To address the foregoing problems we will measure the impact of charged particle irradiation on neuronal anatomy and function using cultures of human neurons grown in the presence and absence of additional cell types known to be critical for proper neuronal function. Studies will also be performed in the presence of antioxidants that can minimize damage from reactive species, providing a useful strategy for gauging the importance of radiation-induced oxidative stress. These cell-based studies will be complemented by animal studies in which similar endpoints will be measured in brain tissue isolated from irradiated mice. One animal model genetically modified to express a neuronal fluorescent marker will be used to measure the subtle structural changes to neurons after irradiation. Another animal model genetically modified to exhibit early onset dementia will be used to gauge how exposure to charged particles found in space might impact the onset and/or severity of neurodegenerative phenotypes.

Collectively, these studies will provide new data regarding the consequences of charged particle irradiation in the CNS, data that will be useful in estimating the uncertainties and risks associated with space travel.

Overall accomplishments

We have now defined many of the underlying causes of charged particle-induced cognitive dysfunction in mice. Mice (6 months of age) have been subjected to an extensive series of cognitive testing 6, 12, and 24 weeks following low dose exposure (5 - 50cGy) to 1H, 4He, 16O, 28Si, and 48Ti HZE (high energy) ions. Behavioral tasks administered at these times reveal marked if not stunning decrements in behavior using 6 different testing paradigms. Each of these paradigms, including Novel Object Recognition (NOR), Object in Place (OiP), Temporal Order (TO), Elevated Plus Maze (EPM), Forced Swim Test (FST), Fear Extinction (FE) tasks interrogate hippocampal and select cortical regions of the brain and have conclusively indicated that deficits and learning and memory along with increased anxiety- and depression-like behavior are associated with exposure to space relevant fluences of these charged particles. Temporally coincident with these decrements are significant reductions in dendritic complexity and spine density along the very neurons that mediate neurotransmission important for each of the selected behavioral tasks. These measurements have also facilitated efforts at defining the relationship between individual performance and specific alterations in structural and/or synaptic integrity. The adverse effects of space radiation are certainly not limited to the structural alterations measured in neurons throughout different regions of the brain, but can also be linked to elevated neuroinflammation and oxidative stress that persist long after exposure. Collectively, our findings have been derived from roughly 2 NASA Space Radiation Laboratory (NSRL) campaigns/year spanning the years of 2013-2016. Results obtained from this funding have laid the groundwork for a deeper understanding of CNS (central nervous system) space radiation effects, and in many respects have been paradigm shifting. Previously very little (if any) information was known concerning the short or long term effects of cosmic radiation exposure on neuronal structure, and these data along with the inclusion of additional behavioral paradigms introduced during this funding cycle have established many of the underlying mechanisms that compromise CNS functionality after radiation exposure. Data derived from this grant has also been instrumental in moving forward with additional mechanistic studies focused on elucidating further how cosmic radiation exposure alters neurotramsmission to adversely impact cognition. We firmly believe that we are now well poised to extend our work and provide NASA more definitive data that quantify the uncertainties and unique risks associated with deep space travel. Lastly, much of this funding will likely prove instrumental in our efforts to develop interventions using mitigating agents, aimed at ameliorating the adverse effects of cosmic radiation exposure on the CNS.

The summation of our published work contains much the critical details outlined above. See Cumulative Bibliography.

ANNUAL REPORTING--NOVEMBER 2016

Scope of Work – General

We have now defined many of the underlying causes of charged particle-induced cognitive dysfunction in mice. Mice (6 months of age) have been subjected to an extensive series of cognitive testing 6, 12, and 24 weeks following low dose exposure (5, 30 cGy) to 16O and 48Ti HZE (high energy) ions. Behavioral tasks administered at these times reveal marked if not stunning decrements in behavior using 5 different testing paradigms that persist 6 months following a single acute dose. Temporally coincident with these decrements are significant reductions in dendritic complexity and spine density along the very neurons that mediate neurotransmission important for each of the selected behavioral tasks. These measurements have also facilitated efforts at defining the relationship between individual performance and specific alterations in structural and/or synaptic integrity. Much of this data has been highlighted in our past progress report and has now resulted in a major manuscript entitled “Cosmic radiation exposure and persistent cognitive dysfunction” published in Scientific Reports in October 2016. Collectively, our findings indicate that HZE ion irradiation elicits significant structural deterioration of neurons that persists and contributes to the progressive dementia found long after exposure. We have many follow up studies that corroborate these findings, extending our work showing that charged particle exposure constitutes a unique risk for developing behavioral decrements and CNS dysfunction. Recent experimental highlights are detailed below:

