Molecular basis of radiation resistance in tardigrades and the medical implications

Abstract

Tardigrades exhibit remarkable resistance to ionizing and ultraviolet radiation, tolerating exposures far beyond the lethal limits for most organisms. Among the tardigrade-derived molecules implicated in this resilience, damage suppressor (Dsup) is the most well-characterized and demonstrates clear radioprotective activity. This mini-review summarizes the molecular mechanisms underlying tardigrade radiation resistance and discusses their potential medical applications. Dsup associates with chromatin, reducing hydroxyl-radical-induced DNA strand breaks and limiting ultraviolet-mediated pyrimidine dimer formation. Human cells expressing Dsup exhibit decreased DNA damage by approximately 40% and enhanced survival following irradiation, indicating that essential protective mechanisms of tardigrades can be functional in mammalian systems. These findings support the exploration of Dsup-based strategies to mitigate radiation-induced cellular injury, improve preservation of biological materials, and enhance resilience in high-radiation environments such as radiotherapy or space missions. Further mechanistic studies of Dsup, DNA repair pathways, and antioxidative systems, along with in vivo evaluations, are essential to fully elucidate the molecular basis of tardigrade radiation resistance. Collectively, these mechanisms provide valuable insights that may guide the development of novel approaches for medical radioprotection.

Key Words: Tardigrade; Damage suppressor; Cryptobiosis; Ionizing radiation; Reactive oxygen species

Core Tip: Tardigrades exhibit extraordinary resistance to ionizing and ultraviolet radiation. Among the tardigrade-derived molecules linked to this resilience, damage suppressor binds to chromatin and mitigates both hydroxyl-radical-induced DNA breaks and ultraviolet-induced pyrimidine dimers. Recent studies have demonstrated that damage suppressor expression in mammalian cells reduces γ-H2AX foci by approximately 40% and prolongs cellular survival after irradiation. In this minireview, we highlight recent advances in understanding tardigrade radiation resistance and explore potential clinical applications for managing radiation-induced cellular injury.

INTRODUCTION

Tardigrades, belonging to the phylum Tardigrada and commonly known as water bears or moss piglets, are microscopic invertebrates typically measuring 0.25-0.50 mm in length[1]. These eight-legged metazoans possess a simple yet distinctive body architecture, characterized by bilateral symmetry, a head region, and four trunk segments, each bearing a pair of short, unjointed legs terminating in claw-like structures[2]. Approximately 1400 species have been identified. Despite their small size, tardigrades possess remarkable biological capabilities[3], particularly their extraordinary resistance to high-energy radiation, far exceeding the tolerance of most other organisms. Notably, compared with humans, for whom the whole-body lethal dose 50% of ionizing radiation is approximately 4-5 Gy, the terrestrial tardigrade species Ramazzottius varieornatus can survive exposure to over 4000 Gy, representing an almost 1000-fold greater tolerance[4]. Tardigrades also withstand extreme dehydration, rapid temperature fluctuations, severe pressure changes, and even direct exposure to outer space. Their lifespan ranges from approximately 3 months to 2 years, depending on species and environmental conditions.

Their exceptional resilience is largely attributed to multiple protective strategies, among which cryptobiosis – a reversible state of suspended animation – is particularly important[5]. Entry into cryptobiosis triggers profound anatomical and physiological remodeling, reducing metabolic activities to virtually undetectable levels and enabling survival in otherwise lethal conditions[6]. During tun formation, tardigrades retract their heads and limbs and contract along the body axis, facilitating the transition to cryptobiosis[7]. During cryptobiosis, oxygen consumption becomes nearly undetectable, and overall metabolism remains profoundly suppressed[8]. However, several tardigrade species also exhibit substantial environmental tolerance even while in their active state, highlighting the importance of studying stress resistance in both the active and cryptobiotic states.

In this minireview, we summarize the remarkable radiation resistance of tardigrades, describe the function of the tardigrade damage suppressor (Dsup) in protecting DNA through chromatin binding, outline additional mechanisms of radiation tolerance, and discuss potential biomedical applications.

