Our mouse studies (Aims 1 and 2) examined how sex differences and multiple genetic risk factors for Alzheimer’s disease modify GCRsim radiation-induced changes in behavior, cognition, disease progression, brain and heart structure, and inflammation in the brain, heart, and kidney. Aim 1 was built upon our previous single-ion (iron and proton) studies but tested low-dose space-like mixed 5-ion GCRsim radiation exposure in the same Alzheimer’s amyloid mouse model and wild type (WT) mice. Aim 2 investigated the effects of a strong vascular Alzheimer’s risk factor Apolipoprotein E4 in another, more physiologically relevant Alzheimer’s “knock-in” mouse model and WT mice in response to low-dose GCRsim radiation. Equal numbers of female and male mice were included in each study. Due to the large number of mice required to achieve a statistically significant result, we divided each of the mouse studies into two staggered cohorts to facilitate breeding, experimentation, and analyses. We performed pre-irradiation and post-irradiation MRI scans of the brain and heart in a subset of mice. The rest of the mice underwent a 10-test behavioral battery that we developed during our first 4 years of funding to evaluate locomotion, strength, fatigue resistance (endurance), motor coordination, sensorimotor effects, psychological state, learning, and memory in mice. In addition, we utilized several novel human brain cell cultures (Aim 3), derived from immortalized progenitor neural cells and induced pluripotent stem cells (iPSCs) differentiated into neurons and glia, to investigate how space-like radiation affects human brain health in the context of specific disease-associated genetic factors. Our collaborators, Dr. Taylor and Dr. Camila Hochman Mendez (Texas Heart Institute, THI), explored the effects of space-like radiation on the heart and kidneys of our mice from Aims 1 and 2, as well as assessing GCRsim effects on heart cell function and maturation from irradiated iPSC-derived cardiomyocytes and endothelial cells in Aim 3. All experiments include additional mice or cell cultures exposed to gamma radiation (similar to x-rays) for comparison with those exposed to the space-like GCRSsim radiation. This aids us in interpreting our findings to understand radiation risk to humans. These studies involve strong collaborations with researchers at the Texas Heart Institute, Massachusetts General Hospital, Massachusetts Institute of Technology, Brookhaven National Laboratory, Duke University, Washington University School of Medicine, NYC School of Medicine, the RIKEN Brain Institute in Japan, the Harvard School of Medicine Mouse Behavior Core, the Brigham & Women’s Hospital Department of Radiology, and the NASA Ames Research Center.

For Aim 1, we completed the irradiation, MRI scanning, behavioral analyses, euthanasia, and tissue collection of all mice. We bred and aged two identical cohorts of 114 mice per cohort, including female and male Alzheimer’s-like transgenic (Tg) mice and WT mice, staggered ~5-6 months apart. Mice underwent pre-irradiation (IRR) MRI scans of the brain and heart at 3.5 months of age, and were transported to Brookhaven National Laboratory (BNL) for low-dose GCRsim IRR (0.5 or 0.75 Gy; WT and Tg) or gamma IRR (0.75 or 2 Gy; WT only) and were returned to BWH where they later underwent behavioral testing and post-IRR MRI scans prior to euthanasia and tissue harvest at 13 months of age. Of the 228 mice at the start of the study, a total of 184 mice survived until the end of the study. Roughly half of the female Tg died prematurely, regardless of radiation exposure, similar to what we and others have previously observed. Male Tg and all WT mice had few, if any, premature deaths. A total of 124 of the mice underwent extensive behavioral testing at 12-13 months of age, while 60 mice underwent follow-up MRI scanning of the brain and heart. In terms of behavior, GCRsim and gamma IRR worsened spatial memory in male WT mice but not females or Tg mice. This is similar to our previous findings with single ion IRR (56-iron or protons) which also worsened memory in male mice but not females and suggests that low doses of space-like radiation-induced sex-specific, long-lasting effects on brain function. Male WT mice had reduced sensorimotor gating compared to female WT mice without radiation, while both GCRsim and gamma IRR reduced sensorimotor gating in female WT mice. In the rotarod test for motor coordination, female Tg mice were able to hold onto the rotating rod longer than all other mice but GCRsim IRR caused them to fall off the rod sooner. Male Tg mice exposed to low-dose GCRsim stayed on the rod longer, suggesting they had improved motor coordination. Gamma IRR had no effect on motor coordination in male or female WT mice. Male Tg mice exposed to GCRsim were better able to resist fatigue (i.e., had better endurance) in the wire hanging test than non-irradiated mice. Importantly, no radiation-specific effects were observed for fear learning and memory, startle response, or anxiety- or depressive-like behaviors in any of the mouse groups.

Comparison of pre- to post-IRR brain MRI scans indicated reduced cortical volume in female and male WT mice exposed to GCRsim and gamma IRR and increased ventricular volume in female WT and male Tg mice exposed to GCRsim; both of which are suggestive of brain atrophy. In contrast, GCRsim IRR increased hippocampal volume in male WT and male Tg mice while gamma IRR increased it in female and male WT mice. Biochemical levels of amyloid-beta, a protein that aggregates and forms plaques in Alzheimer’s disease brain, was higher in female Tg than male Tg mouse brain, as seen previously with this mouse model, but GCRsim IRR did not have any specific effects in females or males. Previously, we found that amyloid-beta levels were increased in male Tg mice exposed to iron radiation, a small component of the GCRsim exposure. Similarly, staining for amyloid plaque deposition in the brain was not altered by GCRsim irradiation.

