Fundamental Biological Features of Spaceflight: Advancing the Field to Enable Deep-Space Exploration.

Research on astronaut health and model organisms have revealed six features of spaceflight biology that guide our current understanding of fundamental molecular changes that occur during space travel. The features include oxidative stress, DNA damage, mitochondrial dysregulation, epigenetic changes (including gene regulation), telomere length alterations, and microbiome shifts. Here we review the known hazards of human spaceflight, how spaceflight affects living systems through these six fundamental features, and the associated health risks of space exploration. We also discuss the essential issues related to the health and safety of astronauts involved in future missions, especially planned long-duration and Martian missions.

Kinase signalling in excitatory neurons regulates sleep quantity and depth.

Progress has been made in the elucidation of sleep and wakefulness regulation at the neurocircuit level1,2. However, the intracellular signalling pathways that regulate sleep and the neuron groups in which these intracellular mechanisms work remain largely unknown. Here, using a forward genetics approach in mice, we identify histone deacetylase 4 (HDAC4) as a sleep-regulating molecule. Haploinsufficiency of Hdac4, a substrate of salt-inducible kinase 3 (SIK3)3, increased sleep. By contrast, mice that lacked SIK3 or its upstream kinase LKB1 in neurons or with a Hdac4S245A mutation that confers resistance to phosphorylation by SIK3 showed decreased sleep. These findings indicate that LKB1-SIK3-HDAC4 constitute a signalling cascade that regulates sleep and wakefulness. We also performed targeted manipulation of SIK3 and HDAC4 in specific neurons and brain regions. This showed that SIK3 signalling in excitatory neurons located in the cerebral cortex and the hypothalamus positively regulates EEG delta power during non-rapid eye movement sleep (NREMS) and NREMS amount, respectively. A subset of transcripts biased towards synaptic functions was commonly regulated in cortical glutamatergic neurons through the expression of a gain-of-function allele of Sik3 and through sleep deprivation. These findings suggest that NREMS quantity and depth are regulated by distinct groups of excitatory neurons through common intracellular signals. This study provides a basis for linking intracellular events and circuit-level mechanisms that control NREMS.

ATF7-Dependent Epigenetic Changes Are Required for the Intergenerational Effect of a Paternal Low-Protein Diet.

Paternal dietary conditions may contribute to metabolic disorders in offspring. We have analyzed the role of the stress-dependent epigenetic regulator cyclic AMP-dependent transcription factor 7 (ATF7) in paternal low-protein diet (pLPD)-induced gene expression changes in mouse liver. Atf7+/- mutations cause an offspring phenotype similar to that caused by pLPD, and the effect of pLPD almost vanished when paternal Atf7+/- mice were used. ATF7 binds to the promoter regions of ∼2,300 genes, including cholesterol biosynthesis-related and tRNA genes in testicular germ cells (TGCs). LPD induces ATF7 phosphorylation by p38 via reactive oxygen species (ROS) in TGCs. This leads to the release of ATF7 and a decrease in histone H3K9 dimethylation (H3K9me2) on its target genes. These epigenetic changes are maintained and induce expression of some tRNA fragments in spermatozoa. These results indicate that LPD-induced and ATF7-dependent epigenetic changes in TGCs play an important role in paternal diet-induced metabolic reprograming in offspring.

Dual-specificity phosphatases 13 and 27 as key switches in muscle stem cell transition from proliferation to differentiation.

Muscle regeneration depends on muscle stem cell (MuSC) activity. Myogenic regulatory factors, including myoblast determination protein 1 (MyoD), regulate the fate transition of MuSCs. However, the direct target of MYOD in the process is not completely clear. Using previously established MyoD knock-in (MyoD-KI) mice, we revealed that MyoD targets dual-specificity phosphatase (Dusp) 13 and Dusp27. In Dusp13:Dusp27 double knock-out (DKO) mice, the ability for muscle regeneration after injury was reduced. Moreover, single-cell RNA sequencing of MyoD-high expressing MuSCs from MyoD-KI mice revealed that Dusp13 and Dusp27 are expressed only in specific populations within MyoD-high MuSCs, which also express Myogenin. Overexpressing Dusp13 in MuSCs causes premature muscle differentiation. Thus, we propose a model where DUSP13 and DUSP27 contribute to the fate transition of MuSCs from proliferation to differentiation during myogenesis.

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Association between idiopathic hypersomnia and a genetic variant in the PER3 gene.

