Mechanistic analysis of radiation-induced microcephaly and cognitive impairment in mice

Mbouombouo Mfossa André

Promoter

Huylebroeck Danny, (KUL), danny.huylebroeck@med.kuleuven.be

SCK•CEN Mentor

Quintens Roel
roel.quintens@sckcen.be
+32 14 33 28 37

SCK•CEN Co-mentor

Benotmane Rafi
rafi.benotmane@sckcen.be
+32 14 33 27 31

Expert group

Radiobiology

PhD started

2015-10-01

Short project description

Radiation exposure of fetuses, especially between weeks 8 to 15 of pregnancy, has been shown to result in neurological defects such as microcephaly, seizures, mental retardation and decreased intelligence. Microcephaly is a congenital disorder which is defined in humans as a brain size at least two standard deviations below the average for age and sex, and has been shown to be related to the degree of radiation-induced mental retardation (Schull and Otake, Teratology 1999). The occurrence of these neurological defects is one of the main reasons why radiation exposure (both for therapy as well as diagnosis) of pregnant women is mostly discouraged.

We and others have been able to recapitulate some of these effects in mice after X-ray irradiation of mice during pregnancy. In particular, we found that prenatal irradiation induced microcephaly (= reduced brain size in mice), while behavioral tests showed cognitive impairments (Verreet et al., accepted and under revision). Furthermore, we have discovered a number of novel target genes of the tumor suppressor protein p53, which are activated within hours after radiation exposure in the embryonic mouse brain (Quintens et al., accepted and under revision). p53, also known as the guardian of the genome, is a critical regulator of the cellular response to radiation by activating its target genes which are responsible for cell cycle arrest, DNA damage repair and apoptosis (cell death). However, we have strong indications that some of the novel p53 target genes are particularly important for neuron differentiation and normal brain development. Our recent findings further indicated that the early, p53-dependent gene activation after radiation exposure may be responsible for the late effects. If so, we hypothesize that inhibition of p53, either genetical or pharmacological, may prevent these detrimental radiation-induced effects.

Objective

This project builds further on previously gathered knowledge within the Radiobiology Unit and aims to establish a mechanistic link between early effects of acute radiation exposure of mouse embryos and late effects in adult mice. The objectives of the project will be to evaluate:

(1) the exact role of p53 and some of its targets in the development of radiation-induced microcephaly;

(2) whether this microcephaly is related to the observed cognitive defects at adult age;

(3) whether these morphological and cognitive defects may be prevented by pharmacological inhibition of p53 at the time of radiation exposure.

The first two aims will be investigated using neuron-specific p53 knock-out mice in comparison with their wild-type littermates. These mice will be bred at the new SCK•CEN animal facility, and will have an inactive p53 gene, specifically in neurons of the cerebral cortex. Using magnetic resonance imaging and behavioral tests, we will evaluate whether they also develop microcephaly and behavioral defects after prenatal radiation exposure. Possible mechanisms will be investigated using immunohistochemistry for apoptosis, neuronal differentiation, and neuronal migration.

Furthermore, we will investigate the function of some recently discovered p53 targets (Quintens et al., accepted and under revision) using primary neuronal cell cultures (in vitro), or zebrafish embryos (in vivo). We will investigate their function in the DNA damage response (cell cycle, DNA repair, apoptosis), neuronal differentiation and brain development, both under normal and DNA damaging conditions. For this, we will silence these genes using either small interfering RNA (in vitro) or morpholino (in vivo) technology.

For the third aim, we will treat pregnant mice with a pharmacological p53 inhibitor, such as alpha-pifithrin (PFT), at the time of irradiation. PFT has already been shown to reduce the genotoxic side effects of radiation treatment on healthy tissue in mice. As for the brain-specific p53 knock-outs, we will firstly investigate the brain size and behavior of the progeny of PFT-treated, irradiated mice. Furthermore, we will assess whether PFT-treated mice have a greater risk for developing radiation-induced cancer in order to evaluate the suitability of PFT as a potential drug for pregnant women undergoing radiation therapy. These results might have important implications for radiation protection of pregnant women and their unborn babies.