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NASA NTRSDate not supplied

Dose, LET, time and strain dependence of radiation-induced 53BP1 foci in 15 mouse strains ex vivo and associations to in vivo radiation susceptibility

We present a comparative analysis on the repair of radiation-induced DNA damage ex vivo in 15 strains of mice, including 5 inbred reference strains and 10 collaborative-cross strains, of both sexes. Non-immortalized primary skin fibroblasts derived from 76 mice were subjected to both low- and high-LET radiation (0.1, 1 and 4 Gy of X rays; 1.1 and 3 particles/100μm2 of 350 MeV/n 40Ar and 600 MeV/n 56Fe). Automated image quantification of 53BP1 radiation-induced foci (RIF) during the first 4-48 h post-irradiation was performed as a function of dose and LET. Similarly to what we had previously reported for immortalized human cell lines [1], we observed a saturation of RIF number with dose at 4h post-irradiation, with more RIF/Gy for lower LET (X rays and 40Ar) compared to 56Fe. However at later time points (24h and above), the trend was inverted with more RIF/Gy for higher LET. Our data suggest that multiple DSBs cluster into RIF: as the linear density of DSBs increases with LET, so does the probability of having more DSBs per RIF, which makes it more difficult for cells to fully resolve high-LET-induced RIF, explaining the hypersensitivity to high-LET radiation despite a low number of RIF. Taking into account the amount of clustering at a given dose and LET, but also the kinetics of DNA damage repair, we introduced a novel mathematical formalism to evaluate the number of remaining RIF over time. We showed that the newly introduced kinetic metrics can be used as surrogate biomarkers for in vivo radiation toxicity, with potential applications in radiotherapy and human space exploration. In particular, we observed an association between the repairable fraction of RIF measured in vitro and survival levels of immune cells collected from irradiated mice. Moreover, the speed of DNA damage repair correlated with spontaneous cancer incidence data collected from the Mouse Tumor Biology database, suggesting a relationship between the efficiency of DSB repair after irradiation and cancer risk. In addition to the efficacy of repair and persistent RIF levels, even the amount of spontaneous foci without irradiation was shown to be strain dependent, indicating that these phenotypes are at least partially driven by genetics, and supporting their potential as indicators of individual radiation sensitivity. [1] Neumaier, T., et al., PNAS, 2012 (8) 109:443

Radiation, DNA damage, repair kinetics↗

NASA NTRSDate not supplied

A Multi Targeted Dietary Supplement as a Potential Countermeasure for Prolonged, Deep Space Exploration

Deep space exploration, particularly to the Moon and Mars, are currently major goals of NASA and other international space agencies. Long-term space flight presents unique challenges to the human physiology from microgravity, social isolation, altered circadian rhythm and radiation. Oxidative stress has been implicated as a crucial factor in space environment induced injury. Elucidating these detrimental effects on the central nervous system and brain is a primary objective, as they may result in adverse changes in astronaut behavior, mood and performance of critical tasks. We were recently funded by HRP Human Factors Behavioral Performance (HFBP) Element to test the hypothesis that Galactic Cosmic Ray Simulation (GCRsim) Ionizing Radiation (IR), microgravity and social isolation combine synergistically to trigger an oxidative stress response that alters immune homeostasis, brain structure and function, and neurobehavioral and cognitive performance. This project will identify potential biomarkers for, and mechanisms underlying, structural and functional changes in the immune and nervous systems leading to behavioral/cognitive performance deficits, and its potential application to develop effective countermeasures to mitigate negative health effects of long duration space habitation. Countermeasures to offset neurological damaged and cognitive deficits are paramount for protecting astronauts during deep space transits, but many countermeasures have been found to be unsuitable in ground-based model studies. We have developed a multi targeted dietary supplement (MTDS) designed to simultaneously ameliorate oxidative stress, inflammatory processes, energetic shortfalls, and membrane and mitochondrial deterioration. Administration of this MTDS has improved cognition and longevity in age accelerated and normal aging mice. Specifically, the MTDS was found to prevent a decline in mitochondrial complex III activity and cell loss, as well as prevent an increase in protein carbonyls and mitochondrial 3-nitrotyrosine in the brain of these mice. Further, administration of the MTDS was also found to reduce bone marrow chromosomal aberrations, DNA damage, lymphocyte apoptosis after whole body exposure of 2 Gy γ radiation. Mice exposed to a high dose cranially (10 Gy) had decreased cognitive responses (seen as increased latency time to uncover buried food and decreased novel object recognition), increased plasma 8-OHdG levels, increased markers of cell stress in the brain, decreased hippocampal brain-derived neurotrophic factor (BDNF) protein and decreased plasma levels of cytokines, all of which were ameliorated with treatment of the MTDS. As we have shown the supplement is protective in models of cognitive damage similar space environment stressors, particularly increased oxidative stress, we propose it may be a suitable countermeasure for upcoming deep space exploration.

