Telomeres protect the chromosome ends from degradation and play crucial roles in cellular aging and disease. Recent studies have additionally found a correlation between psychological stress, telomere length, and health outcome in humans. However, studies have not yet explored the causal relationship between stress and telomere length, or the molecular mechanisms underlying that relationship. Using yeast as a model organism, we show that stresses may have very different outcomes: alcohol and acetic acid elongate telomeres, whereas caffeine and high temperatures shorten telomeres. Additional treatments, such as oxidative stress, show no effect. By combining genome-wide expression measurements with a systematic genetic screen, we identify the Rap1/Rif1 pathway as the central mediator of the telomeric response to environmental signals. These results demonstrate that telomere length can be manipulated, and that a carefully regulated homeostasis may become markedly deregulated in opposing directions in response to different environmental cues.

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.

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Genome-wide systematic screens in yeast have uncovered a large gene network (the telomere length maintenance network or TLM), encompassing more than 400 genes, which acts coordinatively to maintain telomere length. Identifying the genes was an important first stage; the next challenge is to decipher their mechanism of action and to organize then into functional groups or pathways. Here we present a new telomere-length measuring program, TelQuant, and a novel assay, telomere length kinetics assay, and use them to organize tlm mutants into functional classes. Our results show that a mutant defective for the relatively unknown MET7 gene has the same telomeric kinetics as mutants defective for the ribonucleotide reductase subunit Rnr1, in charge of the limiting step in dNTP synthesis, or for the Ku heterodimer, a well-established telomere complex. We confirm the epistatic relationship between the mutants and show that physical interactions exist between Rnr1 and Met7. We also show that Met7 and the Ku heterodimer affect dNTP formation, and play a role in non-homologous end joining. Thus, our telomere kinetics assay uncovers new functional groups, as well as complex genetic interactions between tlm mutants.

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Sus1 is a conserved protein involved in histone H2B de-ubiquitination and mRNA export from the nucleus in eukaryotes. Previous studies implicated Sus1 partners in genome integrity including telomere homeostasis. However, the implication of Sus1 in telomere maintenance remains largely unknown. In this study, we found that yeast Sus1 interacts physically and genetically with factors involved in telomere maintenance and its absence leads to elongated telomeres. Deletion of several of Sus1's partners also leads to longer telomeres. Our results rule out a direct role for Sus1 in recruiting telomerase subunits to telomeres. However, we observe that deletion of SUS1 leads to elongated telomeres even in the presence of mutations like sem1Δ, esc2Δ and rsc2Δ, which cause telomere shortening. We find that rsc2Δ (short telomeres) have reduced levels of mono-ubiquitinated histone H2B at lysine 123 (H2BK123ub), whereas sus1Δ mutants or double-mutants sus1Δ rsc2Δ exhibit longer telomeres and higher H2BK123ub levels. These results suggest that Sus1 activity as a H2B de-ubiquitination modulator plays a role in negatively regulating telomere length. Our results provide solid evidence for a role of Sus1 in negatively regulating telomere length through the modulation of H2BK123 mono-ubiquitination and its interaction with the nuclear pore complex.

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.

Long duration spaceflight poses potential health risks to astronauts during flight and re-adaptation after return to Earth. There is an emerging need for NASA to provide successful and reliable therapeutics for long duration missions when capability for medical intervention will be limited. Clinically relevant, human placenta-derived therapeutic stromal cells (PLX-PAD) are a promising therapeutic alternative. We found that treatment of adult female mice with PLX-PAD near the onset of simulated weightlessness by hindlimb unloading (HU, 30 d) was well-tolerated and partially mitigated decrements caused by HU. Specifically, PLX-PAD treatment rescued HU-induced thymic atrophy, and mitigated HU-induced changes in percentages of circulating neutrophils, but did not rescue changes in the percentages of lymphocytes, monocytes, natural killer (NK) cells, T-cells and splenic atrophy. Further, PLX-PAD partially mitigated HU effects on the expression of select cytokines in the hippocampus. In contrast, PLX-PAD failed to protect bone and muscle from HU-induced effects, suggesting that the mechanisms which regulate the structure of these mechanosensitive tissues in response to disuse are discrete from those that regulate the immune- and central nervous system (CNS). These findings support the therapeutic potential of placenta-derived stromal cells for select physiological deficits during simulated spaceflight. Multiple countermeasures are likely needed for comprehensive protection from the deleterious effects of prolonged spaceflight.