Circadian regulation in aging: Implications for spaceflight and life on earth

Abstract Alterations in the circadian system are characteristic of aging on Earth. With the decline in physiological processes due to aging, several health concerns including vision loss, cardiovascular disorders, cognitive impairments, and muscle mass loss arise in elderly populations. Similar health risks are reported as “red flag” risks among astronauts during and after a long‐term Space exploration journey. However, little is known about the common molecular alterations underlying terrestrial aging and space‐related aging in astronauts, and controversial conclusions have been recently reported. In light of the regulatory role of the circadian clock in the maintenance of human health, we review here the overlapping role of the circadian clock both on aging on Earth and spaceflight with a focus on the four most affected systems: visual, cardiovascular, central nervous, and musculoskeletal systems. In this review, we briefly introduce the regulatory role of the circadian clock in specific cellular processes followed by alterations in those processes due to aging. We next summarize the known molecular alterations associated with spaceflight, highlighting involved clock‐regulated genes in space flown Drosophila, nematodes, small mammals, and astronauts. Finally, we discuss common genes that are altered in terms of their expression due to aging on Earth and spaceflight. Altogether, the data elaborated in this review strengthen our hypothesis regarding the timely need to include circadian dysregulation as an emerging hallmark of aging on Earth and beyond.


| INTRODUC TI ON
Aging is a complex process that involves a time-related decline in several molecular and cellular processes, and ultimately also in physiological functions necessary for survival and fertility in an organism.
Aging phenotype results from alterations in telomere length, epigenetic regulation, and mitochondrial function, along with dysregulations in nine other processes (proteostasis, macroautophagy, cellular senescence, stem cell exhaustion, intercellular communication, chronic inflammation, dysbiosis, genomic instability, and nutrient-sensing), which are altogether referred to as the hallmarks of aging (López-Otín et al., 2023). Interestingly, recent studies associate aging to spaceflight with contradictory conclusions (Biolo et al., 2003;Cannavo et al., 2022;Garrett-Bakelman et al., 2019;Honda et al., 2012;Ma et al., 2015;Malhan et al., 2023;Nwanaji-Enwerem et al., 2020;Otsuka et al., 2019;2022;Wang, 1999), while some of the studies report an anti-aging impact of spaceflight, others observed progressive aging due to spaceflight. Whether the anti-aging impact of spaceflight correlates with the "twin paradox" (Gron, 2006) based on the theory of relativity coined by Albert Einstein, which proposes that an individual in Space ages slower compared to his/her biological twin on Earth, remains to be shown. Increased focus on Space exploration and the required lengthening of Space missions for Deep Space Planetary exploration, increases the need for a better understanding of Space mission-associated alterations on aging-related pathways with impact on the health status of humans in Space.
The HRP (Human Research Program) carried out by the NASA (National Aeronautics and Space Administration) has recently reported five major human spaceflight-related hazards for long Space missions with a focus on future missions to Mars. These include exposure to Space radiation, altered gravity fields, isolation and confinement, closed environment, and distance from Earth (Patel et al., 2020). These hazards are linked with several risks to human health ranked according to their potential impact on the crew's health (red: higher risk, yellow: medium risk, and green: controllable risk).
