Hypothalamic hormone deficiency enables physiological anorexia.

Mammalian hibernators survive prolonged periods of cold and resource scarcity by temporarily modulating normal physiological functions, but the mechanisms underlying these adaptations are poorly understood. The hibernation cycle of thirteen-lined ground squirrels (Ictidomys tridecemlineatus) lasts for 5-7 months and comprises weeks of hypometabolic, hypothermic torpor interspersed with 24-48-hour periods of an active-like interbout arousal (IBA) state. We show that ground squirrels, who endure the entire hibernation season without food, have negligible hunger during IBAs. These squirrels exhibit reversible inhibition of the hypothalamic feeding center, such that hypothalamic arcuate nucleus neurons exhibit reduced sensitivity to the orexigenic and anorexigenic effects of ghrelin and leptin, respectively. However, hypothalamic infusion of thyroid hormone during an IBA is sufficient to rescue hibernation anorexia. Our results reveal that thyroid hormone deficiency underlies hibernation anorexia and demonstrate the functional flexibility of the hypothalamic feeding center.

Human iPSC-derived photoreceptor transplantation in the cone dominant 13-lined ground squirrel.

Several retinal degenerations affect the human central retina, which is primarily comprised of cones and is essential for high acuity and color vision. Transplanting cone photoreceptors is a promising strategy to replace degenerated cones in this region. Although this approach has been investigated in a handful of animal models, commonly used rodent models lack a cone-rich region and larger models can be expensive and inaccessible, impeding the translation of therapies. Here, we transplanted dissociated GFP-expressing photoreceptors from retinal organoids differentiated from human induced pluripotent stem cells into the subretinal space of damaged and undamaged cone-dominant 13-lined ground squirrel eyes. Transplanted cell survival was documented via noninvasive high-resolution imaging and immunohistochemistry to confirm the presence of human donor photoreceptors for up to 4 months posttransplantation. These results demonstrate the utility of a cone-dominant rodent model for advancing the clinical translation of cell replacement therapies.

Neural control of fluid homeostasis is engaged below 10°C in hibernation.

Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) hibernate for several months each winter without access to water,1 but the mechanisms that maintain fluid homeostasis during hibernation are poorly understood. In torpor, when body temperature (TB) reaches 4°C, squirrels decrease metabolism, slow heart rate, and reduce plasma levels of the antidiuretic hormones arginine vasopressin (AVP) and oxytocin (OXT).1 Squirrels spontaneously undergo interbout arousal (IBA) every 2 weeks, temporarily recovering an active-like metabolism and a TB of 37°C for up to 48 h.1,2 Despite the low levels of AVP and OXT during torpor, profound increases in blood pressure and heart rate during the torpor-IBA transition are not associated with massive fluid loss, suggesting the existence of a mechanism that protects against diuresis at a low TB. Here, we demonstrate that the antidiuretic hormone release pathway is activated by hypothalamic supraoptic nucleus (SON) neurons early in the torpor-arousal transition. SON neuron activity, dense-core vesicle release from the posterior pituitary, and plasma hormone levels all begin to increase before TB reaches 10°C. In vivo fiber photometry of SON neurons from hibernating squirrels, together with RNA sequencing and c-FOS immunohistochemistry, confirms that SON is electrically, transcriptionally, and translationally active to monitor blood osmolality throughout the dynamic torpor-arousal transition. Our work emphasizes the importance of the antidiuretic pathway during the torpor-arousal transition and reveals that the neurophysiological mechanism that coordinates the hormonal response to retain fluid is active at an extremely low TB, which is prohibitive for these processes in non-hibernators.

Microvesicle release from inner segments of healthy photoreceptors is a conserved phenomenon in mammalian species.

Many inherited visual diseases arise from mutations that affect the structure and function of photoreceptor cells. In some cases, the pathology is accompanied by a massive release of extracellular vesicles from affected photoreceptors. In this study, we addressed whether vesicular release is an exclusive response to ongoing pathology or a normal homeostatic phenomenon amplified in disease. We analyzed the ultrastructure of normal photoreceptors from both rod- and cone-dominant mammalian species and found that these cells release microvesicles budding from their inner segment compartment. Inner segment-derived microvesicles vary in their content, with some of them containing the visual pigment rhodopsin and others appearing to be interconnected with mitochondria. These data suggest the existence of a fundamental process whereby healthy mammalian photoreceptors release mistrafficked or damaged inner segment material as microvesicles into the interphotoreceptor space. This release may be greatly enhanced under pathological conditions associated with defects in protein targeting and trafficking. This article has an associated First Person interview with the first author of the paper.