Regarding the cardiac studies, the MRI scans revealed no radiation-specific effects on heart structure or function. Our collaborators at THI measured gene expression changes in the heart and kidney. Interestingly, there were strong, long-lasting, radiation-induced gene expression changes in the heart and kidney following low-dose GCRsim exposure, but less so with gamma IRR. In particular, GCRsim IRR reduced the expression of genes in the heart needed to break down fats to use as energy and to transport glucose in WT and Tg mice. It also reduced genes involved in pathological tissue remodeling and fibrosis and inflammatory pathways. In the kidney, GCRsim IRR reduced the expression of genes involved in DNA damage repair and pathological tissue remodeling and fibrosis, and increased expression of a glycoprotein found on immune cells to facilitate their transmigration across endothelial cells. Mouse hearts and kidneys were examined histologically. WT mice irradiated with GCRsim presented increased fibrosis in the kidney compared to sham controls.

For Aim 2, we were somewhat delayed by the COVID-19 pandemic. We bred and aged 272 novel Alzheimer’s-like amyloid knock-in (KI) mice that express either human APOE E3 or E4, the latter of which is the strongest risk factor for Alzheimer’s disease after aging. Mice were divided into 2 cohorts due to the large numbers of animals. Equal numbers of female and male mice (~7 months of age) were subjected to low-dose GCRsim IRR at BNL in May (Cohort 2A) and June (Cohort 2B) 2021. Pre- and post-IRR MRI scans of the brain and heart were performed on a subset of 32 mice from Cohort 2A. 257 out of 272 mice survived until the end of the study; however, a subset of 167 mice underwent behavioral testing. The results from our data show that while there were mostly sex- and genotype-dependent effects in APP;APOE mice irradiated with GCRsim, there were a few radiation-specific effects on behavior in the 2 Gy gamma-irradiated group. In addition, Amyloid-beta ELISA and immunohistochemistry results showed that mice expressing APOE4 had higher levels of Abeta and greater plaque load than mice expressing APOE3. MRI follow-up scans have been conducted and data analysis is currently being finalized, as well as further analysis of blood, brain, heart, and kidney. So far, we have seen that GCRsim can cause reductions in the volume of the cortex in APP;E3F males and APP;E4F females. Because this new mouse model is much heartier than the previous Alzheimer’s mouse model, we had excess mice available for another study. Therefore, we initiated a study in a third cohort (extra littermates ~14 months of age) that was irradiated with low dose GCRsim or gamma irradiation at BNL in Fall 2021. These mice underwent behavioral testing in May 2022, which was followed by euthanasia and tissue harvest, and brain analysis. Fecal samples were collected from each mouse prior to and 24 hours after irradiation and at 145 and 190 days after irradiation for analysis of the gut microbiome. In regards to behavior, APP;E4F mice showed less anxiety, and less locomotor activity than APP;E3F mice. Female mice showed less anxiety, higher motor coordination, a lower motor learning capacity, and mixed results on locomotor activity when compared to male mice. GCRsim irradiated female APP;E4F mice showed higher motor coordination, while GCRsim irradiated male APP;E3F mice showed increased locomotor activity, and a trend for a reduced short-term memory capacity. Mice that traveled to BNL and were sham-irradiated saw less anxiety, a higher motor learning capacity, more locomotor activity, and a better short-term memory than mice who stayed at their home facility. In regards to brain pathology, Expectedly, AD-like mice had more hippocampal fibrillar and total Aß than WT counterparts. The results from the hemosiderin staining run indicate no effect of irradiation on microhemorrhages, travel having mixed results on microhemorrhages, and AD-like mice having more microhemorrhages than their wildtype counterparts. 16S RNAseq analysis of the mices’ fecal samples showed both up and downregulation of a variety of bacteria induced by radiation exposure. Cholesterol and triglyceride ELISAs revealed few radiation effects, including small increases in total cholesterol and triglycerides in female APP;E3F mice with gamma irradiation. On the other hand, GCRsim had small effects on the lowering of total and high-density lipoprotein (HDL) cholesterol in male mice.