The HRP-defined "red flag" risks include SANS (Space-Associated Neuro-Ocular Syndrome), cardiovascular disease, behavioral and cognitive impairment, circadian dysregulation, and muscle wasting due to exposure to microgravity (HRR-Evidence (nasa.gov)).These "red flags" are categorized into the four most affected systems as defined by International Space Agencies like NASA, ESA (European Space Agency), or JAXA (Japan Aerospace Exploration Agency): visual, cardiovascular, central nervous, and musculoskeletal systems.
Similar pathological alterations also occur as a consequence of aging on Earth. As the crew members are at a greater risk of experiencing these health stressors, it is important to understand their specific and combined impact on human performance and health in Space.
One major alteration to which astronauts are subjected during Space missions and which is related to the stressors described above is the disruption of their endogenous biological rhythm generated by the circadian clock (Guo et al., 2014). The circadian clock plays a regulatory role in an optimal adaptation of behavior and physiology to the 24 h Earth's rotation (Olejniczak et al., 2023), thereby generating ~24 h (circadian) rhythms, at the cellular level, in the human body. Cellular clocks are, under normal conditions synchronized with the main clock located in the diencephalic brain (epithalamus and hypothalamus), which in turn is entrained to the external environment (light, temperature), and thus contributes to the regulation of physiological features, for example, the sleep/wake cycle (Yadlapalli et al., 2018). On low Earth orbit, astronauts experience a sunrise or sunset every 45 min, resulting in circadian clock dysregulation with a significant impact on circadian-regulated biological pathways and subsequently on the timing of cellular processes, physiology, and behavior. Previous evidence points to disruptions of the circadian clock being associated to aging and aging hallmarks on Earth (Benitah & Welz, 2020), and increased incidence of age-related diseases such as macular degeneration (Blasiak et al., 2022), cancer (López-Otín et al., 2023), and neurodegenerative disorders (Shen et al., 2023).   (Biolo et al., 2003). Nevertheless, additional supporting evidence is needed to determine if indeed long-duration spaceflight affects human health in the long run, thus possibly affecting aging processes during future extended Space missions, as well as affecting the lifespan of astronauts after returning to Earth.
This becomes more and more relevant the deeper we explore and even colonize other planets requiring long-term exposure to different environments not just in terms of gravity, but also in terms of variable day length durations. The later may even more negatively affect astronauts through circadian disruption, and subsequent requirements for readaptation to new circadian, possibly ultradian (shorter than 24 h) and infradian (longer than 24 h) cycles, for example, in the next generation of extraterrestrial human life.
In this review, we discuss the current knowledge on the circadian clock in both aging on Earth and aging-related alterations during Space travel. Following an overview on the relevance of circadian rhythms and their role in several cellular processes in Section 2.1.
We then describe the impairments of the core-clock system and clock-controlled genes in four major age-related health risks: vision decline, cardiovascular diseases, CNS (central nervous system) damage, and musculoskeletal deterioration associated with aging (Section 2.2), as well with spaceflight (Section 3). Finally, we provide a brief perspective on the future needs in terms of Space exploration to allow for a more mechanistic understanding regarding the aging process and the putative, still largely unstudied, role of circadian (dys)regulation in contribution for its control and overall health management of human life in the future.

