The central nervous system (CNS) is vulnerable to irradiation [1
] and fluid shifts [2
] experienced during short- and long-term spaceflight. An important goal for the National Aeronautics and Space Administration (NASA) is to identify the effects of spaceflight-like conditions on the CNS to better prepare astronauts during long-duration missions to the Moon and Mars. During missions beyond low-earth orbit (LEO), astronauts will be continuously exposed to low-dose ionizing radiation (LDR). While high linear energy transfer (LET) galactic cosmic radiation (GCR) and low LET solar particle event (SPE) radiation contribute large portions of the radiation dose accumulated by astronaut crew members [3
], interactions between GCR and SPE particles and spacecraft release secondary radiation including γ-rays that can deliver a significant fraction of the total mission does [4
]. In addition, altered gravity (hypergravity experienced at launch/landing and microgravity experienced in-flight) affects homeostatic fluid-shifting in the CNS. Studies examining readaptation to Earth’s gravity are of equal importance, as recovery from spaceflight exposure may require medical intervention, in particular within organs known to be sensitive to irradiation and fluid shifts.
Astronauts in spaceflight experience impairments in neurocognition (as measured by altered decision making and problem solving) and neurobehavior (as measured by impaired visual perceptions, motor controls and sleep–wake cycles) [5
]. The NASA Twins study offers some additional insight into post-flight cognitive decline that persists up to 6 months post-flight (end of experimental sampling) [6
]. Longitudinal diffusion magnetic resonance imaging (MRI) data collected from astronauts pre- and post-flight (shuttle missions <30 days and ISS mission <200 days) revealed whole brain shifting within the skull and white matter disruptions, suggesting spaceflight leads to long-term alterations of brain structure in humans [7
Rodent models offer an appropriate alternative to assess the effects of spaceflight-like conditions on brain tissue. Recently, the Rodent Research (RR)-1 mission flew mice to the International Space Station (ISS) for 33 days and measured neurobehavioral outcomes through video monitoring, which indicated 16-week old (at launch) females displayed unique group hyperactivity behavior at 2 weeks post-launch, implying neurological disruptions occur in flight [8
]. Moreover, long-term readaptation to chronic LDR and hindlimb unloading (HLU) (a ground-based analog of spaceflight) resulted in increased aquaporin-4 (AQP4) protein expression, oxidative stress damage, apoptosis, blood–brain barrier (BBB) compromise and an increase in neurobehavioral risk-taking behavior [9
The effects of acute radiation doses on the CNS have been well documented [12
]. However, responses to chronic LDR exposure, as encountered on space missions (<0.04 Gy/y), are not well investigated [13
], particularly in a pan-transcriptome context. Moreover, long-term readaptive outcomes on neurohealth following spaceflight-like conditions are less studied. Due to the need to better understand the transcriptional changes associated with chronic LDR in combination with reduced gravity (unloading) on neurohealth, mice were exposed to chronic LDR γ-ray irradiation (0.04 Gy), HLU, or a combination of HLU+LDR for 21 days, followed by a 4-month readaptation period. Whole transcriptome shotgun sequencing (RNA-seq) data were analyzed for differentially expressed genes (DEG), while data from reduced representation bisulfite sequencing (RRBS) were analyzed to determine differentially methylated promoters (DMP) [14
]. Overall, we found minimal gene alterations at 4 months of readaptation within single treatment conditions of HLU or LDR, namely, in pathways related to the reduced expression of translational machinery, while the combination of HLU+LDR resulted in a wide panel of altered DEG and DMP profiles for tight junctions, aquaporins, neurogenesis markers and neuropeptides. Therefore, exposure to spaceflight-like conditions may lead to mild, long-term neurological consequences, while readaptation is an active process involving neuroplasticity and repair that is cooperatively engaged post-HLU+LDR exposure to maintain neural homeostasis.
Herein, we identified that chronic exposure (21 days) to single or combined HLU and LDR resulted in long-term transcriptional alterations in CNS brain tissue in female mice at 4 months post-exposure. The gene expression changes observed here imply reduced transcriptional machinery, increased neurogenesis and neuropeptide production, and dysregulated cell structure and cell signaling. These studies offer translational value into pathways regulated in humans, as gene ontology analyses identified similar genetic overlap between mice and human profiles, in particular in pathways involved in neurodevelopmental homeostasis and signal transduction. Collectively, brain-related transcriptional changes that are dynamic in readaptation from exposure to spaceflight-like conditions may lead to long-term neurological consequences. Indeed, 21 days of exposure to combined HLU+LDR did not result in immediate neurobehavioral deficits (1 week post-exposure); however, increased risk-taking behavior was observed at 9 months post-exposure [9
], indicating delayed neurological impairments in a combination of HLU+LDR, that was not apparent in HLU or LDR only.
