Mitochondria-Targeted Human Catalase in the Mouse Longevity MCAT Model Mitigates Head-Tilt Bedrest-Induced Neuro-Inflammation in the Hippocampus.

Microgravity (modeled by head-tilt bedrest and hind-limb unloading), experienced during prolonged spaceflight, results in neurological consequences, central nervous system (CNS) dysfunction, and potentially impairment during the performance of critical tasks. Similar pathologies are observed in bedrest, sedentary lifestyle, and muscle disuse on Earth. In our previous study, we saw that head-tilt bedrest together with social isolation upregulated the milieu of pro-inflammatory cytokines in the hippocampus and plasma. These changes were mitigated in a MCAT mouse model overexpressing human catalase in the mitochondria, pointing out the importance of ROS signaling in this stress response. Here, we used a head-tilt model in socially housed mice to tease out the effects of head-tilt bedrest without isolation. In order to find the underlying molecular mechanisms that provoked the cytokine response, we measured CD68, an indicator of microglial activation in the hippocampus, as well as changes in normal in-cage behavior. We hypothesized that hindlimb unloading (HU) will elicit microglial hippocampal activations, which will be mitigated in the MCAT ROS-quenching mice model. Indeed, we saw an elevation of the activated microglia CD68 marker following HU in the hippocampus, and this pathology was mitigated in MCAT mice. Additionally, we identified cytokines in the hippocampus, which had significant positive correlations with CD68 and negative correlations with exploratory behaviors, indicating a link between neuroinflammation and behavioral consequences. Unveiling a correlation between molecular and behavioral changes could reveal a biomarker indicative of these responses and could also result in a potential target for the treatment and prevention of cognitive changes following long space missions and/or muscle disuse on Earth.

Overexpression of catalase in mitochondria mitigates changes in hippocampal cytokine expression following simulated microgravity and isolation.

Isolation on Earth can alter physiology and signaling of organs systems, including the central nervous system. Although not in complete solitude, astronauts operate in an isolated environment during spaceflight. In this study, we determined the effects of isolation and simulated microgravity solely or combined, on the inflammatory cytokine milieu of the hippocampus. Adult female wild-type mice underwent simulated microgravity by hindlimb unloading for 30 days in single or social (paired) housing. In hippocampus, simulated microgravity and isolation each regulate a discrete repertoire of cytokines associated with inflammation. Their combined effects are not additive. A model for mitochondrial reactive oxygen species (ROS) quenching via targeted overexpression of the human catalase gene to the mitochondria (MCAT mice), are protected from isolation- and/or simulated microgravity-induced changes in cytokine expression. These findings suggest a key role for mitochondrial ROS signaling in neuroinflammatory responses to spaceflight and prolonged bedrest, isolation, and confinement on Earth.

Neutrophil-to-Lymphocyte Ratio: A Biomarker to Monitor the Immune Status of Astronauts.

A comprehensive understanding of spaceflight factors involved in immune dysfunction and the evaluation of biomarkers to assess in-flight astronaut health are essential goals for NASA. An elevated neutrophil-to-lymphocyte ratio (NLR) is a potential biomarker candidate, as leukocyte differentials are altered during spaceflight. In the reduced gravity environment of space, rodents and astronauts displayed elevated NLR and granulocyte-to-lymphocyte ratios (GLR), respectively. To simulate microgravity using two well-established ground-based models, we cultured human whole blood-leukocytes in high-aspect rotating wall vessels (HARV-RWV) and used hindlimb unloaded (HU) mice. Both HARV-RWV simulation of leukocytes and HU-exposed mice showed elevated NLR profiles comparable to spaceflight exposed samples. To assess mechanisms involved, we found the simulated microgravity HARV-RWV model resulted in an imbalance of redox processes and activation of myeloperoxidase-producing inflammatory neutrophils, while antioxidant treatment reversed these effects. In the simulated microgravity HU model, mitochondrial catalase-transgenic mice that have reduced oxidative stress responses showed reduced neutrophil counts, NLR, and a dampened release of selective inflammatory cytokines compared to wildtype HU mice, suggesting simulated microgravity induced oxidative stress responses that triggered inflammation. In brief, both spaceflight and simulated microgravity models caused elevated NLR, indicating this as a potential biomarker for future in-flight immune health monitoring.

Circulating miRNA Spaceflight Signature Reveals Targets for Countermeasure Development.

