Yngve Sejersted

Endonuclease VIII-like 3 (Neil3) DNA glycosylase promotes neurogenesis induced by hypoxia-ischemia

Neural stem/progenitor cell proliferation and differentiation are required to replace damaged neurons and regain brain function after hypoxic-ischemic events. DNA base lesions accumulating during hypoxic-ischemic stress are removed by DNA glycosylases in the base-excision repair pathway to prevent cytotoxicity and mutagenesis. Expression of the DNA glycosylase endonuclease VIII-like 3 (Neil3) is confined to regenerative subregions in the embryonic and perinatal brains. Here we show profound neuropathology in Neil3-knockout mice characterized by a reduced number of microglia and loss of proliferating neuronal progenitors in the striatum after hypoxia-ischemia. In vitro expansion of Neil3-deficient neural stem/progenitor cells revealed an inability to augment neurogenesis and a reduced capacity to repair for oxidative base lesions in single-stranded DNA. We propose that Neil3 exercises a highly specialized function through accurate molecular repair of DNA in rapidly proliferating cells.

Hippocampal Adult Neurogenesis Is Maintained by Neil3-Dependent Repair of Oxidative DNA Lesions in Neural Progenitor Cells

Accumulation of oxidative DNA damage has been proposed as a potential cause of age-related cognitive decline. The major pathway for removal of oxidative DNA base lesions is base excision repair, which is initiated by DNA glycosylases. In mice, Neil3 is the main DNA glycosylase for repair of hydantoin lesions in single-stranded DNA of neural stem/progenitor cells, promoting neurogenesis. Adult neurogenesis is crucial for maintenance of hippocampus-dependent functions involved in behavior. Herein, behavioral studies reveal learning and memory deficits and reduced anxiety-like behavior in Neil3(-/-) mice. Neural stem/progenitor cells from aged Neil3(-/-) mice show impaired proliferative capacity and reduced DNA repair activity. Furthermore, hippocampal neurons in Neil3(-/-) mice display synaptic irregularities. It appears that Neil3-dependent repair of oxidative DNA damage in neural stem/progenitor cells is required for maintenance of adult neurogenesis to counteract the age-associated deterioration of cognitive performance.

Oxygenation of the Newborn: A Molecular Approach

In this review oxygenation and hyperoxic injury of newborn infants are described through molecular and genetic levels. Protection and repair mechanisms that may be important for a new understanding of oxidative stress in the newborn are discussed. The research summarized in this article represents a basis for the reduced oxygen supplementation and oxidative load of newborn babies, especially since the turn of the century. The mechanisms discussed may also contribute to an understanding of why hyperoxic resuscitation of the newborn may damage DNA and affect its repair, thus increasing the risk that it may be carcinogenic. Today, term babies should be resuscitated with air rather than 100% oxygen and very and extremely low birth weight infants in need of stabilization or resuscitation at birth should be administered initially 21–30% oxygen and the level should be titrated according to the response, preferably measured by pulse oximetry. In the postnatal period the oxygen saturation should be targeted low <95%; however, saturations between 85 and 89% seem to increase mortality. The optimal oxygen saturation target for these infants postnatally is still unknown.

Loss of Neil3, the major DNA glycosylase activity for removal of hydantoins in single stranded DNA, reduces cellular proliferation and sensitizes cells to genotoxic stress

7,8-Dihydro-8-oxoguanine (8-oxoG) is one of the most common oxidative base lesions in normal tissues induced by a variety of endogenous and exogenous agents. Hydantoins are products of 8-oxoG oxidation and as 8-oxoG, they have been shown to be mutagenic lesions. Oxidative DNA damage has been implicated in the etiology of various age-associated pathologies, such as cancer, cardiovascular diseases, arthritis, and several neurodegenerative diseases. The mammalian endonuclease VIII-like 3 (Neil3) is one of the four DNA glycosylases found to recognize and remove hydantoins in the first step of base excision repair (BER) pathway. We have generated mice lacking Neil3 and by using total cell extracts we demonstrate that Neil3 is the main DNA glycosylase that incises hydantoins in single stranded DNA in tissues. Using the neurosphere culture system as a model to study neural stem/progenitor (NSPC) cells we found that lack of Neil3 impaired self renewal but did not affect differentiation capacity. Proliferation was also reduced in mouse embryonic fibroblasts (MEFs) derived from Neil3(-/-) embryos and these cells were sensitive to both the oxidative toxicant paraquat and interstrand cross-link (ICL)-inducing agent cisplatin. Our data support the involvement of Neil3 in removal of replication blocks in proliferating cells.

