9012 • The Journal of Neuroscience, November 18, 2020 • 40(47):9012–9027 Development/Plasticity/Repair Suppression of DNA Double-Strand Break Formation by DNA Polymerase b in Active DNA Demethylation Is Required for Development of Hippocampal Pyramidal Neurons Akiko Uyeda,1 Kohei Onishi,1 Teruyoshi Hirayama,1,2,3 Satoko Hattori,4 Tsuyoshi Miyakawa,4 Takeshi Yagi,1,2 Nobuhiko Yamamoto,1 and Noriyuki Sugo1 1Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan, 2AMED-CREST, Japan Agency for Medical Research and Development, Suita, Osaka 565-0871, Japan, 3Department of Anatomy and Developmental Neurobiology, Tokushima University Graduate School of Medical Sciences, Kuramoto, Tokushima 770-8503, Japan, and 4Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan Genome stability is essential for brain development and function, as de novo mutations during neuronal development cause psychiatric disorders. However, the contribution of DNA repair to genome stability in neurons remains elusive. Here, we demonstrate that the base excision repair protein DNA polymerase b (Polb) is involved in hippocampal pyramidal neuron fl/fl differentiation via a TET-mediated active DNA demethylation during early postnatal stages using Nex-Cre/Polb mice of ei- ther sex, in which forebrain postmitotic excitatory neurons lack Polb expression. Polb deficiency induced extensive DNA dou- ble-strand breaks (DSBs) in hippocampal pyramidal neurons, but not dentate gyrus granule cells, and to a lesser extent in neocortical neurons, during a period in which decreased levels of 5-methylcytosine and 5-hydroxymethylcytosine were observed in genomic DNA. Inhibition of the hydroxylation of 5-methylcytosine by expression of microRNAs miR-29a/b-1 diminished DSB formation. Conversely, its induction by TET1 catalytic domain overexpression increased DSBs in neocortical neurons. Furthermore, the damaged hippocampal neurons exhibited aberrant neuronal gene expression profiles and dendrite formation, but not apoptosis. Comprehensive behavioral analyses revealed impaired spatial reference memory and contextual fear memory in adulthood. Thus, Polb maintains genome stability in the active DNA demethylation that occurs during early postnatal neuronal development, thereby contributing to differentiation and subsequent learning and memory. Key words: DNA demethylation; DNA double-strand break; DNA repair; hippocampal development; learning and mem- ory; neuronal differentiation Significance Statement Increasing evidence suggests that de novo mutations during neuronal development cause psychiatric disorders. However, strikingly little is known about how DNA repair is involved in neuronal differentiation. We found that Polb , a component of base excision repair, is required for differentiation of hippocampal pyramidal neurons in mice. Polb deficiency transiently led to increased DNA double-strand breaks, but not apoptosis, in early postnatal hippocampal pyramidal neurons. This aberrant double-strand break for- mation was attributed to active DNA demethylation as an epigenetic regulation. Furthermore, the damaged neurons exhibited aber- rant gene expression profiles and dendrite formation, resulting in impaired learning and memory in adulthood. Thus, these findings provide new insight into the contribution of DNA repair to the neuronal genome in early brain development. Received Feb. 3, 2020; revised Oct. 2, 2020; accepted Oct. 16, 2020. Introduction Author contributions: A.U., S.H., T.M., T.Y., N.Y., and N.S. designed research; A.U., K.O., T.H., S.H., and N.S. performed research; A.U., K.O., T.H., S.H., T.M., T.Y., N.Y., and N.S. analyzed data; A.U. and N.S. wrote the first Genome stability is crucial for both genetic and epigenetic regu- draft of the paper; A.U., K.O., T.H., S.H., T.M., T.Y., N.Y., and N.S. edited the paper; A.U., N.Y., and N.S. wrote lation underlying gene expression in the brain throughout life. the paper. DNA repair is essential to maintain genome stability and has This work was supported by Ministry of Education, Culture, Sports, Science and Technology KAKENHI on been well characterized through studies on cancer and immune Dynamic regulation of brain function by Scrap & Build system (No. 16H06460) to N.Y., Japan Society for the Promotion of Science KAKENHI Grants 15K14350 and 17K07109 to N.S., 16H06276 (AdAMS) to N.S. and T.M., cell differentiation in mammals (Lindahl and Wood, 1999; Alt et and Japan Agency for Medical Research and Development–Core Research for Evolutional Science and al., 2013). In the nervous system, mouse models reveal that DNA Technology to T.Y. We thank Dr. K.A. Nave for the Nex-Cre mice; Dr. K. Rajewsky for Polb flox mice; and Dr. repair dysfunction in neural progenitors frequently