NOTE: End date changed to 12/31/2021 per information in PI report (Ed., 8/19/21)

NOTE: End date changed to 9/30/2021 per F. Hernandez/ARC when PI became civil servant summer 2020 and project extended as internal project at NASA Ames; USRA grant 80NSSC20K0125 has official end date of 6/5/2020 (Ed., 9/2/2020)

We propose to utilize for the first time a novel high-throughput human tissue-on-a-chip model for 3D neuronal/astrocyte cultures. We will investigate the morphological and physiological outcomes as well as gene expression changes after simulated space radiation exposure (5-ion simulation of galactic cosmic rays, 500 mGy) and compare them to the responses to gamma radiation in order to establish the relative biological effectiveness. We will also evaluate the necessity and sufficiency of astrocytes in regulating radiation responses by establishing experimental models where astrocytes are either the only cell type that is irradiated, or the only cell type that escapes irradiation. Finally, we will test whether driving astrocytes more towards A1 (inflammation) or towards A2 (scarring) phenotypes may serve as countermeasures by reducing radiation-mediated neuronal damage.

In summary, we will examine whether astrocytes are necessary and sufficient to protect neurons from damage induced by simulated space radiation and evaluate their effectiveness as a target for further countermeasure development. We will also provide a proof of concept for human tissue-on-a-chip use for studying space radiation effects on the CNS.

Our proposal addresses the Part "B. Basic Investigations Opportunity for Investigators New to NASA" of the Appendix B. The new investigator (Principal Investigator) and experienced investigator (CoInvestigator/Institutional Principal Investigator) have combined expertise in space biology, neuroinflammation astrocyte functions, TGFbeta functions in regulating astrocyte phenotypes, and responses to particle radiation. Thus, we have the capacity to complete this 1-year project at NASA Ames and, given sufficient interest, could easily expand it to a) investigate the outcomes of combined exposures to microgravity, stress, and radiation, and b) design an in vivo follow-up study on astrocytic regulation of CNS responses to spaceflight stressors.

2. Scientific. We have discovered that astrocytes are particularly sensitive to ionizing radiation, especially components of galactic cosmic rays. In response to radiation, astrocytes increase oxidative stress and blood-brain barrier permeability and cause immune dysregulation, at least in part via interleukin-1 signaling. These results are important not only for deep space exploration, but also for understanding the outcomes of human central nervous system irradiation for therapeutic purposes (e.g., to treat brain tumors), and results indicate astrocytic interleukin-1 signaling as a potential target for countermeasure/therapeutic development.

NOTE: End date changed to 12/31/2021 per information in PI report (Ed., 8/19/21)

NOTE: End date changed to 9/30/2021 per F. Hernandez/ARC when PI became civil servant summer 2020 and project extended as internal project at NASA Ames; USRA grant 80NSSC20K0125 has official end date of 6/5/2020 (Ed., 9/2/2020)

We propose to utilize for the first time a novel high-throughput human tissue-on-a-chip model for 3D neuronal/astrocyte cultures. We will investigate the morphological and physiological outcomes as well as gene expression changes after simulated space radiation exposure (5-ion simulation of galactic cosmic rays, 500 mGy) and compare them to the responses to gamma radiation in order to establish the relative biological effectiveness. We will also evaluate the necessity and sufficiency of astrocytes in regulating radiation responses by establishing experimental models where astrocytes are either the only cell type that is irradiated, or the only cell type that escapes irradiation. Finally, we will test whether driving astrocytes more towards A1 (inflammation) or towards A2 (scarring) phenotypes may serve as countermeasures by reducing radiation-mediated neuronal damage.

In summary, we will examine whether astrocytes are necessary and sufficient to protect neurons from damage induced by simulated space radiation and evaluate their effectiveness as a target for further countermeasure development. We will also provide a proof of concept for human tissue-on-a-chip use for studying space radiation effects on the CNS.

Our proposal addresses the Part "B. Basic Investigations Opportunity for Investigators New to NASA" of the Appendix B. The new investigator (Principal Investigator) and experienced investigator (CoInvestigator/Institutional Principal Investigator) have combined expertise in space biology, neuroinflammation astrocyte functions, TGFbeta functions in regulating astrocyte phenotypes, and responses to particle radiation. Thus, we have the capacity to complete this 1-year project at NASA Ames and, given sufficient interest, could easily expand it to a) investigate the outcomes of combined exposures to microgravity, stress, and radiation, and b) design an in vivo follow-up study on astrocytic regulation of CNS responses to spaceflight stressors.

2. Scientific. We have discovered that astrocytes are particularly sensitive to ionizing radiation, especially components of galactic cosmic rays. In response to radiation, astrocytes increase oxidative stress and blood-brain barrier permeability and cause immune dysregulation, at least in part via interleukin-1 signaling. These results are important not only for deep space exploration, but also for understanding the outcomes of human central nervous system irradiation for therapeutic purposes (e.g., to treat brain tumors), and results indicate astrocytic interleukin-1 signaling as a potential target for countermeasure/therapeutic development.

