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 RadiationIonizing Radiation

(Energy Library, 2010)

• 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.

Ionizing Radiation and the BrainIonizing 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 ?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

Sha, K. and Boyer, L. A. (2009)

•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)

• 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)

Experimental DesignExperimental Design

Hippocampus Anatomy and Regions

(Sugar et al., 2011)

DG- Dentate GyrusCA- Cornu AmmonisSub- Subiculum

MEA- Medial Entorhinal Cortex LEA- Lateral Entorhinal Cortex

Demethylation of Ventral Hippocampus5mC

5mC

5mC

5hmC

5hmC5hmC

DG

DGDG

DGDG

DG

Decrease of epigenetic marker 5mC

and 5hmC in ventral hippocampus

Epigenetic Profile Decreased in Cingulate Cortex

5mC

5hmC ****

******

********

**

*****

******

****

Control 0.5 Gy X 4

p<0.05 *p<0.01 **p<0.001 ***p<0.0001 ****

5mC

5hmC

Cingulate cortex displayed demethylation as

well as a decrease in DMNT1 activity and

H3K9ac

★

★

N of 4

5mC within the Subgranule Zone

*******

p<0.001 ***p<0.0001 ****

N of 4

Decrease of epigenetic marker 5mC

in subgranule zone (SGZ)

Control 0.5X4 1.5X3

5mC

SGZ

SGZ

SGZSGZ

SGZ

SGZ

DGDGDG

Adult Neurogenesis• Occurs in only two regions of the brain

• Dentate Gyrus• Olfactory bulb

• IR arrest neurogenesis• Frequency and dosage

dependent

********

****

N of 4

p<0.0001 ****

Control

1.5X3

0.5X4

Volume Decrease in Dentate Gyrus

Decrease in size of DG

arm both upper and lowerNo change in brain

weight

Upper Arm

Lower Arm

DG

Control

****

***

****

****

0.5X4

N of 4p<0.001 ***p<0.0001 ****

ConclusionConclusion•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

DiscussionDiscussion 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.

AcknowledgementsAcknowledgements

Zhou LabDr. Feng C. ZhouDr. Chiao-LingDr. Yuanyuan ChenMarisol Resendiz

Mendonca LabDr. Marc S. MendoncaHelen Chin-Sinex

Lossie LabDr. Amy Lossie

Funding•Indiana University Collaborative Research Grant (IUCRG)•W. M Keck Foundation•Diversity Scholars Research Program (DSRP)•Undergraduate Research Opportunity Program (UROP)

Special ThanksLouis Stokes Midwest Center of Excellence

ReferencesReferences

• 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.