Regional and Cellular Distribution of Nuclear Phosphorylated Tau (AT100) in the Frontal Cortex and Cerebellum of Cetaceans

1. Introduction

Brain aging is a complex and multifaceted process involving structural and functional changes that affect cognition, memory, and behavior. In humans, age-related declines in neuroplasticity have been reported and are thought to contribute to cognitive changes associated with normal aging [1,2,3,4]. From a comparative perspective, cetaceans and humans exhibit notable similarities in brain organization, including high levels of encephalization, cortical complexity, and refined neuronal architecture [5,6,7,8]. These convergent traits are associated with complex social behavior and have been proposed to reflect shared principles of neural organization that may extend to aspects of brain development and aging [9,10,11]. In this context, cetaceans constitute valuable models for comparative studies, particularly given the exceptional longevity observed in some species [12].

At the cellular level, tau protein regulates cytoskeletal stability through its interaction with tubulin [13]. Beyond its structural role, tau is implicated in cellular stress responses and age-related neurodegenerative conditions, including Alzheimer’s disease [14,15]. Experimental studies have demonstrated that tau can bind DNA and exert protective effects against oxidative stress [16,17,18,19]. Under physiological conditions, tau phosphorylation is a dynamic process that modulates cytoskeletal organization [20,21]. Among phosphorylated tau species, the AT100 epitope (Thr212/Ser214) is recognized by specific conformational changes [22,23]. In mammalian brains, nuclear localization of AT100 has been associated with neuronal aging and DNA protection [24,25,26]. Conversely, in pathological states, nuclear AT100 immunoreactivity decreases as cytoplasmic aggregation increases [15,24,27]. This duality highlights its relevance as a marker of neuronal state [28]. The present study aimed to evaluate the nuclear localization of AT100 in the frontal cortex and cerebellum of several cetacean species stranded along the Galician coast.

2. Materials and Methods

2.1. Study Subjects

A total of 12 stranded cetaceans from the Galician coast were analyzed between February and August 2025, representing four species: Delphinus delphis ($n=8$), Phocoena phocoena ($n=2$), Tursiops truncatus ($n=1$), and Stenella coeruleoalba ($n=1$).

Samples were provided by the Coordinadora para o Estudo dos Mamíferos Mariños (CEMMA). Individuals were classified by sex and age category based on total body length, external development, and dental eruption patterns. Dental age estimation was performed by counting Growth Layer Groups (GLGs) [29,30,31]. Individuals were grouped into three classes: juveniles (minimal dental wear, incomplete sexual development), subadults (moderate wear, near-maximum body length), and adults (marked dental wear, complete sexual maturity) [32,33]. Frontal cortex and cerebellum samples were available for all specimens as detailed in Table 1.

2.2. Sample Analysis

All biological samples were immediately fixed after collection in 4% (v/v) formaldehyde solution to ensure proper preservation of tissue architecture. Subsequently, tissues were processed using conventional histological techniques, including dehydration through ascending ethanol series and embedding in paraffin blocks. Serial sections were then cut from these blocks using a microtome for histological and molecular analyses, specifically following standardized and validated immunohistochemistry protocols established in this study.

For immunohistochemical studies, sections were deparaffinized in xylene and rehydrated through graded ethanol series. Antigen retrieval was performed by heat-induced epitope retrieval in citrate buffer (pH 6.0) at 95°C for 20 minutes. Endogenous peroxidase activity was subsequently blocked with 3% hydrogen peroxide in methanol for 10 minutes, followed by blocking of nonspecific binding sites with 5% normal goat serum in PBS for 1 hour at room temperature.

Sections were incubated with the primary antibody AT100 (Invitrogen™ Phospho-Tau (Thr212, Ser214) Monoclonal Antibody (clone AT100, cat. MN1060)) at a 1:100 dilution in PBS containing 0.2% Triton X-100 for 2 hours at room temperature, according to the manufacturer’s instructions. Antibody binding was visualized using 0.025% 3,3′-diaminobenzidine (DAB Peroxidase Substrate Kit; Vector Laboratories) with 0.01% H₂O₂ as the catalytic agent. Finally, sections were mounted with Entellan (Merck).

