STEM Today, December 2018, No. 39 by Abhishek Sinha

STEM TODAY December 2018, No. 39

CONTENTS Degen 7: Are there synergistic effects from other spaceflight factors (e.g. altered gravity microgravity), stress, altered immune function, altered circadian rhythms, or other) that modify space radiationinduced degenerative diseases in a clinically significant manner? Exposure to ionizing radiation is associated with an increased risk for development of heart disease, stroke, and other neurovascular and degenerative tissue diseases such as cataracts later in life or well after flight. It is currently unknown whether there are significant synergistic effects from other secondary spaceflight factors (altered gravity (μgravity), stress, immune status, bone loss, etc.) that may alter morbidity and mortality estimates for these late effects resulting from space radiation exposure. Activities todate have included retrospective data mining and flight and ground studies to identify the role of the risk factors outlined above on cardiovascular health.

Editorial Editor: Mr. Abhishek Kumar Sinha Editor / Technical Advisor: Mr. Martin Cabaniss

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Disclaimer ( Non-Commercial Research and Educational Use ) STEM Today is dedicated to STEM Education and Human Spaceflight. This newsletter is designed for Teachers and Students with interests in Human Spaceflight and learning about NASAâ&#x20AC;&#x2122;s Human Research Roadmap. The opinion expressed in this newsletter is the opinion based on fact or knowledge gathered from various research articles. The results or information included in this newsletter are from various research articles and appropriate credits are added. The citation of articles is included in Reference Section. The newsletter is not sold for a profit or included in another media or publication that is sold for a profit. Cover Page Full Moon Photographed From Apollo 11 Spacecraft This outstanding view of the full moon was photographed from the Apollo 11 spacecraft during its trans-Earth journey homeward. When this picture was taken, the spacecraft was already 10,000 nautical miles away. On board Apollo 11 were commander Neil Armstrong, command module pilot Michael Collins and lunar module pilot Buzz Aldrin. While astronauts Armstrong and Aldrin descended in the lunar module Eagle to explore the moon, Collins remained on the command and service module Columbia in lunar orbit. Image Credit: NASA

Back Cover Apollo 11: Catching Some Sun Bright sunlight glints and long dark shadows dramatize this image of the lunar surface taken by Apollo 11 astronaut Neil Armstrong, the first to walk on the Moon. Pictured is the missionâ&#x20AC;&#x2122;s lunar module, the Eagle, and space suited lunar module pilot Buzz Aldrin unfurling a long sheet of foil also known as the Solar Wind Collector. Exposed facing the Sun, the foil trapped atoms streaming outward in the solar wind, ultimately catching a sample of material from the Sun itself. Along with moon rocks and lunar soil samples, the solar wind collector was returned for analysis in earthbound laboratories. Image Credit: NASA

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Editorial Dear Reader

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All young people should be prepared to think deeply and to think well so that they have the chance to become the innovators, educators, researchers, and leaders who can solve the most pressing challenges facing our world, both today and tomorrow. But, right now, not enough of our youth have access to quality STEM learning opportunities and too few students see these disciplines as springboards for their careers. According to Marillyn Hewson, "Our children - the elementary, middle and high school students of today - make up a generation that will change our universe forever. This is the generation that will walk on Mars, explore deep space and unlock mysteries that we can’t yet imagine". "They won’t get there alone. It is our job to prepare, inspire and equip them to build the future." STEM Today will inspire and educate people about Spaceflight and effects of Spaceflight on Astronauts. Editor Mr. Abhishek Kumar Sinha

Editorial Dear Reader The Science, Technology, Engineering and Math (STEM) program is designed to inspire the next generation of innovators, explorers, inventors and pioneers to pursue STEM careers. According to former President Barack Obama, " Science is more than a school subject, or the periodic table, or the properties of waves. It is an approach to the world, a critical way to understand and explore and engage with the world, and then have the capacity to change that world..." STEM Today addresses the inadequate number of teachers skilled to educate in Human Spaceflight. It will prepare , inspire and educate teachers about Spaceflight. STEM Today will focus on NASA’S Human Research Road map. It will research on long duration spaceflight and put together latest research in Human Spaceflight in its monthly newsletter. Editor / Technical Advisor Mr. Martin Cabaniss

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Space Radiation (SR) Degen - 7: Are there synergistic e ects from other space ight factors (e.g. altered gravity (Âľgravity), stress, altered immune function, altered circadian rhythms, or other) that modify space radiation-induced degenerative diseases in a clinically signi cant manner? Exposure to ionizing radiation is associated with an increased risk for development of heart disease, stroke, and other neurovascular and degenerative tissue diseases such as cataracts later in life or well after ight. It is currently unknown whether there are signi cant synergistic e ects from other secondary space ight factors (altered gravity (Âľ-gravity), stress, immune status, bone loss, etc.) that may alter morbidity and mortality estimates for these late e ects resulting from space radiation exposure.