To assess CNS effects, 4-month post-experimental conditions mid/hindbrain (sans cerebellum and brainstem) were collected, sectioned, and total RNA was isolated for RNA-seq analysis. This portion of the brain houses the hippocampus, basal ganglia, substantia nigra, and thalamus, and is a major signaling hub for the neuroendocrine system (hypothalamus and pituitary gland). Mid/hindbrain sections were assessed to localize the transcriptional profile to a region that is central to neurogenesis (hippocampus) and hypothalamic–pituitary axis (HPA) responses, in order to better assess neurofunctional output without potential contamination from the cerebral cortex or cerebellum. The results displayed robust DEG alterations within combination HLU+LDR at 4 months readaptation compared to controls, and marginal DEG alterations in single HLU or LDR at 4 months readaptation. At 4 months post-HLU exposure, DEGs including EphrB3
were altered, suggesting cell morphological and structural changes are active at this timepoint. At 4 months post-LDR exposure, only five DEGs were found, four of which have an unknown function. One DEG, Rps13
, was reduced, suggesting impaired protein synthesis and/or other functional outputs mediated by ribosomes. Interestingly, Rps13
was also reduced in HLU alone at 4 months readaptation along with other protein synthesis-related genes, including Hist1h2bc
, collectively suggesting fundamental translational pathways are altered during chronic HLU and LDR during readaptation. In line with this, another group has reported that chronic LDR whole brain irradiation resulted in protein synthesis impairment [1
] that can affect adult neurogenesis [17
]. Interestingly, no notable protein synthesis pathway deficits were identified in combined HLU+LDR exposures, suggesting distinct responses are generated within each experimental condition at readaptation. Moreover, very limited DEG overlap was observed between experimental conditions, further validating unique responses to each condition and highlighting the importance of implementing combined exposure studies to better assess spaceflight-like readaptation effects on the nervous system. Other spaceflight-like factors such as hypoxia and social isolation were not assessed in this study and merit further investigation. Nonetheless, the usefulness of this model provides a scaffold for future studies to assess the long-term effects of combination HLU+LDR on the health of the nervous system.
Brain structural damage through disruption of the blood–brain barrier (BBB) is associated with neurodegeneration and subsequent behavioral dysfunctions [18
]. Our previous study showed that aquaporin-4 (AQP4), a water-channel protein involved in brain water homeostasis, was up-regulated following combined exposure to irradiation and unloading [9
], suggesting a disturbance in BBB integrity may lead to edema [19
]. Furthermore, AQP4 induction was seen in concert with subtle behavioral changes at 9 months post-exposure [9
], indicating a potential role for BBB dysfunction and neurobehavioral consequences. Although Aqp4
was not identified in this study, Aqp1
gene expression was induced following combination of HLU+LDR at 4 months readaptation, suggesting similar BBB disturbances and possible shifts in brain water homeostasis [19
]. In line with this, the induction of aquaporins may be linked to neuroinflammation [20
], while the upregulation of additional immune activation markers including Cd74
, H2-Ab1, C1ql2
were also identified in combination HLU+LDR at 4 months readaptation, along with hypomethylated Cfi and Mx1
, collectively suggesting chronic neuroinflammation may persist during brain readaptation post-combined HLU+LDR exposure.
In response to extracellular cues (i.e., edema), cells rearrange their structures and ion transport proteins to maintain equilibrium [22
]. Furthermore, neuroplasticity is a homeostatic repair mechanism that involves the formation of new synapses and neural processes to compensate for their communication loss following brain infarct, injury or disease [12
]. These new formations result in brain restructuring and function, which may be occurring during readaptation post-combined HLU+LDR exposures. Indeed, our results fall in line with responses that were observed within brains isolated hours post-LDR (10 cGy) exposure, whereby multiple synaptic ion transport genes were altered [25
]. Herein, multiple cell structural genes, i.e., Drc7
and ion transport genes, i.e., Gria3
, were altered post-combination HLU+LDR conditions. Moreover, hypomethylation analyses indicated altered cytoskeletal organization, i.e., Ndel1
were observed in both LDR and HLU, suggesting cellular structural changes involved in neuroplasticity and readaptation processes are active, in order to restructure and/or maintain brain homeostasis.