radiation↗

NASA NTRSDate not supplied

A MULTI TARGETED DIETARY SUPPLEMENT AS A POTENTIAL COUNTERMEASURE FOR DEEP SPACE EXPLORATION

Future deep space exploration to the Moon and Mars and beyond are currently major goals of NASA. Space flight presents unique challenges to the human physiology from the combined effects of microgravity, social isolation, altered circadian rhythm and radiation. Oxidative stress has been implicated as a crucial factor in space environment induced injury. Elucidating these detrimental effects on the central nervous system and brain is a primary objective, as they may result in adverse changes in astronaut behavior, mood and performance of critical tasks. We were recently funded by HRP Human Factors Behavioral Performance (HFBP) Element to test the hypothesis that Galactic Cosmic Ray Simulation (GCRsim) Ionizing Radiation (IR), microgravity and social isolation combine synergistically to trigger an oxidative stress response that alters immune homeostasis, brain structure and function, and neurobehavioral and cognitive performance. This project will identify potential biomarkers for, and mechanisms underlying, structural and functional changes in the immune and nervous systems leading to behavioral/cognitive performance deficits, and its potential application to develop effective countermeasures to mitigate negative health effects of long duration space habitation. Countermeasures to offset neurological damaged and cognitive deficits are paramount for protecting astronauts during deep space transits, but many countermeasures have been found to be unsuitable in ground-based model studies. We have developed a multi targeted dietary supplement (MTDS) designed to simultaneously ameliorate oxidative stress, inflammatory processes, energetic shortfalls, and membrane and mitochondrial deterioration. Administration of this MTDS has improved cognition and longevity in age accelerated and normal aging mice. Specifically, the MTDS was found to prevent a decline in mitochondrial complex III activity and cell loss, as well as prevent an increase in protein carbonyls and mitochondrial 3-nitrotyrosine in the brain of these mice. Further, administration of the MTDS was also found to reduce bone marrow chromosomal aberrations, DNA damage, lymphocyte apoptosis after whole body exposure of 2 Gy γ radiation. Mice exposed to a high dose cranially (10 Gy) had decreased cognitive responses (seen as increased latency time to uncover buried food and decreased novel object recognition), increased plasma 8-OHdG levels, increased markers of cell stress in the brain, decreased hippocampal brain-derived neurotrophic factor (BDNF) protein and decreased plasma levels of cytokines, all of which were ameliorated with treatment of the MTDS. As we have shown the supplement is protective in models of cognitive damage similar space environment stressors, particularly increased oxidative stress, we propose it may be a suitable countermeasure for upcoming deep space exploration.

Stephanie Puukila↗

NASA NTRSDate not supplied

Multi-Omics Analysis of Mouse Retina Following Low Dose Radiation and/or Hindlimb Unloading