| A LINK B E T WEEN THE CIRC AD IAN CLO CK AND AG ING
As the world's population continues to age, there has been a significant increase in the number of elderly individuals aged 60 years and older from 12% to 22% projected by 2050 (World Health The circadian clock coordinates the timing of several physiological processes including retinal function (Bhoi et al., 2022), cardiac metabolism (Luo et al., 2022;Schroder & Delisle, 2022), neural activity (Reiter et al., 2023), and myogenesis .
Among different biological processes, the circadian clock regulates the expression and activity of mitochondrial metabolic and antioxidant enzymes (Mezhnina et al., 2022;Scrima et al., 2020). To ensure the daily coordination of these processes via environmental cues, diurnal light variations are conveyed by the retina to a central pacemaker, located in the SCN (suprachiasmatic nucleus) in the diencephalic brain hypothalamus (Ono, 2022). Light is a central zeitgeber ("time giver") for entraining the circadian clock to its external environment (Lorber et al., 2022). Besides light, physical exercise (Kim et al., 2023), and food  for example, may serve as zeitgebers. Physiological and organ functions are thus, via the clock, also entrained to the normal daily cycles of life on Earth.

| Visual system and circadian regulation
The circadian clock plays a major role in the maintenance of vision and its disruption can result in retinal degeneration (Jauregui-Lozano F I G U R E 1 Schematic representation of altered biological processes due to aging on Earth (left side) and Space exploration journey (right side).  Storch et al., 2007). Another core-clock gene, Per1 is rhythmically expressed in cells of the inner retina of mice, and is essential for the maintenance of retinal health (Witkovsky et al., 2003).
Besides retina neurons, the so-called "Müller" cells (retinal glial cells) were the first mammalian cell type that were reported, via bioluminescence signal detection, to have circadian expression in isolation from other retinal cells . A circadian system-related hormone, melatonin, synchronizes circadian rhythms and related physiological functions via G-protein-coupled receptors. Melatonin is synthetized in the central pituitary gland of the epithalamus (diencephalon) and stimulated by the SCN via retinal ganglionic axon terminals as a part of a parallel fibre tract, in addition to the main optic nerve and tract fibres that target to the main vision areas in the occipital lobe of the brain. Melatonin synthesis in the pituitary gland (epiphysis) is high during night and usually lowered by normal daylight or other artificial light sources (eye perception) (Zhao et al., 2019). The circadian regulation of melatonin synthesis is mediated by the retino-hypothalamic pathway and the specialized melanopsin-containing neurons directly project the light input to the SCN, suppressing the production of melatonin (Lundmark et al., 2006). In mammals, light is perceived by retinal ganglion cells (ipRGCs), in addition to rods and cones photoreceptors. ipRGCs play an important role in circadian entrainment, and they respond to light through the suppression of nocturnal melatonin (Mure, 2021).
Melatonin is also an effective antioxidant, and several ocular tissues including the corneal epithelium, dopaminergic amacrine neurons contain melatonin receptors, and thus melatonin can protect ocular tissues against oxidative stress (Lundmark et al., 2006). Besides pineal glands, melatonin can be synthesized in various organelles, for example, mitochondria, which is a major source of ROS (reactive oxygen species), and therefore plays a role in redox homeostasis crucial for the maintenance of human health (Melhuish Beaupre et al., 2021). However, melatonin levels decline with aging, thereby weakening its protective effect against oxidative stress in these tissues (Bondy, 2023).

| Cardiovascular system and circadian regulation
In the cardiovascular system, the circadian clock regulates several key mechanisms such as blood pressure (Costello & Gumz, 2021), heart rate (El Jamal et al., 2023), and cardiac contractility (Latimer & Young, 2022). BMAL1 expression is crucial for mitochondrial activities and cardiac function, and its deletion in human embryonic stem F I G U R E 2 Alterations in the core-clock machinery induce expression variations in clock-regulated genes associated with aging on Earth and increased risk of health problems due to spaceflight. Circadian transcriptional-translational feedback loops generate robust rhythms under healthy conditions, which may be dysregulated due to (a) aging on Earth, and (b) extreme environmental conditions such as spaceflight, resulting in increasing health risks if not developing pathophysiologic mechanisms possibly ending up in severe disease states. showed circadian expression in the rat heart and their circadian expression was regulated by NR1D1, essential for myocardial metabolism (Durgan et al., 2005). Several other genes associated with electrophysiological activity including Glut1/4 , and potassium ion channels (Kv1.5 and Kv4.2) showed circadian expression in rat cardiomyocytes (Yamashita et al., 2003).

| Neuronal plasticity and circadian regulation
Previous studies have shown that the core-clock genes Clock, Bmal1, and Per1/2 are involved in regulating behavioral outputs within the CNS (Snider et al., 2018;Vadnie & McClung, 2017). For example, mice with a Clock deletion exhibited altered reward-related behavior and impulsivity, pointing to other mood or behavioral changes such as hyperactivity (Spencer et al., 2012), while Per2 knockout mice displayed impairments in spatial learning and memory, and elevated expression of Drd1 gene (Kim et al., 2022). Bmal1 knockout mice showed altered circadian patterns of locomotor activity and behavioral changes such as impairment to habitual adaptation (Kondratova et al., 2010). Knockout of the core-clock gene Bmal1 or Clock/Npas2 double knockout in mice can lead to impaired neuronal plasticity in the brain hippocampus and cerebral cortex, regions critical for learning and memory (Musiek et al., 2013). The interplay between the Bmal1 and Per2 and CCGs such as GSK3β, a regulator of glucose metabolism and inflammation, was also implicated in regulating neuronal excitability and synaptic plasticity (Besing et al., 2017). Inhibition of GSK3 shortened the period of hippocampal molecular clock evaluated via PER2 reporter activity, whereas its activation resulted in disruption in BMAL1 rhythms suggesting a role for the GSK3 activity on the molecular clock (Besing et al., 2017). These findings suggest that disruption of core-clock genes and CCGs can have a significant impact on behavioural and neural outputs of the CNS.

F I G U R E 3
Graphic representation of the pathophysiological roles of core-clock genes in age-associated diseases. Core-clock genes play a crucial role in the whole-body homeostasis and their loss/gain influence visual, cardiovascular, central nervous system, and musculoskeletal system as depicted in the regulatory networks.  (Qin et al., 2023).