We have identified and validated a spaceflight-associated microRNA (miRNA) signature that is shared by rodents and humans in response to simulated, short-duration and long-duration spaceflight. Previous studies have identified miRNAs that regulate rodent responses to spaceflight in low-Earth orbit, and we have confirmed the expression of these proposed spaceflight-associated miRNAs in rodents reacting to simulated spaceflight conditions. Moreover, astronaut samples from the NASA Twins Study confirmed these expression signatures in miRNA sequencing, single-cell RNA sequencing (scRNA-seq), and single-cell assay for transposase accessible chromatin (scATAC-seq) data. Additionally, a subset of these miRNAs (miR-125, miR-16, and let-7a) was found to regulate vascular damage caused by simulated deep space radiation. To demonstrate the physiological relevance of key spaceflight-associated miRNAs, we utilized antagomirs to inhibit their expression and successfully rescue simulated deep-space-radiation-mediated damage in human 3D vascular constructs.

Skeletal tissue regulation by catalase overexpression in mitochondria.

Accumulation of oxidative damage from excess reactive oxygen species (ROS) may contribute to skeletal aging and mediate adverse responses to physiological challenges. Wild-type (WT) mice and transgenic mice (male, 16 wk of age) with human catalase targeted to the mitochondria (mCAT) were analyzed for skeletal responses to the remodeling stimuli of combined hind-limb unloading and exposure to ionizing radiation (137Cs, 2 Gy). Treatment for 2 wk caused lipid peroxidation in the bones WT but not mCAT mice, showing that transgene expression mitigated oxidative stress. Ex vivo osteoblast colony growth rate was 95% greater in mCAT than WT mice and correlated with catalase activity levels (P < 0.005, r = 0.67), although terminal osteoblast and osteoclast differentiation were unaffected. mCAT mice had lower cancellous bone volume and cortical size than WT mice. Ambulatory control mCAT animals also displayed reduced cancellous and cortical structural properties compared with control WT mice. In mCAT but not WT mice, treatment caused an unexpectedly rapid radial expansion (+8% cortical area, +22% moment of inertia), reminiscent of compensatory bone growth during advancing age. In contrast, treatment caused similar structural deficits in cancellous tissue of mCAT and WT mice. In sum, mitochondrial ROS signaling via H2O2 was important for the acquisition of adult bone structure and catalase overexpression failed to protect cancellous tissue from treatment. In contrast, catabolic stimuli caused radial expansion in mCAT not WT mice, suggesting that mitochondrial ROS in skeletal cells act to suppress tissue turnover in response to remodeling challenges.

Dietary countermeasure mitigates simulated spaceflight-induced osteopenia in mice.

Spaceflight is a unique environment that includes at least two factors which can negatively impact skeletal health: microgravity and ionizing radiation. We have previously shown that a diet supplemented with dried plum powder (DP) prevented radiation-induced bone loss in mice. In this study, we investigated the capacity of the DP diet to prevent bone loss in mice following exposure to simulated spaceflight, combining microgravity (by hindlimb unloading) and radiation exposure. The DP diet was effective at preventing most decrements in bone micro-architectural and mechanical properties due to hindlimb unloading alone and simulated spaceflight. Furthermore, we show that the DP diet can protect osteoprogenitors from impairments resulting from simulated microgravity. Based on our findings, a dietary supplementation with DP could be an effective countermeasure against the skeletal deficits observed in astronauts during spaceflight.

A human mission to Mars: Predicting the bone mineral density loss of astronauts.

A round-trip human mission to Mars is anticipated to last roughly three years. Spaceflight conditions are known to cause loss of bone mineral density (BMD) in astronauts, increasing bone fracture risk. There is an urgent need to understand BMD progression as a function of spaceflight time to minimize associated health implications and ensure mission success. Here we introduce a nonlinear mathematical model of BMD loss for candidate human missions to Mars: (i) Opposition class trajectory (400-600 days), and (ii) Conjunction class trajectory (1000-1200 days). Using femoral neck BMD data (N = 69) from astronauts after 132-day and 228-day spaceflight and the World Health Organization's fracture risk recommendation, we predicted post-mission risk and associated osteopathology. Our model predicts 62% opposition class astronauts and 100% conjunction class astronauts will develop osteopenia, with 33% being at risk for osteoporosis. This model can help in implementing countermeasure strategies and inform space agencies' choice of crew candidates.

