ALBERT

Description: Weightlessness and radiation, two of the unique elements of the space environment, causes a profound decrement in bone mass that mimics aging. This bone loss is thought to result from increased activity of bone-resorbing osteoclasts and functional changes in bone-forming osteoblasts, cells that give rise to mature osteocytes. Our current understanding of the signaling factors and mechanisms underlying bone loss is incomplete. However, it is known that oxidative stress, characterized by the excess production of free radicals, is elevated during radiation exposure. The goals of this study is to examine the response of osteocytes to spaceflight-like radiation and to identify signaling processes that may be targeted to mitigate bone loss in scenarios of space exploration, earth-based radiotherapy and accidental radiation exposure. We hypothesize that (1) oxidative stress, as induced by radiation, decreases osteocyte survival and increases pro-osteoclastogenic signals and that (2) autophagy is one of the key cellular defenses against oxidative stress. Autophagy is the process by which cellular components including organelles and proteins are broken down and recycled. To test our hypothesis, we exposed the osteocyte-like cell line, MLO-Y4, to 0.5, 1, and 2 Gy of simulated space radiation (Iron-56 radiation at 600 MeV/n) and assessed cell numbers, cell growth-associated molecules as well as markers of autophagy and oxidative stress at various time points post-irradiation. We observed a reduction in cell numbers in the groups exposed to 1 and 2 Gy of Iron-56 radiation. Collectively, flow cytometry and gene expression analysis revealed that radiation caused a shift in cell cycle distribution consistent with growth arrest. Compared to sham-treatment, 2 Gy of Iron-56 increased FoxO3, SOD1, and RANKL gene expression yet unexpectedly decreased LC3B-II protein levels at 4 and 24 hours post-IR. Taken together, these findings suggest that simulated space radiation invoke antioxidant, pro-osteoclastogenic, and growth arrest responses in osteocytes. The implications of reduced autophagy flux at the time points examined remain to be elucidated.

Description: Spaceflight has deleterious effects on skeletal structure and function, specifically causingprofound loss in bone mass, density, and strength, as well as changes in expression levels of genes related to oxidative stress [Hyeon et al., Smith et al.]. It is known that bone resorption remains elevated after spaceflight and that bone density and strength fail to recover completely even years following spaceflight [Smith et al., Carpenter et al.]. However, our current understanding of the signaling pathways and molecular mechanisms that control bone loss and that link oxidative stress, bone resorption, and mechanical unloading of skeletal tissue is incomplete. Here, we aim to examine skeletal responses to simulated long-duration spaceflight on bone loss using the ground-based hindlimb unloading (HU) model in adult (9 months old) male rats. We hypothesized that simulated microgravity leads to the temporal regulation of oxidative-defense genes and pro-osteoclastogenic factors, showing progression and eventual plateau during long-term unloading, and that transient changes at early timepoints in these pathways precede skeletal adaptations to long-duration unloading. We will identify oxidativestress and bone resorption-related changes using global gene expression analysis (Affymetrix arrays) for both acute (within 14 days) and long-term timepoints (90 days). We will also use quantitative PCR to examine changes in expression of genes related to oxidative metabolism (e.g. Nrf2, SOD-1), bone turnover (resorption and formation markers, e.g. TRAP, osteocalcin respectively, SOST), and osteoclastogenesis (e.g. RANKL, OPG) at both early and late timepoints. We will then use detailed microarchitectural and structural analysis through microcomputed tomography to relate gene expression changes with structural changes in bone, expecting that plateaus in gene expression correlate with long-term changes in bone microarchitecture.

Description: In this study, we aim to examine skeletal responses to simulated long-duration spaceflight (90 days) and weight-bearing recovery on bone loss using the ground-based hindlimb unloading (HU) model in adolescent (3-month old) male rats. We hypothesized that simulated microgravity leads to the temporal regulation of oxidative defense genes and pro-bone resorption factors, where there is a progression and eventual plateau; furthermore, early transient changes in these pathways precede skeletal adaptations.

Description: Weightlessness and radiation, two unique elements of space, profoundly decreases bone mass. This bone loss is attributed to increased activity of bone-resorbing osteoclasts and functional changes in bone-forming osteoblasts, cells that give rise to mature osteocytes. Our long-term goal is to identify signaling pathways that may be targeted to mitigate bone loss in scenarios of space exploration, radiotherapy and accidental radiation exposure. We have previously shown that exposure of MLO-Y4 osteocyte-like cells to simulated space radiation (56Fe) increased the expression of the pro-osteoclastogenic gene rankl and decreased protein levels of LC3B-II, a key player in autophagy. In this current study, we aimed to further elucidate the role of autophagy in maintaining structural integrity of the skeleton. We hypothesize that loss of autophagy in bone leads to an imbalance in pro-osteoclastogenic and pro-osteogenic signals, resulting in net bone loss. To test our hypothesis we performed global postnatal deletion of Atg12 using tamoxifeninducible Cre recombinase under the control of the CAG promoter. Six-week-old CAGCreERT2/ FloxAtg12 animals were treated daily with Tamoxifen or Vehicle (Control, oil only) for five days and euthanasia performed two weeks after the onset of treatment. Percent change in body weights (prior to treatment and at euthanasia) was not significantly different between treatment groups within the same gender. Compared to Vehicle (Control) groups, Tamoxifen (Atg12 iKO) groups showed decreased LC3B-I to II conversion and increased p62 protein levels, consistent with loss of autophagy. Quantitative PCR revealed increased expression of proosteoclastogenic cytokines mcp1 and rankl in bone and marrow respectively in male iKOs compared to male controls. Expression levels of these genes were not significantly altered in the Atg12 iKO females compared to females controls. Microcomputed tomography of tibiae revealed decreased cortical bone volume, cortical thickness and periosteal perimeter consistent with bone loss; and a longer primary spongiosa in male Atg12 iKOs display compared to male controls. These decrements were less pronounced in the female Atg12 iKOs. Cancellous bone structure was not significantly different between iKOs and controls in both genders. Histological analysis also revealed that compared to male controls, male iKOs showed a profound increase in chondrocyte column length of the growth plate with hyper-expansion of both proliferating and hypertrophic zones. Taken together, these findings indicate that autophagy plays an important role in the maintenance of bone structural integrity by mediating the production of proosteoclastogenic signals and regulating chondrocyte proliferation and differentiation.

Description: Weightlessness and radiation, two unique elements of space, profoundly decreases bone mass. We aimed to elucidate the role of autophagy in maintaining structural integrity of the skeleton. We hypothesize that loss of autophagy in bone leads to an imbalance in pro-osteoclastogenic and pro-osteogenic signals, resulting in net bone loss. To test our hypothesis, we performed global postnatal deletion of Atg12 using tamoxifen-inducible Cre. Compared to Vehicle (Control) groups, Tamoxifen (Atg12 iKO) groups showed decreased LC3B-I to II conversion and increased p62 protein levels, consistent with loss of autophagy. qPCR revealed increased expression of pro-osteoclastogenic cytokines in bone and marrow respectively in male iKOs compared to controls. Microcomputed tomography revealed decreased cortical bone volume, cortical thickness and periosteal perimeter consistent with bone loss; and a longer primary spongiosa in male Atg12 iKOs display compared to male controls. Histology showed that compared to male controls, male iKOs had a profound increase in chondrocyte column length of the growth plate with hyper-expansion of both proliferating and hypertrophic zones. Taken together, these findings indicate that autophagy plays an important role in the maintenance of bone structural integrity by mediating the production of pro-osteoclastogenic signals and regulating chondrocyte proliferation and differentiation.