RADIATION RESISTANCE OF TARDIGRADE

In the active state, tardigrades contain abundant intracellular water, maintain high metabolic activity, and exhibit only baseline resistance to environmental stress, including ionizing radiation (Figure 1). By contrast, during the cryptobiotic (tun) state, they lose most of their water, enter complete metabolic arrest, and display markedly enhanced tolerance to extreme conditions, including a substantially increased resistance to ionizing radiation, particularly in eggs (Figure 1).

Figure 1 Differences between the active and cryptobiotic states of tardigrades.

Ramazzottius varieornatus demonstrates pronounced differences in ultraviolet (UV) tolerance depending on hydration state. In the hydrated state, approximately 80% of individuals survive for 5 days following exposure to 2.5 kJ/m² of UV-C. In the dehydrated state, survival remains around 80% even after exposure to a much higher dose (20 kJ/m²) for up to 13 days, although survival declines thereafter[9]. Reproductive capacity also varies: Dehydrated animals exposed to 2.5 kJ/m² produced 162 eggs in total, whereas higher doses completely abolished egg production[9].

Tardigrades exhibit three remarkable and unexpected characteristics in their resistance to ionizing radiation. First, tardigrades in the hydrated state often tolerate irradiation as well as those in the dehydrated state, which is surprising because indirect radiation damage caused by water-derived radicals should be more severe when water is present[10-12]. Second, adult tardigrades display similar levels of tolerance to both low linear energy transfer (LET) and high LET radiation, and in some cases, they survive better following exposure to high LET radiation. For example, Milnesium tardigradum exposed to high doses of helium ions survived better when hydrated than when dehydrated[11]. Because high LET radiation primarily induces biological damage through direct effects rather than through radical-mediated indirect effects, these observations strongly suggest that tardigrades possess highly efficient DNA protection and repair mechanisms[13,14].

Third, radiation tolerance in tardigrades varies markedly across developmental stages. Embryos are particularly sensitive during early development[15,16]. One proposed explanation for the greater radiation tolerance observed in adults and late-stage embryos, compared with early-stage embryos, is their minimal mitotic activity. Because somatic cell division is scarce in tardigrades and dividing cells are generally more vulnerable to radiation, reduced proliferation may contribute to their higher tolerance[1]. In the earliest embryonic phases, dehydrated eggs of Milnesium tardigradum withstand extraordinary radiation exposure, surviving doses up to approximately 1690 Gy, whereas hydrated eggs tolerate only about 509 Gy[11]. Similarly, reproductive capacity is often impaired at lower radiation doses than those affecting survival. For example, Hypsibius exemplaris can survive and reproduce following exposure to 100 Gy[16]; however, individuals exposed to 500-2000 Gy, near half of the species’ lethal dose 50%, maintain survival but lose reproductive capability[16].

Collectively, these findings indicate that tardigrades employ remarkably effective mechanisms to limit radiation-induced damage across physiological states and developmental stages. Their ability to maintain viability – when conditions permit, reproductive potential – under extreme radiation exposure highlights biological strategies that exceed those observed in other animal groups.

RADIATION RESISTANCE MECHANISM OF DSUP

Dsup is a largely intrinsically disordered DNA-binding protein found exclusively in Ramazzottius varieornatus[4]. Heterologous expression of the Dsup gene in tobacco plants markedly increased resistance to both UV-C and X-ray irradiation, resulting in substantially enhanced cell viability[17]. In HEK293T cells, constitutive Dsup expression markedly reduced X-ray-induced DNA damage, as evidenced by an over 50% decrease in single-strand breaks detected by the alkaline comet assay compared with control cells[4]. Similarly, Dsup expression conferred superior survival following UV-C exposure[18]. Together, these findings suggest that Dsup associates with chromatin and mitigates radiation-induced DNA damage (Figure 2).