We aim to identify genetic markers associated with idiopathic hypersomnia, a disabling orphan central nervous system disorder of hypersomnolence that is still poorly understood. In our study, DNA was extracted from 79 unrelated patients diagnosed with idiopathic hypersomnia with long sleep time at the National Reference Center for Narcolepsy-France according to very stringent diagnostic criteria. Whole exome sequencing on the first 30 patients with idiopathic hypersomnia (25 females and 5 males) allowed the single nucleotide variants to be compared with a control population of 574 healthy subjects from the French Exome project database. We focused on the identification of genetic variants among 182 genes related to the regulation of sleep and circadian rhythm. Candidate variants obtained by exome sequencing analysis were then validated in a second sample of 49 patients with idiopathic hypersomnia (37 females and 12 males). Our study characterised seven variants from six genes significantly associated with idiopathic hypersomnia compared with controls. A targeted sequencing analysis of these seven variants on 49 other patients with idiopathic hypersomnia confirmed the relative over-representation of the A➔C variant of rs2859390, located in a potential splicing-site of PER3 gene. Our findings support a genetic predisposition and identify pathways involved in the pathogeny of idiopathic hypersomnia. A variant of the PER3 gene may predispose to idiopathic hypersomnia with long sleep time.

DNAM-1 regulates Foxp3 expression in regulatory T cells by interfering with TIGIT under inflammatory conditions.

Regulatory T (Treg) cells that express forkhead box P3 (Foxp3) are pivotal for immune tolerance. Although inflammatory mediators cause Foxp3 instability and Treg cell dysfunction, their regulatory mechanisms remain incompletely understood. Here, we show that the transfer of Treg cells deficient in the activating immunoreceptor DNAM-1 ameliorated the development of graft-versus-host disease better than did wild-type Treg cells. We found that DNAM-1 competes with T cell immunoreceptor with Ig and ITIM domains (TIGIT) in binding to their common ligand CD155 and therefore regulates TIGIT signaling to down-regulate Treg cell function without DNAM-1-mediated intracellular signaling. DNAM-1 deficiency augments TIGIT signaling; this subsequently inhibits activation of the protein kinase B-mammalian target of rapamycin complex 1 pathway, resulting in the maintenance of Foxp3 expression and Treg cell function under inflammatory conditions. These findings demonstrate that DNAM-1 regulates Treg cell function via TIGIT signaling and thus, it is a potential molecular target for augmenting Treg function in inflammatory diseases.

Transcriptome Analysis by RNA Sequencing of Mouse Embryonic Stem Cells Stocked on International Space Station for 1584 Days in Frozen State after Culture on the Ground.

As a space project, in "Stem Cells" by the Japan Aerospace Exploration Agency (JAXA), frozen mouse ES cells were stored on the International Space Station (ISS) in the Minus Eighty Degree Laboratory Freezer for ISS (MELFI) for 1584 days. After taking these cells back to the ground, the cells were thawed and cultured, and their gene expressions were comprehensively analyzed using RNA sequencing in order to elucidate the early response of the cells to long-time exposure to space radiation consisting of various ionized particles. The comparisons of gene expression involved in double-stranded break (DSB) repair were examined. The expressions of most of the genes that were involved in homologous recombination (HR) and non-homologous end joining (NHEJ) were not significantly changed between the ISS-stocked cells and ground-stocked control cells. However, the transcription of Trp53inp1 (tumor protein 53 induced nuclear protein-1), Cdkn1a (p21), and Mdm2 genes increased in ISS-stocked cells as well as Fe ion-irradiated cells compared to control cells. This suggests that accumulated DNA damage caused by space radiation exposure would activate these genes, which are involved in cell cycle arrest for repair and apoptosis in a p53-dependent or -independent manner, in order to prevent cells with damaged genomes from proliferating and forming tumors.

Comparing effects of microgravity and amyotrophic lateral sclerosis in the mouse ventral lumbar spinal cord.

