Report on “Radiation Disaster Recovery Studies
Course: Radiation Disaster Medicine
Name: Silvia Natsuko Akutsu•Regarding “Radiation Disaster Recovery Studies”
The “Phoenix Leader Education Program (Hiroshima Initiative) for Renaissance fromRadiation Disaster” was created as a “Program for Leader Graduate Schools” by theMinister of Education, Culture, Sports, Science and Technology of Japan (MEXT) with aninterdisciplinary education to build and cultivate experts in Radiation Disaster Recovery
Studies in a global system.1
Japan had the misfortune to come up against nuclear disaster and incidents; Hiroshimaand Nagasaki atomic bombs (1945), Tokaimura nuclear accident (1999) and Fukushima
Daiichi Nuclear Power Plant (2011). However, those incidents made possible theconcentration of expertise in medical, environmental and social radiation fields. In thePhoenix Program, I belong to the “Radiation Disaster Medical course”, where I had
opportunity to learn with an interdisciplinary broad view of how to recover fromradiation disaster. Mainly, the focus is having the ability to record the lessons learned
documented from previous incidents to avoid and manage future radiation disasters. 1
Phoenix Program has national collaborations, such as Fukushima University, Fukushima
Medical University, Tohoku University, Nagasaki University, Radiation Effects Research
Foundation and the National Institute of Radiological Sciences. In addition, someinternational collaborations, including International Atomic Energy Agency (IAEA),
REAC/TS and Universities. Those links made possible a visiting of expertise to Hiroshima
University and opportunities for me to go to those facilities or institutions for fieldwork,internship, symposium, conference and training courses. Then, the exchange of scientificknowledge, valuable experiences and fruitful discussions enable to develop me as aperson, researcher and future leader for radiation recovery process. 1
During the four years of the doctoral program, I have several lectures in Radiation
Disaster Recovery, special subjects, common course work, fieldwork and internship thatbrought me a broad knowledge. In addition, I could focus in my research theme tounderstand the risks and genetic damage of radiation to the human body.
The studies of Atomic Bomb Casualty Commission (1950)2 reported that the Japaneseatomic bomb survivors who were exposed to the ionizing radiation (IR) in vitro had
affected their brain development. The mental retardation and small head size
(microcephaly) were shown in those individuals. There were also an increase of perinatal
loss, miscarriage, anencephaly and death during infancy due to radiation exposure.
Microcephaly was found in those who were exposed to IR in utero at 8 to 15 weeks ofgestation3. The International Commission on Radiological Protection (ICRP)-60
recommends that pregnant women with occupational radiation exposure should keep
dose below 1mGy due to a high sensitivity of fetus to IR4. The embryonic brain is very
vulnerable to IR. There were several studies in mouse fetus exposure to IR that showedreduction of the brain size formation in a dose-dependent manner, due to a radiationinduces apoptosis of neural progenitors and development of supernumerary
centrosomes causing mitotic catastrophe5.
In my doctoral study, I attempted to identify the microcephaly causing mutations inJapanese patients by whole exome sequencing analysis in order to elucidate thepathological mechanism of microcephaly.
References
1. http://phoenixprogramlp.hiroshima-u.ac.jp/en/
2. http://www.rerf.jp/radefx/genetics_e/birthdef.html
3. Committee on the Biological Effects of Ionizing Radiation (BEIR V), Health Effects ofExposure to Low Levels of Ionizing Radiation: BEIR V. National Research Council ISBN:
0-309-58970-3, 436 pages, 6x9, (1990).
4. ICRP, 1991. 1990 Recommendations of the International Commission onRadiological Protection. ICRP Publication 60. Ann. ICRP 21 (1-3).
5. Shimada M., Matsuzaki F., Kato A. et al. Induction of Excess Centrosomes in Neural
Progenitor Cells during the Development of Radiation- Induced Microcephaly. PLoSOne. 2016.
•Title of Doctoral Thesis:“PLK1-mediated phosphorylation of WDR62/MCPH2 ensures proper mitotic spindleorientation” (PLK1 による WDR62 のリン酸化を介した細胞分裂軸制御機構).
•Summary of Doctoral Thesis
Introduction
The primary microcephaly (MCPH) is an autosomal recessive neurodevelopmental
disorder characterized by reduction of occipitofrontal head circumference at birth. The
MCPH gene products localize to centrosome organelles, which regulates the microtubule(MT) organization during cell division and chromosome segregation. There are about 13
genes described in primary microcephaly development and implicated in spindle poleorientation control. However, the molecular mechanism remains unclear. In this study, I
identified compound heterozygous (missense and non-sense) mutations ofWDR62/MCPH2 gene in two siblings with microcephaly in Japanese family by wholeexome sequencing. Previous studies demonstrated that WDR62 localizes to mitotic
centrosomes to assemble the MT composed of spindles, and that Polo-like kinase 1(PLK1) is a player in stabilizing spindle orientation through astral MT formation. Here, I
demonstrated that PLK1 phosphorylates WDR62 at S897 to develop astral MT, which
stabilize the spindle orientation parallel to the substratum, resulting in symmetrical cell
division.

