Two aims are proposed: aim 1 is to test the experiment in the Biosensor hardware to prepare for a possible lunar lander mission in the future and aim 2 is to use ground-based studies to determine whether SYN or A53T-SYN toxicity is increased by simulated lunar radiation or simulated lunar gravity. The Biosensor hardware has been tested for toxicity studies using yeast and the expression of SYN in Saccharomyces cerevisiae is an established model of Parkinson’s disease. Low SYN levels in yeast are not toxic, but high levels induce apoptosis and Reactive Oxidative Species (ROS) accumulation. We will generate wild-type and RAD51 deleted Saccharomyces cerevisiae strains that can be induced to express SYN or A53T-SYN at low levels by red light. RAD51 deleted yeast are more radiosensitive than wild-type yeast and will increase the sensitivity of detection of SYN toxicity generated by radiation/oxidative stress exposure. Genes known to reduce SYN toxicity in yeast include the gene encoding manganese superoxide dismutase (SOD2), an enzyme that detoxifies superoxide radicals in mitochondria. We will therefore also develop strains that overexpress SOD2 in combination with SYN to determine whether SYN toxicity can be rescued by quenching superoxide radicals. The Biosensor hardware can determine toxicity by the Alamar Blue viability assay, and growth by culture turbidity. We will use these assays in aims 1 and 2 to determine SYN toxicity as well as colony-forming ability in the ground-based studies in aim 2. In aim 2, simulated lunar gravity will be achieved using the random positioning machine (RPM 2.0) at Kennedy Space Center and simulated lunar radiation exposure will be performed at Brookhaven. Dr. Chancellor, a team member, has developed techniques to recreate the polyenergetic radiation spectrum for ground-based studies of galactic cosmic rays, and will use measurements scheduled to occur in early 2022 on the lunar surface to guide exposure of the cultures at Brookhaven in aim 2.

From the work in this proposal, we will be able to study SYN toxicity. We hypothesize that expressing SYN or A53T-SYN in yeast will increase toxicity to simulated lunar radiation or simulated lunar gravity, and toxicity will be greater in yeast expressing A53T-SYN. Toxicity will be decreased by overexpressing SOD2, an enzyme known to protect against SYN toxicity. Understanding how the stresses of living on the moon can enhance SYN toxicity is significant to humans thriving in deep space as extended space missions on the moon could enhance neurodegeneration in astronauts. Knowing the risks will allow NASA to establish countermeasures to protect astronauts on future deep space missions.

Budding yeast (brewer’s or baker’s yeast) is often used as a model to study basic mechanisms that also operate in human cells, because many basic cell processes are the same, but genetic modification and analysis are straightforward and rapid. We are preparing to test whether specific alterations to the yeast genetic makeup make them more or less sensitive to radiation-induced oxidizing conditions. The Parkinson's-related protein, synuclein, is normally present in human cells, but not yeast cells. We have used recombinant DNA methods to alter the yeast to synthesize this protein under conditions we impose. During this year of the project, we constructed a set of genetically modified yeast that will allow us to ask two questions:

-Does the presence of synuclein increase sensitivity to (death from) radiation?

-Do increased levels of a conserved antioxidant protein protect cells (with or without synuclein protein) from radiation effects?

We had, in the previous year, made a set of strains for this purpose, but found that these yeast strains were unable to survive the drying process needed to eventually be tested in lunar or space conditions. Therefore, we identified a better genetic makeup for this purpose and recreated our set of strains using this genetic makeup. Our testing is upcoming for visualizing yeast synthesis of synuclein, as well as testing the effects on radiation survival of making this protein.

Two aims are proposed: aim 1 is to test the experiment in the Biosensor hardware to prepare for a possible lunar lander mission in the future and aim 2 is to use ground-based studies to determine whether SYN or A53T-SYN toxicity is increased by simulated lunar radiation or simulated lunar gravity. The Biosensor hardware has been tested for toxicity studies using yeast and the expression of SYN in Saccharomyces cerevisiae is an established model of Parkinson’s disease. Low SYN levels in yeast are not toxic, but high levels induce apoptosis and Reactive Oxidative Species (ROS) accumulation. We will generate wild-type and RAD51 deleted Saccharomyces cerevisiae strains that can be induced to express SYN or A53T-SYN at low levels by red light. RAD51 deleted yeast are more radiosensitive than wild-type yeast and will increase the sensitivity of detection of SYN toxicity generated by radiation/oxidative stress exposure. Genes known to reduce SYN toxicity in yeast include the gene encoding manganese superoxide dismutase (SOD2), an enzyme that detoxifies superoxide radicals in mitochondria. We will therefore also develop strains that overexpress SOD2 in combination with SYN to determine whether SYN toxicity can be rescued by quenching superoxide radicals. The Biosensor hardware can determine toxicity by the Alamar Blue viability assay, and growth by culture turbidity. We will use these assays in aims 1 and 2 to determine SYN toxicity as well as colony-forming ability in the ground-based studies in aim 2. In aim 2, simulated lunar gravity will be achieved using the random positioning machine (RPM 2.0) at Kennedy Space Center and simulated lunar radiation exposure will be performed at Brookhaven. Dr. Chancellor, a team member, has developed techniques to recreate the polyenergetic radiation spectrum for ground-based studies of galactic cosmic rays, and will use measurements scheduled to occur in early 2022 on the lunar surface to guide exposure of the cultures at Brookhaven in aim 2.

From the work in this proposal, we will be able to study SYN toxicity. We hypothesize that expressing SYN or A53T-SYN in yeast will increase toxicity to simulated lunar radiation or simulated lunar gravity, and toxicity will be greater in yeast expressing A53T-SYN. Toxicity will be decreased by overexpressing SOD2, an enzyme known to protect against SYN toxicity. Understanding how the stresses of living on the moon can enhance SYN toxicity is significant to humans thriving in deep space as extended space missions on the moon could enhance neurodegeneration in astronauts. Knowing the risks will allow NASA to establish countermeasures to protect astronauts on future deep space missions.