Gamma and space irradiation, including 56Fe ions and protons, induce changes in DNA methylation. Of significance for this project, recent studies show that a second form of DNA methylation, termed 5-hyrdroxymethylation (5hmC) is abundant and stable in the brain. The ten eleven translocation (TET) enzymes convert 5mC to 5hmC, which likely play a role in gene regulation because 5hmC is often found at promoters of active genes. In contrast, 5mC is found at the promoters of inactive genes. Changes in 5mC, which are induced by environmental exposures including radiation and behavioral modification, play a role in processes including cancer and neurological disease. A similar role for 5hMC can be assumed due to its abundance in the brain, though 5hmC changes after radiation exposure and in neurological disease has not yet been elucidated. The investigators hypothesized that the detrimental effects of charged particles on hippocampus-dependent cognition are associated with Arc-related changes in the hippocampal networks involved in synaptic plasticity and memory. They further hypothesized that these changes are related to perturbation of 5mC and/or 5hMC in the hippocampus that affect Arc expression and expression of other learning and memory genes in the same signaling pathways and networks.
To test these hypotheses, investigators used the following specific aims:
In year two of the study, investigators finalized the analysis of the novel object recognition data of mice irradiated at BNL and tested 2 and 20 weeks after irradiation. The mice were all six-month-old male C57Bl6/J mice (no retired breeders). The doses of irradiation were: 56Fe (600 MeV/n; 0.1, 0.2, and 0.4 Gy); 28Si (600 MeV/n; 0.3, 0.6, and 0.9 Gy); and protons (150 MeV, 1 Gy). For each radiation condition and time point, 16 mice were cognitively tested. As last cognitive test, eight mice of each experimental radiation condition were exposed twice to the same environment and eight mice of each experimental radiation condition were exposed to two different environments for assessments of hippocampal network stability. In addition to these 16 mice, there were 5 cage controls.
For the novel object recognition test, the mice were habituated to an open field for three subsequent days. On day four, the mice were exposed to the open field containing two objects. On day five, the mice were exposed to the open field with two objects but one familiar object (present on day four) was replaced by a novel one. The time spent with each object was recorded by video and hand-scored by a researcher blinded to the treatment of the mice. The data show impairments in novel object recognition following 56Fe, 28Si, and proton irradiation.
Three days after the last water maze test day, exploration of identical or different environments was used to study the stability of hippocampal networks. Eight mice from each experimental radiation condition were placed individually into a novel environment (A) and allowed to explore for five minutes. Another eight mice of each experimental radiation condition explored environment A for 5 minutes, and 25 minutes later they were placed in a different environment (B), a circular arena 45 cm in diameter, and allowed to explore for 5 minutes (AB Paradigm). Following behavioral testing, the mice were sacrificed by cervical dislocation and the brains quickly removed. One hemisphere was quickly frozen for catFISH and fluorescence immunostaining. Using catFISH, Arc mRNA appear as discrete nuclear foci (recent transcription ~5-10 min), and/or as diffuse mRNA in the cytoplasm (earlier transcription). Nuclear and cytoplasmic Arc can be distinguished using intronic and full-length probe respectively labeled with digoxigenin or fluorescein. Arc staining was classified as: a) None (no Arc staining); b) Arc-foci (containing only the intranuclear foci transcript); c) Arc-cyto (containing only cytoplasmic staining); or d) both Arc-foci/Arc-cyto (containing both foci and cytoplasmic staining). Using the catFISH analysis, we can identify the population of neurons activated during the first (Arc-cyto), the second (Arc-foci), and both exploration sessions (Arc-foci/Arc-cyto) and if irradiation affects these parameters. Furthermore, because of sensitiveness of the catFISH technique, investigators are able to detect possible translocation problems in the movement of Arc mRNA from the nucleus to the cytoplasm. They can determine if the neurons responding (i.e. Arc+) to an initial experience (exploration of environment A) are the same neurons that respond to a second and identical experience as expected based on sham-irradiated animals (AA Paradigm), and if the hippocampal networks activated during exploration of the first environment are statistically independent from those activated by the exploration of a different environment (AB Paradigm). The data shows that 4 weeks following 56Fe and 28Si irradiation, the percentage of Arc-positive cells is not significantly different in the following exposure to the two distinct environments as compared to exposure to twice the same environment as seen in sham-irradiated mice.
Finally the omics-related changes in DNA methylation are tissue specific. Investigators compared DNA methylation levels and metabolomics in the hippocampus and left ventricle 20 weeks following proton irradiation (150 MeV, 1Gy). As expected, 5mC was enriched in poorly expressed regions of the genome. Tissue-dependent DNA methylation, as well as DNA methylation shared in the left ventricle and hippocampus, was revealed. The gene methylation data in the left ventricle reveal significant major pathway changes that are related to known pathophysiologic processes. There was also tissue-specifc radiation-induced changes in metabolomics and in immunoreactivity of the TET2 protein, a methylcytosine dioxygenase that catalyzes the conversion of methylcytosine to 5-hydroxymethylcytosine. These data are very encouraging and indicate that all proposed outcome measures are sensitive to detect effects of relative low dose irradiation at the two-week time point and some proposed outcome measures are sensitive to detect effects of relative low dose irradiation at the 20-week time point. These data are also encouraging as they indicate the anatomical specificity and networks involved in these effects.
No datasets exist for this study. A final report was archived.
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