leads to ge- I. Smith for critical reading of the manuscript. nome instability and neuronal apoptosis during the period of The authors declare no competing financial interests. Correspondence should be addressed to Noriyuki Sugo at [email protected]. neurogenesis (McKinnon, 2013). Genetic diseases related to https://doi.org/10.1523/JNEUROSCI.0319-20.2020 DNA repair also include microcephaly, developmental disorders, Copyright © 2020 the authors and psychiatric disorders (McKinnon, 2013; Madabhushi et al., Uyeda et al. · Genome Stability by Polb in Hippocampal Pyramidal Neurons J. Neurosci., November 18, 2020 • 40(47):9012–9027 • 9013 2014). In addition, accumulation of somatic mutations in neu- Materials and Methods rons during development has been implicated in developmental Animals. All experiments were conducted under the guidelines for brain disorders, such as autism and schizophrenia (Poduri et al., laboratory animals of the Graduate School of Frontier Biosciences, 2013; McConnell et al., 2017). These studies suggest that DNA Osaka University. The protocol was approved by the Animal Care and repair is likely to be critical for normal brain development and Use Committee of the Graduate School of Frontier Biosciences, Osaka 1 function. However, while DNA repair has been characterized in University and Fujita Health University. NexCre/ Polb fl/fl (Nex-Cre/ fl/fl mitotic cells, including neural progenitors, its role in neurons as Polb )miceweregeneratedasdescribedpreviously(Onishi et al., postmitotic cells remains unclear. Thus, it is important to 2017). Both male and female mice were used in all experiments, except uncover novel aspects of DNA repair in neuronal differentiation RNA-seq analysis and the behavioral test. Noon of the day on which the vaginal plug was detected was designated as embryonic day 0.5 (E0.5) and function. and the day of birth was designated as postnatal day 0 (P0). Genotyping Base excision repair (BER) is mainly involved in the removal was performed using the following primers: Polb locus: 59-CCAC of DNA base damage and apurinic/apyrimidinic sites (Wilson et ACCGAAGTCCTCTGAT-39,59-AGGCTGGCCTCAGACTCATA-39 and al., 2000). In addition, recent studies have revealed that BER also 59-CTGGCTCACGTTCTTCTC-39;Crelocus:59-GCAGAACCTGAAG plays a role in the active DNA demethylation process as an epige- ATGTTCGCGAT-39 and 59-AGGTATCTCTGACCAGAGTCATCC-39. netic regulation (Wu and Zhang, 2017). In this process, 5-meth- Cell cultures. Pregnant mice were deeply anesthetized with pentobar- ylcytosine (5mC) is initially oxidized by TET enzymes and is bital (50 mg/kg, i.p.). Neocortices were dissected from E16.5 embryos in converted to 5-hydroxymethylcytosine (5hmC); the modified ice-cold HBSS and then minced with fine scissors in PBS, pH 7.4. The base is finally recognized by thymine DNA glycosylase and minced tissues were incubated with 0.125% trypsin and 0.02% EDTA in replaced with cytosine by DNA polymerase b (Polb )andthe PBS for 5 min at 37°C, and then triturated thoroughly using a fire-pol- Xrcc1/Lig3 complex (Weber et al., 2016). DNA methylation and ished Pasteur pipette. After centrifugation, the cells were resuspended in DMEM/F12 medium (Thermo Fisher Scientific) supplemented with B27 demethylation often play a central role in cell differentiation (Thermo Fisher Scientific) and 5% FBS (Hyclone). A suspension con- (Wu and Zhang, 2017). In the neuronal epigenome, dynamic taining 2.0 Â 105 cells was plated with culture medium on a 12 mm changes in the DNA methylation level are observed during brain micro cover glass (Matsunami) in a multiwell dish (Thermo Fisher development (Lister et al., 2013; Simmons et al., 2013; Sharma et Scientific) coated with 0.1 mg/ml poly-L-ornithine (P3655, Sigma al., 2016) and affect neuronal gene expression, which is impli- Millipore). The cultures were maintained at 37°C in an environment of cated in neurogenesis, maturation, and plasticity (Moretti et al., 5% CO2 and humidified 95% air. 2006; Feng et al., 2010; Sanosaka et al., 2017). This regulation Plasmids. pFN21AE2295, containing HaloTag-human TET1 cDNA, also contributes to learning and memory (Kaas et al., 2013; was purchased from Promega. To generate TET1 catalytic domain Rudenko et al., 2013; Li et al., 2014; Gontier et al., 2018). (TET1CD) expression vector pCAGGS-TET1CD, TET1CD was amplified Studies using conventional Polb -deficient mice show from pFN21AE2295 by PCR with the following primers: TET1CD forward, 59-ATGGAACTGCCCACCTGCAGCTGTCT-39 and TET1CD reverse, increased neuronal apoptosis during the period of neurogenesis 59-TCAGACCCAATGGTTATAGGGCCCCG-39.ThePCRproductwas in the developing nervous system rather than in other tissues, subcloned into pGEM-T Easy vector