Experimental highlights:

1. The Tg (Thy1-EGFP)MJrs/J transgenic mouse strain expresses eGFP in specific subsets of neurons, thereby providing brightly fluorescent neurons for morphometric analyses. Cohorts of 6-month old animals have now been irradiated with 16O and 48Ti ions (600 MeV) at doses of 0, 5, and 30 cGy (NASA Space Radiation Laboratory-NSRL 15B, 15C, 16A). Animals have been analyzed for behavioral deficits at 6, 12, and 24 weeks after exposure. Data sets for 6 weeks are published and data for the 12 & 24 wk time points are provided in this report. Data has now confirmed the presence of significant behavioral deficits for each ion at weeks 12 and 24 using the Novel Object Recognition (NOR), Object in Place (OiP), Temporal Order (TO), Elevated Plus Maze (EPM), Fear Extinction (FE) tasks.

2. All animals described above (i.e., eGFP expressing transgenic mice subjected to 16O and 48Ti ion irradiation) have now been analyzed for alterations in neuronal structure (see data below). These studies highlight persistent and significant reductions in the complexity of the dendritic tree and density of dendritic spines along neurons of the medial prefrontal cortex (mPFC) and within the hippocampal CA1 and dentate gyrus. The majority of this data has now been published and extends our earlier studies demonstrating qualitatively similar findings 6 weeks after exposure.

3. Additional structural and synaptic parameters collected from HZE ion irradiated animals have again been used to provide quantitative readouts of developing behavioral decrements. Discrimination indices routinely decrease with reduced spine density and elevated PSD95 puncta, and validate the utility of our experimental approach for quantifying parameters relevant to the estimation of risk for developing various forms of dementia.

4. We now completed some follow up studies demonstrating the beneficial effects of MCAT expression in the subiculum region of the hippocampus. One month following irradiation of WT and MCAT mice, a range of morphometric parameters were quantified along Golgi-Cox impregnated neurons. Compared to WT mice, subiculum neurons from MCAT mice exhibited increased trends (albeit not statistically significant) toward increased dendritic complexity in both control and irradiated cohorts. However, Sholl analysis of MCAT mice revealed significantly increased arborization of the distal dendritic tree, indicating a protective effect on secondary and tertiary branching. Interestingly, radiation-induced increases in postsynaptic density protein (PSD-95) puncta were not as pronounced in MCAT compared to WT mice, and were significantly lower after the 0.5 Gy dose. As past data has linked radiation exposure to reduced dendritic complexity, elevated PSD-95 and impaired cognition, reductions in mitochondrial oxidative stress have proven useful in ameliorating many of these radiation-induced sequelae.

5. We have started a new line of investigation to extend our structural studies to the level of electron microscopy. In this work, mice subjected to low dose charged particle exposure were analyzed for changes in synapse density and myelination. One month following exposure, mice showed significant decreases in each of these endpoints, demonstrating for the first time that space relevant exposure to charged particles elicits ultrastructural changes detectable by electron microscopy.

To address the foregoing problems we will measure the impact of charged particle irradiation on neuronal anatomy and function using cultures of human neurons grown in the presence and absence of additional cell types known to be critical for proper neuronal function. Studies will also be performed in the presence of antioxidants that can minimize damage from reactive species, providing a useful strategy for gauging the importance of radiation-induced oxidative stress. These cell-based studies will be complemented by animal studies in which similar endpoints will be measured in brain tissue isolated from irradiated mice. One animal model genetically modified to express a neuronal fluorescent marker will be used to measure the subtle structural changes to neurons after irradiation. Another animal model genetically modified to exhibit early onset dementia will be used to gauge how exposure to charged particles found in space might impact the onset and/or severity of neurodegenerative phenotypes.

Collectively, these studies will provide new data regarding the consequences of charged particle irradiation in the CNS, data that will be useful in estimating the uncertainties and risks associated with space travel.

1. The Tg(Thy1-EGFP)MJrs/J transgenic mouse strain expresses eGFP in specific subsets of neurons, thereby providing brightly fluorescent neurons for morphometric analyses. Cohorts of 6-month old animals have now been irradiated with 16O and 48Ti ions (600 MeV) at doses of 0, 5, and 30 cGy (NASA Space Radiation Laboratory NSRL 12-13A-C). Animals have been analyzed for behavioral deficits at 6, 12, and 24 weeks after exposure. Data sets for 6 weeks are published and data for the 12 week data are provided in this report. Data derived from animals irradiated 6 months prior are still under analysis, which is nearly complete. Data has confirmed the presence of significant behavioral deficits for each ion at weeks 6 and 12 using the Novel Object Recognition (NOR) and Object in Place (OiP) tasks.