For Aim 3, we have completed several studies at BNL using human 3D neural cultures derived from immortalized progenitor cells, called ReNcells, some of which express Alzheimer’s disease-associated mutations. This “brain-in-a-dish” paradigm was developed by our collaborators, Drs. Rudy Tanzi and Doo Yong Kim at MGH. We demonstrated the feasibility of transporting these cultures between Boston and Long Island and have collected data on how radiation affects disease progression. In general, we see a consistent increase in amyloid-beta protein levels following gamma IRR but not GCRsim IRR. Amyloid production was not elevated, suggesting that gamma IRR caused an impairment in the breakdown or clearance of amyloid protein. Thus far, we have seen very little change in tau or phosphorylated tau protein (which is one of the hallmarks of Alzheimer’s disease) except under stressful conditions for the cells in which case, radiation caused an elevation in Alzheimer’s disease-related proteins. Bulk RNA sequencing revealed very different responses between GCR-sim-exposed (0.75 Gy) and gamma-exposed (2 Gy) cultures wherein GCR-sim resulted in many more downregulated genes relative to unirradiated controls (~1200 genes) than did gamma rays (~500 genes). This dataset is being made available via NASA GeneLab. Radiation increased the release of inflammatory molecules called cytokines. Adding brain immune cells called microglia to the cultures after GCRsim IRR resulted in migration of the microglia towards the neurons in wildtype, non-Alzheimer’s cell cultures and increased secretion of immune molecules after gamma IRR in Alzheimer’s-like cell cultures. Blocking one of these molecules, CCL2, with an antibody mitigated the increased migration seen with radiation. Cytokines were elevated after adding microglia, regardless of whether the neural cultures had been exposed to radiation or not. In addition, we irradiated human stem cell (iPSC)-derived brain cells with different forms of Apolipoprotein E including APOE4, APOE3, APOE2, and cells lacking APOE. Gamma radiation exposure increased the overall levels of insoluble amyloid-beta but reduced the insoluble (aggregated) amyloid-beta 42/40 ratio in the fluid bathing the E4, E2, and KO cell cultures. In contrast, gamma radiation increased the soluble amyloid-beta 42/40 ratio in all 4 cell lines while GCRsim increased it in the E3 and E2 lines. A subset of these cells was shipped to the NASA Ames Research Center for single-cell RNA sequencing by our collaborators. This dataset, which allows discrimination between radiation type, cell type (neuron or astrocyte), and APOE genotype, is being made available via NASA GeneLab. Lastly, our THI collaborators shipped human stem cell (iPSC)-derived cardiomyocytes and endothelial cells to BNL where we exposed them to GCRsim and gamma radiation in May 2021. A duplicate experiment will be run in June 2021. The cells were shipped back to THI in Houston to look for radiation-induced changes in gene expression, cell morphology and for cardiomyocytes, electrical changes.

Analysis of a natural observation study (i.e., no radiation) was done to look for correlations between plasma and CSF biomarkers with age, biological sex, and cognition in a homologous species. Samples were collected over a 4-year period. Two proteomic platforms were used to detect 114 different proteins in blood and 614 proteins in CSF. Bioinformatics analysis was completed and revealed significant associations. In the CSF we found markers related to growth factor activity, cell signaling, and immune-related inflammation to be implicated in aging, and pathway analysis revealed “regulation of peptidyl-tyrosine phosphorylation” to be a top pathway. Numerous proteins showed sex differences with aging, and several were members of the complement cascade. We also identified clusters of proteins that demonstrate similar trajectories with age, many of which were non-linear, and show wave-like movement. These clusters were enriched for various biological processes and pathways. CSF markers correlated with cognition were involved in glial cell differentiation and several hits were again related to growth factor activity. In the plasma, numerous proteins of aging were involved in lipid metabolism and inflammatory response. Several of these proteins, in addition to others, also showed sex differences. We again identified clusters of proteins with non-linear aging trajectories to demonstrate the complexity of these factors and what that may mean for involved pathways. When looking for plasma proteins associated with cognitive performance, we found a known marker of Alzheimer’s disease to show effects, as well as several other immune-related inflammatory proteins. An interesting part of this study was looking for plasma proteins associated with CSF amyloid-beta, and we were particularly interested in the ratio of amyloid beta 42/40. We found interleukin signaling to be strongly implicated with a worsening ratio and several hits from the cognition analysis overlapped. Overall, we found our model to be translational to humans and provided informative circulating factors that may be used for astronaut monitoring.

The overall goal of our research is to better assess the central nervous system and cardiovascular risks to astronauts during and after deep space travel. To properly understand these risks in the diverse human population, we must account for how sex and genetic differences change the way radiation damage manifests. Our work characterizing these radiation-disease models will also create platforms for testing strategies for mitigating radiation damage to improve the safety and long-term health of the astronauts.

Our current mouse studies (Aims 1 and 2) examine how sex differences and multiple genetic risk factors for Alzheimer’s disease modify GCRsim radiation-induced changes in behavior, cognition, disease progression, brain and heart structure, and inflammation in the brain, heart, and kidney. Aim 1 builds upon our previous single-ion (iron and proton) studies, but tests low-dose space-like mixed 5-ion GCRsim radiation exposure in the same Alzheimer’s amyloid mouse model and wildtype mice. Aim 2 investigates the effects of a strong vascular Alzheimer’s risk factor Apolipoprotein E4 in another, more physiologically relevant Alzheimer’s “knock-in” mouse model and wildtype mice in response to low-dose GCRsim radiation. Equal numbers of female and male mice are included in each study. Due to the large number of mice required to achieve a statistically significant result, we have divided each of the mouse studies into two staggered cohorts to facilitate breeding, experimentation, and analyses. We perform pre-irradiation (IRR) and post-irradiation magnetic resonance imaging (MRI) scans of the brain and heart in a subset of mice. The rest of the mice undergo a 10-test behavioral battery that we developed during our first 4 years of funding to evaluate locomotion, strength, fatigue resistance (endurance), motor coordination, sensorimotor effects, psychological state, learning, and memory in mice. In addition, we utilize several novel human brain cell cultures (Aim 3), derived from immortalized progenitor neural cells and induced pluripotent stem cells (iPSCs) differentiated to neurons and glia, to investigate how space-like radiation affects human brain health in the context of specific disease-associated genetic factors. Our collaborators, Dr. Taylor and Dr. Camila Hochman Mendez (Texas Heart Institute, THI), are exploring the effects of space-like radiation on the heart and kidneys of our mice from Aims 1 and 2, as well as assessing GCRsim effects on heart cell function and maturation from irradiated iPSC-derived cardiomyocytes and endothelial cells in Aim 3. All experiments include additional mice or cell cultures exposed to gamma radiation (similar to X-rays) for comparison with those exposed to the space-like GCRSsim radiation. This aids us in interpreting our findings to understand radiation risk to humans. These studies involve strong collaborations with researchers at the Texas Heart Institute, Massachusetts General Hospital (MGH), Massachusetts Institute of Technology, Brookhaven National Laboratory, Duke University, Washington University School of Medicine, NYU School of Medicine, the RIKEN Brain Institute in Japan, the Harvard School of Medicine Mouse Behavior Core, the Brigham & Women’s Hospital Department of Radiology, and the NASA Ames Research Center.