Characterization of Retinal Development in 13-Lined Ground Squirrels.

Purpose: The cone-dominant, 13-lined ground squirrel (13-LGS) retina mimics the human central retina, but a thorough examination of retinal development in this species has not been reported. Here, the embryonic and postnatal development of the 13-LGS retina was studied to further characterize 13-LGS as a practical alternative animal model for investigating cone-based vision in health and disease. Methods: The spatiotemporal expression of key progenitor and cell type markers was examined in retinas from defined embryonic and postnatal stages using immunohistochemistry. Postnatal gene expression changes were validated by quantitative PCR. Results: The 13-LGS neuroblastic layer expressed key progenitor markers (Sox2, Vsx2, Pax6, and Lhx2) at E18. Sequential cell fate determination evidenced by the first appearance of cell-type-specific marker labeling was at embryonic stage 18 (E18) with ganglion cells (Brn-3A, HuC/D) and microglia (Iba1); at E22.5 with photoreceptor progenitors (Otx2, recoverin) followed shortly by horizontal and amacrine cells (Lhx1, Oc1) at E24 to E25.5; and at postnatal stage 15 (P15) with bipolar cells (Vsx1, CaBP5) and Müller glia cells (GS, Rlbp1). Photoreceptor maturation indicated by opsin-positive outer segments and peanut agglutinin (PNA) labeling of cone sheaths was completed at the time of eye opening (P21-P24). Conclusions: The timeline and order of retinal cell development in the 13-LGS generally matches that recorded from other mammalian models but with a stark variation in the proportion of various cell types due to cone-dense photoreceptors. Translational Relevance: This thorough examination of an emerging translationally relevant cone-dominant specie provides a baseline for future disease modeling and stem cell approach studies of human vision.

Ground squirrels initiate sexual maturation during hibernation.

Adequate nutrition is essential for normal reproductive function, which is vital for species to survive. In humans and other mammals, starvation and undernutrition deplete fat reserves and cause weight loss, attenuating the function of the reproductive axis and causing hypogonadism.1-4 Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) spend 7 months of every year in hibernation without food and water. Hibernating squirrels alternate between periods of torpor and interbout arousal (IBA), when animals temporarily return to an active-like state.5 The physiological significance of IBA is unclear, but it is thought to be essential for hibernation in animals that drop their body temperature to 2°C-4°C during torpor. Here, we report that juvenile male ground squirrels initiate reproductive maturation during their first hibernation season, despite prolonged undernutrition and profound weight loss. We show that the hypothalamic reproductive axis undergoes activation during interbout arousals in the middle of hibernation, triggering production of luteinizing hormone and testosterone, and promoting testicular growth. Initiation of sexual maturation is circannually entrained and is independent of physiological state, ambient temperature, and food availability. Our study suggests a role for interbout arousals during hibernation and uncovers the neurophysiological mechanism of reproductive axis activation during conditions of extreme negative energy balance. VIDEO ABSTRACT.

The PIWI/piRNA response is relaxed in a rodent that lacks mobilizing transposable elements.

Transposable elements (TEs) are genomic parasites that can propagate throughout host genomes. Mammalian genomes are typically dominated by LINE retrotransposons and their associated SINEs, and germline mobilization is a challenge to genome integrity. There are defenses against TE proliferation and the PIWI/piRNA defense is among the most well understood. However, the PIWI/piRNA system has been investigated largely in animals with actively mobilizing TEs and it is unclear how the PIWI/piRNA system functions in the absence of mobilizing TEs. The 13-lined ground squirrel provides the opportunity to examine PIWI/piRNA and TE dynamics within the context of minimal, and possibly nonexistent, TE accumulation. To do so, we compared the PIWI/piRNA dynamics in squirrels to observations from the rabbit and mouse. Despite a lack of young insertions in squirrels, TEs were still actively transcribed at higher levels compared to mouse and rabbit. All three Piwi genes were not expressed, prior to P8 in squirrel testis, and there was little TE expression change with the onset of Piwi expression. We also demonstrated there was not a major expression change in the young squirrel LINE families in the transition from juvenile to adult testis in contrast to young mouse and rabbit LINE families. These observations lead us to conclude that PIWI suppression, was weaker for squirrel LINEs and SINEs and did not strongly reduce their transcription. We speculate that, although the PIWI/piRNA system is adaptable to novel TE threats, transcripts from TEs that are no longer threatening receive less attention from PIWI proteins.