Retina function
Previous studies using mice showed that BMAL1 and CLOCK directly regulate the circadian expression of MyoD, which is a master regulator of myogenesis (Andrews et al., 2010). Moreover, Bmal1 plays a crucial role in muscle regeneration via satellite cell (i.e., typical muscle stem cell type) expansion, and loss of Bmal1 in mice displayed reduced satellite cell growth ex vivo, and lower expression of satellite cell marker Pax7 (Chatterjee et al., 2015). NR1D1, a suppressor of Bmal1 was later reported as a novel inhibitory regulator of muscle regeneration, and loss of Nr1d1 in mice showed enhanced proliferation of satellite cells (Chatterjee et al., 2019). In addition to skeletal muscle, the circadian clock plays a crucial role in bone metabolism (Hirai, 2017). Bone homeostasis is tightly orchestrated by the activity of osteoblasts (bone formation and mineral accretion) and osteoclasts (bone resorption) (Maciel et al., 2023).
The core-clock genes Per2 and Cry2 are shown to affect bone homeostasis in mice via influencing osteoblast and osteoclast activity, respectively (Maronde et al., 2010). Furthermore, several reports showed that bone formation is prevalent during the day, while bone resorption is prevalent during the night, following circadian rhythms (reviewed by (Tian & Ming, 2022)). Loss of Bmal1 displayed low bone mass phenotype in a mouse model (Samsa et al., 2016).
Taken together, these results suggest that the core-clock machinery plays an important role in regulating different components of the musculoskeletal system.

| Aging and functional dysregulation of cellular processes
Twelve hallmarks of aging have been defined, which include cellular and organismal processes dysregulated in aging: mitochondrial dysfunction, genomic instability, dysbiosis, chronic inflammation, al-  et al., 2016), as mentioned in the Introduction (Section 1). In addition, aging is associated with alterations in circadian rhythms, which in turn negatively impact health (Zhu et al., 2022). In the following section, we will focus on the four key systems listed above and introduce literature evidence concerning the dysregulation of circadian rhythms in the context of aging.

| Visual system and aging
Aging has a major impact on the deterioration of vision components. Aging-related alterations in ocular tissues may lead to dry eye disease, macular degeneration, and even blindness (Guymer & Campbell, 2023). Among different known eye pathologies, AMD (age-related macular degeneration), mainly characterized by degeneration in neuroepithelium of macular area, remains a leading cause of vision loss or blindness in different countries (Wong et al., 2014). Despite the increased knowledge on the molecular complexity underlying AMD, little is known about the role of the circadian clock in this pathogenesis. In a genome wide study with 88 AMD cases and 91 controls, APOE polymorphism in E2 allele showed a strong correlation with the increased risk of early AMD (Klaver et al., 1998). Another study showed that the loss of monocyte and macrophage chemoattraction-associated genes Ccl2 and Ccr2 in mice lead to macrophage dysfunction (Table 1), thereby contributing to early AMD pathology (Ambati et al., 2003). Both Ccl2 and Ccr2 expression showed rhythmic profile in the SCN in mice and CCL2 inhibition in the SCN diminished the circadian activation of immunity (Duhart et al., 2016). In a case-control study, ELOVL4 and CFH genes demonstrated a significant association with the status of late AMD (Conley et al., 2005). Another gene from the ELO family, Elovl2 promoter region showed increased methylation with age in mice retina, suggesting a potential role of Elovl2 as a regulator of molecular aging clock in retina .
Moreover, Elovl2 and Elovl4 expression are core-clock regulated as they depicted circadian expression in control rat retina, while their circadian expression was lost or diminished in a diabetic rat retina . Decline in the capacity of antioxidant molecules within RPE (retinal pigment epithelium), the epithelial cell layer within neurosensory area of retina, is a hallmark of AMD (Abokyi et al., 2020). In human RPE cells exposed to arsenite (oxidative stress), ATF4 (activating transcription factor; important for redox processes) lead to a direct increase in VEGF expression, a contributing factor to wet AMD (Roybal et al., 2005). Later, it was shown that the loss of Pten gene, which is important for the maintenance of RPE cell junction integrity, resulted in retinal degeneration in mice via increased oxidative stress (Kim et al., 2008). Nrf2 acts as a master regulator of endogenous antioxidant protection, and a study by Zhao Z et al. reported that the loss of Nrf2 in mice developed an age-dependent degeneration of the RPE cells (Zhao et al., 2011). Another study showed that binding of NRF2 to Cry2 gene, led to the inhibition of CLOCK/BMAL1-mediated transcription in mice hepatocytes (Wible et al., 2018). Furthermore, loss of Nrf2 in mice fibroblasts, hepatocytes, and liver resulted in circadian alterations, depicting a cross-talk between Nrf2 and the circadian clock. In a recent study, Dapl1 deficiency in mice impaired the antioxidant capacity of RPE through inhibition of MITF and its targets (NRF2 and PGC1A) that are needed for antioxidant defence mechanisms (Ma et al., 2023). PGC1α, essential for energy metabolism, is known to regulate different components of the coreclock network including Bmal1 (Liu et al., 2007). Another study TA B L E 1 Association between core-clock regulated genes and aging-related diseases.

