Ionizing Radiation Causes DNA Demethylation in Mouse Brain Darryl S. Watkins, Marc S. Mendonca4, Amy Lossie2, and (Feng C. Zhou1, 3,5). 1Department of Anatomy and Cell Biology, IU School of Medicine; 2Department of Animal Sciences, Purdue University; 3Department of Psychology, IUPUI; 4Department of Radiation Oncology, IU School of Medicine; 5Stark Neuroscience Research Institute, Indianapolis, IN

Ionizing Radiation Ionizing radiation (IR) has enough energy to liberate electrons from atoms or molecules May lead to tissue damage and DNA damage Although potentially hazardous, IR is one of the most common treatments for cancer. (Energy Library, 2010)

Ionizing Radiation and the Brain IR has been known to cause arresting of adult neurogenesis (Drew, M. et al., 2010) IR induced dementia have varied between 11% in one-year brain cancer survivors to 50% in those surviving two years (International Radiosurgery Association, 2011) IR is a known external environmental stressor (Shumei, M. et al., 2010) How fractionated and single dosages of IR compromises neuronal formation and the other deleterious affects on the brain are poorly understood. Epigenetics?

What is the role of Epigenetics ? Chemical coding that can regulate gene transcription without changes to DNA sequence DNA methylation 5-methylcytosine (5mC) 5-hydroxymethylcytosine(5hmC) Me Ac Crucial for embryogenesis & cellular differentiation In our lab it is have been demonstrated that any neuron from embryonic to adult stages go through DNA methylation program (DMP) (Chen et al. 2013) Sha, K. and Boyer, L. A. (2009)

Experimental Design C57/BL6 Postnatal Day P(37) mice Treatments lasted 2 weeks Low to Mild therapeutic doses of IR 0.5 Gy exposed 4 times (2.0 Gy), once a week with 4 day intervals 1.5 Gy exposed 3 times (4.5 Gy), once a week with 5 day intervals 3.0 Gy exposed 2 times (6.0 Gy), once a week with 5 day intervals 4.5 Gy exposed once All animals were anesthetized (including controls) with a ketamine cocktail: ketamine (100-200 mg/kg) + Xylazine (5-16 mg/kg)

Hippocampus Anatomy and Regions  DG- Dentate Gyrus CA- Cornu Ammonis Sub- Subiculum MEA- Medial Entorhinal Cortex LEA- Lateral Entorhinal Cortex (Sugar et al., 2011)

Demethylation of Ventral Hippocampus 5mC 5hmC DG Decrease of epigenetic marker 5mC and 5hmC in ventral hippocampus

Epigenetic Profile Decreased in Cingulate Cortex **** *** ** * Control 0.5 Gy X 4 5mC 5mC ★ 5hmC 5hmC ★ Cingulate cortex displayed demethylation as well as a decrease in DMNT1 activity and H3K9ac N of 4 p<0.05 * p<0.01 ** p<0.001 *** p<0.0001 ****

5mC within the Subgranule Zone Control 0.5X4 1.5X3 5mC SGZ DG *** **** Decrease of epigenetic marker 5mC in subgranule zone (SGZ) N of 4 p<0.001 *** p<0.0001 ****

Adult Neurogenesis Occurs in only two regions of the brain Dentate Gyrus Olfactory bulb IR arrest neurogenesis Frequency and dosage dependent Control **** 0.5X4 N of 4 1.5X3 p<0.0001 ****

Volume Decrease in Dentate Gyrus Control 0.5X4 Decrease in size of DG arm both upper and lower No change in brain weight Upper Arm DG Lower Arm **** **** *** p<0.001 *** p<0.0001 **** N of 4

Conclusion Demethylation in the hippocampus occurred more frequently throughout ventral regions of the hippocampus than the dorsal regions The epigenetic profile of the cingulate cortex was compromised Fractionated exposure (0.5 Gy X 4 ) had a more devastating affect on neurogenesis and the cingulate cortex than any higher fractionated doses and single doses in this study Hippocampal regions (subgranule zone, ventral dentate gyrus), and cortical region the cingulate cortex appear to be more vulnerable to radiation

Discussion It has been suggested that the dorsal and ventral hippocampal regions have distinct differences in functionality, here we show qualitatively that the ventral hippocampal regions exhibit a greater degree of demethylation. Radiation reduced new cell production and inhibited epigenetic programming suggesting a novel epigenetic mechanism for the cellular outcomes of ionizing radiation. Radiation decreased the expression of Ki-67 in the hippocampus, indicating that ionizing radiation compromises adult neurogenesis; however we show the magnitude of the arrest is dosimetric and frequency dependent, this may be due to cellular recovery and repair processes being disrupted. The hippocampus and the cingulate cortex are part of the limbic system, we have shown that both the hippocampus and cingulate cortex are affected by radiation, the limbic system may be more vulnerable to radiation.

Acknowledgements Zhou Lab Mendonca Lab Lossie Lab Funding Dr. Feng C. Zhou Dr. Chiao-Ling Dr. Yuanyuan Chen Marisol Resendiz Mendonca Lab Dr. Marc S. Mendonca Helen Chin-Sinex Lossie Lab Dr. Amy Lossie Funding Indiana University Collaborative Research Grant (IUCRG) W. M Keck Foundation Diversity Scholars Research Program (DSRP) Undergraduate Research Opportunity Program (UROP) Special Thanks Louis Stokes Midwest Center of Excellence

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