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Characterization of gene expression profiles in the mouse brain after 35 days of spaceflight mission

Characterization of gene expression profiles in the mouse brain after 35 days of spaceflight mission www.nature.com/npjmgrav ARTICLE OPEN Characterization of gene expression profiles in the mouse brain after 35 days of spaceflight mission 1 1 1 2 3 1✉ Jacob M. Holley , Seta Stanbouly , Michael J. Pecaut , Jeffrey S. Willey , Michael Delp and Xiao Wen Mao It has been proposed that neuroinflammatory response plays an important role in the neurovascular remodeling in the brain after stress. The goal of the present study was to characterize changes in the gene expression profiles associated with neuroinflammation, neuronal function, metabolism and stress in mouse brain tissue. Ten-week old male C57BL/6 mice were launched to the International Space Station (ISS) on SpaceX-12 for a 35-day mission. Within 38 ± 4 h of splashdown, mice were returned to Earth alive. Brain tissues were TM collected for analysis. A novel digital color-coded barcode counting technology (NanoString ) was used to evaluate gene expression profiles in the spaceflight mouse brain. A set of 54 differently expressed genes (p <0.05) significantly segregates the habitat ground control (GC) group from flight (FLT) group. Many pathways associated with cellular stress, inflammation, apoptosis, and metabolism were significantly altered by flight conditions. A decrease in the expression of genes important for oligodendrocyte differentiation and myelin sheath maintenance was observed. Moreover, mRNA expression of many genes related to anti-viral signaling, reactive oxygen species (ROS) generation, and bacterial immune response were significantly downregulated. Here we report that significantly altered immune reactions may be closely associated with spaceflight-induced stress responses and have an impact on the neuronal function. npj Microgravity (2022) 8:35 ; https://doi.org/10.1038/s41526-022-00217-4 INTRODUCTION tremendous shift in body fluids, an increase in brain fluid and alterations in tissue perfusion . Space microgravity modulates the The spaceflight environment is characterized mainly by ultraviolet expression of cellular molecules, and alters pro-inflammation and ionizing radiation, microgravity, and physiological/psychologi- cytokine secretion .Neuroinflammation is also a central patholo- cal stressors. These conditions present a significant hazard to gical feature of several acute and chronic brain diseases, including spaceflight crews during the course of mission activities. The Alzheimer’s disease (AD), Parkinson disease, amyotrophic lateral susceptibility of the central nervous system (CNS) to spaceflight- sclerosis, and multiple sclerosis . induced changes can be particularly devastating to the health, The CNS is sensitive to oxidative injury due to its high oxygen mission performance, and quality of life of the spaceflight crew 19 20 consumption , the content of oxidizable unsaturated lipids and both acutely and chronically. The health risk from spaceflight- low levels of anti-oxidant defenses . Oxidative injury has been induced neuronal damage and potential adverse neurovascular implicated as a causative or contributory factor in a number of effects are a chief concern, examples are intracranial fluid neurodegenerative conditions, including aging, and ischemic, redistribution results in alteration of brain perfusion, neurovestib- 22–26 1,2 traumatic damage . Our preliminary studies have shown that ular problems, and cognitive alterations in astronauts caused by spaceflight and ionizing radiation cause prolonged oxidative microgravity and/or strong gravitational changes during ascent and stress and endothelial dysfunction that may lead to chronic re-entry. Long-duration spaceflights reportedly induce immune 27–30 inflammation and adverse remodeling . However, our knowl- dysregulation, which is considered a risk to astronaut safety and 3 edge about the mechanism and consequences of spaceflight mission success . Studies to date have demonstrated the altered condition-induced neuroinflammation is very limited. Because of distribution of peripheral leukocytes, a diminished function of the complexity of the processes and pathways that lead to specific leukocyte subpopulations, and skewed cytokine profiles in 3 neuroinflammation, an in-depth “omics” approach will potentially many astronauts . Many biomarkers associated with neuroinflam- provide insight to understanding the impact of environmental mation following space radiation and space stressors in rodents 4–8 insults on the CNS and the immune function that may have a and humans have been identified . However, the precise nature long-lasting impact. of immune dysregulation during spaceflight is not well understood. The goal of this study was to characterize changes in gene Evidence suggests that acute exposure to galactic cosmic rays expression using a neuroinflammatory assay panel to investigate (GCRs) induce detrimental CNS changes including an increased inflammation, neuronal function, growth, metabolism, and stress level of neuroinflammation, neuronal damage and cognitive 9–12 in mouse brain tissue after spaceflight. deficits similar to accelerated aging . Ionizing radiation has been shown to elicit neuroinflammation through direct activation of microglia cells and through the increased infiltration of immune RESULTS and inflammatory cells through the damaged blood-brain barrier Changes of gene expression in neuroinflammation panel (BBB) . Study also showed that both microgravity encountered by astronauts in space and simulated microgravity on earth induce A set of 54 differently expressed genes (DEG) significantly (p < 0.05) by changes in brain structure and function . Microgravity induces a one-way analysis of variance (ANOVA) and Tukey’sHSD (honestly 1 2 Department of Basic Sciences, Division of Biomedical Engineering Sciences (BMES), Loma Linda University School of Medicine, Loma Linda, CA 92350, USA. Department of Radiation Oncology, Wake Forest University, School of Medicine, Winston-Salem, NC 27101, USA. Department of Nutrition, Food and Exercise Sciences, Florida State University, Tallahassee, FL 32306, USA. email: xmao@llu.edu Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; J.M. Holley et al. significant difference) test segregates the ground control (GC) group gene expression that is important for apoptotic cell clearance, from the flight (FLT) group (Table 1). Clusters of genes related to phagocytosis, and proliferation were evident. Genes supporting neuronal function, neuronal cell support, immune function, and innate immune responses were also downregulated regarding anti- cellular growth and stress were significantly altered and organized viral signaling, reactive oxygen species (ROS) generation, and based on their protein’srole. SIN3A(https://www.ncbi.nlm.nih.gov/ bacterial immune response. Expression of genes that are correlated gene/?term=NM_001110350), SLC2A5 (https://www.ncbi.nlm.nih.g with DNA damage and cellular stress were upregulated. Many of ov/gene/?term=NM_019741), MERTK (https://www.ncbi.nlm.nih. these observed changes are in line with our previous findings in the gov/gene/?term=NM_008587), TREM2 (https://www.ncbi.nlm.nih. brains exposed to simulated spaceflight condition. Following gov/gene/?term=NM_025864), RIPK1 (https://www.ncbi.nlm.nih.g combined exposure of simulated microgravity and radiation, path- ov/gene/?term=NM_009068) are listed in multiple tables as they ways involved in neurogenesis, neuroplasticity, the regulation of are found to play various roles. neuropeptides, neuronal structure, stress, and cellular signaling are significantly altered . Gene expression in the brain that was analyzed after 90 days of simulated microgravity showed many Changes of gene expression associated with neuronal genes related to catalytic and oxidoreductase activities were function downregulated . However, in contrary to our findings, many genes Genes directly involved with neuronal function were universally related to stress, immune response, metabolic process, and/or downregulated and are summarized in Table 2 and Fig. 1. Overall, a inflammatory response were significantly upregulated in their study. picture of decreased neuron plasticity and signaling dysfunction is Duration of environmental stress may play a role in different painted. Those genes that are involved in neuron support cell response of gene expression profiles and pathway regulation. function were also found to be broadly downregulated and are We saw uniform downregulation of gene expression in our summarized in Table 3 and Fig. 2. Microglial cell and oligodendrocyte mouse brain samples directly related to neuron function. NRGN dysfunction were most pronounced along with the inhibition of (https://www.ncbi.nlm.nih.gov/gene/?term=NM_022029) coding myelin sheath maintenance. Levels of immune and inflammation for a postsynaptic kinase called neurogranin was found to be related gene expression were grossly downregulated except for two significantly reduced in expression. When neurogranin binds upregulated genes, H2-T23 (https://www.ncbi.nlm.nih.gov/gene/? calmodulin, the concentration of calcium needed to transduce a term=NM_010398)and SOX4 (https://www.ncbi.nlm.nih.gov/gene/? signal is increased . Given the downregulation of NRGN (https:// term=NM_009238). The summary is presented in Table 4 and Fig. 3. www.ncbi.nlm.nih.gov/gene/?term=NM_022029) and possible Generally, we see the impairment of genes related to microbial decrease in availability of neurogranin, neurons may become defense with generalized dysfunction of immunity and disinhibition more sensitive to firing. KCND1(https://www.ncbi.nlm.nih.gov/ of inappropriate inflammation. And finally, genes with roles in cellular gene/?term=NM_008423) codes for a potassium voltage-gated stress and growth are shown in Fig. 4 with downregulation in all channel involved in neurotransmitter release which has been genes with exception of HSPB1 (https://www.ncbi.nlm.nih.gov/ shown to be inhibited by some types of ROS generating gene/?term=NM_013560), GADD45G (https://www.ncbi.nlm.nih 34–36 treatments . Environmental stressor-induced ROS may be .gov/gene/?term=NM_011817), CCNG2 (https://www.ncbi.nlm.n provoking the down regulation of KCND1(https:// ih.gov/gene/?term=NM_007635), SESN1 (https://www.ncbi.nlm. www.ncbi.nlm.nih.gov/gene/?term=NM_008423). DLG4 (https:// nih.gov/gene/?term=NM_001013370), and CDC25A (https:// www.ncbi.nlm.nih.gov/gene/?term=NM_001109752) is tran- www.ncbi.nlm.nih.gov/gene/?term=NM_007658)which were scribed into a protein associated with glutamatergic receptor significantly upregulated. In general, we see an increased signaling while also potentially participating in dendritogenesis . cellular stress response, growth arrest, and a possible trend Its downregulation observed in our assay has the potential to toward more inflammatory-associated cell death. affect glutamatergic neuron signaling, and affect dendritic morphology leading to further dysfunction. NLGN2 (https:// Spaceflight-induced changes of pathways www.ncbi.nlm.nih.gov/gene/?term=NM_198862) codes for a Many pathways associated with cellular stress, inflammation, molecule which selectively mediates inhibitory