Gene Species Mutation/loss/gain Main findings References
Visual system and aging depicted that the knockout of POLDIP2 in human RPE cell line contributes to higher risk of AMD through reduction in mitochondrial superoxidase levels and upregulation of SOD2 gene, which is a mitochondrial superoxidase dismutase (Nguyen et al., 2023).
Polymorphism in several other oxidative stress-associated factors including, HMOX1 and HMOX2 (Synowiec et al., 2012) and alternative complement pathway-associated factor CFD (Stanton et al., 2011) are associated with the occurrence and progression of different forms of AMD (dry and wet) among patients versus controls. Furthermore, BMAL1 in mice regulates the expression of Cldn5, which is required for the functional integrity of inner bloodretina barrier, and loss of Cldn5 is associated with dry AMD pathology (Hudson et al., 2019). In summary, the visual system is highly sensitive to the aging process and is a relevant bridging element between the circadian clock and aging. Several other genes associated with the risk of AMD are regulated by the circadian clock.
On the other hand, important zeitgebers like light require specific cells and paths within the visual system, to allow for entrainment

TA B L E 1 (Continued)
of the endogenous clock with the geophysical time, and the functioning of such cells decline with aging, with a negative impact on circadian regulated cellular and physiological processes.

| Cardiovascular system and aging
Cardiovascular diseases like coronary heart disease and atherosclerosis present a major burden on aging society worldwide (Liberale et al., 2022).
Tuzcu et al. carried out intravascular ultrasound on heart transplant recipients and reported higher prevalence of atherosclerosis among subjects over 50 years old (85%) when compared to subjects under 20 years old (17%) (Tuzcu et al., 2001). In recent years, several genes have been identified in connection with age-associated cardiovascular diseases. Among the different key regulators found, a gene polymorphism in FOXO3 is associated with longevity, and was shown to be important for the maintenance of cardiovascular homeostasis (Zhao & Liu, 2021). Foxo3 was reported to be important to repress cardiac growth, its deletion in mice resulted in a hypertrophic phenotype compared to controls (Ni et al., 2006). Cardiac dysfunction is also associated with an increased expression of KLF5 via FOXO1 and increased oxidative stress among cardiovascular patients with diabetes (Kyriazis et al., 2021).
Age-associated marker Nrf2 is essential to prevent oxidative stress; however, its lack is reported as a protection against the risk of atherosclerosis (reviewed by (Kloska et al., 2019)).

Furthermore, dysregulation among genes including Nox4
(reviewed by (Chen et al., 2012)), Cx43 (reviewed by (Michela et al., 2015)), PGC1α (reviewed by (Oka et al., 2020)), VEGFA (reviewed by (Braile et al., 2020)), and TSP1 (reviewed by (Chistiakov et al., 2017)) are reported with increased risk of cardiovascular diseases. Another important gene associated with age-related cardiovascular disease is KL, its genetic variant in Czech population is associated with a higher risk of cardiovascular diseases (Arking et al., 2005). The aging hallmark gene SIRT1 is essential for the development of cardiomyocytes and protection against oxidative damage through an interplay with the circadian clock (Soni et al., 2021).
In mouse hepatocytes and cultured fibroblasts, SIRT1 accumulation depicted circadian pattern, which was shown to be essential for the transcription of several core-clock genes (Bmal1, Per2, Cry1) (Asher et al., 2008). Higher levels of Sirt1 in mice is associated with cardiomyopathy (Alcendor et al., 2007), and alterations in circadian rhythms also increase the risk of cardiovascular diseases (El Jamal Altogether, these findings highlight the existence of altered clock-associated aging mechanisms as additional risk factors for cardiovascular diseases (Figures 2 and 3).

