elements of neuroinflammation to a greater extent than cytoplasmic SOD deficiency. Identification and investigation of potential pathways and molecules that may be responsible for such observations should provide us the information to determine the significance of these findings.
Given the normal physiologic role of SOD, it seems likely that a major factor in the responses seen in the SOD KO mice relate to superoxide levels. The evolution of the different SOD isoforms must in part be related to the poor diffusion of the superoxide anion across different cellular compartments, as well as the need to remove this mildly reactive molecule from specific intracellular and extracellular spaces. Previous studies with MnSOD deficient mice (Sod2
−/+) showed a significant reduction in reduced GSH and an increase in 8-oxodG in the brain, suggesting an increased steady state level of oxidative stress in SOD2 KO [50
]. Even though similar types of measurements have not been carried out in SOD1 KO (Sod1
−/+) brains, studies focusing on liver and skeletal muscles in homozygous SOD1 KO (Sod1
−/−) mice all showed increased steady state level of oxidative damage [49
]. Therefore, SOD deficiency should increase superoxide levels in cells; but we did not directly measure this in vivo
due to a limited quantity of hippocampal tissue. Therefore, to determine if elevated superoxide has an influence on how neural precursor cells responded to irradiation, we performed an in vitro
study where we could actually subject cells to elevated levels of superoxide prior to irradiation. While cells exposed to xanthine plus xanthine oxidase showed a similar dose response as controls, on average, at the doses used, there was about a 1.5-fold increase in cell number compared to that seen after irradiation only (). There were also increased cell numbers noted in the presence of xanthine alone (), suggesting that there was some endogenous xanthine oxidase activity available that led to intermediate levels of superoxide. While these in vitro
data are limited, they do suggest that the presence of excess superoxide prior to irradiation has significant positive effects on the proliferation and/or survival of multipotent neural precursor cells. Further, the data are qualitatively consistent with our in vivo
studies, and suggest that superoxide may act to stimulate a redox-sensitive pathway/s that ameliorates the inhibition of neurogenesis typically found after irradiation.
In summary, our findings have clearly demonstrated that SOD deficiency has paradoxical effects with respect to hippocampal neurogenesis. In the absence of irradiation, SOD deficient animals exhibited reduced baseline neurogenesis and, presumably, increased basal oxidative stress, the latter of which may have been sufficient to elicit a ‘protective’ or adaptive response following low dose radiation exposure. This effect was independent of specific isoform and whether the deficiency was complete (SOD3) [32
] or partial (SOD1, 2). Further, the effect did not apparently involve compensatory responses in other major antioxidant defenses, nor was it associated with a clear trend in detrimental neuroinflammation. Interestingly, a pilot in vitro
study carried out under excess superoxide levels also revealed beneficial effects in terms of increased survival and/or proliferation. Opposing trends in the yields of newly generated astroglia versus newly generated neurons in unirradiated animals deficient for specific SOD isoforms suggest that factors controlling the phenotypic fate of multipotent hippocampal precursor cells might be restricted to and/or concentrated in specific subcellular compartments. While the precise mechanism behind the neuroprotective effects of SOD deficiency is not known, further studies need to be performed to determine whether adaptation to pre-existing oxidative stress plays a key role. The use of conditional tissue-specific SOD knockout animals [85
] should help delineate the time required to develop any favorable adaptation before exposure to irradiation, and continued in vitro
experimentation will elucidate the redox responsive pathways for conferring the neuroprotective phenotype found in irradiated animals deficient in SOD.