synapses . Its apoptosis, and metabolism were altered by flight condition using downregulation may contribute to dysfunction at inhibitory DEG identified by Nanostring’s Advanced Analysis module path- neuron synapses. Previous study has shown that inflammation way scoring through correlation-guided gene subsetting, which induced loss of inhibitory nerve terminals or a redistribution of functionally annotated groups of genes followed by unsupervised presynaptic machinery in inhibitory neurons may increase risk for clustering of samples . Higher scores compared to normalized developing neurological disease and psychiatric illness, including gene expression general means upregulation while lower scores seizures and schizophrenia . represent downregulation. Figure 5 summarized pathway results Regarding genes involved in neuron function and plasticity, we in a plot of all pathway scores compared to GC controls. Among continue to see universal downregulation in the flight tissue these pathways, scores for angiogenesis, cytokine signaling, compared to GC controls. The gene SIN3A (https:// epigenetic regulation, and notch signaling were significantly www.ncbi.nlm.nih.gov/gene/?term=NM_001110350) behaves as increased which may indicate upregulation of the pathway a transcriptional repressor involved in cortical neuron differentia- (p < 0.05), while scores for innate immune response and oligoden- tion . In past studies the silencing of this gene in mice has drocyte function were greatly reduced in the flight group shown decreased memory consolidation . It is possible that compared to the controls which may indicate downregulation of downregulation may have similar, albeit reduced effects on mice the pathways (p < 0.05) by one-way ANOVA and Tukey’s HSD test in spaceflight. RELN (https://www.ncbi.nlm.nih.gov/gene/?term= (Fig. 6a–f). The score for microglia function was also decreased but NM_011261) codes for an extracellular matrix serine protease with trending significance (p = 0.07) (Fig. 6g). that regulates microtubule function and plays a role in the layering of neurons in the cerebral cortex, among other structures . Its enzymatic activity that is important for cell DISCUSSION adhesion and reduced transcription, as seen in our flight mice, Our data showed robust changes in gene expression profiles after could lead to further reduction of nervous system plasticity. spaceflight. Genes supporting neuronal synaptic signaling and SLC2A5 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_019741) migration were significantly downregulated. Decreased expression in a gene responsible for the GLUT5 (https://www.ncbi.nl of genes important for oligodendrocyte differentiation and myelin m.nih.gov/gene/2700445) fructose transporter that has been sheath maintenance was observed. Downregulation of microglial found in numerous areas including the BBB, and the npj Microgravity (2022) 35 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; J.M. Holley et al. Table 1. Summary of the 54 differentially expressed genes in flight (FLT) mice brains relative to habitat ground controls (GC). Gene name (mRNA) Functions Log2 fold Δ LC limit (log2) UC limit (log2) p value Optn Autophagy, cell cycle, microglia function −0.435 −0.547 −0.324 1.72E−05 Mertk Autophagy, microglia function −0.368 −0.499 −0.237 0.000256 Cd74 Adaptive immune response, inflammatory signaling −1 −1.45 −0.548 0.00146 Plp1 Oligodendrocyte function −0.363 −0.54 −0.187 0.00238 Sox4 Microglia function, Wnt 0.314 0.156 0.473 0.00304 H2-T23 Adaptive immune response, astrocyte function, inflammatory signaling, 0.357 0.165 0.548 0.00447 innate immune response, matrix remodeling Irf7 Apoptosis, inflammatory signaling, innate immune response −1.06 −1.63 −0.487 0.00544 Reln Growth factor signaling, matrix remodeling −0.535 −0.836 −0.235 0.00583 Prkar2b Apoptosis, cell cycle, growth factor signaling −0.32 −0.503 −0.137 0.00641 Agt Astrocyte function 0.432 0.184 0.679 0.00657 Aldh1l1 Astrocyte function −0.345 −0.542 −0.147 0.00659 Brd2 Epigenetic regulation −0.182 −0.286 −0.0773 0.00667 Ccng2 Cell cycle, DNA damage 0.292 0.119 0.465 0.00785 Hspb1 Angiogenesis, astrocyte function, cellular stress, growth factor 0.706 0.284 1.13 0.00825 Dlg4 Adaptive immune response, angiogenesis, cytokine signaling, growth −0.279 −0.45 −0.108 0.00947 factor signaling, neurons and neurotransmission Cd6 Matrix remodeling −0.972 −1.56 −0.388 0.00978 Csk Adaptive immune response, angiogenesis, growth factor −0.236 −0.384 −0.0878 0.0108 F3 Microglia function −0.304 −0.496 −0.113 0.011 Csf1r Cytokine signaling, growth factor signaling, microglia function −0.209 −0.341 −0.0771 0.0111 Uty Epigenetic regulation 0.308 0.113 0.502 0.0112 Kcnd1 Microglia function, neurons and neurotransmission −0.893 −1.45 −0.337 0.0117 Cyp27a1 Inflammatory signaling −0.805 −1.32 −0.292 0.0132 Slc2a5 Microglia function −0.623 −1.02 −0.226 0.0132 Sin3a Epigenetic regulation −0.43 −0.715 −0.146 0.0142 E2f1 Apoptosis, cell cycle, cellular stress, notch −0.709 −1.17 −0.25 0.0143 Egfr Adaptive immune response, angiogenesis, astrocyte function −0.509 −0.841 −0.177 0.0149 Tmem206 Microglia function −0.239 −0.407 −0.0701 0.0196 C5ar1 Inflammatory signaling, neurons and neurotransmission −0.851 −1.44 −0.259 0.0201 Opalin Oligodendrocyte function −0.546 −0.935 −0.157 0.0205 Ncf1 Adaptive immune response, angiogenesis, cellular stress −0.539 −0.918 −0.159 0.0214 Mre11a DNA damage −0.383 −0.665 −0.1 0.0241 Ikbkg Adaptive immune response, apoptosis, inflammatory signaling −0.27 −0.47 −0.0697 0.0246 Prkaca Adaptive immune response, angiogenesis, apoptosis, Wnt −0.144 −0.251 −0.0371 0.0248 Apoe Astrocyte function, cellular stress, lipid metabolism, microglia 0.296 0.072 0.521 0.027 St8sia6 Microglia function −0.479 −0.835 −0.123 0.0272 Gpr62 Oligodendrocyte function −0.418 −0.736 −0.1 0.0276 Hpgds Lipid metabolism −0.527 −0.922 −0.132 0.0282 Sesn1 DNA damage 0.226 0.0509 0.4 0.0298 Cdc25a Cell cycle, DNA damage 0.218 0.0481 0.389 0.0307 Lmna Apoptosis, cell cycle, microglia function −0.284 −0.507 −0.0596 0.0324 Map2k4 Adaptive immune response, apoptosis, cellular stress −0.2 −0.359 −0.041 0.0334 Irf8 Inflammatory signaling, microglia function −0.51 −0.914 −0.106 0.0352 Trem2 Adaptive immune response, inflammatory signaling, microglia −0.461 −0.833 −0.088 0.0359 Mpeg1 Inflammatory signaling −0.292 −0.528 −0.0551 0.0363 Rhoa Angiogenesis, autophagy, growth factor signaling, Wnt 0.196 0.0351 0.357 0.0381 Tarbp2 Epigenetic regulation 0.195 0.0346 0.355 0.0384 Ripk1 Apoptosis, innate immune response, NF-kB −0.63 −1.14 −0.119 0.0389 Gadd45g Cell cycle, DNA damage, growth factor signaling 0.392 0.0686 0.716 0.0389 Brd3 Epigenetic regulation −0.305 −0.557 −0.0526 0.0394 Nrgn Neurons and neurotransmission −0.16 −0.295 −0.0261 0.0413 Nlgn2 Matrix remodeling, neurons and neurotransmission −0.306 −0.562 −0.0486 0.042 Bbc3 Apoptosis, DNA damage −0.538 −0.987 −0.0897 0.0432 Rgl1 Growth factor signaling, microglia function 0.105 0.0146 0.196 0.0462 Creb1 Adaptive immune response, carbohydrate metabolism, DNA damage, 0.235 0.0291 0.44 0.0493 innate immune response, notch LC lower confidence, UC upper confidence. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 35 1234567890():,; J.M. Holley et al. Table 2. Summary of significantly altered gene expressions by spaceflight related to neuronal function. Gene p value Log2 fold Δ Related molecule function NRGN 0.0413 −0.16 A Ca2+ dependent intracellular charge transducer. DLG4 0.00947 −0.279 Postsynaptic density protein associated with glutamatergic receptor signaling. NLGN2 0.042 −0.306 Postsynaptic cell adhesion molecule mediating inhibitory synapses. SIN3A 0.0142 −0.43 Involved in cortical neuron differentiation and callosal axon elongation. RELN 0.00583 −0.535 Regulates microtubule function in neurons and neuronal migration. SLC2A5 0.0132 −0.623 Fructose transporter found in Purkinje cells and the blood-brain barrier. KCND1 0.0117 −0.893 K+ voltage-gated channel involved in neurotransmitter release. transporter, as described previously . With reduced activity, micro- glial cells may have less capacity to utilize fructose as an energy source and may be more prone to suffer from metabolic related dysfunction. Oligodendrocytes are the myelin-forming cells of the CNS. It plays critical roles in axonal metabolic support, myelination, and 57,58 providing nutritional support to neurons . A couple of key genes involved in the oligodendrocyte function have been significantly downregulated in the flight mice. PLP1 (https:// www.ncbi.nlm.nih.gov/gene/?term=NM_011123) codes for a major myelin protein that plays an important role in its formation and maintenance . The implication of significantly downregu- lated PLP1 indicates impaired neuron axonal function with the possibility of long-term neurodegeneration. OPALIN (https:// www.ncbi.nlm.nih.gov/gene/?term=NM_153520) plays a role in oligodendrocyte cytoskeletal remolding and morphology. Down- Fig. 1 Spaceflight-induced changes of gene expression related to regulation of this gene observed in our study may be an indication neuronal function. Bar graph summarizing log2 fold-changes of of decreased activity of oligodendrocytes. These findings are significantly differentially expressed genes (DEG) (p < 0.05) in the further reinforced by a significantly decreased oligodendrocyte flight (FLT) group compared to the ground control (GC) group in function pathway score (Fig. 6a). genes directly related to neuronal function. N = 6/group. p values In terms of gene expression that associated with general are calculated using one-way analysis of variance (ANOVA) and immune functions and inflammation, only two genes were found Tukey’s HSD (honestly significant difference) test. Source data are provided as a Source Data file. to be significantly upregulated in our flight mice population, H2- T23 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_010398) and 43,44 hippocampus . The downregulation of this gene suggests the SOX4 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_009238). decreased ability of cells to utilize fructose as a potential energy H2-T23 expression is essential for immunological protection and source. regulation . HT-T23 dependent T-cell inhibitory interactions aid in Level of gene expression related to neuron support cells, like preventing expansion of autoreactive CD4 T-cells and collateral microglial cells and oligodendrocytes were also seen universally autoimmune diseases . Upregulation of this gene may be a downregulated. Microglia are the resident phagocytes of the innate protective response to generalized inflammation in the flight immune system in the brain and play an important role in cytokine mice. SOX4 directly regulates Innate-like γδ T-cell (Tγδ17), a major production . Their activation is closely associated with environmental source of interleukin-17 (IL-17) (https://www.ncbi.nlm.nih.gov/ stress and microglial dysfunction is implicated in many neurological gene/?term=NM_010552). Increased SOX4 expression may lead 46 62 disorders and diseases .CSF1R (https://www.ncbi.nlm.nih.gov/gene/? to an increase of this cytokine expression in the flight mice . term=NM_001037859) is a gene coding for a cell-surface receptor The gene RIPK1 (https://www.ncbi.nlm.nih.gov/gene/?term=NM which binds to colony stimulating factor-1 (CSF1) (https:// _009068) has been shown to be a promotor of survival, apoptotic 63,64 www.ncbi.nlm.nih.gov/gene/?term=NM_001113529) and interleukin- and inflammatory signaling pathways . However, mice defi- 34 (IL-34) (https://www.ncbi.nlm.nih.gov/gene/?term=NM_0011 cient in RIPK1 demonstrated loss of inhibition of a necroptosis 35100) . CSF1R null mice have been shown to have fewer phagocytic pathway which promoted the release of necroptotic damage- 48 65 cells and reduced survival . Given these findings we suggest that associated molecular patterns . Downregulation of RIPK1 flight mice may have a reduced capacity to generate microglial cells observed in our study may lead to a similar reduction of in the nervous system. MERTK (https://www.ncbi.nlm.nih.gov/gene/? appropriate apoptosis and an increase in necroptosis resulting in term=NM_008587) creates a kinase protein in innate immune cells undesirable inflammation. Overall, taking these findings in which mediates engulfment of apoptotic cells and acts as a negative conjunction with a significantly increased cytokine signaling 49–51 regulator of inflammation . Downregulation of this gene may pathway score (Fig. 6b), inflammatory signaling appears to be contribute to immune dysregulation in our flight mice. TREM2 significantly dysregulated in our spaceflight mice. (https://www.ncbi.nlm.nih.gov/gene/?term=NM_031254)codes fora Immune dysfunction is further illustrated via the downregulation 52,53 regulator of microglial chemotaxis and phagocytosis of apoptotic . of two genes important for antimicrobial defense. MPEG1 (https:// In previous in vivo studies with microglial lacking TREM2, decreased www.ncbi.nlm.nih.gov/gene/?term=NM_010821) is a pore-forming migration to areas of apoptotic neurons was shown .NCF1(https:// bactericidal molecule of the innate immune system. It has been www.ncbi.nlm.nih.gov/gene/?term=NM_001286037) codes for a key shown to be required for the activity of ROS and nitric oxide and 55,56 66,67 subunit of the phagocyte NADPH oxidase system . Downregula- their antibacterial effects . OPTN (https://www.ncbi.nlm.nih.gov/ tion suggests a reduced capacity for microglial cells to generate an gene/?term=NM_181848) codes for a protein that plays a key role effective microbial defense. SLC2A5 codes for the GLUT5 fructose in bacterial autophagic clearance. Deficient mice have also been npj Microgravity (2022) 35 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA J.M. Holley et al. Table 3. Summary of significantly altered gene expressions by spaceflight related to neuronal support cell function. Gene p value Log2 fold Δ Related molecule function CSF1R 0.0111 −0.209 Cell-surface receptor regulating development, proliferation and differentiation. PLP1 0.00283 −0.363 Major myelin protein in the CNS. MERTK 0.000256 −0.368 Signaling protein in innate immune cells mediating engulfment of apoptotic cells. TREM2 0.0359 −0.461 Regulates microglial chemotaxis and phagocytosis of apoptotic neurons. NCF1 0.0214 −0.539 Subunit of NADPH oxidase required for superoxidase production. OPALIN 0.0205 −0.546 Regulator of oligodendrocyte cytoskeletal remolding and morphology. SLC2A5 0.0132 −0.623 Fructose transporter found in microglial cells. proliferation, and stress response. Two upregulated genes play an important part in cell cycle arrest during times of cellular stress. GADD45G (https://www.ncbi.nlm.nih.gov/gene/?term=NM_011817) has been found to play a role in activating checkpoints in the cell cycle following exposure of cells to irradiation .CCNG2 (https:// www.ncbi.nlm.nih.gov/gene/?term=NM_007635) contributes to cell cycle arrest during DNA damage and is upregulated in response to diverse stimuli, including hypoxia . Additionally, expression of CCNG2 is found to be significantly increased in cell cycle-arrested and terminally differentiated cells . Upregulation of these genes may be due to exposure to irradiation, among other stressors, during spaceflight. The downregulation of genes involved in promoting transcrip- tion and cell cycle progression was also seen. BRD2 (https:// www.ncbi.nlm.nih.gov/gene/?term=NM_010238) codes for a Fig. 2 Spaceflight-induced changes of gene expression related to nuclear kinase involved in regulating the expression of cell cycle neuronal support cell function. Bar graph summarizing log2 fold- genes via binding to multiple E2Fs, a family of transcription changes of significantly differentially expressed genes (DEG) factors . E2F1 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_ (p < 0.05) in the flight (FLT) group compared to the ground control 007891) regulates activation of DNA replication and G1/S (GC) group in genes directly related to neuronal supporting cell transition when interacting with BRD2 . BRD2 also interacts with function. N = 6/group. P values are calculated using one-way BRD3, a chromatin reader with roles in regulating transcription . analysis of variance (ANOVA) and Tukey’s HSD (honestly significant BRD3 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_001113573 difference) test. Source data are provided as a Source Data file. ) further regulates transcription by promoting the binding of the transcription factors to their targets . SIN3A (https:// shown to have impaired interferon regulatory factors www.ncbi.nlm.nih.gov/gene/?term=NM_001110350) codes for a (https://www.ncbi.nlm.nih.gov/gene/?term=NM_001159393) sig- transcription factor which regulates cell cycle progression by naling and reduced response to toll-like receptor stimulation .In repressing gene expression for cell cycle inhibitor . It has been summary, these results imply the impaired antibacterial capabilities linked to functional cellular changes in proliferation, cell cycle, and and increased viral susceptibility in the flight mouse brain stem cell function in mice models . Downregulation of these compared to GC group. genes involved in the promotion of cellular proliferation may Our data indicate that the regulation of innate immune further amplify the upregulation of genes with roles in cell cycle response was impaired following spaceflight. The pathway score arrest. These changes are similarly reinforced by a significantly for the innate immune response was significantly reduced in the increased epigenetic regulation pathway score (Fig. 6e) indicating flight group compared to GCs (Fig. 6c). The cell type score for that there may be an increased level of modulating and checks exhausted CD8 cells is also greatly reduced for the flight group being implemented during transcription, due to spaceflight. A compared to controls and may indicate a dysfunctional pheno- recent study has shown that cell cycle regulation is closely linked type in CD8 T-cell response. T-cell exhaustion represents an to hippocampal neurogenesis which plays critical roles in memory adaptive response to conditions of chronic antigen stimulation and learning . Neurogenesis is a dynamic process that involves and inflammation , as well as promoting tissue repair following proliferation and differentiation of stem and progenitor cells, or an inflammatory injury . Functionally exhausted CD8+ cells may survival and maturation of newborn neurons . Environmental result in a severely compromised innate immune response . stressors have been shown to modulate cell cycle progression, Cytochrome P450 27A1 (CYP27A1) (https://www.ncbi.nlm.nih.gov/ particular in the G1 phase . These observed changes of gene gene/?term=NM_024264) plays an important role in the metabolism expression related to the cell cycle in our study may indicate of cholesterol and cholesterol-related compounds .In humans, altered regulation of neurogenesis. complete CYP27A1 deficiency leads to nodule formation in the brain Many downregulated genes were known to be involved in which may lead to dementia, cerebellar ataxia, and spinal cord cellular stress responses. MAP2K4 (https://www.ncbi.nlm.nih.gov/ paresis . This altered cholesterol metabolism closely associates with gene/?term=NM_009157) is a kinase involved with responses to inflammatory responses involved in the pathogenesis of AD cellular stress including apoptosis, inflammation, cellular prolifera- progression .Indeficient mice, a significant increase in cholestanol tion, and neurodegeneration. A deficiency of MAP2K4 has been in the brain is observed . Downregulation of CYP27A1, as seen in shown to result in cellular susceptibility to stress-induced our flight mice, may lead to neurodegeneration and inflammation in more chronic settings or to a milder degree. apoptosis and growth inhibition . However, it has been demon- Fourteen genes that were significantly altered in the flight mice strated that disruption of both MAP2K4 and MAP2K7 (https:// compared to GCs were found to have direct roles in cellular growth, www.ncbi.nlm.nih.gov/gene/?term=NM_001042557) genes was Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 35 J.M. Holley et al. Table 4. Summary of significantly altered gene expressions by spaceflight related to immune function and inflammation. Gene p value Log2 fold Δ Related molecular function H2-T23 0.00447 0.357 Immune regulation and protection through NK and T-cell suppression. SOX4 0.00304 0.314 Transcription factor regulating key genes in Thelper17 cells. CSK 0.0108 −0.236 Negative regulator of T-cell receptor signaling. IKBKG 0.0246 −0.27 Subunit of IKK complex with cardinal role in stimulating NF-κB regulation. MPEG1 0.0363 −0.292 Pore-forming bactericidal effector molecule of the innate immune system. MERTK 0.000256 −0.368 Signaling protein in innate immunity for a negative regulator of inflammation. OPTN 0.0000172 −0.435 Ubiquitin-binding protein important for bacterial autophagic clearance. TREM2 0.0359 −0.461 Inhibitor of neuroinflammation via suppression of NF-kB signaling. ST8SIA6 0.0272 −0.479 A sialyltransferase important for immune suppression and modulation. IRF8 0.0352 −0.51 Transcription factor for anti-viral dendritic cells and IFN-inducible genes. HPGDS 0.0282 −0.527 Enzymatic mediator of prostaglandin D2 creation. RIPK1 0.0389 −0.63 Promotor of survival, apoptotic and inflammatory signaling pathways. CYP27A1 0.0132 −0.805 Cytochrome P450 enzyme important in the metabolism of cholestrol. C5AR1 0.0201 −0.852 Receptor for the chemotactic and inflammatory anaphylatoxin, C5a. CD6 0.00978 −0.972 Stimulatory molecule promoting T-cell activation, proliferation, and adhesion. CD74 0.00146 −1 Chaperone protein involved with MHC class II antigen presentation. IRF7 0.00544 −1.06 Transcriptional regulator of type I interferon dependent immune responses. Fig. 3 Spaceflight-induced changes of gene expression related to immune function and inflammation. Bar graph summarizing log2 fold- changes of significantly differentially expressed genes (DEG) (p < 0.05) in the flight (FLT) group compared to the ground control (GC) group in genes directly related to immune function and inflammation. N= 6/group. p values are calculated using one-way analysis of variance (ANOVA) and Tukey’s HSD (honestly significant difference) test. Source data are provided as a Source Data file. Fig. 4 Spaceflight-induced changes of gene expression related to cellular stress and growth function. Bar graph summarizing log2 fold- changes of significantly differentially expressed genes (DEG) (p < 0.05) in the flight (FLT) group relative to the ground control (GC) group in genes directly related to cellular stress and growth function. N = 6/group. p values are calculated using one-way analysis of variance (ANOVA) and Tukey’s HSD (honestly significant difference) test. Source data are provided as a Source Data file. npj Microgravity (2022) 35 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA J.M. Holley et al. Fig. 5 Spaceflight-induced changes of pathway scores. Summarized pathway scores in flight (FLT) group vs. ground control (GC). *Significantly upregulated pathways (p < 0.05), include: cytokine signaling, Angiogenesis, Epigenetic regulation, and Notch. **Significantly (p < 0.05) or strong trend (p = 0.07) downregulated pathways, include: Oligodendrocyte function, Innate immune response, and Microglia function. p values are by one-way ANOVA and Tukey’s post hoc test. Source data are provided as a Source Data file. Fig. 6 Spaceflight-induced changes in nueroinflammation pathway scores among flight (FLT) and ground controls (GC) groups. Boxplots depict pathway scores on the y-axis and the experimental conditions on the x-axis. N = 5–6 /group. p values are calculated using one-way analysis of variance (ANOVA) and Tukey’s HSD (honestly significant difference) test. a Oligodendrocyte function score p < 0.05, b cytokine signaling score p < 0.05, c innate immune response score p < 0.05, d angiogenesis score p < 0.05, e epigenetic regulation score p < 0.05, f notch score p < 0.01, and g Microglia function score p = 0.07. Boxes are the range between first (25%) and the third (75%) quartile, the center line is the median, the whiskers include the variability those quartiles. Source data are provided as a Source Data file. required to thoroughly block cellular growth caused by environ- protein biomarkers in response to stressors were observed in a 87 41 mental stressors . MRE11A (https://www.ncbi.nlm.nih.gov/gene/? previous flight study . Given this finding, gene expression profiles in term=NM_018736), a part of an exonuclease complex central to specific regions of the brain in response to spaceflight will be the cellular DNA damage response was downregulated. The MRE11 investigated in future studies. More recently, single-cell sequencing complex is essential to vertebrates and defects lead to sensitivity to technologies, including transcriptomics, are available by directly DNA damage and cell cycle checkpoints deficiency .Its down- measuring multiple molecular signatures in specific brain cells, regulation may point to a certain level of dysfunction leading to an providing robust molecular identity of specific cell types . impaired response to cellular stress leaving the flight mice more Brain tissues were dissected and prepared for analysis within 60 h after landing. It is possible that observed changes in gene expression vulnerable to DNA damage. Different brain regions have different vulnerability to environ- profiles in neuroinflammation are a compensatory acute response to mental stressors due to structural and biological heterogeneity of the the acceleration and intense noise of launch or reflect changes to the regions, cell types and molecular networks . Regional differences of combined response of spaceflight environment and the landing. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 35 J.M. Holley et al. In ordertoevaluate theimmuneresponsetospaceflight environ- RNA isolation and gene expression profiling ment, in the further study, mice will be euthanized in the orbit and Isolation of total RNA from the brain tissues was performed using brain tissues will be immediately preserved for analysis on the DNA/RNA/miRNA Universal Kit (Qiagen, # 80224) according to the ground. Furthermore, in order to test the hypothesis that neuroin- manufacturer’s instruction. Briefly, tissue homogenization was flammation plays an important role in developing spaceflight- performed using 1.5 mm beads (Benchmark, # 1032-15) on Minilys induced stress response in the brain, an anti-oxidant or anti- homogenizer (Bertin Technologies) in RLT Plus buffer. The purity and inflammatory compound will be injected to mouse before or during concentration of the eluted RNA were measured using Nanodrop the flight to determine the protective effect against oxidative 2000 (Thermo Fisher Scientific, Waltham, MA) and stored at −80 °C damage in the brain. until further analysis. RNA samples (20 ng/ul) were then shipped to It is noted from our study that among those genes which Nanostring (Technologies Seattle, WA) and gene expression profiling expression were significantly altered by spaceflight, markedly more ® of brain tissues was conducted using the nCounter neuroflamma- gene expressions are being downregulated than upregulated. It is tion pathways panel. The panel includes 757 genes covering the speculated that many genes may be upregulated during spaceflight core pathways and processes that define neuroimmune interactions and the early phase after landing as an adaptation response. During and 13 potential housekeeping genes for normalization. RNA re-adaptation, the expression of these previous upregulated genes samples (100 ng each) on 2 cartridges were used for the Gene could be then downregulated as a compensatory response, or when Expression Assay with Mouse Neuroinflammation panel performed protein synthesis from upregulated genes has been completed, on the nCounter MAX system (https://nanostring.com/wp-content/ these genes expression may be downregulated . uploads/MAN-C0035_nCounter_Analysis_System_MAX_FLEX.pdf), a Collectively, changes noted in our study indicate that exposure multi-channel epifluorescence scanner with Nanostring Advanced to the spaceflight environment induces significant changes in Analysis Module plugin for QC, normalization, and differential gene expression and signaling pathways related to neuronal expression analysis (DE). Data files generated from nCounter system function, immune regulation, growth and metabolic function. were analyzed using nSolver 4.0 software with the Advanced Study showed that chronically dysfunctional and deregulated Analysis module for QC (quality control), normalization, DE analysis, pathways, including cytokine signaling (Fig. 6b), epigenetic and gene-set enrichment analysis. Data normalization included regulation (Fig. 6e), and notch signaling (Fig. 6f) play important 91 2 steps: Positive control normalization to correct platform-associated roles in disease development , and have been associated with variation and Codeset content normalization using the house- several progressive neurodegenerative diseases . Our observed keeping (HK) genes to correct variability of input samples. Geometric changes of a dysregulated inflammatory response, downregulated means of selected HK probes were used to normalize counts of the T-cell response, and reduced microglia signaling (Fig. 6g) might samples and DEGs were generated from the normalized counts. also have an impact on brain structure and function, and further lead to chronic neuroinflammation. It has been shown that here are strong correlations between neuroinflammatory biomarkers, Statistical analysis brain morphology and behavioral outcomes . It may be Gene expression profiling data were analyzed using nSolver hypothesized that chronical changes of observed gene expression analysis and the advanced analysis module software. Advanced profiles could have long-term effects on brain morphology and analysis module software uses open-source R program for organism behavior. pathway scoring and gene-set enrichment analysis. Using the Reactome pathway database annotations, pathway scores are derived by calculating the first principle component of pathway MATERIALS AND METHODS genes’ normalized expression and data are summarized from Flight and ground control conditions changes of a gene set within a given pathway into a single score. Ten-weeks-old male C57BL/6 mice (Jackson Laboratories, Inc. Bar To further analyze the associated pathways of the DEGs, Kyoto Harbor, ME) at the time of launch, were flown for NASA’s ninth Encyclopedia of Genes and Genomes (KEGG) pathways analysis Rodent Research experiment (RR-9) on SpaceX-12 for a 35-day was performed using biological process database to identify the mission and lived in NASA’s Rodent Habitats aboard the annotated sets of genes based on the biological processes in International Space Station. All FLT mice were maintained at an which they participate. Significantly differential expression genes ambient temperature of 26–28 °C with a 12-h light/dark cycle were presented in the tables with individual genes using log during the flight. Habitat GC mice were kept under similar housing (p value) andlog fold change compared to the GC group. conditions, including temperature, humidity and carbon dioxide Pathview module was used to display upregulated genes or (CO ) levels using 48-h delayed telemetry data from the FLT downregulated genes overlaid on KEGG pathways. For DEG and group. Water and food bar diet specifically designed by NASA pathway analysis, p < 0.05 was considered statistically significant were provided ad libitum to FLT and GC groups. All mice received between FLT and GC groups by one-way ANOVA and Tukey’s the same access to food and water. NASA-Ames Research Center HSD test. and KSC Institutional Animal Care and Use Committees approved this flight study. The study has been done in strict accordance Reporting summary with the recommendations in the Guide for the Care and Use of Further information on research design is available in the Nature Laboratory Animals of the National Institute of Health. Research Reporting Summary linked to this article. Mouse brain dissection after spaceflight Within 38 ± 4 h of splashdown, the FLT mice were rapidly DATA AVAILABILITY euthanized in 100% CO . The GC mice were euthanized with the The authors declare that source data supporting the findings of this study with the same method after 38 days of GC housing. Shortly after figures of the article are provided with this paper. euthanasia, brains were removed and bisected along the midline and coronally within the half hemispheres. The right caudal half hemisphere of the brain (containing mid- and hindbrain) from CODE AVAILABILITY each mouse (n = 5–6 per group) was placed in a sterile cryovial, All of the significant gene data obtained in this study were deposited in the NCBI’s snap frozen in liquid nitrogen and kept at −80 °C prior to use. GEO, and are accessible through GEO accession number GSE186278. npj Microgravity (2022) 35 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA J.M. Holley et al. Received: 10 January 2022; Accepted: 15 July 2022; 31. Kwong, L. N. et al. Biological validation of RNA sequencing data from formalin- fixed paraffin-embedded primary melanomas. JCO Precis Oncol. 2018,1–19 (2018). 32. Santucci, D. et al. Evaluation of gene, protein and neurotrophin expression in the brain of mice exposed to space environment for 91 days. PLoS ONE 7, e40112 (2012). REFERENCES 33. Hoffman, L., Chandrasekar, A., Wang, X., Putkey, J. A. & Waxham, M. 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MKK7 is an essential component of the Jnk signal transduction © The Author(s) 2022 pathway activated by proinflammatory cytokines. Genes Dev. 15, 1419–1426 (2001). npj Microgravity (2022) 35 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png npj Microgravity Springer Journals