Rodent models have been used as analogs for studying the effects of spaceflight. NASA’s GeneLab provides access to omics datasets generated from spaceflight and ground-based experiments allowing for additional retrospective analysis. We used GeneLab’s GLDS-203, a dataset generated by researchers at Loma Linda University to study the impact of prolonged unloading and/or low-dose radiation on mouse retina. The purpose of this study was to understand the effect of gamma radiation and/or hindlimb unloading on mice retinas through a multi-omics analysis. In the experiment that generated the omics data, mice were irradiated with gamma-ray and/or subjected to hindlimb unloading for 21 days and multi-omics analysis was performed at 7 days, 1 month, or 4 months post exposure. In the current study, for each of the three timepoints, we compared epigenomic profiles for retinas from exposed mice against timepoint-matched controls. We identified a total of 5,271 differentially methylated loci (DML) and 321 differentially methylated regions (DMR; using a sliding window and step size of 500 bp) with methylation difference > 10% and q-value < 0.05 (sliding linear model corrected p-value) across the nine exposure groups. Highest correlation in methylation difference was seen for significant DMLs (q-value < 0.05) across different conditions at same post exposure timepoint (Figure 1).The location of DMLs and DMRs were characterized with respect to CpG islands and shores, putative promoters, gene body, and intergenic regions (Table 1). We analyzed RNA-seq counts and performed gene set enrichment analysis using differential expression results from comparing each exposure group to its timepoint-matched control group. Significant pathways (adjusted p-value <0.05) enriched in all three microgravity-only groups were related to morphogenesis of a branching epithelium, skeletal muscle cell differentiation, and response to fibroblast growth factor. Common processes across all timepoints in the radiation-only groups were retina homeostasis, synaptic vesicle exocytosis-endocytosis, and chemotaxis. In the combination groups, regulation of trans-synaptic signaling, and Rho protein signal transduction were enriched at all three timepoints. Processes related to purine nucleotide metabolism were enriched in all nine exposure groups, with activation at 1 month, and suppression at 7 days and 4 months. A total of 14 genes contained at least one DML and were differentially expressed at adjusted p-value < 0.05, including genes implicated in cataract development (Sipa1l3, Crybb3) and those involved in cytoskeletal organization (Plec, Flnb, Eef1a1). This analysis is part of a larger effort to understand the molecular mechanisms following spaceflight exposures that can help translate effects observed in animal models to human impacts.

Prachi Kothiyal↗

NASA NTRSDate not supplied

Oxidative Stress and Neuroconsequences of the Spaceflight Environment –A 5-ion GCRsim Dose-response Study

The overall scope of this project is to test the hypothesis that Ionizing Radiation (IR), microgravity and social isolation combine synergistically to trigger an oxidative stress response that alters immune homeostasis, brain structure/function, and neurobehavioral/cognitive performance. Specific Aims for this project are to: (1) Determine dose-response curves for acute ‘Five-Ion GCR Simulation’ exposure for immune, brain and performance responses in crew age-matched adult male and female mice; (2) Determine effects of acute ‘Five-Ion GCR Simulation’ exposure singly and in combination with simulated microgravity and social isolation, on immune, brain and performance responses in crew age-matched male and female mice mimicking deep space missions; and (3) Determine efficacy of the dietary antioxidant, Nicotinamide Mononucleotide (NMN), a key intermediate in nicotinamide adenine dinucleotide (NAD+) biosynthesis. Here we exposed mature (23-24-week-old) male and female mice to simulated 5-Ion GCR (either 0, 5, 15, or 50cGy) at the NASA Space Radiation Laboratory (NSRL). Across ‘Acute’ (IR+72hrs and IR+14 days) and ‘Delayed’ (IR+28 to IR+124 days) phases of the 6-month-long study, we assessed immune, neural, and functional (sensorimotor/behavioral/cognitive) responses anticipated during a transit to Mars. This presentation will report on dose-response curves for 5-ion GCRsim related to potential biomarkers; structural/functional changes in the immune and nervous systems; and behavioral/cognitive performance deficits. This project addresses NASA’s efforts to characterize risks and identify appropriate countermeasures in anticipation of future deep space missions. Ensuring crew health and performance during extended transits necessitates that sensorimotor and cognitive abilities remain strong to avoid potentially catastrophic health and safety outcomes.