Microgravity (MG) exposure and motor neuron diseases, such as amyotrophic lateral sclerosis (ALS), lead to motor deficits, including muscle atrophy and loss of neuronal activity. Abnormalities in motor neurons and muscles caused by MG exposure can be recovered by subsequent ground exercise. In contrast, the degeneration that occurs in ALS is irreversible. A common phenotype between MG exposure and ALS pathology is motor system abnormality, but the causes may be different. In this study, to elucidate the motor system that is affected by each condition, we investigated the effects of MG and the human SOD1 ALS mutation on gene expression in various cell types of the mouse ventral lumbar spinal cord, which is rich in motor neurons innervating the lower limb. To identify cell types affected by MG or ALS pathogenesis, we analyzed differentially expressed genes with known cell-type markers, which were determined from previous single-cell studies of the spinal cord in MG-exposed and SOD1G93A mice, an ALS mouse model. Differentially expressed genes were observed in MG mice in various spinal cord cell types, including neurons, microglia, astrocytes, oligodendrocytes, oligodendrocyte precursor cells, meningeal cells/Schwann cells, and vascular cells. We also examined neuronal populations in the spinal cord. Gene expression in putative excitatory and inhibitory neurons changed more than that in cholinergic motor neurons of the spinal cord in both MG and SOD1G93A mice. Many putative neuron types, especially visceral motor neurons, and axon initial segments (AIS) were affected in MG mice. In contrast, the effect on neurons and AIS in SOD1G93A mice was slight at P30 but progressed with aging. Interestingly, changes in dopaminergic system-related genes were specifically altered in the spinal cord of MG mice. These results indicate that MG and ALS pathology in various cell types contribute to motor neuron degeneration. Furthermore, there were more alterations in neurons in MG-exposed mice than in SOD1G93A mice. A large number of differentially expressed genes (DEGs) in MG mice represent more than SOD1G93A mice with ALS pathology. Elucidation of MG pathogenesis may provide more insight into the pathophysiology of neurodegenerative diseases.

Role of peripheral myelin protein 22 in chronic exercise-induced interactions of muscle and bone in mice.

Exercise is important for the prevention and treatment of sarcopenia and osteoporosis. Although the interactions between skeletal muscles and bone have recently been reported, the myokines linking muscle to bone during exercise remain unknown. We previously revealed that chronic exercise using treadmill running blunts ovariectomy-induced osteopenia in mice. We herein performed an RNA sequence analysis of the gastrocnemius and soleus muscles of male mice with or without chronic exercise to identify the myokines responsible for the effects of chronic exercise on the muscle/bone relationship. We extracted peripheral myelin protein 22 (PMP22) as a humoral factor that was putatively induced by chronic exercise in the soleus and gastrocnemius muscles of mice from the RNA sequence analysis. Chronic exercise significantly enhanced the expression of PMP22 in the gastrocnemius and soleus muscles of female mice. PMP22 suppressed macrophage-colony stimulating factor and receptor activator factor κB ligand-induced increases in the expression of osteoclast-related genes and osteoclast formation from mouse bone marrow cells. Moreover, PMP22 significantly inhibited osteoblast differentiation, alkaline phosphatase activity, and mineralization in mouse osteoblast cultures; however, the overexpression of PMP22 did not affect muscle phenotypes in mouse muscle C2C12 cells. A simple regression analysis revealed that PMP22 mRNA levels in the gastrocnemius and soleus muscles were positively related to cortical bone mineral density at the femurs of mice with or without chronic exercise. In conclusion, we identified PMP22 as a novel myokine induced by chronic exercise in mice. We first showed that PMP22 suppresses osteoclast formation and the osteoblast phenotype in vitro.

MicroRNA-196a-5p in Extracellular Vesicles Secreted from Myoblasts Suppresses Osteoclast-like Cell Formation in Mouse Cells.

Muscle/bone interaction has been recently noted. Extracellular vesicles (EVs) play a vital role in physiological and pathophysiological processes by transferring microRNA (miRNA) to distant tissues. We previously reported that EVs secreted from C2C12 myoblasts (Myo-EVs) suppress osteoclast differentiation. In the present study, we identified 4 miRNAs in Myo-EVs that suppressed osteoclast-like cell formation in Raw264.7 cells using small RNA sequencing analysis. Among them, miR-196a-5p expression was higher in C2C12 cells compared to mouse osteoblasts and bone marrow cells. Transfection of miR-196a-5p mimic suppressed the mRNA levels of osteoclast-related genes and mitochondrial energy metabolism induced by receptor activator of nuclear factor-κB ligand in Raw264.7 cells. In contrast, miR-196a-5p mimic enhanced osteoblastic differentiation in ST-2 cells and MC3T3-E1 cells. In conclusion, we demonstrated that miR-196-5p suppresses osteoclast-like cell formation and mitochondrial energy metabolism in mouse cells, suggesting that it might be a crucial factor for muscle/bone interaction via EVs.

Transcription factor MafB in podocytes protects against the development of focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) is a common cause of steroid-resistant nephrotic syndrome. Spontaneous remission of FSGS is rare and steroid-resistant FSGS frequently progresses to renal failure. Many inheritable forms of FSGS have been described, caused by mutations in proteins that are important for podocyte function. Here, we show that a basic leucine zipper transcription factor, MafB, protects against FSGS. MAFB expression was found to be decreased in the podocytes of patients with FSGS. Moreover, conditional podocyte-specific MafB-knockout mice developed FSGS with massive proteinuria accompanied by depletion of the slit diaphragm-related proteins (Nphs1 and Magi2), and the podocyte-specific transcription factor Tcf21. These findings indicate that MafB plays a crucial role in the pathogenesis of FSGS. Consistent with this, adriamycin-induced FSGS and attendant proteinuria were ameliorated by MafB overexpression in the podocytes of MafB podocyte-specific transgenic mice. Thus, MafB could be a new therapeutic target for FSGS.