Characterization of gene expression profiles in the mouse brain after 35 days of spaceflight mission

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www.nature.com/npjmgrav ARTICLE OPEN Characterization of gene expression profiles in the mouse brain after 35 days of spaceflight mission 1 1 1 2 3 1✉ Jacob M. Holley , Seta Stanbouly , Michael J. Pecaut , Jeffrey S. Willey , Michael Delp and Xiao Wen Mao It has been proposed that neuroinflammatory response plays an important role in the neurovascular remodeling in the brain after stress. The goal of the present study was to characterize changes in the gene expression profiles associated with neuroinflammation, neuronal function, metabolism and stress in mouse brain tissue. Ten-week old male C57BL/6 mice were launched to the International Space Station (ISS) on SpaceX-12 for a 35-day mission. Within 38 ± 4 h of splashdown, mice were returned to Earth alive. Brain tissues were TM collected for analysis. A novel digital color-coded barcode counting technology (NanoString ) was used to evaluate gene expression profiles in the spaceflight mouse brain. A set of 54 differently expressed genes (p <0.05) significantly segregates the habitat ground control (GC) group from flight (FLT) group. Many pathways associated with cellular stress, inflammation, apoptosis, and metabolism were significantly altered by flight conditions. A decrease in the expression of genes important for oligodendrocyte differentiation and myelin sheath maintenance was observed. Moreover, mRNA expression of many genes related to anti-viral signaling, reactive oxygen species (ROS) generation, and bacterial immune response were significantly downregulated. Here we report that significantly altered immune reactions may be closely associated with spaceflight-induced stress responses and have an impact on the neuronal function. npj Microgravity (2022) 8:35 ; https://doi.org/10.1038/s41526-022-00217-4 INTRODUCTION tremendous shift in body fluids, an increase in brain fluid and alterations in tissue perfusion . Space microgravity modulates the The spaceflight environment is characterized mainly by ultraviolet expression of cellular molecules, and alters pro-inflammation and ionizing radiation, microgravity, and physiological/psychologi- cytokine secretion .Neuroinflammation is also a central patholo- cal stressors. These conditions present a significant hazard to gical feature of several acute and chronic brain diseases, including spaceflight crews during the course of mission activities. The Alzheimer’s disease (AD), Parkinson disease, amyotrophic lateral susceptibility of the central nervous system (CNS) to spaceflight- sclerosis, and multiple sclerosis . induced changes can be particularly devastating to the health, The CNS is sensitive to oxidative injury due to its high oxygen mission performance, and quality of life of the spaceflight crew 19 20 consumption , the content of oxidizable unsaturated lipids and both acutely and chronically. The health risk from spaceflight- low levels of anti-oxidant defenses . Oxidative injury has been induced neuronal damage and potential adverse neurovascular implicated as a causative or contributory factor in a number of effects are a chief concern, examples are intracranial fluid neurodegenerative conditions, including aging, and ischemic, redistribution results in alteration of brain perfusion, neurovestib- 22–26 1,2 traumatic damage . Our preliminary studies have shown that ular problems, and cognitive alterations in astronauts caused by spaceflight and ionizing radiation cause prolonged oxidative microgravity and/or strong gravitational changes during ascent and stress and endothelial dysfunction that may lead to chronic re-entry. Long-duration spaceflights reportedly induce immune 27–30 inflammation and adverse remodeling . However, our knowl- dysregulation, which is considered a risk to astronaut safety and 3 edge about the mechanism and consequences of spaceflight mission success . Studies to date have demonstrated the altered condition-induced neuroinflammation is very limited. Because of distribution of peripheral leukocytes, a diminished function of the complexity of the processes and pathways that lead to specific leukocyte subpopulations, and skewed cytokine profiles in 3 neuroinflammation, an in-depth “omics” approach will potentially many astronauts . Many biomarkers associated with neuroinflam- provide insight to understanding the impact of environmental mation following space radiation and space stressors in rodents 4–8 insults on the CNS and the immune function that may have a and humans have been identified . However, the precise nature long-lasting impact. of immune dysregulation during spaceflight is not well understood. The goal of this study was to characterize changes in gene Evidence suggests that acute exposure to galactic cosmic rays expression using a neuroinflammatory assay panel to investigate (GCRs) induce detrimental CNS changes including an increased inflammation, neuronal function, growth, metabolism, and stress level of neuroinflammation, neuronal damage and cognitive 9–12 in mouse brain tissue after spaceflight. deficits similar to accelerated aging . Ionizing radiation has been shown to elicit neuroinflammation through direct activation of microglia cells and through the increased infiltration of immune RESULTS and inflammatory cells through the damaged blood-brain barrier Changes of gene expression in neuroinflammation panel (BBB) . Study also showed that both microgravity encountered by astronauts in space and simulated microgravity on earth induce A set of 54 differently expressed genes (DEG) significantly (p < 0.05) by changes in brain structure and function . Microgravity induces a one-way analysis of variance (ANOVA) and Tukey’sHSD (honestly 1 2 Department of Basic Sciences, Division of Biomedical Engineering Sciences (BMES), Loma Linda University School of Medicine, Loma Linda, CA 92350, USA. Department of Radiation Oncology, Wake Forest University, School of Medicine, Winston-Salem, NC 27101, USA. Department of Nutrition, Food and Exercise Sciences, Florida State University, Tallahassee, FL 32306, USA. email: xmao@llu.edu Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; J.M. Holley et al. significant difference) test segregates the ground control (GC) group gene expression that is important for apoptotic cell clearance, from the flight (FLT) group (Table 1). Clusters of genes related to phagocytosis, and proliferation were evident. Genes supporting neuronal function, neuronal cell support, immune function, and innate immune responses were also downregulated regarding anti- cellular growth and stress were significantly altered and organized viral signaling, reactive oxygen species (ROS) generation, and based on their protein’srole. SIN3A(https://www.ncbi.nlm.nih.gov/ bacterial immune response. Expression of genes that are correlated gene/?term=NM_001110350), SLC2A5 (https://www.ncbi.nlm.nih.g with DNA damage and cellular stress were upregulated. Many of ov/gene/?term=NM_019741), MERTK (https://www.ncbi.nlm.nih. these observed changes are in line with our previous findings in the gov/gene/?term=NM_008587), TREM2 (https://www.ncbi.nlm.nih. brains exposed to simulated spaceflight condition. Following gov/gene/?term=NM_025864), RIPK1 (https://www.ncbi.nlm.nih.g combined exposure of simulated microgravity and radiation, path- ov/gene/?term=NM_009068) are listed in multiple tables as they ways involved in neurogenesis, neuroplasticity, the regulation of are found to play various roles. neuropeptides, neuronal structure, stress, and cellular signaling are significantly altered . Gene expression in the brain that was analyzed after 90 days of simulated microgravity showed many Changes of gene expression associated with neuronal genes related to catalytic and oxidoreductase activities were function downregulated . However, in contrary to our findings, many genes Genes directly involved with neuronal function were universally related to stress, immune response, metabolic process, and/or downregulated and are summarized in Table 2 and Fig. 1. Overall, a inflammatory response were significantly upregulated in their study. picture of decreased neuron plasticity and signaling dysfunction is Duration of environmental stress may play a role in different painted. Those genes that are involved in neuron support cell response of gene expression profiles and pathway regulation. function were also found to be broadly downregulated and are We saw uniform downregulation of gene expression in our summarized in Table 3 and Fig. 2. Microglial cell and oligodendrocyte mouse brain samples directly related to neuron function. NRGN dysfunction were most pronounced along with the inhibition of (https://www.ncbi.nlm.nih.gov/gene/?term=NM_022029) coding myelin sheath maintenance. Levels of immune and inflammation for a postsynaptic kinase called neurogranin was found to be related gene expression were grossly downregulated except for two significantly reduced in expression. When neurogranin binds upregulated genes, H2-T23 (https://www.ncbi.nlm.nih.gov/gene/? calmodulin, the concentration of calcium needed to transduce a term=NM_010398)and SOX4 (https://www.ncbi.nlm.nih.gov/gene/? signal is increased . Given the downregulation of NRGN (https:// term=NM_009238). The summary is presented in Table 4 and Fig. 3. www.ncbi.nlm.nih.gov/gene/?term=NM_022029) and possible Generally, we see the impairment of genes related to microbial decrease in availability of neurogranin, neurons may become defense with generalized dysfunction of immunity and disinhibition more sensitive to firing. KCND1(https://www.ncbi.nlm.nih.gov/ of inappropriate inflammation. And finally, genes with roles in cellular gene/?term=NM_008423) codes for a potassium voltage-gated stress and growth are shown in Fig. 4 with downregulation in all channel involved in neurotransmitter release which has been genes with exception of HSPB1 (https://www.ncbi.nlm.nih.gov/ shown to be inhibited by some types of ROS generating gene/?term=NM_013560), GADD45G (https://www.ncbi.nlm.nih 34–36 treatments . Environmental stressor-induced ROS may be .gov/gene/?term=NM_011817), CCNG2 (https://www.ncbi.nlm.n provoking the down regulation of KCND1(https:// ih.gov/gene/?term=NM_007635), SESN1 (https://www.ncbi.nlm. www.ncbi.nlm.nih.gov/gene/?term=NM_008423). DLG4 (https:// nih.gov/gene/?term=NM_001013370), and CDC25A (https:// www.ncbi.nlm.nih.gov/gene/?term=NM_001109752) is tran- www.ncbi.nlm.nih.gov/gene/?term=NM_007658)which were scribed into a protein associated with glutamatergic receptor significantly upregulated. In general, we see an increased signaling while also potentially participating in dendritogenesis . cellular stress response, growth arrest, and a possible trend Its downregulation observed in our assay has the potential to toward more inflammatory-associated cell death. affect glutamatergic neuron signaling, and affect dendritic morphology leading to further dysfunction. NLGN2 (https:// Spaceflight-induced changes of pathways www.ncbi.nlm.nih.gov/gene/?term=NM_198862) codes for a Many pathways associated with cellular stress, inflammation, molecule which selectively mediates inhibitory synapses . Its apoptosis, and metabolism were altered