Several resident cells of the CNS such as microglia and astrocytes
showed an elevated expression of p16 and p21, which would re- showed an interesting association between the clock and the PARK2 gene, encoding for parkin, which is responsible for half of the autosomal recessive PD phenotype (Lunati et al., 2018). Normal fibroblasts showed rhythmic oscillations of glycolysis and mitochondrial activity whereas these rhythms were disrupted in patient-derived fibroblasts, which were collected from early-onset PD patients with carriers of PARK2 mutation (Pacelli et al., 2019). The expression of core-clock genes CLOCK, PER3 and NR1D1 showed a significant expression variation in patient-derived fibroblasts compared to the controls. Therefore, understanding the mechanisms of circadian dysfunction in these aging-associated pathologies may contribute for developing novel effective treatments and diagnostic strategies for these diseases based on the circadian clock status of the patients.

| Musculoskeletal system and aging
Aging results in a progressive decline in muscle mass and bone quality, thereby affecting mobility (Azzolino et al., 2021).  ., 2023). In a recent study from our group, we reported clock regulated expression of Atf4 in human and mice skeletal muscle (Malhan et al., 2023). A cross-sectional study performed on elderly women identified a specific polymorphism in ACTN3 (R577X), which contributes to a higher risk of developing sarcopenia (Romero-Blanco et al., 2021). Besides ACTN3, NRF2 polymorphism is also associated with sarcopenia risks among elderly (Urzi et al., 2020). A systematic review reported several genes including ACTN3, IGF1, TNFA, CNTF/R, UCP2/3 that showed association with skeletal muscle phenotype, and alterations with aging (Pratt et al., 2019). Interestingly, IGF1 expression is clock regulated, and inhibition of IGF1-signaling via caloric restriction promotes longevity among different species (reviewed by (Acosta-Rodriguez et al., 2021)). Another risk factor for sarcopenia is over-expression of Dkk3, its over-expression in young mice leads to progressive muscle mass loss (Yin et al., 2018). In case of osteoporosis among elderly, genes like CUL1, PTEN, and STAT1 depicted higher expression compared to non-osteoporotic group (Deng et al., 2023). However, the interplay between CUL1, PTEN, STAT1 and the circadian clock remains to be elucidated. Taken together, these findings point to a dysregulation of molecular pathways (IGF1, TNFA signaling) resulting in, at least, partial deterioration of musculoskeletal system with aging.

| ALTER ATI ON OF CIRC AD IAN CLO CK AND S PACEFLI G HT: AN ( ANTI)AG ING EFFEC T ?
Space environment either promotes or even prevents aging processes. However, the exact duration of such an exposure to microgravity in Space among astronauts is still unclear, and when or to what extent pathophysiologic effects are actually detectable in an astronaut flown to Space remains a point of debate. Some of the mentioned dysfunctions seem to happen even faster in Space, a dramatic example is the reported loss of functional capacity of the cardiovascular system (e.g., cephalad fluid shift, heart rate, and stroke volume reduction), and musculoskeletal system (disuse atrophy), that proceeds approximately 10 times faster in Space than due to normal aging on Earth (Vernikos & Schneider, 2010). For example, a study using rat neuronal cells (PC12 cells) under simulated microgravity condition showed partial cell cycle G1 phase arrest, and activation of p53 and p16 pathways, that leads to cellular senescence and aging in vitro (Wang et al., 2009). In tail-suspended mice (analogue to microgravity unloading), mimicking spaceflight condition, a decreased lymphocyte B-cell differentiation was observed resembling agingrelated modifications (Lescale et al., 2015).
A number of bedrest studies with otherwise healthy participants (analogue to human spaceflight) have been used to simulate physiological alterations related to acute or chronic muscle disuse, similar to microgravity environments as experienced during short-, medium-, and long-duration spaceflights, were reported to lead to aging-related phenotypes, such as deterioration of cardiovascular function and affecting musculoskeletal health (reviewed by (Kehler et al., 2019)).

