Anabolic effects of IGF-1 signaling on the skeleton

This review focuses on the anabolic effects of IGF-1 signaling on the skeleton, emphasizing the requirement for IGF-1 signaling in normal bone formation and remodeling. We first discuss the genomic context, splicing variants, and species conservation of the IGF-1 locus. The modulation of IGF-1 action by growth hormone (GH) is then reviewed while also discussing the current model which takes into account the GH-independent actions of IGF-1. Next, the skeletal phenotypes of IGF-1-deficient animals are described in both embryonic and postnatal stages of development, which include severe dwarfism and an undermineralized skeleton. We then highlight two mechanisms by which IGF-1 exerts its anabolic action on the skeleton. Firstly, the role of IGF-1 signaling in the modulation of anabolic effects of parathyroid hormone (PTH) on bone will be discussed, presenting in vitro and in vivo studies that establish this concept and the proposed underlying molecular mechanisms involving Indian hedgehog (Ihh) and the ephrins. Secondly, the crosstalk of IGF-1 signaling with mechanosensing pathways will be discussed, beginning with the observation that animals subjected to skeletal unloading by hindlimb elevation are unable to mitigate cessation of bone growth despite infusion with IGF-1 and the failure of IGF-1 to activate its receptor in bone marrow stromal cell cultures from unloaded bone. Disrupted crosstalk between IGF-1 signaling and the integrin mechanotransduction pathways is discussed as one of the potential mechanisms for this IGF-1 resistance. Next, emerging paradigms on bone-muscle crosstalk are examined, focusing on the potential role of IGF-1 signaling in modulating such interactions. Finally, we present a future outlook on IGF research

Dried Plum as a Candidate Radiomitigant

This presentation will focus on data obtained from that authors' studies that test the efficacy of dried plum as a countermeasure against radiation-induced bone loss

Neutrophil to Lymphocyte Ratio: A Prognostic Indicator for Astronaut Health

Short-term and long-term spaceflight missions can cause immune system dysfunction in astronauts. Recent studies indicate elevated white blood cells (WBC) and polymorphonuclear neutrophils (PMN) in astronaut blood, along with unchanged or reduced lymphocyte counts, and reduced T cell function, during short-(days) and long-(months) term spaceflight. A high PMN to lymphocyte ratio (NLR) can acts as a strong predictor of poor prognosis in cancer, and as a biomarker for subclinical inflammation in humans and chronic stress in mouse models, however, the NLR has not yet been identified as a predictor of astronaut health during spaceflight. For this, complete blood cell count data collected from astronauts and rodents that have flown for short- and long-term missions on board the International Space Station (ISS) was repurposed to determine the NLR pre-, in-, and post-flight. The results displayed that the NLR progressively increased during spaceflight in both human and mice, while a spike in the NLR was observed at post-flight landing, suggesting stress-induced factors may be involved. In addition, the ground-based chronic microgravity analog, hindlimb unloading in mice, indicated an increased NLR, along with induced myeloperoxidase expression, as measured by quantitative (q)PCR. The mechanism for increased NLR was further assessed in vitro using the NASA-developed rotating wall vessel (RWV) cell culture suspension system with human WBCs. The results indicated that simulated microgravity led to increased mature PMN counts, NLR profiles, and production of reactive oxygen species (ROS). Collectively, these studies show that an increased NLR is observed in spaceflight missions, and in chronic microgravity-analog simulation in mice, and that this effect may be potentiated by the oxidative stress response in blood cells under microgravity conditions. Furthermore, these results suggest that a disrupted NLR profile in spaceflight may further disrupt immune homeostasis, potentially causing chronic immune-mediated inflammatory diseases. Thus, we propose that the health status of astronauts during short- and long-term space missions can be monitored by their NLR profile, in addition to utilizing this measurement as a tool for interventions and countermeasure development to restore homeostatic immunity

Effects of Simulated Spaceflight on Mitochondrial Oxidative Stress in Bone Remodelling

Microgravity and ionizing radiation may contribute to cellular stress; resulting in increased generation of reactive oxygen species (ROS), DNA damage, cell cycle arrest, and cell death. We hypothesized that suppression of excess ROS in osteoblasts and osteoclasts will improve bone microarchitecture. To test our hypothesis, we used irradiated transgenic mCAT mice overexpressing human anti-oxidant catalase gene targeted to the mitochondria (main site for ROS production). mCAT mice expressed the transgene and displayed elevated catalase activity in bone and ex vivo osteoblast and osteoclast cultures. Treated bone from wildtype mice showed elevated levels of oxidative damage whereas mCAT mice did not. Also, increased catalase activity correlated with decreased MDA levels and that increased oxidative damage correlated with decreased % bone volume. Ex-vivo osteoblast colony growth positively correlated with osteoblast catalase activity. mCAT mice displayed reduced % bone volume. Treatment caused significant bone loss in wildtype mice. Treatment also caused slight deficits in microarchitecture of mCAT mice. In conclusion, ROS signaling in both osteoblast and osteoclast lineage cells contribute to skeletal development and remodeling and quenching oxidative damage could play a role in bone loss prevention