2. All animals described above (i.e., eGFP expressing transgenic mice subjected to 16O and 48Ti ion irradiation) are being processed for the micromorphometric analysis of neurons (see data below). To initiate these labor-intensive studies we have selected to analyze neurons within the medial prefrontal cortex (mPFC) and hippocampal Ca1 and dentate gyrus. These neurons are involved in mediating neurotransmission between cortical and hippocampal circuits and impact performance on the selected behavioral tasks above. New data presented in this progress report illustrates some of the structural changes found after 6 weeks after titanium ion exposure in the CA1 region of the hippocampus.

3. Concurrent with the foregoing analyses, the levels of specific synaptic proteins have now been quantified. Levels of presynaptic synaptophysin are significantly depressed at all times analyzed while the opposite holds true for the level of postsynaptic density-95 (PSD95) protein. These alterations underscore the persistent deficits in the synaptic machinery that occur at relatively low particle fluences, changes that likely elevate the risk of developing impaired cognition that could compromise mission critical activities.

4. Structural and synaptic parameters collected from HZE (high energy) ion irradiated animals have now been used to provide quantitative readouts of developing behavioral decrements. Discrimination indices routinely decrease with reduced spine density and elevated PSD95 puncta, and validate the utility of our experimental approach for quantifying parameters relevant to the estimation of risk for developing various forms of dementia.

5. Genetic strategies designed to ameliorate oxidative stress have been found to minimize the adverse effects of proton irradiation in the brain. Mice that overexpress human catalase targeted to the mitochondria (MCAT) were found to show significantly improved cognition following proton irradiation (0.5, 2 Gy) compared to wild type (WT) mice. Improved cognition was coincident with a preservation of host neuronal morphology, suggesting a mechanism for the neuroprotective phenotype in MCAT mice.

6. We have also undertaken some follow up studies demonstrating the beneficial effects of MCAT expression in the subiculum region of the hippocampus. One month following irradiation of WT and MCAT mice, a range of morphometric parameters were quantified along Golgi-Cox impregnated neurons. Compared to WT mice, subiculum neurons from MCAT mice exhibited increased trends (albeit not statistically significant) toward increased dendritic complexity in both control and irradiated cohorts. However, Sholl analysis of MCAT mice revealed significantly increased arborization of the distal dendritic tree, indicating a protective effect on secondary and tertiary branching. Interestingly, radiation-induced increases in postsynaptic density protein (PSD-95) puncta were not as pronounced in MCAT compared to WT mice, and were significantly lower after the 0.5 Gy dose. As past data has linked radiation exposure to reduced dendritic complexity, elevated PSD-95 and impaired cognition, reductions in mitochondrial oxidative stress have proven useful in ameliorating many of these radiation-induced sequelae.

To address the foregoing problems we will measure the impact of charged particle irradiation on neuronal anatomy and function using cultures of human neurons grown in the presence and absence of additional cell types known to be critical for proper neuronal function. Studies will also be performed in the presence of antioxidants that can minimize damage from reactive species, providing a useful strategy for gauging the importance of radiation-induced oxidative stress. These cell-based studies will be complemented by animal studies in which similar endpoints will be measured in brain tissue isolated from irradiated mice. One animal model genetically modified to express a neuronal fluorescent marker will be used to measure the subtle structural changes to neurons after irradiation. Another animal model genetically modified to exhibit early onset dementia will be used to gauge how exposure to charged particles found in space might impact the onset and/or severity of neurodegenerative phenotypes.

Collectively, these studies will provide new data regarding the consequences of charged particle irradiation in the CNS, data that will be useful in estimating the uncertainties and risks associated with space travel.

Collectively our studies have made significant strides at addressing our overarching goal aimed at determining if/how low dose charged particle irradiation elicits changes in structural and synaptic plasticity that compromise the functionality of the CNS.

To address the foregoing problems we will measure the impact of charged particle irradiation on neuronal anatomy and function using cultures of human neurons grown in the presence and absence of additional cell types known to be critical for proper neuronal function. Studies will also be performed in the presence of antioxidants that can minimize damage from reactive species, providing a useful strategy for gauging the importance of radiation-induced oxidative stress. These cell-based studies will be complemented by animal studies in which similar endpoints will be measured in brain tissue isolated from irradiated mice. One animal model genetically modified to express a neuronal fluorescent marker will be used to measure the subtle structural changes to neurons after irradiation. Another animal model genetically modified to exhibit early onset dementia will be used to gauge how exposure to charged particles found in space might impact the onset and/or severity of neurodegenerative phenotypes.