| Effects of microgravity on organ systems
As circadian rhythms on Earth are governed by natural diurnal light-dark cycles, it will be of utmost importance to better under- a lower blood production and abnormal blood pressure resulting in a decreased stroke volume and diastolic blood pressure with a lower heart rate (Norsk et al., 2015;Otsuka et al., 2015). With less volume to pump, the heart muscle adapts accordingly with size reduction of the left ventricle wall and lower contractility (Perhonen et al., 2001). Due to microgravity-induced functional adaptation of the cardiovascular system after the first 1 or 2 weeks in Space, though more or less suffering from acute "space-sickness" during initial microgravity adaptation, the astronaut may be able to still maintain performance and mission duties during their entire missions (Goswami, 2017). However, when re-entering gravitational forces, astronauts suffer from orthostatic intolerance with risk of syncope shortly after landing (Goswami, 2017). While muscle atrophy, bone loss and blood volume may be partly prevented by strict exercise as an inflight countermeasure (Loehr et al., 2015) and thus may allow for improved mission success following longer stays in Space, other impairments such as SANS create an even greater problem for extended Space missions over several weeks or months. SANS includes optic disc edema, posterior globe flattening, chorioretinal folds, and retinal nerve fiber layer thickening (Ong et al., 2022). Pathogenesis of SANS is still unknown, but un- Another space-related challenge for astronauts is sleep disruption. Astronauts experience sleep fragmentation (i.e., impaired sleep architecture), reduced daytime alertness, and reduced nocturnal sleep tendency (Czeisler et al., 1991).

| Clock genes and gravitational unloading in animal and human research
Microgravity-induced biological dysregulations discussed in the previous section may be explained by disruptions in the circadian clock (Czeisler et al., 1991). During spaceflight many environmental factors that have been reported as possible zeitgebers are altered (e.g., 24 h light-dark cycle, and feeding habits). The expression of clock-associated genes may be changed by many other factors related to the microgravity environment in Space (e.g., electromagnetic field, radiation, hypoactivity, and heat stress) via lack of sweat evaporation required for body core temperature control, (see (Stahn et al., 2017)), as well as unique conditions onboard the ISS (International Space Station) (e.g., lighting conditions, shift-work schedule) (Guo et al., 2014). Although the effect of spaceflight on the circadian rhythm is known for more than has a key role in muscle growth after disuse atrophy (Washington et al., 2011). In a study with Igf1 knockout mice, the authors reported an equal muscle mass loss after hindlimb suspension between knockout mice, their controls (knockout without hindlimb suspension) and wild type (no knockout with hindlimb suspension).
The loss of muscle strength was greatest in Igf1-deleted mice following disuse. The return of muscle mass and strength in recovery seemed independent of Igf1 concluding Igf1 as one of the key players in muscle degradation (Spradlin et al., 2021). Vogel et al.
reports on human cells (Jurkat T-cells, U937) that were exposed to altered gravity within short duration gravitational transitions in parabolic flight (Vogel et al., 2019). The expression of HIF1A, a regulator of T-cell response, was downregulated in microgravity, as well as pro-inflammatory cytokine IL1b. HIF1-related genes (involved in HIF1 signaling) were upregulated during the hypergravity phase, but not reversed or otherwise changed during microgravity phase (Vogel et al., 2019). A previous study of the same working group discovered altered regulatory genes in microgravity phase (ATP6V1, LIN00837, IGHD3) (Thiel et al., 2017).
To obtain a comprehensive view of human body's response to Space environment, the NASA carried out a twin's study in 2015-2016 where one identical twin astronaut was monitored before, during, and after a one-year mission while the other twin counterpart served as a genetically matched ground control (Garrett-Bakelman et al., 2019). The results from the twin's study revealed multiple changes including, increased retinal thickness, and changes in artery dimensions due to Space environment, that reversed to their normal levels after 6 months upon return to Earth.
Interestingly, telomere lengthening (sign of anti-aging) was observed while the astronaut was in Space, which reversed upon his return to Earth. Following 6 months after return to Earth, the twin also showed increased number of shorter telomeres (sign of aging). Moreover, increased DNA damage, and altered cognitive function were observed after 6 months on Earth (Garrett-Bakelman et al., 2019). An interesting anti-aging effect of spaceflight was reported in the DNA methylation data of astronauts on Space mission for 520 days (Nwanaji-Enwerem et al., 2020). In another study, space flown Caenorhabditis elegans exhibited the signs of slower aging through neuronal and endocrine response (Honda et al., 2012). Similarly, Drosophila melanogaster post 13 days in Space showed longer lifespan versus their ground controls . Furthermore, Otsuka K et al. reported anti-aging effect of spaceflight through evaluation of heart rate variability among astronauts during 6-month mission (Otsuka et al., 2019), and during 1-year mission (Otsuka et al., 2022). In vitro culture of human skeletal muscle myoblasts (which can fusion to multinucleated myotubes/myofibers and eventually differentiate into young and adult muscle fibers) under simulated microgravity resulted in decreased cell proliferation and cytoskeleton enlargement of cells similar to changes observed during normal aging . Also in vitro experiments in Yeast fungi, Saccharomyces cerevisiae, seem to point to a correlation between aging-induced phenotype due to altered gravity conditions, and signs of accelerated aging including oxidative stress and heat shock protein expression under simulated microgravity (Fukuda et al., 2021).

