Extracellular superoxide dismutase is important for hippocampal neurogenesis and preservation of cognitive functions after irradiation.

Cranial irradiation is widely used in cancer therapy, but it often causes cognitive defects in cancer survivors. Oxidative stress is considered a major cause of tissue injury from irradiation. However, in an earlier study mice deficient in the antioxidant enzyme extracellular superoxide dismutase (EC-SOD KO) showed reduced sensitivity to radiation-induced defects in hippocampal functions. To further dissect the role of EC-SOD in neurogenesis and in response to irradiation, we generated a bigenic EC-SOD mouse model (OE mice) that expressed high levels of EC-SOD in mature neurons in an otherwise EC-SOD-deficient environment. EC-SOD deficiency was associated with reduced progenitor cell proliferation in the subgranular zone of dentate gyrus in KO and OE mice. However, high levels of EC-SOD in the granule cell layer supported normal maturation of newborn neurons in OE mice. Following irradiation, wild-type mice showed reduced hippocampal neurogenesis, reduced dendritic spine densities, and defects in cognitive functions. OE and KO mice, on the other hand, were largely unaffected, and the mice performed normally in neurocognitive tests. Although the resulting hippocampal-related functions were similar in OE and KO mice following cranial irradiation, molecular analyses suggested that they may be governed by different mechanisms: whereas neurotrophic factors may influence radiation responses in OE mice, dendritic maintenance may be important in the KO environment. Taken together, our data suggest that EC-SOD plays an important role in all stages of hippocampal neurogenesis and its associated cognitive functions, and that high-level EC-SOD may provide protection against irradiation-related defects in hippocampal functions.

Oxidative stress and adult neurogenesis--effects of radiation and superoxide dismutase deficiency.

Hippocampus plays an important role in learning and memory and in spatial navigation. Production of new neurons that are functionally integrated into the hippocampal neuronal network is important for the maintenance of functional plasticity. In adults, production of new neurons in the hippocampus takes place in the subgranular zone (SGZ) of dentate gyrus. Neural progenitor/stem cells go through processes of proliferation, differentiation, migration, and maturation. This process is exquisitely sensitive to oxidative stress, and perturbation in the redox balance in the neurogenic microenvironment can lead to reduced neurogenesis. Cranial irradiation is an effective treatment for primary and secondary brain tumors. However, even low doses of irradiation can lead to persistent elevation of oxidative stress and sustained suppression of hippocampal neurogenesis. Superoxide dismutases (SODs) are major antioxidant enzymes for the removal of superoxide radicals in different subcellular compartments. To identify the subcellular location where reactive oxygen species (ROS) are continuously generated after cranial irradiation, different SOD deficient mice have been used to determine the effects of irradiation on hippocampal neurogenesis. The study results suggest that, regardless of the subcellular location, SOD deficiency leads to a significant reduction in the production of new neurons in the SGZ of hippocampal dentate gyrus. In exchange, the generation of new glial cells was significantly increased. The SOD deficient condition, however, altered the tissue response to irradiation, and SOD deficient mice were able to maintain a similar level of neurogenesis after irradiation while wild type mice showed a significant reduction in the production of new neurons.

Zinc deficiency regulates hippocampal gene expression and impairs neuronal differentiation.

OBJECTIVES: Proliferating adult stem cells in the subgranular zone of the dentate gyrus have the capacity not only to divide, but also to differentiate into neurons and integrate into the hippocampal circuitry. The present study identifies several hippocampal genes putatively regulated by zinc and tests the hypothesis that zinc deficiency impairs neuronal stem cell differentiation. METHODS: Genes that regulate neurogenic processes were identified using microarray analysis of hippocampal mRNA isolated from adult rats fed zinc-adequate or zinc-deficient (ZD) diets. We directly tested our hypothesis with cultured human neuronal precursor cells (NT2), stimulated to differentiate into post-mitotic neurons by retinoic acid (RA), along with immunocytochemistry and western analysis. RESULTS: Microarray analysis revealed the regulation of genes involved in cellular proliferation. This analysis also identified a number of genes known to be involved in neuronal differentiation, including the nuclear RA receptor, retinoid X receptor (RXR), doublecortin, and a transforming growth factor-beta (TGF-ß) binding protein (P < 0.05). Zinc deficiency significantly reduced RA-induced expression of the neuronal marker proteins doublecortin and ß-tubulin type III (TuJ1) to 40% of control levels (P < 0.01). This impairment of differentiation may be partially mediated by alterations in TGF-ß signaling. The TGF-ß type II receptor, responsible for binding TGF-ß during neuronal differentiation, was increased 14-fold in NT2 cells treated with RA (P < 0.001). However, this increase was decreased by 60% in ZD RA-treated cells (P < 0.001). DISCUSSION: This research identifies target genes that are involved in governing neurogenesis under ZD conditions and suggests an important role for TGF-ß and the trace metal zinc in regulating neuronal differentiation.

