IMMUNOCYTOCHEMICAL LOCALIZATION OF DNA DOUBLE-STRAND BREAKS IN HUMAN AND RAT BRAINS G. TORRES, * J. R. LEHESTE AND R. L. RAMOS Department of Biomedical Sciences, New York Institute of Technology, College of Osteopathic Medicine, Old Westbury, NY 11568, USA Abstract—Post-mitotic neurons are particularly susceptible to DNA double-strand breaks during their relatively long lifespan. Here, we report the anatomical distribution and subcellular localization of a molecule first identified as a DNA damage checkpoint protein. Immunocytochemical analysis of 53BP1 showed that this nuclear molecule is widely expressed in adult human and rat brains. Further, we showed that 53BP1 routinely co-clusters with c-amino- butyric acid neurons throughout the rat neuraxis. Notably, 53BP1 is only expressed in neuronal cells as the DNA dam- age checkpoint protein was virtually absent from glial cells. Finally, we found that human neural progenitors showed a differential index of DNA fragmentation at different stages of cellular differentiation. These data provide additional and important anatomical findings for the distribution and phenotype of DNA double-strand breaks in the mammalian brain, and suggest that DNA fragmentation is a spontaneous event routinely occurring in neural progenitors and adult neurons. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: anterior hypothalamus, cortex; GABA cells; glial cells; inferior colliculus; neural progenitors. INTRODUCTION Neurons of the mammalian brain are particularly susceptible to continual DNA damage because of their post-mitotic state, high rates of oxygen metabolism and relative long lifespan (Brochier and Langley, 2013). For instance, double-stranded breaks occur during brain development as a result of spontaneous neuronal activity or during DNA replication of early-born neural progenitors (McKinnon, 2013; Suberbielle et al., 2013). To compen- sate for this damage, several DNA repair response path- ways exist in the nervous system to resolve specific DNA lesions and maintain genome stability (Santivasi and Xia, 2013). One of these repairing pathways is the so-called non-homologous end-joining (NHEJ) pathway which repairs helix-distorting lesions such as those induced by ultraviolet radiation (McKinnon, 2013). If NHEJ is dis- rupted during neurogenesis, neurodevelopmental defects arise in the form of microcephaly and/or high-grade gliomas (Gilmore and Walsh, 2013). Another repairing pathway operating in the nervous system is ATM (ataxia telangiectasia, mutated) which responds to DNA dam- age-responsive kinases. Of interest, disruption of ATM may lead to neurodegenerative disorders causing widespread synaptic signal dysfunction throughout the brain (Suberbielle et al., 2013). Indeed, the importance of the aforementioned repair pathways is underscored by the consequences of their failure to respond to DNA fragmentation in a number of disorders with neurodegen- erative phenotypes, including Xeroderma Pigmentosum and the Cockayne syndrome (Jeppesen et al., 2011; McKinnon, 2013). To fully appreciate the biological contributions of DNA double-strand breaks in the mammalian brain, we must first expand our knowledge of the anatomical distribution of such a DNA damage signaling focus within the adult human nervous system and to compare whether its expression and distribution pattern is phenotypically similar to that of animal models of psychopathology. In this regard, a detailed characterization of DNA double- strand breaks in neurons and non-neuronal cells (i.e., glia) is lacking. To accomplish this experimental goal, we have carried out a detailed immunocytochemical localization of DNA double-strand breaks in human and rat brains using a primary antibody (53BP1, tumor 53-binding protein 1) whose specificity has been documented in several prior investigations, including the demonstration that exploration of novel environments causes DNA double-strand breaks in neurons of adult wild-type mice (Suberbielle et al., 2013). In addition, we tested whether DNA-strand breaks would also occur in specific immature and mature human cell lines (i.e., SH- SY5Y) to complement our studies of the adult nervous system. This particular (SH-SY5Y) cell line can be differ- entiated into functional mature neuronal phenotypes, including dopamine cells which are remarkably similar to primary mesencephalic neurons (Biedler et al., 1973). Thus, combining animal and human cell lines would be a meaningful way to exploit the unique viewpoint that each model yields for describing the localization of DNA double-strand breaks in the mammalian brain. http://dx.doi.org/10.1016/j.neuroscience.2015.01.027 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. * Corresponding author. Tel: +1-516-686-3806; fax: +1-516-686- 1454. E-mail address: torresg@nyit.edu (G. Torres). Abbreviations: ATM, ataxia telangiectasia, mutated; EDTA, ethylenediaminetetraacetic acid; GAD-67, glutamic acid decarboxylase 67; GFAP, glial fibrillary acidic protein; KPBS, potassium phosphate buffer saline; NHEJ, non-homologous end- joining. Neuroscience 290 (2015) 196–203 196