2. ASTROCYTES EXACERBATE IMMUNE DYSFUNCTION CAUSED BY SIMULATED DEEP SPACE RADIATION. One of the main functions of astrocytes is regulating CNS immune responses in health and disease. Therefore, we used astrocyte and mixed endothelial cell-astrocyte neurovascular models to investigate the effects of simulated deep space radiation on astrocyte immunoregulatory characteristics. Secreted immune cytokines revealed a dysfunction in multiple pathways in astrocytes alone and in neurovascular models. Specifically, our results suggest that radiation disrupts astrocyte immunoregulatory functions, which exacerbates the effects of radiation on endothelial cell immune cytokine production, leading to neurovascular leakiness, increased inflammation and immune dysfunction.

3. ONGOING INVESTIGATIONS: NEUROVASCULAR RESPONSES TO 5-ION SIMPLIFIED SIMULATED GALACTIC COSMIC RAYS. Finally, during NASA Space Radiation Laboratory (NSRL) 21B campaign (June 2021), we exposed astrocyte-endothelial cell and astrocyte-neuron-endothelial cell combinations to 5-ion simplified simulated GCRs (SimGCRSim) and repeated 600 MeV/n 56Fe exposure to validate the results obtained during the previous irradiation experiment. Live imaging of neurovascular permeability was performed and samples were collected at different time points days post irradiation. Supernatants were frozen for oxidative stress and immune cytokine measurements, and neurovascular organ models were fixed and stained for immunohistochemistry. All sample analysis is ongoing.

We propose to utilize for the first time a novel high-throughput human tissue-on-a-chip model for 3D neuronal/astrocyte cultures. We will investigate the morphological and physiological outcomes as well as gene expression changes after simulated space radiation exposure (5-ion simulation of galactic cosmic rays, 500 mGy) and compare them to the responses to gamma radiation in order to establish the relative biological effectiveness. We will also evaluate the necessity and sufficiency of astrocytes in regulating radiation responses by establishing experimental models where astrocytes are either the only cell type that is irradiated, or the only cell type that escapes irradiation. Finally, we will test whether driving astrocytes more towards A1 (inflammation) or towards A2 (scarring) phenotypes may serve as countermeasures by reducing radiation-mediated neuronal damage.

In summary, we will examine whether astrocytes are necessary and sufficient to protect neurons from damage induced by simulated space radiation and evaluate their effectiveness as a target for further countermeasure development. We will also provide a proof of concept for human tissue-on-a-chip use for studying space radiation effects on the CNS.

Our proposal addresses the Part "B. Basic Investigations Opportunity for Investigators New to NASA" of the Appendix B. The new investigator (Principal Investigator) and experienced investigator (CoInvestigator/Institutional Principal Investigator) have combined expertise in space biology, neuroinflammation astrocyte functions, TGFbeta functions in regulating astrocyte phenotypes, and responses to particle radiation. Thus, we have the capacity to complete this 1-year project at NASA Ames and, given sufficient interest, could easily expand it to a) investigate the outcomes of combined exposures to microgravity, stress, and radiation, and b) design an in vivo follow-up study on astrocytic regulation of CNS responses to spaceflight stressors.

We have established a robust and reproducible model of human CNS-on-a-chip that includes blood-brain barrier components in addition to astrocytes. We used this model to discover that GCR components (e.g., 600 MeV/n Fe particles) selectively damage astrocytes and increase blood-brain barrier permeability in an astrocyte-specific manner. In addition, based on our X-ray irradiation experiments, radiation-mediated increase in blood-brain barrier permeability is associated with damage to endothelial cells and tight junctions, release of inflammatory cytokines, and increased oxidative stress.

Our 5-ion simplified simulated GCR exposure experiments were canceled due to Covid-19; therefore, we have requested a costed extension to be able to finish this work once Covid-19 mediated shelter-in-place is over, and to perform additional experiments that aim to demonstrate the molecular and cellular correlates of responses to GCR irradiation, and also evaluate whether astrocytes could be targeted for radioprotection.

We propose to utilize for the first time a novel high-throughput human tissue-on-a-chip model for 3D neuronal/astrocyte cultures. We will investigate the morphological and physiological outcomes as well as gene expression changes after simulated space radiation exposure (5-ion simulation of galactic cosmic rays, 500 mGy) and compare them to the responses to gamma radiation in order to establish the relative biological effectiveness. We will also evaluate the necessity and sufficiency of astrocytes in regulating radiation responses by establishing experimental models where astrocytes are either the only cell type that is irradiated, or the only cell type that escapes irradiation. Finally, we will test whether driving astrocytes more towards A1 (inflammation) or towards A2 (scarring) phenotypes may serve as countermeasures by reducing radiation-mediated neuronal damage.

In summary, we will examine whether astrocytes are necessary and sufficient to protect neurons from damage induced by simulated space radiation and evaluate their effectiveness as a target for further countermeasure development. We will also provide a proof of concept for human tissue-on-a-chip use for studying space radiation effects on the CNS.

Our proposal addresses the Part "B. Basic Investigations Opportunity for Investigators New to NASA" of the Appendix B. The new investigator (Principal Investigator) and experienced investigator (CoInvestigator/Institutional Principal Investigator) have combined expertise in space biology, neuroinflammation astrocyte functions, TGFbeta functions in regulating astrocyte phenotypes, and responses to particle radiation. Thus, we have the capacity to complete this 1-year project at NASA Ames and, given sufficient interest, could easily expand it to a) investigate the outcomes of combined exposures to microgravity, stress, and radiation, and b) design an in vivo follow-up study on astrocytic regulation of CNS responses to spaceflight stressors.