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 radiations found in space (protons, 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 space flight 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 proposal will evaluate how low dose proton exposure will impact 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. Three aims are proposed:

Specific Aim 1: To determine if/how low dose proton irradiation elicits radioadaptive changes in neural precursor cells from the CNS that depend on oxidative stress.

Specific Aim 2: To determine the acute dose response of satellite cells to proton irradiation with respect to survival, phenotypic fate and oxidative stress.

Specific Aim 3: Elucidate how low dose irradiation impacts neurogenesis in mice expressing human catalase targeted to the mitochondria (MCAT).

In aims 1 and 2, a range of biochemical, oxidative, and immunohistochemical approaches will be used that involve the quantification of reactive oxygen and nitrogen species and the detection of oxidative damage in neural precursor and satellite cells. Fluorogenic dyes will be used in conjunction with FACS analysis and damage specific antibodies will be used for immunostaining of irradiated cells to detect and measure oxidative stress. Similar approaches will be used to determine the role of the mitochondria in oxidative processes and how such stress impacts differentiation of these multipotent cells. Lastly, we will utilize mice genetically modified to minimize mitochondrial hydrogen peroxide to determine how reducing intracellular oxidants impacts neurogenesis. Dual staining immunohistochemistry will be used to quantify the numbers of differentiated cell types in tissue sections derived from irradiated animals labeled with bromodeoxyuridine as a marker for proliferation. Completion of these studies will help establish whether adaptation occurs after low dose proton irradiation, and whether stem cells and oxidative stress represent logical targets for countermeasure strategies.

The key findings thus far are that at low doses of proton irradiation of 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 irradiation 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.

The main impact of these finding 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 towards 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 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, beginning in the early fall quarter at Loma Linda and continued when more irradiations can be scheduled as well as if scheduling permits at Brookhaven. Irradiation of MCAT 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.

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 wild type (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 RNS production in live cells. Changes in ROS and RNS following proton irradiation may prove be similar to what we have observed in either neural precursors exposed to the same irradiation at the same doses suggesting a common radioresponse pathway or 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.

Significant progress has been made with respect to becoming proficient at working with and maintaining mouse neural precursor cells (NPCs) as neurospheres. Also, generation of new primary culture neurospheres has been achieved for wild type and transgenic mice either from the early postnatal or the embryonic setting which will increase neurosphere yields.

Further, proficiency has been achieved at assaying for ROS, RNS and mitochondrial superoxide levels using flow cytometry. This technique has been used to examine ROS and RNS levels following low doses as well as low dose rates of proton irradiation on neural precursor cells. The ROS and RNS levels fluctuate more rapidly over time following a clinical radiotherapy dose rate whereas a lower dose rate elicits a detectable but subdued response. Superoxide levels along with other measures of mitochondrial function, apoptosis, survival, redox responsive signaling and phenotypic fate will be examined in the near future. Initial experiments also suggest that a lower dose rate priming dose may be more beneficial than a corresponding higher rate irradiation at the same total dose with respect to eliciting a radioadaptive response when given a subsequent high dose challenge. Clearly the differences in radiobiology of varying dose rates warrant further study. There were initial difficulties with the yields of satellite cells extracted from animals with respect to viable cell numbers. Discussions with experts in the field have been fruitful and reasonable numbers of these cells can now be isolated on a consistent basis. Gamma irradiation of these cells to examine ROS, RNS and superoxide levels have been performed with results similar to the neural precursor cells in terms of a fluctuating response for ROS and RNS. A gradual increase in superoxide levels is observed over time. Proton irradiation experiments on these cells will commence imminently.

MCAT mice have been acquired and are breeding so that sufficient numbers can be irradiated at the correct age so that statistical significance can be achieved for examining neurogenesis. Further, neurosphere cultures have been successfully prepared from these animals and are a viable option for use in future experiments.

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 proposal will evaluate how low dose proton exposure will impact 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:

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.

To determine the acute dose response of satellite cells to proton irradiation with respect to survival, phenotypic fate and oxidative stress.

Elucidate how low dose irradiation impacts neurogenesis in mice expressing human catalase targeted to the mitochondria.

In aims 1 and 2, a range of biochemical, oxidative and immunohistochemical approaches will be used that involve the quantification of reactive oxygen and nitrogen species and the detection of oxidative damage in neural precursor and satellite cells. Fluorogenic dyes will be used in conjunction with fluorescent activated cell cytometer/sorter analysis, and damage-specific antibodies will be used for immunostaining of irradiated cells to detect and measure oxidative stress. Similar approaches will be used to determine the role of the mitochondria in oxidative processes and how such stress impacts differentiation of these multipotent cells. Lastly, we will utilize mice genetically modified to minimize mitochondrial hydrogen peroxide to determine how reducing intracellular oxidants impacts neurogenesis. Dual staining immunohistochemistry will be used to quantify the numbers of differentiated cell types in tissue sections derived from irradiated animals labeled with bromodeoxyuridine as a marker for proliferation. Completion of these studies will help establish whether adaptation occurs after low dose proton irradiation, and whether stem cells and oxidative stress represent logical targets for countermeasure strategies.