Neil3 DNA Glycosylase in Maintenance and Repair of the Mammalian Brain
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AbstractThe DNA molecule is susceptible to damage induced by cellular metabolites and exogenous DNA damaging agents. Reactive oxygen species (ROS), generated as respiration by-products or arising from inflammatory reactions, are the most abundant endogenous DNA damaging agents and lead to the formation of a number of mutagenic and cytotoxic DNA lesions. To preserve DNA integrity and allow correct transmission of genetic information, several pathways for the repair of such lesions have evolved. Among these, base excision repair (BER) is the main pathway responsible for the repair of non-helix distorting base lesions arising from oxidation, deamination, and alkylation, in addition to single-strand breaks (SSB) and apurinic/apyrimidinic (AP) sites. BER is a multi-step pathway, which is initiated by DNA glycosylases that recognize specific base lesions and subsequently remove them from the DNA backbone. Five different DNA glycosylases initiate repair of oxidative base lesions, including the Endonuclease VIII-like (NEIL) paralogs 1, 2, and 3. NEIL1 and NEIL2 have been thoroughly characterized and shown to initiate BER of diverse oxidized DNA lesions, with preference for pyrimidine modifications in both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA). Less has been known about NEIL3 due to difficulties in expression and purification, although recent publications have confirmed DNA glycosylase activity and a preference for oxidized DNA base lesions. In mice, Neil1 and Neil2 show ubiquitous expression in the brain, whereas the expression of Neil3 is confined in space and time.
Supplemental oxygen during resuscitation of newborn babies is proposed to be an exogenous source of DNA damage; such treatment is associated with increased risk of childhood cancers. In the present study we have attempted to investigate supplemental oxygen as a source of DNA damage and to decipher Neil3 function in the mouse brain. For this, we have employed perinatal mouse models of increased oxidative stress, including ischemia, hypoxia, and hyperoxia, to induce DNA damage and trigger BER.
In the first part of the study, we performed experiments with hyperoxic resuscitation after global hypoxia. This resulted in accumulation of oxidative base lesions in liver DNA, but no effect, either in oxidative lesions or DNA glycosylase activity, was found in the brain. In the second part of the study we describe the construction and phenotype of a novel Neil3-/- mouse. We employed a model of combined cerebral ischemia and transient hypoxia to aid the search for Neil3- dependent phenotypic expressivity. Perinatal Neil3-/- mice displayed a lack of proliferative response in neuronal progenitor cells and neuronal tissue regeneration. Neil3-/- neural stem/progenitor cells (NSPCs) propagated as neurospheres in vitro displayed reduced growth, skewed differentiation, and diminished repair activity for hydantoin lesions in ssDNA. Additionally, Neil3-/- NSPCs accumulated more DNA strand breaks than Neil3+/+ spheres when exposed to a genotoxic agent. In aged Neil3- /- mice we found aberrant hippocampus-dependent behaviors, and also alterations in the presence and composition of hippocampal synapses. NSPC growth and repair activity for hydantoin lesions in vitro was impaired also in the aged mice. In sum, the findings herein implicate involvement of specific oxidative DNA base lesion recognition and repair by Neil3 in regulation of NSPC proliferation, differentiation, and integration of new neurons to maintain and repair the rodent brain.
List of papers. The papers are removed from the thesis due to copyright restrictions.
Paper I Yngve Sejersted, Anne Leikny Aasland, Magnar Bjørås, Lars Eide, and Ola Didrik Saugstad. Accumulation of 8-oxoguanine in liver DNA during hyperoxic resuscitation of newborn mice. Pediatric Research 2009 Nov;66(5):533-8 doi:10.1203/PDR.0b013e3181ba1a42
Paper II Yngve Sejersted, Gunn Annette Hildrestrand, David Kunke, Veslemøy Rolseth, Silje Zandstra Krokeide, Christine Gran Neurauter, Rajikala Suganthan, Monica Atneosen-Åsegg, Aaron M. Fleming, Ola Didrik Saugstad, Cynthia J. Burrows, Luisa Luna, and Magnar Bjørås. Endonuclease VIII-like 3 (Neil3) DNA glycosylase promotes neurogenesis induced by hypoxiaischemia. Proceedings of the National Academy of Sciences of the United States of America 2011 Nov;108(46):18802-18807 doi:10.1073/pnas.1106880108
Paper III Christine Elisabeth Regnell, Gunn Annette Hildrestrand, Yngve Sejersted, Tirill Medin, Olve Moldestad, Veslemøy Rolseth, Silje Zandstra Krokeide, Luisa Luna, Magnar Bjørås, and Linda H. Bergersen. Hippocampal adult neurogenesis is maintained by Neil3-dependent repair of oxidative DNA lesions in neural progenitor cells. Cell Rep. 2012 Sep 27;2(3):503-10. doi:10.1016/j.celrep.2012.08.008