Neuronal nuclei in the frontal cortex and cerebellum were quantified independently by two observers from the Pathology Service of the Veterinary Clinical Hospital of Universidad Alfonso X El Sabio (HCV-UAX) to minimize counting errors. Both observers were blinded to the age and sex of the individuals. Neuronal nuclei were specifically counted in each sample of frontal cortex and cerebellum. Five fields per immunohistochemical preparation were analyzed, using three samples per animal, at $600\times$ magnification in sections of the frontal cortex and lateral cerebellar hemispheres.

2.3. Statistical Analysis

Due to the small sample size ($n=12$) and the proportional nature of AT100 expression data (percent values ranging from 0 to 100%), Bayesian statistical methods were employed. These are particularly suitable for limited datasets as they allow the incorporation of prior biological knowledge and provide full uncertainty quantification via credible intervals [34,35]. It should be noted, however, that with small samples, posterior estimates remain sensitive to prior specification; poorly chosen priors can introduce bias that may exceed that of frequentist alternatives. To address this concern, informative priors were grounded in published mammalian tau literature, and a formal sensitivity analysis was conducted (Section 3.6) to confirm that the direction and magnitude of the estimated effects were robust across a range of prior specifications.

2.3.1. Bayesian Beta Regression Models

AT100 expression data were modeled using Bayesian Beta regression, appropriate for proportional data bounded between 0 and 1 [36]. The Beta distribution naturally accommodates the constraints of percentage data and the various distributional shapes commonly observed in biological proportions. Separate models were fitted for AT100 expression in the cerebral cortex and cerebellum:

$${\mathrm{AT}100}_i\sim \mathrm{Beta}({\mu }_i,\kappa )$$

(1)

$$\mathrm{logit}\left({\mu }_i\right)=\alpha +{\beta }_{\mathrm{age}}·{\mathrm{age}}_i+{\beta }_{\mathrm{sex}}·{\mathrm{Sex}}_i$$

(2)

where $\mu$ represents the expected proportion of AT100-positive neurons, $\kappa$ is the concentration parameter (inversely related to variance), Age is an ordinal variable ($1=$ juvenile, $2=$ subadult, $3=$ adult), and Sex is a binary indicator ($1=$ male, $0=$ female).

2.3.2. Prior Specification

Informative priors were defined based on existing literature on tau protein expression in mammalian brains [15,38]. For cortex models, the prior for the intercept was set as $\mathcal{N}\left(\mathrm{logit}\right(0.6),0.5)$, reflecting expected baseline AT100 levels of ∼60% in young cetaceans. For cerebellum models, a slightly lower baseline was assumed: $\mathcal{N}\left(\mathrm{logit}\right(0.5),0.5)$. Age effects received weakly informative positive priors: $\mathcal{N}(0.1,0.2)$, based on literature suggesting increased tau phosphorylation with age. Sex effects were assigned noninformative priors centered at zero: $\mathcal{N}(0,0.2)$. The concentration parameter $\kappa$ was assigned an $\mathrm{Exponential}\left(0.5\right)$ prior, favoring moderate dispersion while allowing high variability if supported by the data.

2.3.3. Model Fitting and Diagnostics

Models were fitted using PyMC (version 5.0+) with No-U-Turn Sampling (NUTS), a Hamiltonian Monte Carlo variant effective for complex hierarchical models [37]. Four chains were run with 2,000 warm-up and 2,000 posterior samples each, yielding a total of 8,000 posterior samples. Convergence was assessed via the Gelman–Rubin statistic ($\widehat{R}<1.01$) and effective sample sizes ($\mathrm{ESS}>400$). Model fit was evaluated through posterior predictive checks, comparing observed data distributions with distributions predicted from posterior parameter samples.

2.3.4. Interpretation Framework

Bayesian results were interpreted probabilistically rather than via classical null hypothesis testing. Posterior means with 95% credible intervals (CrI) are reported, representing the range containing the true parameter with 95% probability given the data and priors. The probability of positive effects, $P(\beta >0)$, is also reported, providing direct measures of evidence for age-related increases in AT100 expression. Effect sizes are reported on the logit scale, where positive values indicate increases in AT100 proportion and negative values indicate decreases.