by flight condition using downregulation may contribute to dysfunction at inhibitory DEG identified by Nanostring’s Advanced Analysis module path- neuron synapses. Previous study has shown that inflammation way scoring through correlation-guided gene subsetting, which induced loss of inhibitory nerve terminals or a redistribution of functionally annotated groups of genes followed by unsupervised presynaptic machinery in inhibitory neurons may increase risk for clustering of samples . Higher scores compared to normalized developing neurological disease and psychiatric illness, including gene expression general means upregulation while lower scores seizures and schizophrenia . represent downregulation. Figure 5 summarized pathway results Regarding genes involved in neuron function and plasticity, we in a plot of all pathway scores compared to GC controls. Among continue to see universal downregulation in the flight tissue these pathways, scores for angiogenesis, cytokine signaling, compared to GC controls. The gene SIN3A (https:// epigenetic regulation, and notch signaling were significantly www.ncbi.nlm.nih.gov/gene/?term=NM_001110350) behaves as increased which may indicate upregulation of the pathway a transcriptional repressor involved in cortical neuron differentia- (p < 0.05), while scores for innate immune response and oligoden- tion . In past studies the silencing of this gene in mice has drocyte function were greatly reduced in the flight group shown decreased memory consolidation . It is possible that compared to the controls which may indicate downregulation of downregulation may have similar, albeit reduced effects on mice the pathways (p < 0.05) by one-way ANOVA and Tukey’s HSD test in spaceflight. RELN (https://www.ncbi.nlm.nih.gov/gene/?term= (Fig. 6a–f). The score for microglia function was also decreased but NM_011261) codes for an extracellular matrix serine protease with trending significance (p = 0.07) (Fig. 6g). that regulates microtubule function and plays a role in the layering of neurons in the cerebral cortex, among other structures . Its enzymatic activity that is important for cell DISCUSSION adhesion and reduced transcription, as seen in our flight mice, Our data showed robust changes in gene expression profiles after could lead to further reduction of nervous system plasticity. spaceflight. Genes supporting neuronal synaptic signaling and SLC2A5 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_019741) migration were significantly downregulated. Decreased expression in a gene responsible for the GLUT5 (https://www.ncbi.nl of genes important for oligodendrocyte differentiation and myelin m.nih.gov/gene/2700445) fructose transporter that has been sheath maintenance was observed. Downregulation of microglial found in numerous areas including the BBB, and the npj Microgravity (2022) 35 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; J.M. Holley et al. Table 1. Summary of the 54 differentially expressed genes in flight (FLT) mice brains relative to habitat ground controls (GC). Gene name (mRNA) Functions Log2 fold Δ LC limit (log2) UC limit (log2) p value Optn Autophagy, cell cycle, microglia function −0.435 −0.547 −0.324 1.72E−05 Mertk Autophagy, microglia function −0.368 −0.499 −0.237 0.000256 Cd74 Adaptive immune response, inflammatory signaling −1 −1.45 −0.548 0.00146 Plp1 Oligodendrocyte function −0.363 −0.54 −0.187 0.00238 Sox4 Microglia function, Wnt 0.314 0.156 0.473 0.00304 H2-T23 Adaptive immune response, astrocyte function, inflammatory signaling, 0.357 0.165 0.548 0.00447 innate immune response, matrix remodeling Irf7 Apoptosis, inflammatory signaling, innate immune response −1.06 −1.63 −0.487 0.00544 Reln Growth factor signaling, matrix remodeling −0.535 −0.836 −0.235 0.00583 Prkar2b Apoptosis, cell cycle, growth factor signaling −0.32 −0.503 −0.137 0.00641 Agt Astrocyte function 0.432 0.184 0.679 0.00657 Aldh1l1 Astrocyte function −0.345 −0.542 −0.147 0.00659 Brd2 Epigenetic regulation −0.182 −0.286 −0.0773 0.00667 Ccng2 Cell cycle, DNA damage 0.292 0.119 0.465 0.00785 Hspb1 Angiogenesis, astrocyte function, cellular stress, growth factor 0.706 0.284 1.13 0.00825 Dlg4 Adaptive immune response, angiogenesis, cytokine signaling, growth −0.279 −0.45 −0.108 0.00947 factor signaling, neurons and neurotransmission Cd6 Matrix remodeling −0.972 −1.56 −0.388 0.00978 Csk Adaptive immune response, angiogenesis, growth factor −0.236 −0.384 −0.0878 0.0108 F3 Microglia function −0.304 −0.496 −0.113 0.011 Csf1r Cytokine signaling, growth factor signaling, microglia function −0.209 −0.341 −0.0771 0.0111 Uty Epigenetic regulation 0.308 0.113 0.502 0.0112 Kcnd1 Microglia function, neurons and neurotransmission −0.893 −1.45 −0.337 0.0117 Cyp27a1 Inflammatory signaling −0.805 −1.32 −0.292 0.0132 Slc2a5 Microglia function −0.623 −1.02 −0.226 0.0132 Sin3a Epigenetic regulation −0.43 −0.715 −0.146 0.0142 E2f1 Apoptosis, cell cycle, cellular stress, notch −0.709 −1.17 −0.25 0.0143 Egfr Adaptive immune response, angiogenesis, astrocyte function −0.509 −0.841 −0.177 0.0149 Tmem206 Microglia function −0.239 −0.407 −0.0701 0.0196 C5ar1 Inflammatory signaling, neurons and neurotransmission −0.851 −1.44 −0.259 0.0201 Opalin Oligodendrocyte function −0.546 −0.935 −0.157 0.0205 Ncf1 Adaptive immune response, angiogenesis, cellular stress −0.539 −0.918 −0.159 0.0214 Mre11a DNA damage −0.383 −0.665 −0.1 0.0241 Ikbkg Adaptive immune response, apoptosis, inflammatory signaling −0.27 −0.47 −0.0697 0.0246 Prkaca Adaptive immune response, angiogenesis, apoptosis, Wnt −0.144 −0.251 −0.0371 0.0248 Apoe Astrocyte function, cellular stress, lipid metabolism, microglia 0.296 0.072 0.521 0.027 St8sia6 Microglia function −0.479 −0.835 −0.123 0.0272 Gpr62 Oligodendrocyte function −0.418 −0.736 −0.1 0.0276 Hpgds Lipid metabolism −0.527 −0.922 −0.132 0.0282 Sesn1 DNA damage 0.226 0.0509 0.4 0.0298 Cdc25a Cell cycle, DNA damage 0.218 0.0481 0.389 0.0307 Lmna Apoptosis, cell cycle, microglia function −0.284 −0.507 −0.0596 0.0324 Map2k4 Adaptive immune response, apoptosis, cellular stress −0.2 −0.359 −0.041 0.0334 Irf8 Inflammatory signaling, microglia function −0.51 −0.914 −0.106 0.0352 Trem2 Adaptive immune response, inflammatory signaling, microglia −0.461 −0.833 −0.088 0.0359 Mpeg1 Inflammatory signaling −0.292 −0.528 −0.0551 0.0363 Rhoa Angiogenesis, autophagy, growth factor signaling, Wnt 0.196 0.0351 0.357 0.0381 Tarbp2 Epigenetic regulation 0.195 0.0346 0.355 0.0384 Ripk1 Apoptosis, innate immune response, NF-kB −0.63 −1.14 −0.119 0.0389 Gadd45g Cell cycle, DNA damage, growth factor signaling 0.392 0.0686 0.716 0.0389 Brd3 Epigenetic regulation −0.305 −0.557 −0.0526 0.0394 Nrgn Neurons and neurotransmission −0.16 −0.295 −0.0261 0.0413 Nlgn2 Matrix remodeling, neurons and neurotransmission −0.306 −0.562 −0.0486 0.042 Bbc3 Apoptosis, DNA damage −0.538 −0.987 −0.0897 0.0432 Rgl1 Growth factor signaling, microglia function 0.105 0.0146 0.196 0.0462 Creb1 Adaptive immune response, carbohydrate metabolism, DNA damage, 0.235 0.0291 0.44 0.0493 innate immune response, notch LC lower confidence, UC upper confidence. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 35 1234567890():,; J.M. Holley et al. Table 2. Summary of significantly altered gene expressions by spaceflight related to neuronal function. Gene p value Log2 fold Δ Related molecule function NRGN 0.0413 −0.16 A Ca2+ dependent intracellular charge transducer. DLG4 0.00947 −0.279 Postsynaptic density protein associated with glutamatergic receptor signaling. NLGN2 0.042 −0.306 Postsynaptic cell adhesion molecule mediating inhibitory synapses. SIN3A 0.0142 −0.43 Involved in cortical neuron differentiation and callosal axon elongation. RELN 0.00583 −0.535 Regulates microtubule function in neurons and neuronal migration. SLC2A5 0.0132 −0.623 Fructose transporter found in Purkinje cells and the blood-brain barrier. KCND1 0.0117 −0.893 K+ voltage-gated channel involved in neurotransmitter release. transporter, as described previously . With reduced activity, micro- glial cells may have less capacity to utilize fructose as an energy source and may be more prone to suffer from metabolic related dysfunction. Oligodendrocytes are the myelin-forming cells of the CNS. It plays critical roles in axonal metabolic support, myelination, and 57,58 providing nutritional support to neurons . A couple of key genes involved in the oligodendrocyte function have been significantly downregulated in the flight mice. PLP1 (https:// www.ncbi.nlm.nih.gov/gene/?term=NM_011123) codes for a major myelin protein that plays an important role in its formation and maintenance . The implication of significantly downregu- lated PLP1 indicates impaired neuron axonal function with the possibility of long-term neurodegeneration. OPALIN (https:// www.ncbi.nlm.nih.gov/gene/?term=NM_153520) plays a role in oligodendrocyte cytoskeletal remolding and morphology. Down- Fig. 1 Spaceflight-induced changes of gene expression related to regulation of this gene observed in our study may be an indication neuronal function. Bar graph summarizing log2 fold-changes of of decreased activity of oligodendrocytes. These findings are significantly differentially expressed genes (DEG) (p < 0.05) in the further reinforced by a significantly decreased oligodendrocyte flight (FLT) group compared to the ground control (GC) group in function pathway score (Fig. 6a). genes directly related to neuronal function. N = 6/group. p values In terms of gene expression that associated with general are calculated using one-way analysis of variance (ANOVA) and immune functions and inflammation, only two genes were found Tukey’s HSD (honestly significant difference) test. Source data are provided as a Source Data file. to be significantly upregulated in our flight mice population, H2- T23 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_010398) and 43,44 hippocampus . The downregulation of this gene suggests the SOX4 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_009238). decreased ability of cells to utilize fructose as a potential energy H2-T23 expression is essential for immunological protection and source. regulation . HT-T23 dependent T-cell inhibitory interactions aid in Level of gene expression related to neuron support cells, like preventing expansion of autoreactive CD4 T-cells and collateral microglial cells and oligodendrocytes were also seen universally autoimmune diseases . Upregulation of this gene may be a downregulated. Microglia are the resident phagocytes of the innate protective response to generalized inflammation in the flight immune system in the brain and play an important role in cytokine mice. SOX4 directly regulates Innate-like γδ T-cell (Tγδ17), a major production . Their activation is closely associated with environmental source of interleukin-17 (IL-17) (https://www.ncbi.nlm.nih.gov/ stress and microglial dysfunction is implicated in many neurological gene/?term=NM_010552). Increased SOX4 expression may lead 46 62 disorders and diseases .CSF1R (https://www.ncbi.nlm.nih.gov/gene/? to an increase of this cytokine expression in the flight mice . term=NM_001037859) is a gene coding for a cell-surface receptor The gene RIPK1 (https://www.ncbi.nlm.nih.gov/gene/?term=NM which binds to colony stimulating factor-1 (CSF1) (https:// _009068) has been shown to be a promotor of