Klf5 maintains the balance of primitive endoderm versus epiblast specification during mouse embryonic development by suppression of Fgf4.

The inner cell mass of the mouse blastocyst gives rise to the pluripotent epiblast (EPI), which forms the embryo proper, and the primitive endoderm (PrE), which forms extra-embryonic yolk sac tissues. All inner cells coexpress lineage markers such as Nanog and Gata6 at embryonic day (E) 3.25, and the EPI and PrE precursor cells eventually segregate to exclusively express Nanog and Gata6, respectively. Fibroblast growth factor (FGF)-extracellular signal-regulated kinase (ERK) signalling is involved in segregation of the EPI and PrE lineages; however, the mechanism involved in Fgf4 regulation is poorly understood. Here, we identified Klf5 as an upstream repressor of Fgf4Fgf4 was markedly upregulated in Klf5 knockout (KO) embryos at E3.0, and was downregulated in embryos overexpressing Klf5 Furthermore, Klf5 KO and overexpressing blastocysts showed skewed lineage specification phenotypes, similar to FGF4-treated preimplantation embryos and Fgf4 KO embryos, respectively. Inhibitors of the FGF receptor (Fgfr) and ERK pathways reversed the skewed lineage specification of Klf5 KO blastocysts. These data demonstrate that Klf5 suppresses Fgf4-Fgfr-ERK signalling, thus preventing precocious activation of the PrE specification programme.

Stress-induced and ATF7-dependent epigenetic change influences cellular senescence.

Cellular senescence plays an important role in aging and is induced by cyclin-dependent kinase (Cdk) inhibitors that accumulate following stresses during aging. However, the underlying mechanism remains elusive. Herein, we demonstrate that activating transcription factor 7 (ATF7), the stress-responsive recruiter of histone H3K9 di- and trimethyltransferases, functions in the accumulation of Cdk inhibitors. Atf7-deficient (Atf7-/- ) mice have a shorter lifespan than wild-type (WT) mice. Levels of p16Ink4a Cdk inhibitor mRNA increased with age more rapidly in Atf7-/- mice than in WT animals. ATF7 binds to the p16Ink4a gene promoter and was released with age. Consistently, histone H3K9me2 levels on the p16Ink4a gene promoter were lower in Atf7-/- mice than in WT animals. Similar results were obtained when Atf7-/- and WT mouse embryonic fibroblasts (MEFs) were cultured under 20% oxygen conditions, which induces cellular senescence via oxidative stress. Phosphorylation of ATF7 by p38 was also increased with the passage of MEFs, consistent with previous observations that ATF7 phosphorylation by p38 induces its release from chromatin. These results indicate that stress-induced and ATF7-dependent epigenetic changes on p16Ink4a genes play an important role in cellular senescence.

Carcinogen-induced tumors in SFN-transgenic mice harbor a characteristic mutation spectrum of human lung adenocarcinoma.

The landscape of genetic alterations in disease models such as transgenic mice or mice with carcinogen-induced tumors has provided a huge amount of information that has shed light on the process of tumorigenesis in human non-small-cell lung cancer (NSCLC). We have previously identified stratifin (SFN) as a potent oncogene, and generated SFN-transgenic (Tg-SPC-SFN+/- ) mice, which express human SFN (hSFN) only in the lung. Here, we have found that carcinogen nicotine-derived nitrosaminoketone (NNK)-induced tumors developing in Tg-SPC-SFN+/- mice show a similar histology to human lung adenocarcinoma and exhibit high hSFN expression. In order to compare the genetic characteristics of Tg-SPC-SFN+/- tumors and human lung adenocarcinoma, the former were subjected to whole-exome sequencing. Interestingly, Tg-SPC-SFN+/- tumors showed the distinct distribution of exonic mutations and high number of mutated genes and transversion. Moreover, Tg-SPC-SFN+/- tumors showed 73 genes that were commonly detected in more than 2 tumors, mutations of which were also found in human lung adenocarcinoma. The expression levels of some of these genes were significantly associated with the clinical outcome of lung adenocarcinoma patients. Additionally, mutated genes in Tg-SPC-SFN+/- tumors were closely associated with key canonical pathways such as PI3K/AKT signaling and apoptosis signaling. These results suggest that SFN overexpression is a universal abnormality in human lung adenocarcinogenesis and Tg-SPC-SFN+/- tumors recapitulate key features of major human lung adenocarcinoma. Therefore, Tg-SPC-SFN+/- mice provide a useful model for clarifying the molecular mechanism underlying lung adenocarcinogenesis.