Proteomic Analysis of Mouse Brain Subjected to Spaceflight

Xiao Wen Mao, Lawrence B. Sandberg, Daila S. Gridley, E. Clifford Herrmann, Guangyu Zhang, Ravi Raghavan, Roman A. Zubarev, Bo Zhang, Louis S. Stodieck, Virginia L. Ferguson, Ted A. Bateman and Michael J. Pecaut studied about the proteomic changes following spaceflight in mouse brain and published their findings in the paper "Proteomic Analysis of Mouse Brain Subjected to Spaceflight". Long-term deep space missions expose astronauts to an environment that is characterized mainly by ultraviolet and ionizing radiation, microgravity, and physiological/psychological stressors. These conditions present a significant hazard to spaceflight crews during and after the course of mission activities. The hazards posed to normal tissues, such as the central nervous system (CNS), are not fully understood.

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Brain damage and degeneration can be promoted by many factors including aging, ischemia, fluctuation in oxygen tension, oxidative stress, and increased intraocular pressure. There is some evidence that low-dose, space-relevant radiation induces changes in neuronal functions. Microgravity induces intraocular pressure and vascular changes and promotes apoptosis of astrocytes. Spaceflight also induces cognitive and perceptual motor performance deterioration under stress. Studies have shown that exposure to spaceflights has a strong impact on metabolic and stress response. Collective evidence indicates that exposure to stressful spaceflight environments might induce changes in brain neuronal structure and function. However, the pathophysiological consequences and role of cellular mechanisms of stress stimuli, especially those stemming from the spaceflight environment, in facilitating brain damage and neurodegeneration have been studied less and remain unclear.

Gray and White matter Gray matter consists of neurons (i.e., it contains the cell bodies, dendrites and axon terminals of neurons), nerve fibers, astrocytes, microglia, and capillaries. Gray matter is closely associated with the functional domains of performance, locomotion, learning, memory and coordination. On the other hand, white matter consists mostly of oligodendroglial cells, myelinated axons and capillaries. White matter allows communication to and from gray matter areas, and between gray matter and the other parts of the body. It functions by transmitting the information from the different parts of the body towards the cerebral cortex. It modulates the distribution of action potentials, acting as a relay and coordinating communication between different brain regions. Changes in gray matter are known to be primarily associated with Alzheimerâ&#x20AC;&#x2122;s disease and other neurodegenerative diseases, with secondary effects on the white matter. The deficits range from language ability to delayed memory and visuospatial construction. Disrupted white matter organization has been linked to poorer motor performance. Studies have shown altered expressions of a number of genes and proteins involving a wide spectrum of biological functions following exposure to space environments. These alterations induced distinct changes specific to the regions of the brain. Regional difference in stress response was also documented following simulated microgravity in human brain gray matter and white matter.

Space Shuttle Atlantis (STS-135), was launched from the Kennedy Space Center (KSC) on a 13-day mission in July of 2011. Female C57BL/6 mice (Charles River Laboratories, Inc., Hollister, CA, USA) were flown in STS-135 using NASAâ&#x20AC;&#x2122;s animal enclosure modules (AEMs). Mice were housed in two groups of five per AEM, separated by mesh wire. A set of ground controls (Ground AEMs) was housed at the Space Life Science Laboratory (SLSL) at the KSC. Ground AEM control mice were placed into the same housing hardware used in flight and environmental parameters such as temperature and carbon dioxide (CO2 ) levels were matched as closely as possible based on 48-h delayed telemetry data. All mice were under ambient temperatures of 26-28â&#x2014;Ś C with a 12-h day/night cycle during the flight. The mid deck CO2 levels that the mice were exposed to averaged 2150 parts per million (ppm) and ranged from a few 4

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hundred ppm while on the ground, before installation in the shuttle, to a maximum level of 3480 ppm in the shuttle during the mission. AEM controls were fed a special NASA food bar diet, the same as the space-flown mice. All mice received the same access to food and water ad libitum.