Pre-retinal delivery of recombinant adeno-associated virus vector significantly improves retinal transduction efficiency.

Intravitreal injection is the most widely used injection technique for ocular gene delivery. However, vector diffusion is attenuated by physical barriers and neutralizing antibodies in the vitreous. The 13-lined ground squirrel (13-LGS), as in humans, has a larger relative vitreous body volume than the more common rodent models such as rats and mice, which would further reduce transduction efficiency with the intravitreal injection route. We report here a "pre-retinal" injection approach that leads to detachment of the posterior hyaloid membrane and delivers vector into the space between vitreous and inner retina. Vectors carrying a ubiquitously expressing mCherry reporter were injected into the deep vitreous or pre-retinal space in adult wild-type 13-LGSs. Then, adeno-associated virus (AAV)-mediated mCherry expression was evaluated with non-invasive imaging, immunofluorescence, and flow cytometry. Compared to deep vitreous delivery, pre-retinal administration achieved pan-retinal gene expression with a lower vector dose volume and significantly increased the number of transduced cone photoreceptors. These results suggest that pre-retinal injection is a promising tool in the development of gene therapy strategies in animal models and is a potential approach for use in human research, particularly in younger individuals with an intact posterior hyaloid membrane and stable vitreous.

Cone photoreceptor reflectance variation in the northern tree shrew and thirteen-lined ground squirrel.

In vivo images of human cone photoreceptors have been shown to vary in their reflectance both spatially and temporally. While it is generally accepted that the unique anatomy and physiology of the photoreceptors themselves drives this behavior, the exact mechanisms have not been fully elucidated as most studies on these phenomena have been limited to the human retina. Unlike humans, animal models offer the ability to experimentally manipulate the retina and perform direct in vivo and ex vivo comparisons. The thirteen-lined ground squirrel and northern tree shrew are two emerging animal models being used in vision research. Both models feature cone-dominant retinas, overcoming a key limitation of traditional rodent models. Additionally, each possesses unique but well-documented anatomical differences in cone structure compared to human cones, which can be leveraged to further constrain theoretical models of light propagation within photoreceptors. Here we sought to characterize the spatial and temporal reflectance behavior of cones in these species. Adaptive optics scanning light ophthalmoscopy (AOSLO) was used to non-invasively image the photoreceptors of both species at 5 to 10 min intervals over the span of 18 to 25 min. The reflectance of individual cone photoreceptors was measured over time, and images at individual time points were used to assess the variability of cone reflectance across the cone mosaic. Variability in spatial and temporal photoreceptor reflectance was observed in both species, with similar behavior to that seen in human AOSLO images. Despite the unique cone structure in these animals, these data suggest a common origin of photoreceptor reflectance behavior across species. Such data may help constrain models of the cellular origins of photoreceptor reflectance signals. These animal models provide an experimental platform to further explore the morphological origins of light capture and propagation.

Optical Coherence Tomography Angiography in the Thirteen-Lined Ground Squirrel.

Purpose: To assess the performance of two spectral-domain optical coherence tomography-angiography systems in a natural model of hypoperfusion: the hibernating thirteen-lined ground squirrel (13-LGS). Methods: Using a high-speed (130 kHz) OCT-A system (HS-OCT-A) and a commercial OCT (36 kHz; Bioptigen Envisu; BE-OCT-A), we imaged the 13-LGS retina throughout its hibernation cycle. Custom software was used to extract the superior, middle, and deep capillary plexus (SCP, MCP, and DCP, respectively). The retinal vasculature was also imaged with adaptive optics scanning light ophthalmoscopy (AOSLO) during torpor to visualize individual blood cells. Finally, correlative histology with immunolabeled or DiI-stained vasculature was performed. Results: During euthermia, vessel density was similar between devices for the SCP and MCP (P = 0.88, 0.72, respectively), with a small difference in the DCP (-1.63 ± 1.54%, P = 0.036). Apparent capillary dropout was observed during torpor, but recovered after forced arousal, and this effect was exaggerated in high-speed OCT-A imaging. Based on cell flux measurements with AOSLO, increasing OCT-A scan duration by ∼1000× would avoid the apparent capillary dropout artifact. High correspondence between OCT-A (during euthermia) and histology enabled lateral scale calibration. Conclusions: While the HS-OCT-A system provides a more efficient workflow, the shorter interscan interval may render it more susceptible to the apparent capillary dropout artifact. Disambiguation between capillary dropout and transient ischemia can have important implications in the management of retinal disease and warrants additional diagnostics. Translational Relevance: The 13-LGS provides a natural model of hypoperfusion that may prove valuable in modeling the utility of OCT-A in human pathologies associated with altered blood flow.

