In this study, we used a targeted IP-MS method to quantify phosphorylated and non-phosphorylated tau peptides in plasma from free-ranging brown bears during summer and hibernation and validated the results in hamster brains during torpor and euthermy. Hibernation-linked increases in plasma levels of p-tau181, p-tau205, p-tau217, and p-tau231, with unchanged p-tau199 and p-tau202 levels, resemble a pattern seen in AD patients [11]. We observed the greatest increases in plasma p-tau181 and p-tau217, biomarkers strongly associated with AD neuropathology [18, 25]. These findings align with a previous study on hibernating American black bears that reported, with immunoblotting and immunohistochemistry, neuropathological changes in phospho-sites Thr181, Thr205, Thr217, and Thr231 [14]. While that study reported Ser202 phosphorylation changes and no total-tau alterations, we did not observe p-tau202 changes and did not measure total-tau. Instead, we measured non-phosphorylated fragments (tau195-209 and tau212-221), which were markedly increased during hibernation. While total-tau, measured by immunoassays, is traditionally considered a marker of neurodegeneration, less evidence is available on mass spectrometry-based techniques quantifying smaller non-phosphorylated fragments. Nevertheless, a recent study shows that tau212-221 measured by MS correlates tightly with total-tau measured by the Lumipulse immunoassay (r = 0.99) [26], which could explain why we observed increases in tau195-209 and tau212-221 in a similar direction as with their phospho-counterparts, p-tau205 and p-tau217. Given the cleavage peptides undergo before quantification, it is also possible that part of the two non-phosphorylated fragments we measured come from molecules highly phosphorylated at other sites, due to global increases in tau secretion from neurons, a process that may be increased in hibernation. As hibernating animals do not experience neurodegeneration, the observed increases in these non-phosphorylated fragments may reflect hibernation-linked tau processing alterations rather than neurodegeneration [1, 12, 14]. Additionally, the ratios of p-tau205 and p-tau217 to their non-phosphorylated counterparts did not show greater increases than p-tau variants alone. We interpret the changes in plasma p-tau levels during hibernation as indicative of the tau hyperphosphorylation process reported in hibernating animals. While it could be argued that protein levels are increasing due to the hemoconcentration that occurs during hibernation, the previous work from the Scandinavian Brown Bear Project estimates that hemoconcentration is limited to a 10–30% magnitude, which would not account for the > 300% increases we found for plasma p-tau181 and p-tau217 [27]. Additionally, prominent and consistent cerebral Aβ deposition has not been robustly documented in hibernating species—unlike humans where increased p-tau levels are closely associated with Aβ pathology—suggesting that hibernation-linked tau hyperphosphorylation occurs independently of Aβ abnormalities [1].

Our IP-MS findings on brain tau levels in golden Syrian hamsters during hibernation align with bear plasma results obtained via IP-MS and immunoassays. Brain p-tau181, p-tau217, and p-tau231 levels increased, in varying magnitudes, in the torpor group in both TBS and SI fractions (although p-tau181 not significant in SI), consistent with prior brain tissue studies on small hibernators showing high tau phosphorylation rates via immunohistochemistry and immunoblotting [12,13,14]. Similarly, the observed increases in bear plasma p-tau181 corroborate recent findings in hamsters, where plasma p-tau181 levels were also higher during torpor than arousal when measured with a non-clinically validated biofunctionalization method [28]. This converging evidence highlights the importance of evaluating this hibernation-linked hyperphosphorylation through different species, biological matrices, and analytical methods.

These tau phosphorylation changes may, in part, reflect a passive consequence of lowered body temperature. Planel and colleagues previously proposed that tau hyperphosphorylation under hypothermic conditions results from differential temperature sensitivity of kinases and phosphatases, particularly the inhibition of protein phosphatase 2A [29]. Our findings are compatible with this mechanism, but also align with results from hibernating mammals suggesting an additional hibernation-state-specific regulatory component. In particular, Stieler et al. demonstrated that although low temperatures facilitate tau phosphorylation, tissue from torpid animals shows enhanced phosphate incorporation compared to euthermic controls at similar temperatures, indicating active, regulated mechanisms beyond temperature alone [14]. Thus, hibernation-linked tau phosphorylation likely involves both passive thermodynamic effects and active, reversible processes associated with the hypometabolic state.

It is well established that hibernators do not develop AD-like fibrillar tangle pathology despite seasonal tau hyperphosphorylation [1]. While it has been reported aging bears may form pre-tangle tau aggregates, such formations are rare and not observed in all aged bears [14, 30]. The MTBR domain of tau is a key component of insoluble neurofibrillary tangles, and has even been proposed as a tangle-specific biomarker in biofluids [31, 32]. In brain tissue from AD patients, where tangle pathology is present, MTBR tau fragments showed dramatic increases, compared with controls without AD pathology. In the SI fraction, containing insoluble aggregates, MTBR tau354-369, in particular, was increased by ~ 20,000% and still showed a ~ 238% increase in the soluble TBS fraction, highlighting the substantial tangle burden in AD brains. In hibernating hamsters, however, unlike for p-tau variants, brain levels of MTBR tau243-254 and tau354-369 were not increased compared with euthermy in the SI fraction, and even slightly reduced (-6%) in the TBS fraction. This may be further reflective of the fact that hibernation-linked tau hyperphosphorylation does not lead to tau aggregation with neurofibrillary tangle formation. Additionally, recent evidence suggests that p-tau205, measured in plasma or CSF, may be the p-tau variant with the strongest relation to tau tangles [10, 11, 25]. Interestingly, p-tau205 levels showed no increase in torpor hamster brains, in both SI and TBS fractions, and only modest increases in bear plasma.