The Loma Linda University (LLU) Institutional Animal Care and Use Committee (IACUC) was consulted but no protocol was required since tissues were only obtained after euthanasia. However, it should be noted that all NASA research with vertebrate animals is done in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institute of Health (NIH). The primary science team responsible for running the project obtained approval from the NASA Ames Research Center ACUC (NAS-11002-Y1) on 5/31/2011. Upon return to Earth, animals were removed from the AEM nursing facility and assessed for survival and health. It was reported that all the mice survived the 13-day space mission. All animals were described by the inspecting personnel as being in good condition. Results There were nine and 17 proteins that were significantly altered after spaceflight in the white (Table 1) and gray (Table 2) matter, respectively (p < 0.05, log fold change >1.0 or <-1.0). In general, proteins that were significantly altered were upregulated in both areas of the brain. However, there was no overlap between the brain area data sets. If log fold change constraints were reduced to >0.5, the number of proteins increased to 16 and 25 for white and gray matter, respectively. There were no significant changes in canonical pathways or upstream regulators in the pathway analysis for either the white or gray matter proteins. However, there were strong trends for changes in functionally related proteins in both brain regions (Table 3).

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In the white matter, there was a strong trend for a downregulation of functions related to the formation of cellular protrusions (Z = -1.98). In the gray matter, there was a significant downregulation of functions related to overall organismal death and organ degeneration (Z < -2.0). There were also strong trends for downregulation in cellular and neural degenerative functions (Z = -1.98). Interestingly, there was a significant upregulation in functions related to viral infection (Z > 2.0). While there do not appear to be enough significantly different proteins in either white or gray matter in this analysis to appear as a significantly activated canonical pathway, there still appear to be common functional themes: (1) synaptic function, (2) intracellular communication, (3) metabolism, (4) oxidative stress and tissue damage responses, and (5) activation of catecholamines.

Synaptic Function: Plasticity, Vesicles and Dendritic Spines In the white matter there were three proteins related to synaptic plasticity that were upregulated. Calcium voltage-gated channel auxiliary subunit α2δ1 (CACNA2D1) is intimately involved in calcium channel trafficking and regulates excitatory synapse formation during development or after injury. PTPRF interacting protein α3 (PPFIA3, also known as Liprin-α-3) is typically found to be co-expressed with 6

a variety of pre-synaptic proteins in neurons but has also been found in astrocytes. It is thought to be involved in presynaptic plasticity and synaptic vesicle release, particularly in excitatory synapses. Myosin VA (MYO5A) is an F-actin-based motor protein that is also important in the generation and movement of synaptic vesicles. It has been found in dendritic spines and synaptic vesicles and appears to be critical for synaptic plasticity and organelle transport. Several proteins associated with synaptic function that are upregulated in the gray matter after flight involve vesicle formation, exocytosis and endocytosis. Syntaxin 1A (STX1A) is a soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) protein that is expressed in most neurons and is a critical component of synaptic vesicle formation and exocytosis. DNAJ heat shock protein family (Hsp40) member C5 (DNAJC5) is a pre-synaptic DNAJ C-class Hsp40 cochaperone that is primarily expressed in the brain and retina. It is part of a complex of proteins that resides on synaptic vesicles and chaperones pre-synaptic SNARE proteins, making it critical during repeated synaptic vesicle cycles. Indeed, DNAJC5 knockout mice have progressive, age-dependent sensorimotor deficits and the protein appears to be critical to preventing pre-synaptic degeneration via deficits in endocytosis.