Influence of Social Isolation During Prolonged Simulated Weightlessness by Hindlimb Unloading.

The hindlimb unloading (HU) model has been used extensively to simulate the cephalad fluid shift and musculoskeletal disuse observed in spaceflight with its application expanding to study immune, cardiovascular and central nervous system responses, among others. Most HU studies are performed with singly housed animals, although social isolation also can substantially impact behavior and physiology, and therefore may confound HU experimental results. Other HU variants that allow for paired housing have been developed although no systematic assessment has been made to understand the effects of social isolation on HU outcomes. Hence, we aimed to determine the contribution of social isolation to tissue responses to HU. To accomplish this, we developed a refinement to the traditional NASA Ames single housing HU system to accommodate social housing in pairs, retaining desirable features of the original design. We conducted a 30-day HU experiment with adult, female mice that were either singly or socially housed. HU animals in both single and social housing displayed expected musculoskeletal deficits versus housing matched, normally loaded (NL) controls. However, select immune and hypothalamic-pituitary-adrenal (HPA) axis responses were differentially impacted by the HU social environment relative to matched NL controls. HU led to a reduction in % CD4+ T cells in singly housed, but not in socially housed mice. Unexpectedly, HU increased adrenal gland mass in socially housed but not singly housed mice, while social isolation increased adrenal gland mass in NL controls. HU also led to elevated plasma corticosterone levels at day 30 in both singly and socially housed mice. Thus, musculoskeletal responses to simulated weightlessness are similar regardless of social environment with a few differences in adrenal and immune responses. Our findings show that combined stressors can mask, not only exacerbate, select responses to HU. These findings further expand the utility of the HU model for studying possible combined effects of spaceflight stressors.

Dose- and Ion-Dependent Effects in the Oxidative Stress Response to Space-Like Radiation Exposure in the Skeletal System.

Space radiation may pose a risk to skeletal health during subsequent aging. Irradiation acutely stimulates bone remodeling in mice, although the long-term influence of space radiation on bone-forming potential (osteoblastogenesis) and possible adaptive mechanisms are not well understood. We hypothesized that ionizing radiation impairs osteoblastogenesis in an ion-type specific manner, with low doses capable of modulating expression of redox-related genes. 16-weeks old, male, C57BL6/J mice were exposed to low linear-energy-transfer (LET) protons (150 MeV/n) or high-LET 56Fe ions (600 MeV/n) using either low (5 or 10 cGy) or high (50 or 200 cGy) doses at NASA's Space Radiation Lab. Five weeks or one year after irradiation, tissues were harvested and analyzed by microcomputed tomography for cancellous microarchitecture and cortical geometry. Marrow-derived, adherent cells were grown under osteoblastogenic culture conditions. Cell lysates were analyzed by RT-PCR during the proliferative or mineralizing phase of growth, and differentiation was analyzed by imaging mineralized nodules. As expected, a high dose (200 cGy), but not lower doses, of either 56Fe or protons caused a loss of cancellous bone volume/total volume. Marrow cells produced mineralized nodules ex vivo regardless of radiation type or dose; 56Fe (200 cGy) inhibited osteoblastogenesis by more than 90% (5 weeks and 1 year post-IR). After 5 weeks, irradiation (protons or 56Fe) caused few changes in gene expression levels during osteoblastogenesis, although a high dose 56Fe (200 cGy) increased Catalase and Gadd45. The addition of exogenous superoxide dismutase (SOD) protected marrow-derived osteoprogenitors from the damaging effects of exposure to low-LET (137Cs γ) when irradiated in vitro, but had limited protective effects on high-LET 56Fe-exposed cells. In sum, either protons or 56Fe at a relatively high dose (200 cGy) caused persistent bone loss, whereas only high-LET 56Fe increased redox-related gene expression, albeit to a limited extent, and inhibited osteoblastogenesis. Doses below 50 cGy did not elicit widespread responses in any parameter measured. We conclude that high-LET irradiation at 200 cGy impaired osteoblastogenesis and regulated steady-state gene expression of select redox-related genes during osteoblastogenesis, which may contribute to persistent bone loss.

Effects of High-LET Radiation Exposure and Hindlimb Unloading on Skeletal Muscle Resistance Artery Vasomotor Properties and Cancellous Bone Microarchitecture in Mice.