For Aim 1, we have now completed the irradiation, MRI scanning, behavioral analyses, sacrifice, and tissue collection of all mice. We bred and aged two identical cohorts of 114 mice per cohort, including female and male Alzheimer’s-like transgenic (Tg) mice and wildtype (WT) mice, staggered ~5-6 months apart. Mice underwent pre-irradiation MRI scans of brain and heart at 3.5 months of age, were transported to Brookhaven National Laboratory (BNL) for low-dose GCRsim IRR (0.5 or 0.75 Gy; WT and Tg) or gamma IRR (0.75 or 2 Gy; WT only) and were returned to Brigham & Women’s Hospital (BWH) where they later underwent behavioral testing and post-IRR MRI scans prior to sacrifice and tissue harvest at 13 months of age. Of the 228 mice at the start of the study, a total of 184 mice survived until the end of the study. Roughly half of the female Tg died prematurely, regardless of radiation exposure, similar to what we and others have previously observed. Male Tg and all WT mice had few, if any, premature deaths. A total of 124 of the mice underwent extensive behavioral testing at 12-13 months of age, while 60 mice underwent follow-up MRI scanning of the brain and heart. In terms of behavior, GCRsim and gamma IRR worsened spatial memory in male WT mice but not females or Tg mice. This is similar to our previous findings with single ion IRR (56-iron or protons) which also worsened memory in male mice but not females, and suggests that low doses of space-like radiation induced sex-specific, long-lasting effects on brain function. Male WT mice had reduced sensorimotor gating compared to female WT mice without radiation, while both GCRsim and gamma IRR reduced sensorimotor gating in female WT mice. In the rotarod test for motor coordination, female Tg mice were able to hold onto the rotating rod longer than all other mice but GCRsim IRR caused them to fall off the rod sooner. Male Tg mice exposed to low-dose GCRsim stayed on the rod longer, suggesting they had improved motor coordination. Gamma IRR had no effect on motor coordination in male or female WT mice. Male Tg mice exposed to GCRsim were better able to resist fatigue (i.e., had better endurance) in the wire hanging test than non-irradiated mice. Importantly, no radiation-specific effects were observed for fear learning and memory, startle response, or anxiety- or depressive-like behaviors in any of the mouse groups.

Comparison of pre- to post-IRR brain MRI scans indicated reduced cortical volume in female and male WT mice exposed to GCRsim and gamma IRR, and increased ventricular volume in female WT and male Tg mice exposed to GCRsim; both of which are suggestive of brain atrophy. In contrast, GCRsim IRR increased hippocampal volume in male WT and male Tg mice, while gamma IRR increased it in female and male WT mice. Biochemical levels of amyloid-beta, a protein that aggregates and forms plaques in Alzheimer’s disease brain, was higher in female Tg than male Tg mouse brain, as seen previously with this mouse model, but GCRsim IRR did not have any specific effects in females or males. Previously, we found that amyloid-beta levels were increased in male Tg mice exposed to iron radiation, a small component of the GCRsim exposure. Similarly, staining for amyloid plaque deposition in brain was not altered by GCRsim irradiation. Radiation had no effect on microbleeds in wildtype mice; Alzheimer’s mouse brains are now being analyzed for microbleeds.

Regarding the cardiac studies, the MRI scans revealed no radiation-specific effects on heart structure or function. Our collaborators at THI measured gene expression changes in the heart and kidney. Interestingly, there were strong, long-lasting, radiation-induced gene expression changes in the heart and kidney following low-dose GCRsim exposure, but less so with gamma IRR. In particular, GCRsim IRR reduced the expression of genes in the heart needed to break down fats to use as energy and to transport glucose in WT and Tg mice. It also reduced genes involved in pathological tissue remodeling and fibrosis and inflammatory pathways. In the kidney, GCRsim IRR reduced the expression of genes involved in DNA damage repair and pathological tissue remodeling and fibrosis, and increased expression of a glycoprotein found on immune cells to facilitate their transmigration across endothelial cells. Mouse hearts and kidneys are being examined histologically.

For Aim 2, we were somewhat delayed by the COVID-19 pandemic. We have now bred and aged 272 novel Alzheimer’s-like amyloid knock-in (KI) mice that express either human Apolipoprotein E (APOE) E3 or E4, the latter of which is the strongest risk factor for Alzheimer’s disease after aging. Mice were divided into 2 cohorts due to the large numbers of animals. Equal numbers of female and male mice (~7 months of age) were subjected to low-dose GCRsim IRR at BNL in May (Cohort 2A) and June (Cohort 2B) 2021. Pre- and post-IRR MRI scans of the brain and heart were performed on a subset of 32 mice from Cohort 2A. Behavioral testing, MRI follow-up scans, and analysis of blood, brain, heart, and kidney are currently underway. Because this new mouse model is much heartier than the previous Alzheimer’s mouse model, we had excess mice available for another study. Therefore, we initiated a study in a third cohort (extra littermates ~12-13 months of age) that were irradiated with low dose GCRsim or gamma irradiation at BNL in Fall 2021. These mice are now undergoing behavioral testing which will be followed by brain analysis. Fecal samples were collected from each mouse prior to and 24 hours after irradiation and at 2 later timepoints for analysis of the gut microbiome. Upon completion of the study, we will look for correlations of the effects of space radiation between the brain, gut, and cognition.

