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Ionizing Radiation Causes DNA Demethylation in Mouse Brain

This study explores the effects of ionizing radiation on DNA demethylation in the mouse brain, specifically the hippocampus and cingulate cortex. Fractionated exposure had a more devastating effect on neurogenesis and the cingulate cortex compared to higher fractionated doses and single doses.

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Ionizing Radiation Causes DNA Demethylation in Mouse Brain

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  1. 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

  2. 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)

  3. 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?

  4. 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) • 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) Ac Me Sha, K. and Boyer, L. A. (2009)

  5. 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)

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

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

  8. 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 ****

  9. 5mC within the Subgranule Zone 5mC DG DG DG SGZ SGZ SGZ SGZ SGZ SGZ • Decrease of epigenetic marker 5mC in subgranule zone (SGZ) *** **** N of 4 Control 0.5X4 1.5X3 p<0.001 *** p<0.0001 ****

  10. 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 ****

  11. **** 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

  12. 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

  13. 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.

  14. Acknowledgements Zhou Lab 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

  15. References • The Energy Library, 2010. “Ionizing Radiation”www.theenergeylibrary.com • International Radiosurgery Association, 2011 “Metastatic Brain Tumors”www.irsa.org • Chen, Y., et al. (2013). “DNA Methylation Program in Developing Hippocampus and Its Alteration by Alcohol.” PLoS ONE 8(3): e60503 • Drew, M., et al. (2010). “Arrest of adult hippocampal neurogenesis in mice impairs single-but not multiple-trial contextual fear conditioning.” Behav Neurosci 124(4): 446-54 • Sha, K. and Boyer, L. A. The chromatin signature of pluripotent cells (May 31, 2009), StemBook, ed. The Stem Cell Research Community, StemBook, doi/10.3824/stembook.1.45.1. http://www.stembook.org/node/585 • Shumei, M., et al. (2010). “Low-dose radiation-induced responses: Focusing on epigenetic regulation.” Int J Of Rad Biol, 86(7), 517-528. • Sugar, J., et al., (2011). “The retrosplenial cortex: intrinsic connectivity and connections with the (para)hippocampal region in the rat. An interactive connectome.” Frontiers in Neuroinformatics 5: 7.

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