Figure 2 Chromatin protection and reduction of radiation-induced DNA damage by damage suppressor. Dsup: Damage suppressor.

Dsup comprises 445 amino acids and contains a long central α-helix flanked by flexible, intrinsically disordered regions enriched in basic residues. Its C-terminal region harbors a defined nuclear localization signal (residues 383-404) and a short-conserved motif (residues 363-370) resembling the nucleosome-binding module of vertebrate high-mobility group N proteins. Proteins homologous to Dsup have been identified only in another tardigrade species, Hypsibius exemplaris, and share approximately 24% sequence identity with the canonical Dsup sequence[19]. Despite this low overall identity, several short sequence motifs are highly conserved and are directly associated with Dsup function[19]. Dsup exhibits a pronounced electrostatic bias, containing substantially more basic than acidic residues and carrying an estimated net positive charge of +23. The sequence is enriched in lysine and arginine, whereas acidic residues are relatively scarce. In addition, the protein is dominated by simple nonpolar (notably glycine and alanine) and polar (such as serine and threonine) residues, yielding a composition that is both charge-rich and structurally flexible[19]. A further notable characteristic of Dsup is its unusual amino acid composition. The protein contains no cysteine residues and only very few aromatic amino acids. Long side-chain hydrophobic residues are also rare, with only a few isoleucines and a single leucine present. This composition is consistent with intrinsically disordered behavior, as aromatic and bulky hydrophobic residues promote hydrophobic aggregation and cysteine enables disulfide bond formation, both of which would restrict conformational flexibility. Consistent with this property, Dsup binds DNA with high affinity through multivalent interactions and induces conformational changes in the nucleic acid molecule that may reduce its susceptibility to radiation-induced damage[20].

Recent findings indicate that Dsup also contributes to shielding genomic DNA from damage induced by reactive oxygen species (ROS), including hydroxyl radicals, which are cytotoxic intermediates produced during radiation exposure (with biochemical studies suggesting binding of up to approximately 4 Dsup molecules per nucleosome)[18,21-24]. To evaluate its effects on oxidative stress resistance and whole-organism physiology, Drosophila melanogaster lines were engineered to express the tardigrade Dsup protein[25]. In these transgenic flies, Dsup markedly attenuated hydrogen peroxide–induced oxidative damage and improved survival measures, including median and maximum lifespan as well as the age at 90% mortality. Similarly, expression of the Ramazzottius varieornatus Dsup gene in Caenorhabditis elegans conferred tolerance to X-ray irradiation and oxidative stress without detectable toxicity and produced a marked extension of lifespan, which was associated with a substantial reduction in mitochondrial respiration[26]. However, whether these protective effects generalize across diverse cell types and species remains unresolved. Furthermore, because CRISPR-Cas9-mediated gene knockout has not yet been established in tardigrades[27], direct experimental evidence demonstrating the essential role of Dsup in their exceptional resistance to ionizing radiation is still lacking.

Collectively, the available evidence identifies Dsup as a key modulator of DNA integrity, although its biological functions appear more complex than currently understood. Elucidating how Dsup contributes to the extraordinary radiation tolerance of tardigrades will require future investigations examining its roles at the molecular, cellular, and organismal levels.

OTHER RADIATION RESISTANCE MECHANISMS

Beyond the efficient DNA protection mediated by Dsup, enhanced DNA repair and a robust antioxidative defense system have been proposed as key contributors to the high radiation tolerance observed among tardigrades. Table 1 presents the radiation resistance mechanisms other than Dsup[4,28-33].

Table 1 Radiation resistance genes other than damage suppressor.