deep space↗

NASA NTRSDate not supplied

Looking into Ocular Risks of Spaceflight through the Mouse Retina

Ocular alterations have been observed at anatomical levels in astronauts on long duration spaceflight missions, such as what would be required for missions to Mars. These alterations cause an array of signs which together constitute the Spaceflight-Associated Neuro-ocular Syndrome (SANS), one of the top risk priorities of the NASA Human Research Program. Not much is known about SANS at the cellular and molecular level, but studies in mice and rats have recently begun to yield observations on how the spaceflight environment might affect the eye’s biology. Preliminary data from shuttle mouse experiments, and more recently experiments on ISS, have shown changes in retinal physiology via histology and gene expression analysis. This study utilizes samples from the CASIS sponsored Rodent Research 8 Experiment (RRRM-1) tissue sharing opportunity, delivered to the ISS by SpaceX CRS-16 on 12/08/2018. Female BALB/cAnNTac mice were on the ISS for 45 days, while ground controls consisted ofa standard vivarium group and spaceflight habitat group. Here we investigate the molecular response of the mouse retina to identify genes and pathways affected by spaceflight conditions using histology and transcriptomic RNAseq data. This Differentially Expressed Gene (DEG) data was used for pathway analysis with Galaxy (Genelab) and Ingenuity Pathway Analysis (IPA). We identified pathways related to neuronal differentiation, cellular transport/movement, and wound healing. Some of the top DEGs have known relation to ophthalmic diseases. Though there were DEGs throughout the comparisons we tested, there was no clear effect of spaceflight. This could be due to sample processing, which required mice to be returned to Earth about a day before they were sacrificed, possibly allowing for readaptation affecting the retinal transcriptome. However, there was a clear effect of age, between the young (10-12 weeks) and old (32 weeks) groups, and between the baseline and end of experiment, about 46 days.

SANS↗

NASA NTRSDate not supplied

Neuro-behavioral Consequences of Low Dose Radiation Social Isolation and Sex Differences in the Longevity MCAT Mouse Model

The physiological responses to spaceflight elicit wide-ranging consequences and resemble aspects of aging on Earth. Previous studies have shown that oxidative damage via reactive oxygen species (ROS), contributes to aging-related pathologies. Our study uses 1-year old C57BL/6NJ male and female mice (astronaut-relevant age) that underwent exposure to 0.5 gray of gamma radiation together with social isolation and were euthanized 12 weeks after. We used the longevity MCAT mouse model in which human catalase is overexpressed in the mitochondria, for ROS quenching. We aimed to determine whether in older mice quenching ROS, will mitigate the neuro-behavioral consequences of low dose ionizing radiation and/or social isolation and whether the outcomes will differ in males and females. We have performed five mission relevant behavioral tests which focused on performance, memory, physical stance, and stress. We have detected both sex and radiation effects; the older females look physically better are faster and perform better almost in all behavioral tests compared to their male counterparts. On the other hand, they are more sensitive to low dose radiation in many cases, in some cases this effect was indeed mitigated in the MCAT mice, pointing out to the importance of ROS in response to radiation stress and social isolation. We have measured plasma (7- and 90-days post radiation), cytokines, corticosterone and hippocampal cytokine and microglial activation at the end of the experiment. We saw significant changes in the plasma markers due to radiation, sex, and genotype in both short and long post radiation period and detected long term sex and radiation effects in the brain. Our focus is now on applying advanced statistical modeling to corelate the behavioral tests with our recent molecular findings to look for specific biomarkers that could predict behavioral deficits.

radiation↗

NASA NTRSDate not supplied

Evaluating the Feasibility of Data Simulation for Radiation Research: Estimating RBE Values for HZE Radiation

As the drive for deep space exploration intensifies, a comprehensive understanding of the health effects of radiation exposure becomes paramount to the future of human space flight. However, epidemiological data for radiation exposure, particularly to high-energy (HZE) ions, is limited, partially due to the financial and logistical costs of radiation studies. As an alternative, this study aims to assess the viability of data simulation strategies to accurately model potential study parameters prior to utilizing laboratory conditions. This study estimates a relative biological effectiveness (RBE) factor based on the solid tumor data for outbred mice provided by Edmundson et al. 2020. Excess relative risk (ERR) models for HZE-Fe ions and gamma radiation were estimated using Poisson regression with Weibull models to represent the background solid tumor hazard without radiation. RBE values were calculated from the ratio of the heavy ion linear slope to the gamma linear slope. The parameters from these models were then applied to simulate iterations of 300 datasets across HZE-Fe doses of 0.05, 0.2, 0.4, and 0.75 Gy; gamma radiation doses of 0.75, 2, and 3 Gy, as well as an unirradiated control group. Sample size per dose varied from 100-500 mice across simulations (800-4000 mice total per dataset). 1500 datasets were generated total (300 each for sample sizes 800, 1600, 3200, and 4000). For each dataset, ERR per radiation type and RBE for HZE-Fe were calculated. The RBE from the Edmondson data was calculated to be 5.55. The RBEs from the simulated data converged around this value across the increasing sample sizes. For n = 4000, the mean RBE across the 300 datasets was 5.71 (95% CI: 5.47-5.95). These results suggest that RBEs calculated from simulated data are credible. Based on this exploration, data simulation is a viable method of testing radiation studies. It provides a method of testing study assumptions and refining research questions prior to embarking on costly laboratory experiments.

