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

SummaryAccumulation 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

Biochemical characterization of mammalian NEIL3 and involvement in repair of hydantoin lesions in proliferative tissue.

Oxidative damage is a major threat to the integrity of our genome and can lead to mutations or block replication if not repaired. Base excision repair (BER) is the main pathway for repair of oxidative lesions and is initiated by the action of a DNA glycosylase. A monofunctional glycosylase recognizes and excises the modified nucleotide, while a bifunctional glycosylase in addition possesses an intrinsic apurinic/apyrimidinic (AP) lyase activity generating a nick in the DNA strand. There are five DNA glycosylases acting on oxidative damage in eukaryotic cells; 8-oxoguanine DNA glycosylase (OGG1), endonuclease III (NTH1), and endonuclease VIII-like 1, 2 and 3 (NEIL1, NEIL2 and NEIL3). These enzymes are bifunctional with overlapping substrate specificity and knockout mouse studies reveals that none of them are essential for life. NEIL3 is sequentially related to the well characterized enzymes NEIL1 and 2, but less is known about its biochemical properties. Paper I of this thesis includes an expression and purification protocol for human NEIL3. NEIL3 recognizes and excises guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) from the genome. In paper II we elucidate the amino acids important for activity. NEIL1 and NEIL2 have tightly coupled DNA glycosylase/AP lyase activities and perform β,δ-elimination mechanism to incise DNA. NEIL3 has uncoupled activities, where the base excision is more efficient than the strand incision. We demonstrate that a V2P mutation changes the catalytic properties of NEIL3 to be similar to the activity of NEIL1 and 2. While OGG1, NTH1, NEIL1 and NEIL2 are expressed in most tissues throughout the organism, NEIL3 expression is limited to some lymphatic organs and compartments of the brain harboring neural stem cells, indicating a role in highly proliferative tissue. Paper III shows that mouse Neil3 is responsible for repair of Gh and Sp lesions in single-stranded DNA (ssDNA) in thymus and spleen, where repair activity correlates with Neil3 expression level. In paper IV of this study, we expose young Neil3- deficient mice to hypoxia-ischemia, to introduce cell death and activate proliferation of neural tissue. We find that the Neil3 deficit leads to reduced regeneration of neural tissue in the striatum. Further, in vitro propagated Neil3-deficient neural stem cells have a depressed neurogenesis and impaired ability to repair Gh and Sp lesions in ssDNA. Taken together, we suggest that NEIL3 has a unique role in removing hydantoin lesions from ssDNA and thereby promoting replication of proliferating cells