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Upregulated within gray matter, dynamin 3 (DNM3) is expressed in the dendritic spines and is primarily associated with regulating synaptic vesicle endocytosis and recycling. There are three isoforms of dynamin that share about 80% overall homology and have mostly redundant roles in clathrin-mediated endocytosis and membrane fission. The SH3 domain containing GRB2-like 2, endophilin A1 (SH3GL2, also known as endophilin-1) is a potential tumor suppressor gene that is highly expressed in the brain, particularly in presynaptic ganglion. However, the primary function of this protein is to regulate clathrin-mediated endocytosis. Finally, while not directly related to neuronal communication, ATPase H+ transporting V0 subunit A1 (ATP6V0A1) involves a form of endocytosis in microglia. This protein that was upregulated in gray matter, is involved with the merging of lysosomes and phagosomes during phagocytosis in the brain. Interestingly, clathrin-mediated endocytosis was significantly and highly upregulated in the liver of these mice as well, suggesting a systemic response. In addition to vesicle formation, several of the proteins upregulated in gray matter after spaceflight have been implicated in neurite and dendritic spine formation. Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein epsilon (YWHAE) is believed to play a critical role during neuronal development and migration and neurite formation during cortical development. Over expression of this gene disrupts neurite formation through the microtubule binding protein, doublecortin. Enolase 2 (ENO2) has also been shown to have neurotrophic activity and is involved in cytoskeletal remodeling and neurite regeneration. Similarly, both dynamin 3 (DNM3) and SH3 domain containing GRB2-like 2, endophilin A1 (SH3GL2) that were upregulated in gray matter are involved with dendritic spine morphogenesis and stability. Another upregulated protein in the gray matter is actinin alpha 1 (ACTN1). Expressed in the dendritic spines of the post-synaptic density (PSD) , this protein is an actin-crosslinking protein that is involved with synaptic plasticity. This is interesting because changes in dendrite activity might be related to synaptic plasticity. Consistent with the upregulation of proteins related to neurite and dendrite growth is the downregulation of SEC22B (SEC22 homolog B, a vesicle trafficking protein) in the gray matter. Knocking down this protein using siRNA reduced neurite length had no impact on neuronal migration. Intracellular Communication: Myelination and Protein/Organelle Transport Another broad category impacted by spaceflight is involved in intracellular communication. Specifically, (1) axonal signaling that is "insulated" via myelin and (2) protein and organelle transport. Downregulated in the murine white matter, myelin basic protein (MBP) has an important role in the process of myelination of axons, particularly in the adhesion of myelin layers between cytosolic surfaces. MBP is implicated in auto-immune responses within the human CNS, and is thought to be a target for T cell activity in multiple sclerosis and other demyelinating or degenerative disorders. Its reduction over an extended period is usually associated with glial inflammation activation and proliferation, leading to reactive astrocytosis. Interestingly, three important factors found in oligodendrocytes and involved in myelin formation were upregulated in the gray matter: acyl-coenzyme A, a cholesterol acyltransferase 1 (ACAT1); 2â&#x20AC;&#x2122;,3â&#x20AC;&#x2122;-cyclic nucleotide 3â&#x20AC;&#x2122;-phosphodiesterase (CNP); and neurofilament light (NEFL). Two components 7

of protein/organelle transport were upregulated in the white matter. As stated previously, myosin VA (MYO5A) is important for organelle transport. Similarly, dynein light chain LC8-type 2 (DYNLL2, also known as DLC2), originally identified as part of the microtubule-based motor protein dynein, is involved with transporting mitochondria along the axons of neurons in response to local energy and metabolic requirements. However, DYNLL2 has also been shown to have a variety of other targets including nNOS, post synaptic scaffolding proteins, and pro-apoptotic proteins. However, there were also two factors involved in protein transport that were downregulated in white matter. Vacuolar protein sorting 35 ortholog (VPS35) is a component of the "cargo recognition complex" of the retromer complex responsible for the retrograde transport of proteins from endosomes to the trans-Golgi network or the plasma membrane. Already mentioned above as an important component of myelin, MBP also interacts with the cytoskeleton and/or tight junctions, making it critical for communicating extracellular signaling to the inside of the cell. Decreases in MBP have been associated with glial activation.

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Metabolism: Glycolysis and Mitochondrial Function Consistent with author’s previous results, in the liver and skin of the same mice in which they found that spaceflight had a major impact on metabolism, the next broad category involved the impact of spaceflight on the brain, including glycolysis and metabolism. Two proteins involved in glycolysis or metabolism were altered by spaceflight in the white matter. This first one was upregulated. Phosphatidylinositol transfer protein α(PITPNA) is involved with coordinating lipid metabolism and signaling, transferring phospholipids out of the endoplasmic reticulum and into other membranes. Interestingly, an increase in oxidative stress has been shown to cause a decrease in this protein, particularly in the brains of aged or Parkinson’s disease models. This lack has been associated with neurodegenerative disease, which has been linked to changes in glucose homeostasis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is an abundant enzyme in brain tissue that is critical to energy metabolism and glycolysis. In conditions of oxidative stress, GAPDH activity is impaired, leading to cellular aging and apoptosis. This enzyme can undergo sulfhydration, S-nitrosylation and oxidation, which, in turn, can lead to memory loss, apoptotic cell death and neurodegeneration, noted in ischemia and Alzheimer’s disease models. In the gray matter, enolase 2 (ENO2) was upregulated after flight, also suggesting glycolysis may be impacted. ENO2 is a glycolytic enzyme found in neurons, neuroendocrine cells and microglia. However, one of the few proteins that were downregulated in the gray matter, SEC22B (SEC22 homolog B, vesicle trafficking protein), also complexes with SNARE proteins in the endoplasmic reticulum (ER). Knocking down this protein using siRNA had no impact on exocytosis. Instead, SEC22B interacted with lipid transfer proteins, a factor which, when inhibited, has been shown to result in changes in lipid metabolism and transfer. Simultaneously, four proteins critical to mitochondrial function were also upregulated in the gray matter. The mitochondrial localized enzyme, acetyl-CoA acetyltransferase 1 (ACAT1), has been linked to cholesterol homeostasis and metabolism and can be found in axons of the cerebral cortex and hippocampus. Dynamin 1-like (DNM1L) is critical to mitochondrial fission. This protein drives mitochondrial division by self-assembling into filaments that constrict around the organelle. The 2’,3’-cyclic nucleotide 3’-phosphodiesterase (CNP) can also be found in the inner membranes of mitochondria and is important for Ca2+ transport. Interestingly CNP levels decreased in non-synaptic mitochondria in the brains of old rats. An increase in CNP release suggests mitochondria may be in a calcium-overloaded condition. Inner membrane mitochondrial protein (IMMT), also known as MIC60 and mitofilin, is important for protein translocation across the mitochondrial membrane, regulating both morphology and protein biogenesis. Even though the tissues were collected and prepared for analysis less than five hours after landing, it is possible that the changes in proteins related to metabolism are simply a response to the landing and do not reflect changes in the spaceflight environment. To confirm or deny this possibility would require that mice be euthanized in orbit and tissues immediately preserved for analysis on the ground. Indeed, such studies have already been planned. However, in author’s previous study examining the livers of these same mice, there were significant changes 8

