Astronauts experience constant exposure to altered gravity (AG) and elevated CO2 levels during spaceflight, yet the combined effects of AG and elevated CO2 on CNS remain poorly understood.

Our study is designed to address this gap by subjecting adult flies aged 3 days to chronic HG (1.2g and 3g) and/or elevated CO2 (4000 ppm) for 15 days. The selection of 1.2g and 3g as gravitational loads were based on preliminary findings, with 3g chosen to evaluate CNS responses across a gravity continuum. The CO2 concentration of 4000 ppm reflects average levels observed on the ISS during our MVP-Fly-01 spaceflight mission. Our treatment paradigm allows for the longitudinal study of CNS responses to altered gravity, with data collection at immediate post-treatment (R+0), five days post-treatment (R+5), ten days post-treatment (R+10), and twenty-five days post-treatment (R+25). This comprehensive approach will provide valuable insights into the combined effects of altered gravity and elevated CO2 on CNS function, shedding light on the challenges faced by astronauts during space missions and informing strategies to mitigate potential adverse effects.

Neurobehavioral changes were examined under both single and combinatorial stressor conditions using the climbing assay, which manipulates the negative geotaxis reflex in Drosophila melanogaster. At the R+0 timepoint, dose-dependent alterations in climbing ability were evident in both male and female flies, indicating dysfunction in locomotive pathways within the brain-body axis. By the R+5 timepoint, full acclimation to both 1.2g and 3g treatments was observed, with climbing ability restored in both sexes. However, in the case of elevated CO2 exposure, males exhibited a trend of reduced climbing ability at R+0, though not statistically significant between 1g and 1gCO2 males. Notably, 3gCO2 males displayed a statistically significant decrease in climbing ability (p=0.0033). Conversely, females showed a dose-dependent response, with 3gCO2 females exhibiting significantly greater climbing deficits compared to 1gCO2 and 1g controls (p<0.0001). Ongoing investigation will evaluate behavioral responses at subsequent longitudinal time points (R+5, R+10, and R+25) under both single and combinatorial stressor conditions, providing further insights into the persistence and evolution of neurobehavioral changes in response to altered gravity and elevated CO2 levels.

In addition to behavioral assessments, bioenergetic assays were conducted to evaluate various parameters including mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) quantification, Reactive Oxygen Species (ROS) levels, and mitotracker (indicative of mitochondrial abundance). Preliminary findings from these assays indicate notable alterations in ROS levels, mitochondrial membrane potential, and ATP production. Specifically, increased ROS levels suggest heightened oxidative stress within the cellular environment. Additionally, changes in mitochondrial membrane potential indicate potential disruptions in mitochondrial function, which can impact cellular energy metabolism and overall cellular health. Moreover, variations in ATP production suggest alterations in cellular energy production pathways, which may reflect adaptive responses to the imposed stressors. Further analysis and validation of these findings will provide deeper insights into the cellular responses to altered gravity and elevated CO2 levels. Understanding the bioenergetic implications of these stressors is critical for elucidating their effects on cellular physiology and may inform strategies for mitigating potential adverse outcomes in organisms exposed to such environmental conditions.

Our preliminary findings indicate an increase in dopamine (DA) neuron loss and elevated cell death in chronic hypergravity (HG) conditions and spaceflight. Additionally, both spaceflight-exposed and terrestrial flies exposed to elevated CO2 demonstrate heightened oxidative stress, as evidenced by increased 8-oxo-dG puncta in the brain. To further investigate the effects of HG with or without CO2 on brain structure, we employ multiple GAL4 fly lines for brain morphology assessment. In all fly lines, we assess cell death and oxidative damage using anti-CC3 antibody for cell death detection and anti-8-oxo-dG antibody for oxidative damage assessment. At the R+0 timepoint, brain dissections and staining have been completed for all fly lines. The samples are currently at various stages of imaging (confocal) and subsequent analysis. Additionally, brain dissections for other time points have been conducted and are ready for further analysis. This comprehensive approach allows us to investigate the structural and cellular changes in the brain in response to HG and elevated CO2 exposure, providing insights into the underlying mechanisms of neurobiological adaptation and potential impacts on cognitive function and neuronal health.