Gene	Study model	Key findings	Ref.
DNA repair–related genes	Ramazzottius varieornatus exposed to γ-radiation (500 Gy) with transcriptomic analysis	Upregulation of DNA repair pathways, including those of homologous recombination and base excision repair, following γ-irradiation	Yoshida et al[28]
	Hypsibius exemplaris exposed to γ-radiation (100-2180 Gy) with transcriptomic analysis	Upregulation of multiple DNA repair pathways following γ-irradiation	Clark-Hachtel et al[29]
Antioxidative defense genes	Ramazzottius varieornatus genome analysis with desiccation-related transcriptomic analysis	Expansion of SOD genes (including as CuZn-type SODs and Mn-type SODs) and presence of bacterial-type catalases, suggesting enhanced ROS detoxification capacity	Hashimoto et al[4]
	Echiniscoides cf. sigismundi and Ramazzottius cf. coronifer with transcriptomic analysis	Antioxidative defense involving SOD, catalase, and GST, suggesting enhanced ROS detoxification capacity	Kamilari et al[30]
	Milnesium tardigradum with proteomic analysis	Presence of antioxidative enzymes, including SOD, catalase-related peroxidases, and GST, indicating ROS detoxification capacity	Schokraie et al[31]
Dihydroxyphenylalanine dioxygenase 1	Hypsibius henanensis exposed to heavy-ion radiation (200 Gy and 2000 Gy) with multi-omics analysis	Activation of a tyrosine–dihydroxyphenylalanine-betalain metabolic pathway limiting ROS	Li et al[32]
Tardigrade-specific radiation-induced disordered protein 1		Radiation-induced liquid–liquid phase separation concentrating 53BP1 and Ku70 at DNA double-strand breaks
NADH dehydrogenase (ubiquinone) 1 beta subcomplex subunit 8/ubiquinol-cytochrome C reductase synthesis		Enhanced mitochondrial oxidative phosphorylation with nicotinamide adenine dinucleotide regeneration
Tardigrade DNA damage response 1	Hypsibius exemplaris exposed to γ-radiation (500 Gy)	DNA binding and chromatin compaction with reduced phospho-H2AX levels after γ-irradiation	Anoud et al[33]

GST: Glutathione S-transferase; ROS: Reactive oxygen species; SOD: Superoxide dismutase.

With respect to DNA repair, tardigrades exhibit pronounced transcriptional responses that likely contribute to their exceptional radio-resistance. In Milnesium cf. tardigradum, ionizing radiation causes developmental delay during embryogenesis, which suggests that DNA repair processes have been activated[15], and similar radiation-responsive regulation of DNA repair genes has been reported in Ramazzottius varieornatus[28]. Hypsibius exemplaris sustains DNA damage after γ-irradiation but rapidly repairs it through an exceptionally strong radiation-induced upregulation of DNA repair gene expression, with some transcripts becoming among the most abundant in the organism[29]. Analysis of the chromosome and nuclear architecture in early embryos of Hypsibius exemplaris revealed that chromosomes remain individualized and are housed in lamin-lined compartments throughout the cell cycle, suggesting a mechanism that may limit chromosomal rearrangements during DNA damage repair[34].

Superoxide dismutases (SODs) are key antioxidative enzymes that eliminate superoxide radicals by converting them into molecular oxygen and hydrogen peroxide. Genomic comparisons revealed that tardigrades possess an expanded set of stress-responsive gene families, particularly those encoding SODs[3,4,30]. Ramazzottius varieornatus contains approximately 16 putative SOD genes, which are predicted to localize to the mitochondria, cytosol, and possibly peroxisomes, and this expansion appears to be characteristic of tardigrades[30]. Eutardigrade species, in general, harbor approximately 12-16 putative SOD genes, whereas heterotardigrades, such as Echiniscus sigismundi, possess only about seven[30]. In the eutardigrade Milnesium tardigradum, both CuZn-type SODs (six contigs) and Mn-type SODs (two contigs) have been identified. Ramazzottius varieornatus shows particularly strong basal expression of CuZn-type SODs, whereas Echiniscus sigismundi exhibits a more modest induction of these genes following exposure to ionizing radiation or bleomycin[31]. Catalase, which decomposes hydrogen peroxide, and glutathione S-transferases, which mediate glutathione-dependent detoxification reactions, are also present in tardigrades and show stress-responsive expression[4,30,31]. However, their gene families do not display the pronounced expansion observed in SODs, and detailed functional studies remain limited[3].