Recent work by Lövestam et al. provides compelling evidence that tau hyperphosphorylation at specific residues is sufficient to drive aggregation into Alzheimer-type paired helical filaments. Using 12 phosphomimetic mutations (PAD12) that mimic phosphorylation at sites, including T181, T205, T217, and T231, the authors demonstrated spontaneous in vitro assembly of recombinant full-length tau into filaments structurally indistinguishable from those found in AD brains [33]. These findings establish a mechanistic link between site-specific hyperphosphorylation and the adoption of the AD-specific tau folding, suggesting that tau phosphorylation may be a key driver of filament formation. In our hibernating animal models, we observed reversible phosphorylation at several of these same residues without detectable tau aggregation, suggesting that while phosphorylation can be sufficient under defined experimental conditions and likely also in AD, its capacity to trigger filament formation in vivo may still depend on additional factors not present in hibernation, such as the very long duration of asymptomatic and symptomatic duration of AD, altered clearance, or local biochemical milieu.

Beyond the clear differences in changes in MTBR tau fragments in the brains of AD patients vs controls and lack of changes in hibernating hamster brain tissue, magnitudes of brain tissue phosphorylation were also notably distinct in AD. For instance, in the SI fraction, p-tau217 and p-tau231 were increased by ~ 50,000% in AD vs controls. Even in the TBS fraction, p-tau217 showed an increase of ~ 400% in AD. In contrast, in hibernating hamster brain tissue, p-tau217 showed increases of ~ 200% and ~ 90% in the SI and TBS fractions, respectively, compared with euthermal hamsters. This may underscore a fundamental difference between the regulated, reversible tau phosphorylation observed in hibernation and the pathological tau accumulation and aggregation characteristic of AD. While hamster brain tissue results were remarkably similar between TBS and SI fractions with modest increases in p-tau217 and p-tau231, human results showed much more pronounced increases in all measured tau peptides in SI compared with TBS, possibly suggesting that the shift from soluble phosphorylated tau to insoluble aggregated tau, as observed in AD, does not occur during hibernation. These results, alongside the absence of MTBR tau accumulation in torpor, even in the SI fraction, supports the notion that hibernation-associated hyperphosphorylation does not progress to tangle formation, further suggesting hibernation as a non-pathological model for studying tau biology.

Interestingly, the non-phosphorylated tau195-209 and tau212-221 peptides were decreased by around ~ 20–30% in the hamster brain tissue torpor group. This reduction in non-phospho-peptides was also observed for TBS results in AD brains compared with controls. Unlike with plasma levels, which reflect soluble tau processing in a more dynamic way, the direct quantification of tau in the brain tissue may allow for interpreting that these decreases could be at least partly attributed to increases in phosphorylation rates. The reduction in these non-phosphorylated peptides in brain tissue, combined with their increase in blood levels and the slight decreases in non-phosphorylated MTBR tau fragments, may reflect uncharacterized aspects of tau processing during hibernation.

This work is not exempt from limitations, mostly associated with the high complexity of carrying out longitudinal monitoring in free-ranging animals. Our sample size for hibernators (bears: n = 10; hamsters: n = 10) is relatively small, but similar to that of other studies in the interface between AD pathology and hibernation [12, 14, 30]. Also, due to ethical considerations and animal welfare, sampling was restricted to plasma and limited to two occasions, preventing us from quantifying tau proteins in the CSF and from conducting a more detailed repeated-measures analysis of p-tau dynamics throughout the year. While older bears may serve as a more suitable model for aging human disease, the sub-adult (2–3 years old) bears in our study still exhibited marked changes in plasma p-tau levels. Also, studying older free-ranging bears presents significant challenges, including increased potential for comorbidities and, especially, greater risks to the research team. Brain tissue is not collected within the Scandinavian Brown Bear Research Project, preventing neuropathological analyses in bears, and hamster plasma analysis was not possible due to the high-volume needed by the IP-MS plasma method, which is considerably larger than the small volume of blood usually collected from each hamster. When using methods based on centrifugation and relative detergent solubility, it is possible that some degree of cross-contamination may occur across fractions. Thus, we cannot exclude that the low levels measure here in hamster brain may be tau proteins that pelleted or fragments of non-aggregated insoluble tau (previously reported in hibernation), despite not being assembled in AD-like NFTs [14]. At the same time, it is evident from the results of our study that the differences for the key p-tau species measured (the best example being pTau217) change in the same direction as in AD and hamster brain in both SI and TBS fractions (as well as AD CSF/plasma from the literature), and even in plasma from hibernating bears. However, other tau variants which would be expected in the presence of NFT pathology, such as the MTBR species and p-tau205, do not change. We understand that this speaks against the fact that cross-contamination could be driving the results, and that there is a consistent between-species consistent biological phenomenon of tau phosphorylation during hibernation.

To the best of our knowledge, our study provides the first evidence that hibernation-linked tau hyperphosphorylation is reflected in increases in plasma p-tau as measured by validated assays, at the same phospho-sites which are increased in AD patients, as well as the first mass-spectrometry characterization of tau processing in brain tissue during hibernation. The pattern of brain and plasma tau levels is consistent with the lack of tangle formation during hibernation. Further translational studies of this phenomenon may provide insights into the biology of p-tau as well as identify novel strategies to prevent p-tau accumulation in the human brain, taking into account the dynamics of both soluble and insoluble tau. Our results also emphasize the need for more research on hibernation as a translational model for advancing our knowledge on aging-related human diseases.