| Outlook to future research
Hence, the results presented in space-analogue studies, space flown mice or isolated cells housed in specialized animal habitats or single-cell culture modules, have shown that microgravity alters circadian clock genes in various organisms from cells ( Table 2)

Human
Overall mortality is higher in astronauts probably due to higher risk of spacecraft accidents; astronauts have a higher risk of developing cataract as they are exposed to a higher dose of space radiation.  et al., 2023). Alternatively, they can be studied in an expedient way using small biopsy samples obtained directly from a tissue of interest (tissue-specific approach) such as in a particular postural and gravity-sensitive functional leg skeletal muscle as recently reported from Space omics datasets of astronauts Rittweger et al., 2018).

Institute of Medicine
New findings from this still largely unstudied area will help to improve countermeasures for future Space missions, and at the same time increase our understanding toward life and aging on Earth.

| CON CLUS IONS
Aging is a complex process that is influenced by a multitude of factors, including circadian regulation. The disruption of circadian rhythms can have profound consequences for human health, particularly during spaceflight where astronauts are exposed to extreme conditions that can disrupt their normal daily mission duties including impaired sleep-wake cycles. Furthermore, aging on Earth is also associated with a decline in circadian regulation, which can contribute to the development of various age-related diseases such as visual decline, cardiovascular diseases, neurodegeneration, and musculoskeletal deterioration.
Understanding the mechanisms underlying circadian regulation in aging is thus essential for developing interventions that can improve health outcomes for both astronauts and elderly population on Earth. For instance, promoting healthy sleep patterns through the use of light therapy or other non-pharmacological interventions may help to mitigate the negative effects of circadian disruption during spaceflight. Melatonin supplementation may help to minimize the impact of oxidative stress seen due to aging or due to extreme environmental conditions. Interventions that target circadian regulation in aging individuals on Earth may help to reduce the risk of age-related diseases and improve overall health and well-being.
Moving forward, it will be important to continue to explore the relationship between aging, circadian regulation, and spaceflight.
Advances in technology and data analytics are providing new opportunities to monitor and manipulate circadian rhythms in real-

time, which could have important implications for human health in
Space and on Earth. Additionally, the development of personalized interventions that specifically target circadian rhythms may hold promise for improving health outcomes among elderly. By better understanding the complex interplay between these factors, from single cells and tissues to multicellular organs and systems level, but also from various organisms in evolution up to higher vertebrates and humans, we may be able to develop new strategies for improving health outcomes and promoting healthy aging for all individuals.

ACK N OWLED G M ENTS
We are grateful to all members of Relógio group for their critical remarks and feedback. Vector icons used in this manuscript were obtained from Flaticon (https://www.flati con.com/), Freepik (https:// de.freep ik.com/) and modified accordingly.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors have no conflict of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analyzed in this study.