Electroretinogram of the Cone-Dominant Thirteen-Lined Ground Squirrel during Euthermia and Hibernation in Comparison with the Rod-Dominant Brown Norway Rat.

Purpose: The majority of small animal species used in research are nocturnal, with retinae that are anatomically and functionally dissimilar from humans, complicating their use as disease models. Herein we characterize the retinal structure and electrophysiological function of the diurnal, cone-dominant 13-lined ground squirrel (13-LGS) retina during euthermia and in hibernation. Methods: Full-field electroretinography (ERG) was performed in 13-LGS and Brown Norway (BN) rat models to establish baseline values for retinal function in each species, including following intravitreal injection of pharmacologic agents to selectively block the contributions of ON- and OFF-bipolar cells. The effect of hibernation-associated retinal remodeling on electrophysiological function was assessed in 13-LGS during torpor and emergence, with correlative histology performed using transmission electron microscopy. Results: Under light-adapted conditions, the a-, b-, and d-wave amplitude of the 13-LGS was significantly greater than that of the BN rat. Retinal function was absent in the 13-LGS during hibernation and correlated to widespread disruption of photoreceptor and RPE structure. Remarkably, both retinal function and structure recovered rapidly on emergence from hibernation, with ERG responses reaching normal amplitude within 6 hours. Conclusions: ERG responses for both BN rats and 13-LGS reflect the relative proportions of cone photoreceptors present within the retinae, indicating that the cone-dominant 13-LGS may be a potentially useful model for studying human central retinal function and disease. That retinal remodeling and restoration of electrophysiological function occurs rapidly on emergence from hibernation implies the 13-LGS may also be a useful tool for studying aspects of retinal physiology and recovery from injury.

CNGA3 acts as a cold sensor in hypothalamic neurons.

Most mammals maintain their body temperature around 37°C, whereas in hibernators it can approach 0°C without triggering a thermogenic response. The remarkable plasticity of the thermoregulatory system allowed mammals to thrive in variable environmental conditions and occupy a wide range of geographical habitats, but the molecular basis of thermoregulation remains poorly understood. Here we leverage the thermoregulatory differences between mice and hibernating thirteen-lined ground squirrels (Ictidomys tridecemlineatus) to investigate the mechanism of cold sensitivity in the preoptic area (POA) of the hypothalamus, a critical thermoregulatory region. We report that, in comparison to squirrels, mice have a larger proportion of cold-sensitive neurons in the POA. We further show that mouse cold-sensitive neurons express the cyclic nucleotide-gated ion channel CNGA3, and that mouse, but not squirrel, CNGA3 is potentiated by cold. Our data reveal CNGA3 as a hypothalamic cold sensor and a molecular marker to interrogate the neuronal circuitry underlying thermoregulation.

Diurnal rodents as pertinent animal models of human retinal physiology and pathology.

This presentation will survey the retinal architecture, advantages, and limitations of several lesser-known rodent species that provide a useful diurnal complement to rats and mice. These diurnal rodents also possess unusually cone-rich photoreceptor mosaics that facilitate the study of cone cells and pathways. Species to be presented include principally the Sudanian Unstriped Grass Rat and Nile Rat (Arvicanthis spp.), the Fat Sand Rat (Psammomys obesus), the degu (Octodon degus) and the 13-lined ground squirrel (Ictidomys tridecemlineatus). The retina and optic nerve in several of these species demonstrate unusual resilience in the face of neuronal injury, itself an interesting phenomenon with potential translational value.

Genetic variation drives seasonal onset of hibernation in the 13-lined ground squirrel.