For Aim 3, we have completed several studies at BNL using human 3D neural cultures derived from immortalized progenitor cells, called ReNcells, some of which express Alzheimer’s disease-associated mutations. This “brain-in-a-dish” paradigm was developed by our collaborators, Drs. Rudy Tanzi and Doo Yong Kim at MGH. We demonstrated the feasibility of transporting these cultures between Boston and Long Island and have collected data on how radiation affects disease progression. In general, we see a consistent increase in amyloid-beta protein levels following gamma IRR but not GCRsim IRR. Amyloid production was not elevated, suggesting that gamma IRR caused an impairment in breakdown or clearance of amyloid protein. Thus far, we have seen very little change in tau or phosphorylated tau protein (which is one of the hallmarks of Alzheimer’s disease) except under stressful conditions for the cells; in which case, radiation caused an elevation in Alzheimer’s disease-related proteins. Radiation increased the release of inflammatory molecules called cytokines. Adding brain immune cells called microglia to the cultures after GCRsim IRR resulted in migration of the microglia towards the neurons in wildtype, non-Alzheimer’s cell cultures and increased secretion of immune molecules after gamma IRR in Alzheimer’s-like cell cultures. Blocking one of these molecules, CCL2, with an antibody mitigated the increased migration seen with radiation. Cytokines were elevated after adding microglia, regardless of whether the neural cultures had been exposed to radiation or not. In addition, we irradiated human stem cell (iPSC)-derived brain cells with different forms of Apolipoprotein E including APOE4, APOE3, APOE2, and cells lacking APOE. Gamma radiation exposure increased the overall levels of insoluble amyloid-beta, but reduced the insoluble (aggregated) amyloid-beta 42/40 ratio in the fluid bathing the E4, E2, and KO cell cultures. In contrast, gamma radiation increased the soluble amyloid-beta 42/49 ratio in all 4 cell lines while GCRsim increased it in the E3 and E2 lines. A subset of these cells was shipped to the NASA Ames Research Center for single cell RNA sequencing by our collaborators. Lastly, our THI collaborators shipped human stem cell (iPSC)-derived cardiomyocytes and endothelial cells to BNL where we exposed them to GCRsim and gamma radiation in May 2021. A duplicate experiment will be run in June 2021. The cells were shipped back to THI in Houston to look for radiation-induced changes in gene expression, cell morphology, and for cardiomyocytes, electrical changes.

Analysis of a natural observation study (i.e., no radiation) is underway to look for correlations between plasma and cerebrospinal fluid (CSF) biomarkers with age, biological sex, and cognition in a homologous species. Samples were collected over a 4-year period. Two proteomic platforms were used to detect 114 different proteins in blood and 650 proteins in CSF. Bioinformatics analysis is ongoing.

The overall goal of our research is to better assess the central nervous system and cardiovascular risks to astronauts during and after deep space travel. To properly understand these risks in the diverse human population, we must account for how sex and genetic differences change the way radiation damage manifests. Our work characterizing these radiation-disease models will also create platforms for testing strategies for mitigating radiation damage to improve the safety and long-term health of the astronauts.

Our current mouse studies (Aims 1 and 2) examine how sex differences and multiple genetic risk factors for Alzheimer’s disease modify GCRsim radiation-induced changes in behavior, cognition, disease progression, brain and heart structure, and inflammation in the brain, heart, and kidney. Aim 1 builds upon our previous single-ion (iron and proton) studies but tests low-dose space-like mixed 5-ion GCRsim radiation exposure in the same Alzheimer’s amyloid mouse model and wildtype mice. Aim 2 investigates the effects of a strong vascular Alzheimer’s risk factor Apolipoprotein E4 in another, more physiologically relevant Alzheimer’s “knock-in” mouse model and wildtype mice in response to low-dose GCRsim radiation. Equal numbers of female and male mice are included in each study. Due to the large number of mice required to achieve a statistically significant result, we have divided each of the mouse studies into two staggered cohorts to facilitate breeding, experimentation and analyses. We perform pre-irradiation and post-irradiation MRI scans of the brain and heart in a subset of mice. The rest of the mice undergo a 10-test behavioral battery that we developed during our first 4 years of funding to evaluate locomotion, strength, fatigue resistance (endurance), motor coordination, sensorimotor effects, psychological state, learning, and memory in mice. In addition, we utilize several novel human brain cell cultures (Aim 3), derived from immortalized progenitor neural cells and induced pluripotent stem cells (iPSCs) differentiated to neurons and glia, to investigate how space-like radiation affects human brain health in the context of specific disease-associated genetic factors. Our collaborators, Dr. Taylor and Dr. Camila Hochman Mendez (Texas Heart Institute, THI), are exploring the effects of space-like radiation on the heart and kidneys of our mice from Aims 1 and 2, as well as assessing GCRsim effects on heart cell function and maturation from irradiated iPSC-derived cardiomyocytes and endothelial cells in Aim 3. All experiments include additional mice or cell cultures exposed to gamma radiation (similar to x-rays) for comparison with those exposed to the space-like GCRSim radiation. This aids us in interpreting our findings to understand radiation risk to humans. These studies involve strong collaborations with researchers at the Texas Heart Institute, Massachusetts General Hospital, Massachusetts Institute of Technology, Brookhaven National Laboratory (BNL), Duke University, Washington University School of Medicine, NYU School of Medicine, the RIKEN Brain Institute in Japan, the Harvard School of Medicine Mouse Behavior Core, the Brigham & Women’s Hospital Department of Radiology, and the NASA Ames Research Center.