Sneha Mehta↗

NASA NTRSDate not supplied

Metadata Entry Optimization for NASA's Biological Institutional Scientific Collection (NBISC)

The NASA Biological Institutional Sample Collection (NBISC) at NASA’s Ames Research Center is a critical resource housing non-human samples collected from spaceflight missions and ground analog studies, primarily consisting of specimens from rats, mice, and select microbes. The primary objective of NBISC is to systematically receive, document, preserve, and facilitate access to these samples for the global scientific community. NBISC promotes international collaboration and maximizes the return on investment for precious tissues from spaceflight and analog experiments. Researchers can request physical samples through an online request form and subsequent written proposal review process. This study addresses two core research objectives: streamlining the NBISC sample lifecycle processes and strategizing for managing an influx of 50,000 tissue samples from a series of cosmic radiation analog experiments carried out at the NASA Space Radiation Laboratory (NSRL) by Drs. Eleanor Chang (Lawrence Berkeley Laboratory) and Polly Blakely (SRI). The Chang/Blakely studies investigated Harderian gland (HG) tumorigenesis in mice exposed to low dose and LET radiation comprising 8 different exposure protocols in over 4000 mice. NBISC sample metadata is stored in a Laboratory Information Management System (SLIMS). To streamline sample data entry, we customize python scripts using information extracted from the individual experimental protocols. The scripts automate entry into multiple SLIMS data fields including protocol name, unique sample barcode, tissue and sub-tissue information, freezer location, sample preservation method, etc. The semi-automated procedure significantly decreases the time spent on data entry by several orders of magnitude. Automation and data organization are essential, as they free up time for curation and promotion of the collection which, in turn, increase the accessibility of samples to the broader research community. NBISC benefits from streamlined data ingestion, and the methodologies developed here are applicable to other projects which use SLIMS including the NASA Biospecimen Sharing Program and GeneLab. As of Fall 2023, plans include transferring sample data from SLIMS to public facing repositories (OSDR and NLSP), expanding the reach of the Chang/Blakely sample collection. The Human Research Program Space Radiation Element plans to transfer non-human tissues from many more investigations to NBISC in the coming year.

Sample Repository↗

NASA NTRSDate not supplied

Metadata Entry Optimization For NASA's Biological Institutional Scientific Collection (NBISC)

Biospecimen↗

NASA NTRS2003

Dual reporter transgene driven by 2.3Col1a1 promoter is active in differentiated osteoblasts

AIM: As quantitative and spatial analyses of promoter reporter constructs are not easily performed in intact bone, we designed a reporter gene specific to bone, which could be analyzed both visually and quantitatively by using chloramphenicol acetyltransferase (CAT) and a cyan version of green fluorescent protein (GFPcyan), driven by a 2.3-kb fragment of the rat collagen promoter (Col2.3). METHODS: The construct Col2.3CATiresGFPcyan was used for generating transgenic mice. Quantitative measurement of promoter activity was performed by CAT analysis of different tissues derived from transgenic animals; localization was performed by visualized GFP in frozen bone sections. To assess transgene expression during in vitro differentiation, marrow stromal cell and neonatal calvarial osteoblast cultures were analyzed for CAT and GFP activity. RESULTS: In mice, CAT activity was detected in the calvaria, long bone, teeth, and tendon, whereas histology showed that GFP expression was limited to osteoblasts and osteocytes. In cell culture, increased activity of CAT correlated with increased differentiation, and GFP activity was restricted to mineralized nodules. CONCLUSION: The concept of a dual reporter allows a simultaneous visual and quantitative analysis of transgene activity in bone.