Comprehensive integration of multi-omics analyses with functional evidence has shown that dihydroxyphenylalanine dioxygenase 1 activates an amino acid metabolic pathway involving tyrosine, dihydroxyphenylalanine, and betalains, which helps limit ROS. Tardigrade-specific radiation-induced disordered protein 1 promotes liquid-liquid phase separation, which increases the efficiency of DNA double-strand break repair by approximately 1.5-2.0-fold and contributes to radiation tolerance[32]. Additionally, NADH dehydrogenase (ubiquinone) 1 beta subcomplex subunit 8 and ubiquinol-cytochrome C reductase synthesis have been implicated in enhancing mitochondrial oxidative phosphorylation and nicotinamide adenine dinucleotide regeneration, thereby sustaining energy metabolism under stress conditions[32].

Tardigrade DNA damage response 1 (TDR1) is a widely conserved tardigrade protein that appears among the transcripts with exceptionally abundant expression[33]. The abundance of basic residues in TDR1 suggests a strong affinity for DNA, with binding estimated at approximately one protein molecule per three base pairs. This property is thought to promote the formation of extensive DNA-protein assemblies that provide direct protection against ROS. TDR1 exhibits robust DNA association in vitro, a feature it shares with Dsup, despite the absence of detectable sequence homology and reliance solely on their richness in basic residues[33]. TDR1 expression reduced bleomycin-induced γ-H2AX foci by approximately 40%-50%, indicating a substantial decrease in DNA double-strand break damage[33].

These findings indicate that the extraordinary radiation tolerance of tardigrades arises from a coordinated network of protective strategies that extend far beyond Dsup alone. Continued investigation of these diverse molecular defenses will be essential for understanding how tardigrades maintain genomic stability under extreme environmental stress.

POTENTIAL APPLICATION IN MEDICINE

Kirtane et al[35] demonstrated that localized and transient expression of Dsup reduces radiation-induced DNA damage in oral and rectal epithelial tissues. Using ionizable lipid nanoparticles supplemented with biodegradable cationic polymers for efficient mRNA delivery, they achieved radioprotection of normal tissues in a mouse orthotopic oral cancer model without compromising antitumor efficacy, highlighting the potential broad applicability of this approach.

Chen et al[36] reported that expression of Dsup in mesenchymal stem cells (MSCs) confers radioprotective effects on the hematopoietic microenvironment. Dsup expression did not impair MSC stemness or differentiation. In both hematopoietic stem and progenitor cell (HSPC)-MSC co-culture systems and irradiated mouse models, Dsup-modified MSCs promoted faster hematopoietic recovery, increased HSPC numbers, and preserved differentiation capacity. Consistent with these findings, administration of Dsup-modified MSCs in mouse models significantly improved the survival and recovery of residual HSPCs after irradiation, indicating a promising MSC-based radioprotective strategy for acute radiation syndrome by protecting the hematopoietic niche.

Radiotherapy – while one of the most effective cancer treatment modalities – frequently induces immune cell loss and dysfunction, as multiple immune cell populations, including lymphocytes, monocytes, and natural killer cells, are highly radiosensitive[37,38]. This limitation is particularly relevant in brain tumors, where monotherapy remains challenging, and combination strategies are increasingly pursued. Radiotherapy combined with adoptive cell therapy (ACT) has therefore emerged as a promising approach to enhance therapeutic efficacy. An emerging review by Groth et al[39] highlighted immune cell radioprotection as a key strategy in neuro-oncology, proposing the use of chimeric antigen receptor T cells expressing tardigrade-derived molecules to preserve immune function and enhance antitumor activity during radiotherapy. The review further discusses approaches for identifying radioprotective molecules, as well as formulation and delivery systems suitable for clinical translation. However, direct experimental evidence evaluating the effects of tardigrade-derived molecules on immune cells remains limited, underscoring the need for further studies to assess their efficacy, safety, and long-term genomic stability before immune cell radioprotection strategies based on tardigrade-derived molecules can be translated into clinical ACT settings. If validated in future studies, Dsup could potentially enable not only combined treatment with radiotherapy and ACT but also other radiotherapeutic approaches (e.g., the delivery of higher radiotherapy doses in image-guided treatments), while minimizing normal tissue toxicity.

