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.DNA damage | formamidopyrimidine-DNA glycosylase/endonuclease VIII | hydantoins | neural stem cells | neuronal progenitor cells T he base-excision repair pathway (BER) maintains genomic integrity by removing base lesions caused by oxidation, alkylation, and deamination. DNA base lesions frequently are cytotoxic or mutagenic if not removed. BER is initiated by DNA glycosylases that recognize modified bases and catalyze cleavage of the N-glycosidic bond, creating an apurinic or apyrimidinic (AP) site. The exposed DNA backbone is cleaved by the AP lyase activity of bifunctional DNA glycosylases or by an AP endonuclease. Repair synthesis is completed by gap filling and ligation (1, 2).Endonuclease VIII-like 3 (NEIL3) and endonuclease VIII-like 1 (NEIL1) are mammalian oxidized base-specific DNA glycosylases (3, 4). The function of NEIL3 has remained enigmatic, but recently the mouse ortholog was shown to remove a broad spectrum of oxidative base lesions on single-stranded DNA substrates with preference for spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh), which are further oxidation products of one of the most common base lesions, 8-oxo-7,8-dihydroguanine (8ohG) (5). These findings suggest that NEIL3 serves as a DNA glycosylase to prevent accumulation of cytotoxic and mutagenic DNA lesions in mammalian cells, although the activity of NEIL1 far exceeds that of NEIL3 on most substrates.In the late postnatal and adult brain, newborn neurons arise from neural stem/progenitor cells (NSPCs) in both the subgranular zone (SGZ) of the hippocampal dentate gyrus and in the subventricular zone (SVZ) (6). We previously reported a discrete expression pattern of Neil3 in the rodent SGZ and SVZ, confined to the embryonic and perinatal stages (7,8). These observations indicate a role for Neil3 in proliferating cells in the brain. However, naïve Neil3-knockout mice generated by us and others (4) appear phenotypically normal. After perinatal hypoxic-ischemic (HI) and adult ischemic stroke, proliferation of SVZ NSPCs is enhanced, and differentiating p...

The mitochondrial DNA (mtDNA) of neural stem cells (NSCs) is vulnerable to oxidation damage. Subtle manipulations of the cellular redox state affect mtDNA integrity in addition to regulating the NSC differentiation lineage, suggesting a molecular link between mtDNA integrity and regulation of differentiation. Here we show that 8-oxoguanine DNA glycosylase (OGG1) is essential for repair of mtDNA damage and NSC viability during mitochondrial oxidative stress. Differentiating neural cells from ogg1 Ϫ/Ϫ knock-out mice spontaneously accumulate mtDNA damage and concomitantly shift their differentiation direction toward an astrocytic lineage, similar to wt NSCs subjected to mtDNA damaging insults. Antioxidant treatments reversed mtDNA damage accumulation and separately increased neurogenesis in ogg1 Ϫ/Ϫ cells. NSCs from a transgenic ogg1 Ϫ/Ϫ mouse expressing mitochondrially targeted human OGG1 were protected from mtDNA damage during differentiation, and displayed elevated neurogenesis. The underlying mechanisms for this shift in differentiation direction involve the astrogenesis promoting Sirt1 via an increased NAD/NADH ratio in ogg1 Ϫ/Ϫ cells. Redox manipulations to alter mtDNA damage level correspondingly activated Sirt1 in both cell types. Our results demonstrate for the first time the interdependence between mtDNA integrity and NSC differentiation fate, suggesting that mtDNA damage is the primary signal for the elevated astrogliosis and lack of neurogenesis seen during repair of neuronal injury.

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.

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