Multipotent stem cells exist throughout the body and maintain the health of a given tissue by replenishing cells lost or damaged throughout life. These unique cells retain the capability to proliferate, migrate and differentiate to return functionality to a tissue compromised by age, disease or a variety of genotoxic/cytotoxic stressors such as ionizing radiation.

Our laboratory has accumulated a wealth of data showing that relatively low doses of radiation found in space, such as protons and heavy ions, can compromise the function of specific tissues and organs through a variety of biochemical mechanisms that involve oxidative stress. While the details vary, oxidative stress that persists after irradiation has been shown to reduce bone density, impair muscle regrowth and inhibit neurogenesis. These acute, functional decrements could severely compromise the capability of astronauts to complete mission-critical tasks, where accelerated muscle fatigue and confusion caused by muscle and neural stem cell loss could result from relatively mild exposures to the space radiation environment. Each of these debilitating conditions can, in part, be caused by radiation-induced oxidative stress, a condition we have shown to adversely impact the function of tissue-specific stem cell populations. Our data reinforces the importance of these multipotent cells and suggests they represent critical targets for countermeasures aimed at ameliorating radiation and spaceflight-related sequelae.

To expand on our past space-related research, we propose in vitro and in vivo experiments to determine if/how relatively low doses of protons can initiate radioadaptive changes in stem cells found in the brain and skeletal muscle. Our project is evaluating how low-dose proton exposure impacts stem cell proliferation, differentiation and survival and whether such endpoints can be altered by prior exposure. We hypothesize that stem cells incurring particle traversals during spaceflight can undergo adaptive changes that attenuate their response to subsequent larger exposures (e.g., from a solar flare). We will also investigate whether the mechanistic basis of adaptation involves changes in the redox environment such as oxidative stress.

Specific Aims

1. To determine if/how low-dose proton irradiation elicits radioadaptive changes in neural precursor cells from the central nervous system that depend on oxidative stress.
2. To determine the acute dose response of satellite cells to proton irradiation with respect to survival, phenotypic fate and oxidative stress.
3. Elucidate how low-dose irradiation impacts neurogenesis in mice expressing human catalase targeted to the mitochondria.

Key Findings
Thus far, the key findings are that at low doses of proton irradiation of neutron proportional counters (NPCs), ROS and RNS levels fluctuate over time, and these fluctuations are more rapid when the NPCs are exposed to a higher dose rate such as that of a clinical setting, as opposed to a lower dose rate such as that which is more likely to mimic the space environment. These fluctuations were observed in satellite cells exposed to gamma irridation for ROS and RNS levels but mitochondrial superoxide levels showed a more prolonged and steady increase over time. In addition to low dose, low dose rate may be a key component of radioadaption in NPCs when examining survival and cell proliferation following low dose or dose rate priming and a high dose challenge.

Plans for Coming Year
The main impact of these findings is that the aims will be modified to examine the effect of both low dose and low dose rate proton irradiation on the NPCs and satellite cells. The plan for the coming year is to continue the dose and dose rate studies in the NPCs but with a move toward examining mitochondrial function, survival and apoptosis, redox sensitive signal and phenotypic fate. Adaptive responses will be examined at the low doses and low dose rates which can be achieved at Loma Linda University and potentially at even lower dose rates at Brookhaven National Lab. For the satellite cells, proton irradiation experiments involving ROS, RNS, mitochondrial function, survival and apoptosis as well as phenotypic fate will be conducted throughout the year. They will begin in the early fall quarter at Loma Linda and will continue when more irradiations can be scheduled and if scheduling permits at Brookhaven. Irradiation of mitochondrion animals is currently scheduled for the fall quarter at Loma Linda, and the neurogenesis studies will occur in the following months. Subsequent irradiation dates will be scheduled as sufficient mouse numbers are generated. Neurosphere cultures can be generated from these animals and assayed as proposed for the wild type NPCs with respect to ROS, RNS and mitochondrial function.

Earth-based Applications of Research Project
Very little is known about the low dose and low dose rate radiobiology of neural precursors cells. These studies will yield important information regarding how this radio-response differs from the response to higher doses of ionizing radiation. Further, increased reactive oxygen species (ROS) can be damaging by challenging cellular repair and renewal processes, but it can also activate redox sensitive signaling pathways which may underlie an adaptive response in neural precursors. Our recent work has begun to elucidate the effectiveness of low dose rate irradiation on the radioadaption of neural precursor cells, and it may prove to be quite beneficial as these dose rates better simulate the space radiation environment. Knowledge gained from understanding the mechanisms of change in oxidative stress, mitochondrial function and redox sensitive signaling in these experiments will provide the rationale for potentially effective countermeasure design for both acute and chronic space radiation exposure.

Ionizing radiation significantly reduces dentate neurogenesis, and such changes are dose dependent and persistent. These effects are linked to alterations in the neurogenic microenvironment, including inflammatory changes and oxidative stress although the precise mechanisms responsible are not yet known. While ROS have often been considered to be hostile or destructive entities, they also have been shown to have beneficial effects, at least in part due to their role as signaling moieties. It has recently been shown that a persistent level of oxidative stress in extracellular superoxide dismutase knockout mice (EC-SOD KO) was associated with a lower baseline level of neurogenesis relative to wildtype (WT) mice. However, when those same mice were subjected to a modest dose of x-rays (5 Gy), there was no effect on neurogenesis in KO mice but a significant reduction in WT mice. Thus, we saw both negative (baseline neurogenesis) and positive (adaptive) effects in the EC-SOD KO mice, presumably as a result of mechanisms associated with redox balance.

Also, we have also been able to demonstrate the paradoxical nature of oxidative species in vitro using cultured neural precursor cells, where excess hydrogen peroxide reduces survival while excess superoxide increases survival. These paradoxical effects highlight the potential importance of adaptive responses in the context of the delicate balance in redox homeostasis, and how that may ultimately affect cell or tissue function. We believe that low doses of irradiation will elevate the level of oxidative stress in the neurogenic microenvironment and that this may have a beneficial effect when buffered by enhanced catalase activity with respect to dentate neurogenesis. These experiments can be conducted using a transgenic mouse with targeted expression of human catalase in the mitochondria (MCAT). Given the association between neurogenesis and cognitive function after irradiation, this modulation of oxidative stress via catalase could ultimately be developed into a countermeasure to maintain behavioral performance while engaged in space exploration.

With the capability to culture satellite cells in a multipotent state, we can now investigate the mechanistic details of ROS and reactive nitrogen species (RNS) production in live cells. Changes in ROS and RNS following proton irradiation may prove to be similar to what we have observed in either neural precursors exposed to the same irradiation at the same doses suggesting a common radio-response pathway or, the changes may prove to be quite different, suggesting a more cell-type specific response. Similarly, mitochondrial redox function via superoxide levels can be readily assayed in these. Understanding these effects of irradiation on myogenic oxidative stress, proliferation and differentiation will help to provide the basis in order to design countermeasures to maintain muscle function during space exploration.

Project Description
NASA Task Book Entry