2Methods and Results
To better understand the role of WDR62 protein, we introduced CRISPR/Cas9 expression
vector and ssODN with WDR62 c.731 C>T variant (missense mutation), silent mutation
mediated BamHI restriction enzyme site into HCT116 cell lines to generate WDR62 invitro model cells. The mRNA and protein levels of WDR62 were significantly reduced inthose cells. The immunostaining analysis with anti-pericentrin and alpha-tubulin
antibodies of WDR62 mutant cells detected a bipolar spindle mis-orientated by different
confocal z-section planes and a significant reduction of astral microtubule formation. Thesame phenomena were observed in WDR62S244L/S244L HEK293T cells generated by thesame procedure. These results indicated that WDR62 regulates the correct spindleorientation by promoting astral MT assembly.
The immunoprecipitation analysis demonstrated that PLK1 physically interacts with
WDR62 in a Polo-box domain dependent manner. According to the consensus
phosphorylation motifs of PLK1, I constructed the same AcGFP tagged alaninesubstituted mutants of WDR62 (T575A, S897A, S987A and S1038A), in order to identify
the essential residue on WDR62 for PLK1 dependent spindle orientation. The
introduction of S897A mutant did not rescue the proper spindle orientation in WDR62
null cells. To confirm whether this residue is indeed the site of PLK1 phosphorylation, I
generated anti-phospho WDR62(S897) polyclonal antibody and performed an in vitro
kinase assay. The results confirmed that PLK1 phosphorylate WDR62 at Ser897.
Moreover, exogenous WDR62 S897E enable to maintain the spindle orientation andastral MT development in the WDR62+/+ cells treated by PLK1 inhibition.
Conclusion
I demonstrated that the whole exome analysis of Japanese MCPH family is a useful tool
to identify novel mutations of the WDR62/MCPH gene. The use of genome editing
technology to generate human disease model cell lines is important to understand thebiological mechanism. The PLK1 phosphorylates WDR62 at S897 to assemble astral MTand ensure the proper spindle orientation.

•Other theses published in academic research journals

1. Tatsuo Miyamoto, Silvia Natsuko Akutsu, Shinya Matsuura. Update summary of genome
editing technology in human cultured cells linked to human genetics studies. Journal ofHuman Genetics. 2017, in press.
2. Ekaterina Royba, Tatsuo Miyamoto, Silvia Natsuko Akutsu, Kosuke Hosoba, Hiroshi Tauchi,
Yoshiki Kudo, Satoshi Tashiro, Takashi Yamamoto, Shinya Matsuura. Evaluation of ATM
heterozygous mutations underlying individual differences in radiosensitivity using
genome editing in human cultured cells. Scientific Reports. 20;7(1):5996, 2017.

33. Shinya Matsuura, Ekaterina Royba, Silvia Natsuko Akutsu, Hiromi Yanagihara, Hiroshi Ochiai,
Kudo Y, Satoshi Tashiro, Tatsuo Miyamoto. Analysis of individual differences inradiosensitivity using genome editing. Annals of the ICRP. 45: 290-296, 2016.

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