For Aim 1, we have now completed the irradiation, MRI scanning, behavioral analyses, sacrifice, and tissue collection of all mice. We bred and aged two identical cohorts of 114 mice per cohort, including female and male Alzheimer’s-like transgenic (Tg) mice and wildtype (WT) mice, staggered ~5-6 months apart. Mice underwent pre-irradiation (IRR) MRI scans of brain and heart at 3.5 months of age, were transported to Brookhaven National Laboratory (BNL) for low-dose GCRsim IRR (0.5 or 0.75 Gy; WT and Tg) or gamma IRR (0.75 or 2 Gy; WT only), and were returned to BWH where they later underwent behavioral testing and post-IRR MRI scans prior to sacrifice and tissue harvest at 13 months of age. Of the 228 mice at the start of the study, a total of 184 mice survived until the end of the study. Roughly half of the female Tg died prematurely, regardless of radiation exposure, similar to what we and others have previously observed. Male Tg and all WT mice had few, if any, premature deaths. A total of 124 of the mice underwent extensive behavioral testing at 12-13 months of age, while 60 mice underwent follow-up MRI scanning of the brain and heart. In terms of behavior, GCRsim and gamma IRR worsened spatial memory in male WT mice but not females or Tg mice. This is similar to our previous findings with single ion IRR (56-iron or protons) which also worsened memory in male mice but not females and suggests that low doses of space-like radiation induced sex-specific, long-lasting effects on brain function. Male WT mice had reduced sensorimotor gating compared to female WT mice without radiation, while both GCRsim and gamma IRR reduced sensorimotor gating in female WT mice. In the rotarod test for motor coordination, female Tg mice were able to hold onto the rotating rod longer than all other mice but GCRsim IRR caused them to fall off the rod sooner. Male Tg mice exposed to low-dose GCRsim stayed on the rod longer, suggesting they had improved motor coordination. Gamma IRR had no effect on motor coordination in male or female WT mice. Male Tg mice exposed to GCRsim were better able to resist fatigue (i.e., had better endurance) in the wire hanging test than non-irradiated mice. Importantly, no radiation-specific effects were observed for fear learning and memory, startle response, or anxiety- or depressive-like behaviors in any of the mouse groups.

Comparison of pre- to post-IRR brain MRI scans indicated reduced cortical volume in female and male WT mice exposed to GCRsim and gamma IRR and increased ventricular volume in female WT and male Tg mice exposed to GCRsim, both of which are suggestive of brain atrophy. In contrast, GCRsim IRR increased hippocampal volume in male WT and male Tg mice while gamma IRR increased it in female and male WT mice. We will stain brain sections to confirm hippocampal volume changes and look for signs of neurogenesis or peripheral cell infiltration. Amyloid-beta, a protein that aggregates and forms plaques in Alzheimer’s disease brain, was higher in female Tg than male Tg mouse brain, as seen previously with this mouse model, but GCRsim IRR had no effect. Previously, we found that Abeta levels were increased in male Tg mice exposed to iron IRR, a small component of the GCRsim exposure. Further brain analyses are underway. Regarding the cardiac studies, the MRI scans revealed no radiation-specific effects on heart structure or function. Our collaborators at THI measured gene expression changes in the heart and kidney. Interestingly, there were strong, long-lasting, radiation-induced gene expression changes in the heart and kidney following low-dose GCRsim exposure, but less so with gamma IRR. In particular, GCRsim IRR reduced the expression of genes in the heart needed to break down fats to use as energy and to transport glucose in WT and Tg mice. It also reduced genes involved in pathological tissue remodeling and fibrosis and inflammatory pathways. In kidney, GCRsim IRR reduced the expression of genes involved in DNA damage repair and pathological tissue remodeling and fibrosis, and increased expression of a glycoprotein found on immune cells to facilitate their transmigration across endothelial cells. Mouse hearts and kidneys are being examined histologically.

For Aim 2, we were somewhat delayed by the COVID-19 pandemic. We have now bred and aged 272 novel Alzheimer’s-like amyloid knock-in (KI) mice that express either human APOE E3 or E4, the latter of which is the strongest risk factor for Alzheimer’s disease after aging. Equal numbers of female and male mice were subjected to low-dose GCRsim IRR at BNL in May 2021. A second cohort will be subjected to GCRsim and gamma IRR at BNL in June 2021. Pre-IRR MRI scans were performed on a subset of 32 mice. Behavioral testing, MRI follow-up scans, and analysis of blood, brain, heart and kidney will begin in Spring 2022.