Weightlessness during spaceflight leads to functional changes in resistance arteries and loss of cancellous bone, which may be potentiated by radiation exposure. The purpose of this study was to assess the effects of hindlimb unloading (HU) and total-body irradiation (TBI) on the vasomotor responses of skeletal muscle arteries. Male C57BL/6 mice were assigned to control, HU (13-16 days), TBI (1 Gy (56)Fe, 600 MeV, 10 cGy/min) and HU-TBI groups. Gastrocnemius muscle feed arteries were isolated for in vitro study. Endothelium-dependent (acetylcholine) and -independent (Dea-NONOate) vasodilator and vasoconstrictor (KCl, phenylephrine and myogenic) responses were evaluated. Arterial endothelial nitric oxide synthase (eNOS), superoxide dismutase-1 (SOD-1) and xanthine oxidase (XO) protein content and tibial cancellous bone microarchitecture were quantified. Endothelium-dependent and -independent vasodilator responses were impaired in all groups relative to control, and acetylcholine-induced vasodilation was lower in the HU-TBI group relative to that in the HU and TBI groups. Reductions in endothelium-dependent vasodilation correlated with a lower cancellous bone volume fraction. Nitric oxide synthase inhibition abolished all group differences in endothelium-dependent vasodilation. HU and HU-TBI resulted in decreases in eNOS protein levels, while TBI and HU-TBI produced lower SOD-1 and higher XO protein content. Vasoconstrictor responses were not altered. Reductions in NO bioavailability (eNOS), lower anti-oxidant capacity (SOD-1) and higher pro-oxidant capacity (XO) may contribute to the deficits in NOS signaling in skeletal muscle resistance arteries. These findings suggest that the combination of insults experienced in spaceflight leads to impairment of vasodilator function in resistance arteries that is mediated through deficits in NOS signaling.

Galactic cosmic ray simulation at the NASA Space Radiation Laboratory.

Most accelerator-based space radiation experiments have been performed with single ion beams at fixed energies. However, the space radiation environment consists of a wide variety of ion species with a continuous range of energies. Due to recent developments in beam switching technology implemented at the NASA Space Radiation Laboratory (NSRL) at Brookhaven National Laboratory (BNL), it is now possible to rapidly switch ion species and energies, allowing for the possibility to more realistically simulate the actual radiation environment found in space. The present paper discusses a variety of issues related to implementation of galactic cosmic ray (GCR) simulation at NSRL, especially for experiments in radiobiology. Advantages and disadvantages of different approaches to developing a GCR simulator are presented. In addition, issues common to both GCR simulation and single beam experiments are compared to issues unique to GCR simulation studies. A set of conclusions is presented as well as a discussion of the technical implementation of GCR simulation.

Mechanical loading causes site-specific anabolic effects on bone following exposure to ionizing radiation.

During spaceflight, astronauts will be exposed to a complex mixture of ionizing radiation that poses a risk to their health. Exposure of rodents to ionizing radiation on Earth causes bone loss and increases osteoclasts in cancellous tissue, but also may cause persistent damage to stem cells and osteoprogenitors. We hypothesized that ionizing radiation damages skeletal tissue despite a prolonged recovery period, and depletes the ability of cells in the osteoblast lineage to respond at a later time. The goal of the current study was to test if irradiation prevents bone accrual and bone formation induced by an anabolic mechanical stimulus. Tibial axial compression was used as an anabolic stimulus after irradiation with heavy ions. Mice (male, C57BL/6J, 16 weeks) were exposed to high atomic number, high energy (HZE) iron ions ((56)Fe, 2 Gy, 600 MeV/ion) (IR, n=5) or sham-irradiated (Sham, n=5). In vivo axial loading was initiated 5 months post-irradiation; right tibiae in anesthetized mice were subjected to an established protocol known to stimulate bone formation (cyclic 9N compressive pulse, 60 cycles/day, 3 day/wk for 4 weeks). In vivo data showed no difference due to irradiation in the apparent stiffness of the lower limb at the initiation of the axial loading regimen. Axial loading increased cancellous bone volume by microcomputed tomography and bone formation rate by histomorphometry in both sham and irradiated animals, with a main effect of axial loading determined by two-factor ANOVA with repeated measure. There were no effects of radiation in cancellous bone microarchitecture and indices of bone formation. At the tibia diaphysis, results also revealed a main effect of axial loading on structure. Furthermore, irradiation prevented axial loading-induced stimulation of bone formation rate at the periosteal surface of cortical tissue. In summary, axial loading stimulated the net accrual of cancellous and cortical mass and increased cancellous bone formation rate despite prior exposure to ionizing radiation, in this case, HZE particles. Our findings suggest that mechanical stimuli may prove an effective treatment to improve skeletal structure following exposure to ionizing radiation.