in lipid metabolism indicative of a pre-diabetic state that seems to suggest a long-term effect rather than an acute response due to landing.

Oxidative Stress and Tissue Damage Responses The changes noted in metabolism are likely related to increases in proteins involved with oxidative stress and inflammation in the white matter. Arginase 1 (ARG1), which was highly upregulated, is an important enzyme of the urea cycle that is generally found in the liver and is critical to removing ammonia from the body. However, ARG1+ is also commonly expressed by alternatively-activated macrophages and microglia, which tend to be anti-inflammatory. In the brain, ARG1+ microglia have been implicated in amyloid beta plaque removal. Furthermore, it is important to nitric oxide (NO)-mediated vasodilation in microvascular endothelial cells. In activated macrophages, ARG1+ competes with NO synthase (NOS) for their common substrate, Larginine, leading to a reduction in NO production.

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Not surprisingly, many of the mitochondrial proteins that were upregulated in the gray matter are also involved in the oxidative stress response. ACAT1 expression has been shown to be elevated during conditions of oxidative stress. Mutations in DNM1L often result in death associated with oxidative stress-induced neurodegeneration. In vitro, deleting DNML1 from Purkinje cells resulted in increased oxidative damage via the peroxidation of proteins and lipids. Decreases in IMMT appear to result in increases in ROS levels and seem to be an antiapoptotic protein important for the regulation of cytochrome c release. Interestingly, the oxidative damage due to lipid peroxidation found in the interfibrillar mitochondria of diabetic heart tissue was reduced by overexpressing IMMT. Increases in the above oxidative stress factors are consistent with the decreases noted in another mitochondrial protein, UQCRB (ubiquinol-cytochrome c reductase binding protein), a subunit of complex III in the mitochondrial respiratory chain of the gray matter. UQCRB is important in mediating mitochondrial-derived reactive oxygen species (ROS) that are both independent of NADPH oxidase and important for angiogenesis. Drugs which inhibit the activity of UQCRB reduce the ROS produced by the mitochondria. Although not found in mitochondria, Quinonoid dihydropteridine reductase (QDPR) is also upregulated in the gray matter. QDPR is primarily associated with the regeneration of tetrahydrobiopterin (BH4) from quinonoid dihydrobiopterin (qBH2). This is important because BH4 is a critical co-factor in the generation of all three NO synthases, iNOS, nNOS, and eNOS. In quinonoid dihydropteridine reductase (QDPR)â&#x2C6;&#x2019;/â&#x2C6;&#x2019; knockout mice, the biomarkers of folate-dependent oxidative stress such as ophthalamate, spermine, and Îł-Glu-Cys all appeared to be elevated. Given the changes in markers indicative of oxidative stress, it should not be surprising that there were also changes in proteins related to cell damage and death. In the gray matter, at least four upregulated proteins dealing with intracellular damage and/or cell death appeared to be involved. This is consistent with the IPA analysis that found changes in proteins related to organismal death, degeneration of cells, and neurodegeneration. ATP6V0A1 is critically important in mediating autophagosome-lysosome fusion. DNAJC5 appears to have some influence on protein folding and endosomal autophagy, depending on the presence of SGT and Hsc70. ACAT1 is instrumental in induction of necroptosis through lipid droplet formation that has been demonstrated to be the initial key event in cell death. Finally, CNP appears to play a role with caspase-independent apoptosis. Activation of Catecholamines Finally, there were several changes in the white matter involved in sympathetic activity and catecholamine production. As mentioned previously, CACNA2D1 was upregulated in the white matter. Although we did not specifically look at areas within the brain, this protein is active in the periventricular nucleus (PVN) and is involved with sympathetic outflow. This is interesting because two proteins involved with sympathetic responses were also upregulated in the gray matter. Quinonoid dihydropteridine reductase (QDPR) is primarily associated with the regeneration of tetrahydrobiopterin (BH4) from quinonoid dihydrobiopterin (qBH2). BH4 is a critical co-factor in the biosynthesis of the neurotransmitters dopamine and serotonin. Syntaxin 1A (STX1A) is expressed in endocrine cells and STX1A knockout mice had decreased circulating levels of the stress hormones, CRH and ACTH, as well as serotonergic precursors.