Although Dsup is well recognized for its capacity to protect DNA from radiation damage, recent evidence shows that its expression in primary cortical neurons derived from embryonic rat brain induces neurotoxicity, promotes DNA double-strand breaks, and disrupts chromatin structure rather than providing protection[40]. These findings raise concerns that Dsup may exert harmful effects not only in neurons but also in other highly differentiated tissues, highlighting the need for careful safety evaluation before considering translational applications. Therefore, the possible tissue-specific effects of Dsup (e.g., potentially safe in epithelial or hematopoietic cells but neurotoxic in neurons) require careful evaluation across different cell types.

Collectively, efforts to translate tardigrade biology into practical radioprotection are rapidly expanding. Current strategies under investigation include recombinant and cell-penetrating forms of tardigrade-derived molecules, messenger RNA therapeutics encoding stress-tolerant proteins, peptidomimetic versions of intrinsically disordered motifs, and biomimetic nanomaterials inspired by vitrification[41,42]. These approaches aim to reproduce the remarkable molecular safeguards that preserve cellular integrity under extreme irradiation, with potential applications in radiotherapy and protecting against radiation exposure in scenarios such as nuclear accidents and space missions.

Several tardigrade-derived molecular systems may also hold promise for biomedical applications. The expanded network of antioxidants (including multiple SODs) observed in tardigrades may help to mitigate radiation-induced oxidative stress, thereby protecting normal tissues during radiotherapy[42]. Such mechanisms could be particularly relevant for reducing damage to radiosensitive organs, including the hematopoietic system and gastrointestinal epithelium. Furthermore, the strong induction of radiation-responsive DNA repair pathways, including that of homologous recombination factors such as Rad51 and the phase-separation-mediated repair activity of tardigrade-specific radiation-induced disordered protein 1, suggest potential strategies for enhancing genome stability under genotoxic stress[32,42]. The transient activation of these repair systems could potentially protect hematopoietic stem cells from radiation-induced or chemotherapy-induced DNA damage, thereby improving treatment tolerability. Additionally, tardigrade proteins associated with mitochondrial function [e.g., NADH dehydrogenase (ubiquinone) 1 beta subcomplex subunit 8 and ubiquinol-cytochrome C reductase synthesis] may contribute to maintaining oxidative phosphorylation and cellular energy balance under stress conditions, offering possible strategies to enhance cellular resilience under oxidative or metabolic stress.

However, important challenges remain, including large-scale production, maintaining molecular stability, ensuring immune safety in vivo, and achieving precise delivery to vulnerable tissues. Overcoming these obstacles will require coordinated advances in molecular biology, materials science, and clinical innovation, ultimately determining whether tardigrade-inspired radioprotectants can transition from experimental promise to practical therapeutic applications.

CONCLUSION

Tardigrades exhibit exceptional molecular strategies for surviving extreme radiation, offering valuable insights for the future of radiation medicine. A notable component is Dsup, which associates with chromatin, suppresses radiation-induced DNA strand breaks, and enhances the efficiency of cellular repair responses. Rapid advances in protein engineering and messenger RNA-based therapeutics have provided realistic prospects for clinical translation. Tardigrade-inspired strategies have the potential to protect patients undergoing radiotherapy, safeguard astronauts during deep-space missions, and strengthen preparedness for nuclear emergencies, transforming an extraordinary organism into a practical foundation for innovative radiation defense.

ACKNOWLEDGEMENTS

We thank Fukushima K for assistance with administrative tasks.