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

Exposure to 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 exposure to ionizing radiation impairs osteoblastogenesis in an ion-type specific manner, with low doses capable of modulating expression of redox-related genes. 16-week old, male, C57BL6/J mice were exposed to low linear-energy-transfer (LET) protons (150 mega electron volts per nucleon) or high-LET (sup 56) Fe ions (600 mega electron volts per nucleon) using either low (5 or 10 centigrays) or high (50 or 200 centigrays) doses at NASAs Space Radiation Lab at Brookhaven National Lab (NSRL/BNL). Tissues were harvested 5 weeks or 1 year after irradiation and bones were analyzed by microcomputed tomography for cancellous microarchitecture and cortical geometry. Marrow-derived, adherent cells were grown under osteoblastogenic culture conditions. Cell lysates were analyzed for select groups by RT-PCR (Reverse Transcription-Polymerase Chain Reaction) during the proliferative phase or the mineralizing phase, and differentiation was analyzed by imaging mineralized nodules (percentage surface area). Representative genes were selected for expression analyses, including cell proliferation (PCNA, Cdk2, p21, p53), differentiation (Runx2, Alpl, Bglap), oxidative metabolism (Catalase, GPX, MnSOD, CuZnSOD, iNos, Foxo1), DNA-damage repair (Gadd45), or apoptosis (Caspase 3). As expected, a high dose (200 centigrays), but not low doses, of either (sup 56) Fe or protons caused a loss of cancellous bone volume per total volume. Marrow cells produced mineralized nodules ex vivo regardless of radiation type or dose; (sup 56) Fe (200 centigrays) inhibited median nodule area by more than 90 percent at 5 weeks and 1 year post-irradiation, compared to controls. At 5 weeks post exposure, irradiation with protons or (sup 56) Fe caused few changes in gene expression levels during osteoblastogenesis, although a high dose of (sup 56) Fe (200 centigrays) increased levels of Catalase and Gadd45. In addition, supplementing cell culture media with SOD protected marrow-derived osteoprogenitors from the damaging effects of exposure to low-LET ((sup 137) Cs gamma) if irradiated in vitro, but had limited protective effects on high-LET (sup 56) Fe-exposed cells. In sum, exposure of mice to either protons or (sup 56) Fe at a relatively high dose (200 cGy) caused persistent bone loss, whereas only high-LET (sup 56) Fe increased expression of redox-related genes and inhibited osteoblastogenesis, albeit to a limited extent. We conclude that high-LET irradiation impaired osteoblastogenesis and regulated steady-state gene expression of select redox-related genes during osteoblastogenesis, which may contribute to persistent bone loss

Potential Dietary Countermeasure Against Spaceflight-Induced Bone Loss

As humans venture further into space and beyond low Earth orbit, space radiation is one of the main challenges for astronauts' health. Radiation-induced bone loss is a potential health problem for long duration habitation in space. We showed that a dietary countermeasure prevents bone loss in mice exposed to total body irradiation (TBI). We used a range of ionizing radiation, gamma (137Cs), proton (1H), iron (56Fe), and a combination of sequential proton and iron beam (1H/56Fe/1H) to evaluate skeletal responses. These TBI cover a range of linear energy transfer (LET), from low-LET such as proton, to high-LET such as 56Fe (HZE: high Z- high energy) at doses between 1-2 Gy. The countermeasure diet, composed of 25% Dried Plum (DP) was effective at preventing radiation-induced cancellous bone loss in appendicular bone (tibia). Furthermore, exposing mice to HZE radiation, such as 56Fe (1Gy), impaired ex vivo growth of marrow-derived, bone-forming osteoblasts, which led to reduced mineralization capacity (-77%). In contrast, mice fed the DP diet did not display these deficits, showing the diet's capacity to protect marrow-derived osteoprogenitors. Dietary DP prevented the increase of bone resorbing osteoclast cells, inflammation and oxidative stress, while protecting the osteoprogenitors and mesenchymal stem cells, which few drugs against osteoporosis may achieve. Spaceflight is a combination of multiple factors including microgravity, in addition to space radiation. Therefore, we conducted additional studies to determine if the DP diet could prevent simulated spaceflight (simulated microgravity and radiation combined) bone loss. Mice were exposed to gamma (TBI, 137Cs, 2 Gy), simulated microgravity (using the hindlimb unloading system, HU) or TBI+HU. While we observed bone loss in mice fed the control diet (CD) due to both treatments (TBI=14%, HU=20%), and a worse effect with combined treatments (TBI+HU=25%), mice fed the DP diet did not sustain significant bone loss relative to untreated controls. The DP diet prevented microarchitectural decrements in both appendicular bone (tibia) and axial bone (vertebrae). In addition, the DP diet mitigated HU-induced deficits in osteoblastogenesis. Interestingly, lower doses of DP diet (5%, 10%) did not appear to prevent cancellous bone loss, which shows the importance of identifying the active component(s) of DP. Finally, we have preliminary data showing the potential of DP to prevent radiation-induced damage at a systematic level.. In summary, this novel dietary countermeasure is a promising candidate nutritional countermeasure for spaceflight-induced bone loss and tissue damage

