OBJECTIVES:
NASA is preparing for space exploration beyond low earth orbit (LEO). Space exploration beyond LEO poses threats to astronauts’ mental and physical health due to chronic exposure to microgravity and space radiation. Exposure to space radiation has four primary biomedical risks: degenerative tissue effects, neurological effects, carcinogenesis, and acute radiation syndrome. Exposure to microgravity compounds these risks. Modifications to cellular and molecular mechanisms because of exposure to the space environment must be understood in order to mitigate the consequences.
Endogenous Retroviruses (ERVs) are genetic elements that comprise approximately eight percent of the human genome and are considered to play a major role in human evolution. ERVs are genomic instability markers that are maintained in a dormant state by epigenetic mechanisms. However, environmental stressors can inadvertently activate expression of ERVs. Activation of ERVs have been associated with the development of cancer, autoimmune diseases, and neurodegenerative disease in humans. ERV activation may have detrimental health effects on astronauts including biological, mental, and reproductive consequences. Astronauts are exposed to exogenous stressors, microgravity and space radiation, which can cause ERV activation through genomic modifications.
The goal of this experiment is to address if microgravity and radiation can influence expression of ERVs and retroviral elements in human stem/progenitor cells in vitro and in mouse tissues in vivo. This study has the following specific aims:
- Evaluate human cells grown in a microgravity environment for evidence of ERV activation.
- Evaluate mouse tissues that have been exposed to space radiation for evidence of ERV activation.
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APPROACH:
Bone marrow-derived mesenchymal stem cells (BM-MSCs) will be obtained and maintained under normal growth conditions. Cells under normal gravity will be plated on tissue culture flasks while cells in microgravity will be seeded onto Cytodex-3 microcarrier beads and then transferred to rotating wall vessels (RWV). Cell adhesion will be enhanced by allowing the cell-bead mixture to acclimate for two hours before rotation. Both the cells in normal gravity and microgravity will be cultured for 0, 6, 12, 24, 48, 72, 96, and 120 hours to determine if simulated microgravity causes kinetic changes. The chronic effects of microgravity will be determined by maintaining BM-MSCs under normal gravity and microgravity for two weeks, one month, and three months. The BM-MSCs that are exposed to acute and chronic microgravity exposure will undergo immunofluorescence microscopy and flow cytometry.
A bio-bank of male C57BL/6J mice tissues that have been exposed to different doses of gamma, 56F, or 16O radiation will be compared to control mice that were not exposed to radiation. The tissues to be tested will be representative of all major organ systems including the heart, lung, brain, spleen, lymph nodes, liver, kidney, intestines, and testes. The tissues will be assayed for ERVs and/or retrotransposable elements by immunohistochemistry. Immunohistochemistry screening will ensure the identification of potentially rare cell populations that express ERV RNAs. Duke University histology core facility will process and probe all tissues. The tissues will be intracellular stained with antibodies specific for double-stranded RNA. This will allow for the detection of ERV RNAs and RNAs from other retrotransposable elements.
RESULTS:
The experiment is in progress. Results will be available at a later date.
PLANNED SCHEDULE:
Specific Aim 1:
- Purchase Synthecon rotator and necessary reagents. (1st Quarter)
- Normal gravity and microgravity cells will be cultured for 0, 6, 2, 24, 49, 72, 96, and 120 hours. (1st Quarter)
- Bone-marrow derived mesenchymal stem cells (BM-MSCs) will be maintained under normal gravity and microgravity for two weeks, one month, and three months. (2nd and 3rd Quarter)
- Prepare BM-MSCs for flow cytometry and immunofluorescence microscopy. (2nd and 3rd Quarter)
- Electron microscopy (EM) ultrastructural studies (2nd and 3rd Quarter)
- Data assessment, manuscript generation, and application for follow on funding (3rd and 4th Quarter)
Specific Aim 2:
- Procurement of tissue samples from storage and provide tissue samples to histology core for sectioning and staining. (1st and 2nd Quarter)
- Analysis of tissue slides and EM ultrastructural studies (3rd Quarter)
- Data assessment, manuscript generation, and application for follow on funding (3rd and 4th Quarter)
Hematopoietic
RNA, endogenous retroviruses (ERV)-encoded, expression (Human)
RNA, endogenous retroviruses (ERV)-encoded, expression (Mice)
RNA, retrotransposable element (Human)
RNA, retrotransposable element (Mice)
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