For Aim 3, we have completed several studies at BNL using human 3D neural cultures derived from immortalized progenitor cells, called ReNcells, some of which express Alzheimer’s disease-associated mutations. This “brain-in-a-dish” paradigm was developed by our collaborators, Drs. Rudy Tanzi and Doo Yong Kim at MGH. We demonstrated the feasibility of transporting these cultures between Boston and Long Island and have collected data on how radiation affects disease progression. In general, we see a consistent increase in amyloid-beta protein levels following gamma IRR but not GCRsim IRR. Amyloid production was not elevated, suggesting that gamma IRR caused an impairment in breakdown or clearance of amyloid protein. Thus far, we have seen very little change in tau or phosphorylated tau protein (which is one of the hallmarks of Alzheimer’s disease). Radiation increased the release of inflammatory molecules called cytokines. Adding brain immune cells called microglia to the cultures after GCRsim IRR resulted in migration of the microglia towards the neurons in wildtype, non-Alzheimer’s cell cultures and increased secretion of immune molecules after gamma IRR in Alzheimer’s-like cell cultures. Cytokines were elevated after adding microglia, regardless of whether the neural cultures had been exposed to radiation or not. In addition, we recently irradiated human stem cell (iPSC)-derived brain cells with different forms of Apolipoprotein E including APOE4, APOE3, APOE2, and cells lacking APOE. Gamma radiation exposure increased the overall levels of insoluble amyloid-beta but reduced the insoluble (aggregated) amyloid-beta 42/40 ratio in the fluid bathing the E4, E2, and KO cell cultures. In contrast, gamma radiation increased the soluble amyloid-beta 42/49 ratio in all 4 cell lines while GCRsim increased it in the E3 and E2 lines. A subset of these cells was shipped to the NASA Ames Research Center for single cell RNA sequencing by our collaborators. Lastly, our THI collaborators shipped human stem cell (iPSC)-derived cardiomyocytes and endothelial cells to BNL where we exposed them to GCRsim and gamma radiation in May 2021. A duplicate experiment will be run in June 2021. The cells were shipped back to THI in Houston to look for radiation-induced changes in gene expression, cell morphology and for cardiomyocytes, electrical changes.

The overall goal of our research is to better assess the central nervous system and cardiovascular risks to astronauts during and after deep space travel. To properly understand these risks in the diverse human population, we must account for how sex and genetic differences change the way radiation damage manifests. Our work characterizing these radiation-disease models will also create platforms for testing strategies for mitigating radiation damage to improve the safety and long-term health of the astronauts.

Our current mouse studies (Aims 1 and 2) examine how sex differences and multiple genetic risk factors for Alzheimer’s disease modify GCRsim radiation-induced changes in behavior, cognition, disease progression, brain and heart structure, and inflammation in the brain, heart, and kidney. Aim 1 builds upon our previous single-ion (iron and proton) studies but uses space-like mixed 5-ion GCRsim radiation exposure in the same Alzheimer’s mouse model and wildtype mice. Aim 2 investigates the effects of a strong vascular Alzheimer’s risk factor Apolipoprotein E4 in the same Alzheimer’s mouse model and wildtype mice in response to GCRsim radiation. Equal numbers of female and male mice are included in each study. Due to the large number of mice required to achieve a statistically significant result, we have divided each of the mouse studies into two staggered cohorts to facilitate breeding, experimentation, and analyses. We use a 10-test behavioral battery that we developed during our first 4 years of funding to evaluate locomotion, strength, fatigue resistance, motor coordination, sensorimotor effects, psychological state, learning, and memory in mice. In addition, we utilize several novel human brain cell cultures (Aim 3), derived from immortalized progenitor neural cells and induced pluripotent stem cells (iPSCs) differentiated to neurons and glia, to investigate how space-like radiation affects human brain health in the context of specific disease-associated genetic factors.

Our collaborators, Dr. Taylor and Dr. Camila Hochman Mendez (THI), are exploring the effects of space-like radiation on the heart and kidneys of our mice from Aims 1 and 2, as well as assessing GCRsim effects on heart cell function and maturation from irradiated iPSCs in Aim 3. All experiments include additional mice or cell cultures exposed to gamma radiation for comparison with those exposed to the space-like GCRSsim radiation. This aids us in interpreting our findings to understand radiation risk to humans. These studies involve strong collaborations with researchers at the Texas Heart Institute, Massachusetts General Hospital, Massachusetts Institute of Technology, Brookhaven National Laboratory, Duke University, Washington University School of Medicine, NYU School of Medicine, the RIKEN Brain Institute in Japan, the Harvard School of Medicine Mouse Behavior Core, and the Brigham & Women’s Hospital Department of Radiology. To date, we have performed 2 in vivo radiation studies in mice for Aim 1. We bred and aged two identical cohorts of 114 mice, including female and male Alzheimer’s-like mice and wildtype (WT) mice, staggered ~5-6 months apart. Aim 1A mice underwent pre-irradiation (IRR) MRI scans of brain and heart at 3.5 months of age, were transported to BNL for irradiationin April 2019, and were returned to BWH where they later underwent behavioral imaging and post-IRR MRI scans prior to sacrifice and tissue harvest at 12 months of age in December 2019. A total of 22 mice died prior to the end of the study, with female Alzheimer’s mice having the highest attrition rate. The brains, hearts, and kidneys of the remaining 92 mice are currently undergoing analyses. Thus far, the preliminary data analysis of our behavioral studies from the first of two cohorts (and combining sexes) has revealed only a few effects of radiation. Locomotion was unaltered by GCRsim or gamma radiation. AD-like mice irradiated with 0.75 Gy GCRsim appeared to be less anxious whereas WT mice exposed to 2 Gy gamma radiation showed a tendency to be more anxious than non-irradiated mice. GCRsim irradiation had no effect on grip strength but 0.75 Gy gamma irradiation reduced grip strength in WT mice. Radiation, in general, had no effect on fatigue resistance but WT mice irradiated with 0.5 Gy GCRsim showed a hint of reduced fatigue resistance in one measure. WT mice that were irradiated with 0.75 Gy GCRsim had deficits in spatial memory, whereas gamma radiation had no effect. Depressive-like behaviors were not altered by radiation. As expected, brain levels of the Alzheimer’s protein Ab42 were higher in female than male 12 month-old Alzheimer’s-like mice, as previously demonstrated; however, no radiation-specific effects were observed. Cardiac MRI analysis revealed no genotype or radiation effects on heart structure or function. Heart and kidney tissues are being examined for radiation-induced changes gene expression and proteins related to fibrosis, inflammation, and cardiovascular function. Brain MRI analysis is ongoing. Pathological analyses will begin upon re-opening of the lab following the COVID-19 shutdown. Aim 1B mice, identical to Aim 1A, underwent pre-IRR MRI scans of brain and heart and irradiation at BNL in Oct 2019. These mice will undergo behavioral testing and post-IRR MRI scans at BWH in June 2020, followed by sacrifice and tissue harvest.