The 'Effects of Transfusion Thresholds on Neurocognitive Outcome of Extremely Low Birth-Weight Infants (ETTNO)' Study: Background Aims, and Study Protocol

Background: Infants with extremely low birth weight uniformly develop anemia of prematurity and frequently require red blood cell transfusions (RBCTs). Although RBCT is widely practiced, the indications remain controversial in the absence of conclusive data on the long-term effects of RBCT. Objectives: To summarize the current equipoise and to outline the study protocol of the ‘Effects of Transfusion Thresholds on Neurocognitive Outcome of extremely low birth-weight infants (ETTNO)’ study. Methods: Review of the literature and design of a large pragmatic randomized controlled trial of restrictive versus liberal RBCT guidelines enrolling 920 infants with birth weights of 400–999 g with long-term neurodevelopmental follow-up. Results and Conclusions: The results of ETTNO will provide definite data about the efficacy and safety of restrictive versus liberal RBCT guidelines in very preterm infants.

Accumulation of 8-oxoguanine in liver DNA during hyperoxic resuscitation of newborn mice.

Supplementary oxygen during resuscitation of the asphyxiated newborn is associated with long-term detrimental effects including increased risk of childhood cancer. It is suspected that the resuscitation procedure results in accumulated DNA damage and mutagenesis. Base excision repair (BER) is the major pathway for repair of premutagenic oxidative DNA lesions. This study addresses DNA base damage and BER in brain, lung, and liver in neonatal mice (P7) after hyperoxic resuscitation. Mice were randomized to 8% oxygen or room air for 60 min in a closed chamber and subsequent reoxygenation with 100% oxygen for 0 to 90 min. During this treatment, 8-oxoguanine accumulated in liver but not in lung or cerebellum. We observed a linear relation between 8-oxoguanine and reoxygenation time in liver DNA from hypoxic animals (n = 28; B = 0.011 [0.001, 0.020]; p = 0.037). BER activity was not significantly changed during resuscitation. Our data suggest that after hypoxia, the capacity for immediate repair in liver tissue is inadequate to meet increasing amounts of DNA damage. The duration of supplementary oxygen use during resuscitation should be kept as short as justifiable to minimize the risk of genetic instability.

Transcriptome profiling of the newborn mouse brain after hypoxia-reoxygenation: hyperoxic reoxygenation induces inflammatory and energy failure responsive genes

Background:Supplemental oxygen used during resuscitation can be detrimental to the newborn brain. The aim was to determine how different oxygen therapies affect gene transcription in a hypoxia–reoxygenation model.Methods:C57BL/6 mice (n = 56), postnatal day 7, were randomized either to 120 min of hypoxia 8% O2 followed by 30 min of reoxygenation with 21, 40, 60, or 100% O2, or to normoxia followed by 30 min of 21 or 100% O2. Affymetrix 750k expression array was applied with RT-PCR used for validation. Histopathology and immunohistochemistry 3 d after hypoxia–reoxygenation compared groups reoxygenated with 21 or 100% O2 with normoxic controls (n = 22).Results:In total, ~81% of the gene expression changes were altered in response to reoxygenation with 60 or 100% O2 and constituted many inflammatory-responsive genes (i.e., C5ar2, Stat3, and Ccl12). Oxidative phosphorylation was downregulated after 60 or 100% O2. Iba1+ cells were significantly increased in the striatum and hippocampal CA1 after both 21 and 100% O2.Conclusion:In the present model, hypoxia–reoxygenation induces microglial accumulation in subregions of the brain. The transcriptional changes dominating after applying hyperoxic reoxygenation regimes include upregulating genes related to inflammatory responses and suppressing the oxidative phosphorylation pathway.

Transcriptome profiling of the newborn mouse lung after hypoxia and reoxygenation: hyperoxic reoxygenation affects mTOR signaling pathway, DNA repair, and JNK-pathway regulation