Chronic caloric restriction reduces tissue damage and improves spatial memory in a rat model of traumatic brain injury.

Although it has been known for some time that chronic caloric or dietary restriction reduces the risk of neurodegenerative disorders and injury following ischemia, the possible role of chronic restriction in improving outcomes after traumatic brain injury (TBI) has not been previously studied. Therefore, 2-month-old male Sprague-Dawley rats were divided into two dietary groups, an ad libitum fed group (AL) and a caloric-restriction group (CR) that was provided with 70% of the food intake of AL rats (n = 10/group). After 4 months, a weight-drop device (300 g) was used to produce a 2-mm bilateral medial frontal cortex contusion following craniotomy. Additional animals in each dietary group (n = 10) were used as sham-operated controls. The CR diet resulted in body weights that were reduced by 30% compared with AL controls. Not only did CR decrease the size of the cortical lesion after injury, there were marked improvements in spatial memory as measured by Morris water maze that included an increase in the number of animals successfully finding the platform as well as significantly reduced time to finding the hidden platform. Western analysis, used to examine the expression of proteins that play a role in neuronal survival, revealed significant increases in brain-derived neurotrophic factor (BDNF) in the cortical region around the site of injury and in the hippocampus in CR rats after injury. These findings suggest that molecular mechanisms involved in cell survival may play a role in reducing tissue damage and improving cognition after TBI and that these mechanisms can be regulated by dietary interventions.

Zinc deficiency impairs neuronal precursor cell proliferation and induces apoptosis via p53-mediated mechanisms.

The potential importance of stem cells in the adult central nervous system (CNS) that cannot only divide, but also participate in neurogenesis, is now widely appreciated. While we know that the trace element zinc is needed for brain development, the role of this essential nutrient in adult stem cell proliferation and neurogenesis has not been investigated. Adult male rats fed a zinc-restricted diet had approximately 50% fewer Ki67-positive stem cells in the subgranular zone (SGZ) and granular cell layer of the dentate gyrus compared to both zinc-adequate and pair-fed controls (p<0.05). Zinc-deficient rats also had a significant increase the number of TUNEL-labeled cells in the SGZ compared to pair-fed rats (p<0.05). To explore the mechanisms responsible for the effects of zinc deficiency, cultured human Ntera-2 (NT2) neuronal precursor cells were deprived of zinc using the chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). Consistent with the effects of deficiency in vivo, TPEN treatment resulted in a significant decrease in cellular proliferation, as measured by bromodeoxyuridine (BrdU) uptake, and an increase in caspase3/7-dependent apoptosis. These changes were accompanied by increases in nuclear p53. Oligonucleotide arrays, coupled with use of a dominant-negative p53 construct in NT2 cells, identified 14 differentially regulated p53 target genes. In the early phases zinc deficiency, p53 targets responsible for cell cycle arrest were induced. Continuation of deficiency resulted in the induction of a variety of pro-apoptotic genes such as transforming growth factor-beta (TGF-beta) and retinoblastoma-1 (Rb-1), as well as cellular protection genes such as glutathione peroxidase (GPx). These data suggest that zinc plays a role in neurogenesis by regulating p53-dependent molecular mechanisms that control neuronal precursor cell proliferation and survival.

Zinc deficiency induces depression-like symptoms in adult rats.