Non-NASA Center↗

NASA NTRS2002

Behavioral consequences of radiation exposure to simulated space radiation in the C57BL/6 mouse: open field, rotorod, and acoustic startle

Two experiments were carried out to investigate the consequences of exposure to proton radiation, such as might occur for astronauts during space flight. C57BL/6 mice were exposed, either with or without 15-g/cm2 aluminum shielding, to 0-, 3-, or 4-Gy proton irradiation mimicking features of a solar particle event. Irradiation produced transient direct deficits in open-field exploratory behavior and acoustic startle habituation. Rotorod performance at 18 rpm was impaired by exposure to proton radiation and was impaired at 26 rpm, but only for mice irradiated with shielding and at the 4-Gy dose. Long-term (>2 weeks) indirect deficits in open-field activity appeared as a result of impaired experiential encoding immediately following exposure. A 2-week recovery prior to testing decreased most of the direct effects of exposure, with only rotorod performance at 26 rpm being impaired. These results suggest that the performance deficits may have been mediated by radiation damage to hippocampal, cerebellar, and possibly, forebrain dopaminergic function.

Non-NASA Center↗

NASA NTRS2003

Bayesian estimation of dose thresholds

An example is described of Bayesian estimation of radiation absorbed dose thresholds (subsequently simply referred to as dose thresholds) using a specific parametric model applied to a data set on mice exposed to 60Co gamma rays and fission neutrons. A Weibull based relative risk model with a dose threshold parameter was used to analyse, as an example, lung cancer mortality and determine the posterior density for the threshold dose after single exposures to 60Co gamma rays or fission neutrons from the JANUS reactor at Argonne National Laboratory. The data consisted of survival, censoring times and cause of death information for male B6CF1 unexposed and exposed mice. The 60Co gamma whole-body doses for the two exposed groups were 0.86 and 1.37 Gy. The neutron whole-body doses were 0.19 and 0.38 Gy. Marginal posterior densities for the dose thresholds for neutron and gamma radiation were calculated with numerical integration and found to have quite different shapes. The density of the threshold for 60Co is unimodal with a mode at about 0.50 Gy. The threshold density for fission neutrons declines monotonically from a maximum value at zero with increasing doses. The posterior densities for all other parameters were similar for the two radiation types.

NASA Discipline Radiation Health↗

NASA NTRS2003

Polycystins 1 and 2 mediate mechanosensation in the primary cilium of kidney cells

Several proteins implicated in the pathogenesis of polycystic kidney disease (PKD) localize to cilia. Furthermore, cilia are malformed in mice with PKD with mutations in TgN737Rpw (encoding polaris). It is not known, however, whether ciliary dysfunction occurs or is relevant to cyst formation in PKD. Here, we show that polycystin-1 (PC1) and polycystin-2 (PC2), proteins respectively encoded by Pkd1 and Pkd2, mouse orthologs of genes mutated in human autosomal dominant PKD, co-distribute in the primary cilia of kidney epithelium. Cells isolated from transgenic mice that lack functional PC1 formed cilia but did not increase Ca(2+) influx in response to physiological fluid flow. Blocking antibodies directed against PC2 similarly abolished the flow response in wild-type cells as did inhibitors of the ryanodine receptor, whereas inhibitors of G-proteins, phospholipase C and InsP(3) receptors had no effect. These data suggest that PC1 and PC2 contribute to fluid-flow sensation by the primary cilium in renal epithelium and that they both function in the same mechanotransduction pathway. Loss or dysfunction of PC1 or PC2 may therefore lead to PKD owing to the inability of cells to sense mechanical cues that normally regulate tissue morphogenesis.

Non-NASA Center↗

NASA NTRS2003

Genetic models in applied physiology: selected contribution: effects of spaceflight on immunity in the C57BL/6 mouse. I. Immune population distributions

There are several aspects of the spaceflight environment that may lead to changes in immunity: mission-related psychological stress, radiation, and changes in gravity. On December 5, 2001, the space shuttle Endeavor launched for a 12-day mission to examine these effects on C57BL/6 mice for the first time. On their return, assays were performed on the spleen, blood, and bone marrow. In response to flight, there were no significant differences in the general circulating leukocyte proportions. In contrast, there was an increase in splenic lymphocyte percentages, with a corresponding decrease in granulocytes. There was an overall shift in splenic lymphocytes away from T cells toward B cells, and a decrease in the CD4-to-CD8 ratios due to a decrease in T helpers. In contrast, there were proportional increases in bone marrow T cells, with decreases in B cells. Although the blast percentage and count were decreased in flight mice, the CD34(+) population was increased. The data were more consistent with a shift in bone marrow populations rather than a response to changes in the periphery. Many of the results are similar to those using other models. Clearly, spaceflight can influence immune parameters ranging from hematopoiesis to mature leukocyte mechanisms.