Background:The use of oxygen in acute treatment of asphyxiated term newborns is associated with increased mortality. It is unclear how hyperoxic reoxygenation after hypoxia affects transcriptional changes in the newborn lung.Methods:On postnatal day 7, C57BL/6 mice (n = 62) were randomized to 120-min hypoxia (fraction of inspired oxygen (FiO2) 0.08) or normoxia. The hypoxia group was further randomized to reoxygenation for 30 min with FiO2 0.21, 0.40, 0.60, or 1.00, and the normoxia group to FiO2 0.21 or 1.00. Transcriptome profiling was performed on homogenized lung tissue using the Affymetrix 750k expression array, and validation was carried out by real-time polymerase chain reaction and enzyme-linked immunosorbent assay.Results:The hypoxia–reoxygenation model induced hypoxia-inducible factor 1 (HIF-1) targets like Vegfc, Adm, and Aqp1. In total, ~70% of the significantly differentially expressed genes were detected in the two high hyperoxic groups (FiO2 0.60 and 1.00). Reoxygenation with 100% oxygen after hypoxia uniquely upregulated Gadd45g, Dusp1, Peg3, and Tgm2. Pathway analysis identified mammalian target of rapamycin (mTOR) signaling pathway, DNA repair, c-jun N-terminal kinase (JNK)-pathway regulation, and cell cycle after hyperoxic reoxygenation was applied.Conclusion:Acute hypoxia induces HIF-1 targets independent of the reoxygenation regime applied. Hyperoxic reoxygenation affects pathways regulating cell growth and survival. DNA-damage–responsive genes are restricted to reoxygenation with 100% oxygen.

Early Upregulation of NLRP3 in the Brain of Neonatal Mice Exposed to Hypoxia-Ischemia: No Early Neuroprotective Effects of NLRP3 Deficiency.

Background: The NLRP3 inflammasome acts as an early mediator of inflammation by cleaving and releasing IL-1β and IL-18 from their proforms. Objective: The aim of this study was to describe NLRP3 activation and evaluate whether deficiency of NLRP3 protects against neonatal hypoxic ischemic brain damage. Methods: C57BL/6 and NLRP3-/- mice at P9 were subjected to unilateral common carotid ligation followed by hypoxia. RT-PCR was used on mRNA in five different subregions of the brain. Brain infarction was evaluated by histopathology and 2,3,5-triphenyltetrazolium chloride staining. Plasma levels of IL-18 were measured by ELISA. Double labeling immunohistochemistry was used to examine cell-specific NLRP3 expression. Results: NLRP3 was upregulated 24 h after hypoxia-ischemia (HI) in the hippocampus (2.6-fold), striatum (2.2-fold) and thalamus (2.3-fold). Brain infarction volumes were not statistically significantly different in NLRP3-/- mice compared to WT mice 24 h after HI, accompanied by no significant changes in plasma IL-18 levels. Three hours after HI, NLRP3 expression occurred in astrocytes located in the hippocampus and habenular nucleus of the thalamus. Microglia only showed scarce expression at this time point, but prominent NLRP3 expression 72 h after HI. Conclusion: Astrocytes are early mediators of NLRP3 activity. No early neuroprotective effect of NLRP3 deficiency in neonatal HI brain damage was shown.

Hypoxia-reoxygenation affects whole-genome expression in the newborn eye.

PURPOSE Resuscitation of newborns is one of the most frequent procedures in neonatal medicine. The use of supplementary oxygen during resuscitation of the asphyxiated newborn has been shown to be detrimental to vulnerable tissues. We wanted to assess transcriptional changes in ocular tissue after the acute use of oxygen in the delivery room in a hypoxia-reoxygenation model of the newborn mouse. METHODS C57BL/6 mice (n = 57), postnatal day 7, were randomized to receive either 120 minutes of hypoxia, at 8% O2, followed by 30 minutes of reoxygenation with 21, 40, 60, or 100% O2 or to normoxia followed by 30 minutes of 21% or 100% O2. Whole ocular homogenates were analyzed by Affymetrix 750k expression array, and RT-PCR was performed for validation. Bayesian analysis of variance for microarray data (BAMarray) was used to identify single significant genes, and Gene Set Enrichment Analysis (GSEA) was applied to reveal significant pathway systems. RESULTS In total, ∼ 92% of the gene expression changes were altered in response to reoxygenation with 60% or 100% O2 compared to expression at the lower percentages of 21% and 40%. After 100% O2 treatment, genes involved in inflammation (Ccl12), angiogenesis (Igfr1, Stat3), and metabolism (Hk2) were upregulated. Pathway analyses after hypoxia-reoxygenation revealed significant alterations of six pathways which included apoptosis, TGF-beta signaling, oxidative phosphorylation, voltage-gated calcium channel complex, mitochondrion, and regulation of RAS protein signal transduction. CONCLUSIONS Hypoxia-reoxygenation can induce immediate transcriptional responses in ocular tissue involving inflammation, angiogenesis, energy failure, and Ras signaling.