Changes in protein expression profiles and oxidative stress-associated apoptosis in mouse ocular tissue after spaceflight

Approximately 30% of astronauts on short-term (∼2 week) Space Shuttle flights and 60% on long-duration (∼6 month) missions to the International Space Station (ISS) are reported to have experienced some impairment in distant or near visual acuity. Previous studies of rodents also show that environmental conditions associated with spaceflight and simulated microgravity induce alterations in retinal structure and function. Retinal damage and degeneration can occur as a result of multiple factors, including aging, ischemia, fluctuation in oxygen tension, oxidative stress, and increased intraocular pressure. The previous data from Space Shuttle studies showed a significant increase of apoptosis in retinal cells of flight mice compared to ground controls, which could lead to morphologic changes and impairment of retinal function. However, the pathophysiological consequences and cellular mechanisms of stress stimuli, especially those associated with a spaceflight environment, in facilitating retinal damage are less studied and remain unclear.

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Data on the response of biochemical systems to altered gravity is very limited in animal studies. Some lines of evidence suggest that one of the mechanisms involved in response to spaceflight, including changes in the gravity vector, is likely due to oxidative stress. Studies have shown that exposure to microgravity during spaceflights is associated with elevations in oxidative stress and production of lipid peroxidation in both humans and rodents. However, the knowledge about the duration or dose of microgravity exposure needed to induce oxidative stress or alterations in retinal structure and function is extremely limited. For example, one study has shown that even transient exposure to microgravity produced by parabolic flight can cause alterations in retinal vasculature to occur. One means to address the impact of microgravity conditions is through the application of artificial gravity (AG) during spaceflight. Creating AG by centrifugal force makes it possible to have a partial gravity or Earth-like gravity (1 g) control in space, where all other environmental conditions associated with spaceflight are present. A newly developed mouse habitat cage unit (HCU) by Japan Aerospace Exploration Agency (JAXA) has been installed in the Centrifuge-equipped Biological Experiment Facility (CBEF) on board the ISS. This rodent centrifugation capability provides opportunities to compare the effectiveness of AG prescription under spaceflight conditions. Since the HCU can reproduce Earth-like gravity, it can be used to test the effectiveness of AG to mitigate the effects of microgravity on physiological systems. The purpose of this study was to investigate the effects of spaceflight on oxidative stress and apoptosis in retinal endothelial cells and to identify spaceflight-induced changes in protein expression profiles in mouse ocular tissue. Twelve male 9-week old C57BL/6 male mice, obtained from a US breeding colony, were launched 18 July 2016, at the Kennedy Space Center (KSC) on a SpaceX-9 rocket for the 35-day MHU-1 mission to the ISS. The animals were housed in the mouse HCU located in the JAXA "Kibo" facility on the ISS. The 12 flight mice were subdivided into two groups. The first group of flight mice (n = 6) were exposed to ambient microgravity conditions (µg group), while the second group of flight mice (n = 6) were exposed to continuous artificial Earth gravity (µg + 1 g group) while they were in the HCU. AG was achieved through the use of a short-arm centrifuge for the duration of their stay on the ISS. The flight mice were then returned live to Earth and splashed down in the Pacific Ocean on 26 August 2016. It took approximately 40 h for the mice to be recovered in the Pacific Ocean, brought to shore and transported to the testing and processing laboratory located in San Diego, California on 28 August 2016. The spaceflight mice were then euthanized and their eyes were removed and prepared for analysis. Ground control mouse studies were completed in Japan after the return of the flight mice. Control mice (habitat controls, n = 6; vivarium controls, n = 6) were acquired on 31 August 2016 from a breeding colony in Japan and shipped to the JAXA animal facility in Tsukuba, Japan. HC mice were acclimated to the water lixit system and the same special food bar diet as the space flown mice were fed. They were first housed in the transportation cage unit (TCU) to simulate launch and flight to the ISS, and then placed in the HCUs to simulate the housing conditions experienced by µg mice on the ISS. They were again placed in the TCU to simulate the return 10

to Earth flight. The control mouse dissections took place on 3 November 2016. Control mouse eye tissue was then shipped to the US for analysis. All mice received the same ad libitum access to food and water. A detailed description of the flight schedule and mouse information has been previously reported.