Spd2 assists Spd1 in the modulation of ribonucleotide reductase architecture but does not regulate deoxynucleotide pools.

In yeasts, small intrinsically disordered proteins (IDPs) modulate ribonucleotide reductase (RNR) activity to ensure an optimal supply of dNTPs for DNA synthesis. The Schizosaccharomyces pombe Spd1 protein can directly inhibit the large RNR subunit (R1), import the small subunit (R2) into the nucleus and induce an architectural change in the R1-R2 holocomplex. Here, we report the characterization of Spd2, a protein with sequence similarity to Spd1. We show that Spd2 is a CRL4(Cdt2)-controlled IDP that functions together with Spd1 in the DNA damage response and in modulation of RNR architecture. However, Spd2 does not regulate dNTP pools and R2 nuclear import. Furthermore, deletion of spd2 only weakly suppresses the Rad3(ATR) checkpoint dependency of CRL4(Cdt2) mutants. However, when we raised intracellular dNTP pools by inactivation of RNR feedback inhibition, deletion of spd2 could suppress the checkpoint dependency of CRL4(Cdt2) mutant cells to the same extent as deletion of spd1. Collectively, these observations suggest that Spd1 on its own regulates dNTP pools, whereas in combination with Spd2 it modulates RNR architecture and sensitizes cells to DNA damage.

Regulation of ribonucleotide reductase by Spd1 involves multiple mechanisms.

The correct levels of deoxyribonucleotide triphosphates and their relative abundance are important to maintain genomic integrity. Ribonucleotide reductase (RNR) regulation is complex and multifaceted. RNR is regulated allosterically by two nucleotide-binding sites, by transcriptional control, and by small inhibitory proteins that associate with the R1 catalytic subunit. In addition, the subcellular localization of the R2 subunit is regulated through the cell cycle and in response to DNA damage. We show that the fission yeast small RNR inhibitor Spd1 is intrinsically disordered and regulates R2 nuclear import, as predicted by its relationship to Saccharomyces cerevisiae Dif1. We demonstrate that Spd1 can interact with both R1 and R2, and show that the major restraint of RNR in vivo by Spd1 is unrelated to R2 subcellular localization. Finally, we identify a new behavior for RNR complexes that potentially provides yet another mechanism to regulate dNTP synthesis via modulation of RNR complex architecture.

[Clinical and molecular consequences of microsatellite instability in human cancers]. / Conséquences cliniques et moléculaires de l'instabilité des microsatellites dans les cancers humains.

During each cell division, DNA polymerase makes mistakes while copying DNA. These errors, more frequent at the level of repeated sequences called microsatellites are normally repaired by a system called MMR (mismatch repair). Tumors defective in their MMR system accumulate mutations (deletions and insertions of some nucleotides) at the level of microsatellites and are called MSI (microsatellite instability). Microsatellites are numerous and scattered throughout the genome, in coding and non-coding regions. The instability of non-coding microsatellites is not known to have a major role in the process of cell transformation, but is a good indicator of the MSI status. On the other hand, instability by deletion or insertion in a coding region leads to a frameshift within the gene containing the repeat. The consequence is, the more often, the inactivation of this gene that potentially plays a role in initiation and/or MSI tumor progression. The MSI phenotype was first described in about 15 % of colorectal cancers that maybe of sporadic or hereditary (Lynch syndrome, or HNPCC for hereditary non-polyposis colorectal cancer) origin. It is also associated with about 15 % of gastric and endometrial tumors, and to a lesser extent with other human tumors. Besides a fundamental interest because of its original transformation mechanism, the analysis of MSI tumors is also important for clinical reasons. It was indeed shown that MSI tumors were associated with a better prognosis than non-MSI (also called MSS for microsatellite stable) tumors, and responded differently to conventional chemotherapeutic drugs used for the management of colorectal cancers. All these points will be discussed in details in the present review.