There is mounting evidence suggesting a link between serum zinc levels and clinical depression. Not only is serum zinc negatively correlated with the severity of symptoms, but zinc levels appear to be lowest in patients who do not respond to antidepressant drug therapy. It is not known if reduced zinc levels are contributing to depression, or the result of dietary or other factors associated with major depression. Thus, we designed this study to test the hypothesis that dietary zinc deficiency would induce depression-like behaviors in rats. Two-month-old male rats were fed zinc adequate (ZA, 30 ppm), deficient (ZD, 1 ppm), or supplemented (ZS, 180 ppm) diets for 3 weeks. Consistent with the development of depression, ZD rats displayed anorexia (p<0.001), anhedonia (reduced saccharin:water intake, p< 0.001), and increased anxiety-like behaviors in a light-dark box test (p<0.05). Furthermore, the antidepressant drug fluoxetine (10 mg/kg body wt) reduced behavioral despair, as measured by the forced swim test, in rats fed the ZA and ZS rats (p<0.05), but was ineffective in ZD rats. Together these studies suggest that zinc deficiency leads to the development of depression-like behaviors that may be refractory to antidepressant treatment.

Zinc deficiency induces apoptosis via mitochondrial p53- and caspase-dependent pathways in human neuronal precursor cells.

Previous studies have shown that zinc deficiency leads to apoptosis of neuronal precursor cells in vivo and in vitro. In addition to the role of p53 as a nuclear transcription factor in zinc deficient cultured human neuronal precursors (NT-2), we have now identified the translocation of phosphorylated p53 to the mitochondria and p53-dependent increases in the pro-apoptotic mitochondrial protein BAX leading to a loss of mitochondrial membrane potential as demonstrated by a 25% decrease in JC-1 red:green fluorescence ratio. Disruption of mitochondrial membrane integrity was accompanied by efflux of the apoptosis inducing factor (AIF) from the mitochondria and translocation to the nucleus with a significant increase in reactive oxygen species (ROS) after 24h of zinc deficiency. Measurement of caspase cleavage, mRNA, and treatment with caspase inhibitors revealed the involvement of caspases 2, 3, 6, and 7 in zinc deficiency-mediated apoptosis. Down-stream targets of caspase activation, including the nuclear structure protein lamin and polyADP ribose polymerase (PARP), which participates in DNA repair, were also cleaved. Transfection with a dominant-negative p53 construct and use of the p53 inhibitor, pifithrin-µ, established that these alterations were largely dependent on p53. Together these data identify a cascade of events involving mitochondrial p53 as well as p53-dependent caspase-mediated mechanisms leading to apoptosis during zinc deficiency.

Paradoxical relationship between Mn superoxide dismutase deficiency and radiation-induced cognitive defects.

Radiation therapy of the CNS, even at low doses, can lead to deficits in neurocognitive functions. Reduction in hippocampal neurogenesis is usually, but not always, associated with cognitive deficits resulting from radiation therapy. Generation of reactive oxygen species is considered the main cause of radiation-induced tissue injuries, and elevated levels of oxidative stress persist long after the initial cranial irradiation. Consequently, mutant mice with reduced levels of the mitochondrial antioxidant enzyme, Mn superoxide dismutase (MnSOD or Sod2), are expected to be more sensitive to radiation-induced changes in hippocampal neurogenesis and the related functions. In this study, we showed that MnSOD deficiency led to reduced generation of immature neurons in Sod2-/+ mice even though progenitor cell proliferation was not affected. Compared to irradiated Sod2+/+ mice, which showed cognitive defects and reduced differentiation of newborn cells towards the neuronal lineage, irradiated Sod2-/+ mice showed normal hippocampal-dependent cognitive functions and normal differentiation pattern for newborn neurons and astroglia. However, we also observed a disproportional decrease in newborn neurons in irradiated Sod2-/+ following behavioral studies, suggesting that MnSOD deficiency may render newborn neurons more sensitive to stress from behavioral trainings following cranial irradiation. A positive correlation between normal cognitive functions and normal dendritic spine densities in dentate granule cells was observed. The data suggest that maintenance of synaptic connections, via maintenance of dendritic spines, may be important for normal cognitive functions following cranial irradiation.