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Results Apoptosis in Retinal Endothelial Cells Following Spaceflight Immunocytochemical analysis by terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) assay showed that spaceflight conditions induced significant apoptosis in the retinal endothelial cells (Figure 1A) relative to that in the habitat and vivarium control conditions and in the AG (Âľg + 1 g) condition. Our quantitative assessment revealed that the density of apoptotic cells in the retina was the highest in the Âľg group, and was 64% greater than that in the habitat control group (Figure 1B).

4-Hydroxynonenal (4-HNE) Immunoreactivity Following Spaceflight Reactive oxygen species (ROS) are involved in lipid peroxidation and membrane lipids are among the major targets of ROS. The occurrence of lipid peroxidation was evaluated with immunohistochemistry with an antibody against 4-HNE, which is an indicative marker of oxidative damage to the retina (Figure 1C). There were no significant differences among groups in the level of 4-HNE immunoreactivities (Figure 1D).

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Proteomics on Mouse Ocular Tissue Protein expression profile analysis were focused on comparisons between flight groups vs habitat control (HC). Analyzing the proteomic changes induced by flight conditions vs the HC group is more relevant for determin-

ing the effects of weightlessness and AG since HC mice were placed in the same flight hardware (cages) used in flight and environmental parameters such as temperature, humidity and carbon dioxide (CO2 ) levels were matched to that during spaceflight. Five micrograms of protein from each eyecup sample was resolved by 4-20% sodium dodecyl sulfate (SDS) Tris-Gly gel electrophoresis, visualized by Coomassie stain, in-gel trypsin digested, and analyzed by LC/MS on an LTQ Orbitrap Velos mass spectrometer. Figure2A shows a gel image depicting one representative sample for each of the three sample groups.

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A MaxQuant database search (restricted to Mus musculus) identified a total of 4179 proteins from all 18 samples. A Venn diagram indicates that we identified 3174 (76%) common proteins in all groups and, therefore, differences between the groups are reflective of the intensities of the proteins detected (Figure 2B). A small percentage of proteins were uniquely identified in each of the µg + 1 g, µg, and HC samples and may reflect specific changes within each group. The MS1 precursor intensities were converted to relative iBAQ intensities by dividing the iBAQ intensity by the sum of all iBAQ intensities in the sample. The samples were normalized based on the median relative iBAQ intensities for each sample, log2 transformed, and missing values were imputed based on the normal distribution. The log2 normalized iBAQ intensities were used for analyses.

Author’s identified 250 and 171 significantly differentiating proteins comparing the µg versus HC and µg + 1 g versus HC sample groups, respectively, based on a Mann-Whitney U test with a false discovery rate (FDR) corrected p-value < 0.05. A hierarchical cluster was generated for both comparisons (Figure 3) and visually representing the significant protein intensities for each sample group. They further filtered the list of significant proteins by using a fold change >2 threshold. By using a p-value and a protein expression threshold, we were able to identify the top significant proteins. These top significant

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proteins are seen in the heat maps as the clusters of proteins with the largest changes in intensity (yellow to blue) (Figure3). Volcano plots were used to visualize all identified proteins and highlight the significant proteins for each comparison in the study using R studio (Figure 4).

The y-axis consists of -log10 p-values based on the Mann-Whitney U FDR adjusted p-values, while the x-axis consists of the log2 fold change. The vertical lines indicate up- and down expression using a fold change >2 threshold. The horizontal line indicates a p-value of 0.05. They identified 77 and 23 significant proteins based on these two criteria for the µg versus HC and µg + 1 g versus HC, respectively. These differentially expressed proteins were then analyzed using Ingenuity Pathway Analysis. There were significant changes in canonical pathways and upstream regulators in the pathway analysis for flight groups compared to the HC controls. Author’s proteomic analysis identified only 33 upstream regulator proteins based on the Z-scores used to predict either activation (Z-score > 0) or inhibition (Z-score < 0) in the µg + 1 g animals compared to HC mice that involves disease development, molecular/cellular function and cell signaling, while 60 regulators were identified in the µg group compared to HC mice. IPA analysis revealed many key pathways are affected that are responsible for cell death, cell repair, inflammation, carbohydrate metabolism, mitochondrial function, fatty acid metabolism, and oxidative phosphorylation when comparing the µg group with HC group. Pathways were considered significant based on the Fisher exact test with a -log10 p-value > 1.3 (corresponds to a p-value < 0.05). Significantly affected canonical pathways between µg + 1 g vs. HC or µg group vs HC or are shown in Figure 5A,B, respectively. There were also significant changes in regulated protein expression in the pathway analysis for µg or µg + 1 g groups compared to

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HC group or µg group vs. µg + 1 g group.