Genetic evidence implies that primary and relapsed tumors arise from common precursor cells in primary central nervous system lymphoma.

Primary central nervous system lymphoma (PCNSL) is a rare subtype of lymphoma that arises within the brain or the eyes. PCNSL recurs within the central nervous system (CNS) in most relapsed cases, whereas extra-CNS relapse is experienced in rare cases. The present study aimed at identifying the presence of common precursor cells (CPC) for primary intra- and relapsed extra-CNS tumors, and further assessing the initiating events in bone marrow (BM). Targeted deep sequencing was carried out for five paired primary intra- and relapsed extra-CNS tumors of PCNSL. Two to five mutations were shared by each pair of intra- and extra-CNS tumors. In particular, MYD88 mutations, L265P in three and P258L in one, were shared by four pairs. Unique somatic mutations were observed in all five intra-CNS tumors and in four out of five extra-CNS tumors. Remarkably, IgH clones in the intra- and the extra-CNS tumors in two pairs were distinct from each other, whereas one pair of tumors shared identical monoclonal IgH rearrangement. In a cohort of 23 PCNSL patients, L265P MYD88 mutations were examined in tumor-free BM mononuclear cells (MNC) in which the PCNSL tumors had L265P MYD88 mutations. L265P MYD88 mutations were detected by a droplet digital PCR method in nine out of 23 bone marrow mononuclear cells. These results suggest that intra- and extra-tumors are derived from CPC with MYD88 mutations in most PCNSL, arising either before or after IgH rearrangement. The initiating MYD88 mutations may occur during B-cell differentiation in BM.

Comprehensive benchmarking reveals H2BK20 acetylation as a distinctive signature of cell-state-specific enhancers and promoters.

Although over 35 different histone acetylation marks have been described, the overwhelming majority of regulatory genomics studies focus exclusively on H3K27ac and H3K9ac. In order to identify novel epigenomic traits of regulatory elements, we constructed a benchmark set of validated enhancers by performing 140 enhancer assays in human T cells. We tested 40 chromatin signatures on this unbiased enhancer set and identified H2BK20ac, a little-studied histone modification, as the most predictive mark of active enhancers. Notably, we detected a novel class of functionally distinct enhancers enriched in H2BK20ac but lacking H3K27ac, which was present in all examined cell lines and also in embryonic forebrain tissue. H2BK20ac was also unique in highlighting cell-type-specific promoters. In contrast, other acetylation marks were present in all active promoters, regardless of cell-type specificity. In stimulated microglial cells, H2BK20ac was more correlated with cell-state-specific expression changes than H3K27ac, with TGF-beta signaling decoupling the two acetylation marks at a subset of regulatory elements. In summary, our study reveals a previously unknown connection between histone acetylation and cell-type-specific gene regulation and indicates that H2BK20ac profiling can be used to uncover new dimensions of gene regulation.

Expression Profile of Cell Cycle-Related Genes in Human Fibroblasts Exposed Simultaneously to Radiation and Simulated Microgravity.

Multiple unique environmental factors such as space radiation and microgravity (µG) pose a serious threat to human gene stability during space travel. Recently, we reported that simultaneous exposure of human fibroblasts to simulated µG and radiation results in more chromosomal aberrations than in cells exposed to radiation alone. However, the mechanisms behind this remain unknown. The purpose of this study was thus to obtain comprehensive data on gene expression using a three-dimensional clinostat synchronized to a carbon (C)-ion or X-ray irradiation system. Human fibroblasts (1BR-hTERT) were maintained under standing or rotating conditions for 3 or 24 h after synchronized C-ion or X-ray irradiation at 1 Gy as part of a total culture time of 2 days. Among 57,773 genes analyzed with RNA sequencing, we focused particularly on the expression of 82 cell cycle-related genes after exposure to the radiation and simulated µG. The expression of cell cycle-suppressing genes (ABL1 and CDKN1A) decreased and that of cell cycle-promoting genes (CCNB1, CCND1, KPNA2, MCM4, MKI67, and STMN1) increased after C-ion irradiation under µG. The cell may pass through the G1/S and G2 checkpoints with DNA damage due to the combined effects of C-ions and µG, suggesting that increased genomic instability might occur in space.