Hibernation in sciurid rodents is a dynamic phenotype timed by a circannual clock. When housed in an animal facility, 13-lined ground squirrels exhibit variation in seasonal onset of hibernation, which is not explained by environmental or biological factors. We hypothesized that genetic factors instead drive variation in timing. After increasing genome contiguity, here, we employ a genotype-by-sequencing approach to characterize genetic variation in 153 ground squirrels. Combined with datalogger records (n = 72), we estimate high heritability (61-100%) for hibernation onset. Applying a genome-wide scan with 46,996 variants, we identify 2 loci significantly (p < 7.14 × 10-6), and 12 loci suggestively (p < 2.13 × 10-4), associated with onset. At the most significant locus, whole-genome resequencing reveals a putative causal variant in the promoter of FAM204A. Expression quantitative trait loci (eQTL) analyses further reveal gene associations for 8/14 loci. Our results highlight the power of applying genetic mapping to hibernation and present new insight into genetics driving its onset.

Osmolyte Depletion and Thirst Suppression Allow Hibernators to Survive for Months without Water.

Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) are obligatory hibernators who can survive over 6 months of the year in underground burrows or laboratory hibernaculum without access to food or water [1]. Hibernation consists of prolonged periods of torpor, lasting up to 18 days, which are characterized by low body temperature and suppressed metabolism. This torpidity is interspersed with short periods of interbout arousal, lasting up to 48 h, during which squirrels temporarily return to an active-like state and lose small amounts of water to urination and evaporation [2]. Water is also lost during torpor due to a positive vapor pressure difference created by the slightly higher temperature of the body compared to its surroundings [2, 3]. Here, we investigate the physiological mechanism of survival during prolonged water loss and deprivation throughout hibernation. By measuring hydration status during hibernation, we show that squirrels remain hydrated during torpor by depleting osmolytes from the extracellular fluid. During brief periods of arousal, serum osmolality and antidiuretic hormone levels are restored, but thirst remains suppressed. This decoupling of thirst and diuresis enables water retention by the kidney while suppressing the drive to leave the safety of the underground burrow in search of water. An acute increase in serum osmolality reinstates water-seeking behavior, demonstrating preservation of the physiological thirst circuit during hibernation. Better mechanistic understanding of internal osmolyte regulation and thirst suppression could translate to advancements in human medicine and long-term manned spaceflight. VIDEO ABSTRACT.

Evaluating seasonal changes of cone photoreceptor structure in the 13-lined ground squirrel.

Cone photoreceptors of the 13-lined ground squirrel (13-LGS) undergo reversible structural changes during hibernation, including cone outer segment disc degeneration and inner segment mitochondria depletion. Here, we evaluated cone structure with adaptive optics scanning light ophthalmoscopy (AOSLO) before, during, and after hibernation. Also, intra-animal comparisons of cone structure were made at distinct physiological states (pre-hibernation, torpor, interbout euthermia, and post-hibernation) with AOSLO and transmission electron microscopy. Our results indicate that the 13-LGS cone mosaic is only transiently affected by structural remodeling during hibernation. Outer segment remodeling starts during torpid states during a period of fall transition in room temperature, with more severe structural changes during bouts of torpor in cold temperature. Cones return to euthermic-like structure during brief periods of interbout euthermia and recover normal waveguiding properties as soon as 24 h post-hibernation. Cone structure is visible with split-detector AOSLO throughout hibernation, providing evidence that intact outer segments are not necessary to visualize cones with this technique. Despite the changes to cone structure during hibernation, cone density and packing remained unchanged throughout the seasonal cycle. Pairing non-invasive imaging with ultrastructural assessment may provide insight to the biological origins of cone photoreceptor signals observed with AOSLO.

Somatosensory Neurons Enter a State of Altered Excitability during Hibernation.

Hibernation in mammals involves prolonged periods of inactivity, hypothermia, hypometabolism, and decreased somatosensation. Peripheral somatosensory neurons play an essential role in the detection and transmission of sensory information to CNS and in the generation of adaptive responses. During hibernation, when body temperature drops to as low as 2°C, animals dramatically reduce their sensitivity to physical cues [1, 2]. It is well established that, in non-hibernators, cold exposure suppresses energy production, leading to dissipation of the ionic and electrical gradients across the plasma membrane and, in the case of neurons, inhibiting the generation of action potentials [3]. Conceivably, such cold-induced elimination of electrogenesis could be part of a general mechanism that inhibits sensory abilities in hibernators. However, when hibernators become active, the bodily functions-including the ability to sense environmental cues-return to normal within hours, suggesting the existence of mechanisms supporting basal functionality of cells during torpor and rapid restoration of activity upon arousal. We tested this by comparing properties of somatosensory neurons from active and torpid thirteen-lined ground squirrels (Ictidomys tridecemlineatus). We found that torpid neurons can compensate for cold-induced functional deficits, resulting in unaltered resting potential, input resistance, and rheobase. Torpid neurons can generate action potentials but manifest markedly altered firing patterns, partially due to decreased activity of voltage-gated sodium channels. Our results provide insights into the mechanism that preserves somatosensory neurons in a semi-active state, enabling fast restoration of sensory function upon arousal. These findings contribute to the development of strategies enabling therapeutic hypothermia and hypometabolism.