Collectively, these studies will provide new data regarding the consequences of charged particle irradiation in the CNS, data that will be useful in estimating the uncertainties and risks associated with space travel.

2. The experiments detailed above have been replicated in the presence of free radical scavenger N-acetyl cysteine (NAC) at 20 µM in efforts to determine whether reducing the level of radiation-induced oxidative stress would spare disruptions to neuronal anatomy. Analysis of these samples is currently underway. These studies directly address specific aim 1.

3. Log-phase cultures of multipotent human neural stem cells subjected to the irradiation conditions described above were analyzed for changes in ATP levels and the induction of an antioxidant response element containing the redox-sensitive Nrf1 and Nrf2 promoter. Cells lysates were prepared 3 and 7 days after irradiation and are being analyzed against normalized standard curves to determine whether charged particle irradiation caused changes in the antioxidant and energy profiles of hNSCs. These studies directly address specific aim 1.

4. The Tg(Thy1-EGFP)MJrs/J transgenic mouse strain expresses eGFP in specific subsets of neurons, thereby providing brightly fluorescent neurons for morphometric analyses. Cohorts of 2-month old animals have now been irradiated with 16O and 48Ti ions (600 MeV) at doses of 0, 5, and 30 cGy (NSRL 12C). Animals have been analyzed for behavioral deficits at 6 and 12 weeks and will be analyzed at 24 weeks post-irradiation (pending). At 6 weeks post-IR, significant radiation-induced behavioral deficits based on novel object and place recognition have been found for both heavy ions. Behavioral data is being analyzed for the 12-week time point while data for the 24-week time point has yet to be collected. These studies directly address specific aim 3.

5. All animals described above (i.e. eGFP expressing transgenic mice subjected to 16O and 48Ti ion irradiation) are being processed for the micromorphometric analysis of neurons (see data below). These time intensive studies will continue throughout year 2. These studies directly address specific aim 3.

6. We have now successfully bred cohorts of 6-month old Tg(Thy1-EGFP)MJrs/J transgenic mice of sufficient size (n=60) for the NSRL13 campaigns. These older 6-month old mice were irradiated with 16O (600 MeV) at doses of 0, 5, and 30 cGy (NSRL 13A). Mice of this same age (6-month) will also be irradiated with 48Ti ions (600 MeV) at doses of 0, 5, and 30 cGy (upcoming NSRL 13C). Follow up studies analyzing these older mice for potential behavioral deficits and disruptions to the ultrastructural features of neurons will continue throughout the funding period. These studies directly address specific aim 3.

7. Studies relevant to specific aim 2, utilizing microfluidic chambers to grow 3D models of the neurovascular unit will be initiated in year 2, and we have received approval for 10h of beam time in the Spring of 2014 (NSRL14A) to begin this work.

Publications:

1. Parihar, V.K. and Limoli, C.L. Cranial irradiation compromises neuronal architecture in the hippocampus. Proc. Natl. Acad. Sci. U.S.A. In Press (2013).

Presentations:

Sept. 2013 Symposia & Chair: CNS Effects of Radiation Damage, 59th Annual Meeting Radiation Research Society, New Orleans, LA.

Sept. 2013 Scholars and Training workshop: The adverse effects of exposure to the space radiation environment, where in vitro and in vivo models are used to define biological responses to charged particle irradiation, 59th Annual Meeting Radiation Research Society, New Orleans, LA.

June 2013 Scripps Proton Therapy Center, Causes, consequences and potential remedies for radiation injury in the CNS. San Diego, CA.

Dec. 2012 NASA/ASL seminar series: Assessing radiation effects in the CNS: From the clinic to Mars and beyond. NASA Ames Research Center, Moffett Field, CA.

To address the foregoing problems we will measure the impact of charged particle irradiation on neuronal anatomy and function using cultures of human neurons grown in the presence and absence of additional cell types known to be critical for proper neuronal function. Studies will also be performed in the presence of antioxidants that can minimize damage from reactive species, providing a useful strategy for gauging the importance of radiation-induced oxidative stress. These cell-based studies will be complemented by animal studies in which similar endpoints will be measured in brain tissue isolated from irradiated mice. One animal model genetically modified to express a neuronal fluorescent marker will be used to measure the subtle structural changes to neurons after irradiation. Another animal model genetically modified to exhibit early onset dementia will be used to gauge how exposure to charged particles found in space might impact the onset and/or severity of neurodegenerative phenotypes.

Collectively, these studies will provide new data regarding the consequences of charged particle irradiation in the CNS, data that will be useful in estimating the uncertainties and risks associated with space travel.