There was very little overlap of significantly regulated protein between µg vs. HC and µg + 1 g vs. HC. In the µg group, there was significant alteration in functions related to overall organismal survival, cellular assembly and metabolism. Significant increased or decreased protein expression levels are summarized and listed in Tables 1-3. This study demonstrates that spaceflight induces apoptosis in retinal vascular endothelial cells. Authors also identified spaceflight-induced changes in proteomic profiles and pathways in the ocular tissue. The results indicate that spaceflight induces changes in neuronal structure, cellular organization, mitochondrial function and oxidative phosphorylation and inflammation which, in turn, may lead to tissue injury and late neurodegeneration. This study is the first to investigate the role of AG provided by centrifugation during spaceflight as a countermeasure for mitigating putative effects of microgravity on ocular structure and function. The several oxidative stress-related signaling pathways are significantly altered in the µg group, which includes fatty acid β-oxidation I, fatty acid activation and α-adrenergic pathway. However, they did not find a significant change in an oxidative stress marker for lipid peroxidation, 4-HNE when measured two days after splash-down. In author’s previous space shuttle Atlantis (STS-135) study, the level of HNE protein was significantly elevated in the retina after spaceflight compared to controls. This is not surprising as these two flight conditions are different. Female C57BL/6 mice were flown in the STS-135 for 13-day mission, and within 3-5 h of landing, eyes were removed for analysis. In this study, male mice were flown to the ISS for a 35-day mission, and the mice were euthanized at about 40 h after splashing down. Differences in gender, mission duration and recovery time might contribute to their dynamic stress response. The level of oxidative stress can be affected by their stress reaction to a prolonged flight in space and in the period of re-adaptation to the normal gravity after landing. Another possible explanation for the lack of significant change in 4-HNE in the current study is that spaceflight might elicit protective effects in the central nervous system (CNS) by inhibiting some gravity-initiated stress signaling pathway. Of all the proteins significantly altered by spaceflight, the Cap-Gly domain containing 15

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linker protein 2 (CLIP2), also known as CLIP-115, was significantly down-regulated in both µg and µg + 1 g groups.

This protein is found predominantly in the CNS, where it likely plays a role in the normal structure and function of nerve cells. Within cells, this protein is thought to regulate cell cytoskeleton function that helps to determine cell shape, size, and movement. Figure 6A illustrates CLIP2 in the network of proteins responsible for cellular assembly and organization, cell signaling and interaction as defined by IPA in response to spaceflight. Methyl-CpG-binding protein 2 (MECP2) had the highest fold changes in flight mice (µg group) compared to HC. This protein binds to methylated DNA and regulates chromatin structure and transcription. MECP2 is most abundantly produced in the brain where it is expressed primarily in post-mitotic neurons. Previous work has also shown MECP2 mediated epigenetic regulation of senescent endothelial progenitor cells and promoted apoptosis. Furthermore, a recent study provides evidence that elevated MECP2 in mice causes neurodegeneration. Figure 6B shows the regulative role of MECP2 in a network of proteins that involves cell death and survival. The data from this study indicates that adding 1 g on the ISS is effective in reducing the endothelial cell damage and increasing cellular organization and function compared to the µg group. Another study of the same animals used in this study also showed that 1 g AG served to maintain femur bone density and soleus/gastrocnemius muscle mass similar to that in the ground control group, whereas significant decreases in bone density and muscle mass occurred in the flight mice without centrifuge.

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These studies provide evidence that in-flight AG can mitigate some of the effects of weightlessness during spaceflight. Further investigation will be needed to define the relationship between gravitational dose/time and physiological response by assessing retinal physiological endpoints and function. More rodent centrifugation studies on board the ISS are also needed to provide comprehensive information to compare the effectiveness of the AG prescription in other physiological systems during weightless conditions.

Operational overview of mouse rearing experiment

Development of Mouse Habitat Unit for Use in "KIBO" Japanese Experiment Module on International Space Station ,Mitsubishi Heavy Industries Technical Review Vol. 53 No. 4 (December 2016).

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