survival, apoptotic 63,64 www.ncbi.nlm.nih.gov/gene/?term=NM_001113529) and interleukin- and inflammatory signaling pathways . However, mice defi- 34 (IL-34) (https://www.ncbi.nlm.nih.gov/gene/?term=NM_0011 cient in RIPK1 demonstrated loss of inhibition of a necroptosis 35100) . CSF1R null mice have been shown to have fewer phagocytic pathway which promoted the release of necroptotic damage- 48 65 cells and reduced survival . Given these findings we suggest that associated molecular patterns . Downregulation of RIPK1 flight mice may have a reduced capacity to generate microglial cells observed in our study may lead to a similar reduction of in the nervous system. MERTK (https://www.ncbi.nlm.nih.gov/gene/? appropriate apoptosis and an increase in necroptosis resulting in term=NM_008587) creates a kinase protein in innate immune cells undesirable inflammation. Overall, taking these findings in which mediates engulfment of apoptotic cells and acts as a negative conjunction with a significantly increased cytokine signaling 49–51 regulator of inflammation . Downregulation of this gene may pathway score (Fig. 6b), inflammatory signaling appears to be contribute to immune dysregulation in our flight mice. TREM2 significantly dysregulated in our spaceflight mice. (https://www.ncbi.nlm.nih.gov/gene/?term=NM_031254)codes fora Immune dysfunction is further illustrated via the downregulation 52,53 regulator of microglial chemotaxis and phagocytosis of apoptotic . of two genes important for antimicrobial defense. MPEG1 (https:// In previous in vivo studies with microglial lacking TREM2, decreased www.ncbi.nlm.nih.gov/gene/?term=NM_010821) is a pore-forming migration to areas of apoptotic neurons was shown .NCF1(https:// bactericidal molecule of the innate immune system. It has been www.ncbi.nlm.nih.gov/gene/?term=NM_001286037) codes for a key shown to be required for the activity of ROS and nitric oxide and 55,56 66,67 subunit of the phagocyte NADPH oxidase system . Downregula- their antibacterial effects . OPTN (https://www.ncbi.nlm.nih.gov/ tion suggests a reduced capacity for microglial cells to generate an gene/?term=NM_181848) codes for a protein that plays a key role effective microbial defense. SLC2A5 codes for the GLUT5 fructose in bacterial autophagic clearance. Deficient mice have also been npj Microgravity (2022) 35 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA J.M. Holley et al. Table 3. Summary of significantly altered gene expressions by spaceflight related to neuronal support cell function. Gene p value Log2 fold Δ Related molecule function CSF1R 0.0111 −0.209 Cell-surface receptor regulating development, proliferation and differentiation. PLP1 0.00283 −0.363 Major myelin protein in the CNS. MERTK 0.000256 −0.368 Signaling protein in innate immune cells mediating engulfment of apoptotic cells. TREM2 0.0359 −0.461 Regulates microglial chemotaxis and phagocytosis of apoptotic neurons. NCF1 0.0214 −0.539 Subunit of NADPH oxidase required for superoxidase production. OPALIN 0.0205 −0.546 Regulator of oligodendrocyte cytoskeletal remolding and morphology. SLC2A5 0.0132 −0.623 Fructose transporter found in microglial cells. proliferation, and stress response. Two upregulated genes play an important part in cell cycle arrest during times of cellular stress. GADD45G (https://www.ncbi.nlm.nih.gov/gene/?term=NM_011817) has been found to play a role in activating checkpoints in the cell cycle following exposure of cells to irradiation .CCNG2 (https:// www.ncbi.nlm.nih.gov/gene/?term=NM_007635) contributes to cell cycle arrest during DNA damage and is upregulated in response to diverse stimuli, including hypoxia . Additionally, expression of CCNG2 is found to be significantly increased in cell cycle-arrested and terminally differentiated cells . Upregulation of these genes may be due to exposure to irradiation, among other stressors, during spaceflight. The downregulation of genes involved in promoting transcrip- tion and cell cycle progression was also seen. BRD2 (https:// www.ncbi.nlm.nih.gov/gene/?term=NM_010238) codes for a Fig. 2 Spaceflight-induced changes of gene expression related to nuclear kinase involved in regulating the expression of cell cycle neuronal support cell function. Bar graph summarizing log2 fold- genes via binding to multiple E2Fs, a family of transcription changes of significantly differentially expressed genes (DEG) factors . E2F1 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_ (p < 0.05) in the flight (FLT) group compared to the ground control 007891) regulates activation of DNA replication and G1/S (GC) group in genes directly related to neuronal supporting cell transition when interacting with BRD2 . BRD2 also interacts with function. N = 6/group. P values are calculated using one-way BRD3, a chromatin reader with roles in regulating transcription . analysis of variance (ANOVA) and Tukey’s HSD (honestly significant BRD3 (https://www.ncbi.nlm.nih.gov/gene/?term=NM_001113573 difference) test. Source data are provided as a Source Data file. ) further regulates transcription by promoting the binding of the transcription factors to their targets . SIN3A (https:// shown to have impaired interferon regulatory factors www.ncbi.nlm.nih.gov/gene/?term=NM_001110350) codes for a (https://www.ncbi.nlm.nih.gov/gene/?term=NM_001159393) sig- transcription factor which regulates cell cycle progression by naling and reduced response to toll-like receptor stimulation .In repressing gene expression for cell cycle inhibitor . It has been summary, these results imply the impaired antibacterial capabilities linked to functional cellular changes in proliferation, cell cycle, and and increased viral susceptibility in the flight mouse brain stem cell function in mice models . Downregulation of these compared to GC group. genes involved in the promotion of cellular proliferation may Our data indicate that the regulation of innate immune further amplify the upregulation of genes with roles in cell cycle response was impaired following spaceflight. The pathway score arrest. These changes are similarly reinforced by a significantly for the innate immune response was significantly reduced in the increased epigenetic regulation pathway score (Fig. 6e) indicating flight group compared to GCs (Fig. 6c). The cell type score for that there may be an increased level of modulating and checks exhausted CD8 cells is also greatly reduced for the flight group being implemented during transcription, due to spaceflight. A compared to controls and may indicate a dysfunctional pheno- recent study has shown that cell cycle regulation is closely linked type in CD8 T-cell response. T-cell exhaustion represents an to hippocampal neurogenesis which plays critical roles in memory adaptive response to conditions of chronic antigen stimulation and learning . Neurogenesis is a dynamic process that involves and inflammation , as well as promoting tissue repair following proliferation and differentiation of stem and progenitor cells, or an inflammatory injury . Functionally exhausted CD8+ cells may survival and maturation of newborn neurons . Environmental result in a severely compromised innate immune response . stressors have been shown to modulate cell cycle progression, Cytochrome P450 27A1 (CYP27A1) (https://www.ncbi.nlm.nih.gov/ particular in the G1 phase . These observed changes of gene gene/?term=NM_024264) plays an important role in the metabolism expression related to the cell cycle in our study may indicate of cholesterol and cholesterol-related compounds .In humans, altered regulation of neurogenesis. complete CYP27A1 deficiency leads to nodule formation in the brain Many downregulated genes were known to be involved in which may lead to dementia, cerebellar ataxia, and spinal cord cellular stress responses. MAP2K4 (https://www.ncbi.nlm.nih.gov/ paresis . This altered cholesterol metabolism closely associates with gene/?term=NM_009157) is a kinase involved with responses to inflammatory responses involved in the pathogenesis of AD cellular stress including apoptosis, inflammation, cellular prolifera- progression .Indeficient mice, a significant increase in cholestanol tion, and neurodegeneration. A deficiency of MAP2K4 has been in the brain is observed . Downregulation of CYP27A1, as seen in shown to result in cellular susceptibility to stress-induced our flight mice, may lead to neurodegeneration and inflammation in more chronic settings or to a milder degree. apoptosis and growth inhibition . However, it has been demon- Fourteen genes that were significantly altered in the flight mice strated that disruption of both MAP2K4 and MAP2K7 (https:// compared to GCs were found to have direct roles in cellular growth, www.ncbi.nlm.nih.gov/gene/?term=NM_001042557) genes was Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 35 J.M. Holley et al. Table 4. Summary of significantly altered gene expressions by spaceflight related to immune function and inflammation. Gene p value Log2 fold Δ Related molecular function H2-T23 0.00447 0.357 Immune regulation and protection through NK and T-cell suppression. SOX4 0.00304 0.314 Transcription factor regulating key genes in Thelper17 cells. CSK 0.0108 −0.236 Negative regulator of T-cell receptor signaling. IKBKG 0.0246 −0.27 Subunit of IKK complex with cardinal role in stimulating NF-κB regulation. MPEG1 0.0363 −0.292 Pore-forming bactericidal effector molecule of the innate immune system. MERTK 0.000256 −0.368 Signaling protein in innate immunity for a negative regulator of inflammation. OPTN 0.0000172 −0.435 Ubiquitin-binding protein important for bacterial autophagic clearance. TREM2 0.0359 −0.461 Inhibitor of neuroinflammation via suppression of NF-kB signaling. ST8SIA6 0.0272 −0.479 A sialyltransferase important for immune suppression and modulation. IRF8 0.0352 −0.51 Transcription factor for anti-viral dendritic cells and IFN-inducible genes. HPGDS 0.0282 −0.527 Enzymatic mediator of prostaglandin D2 creation. RIPK1 0.0389 −0.63 Promotor of survival, apoptotic and inflammatory signaling pathways. CYP27A1 0.0132 −0.805 Cytochrome P450 enzyme important in the metabolism of cholestrol. C5AR1 0.0201 −0.852 Receptor for the chemotactic and inflammatory anaphylatoxin, C5a. CD6 0.00978 −0.972 Stimulatory molecule promoting T-cell activation, proliferation, and adhesion. CD74 0.00146 −1 Chaperone protein involved with MHC class II antigen presentation. IRF7 0.00544 −1.06 Transcriptional regulator of type I interferon dependent immune responses. Fig. 3 Spaceflight-induced changes of gene expression related to immune function and inflammation. Bar graph summarizing log2 fold- changes of significantly differentially expressed genes (DEG) (p < 0.05) in the flight (FLT) group compared to the ground control (GC) group in genes directly related to immune function and inflammation. N= 6/group. p values are calculated using one-way analysis of variance (ANOVA) and Tukey’s HSD (honestly significant difference) test. Source data are provided as a Source Data file. Fig. 4 Spaceflight-induced changes of gene expression related to cellular stress and growth function. Bar graph summarizing log2 fold- changes of significantly differentially expressed genes (DEG) (p < 0.05) in the flight (FLT) group relative to the ground control (GC) group in genes directly related to cellular stress and