Non-NASA Center↗

NASA NTRS2002

Expression of a calpastatin transgene slows muscle wasting and obviates changes in myosin isoform expression during murine muscle disuse

Muscle wasting is a prominent feature of several systemic diseases, neurological damage and muscle disuse. The contribution of calpain proteases to muscle wasting in any instance of muscle injury or disease has remained unknown because of the inability to specifically perturb calpain activity in vivo. We have generated a transgenic mouse with muscle-specific overexpression of calpastatin, which is the endogenous inhibitor of calpains, and induced muscle atrophy by unloading hindlimb musculature for 10 days. Expression of the transgene resulted in increases in calpastatin concentration in muscle by 30- to 50-fold, and eliminated all calpain activity that was detectable on zymograms. Muscle fibres in ambulatory, transgenic mice were smaller in diameter, but more numerous, so that muscle mass did not differ between transgenic and non-transgenic mice. This is consistent with the role of the calpain-calpastatin system in muscle cell fusion that has been observed in vitro. Overexpression of calpastatin reduced muscle atrophy by 30 % during the 10 day unloading period. In addition, calpastatin overexpression completely prevented the shift in myofibrillar myosin content from slow to fast isoforms, which normally occurs in muscle unloading. These findings indicate that therapeutics directed toward regulating the calpain-calpastatin system may be beneficial in preventing muscle mass loss in muscle injury and disease.

Non-NASA Center↗

NASA NTRS2002

Leptin regulates bone formation via the sympathetic nervous system

We previously showed that leptin inhibits bone formation by an undefined mechanism. Here, we show that hypothalamic leptin-dependent antiosteogenic and anorexigenic networks differ, and that the peripheral mediators of leptin antiosteogenic function appear to be neuronal. Neuropeptides mediating leptin anorexigenic function do not affect bone formation. Leptin deficiency results in low sympathetic tone, and genetic or pharmacological ablation of adrenergic signaling leads to a leptin-resistant high bone mass. beta-adrenergic receptors on osteoblasts regulate their proliferation, and a beta-adrenergic agonist decreases bone mass in leptin-deficient and wild-type mice while a beta-adrenergic antagonist increases bone mass in wild-type and ovariectomized mice. None of these manipulations affects body weight. This study demonstrates a leptin-dependent neuronal regulation of bone formation with potential therapeutic implications for osteoporosis.

NASA Discipline Regulatory Physiology↗

NASA NTRS2001

Proliferation, differentiation and apoptosis in connexin43-null osteoblasts

Osteoblasts are highly coupled by gap junctions formed primarily by connexin43 (Cx43). We have shown that interference with Cx43 expression or function disrupts transcriptional regulation of osteoblast genes, and that deletion of Cx43 in the mouse causes skeletal malformations, delayed mineralization, and osteoblast dysfunction. Here, we studied the mechanisms by which genetic deficiency of Cx43 alters osteoblast development. While cell proliferation rates were similar in osteoblastic cells derived from calvaria of Cx43-null and wild type mice, camptothecin-induced apoptosis was 3-fold higher in mutant compared to wild type osteoblasts. When grown in mineralizing medium, Cx43-null cells were able to produce mineralized matrix but it took one week longer to reach the same mineralization levels as in normal cells. Likewise, expression of alkaline phosphatase activity per cell--a marker of osteoblast differentiation--was maximal only 2 weeks later in Cx43-null relative to wild-type cells. These observations suggest that Cx43 is important for a normal and timely development of the osteoblastic phenotype. Delayed differentiation and increase programmed cell death may explain the skeletal phenotype of Cx43-null mice.

NASA Discipline Cell Biology↗