Differential composition of DHA and very-long-chain PUFAs in rod and cone photoreceptors.

Long-chain PUFAs (LC-PUFAs; C20-C22; e.g., DHA and arachidonic acid) are highly enriched in vertebrate retina, where they are elongated to very-long-chain PUFAs (VLC-PUFAs; C 28) by the elongation of very-long-chain fatty acids-4 (ELOVL4) enzyme. These fatty acids play essential roles in modulating neuronal function and health. The relevance of different lipid requirements in rods and cones to disease processes, such as age-related macular degeneration, however, remains unclear. To better understand the role of LC-PUFAs and VLC-PUFAs in the retina, we investigated the lipid compositions of whole retinas or photoreceptor outer segment (OS) membranes in rodents with rod- or cone-dominant retinas. We analyzed fatty acid methyl esters and the molecular species of glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine) by GC-MS/GC-flame ionization detection and ESI-MS/MS, respectively. We found that whole retinas and OS membranes in rod-dominant animals compared with cone-dominant animals had higher amounts of LC-PUFAs and VLC-PUFAs. Compared with those of rod-dominant animals, retinas and OS membranes from cone-dominant animals also had about 2-fold lower levels of di-DHA (22:6/22:6) molecular species of glycerophospholipids. Because PUFAs are necessary for optimal G protein-coupled receptor signaling in rods, these findings suggest that cones may not have the same lipid requirements as rods.

Assessment of Outer Retinal Remodeling in the Hibernating 13-Lined Ground Squirrel.

Purpose: We examined outer retinal remodeling of the euthermic and torpid cone-dominant 13-lined ground squirrel (13-LGS) retina using optical coherence tomography (OCT) imaging and histology. Methods: Retinas and corneas of living 13-LGSs were imaged during euthermic and torpid physiological states using OCT. Retinal layer thickness was measured at the visual streak from registered and averaged vertical B-scans. Following OCT, some retinas were collected immediately for postmortem histologic comparison using light microscopy, immunofluorescence, or transmission electron microscopy. Results: Compared to OCT images from euthermic retinae, OCT images of torpid retinae revealed significantly thicker inner and outer nuclear layers, as well as increases in the distances between outer retinal reflectivity bands 1 and 2, and bands 3 and 4. A significant decrease in the distance between bands 2 and 3 also was seen, alongside significant thinning of the choriocapillaris and choroid. OCT image quality was reduced in torpid eyes, partly due to significant thickening of the corneal stroma during this state. Conclusions: The torpid retina of the hibernating 13-LGS undergoes structural changes that can be detected by OCT imaging. Comparisons between in vivo OCT and ex vivo histomorphometry may offer insight to the origin of hyperreflective OCT bands within the outer retina of the cone-dominant 13-LGS.

iPSCs from a Hibernator Provide a Platform for Studying Cold Adaptation and Its Potential Medical Applications.

Hibernating mammals survive hypothermia (<10°C) without injury, a remarkable feat of cellular preservation that bears significance for potential medical applications. However, mechanisms imparting cold resistance, such as cytoskeleton stability, remain elusive. Using the first iPSC line from a hibernating mammal (13-lined ground squirrel), we uncovered cellular pathways critical for cold tolerance. Comparison between human and ground squirrel iPSC-derived neurons revealed differential mitochondrial and protein quality control responses to cold. In human iPSC-neurons, cold triggered mitochondrial stress, resulting in reactive oxygen species overproduction and lysosomal membrane permeabilization, contributing to microtubule destruction. Manipulations of these pathways endowed microtubule cold stability upon human iPSC-neurons and rat (a non-hibernator) retina, preserving its light responsiveness after prolonged cold exposure. Furthermore, these treatments significantly improved microtubule integrity in cold-stored kidneys, demonstrating the potential for prolonging shelf-life of organ transplants. Thus, ground squirrel iPSCs offer a unique platform for bringing cold-adaptive strategies from hibernators to humans in clinical applications. VIDEO ABSTRACT.