For Aim 2, we have obtained approval and breeding pairs of the specific type of mice needed to assess the role of the Apo E4 in a novel Alzheimer’s-like mouse model in the radiation response. Breeding is now underway. We will breed 2 staggered cohorts, similar to Aim 1 and expect to irradiate the first cohort of Aim 2 mice with GCRsim at BNL in Spring 2021. Equal numbers of female and male mice will be included.

Regarding our cell culture experiments of Aim 3, we have completed a pilot irradiation study (May 2018) and two full irradiation studies (Nov 2018 and Apr 2019) at BNL using human neural cultures derived from immortalized progenitor cells that have been induced with Alzheimer’s disease-associated mutations. We demonstrated the feasibility of transporting these cultures between Boston and Long Island with the pilot study and have collected data on how radiation affects disease progression with the full experiment. So far, we have found that a single 2 Gy dose of gamma radiation consistently increased Ab levels 1 week and 6 weeks post-IRR, whereas GCRsim had no effect in human neural cultures bearing Alzheimer’s disease-associated mutations. We also ran a pilot study at BNL in Oct 2019 in which upon return, we plated human microglia immune cells on top of irradiated and non-irradiated human neural cells. We are currently optimizing the protocol for a future study later this year. We are also preparing for the first of the iPSC model experiments to compare the effects of GCRsim and gamma radiation in human iPSC-derived brain cells with different forms of Apolipoprotein E, including Apo E4, a major risk factor of Alzheimer’s disease. We hope to run this experiment at BNL in the Fall 2020 campaign.

Our current mouse studies (Aims 1 and 2) will examine how sex differences and multiple genetic risk factors for cardiovascular and Alzheimer’s disease modify radiation-induced changes in behavior, cognition, disease progression, brain and heart structure, and inflammation in the brain, heart, and kidney. We will continue to use an 11-test behavioral battery that we developed during our first 4 years of funding to evaluate general health, strength, fatigue resistance, motor coordination, sensorimotor effects, psychological state, learning, and memory in mice. In addition, we will utilize several novel human brain cell cultures (Aim 3), derived from immortalized progenitor cells and induced pluripotent stem cells (iPSCs), to investigate how space-like radiation affects human brain health in the context of specific disease-associated genetic factors. Dr. Taylor’s lab will assess the effects of this radiation on heart cell function and development from irradiated iPSCs. All experiments will include additional mice or cell cultures exposed to gamma radiation for comparison with those exposed to the space-like radiation. This will aid us in interpreting our findings to understand radiation risk in humans. These studies involve strong collaborations with researchers at the Texas Heart Institute, Massachusetts General Hospital, Massachusetts Institute of Technology, Brookhaven National Laboratory (BNL), Duke University, Washington University School of Medicine, NYC School of Medicine, the Harvard School of Medicine Mouse Behavior Lab, and the Brigham & Women’s Hospital Department of Radiology.

Thus far, we have prepared mice for the first of our radiation experiments at BNL and are currently performing pre-irradiation imaging of their brains and hearts. We have also completed requirements for obtaining the specific type of mice needed to assess the role of the Apo E allele in the radiation response in the second half of our mouse experiments. Regarding the cell culture experiments, we have completed a pilot irradiation (Spring 2018 campaign) and the first full irradiation (Fall 2018 campaign) at BNL with the human neural cultures derived from immortalized progenitor cells that have been induced with Alzheimer’s disease-associated mutations. We have demonstrated the feasibility of transporting these cultures between Boston and Long Island with the pilot study and have collected initial data on how radiation affects disease progression with the full experiment. So far, we have found that non-mutant cultures show a radiation-induced reduction in a specific type of neuronal structural protein that accumulates in Alzheimer’s disease whereas the mutant cultures did not show this reduction, and we are continuing to collect more data.

We are currently growing cultures for the second full irradiation experiment in which we will let the cultures grow longer after irradiation before analyzing them and expect to detect stronger differences in Alzheimer’s disease brain changes. In addition, we will examine the response of brain immune cells to irradiated neural cells in the Fall 2019 campaign. We are also preparing for the first of the iPSC model experiments in the Spring 2020 campaign.