growth function. N = 6/group. p values are calculated using one-way analysis of variance (ANOVA) and Tukey’s HSD (honestly significant difference) test. Source data are provided as a Source Data file. npj Microgravity (2022) 35 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA J.M. Holley et al. Fig. 5 Spaceflight-induced changes of pathway scores. Summarized pathway scores in flight (FLT) group vs. ground control (GC). *Significantly upregulated pathways (p < 0.05), include: cytokine signaling, Angiogenesis, Epigenetic regulation, and Notch. **Significantly (p < 0.05) or strong trend (p = 0.07) downregulated pathways, include: Oligodendrocyte function, Innate immune response, and Microglia function. p values are by one-way ANOVA and Tukey’s post hoc test. Source data are provided as a Source Data file. Fig. 6 Spaceflight-induced changes in nueroinflammation pathway scores among flight (FLT) and ground controls (GC) groups. Boxplots depict pathway scores on the y-axis and the experimental conditions on the x-axis. N = 5–6 /group. p values are calculated using one-way analysis of variance (ANOVA) and Tukey’s HSD (honestly significant difference) test. a Oligodendrocyte function score p < 0.05, b cytokine signaling score p < 0.05, c innate immune response score p < 0.05, d angiogenesis score p < 0.05, e epigenetic regulation score p < 0.05, f notch score p < 0.01, and g Microglia function score p = 0.07. Boxes are the range between first (25%) and the third (75%) quartile, the center line is the median, the whiskers include the variability those quartiles. Source data are provided as a Source Data file. required to thoroughly block cellular growth caused by environ- protein biomarkers in response to stressors were observed in a 87 41 mental stressors . MRE11A (https://www.ncbi.nlm.nih.gov/gene/? previous flight study . Given this finding, gene expression profiles in term=NM_018736), a part of an exonuclease complex central to specific regions of the brain in response to spaceflight will be the cellular DNA damage response was downregulated. The MRE11 investigated in future studies. More recently, single-cell sequencing complex is essential to vertebrates and defects lead to sensitivity to technologies, including transcriptomics, are available by directly DNA damage and cell cycle checkpoints deficiency .Its down- measuring multiple molecular signatures in specific brain cells, regulation may point to a certain level of dysfunction leading to an providing robust molecular identity of specific cell types . impaired response to cellular stress leaving the flight mice more Brain tissues were dissected and prepared for analysis within 60 h after landing. It is possible that observed changes in gene expression vulnerable to DNA damage. Different brain regions have different vulnerability to environ- profiles in neuroinflammation are a compensatory acute response to mental stressors due to structural and biological heterogeneity of the the acceleration and intense noise of launch or reflect changes to the regions, cell types and molecular networks . Regional differences of combined response of spaceflight environment and the landing. Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2022) 35 J.M. Holley et al. In ordertoevaluate theimmuneresponsetospaceflight environ- RNA isolation and gene expression profiling ment, in the further study, mice will be euthanized in the orbit and Isolation of total RNA from the brain tissues was performed using brain tissues will be immediately preserved for analysis on the DNA/RNA/miRNA Universal Kit (Qiagen, # 80224) according to the ground. Furthermore, in order to test the hypothesis that neuroin- manufacturer’s instruction. Briefly, tissue homogenization was flammation plays an important role in developing spaceflight- performed using 1.5 mm beads (Benchmark, # 1032-15) on Minilys induced stress response in the brain, an anti-oxidant or anti- homogenizer (Bertin Technologies) in RLT Plus buffer. The purity and inflammatory compound will be injected to mouse before or during concentration of the eluted RNA were measured using Nanodrop the flight to determine the protective effect against oxidative 2000 (Thermo Fisher Scientific, Waltham, MA) and stored at −80 °C damage in the brain. until further analysis. RNA samples (20 ng/ul) were then shipped to It is noted from our study that among those genes which Nanostring (Technologies Seattle, WA) and gene expression profiling expression were significantly altered by spaceflight, markedly more ® of brain tissues was conducted using the nCounter neuroflamma- gene expressions are being downregulated than upregulated. It is tion pathways panel. The panel includes 757 genes covering the speculated that many genes may be upregulated during spaceflight core pathways and processes that define neuroimmune interactions and the early phase after landing as an adaptation response. During and 13 potential housekeeping genes for normalization. RNA re-adaptation, the expression of these previous upregulated genes samples (100 ng each) on 2 cartridges were used for the Gene could be then downregulated as a compensatory response, or when Expression Assay with Mouse Neuroinflammation panel performed protein synthesis from upregulated genes has been completed, on the nCounter MAX system (https://nanostring.com/wp-content/ these genes expression may be downregulated . uploads/MAN-C0035_nCounter_Analysis_System_MAX_FLEX.pdf), a Collectively, changes noted in our study indicate that exposure multi-channel epifluorescence scanner with Nanostring Advanced to the spaceflight environment induces significant changes in Analysis Module plugin for QC, normalization, and differential gene expression and signaling pathways related to neuronal expression analysis (DE). Data files generated from nCounter system function, immune regulation, growth and metabolic function. were analyzed using nSolver 4.0 software with the Advanced Study showed that chronically dysfunctional and deregulated Analysis module for QC (quality control), normalization, DE analysis, pathways, including cytokine signaling (Fig. 6b), epigenetic and gene-set enrichment analysis. Data normalization included regulation (Fig. 6e), and notch signaling (Fig. 6f) play important 91 2 steps: Positive control normalization to correct platform-associated roles in disease development , and have been associated with variation and Codeset content normalization using the house- several progressive neurodegenerative diseases . Our observed keeping (HK) genes to correct variability of input samples. Geometric changes of a dysregulated inflammatory response, downregulated means of selected HK probes were used to normalize counts of the T-cell response, and reduced microglia signaling (Fig. 6g) might samples and DEGs were generated from the normalized counts. also have an impact on brain structure and function, and further lead to chronic neuroinflammation. It has been shown that here are strong correlations between neuroinflammatory biomarkers, Statistical analysis brain morphology and behavioral outcomes . It may be Gene expression profiling data were analyzed using nSolver hypothesized that chronical changes of observed gene expression analysis and the advanced analysis module software. Advanced profiles could have long-term effects on brain morphology and analysis module software uses open-source R program for organism behavior. pathway scoring and gene-set enrichment analysis. Using the Reactome pathway database annotations, pathway scores are derived by calculating the first principle component of pathway MATERIALS AND METHODS genes’ normalized expression and data are summarized from Flight and ground control conditions changes of a gene set within a given pathway into a single score. Ten-weeks-old male C57BL/6 mice (Jackson Laboratories, Inc. Bar To further analyze the associated pathways of the DEGs, Kyoto Harbor, ME) at the time of launch, were flown for NASA’s ninth Encyclopedia of Genes and Genomes (KEGG) pathways analysis Rodent Research experiment (RR-9) on SpaceX-12 for a 35-day was performed using biological process database to identify the mission and lived in NASA’s Rodent Habitats aboard the annotated sets of genes based on the biological processes in International Space Station. All FLT mice were maintained at an which they participate. Significantly differential expression genes ambient temperature of 26–28 °C with a 12-h light/dark cycle were presented in the tables with individual genes using log during the flight. Habitat GC mice were kept under similar housing (p value) andlog fold change compared to the GC group. conditions, including temperature, humidity and carbon dioxide Pathview module was used to display upregulated genes or (CO ) levels using 48-h delayed telemetry data from the FLT downregulated genes overlaid on KEGG pathways. For DEG and group. Water and food bar diet specifically designed by NASA pathway analysis, p < 0.05 was considered statistically significant were provided ad libitum to FLT and GC groups. All mice received between FLT and GC groups by one-way ANOVA and Tukey’s the same access to food and water. NASA-Ames Research Center HSD test. and KSC Institutional Animal Care and Use Committees approved this flight study. The study has been done in strict accordance Reporting summary with the recommendations in the Guide for the Care and Use of Further information on research design is available in the Nature Laboratory Animals of the National Institute of Health. Research Reporting Summary linked to this article. Mouse brain dissection after spaceflight Within 38 ± 4 h of splashdown, the FLT mice were rapidly DATA AVAILABILITY euthanized in 100% CO . The GC mice were euthanized with the The authors declare that source data supporting the findings of this study with the same method after 38 days of GC housing. Shortly after figures of the article are provided with this paper. euthanasia, brains were removed and bisected along the midline and coronally within the half hemispheres. The right caudal half hemisphere of the brain (containing mid- and hindbrain) from CODE AVAILABILITY each mouse (n = 5–6 per group) was placed in a sterile cryovial, All of the significant gene data obtained in this study were deposited in the NCBI’s snap frozen in liquid nitrogen and kept at −80 °C prior to use. GEO, and are accessible through GEO accession number GSE186278. npj Microgravity (2022) 35 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA J.M. Holley et al. Received: 10 January 2022; Accepted: 15 July 2022; 31. Kwong, L. N. et al. Biological validation of RNA sequencing data from formalin- fixed paraffin-embedded primary melanomas. JCO Precis Oncol. 2018,1–19 (2018). 32. Santucci, D. et al. Evaluation of gene, protein and neurotrophin expression in the brain of mice exposed to space environment for 91 days. PLoS ONE 7, e40112 (2012). REFERENCES 33. Hoffman, L., Chandrasekar, A., Wang, X., Putkey, J. A. & Waxham, M. 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MKK7 is an essential component of the Jnk signal transduction © The Author(s) 2022 pathway activated by proinflammatory cytokines. Genes Dev. 15, 1419–1426 (2001). npj Microgravity (2022) 35 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA

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