Mice Exposed to Combined Chronic Low-Dose Irradiation and Modeled Microgravity Develop Long-Term Neurological Sequelae

Spaceflight poses many challenges for humans. Ground-based analogs typically focus on single parameters of spaceflight and their associated acute effects. This study assesses the long-term transcriptional effects following single and combination spaceflight analog conditions using the mouse model: simulated microgravity via hindlimb unloading (HLU) and/or low-dose γ-ray irradiation (LDR) for 21 days, followed by 4 months of readaptation. Changes in gene expression and epigenetic modifications in brain samples during readaptation were analyzed by whole transcriptome shotgun sequencing (RNA-seq) and reduced representation bisulfite sequencing (RRBS). The results showed minimal gene expression and cytosine methylation alterations at 4 months readaptation within single treatment conditions of HLU or LDR. In contrast, following combined HLU+LDR, gene expression and promoter methylation analyses showed multiple altered pathways involved in neurogenesis and neuroplasticity, the regulation of neuropeptides, and cellular signaling. In brief, neurological readaptation following combined chronic LDR and HLU is a dynamic process that involves pathways that regulate neuronal function and structure and may lead to late onset neurological sequelae

Diurnal Immune Cell Migration Patterns Characterized in the Spaceflight Environment

Daily diurnal immune rhythm shapes biological pathways of organisms and closely aligns with optimizing energy usage in response to environmental light-dark cycles. Immune mobilization depends on diurnal signals to regulate immunity. In spaceflight, disrupted circadian rhythms and immune systems are noted. However, crosstalk between these systems has not been fully characterized. To fill this knowledge gap, we utilized a ground-based model of spaceflight to phenotype diurnal immunity in mice. For this, 24-week-old male and female mice were exposed to a combination of single-housed, acute 15cGy 5-ion GCRsim irradiation and continuous hindlimb unloading for 2 weeks on a light:dark [12hr:12hr] cycle throughout. Blood was collected at 24 hours and 2 weeks post irradiation and flow cytometrically profiled. Additionally, ribo-depleted, bulk RNA sequencing characterized unique, diurnal and sex-specific biosignatures. This work expands our understanding of diurnal immunity which is important to consider for personalized medicine directives for astronauts. This work was supported in part by the NASA Human Research Program (HRP) Human Factors Behavioral Performance Element Grant 18 18FLAG 2 0028 to AER and Embry-Riddle Start-up grant to Dr. Amber Paul

Efficient integration of transgenes into a defined locus in human embryonic stem cells

Random integration is one of the more straightforward methods to introduce a transgene into human embryonic stem (ES) cells. However, random integration may result in transgene silencing and altered cell phenotype due to insertional mutagenesis in undefined gene regions. Moreover, reliability of data may be compromised by differences in transgene integration sites when comparing multiple transgenic cell lines. To address these issues, we developed a genetic manipulation strategy based on homologous recombination and Cre recombinase-mediated site-specific integration. First, we performed gene targeting of the hypoxanthine phosphoribosyltransferase 1 (HPRT) locus of the human ES cell line KhES-1. Next, a gene-replacement system was created so that a circular vector specifically integrates into the targeted HPRT locus via Cre recombinase activity. We demonstrate the application of this strategy through the creation of a tetracycline-inducible reporter system at the HPRT locus. We show that reporter gene expression was responsive to doxycycline and that the resulting transgenic human ES cells retain their self-renewal capacity and pluripotency

Mammalian and Invertebrate Models as Complementary Tools for Gaining Mechanistic Insight on Muscle Responses to Spaceflight

Bioinformatics approaches have proven useful in understanding biological responses to spaceflight. Spaceflight experiments remain resource intensive and rare. One outstanding issue is how to maximize scientific output from a limited number of omics datasets from traditional animal models including nematodes, fruitfly, and rodents. The utility of omics data from invertebrate models in anticipating mammalian responses to spaceflight has not been fully explored. Hence, we performed comparative analyses of transcriptomes of soleus and extensor digitorum longus (EDL) in mice that underwent 37 days of spaceflight. Results indicate shared stress responses and altered circadian rhythm. EDL showed more robust growth signals and Pde2a downregulation, possibly underlying its resistance to atrophy versus soleus. Spaceflight and hindlimb unloading mice shared differential regulation of proliferation, circadian, and neuronal signaling. Shared gene regulation in muscles of humans on bedrest and space flown rodents suggest targets for mitigating muscle atrophy in space and on Earth. Spaceflight responses of C. elegans were more similar to EDL. Discrete life stages of D. melanogaster have distinct utility in anticipating EDL and soleus responses. In summary, spaceflight leads to shared and discrete molecular responses between muscle types and invertebrate models may augment mechanistic knowledge gained from rodent spaceflight and ground-based studies