Tight junctions, including claudins, connexins, and occludins contribute to BBB permeability and maintenance. A reduction in these molecules has been implicated in acute BBB disruption in neuroviral [26
] and neuroinflammatory diseases [27
]. In line with this, Cdh12
was downregulated, whereas, Cldn2
was induced in combination of HLU+LDR. Furthermore, Cdhr4
was hypomethylated in both LDR alone and HLU+LDR at 4 months readaptation, while Cdhr2
was hypomethylated in LDR and HLU alone, but not in combination HLU+LDR at 4 months readaptation, suggesting BBB restructuring, and further indicating the dynamic process of brain physiology during readaptation.
Adult neurogenesis is a process of neural and glia self-renewal and the generation of progenitor cells from radial glial cells (RGCs) within the subventricular and subgranular zones of the hippocampus [28
]. RGCs are quiescent and are activated to initiate neurogenesis in response to a stimulus [29
], such as LDR [17
] or models of increased intracranial pressure [31
]. Indeed, the majority of highly upregulated DEGs post-combination HLU+LDR were involved in HPA signaling, neuropeptide growth factors, and neurogenesis, i.e., Otp
, and Hcrt
. While the magnitude of gene expression does not always result in observable phenotypes, these notable DEGs may serve an important purpose in neuromaintenance during readaptation. Interestingly, Crh
, a stress-induced hormone, was hypermethylated post-exposure to LDR and combination HLU+LDR at 4 months readaptation, implying transcriptional silencing of Crh
may be engaged to suppress robust HPA activity during readaptation, which warrants further studies.
Readaptation results from combined exposures to HLU+LDR are in agreement with literature that spaceflight can alter neurofunction. Indeed, neurocognitive and behavioral effects via altered decision making, problem solving, visual perceptions, motor controls, and sleep–wake cycles have all been identified in flight [5
]. While “space-brain” characteristics have suggested astronauts are at risk of developing neurological sequelae in flight [33
] and post-flight [6
]. Due to the limitations of human neurological studies, mouse models provide an excellent alternative, with the caveat that fluid shifts in mice are marginal in comparison to their human counterparts. Therefore, follow-up studies utilizing larger mammals, i.e., rats, may provide more robust responses. Further, readaptation effects from radiation have been described to cause differential nervous system responses in sex, age, type of irradiation, duration of exposure and total doses [1
], while the readaptation effects of prolonged HLU on the neurohealth of different sexes and age groups are still unclear. Therefore, future studies approaching these additional demographics, both modeled in mice or rats, are required. Nonetheless, our readaptation study in mice exposed to combined HLU+LDR displayed a panel of neurological changes that were translational to humans, suggesting this is a suitable animal model to study spaceflight-related readaptation effects on the nervous system. Additionally, future studies should expand the types of radiation exposure animals experience in order to fully capture the spectrum of radiation that bombards astronauts during spaceflight. The space radiation environment beyond LEO contains several types of ionizing radiation. Galactic cosmic radiation (GCR) with relatively high linear energy transfer (LET) and proton radiation due to solar particle events (SPEs) contribute a significant portion of the radiation dose accumulated by astronaut crew members [3
]. High-energy heavy ions (HZE radiation) can produce distinct patterns of energy deposition in cells and tissues [13
]. However, secondary particles produced by the interaction of SPE protons and heavy-charged GCR particles with the spacecraft structure include γ-rays and x-rays [3
]. These radiation sources can also deliver a significant fraction of the total mission dose and pose a hazard to astronauts’ health [15
]. For chronic irradiation exposure, we used flood sources consisting of plastic sheets embedded with the isotope 57
Co that emits medium energy (122 and 136 keV) γ-rays. Additional research is needed to elucidate the CNS damage and readaptation response induced by the full spectrum of space radiation.
In summary, a multitude of neurological effects and persistent brain transcriptional alterations were observed at 4 months post-HLU+LDR in 21-day exposures. Changes included reduced transcriptional machinery, increased neurogenesis and neuropeptide production, and dysregulated cell structure and signaling genes. Thus, brain-related transcriptional changes are dynamic and plastic during readaptation from exposure to spaceflight-like conditions, and while these exposures may lead to long-term neurological consequences, the active processes of neuroplasticity and repair are engaged to maintain neural homeostasis.