3. Results

3.1. Immunohistochemical Analysis and AT100 Quantification

Manual quantitative evaluation of AT100-positive nuclei is summarized in Table 2.

The immunohistochemical analysis of AT100 protein generally revealed age-related differences in its subcellular distribution in both frontal cortex and cerebellum. In the cortex, quantification of positive nuclei showed a higher proportion of nuclear AT100 immunoreactivity in adult individuals, followed by subadults, whereas juveniles exhibited lower nuclear immunoreactivity and a more restricted distribution (Figure 1 and Figure 2).

As previously mentioned, the cerebellum exhibited the same age-dependent pattern, with adults showing the highest proportion of AT100 positive nuclei, followed by subadults, while juveniles displayed the lowest nuclear expression. Although quantification of AT100 in the cerebellum, as in the cortex, showed a progressive increase with age, the values obtained in the cerebellum were generally lower than those recorded in the cortex for the same age groups. This finding indicates that, while the age-associated increase pattern is maintained in both structures, the magnitude of nuclear staining in the cerebellum is comparatively lower than in the cortex. AT100 immunolabeling in the cerebellum revealed phosphorylated tau predominantly in granule cells and, to a lesser extent, in Purkinje cells (Figure 3 and Figure 4).

Overall, these results indicate a progressive increase in nuclear AT100 localization associated with advancing age in both analyzed brain regions.

3.2. Atypical AT100 Expression in Two Subadult D. delphis

Two subadult male D. delphis exhibited nuclear AT100 immunoreactivity levels indistinguishable from those of adult individuals: the first reached 96.55% in CC and 62.41% in Cb; the second, 88.7% in CC and 78.4% in Cb (Table 2, Figure 5). These values lie 1.4–2.1 standard deviations above the subadult group mean and overlap entirely with the adult range. One of these individuals presented macroscopic cerebral necrosis at necropsy. Necrotic processes trigger energy failure, ionic imbalance, and activation of stress-sensitive kinases (GSK-3$\beta$, CDK5), all of which promote tau hyperphosphorylation and nuclear redistribution independently of age [20,21]. The second atypical individual showed no gross neuropathology, raising the possibility of subclinical injury or individual biological precocity. Taken together, these cases suggest that nuclear AT100 accumulation reflects both a physiological aging trajectory and an acute neuropathological response, supporting its dual utility as an aging marker and a potential indicator of neuronal distress.

3.3. Descriptive Statistics

AT100 expression was successfully quantified in all 12 cetacean individuals across both brain regions. In the cerebral cortex, AT100 expression ranged from 17.9% to 96.7% (mean ± SD: $57.3\pm 28.4$%), while in the cerebellum it ranged from 19.5% to 91.7% (mean ± SD: $49.0\pm 23.8$%). The sample included 3 juveniles, 5 subadults, and 4 adults, with 7 males and 5 females distributed across four cetacean species, predominantly Delphinus delphis ($n=8$).

A notably strong positive correlation was observed between AT100 expression in the cerebral cortex and cerebellum ($r=0.932$; 95% CI: 0.82–0.98), indicating highly coordinated patterns of tau phosphorylation across brain regions within individuals. This correlation suggests systemic rather than region-specific mechanisms underlying AT100 expression in cetacean brains. The distribution of individual values, differentiated by sex and age category, is illustrated in Figure 6, showing that the cortex–cerebellum relationship remains consistent across all age groups and both sexes.

3.4. Bayesian Regression Results

3.4.1. Model Convergence and Fit

Both models (cerebral cortex and cerebellum) showed excellent convergence diagnostics. All $\widehat{R}$ values were $\le 1.001$, well below the conventional threshold of 1.05, and effective sample sizes exceeded 4,000 for all parameters, indicating robust sampling of the posterior distribution. Posterior predictive checks confirmed good model fit, with predicted distributions closely matching observed patterns in both brain regions.

3.4.2. Age Effects on AT100 Expression

Bayesian analysis revealed consistent positive age effects in both brain regions, providing credible probabilistic evidence of age-related increases in AT100 expression in cetaceans. In the cerebral cortex, the estimated age effect was ${\widehat{\beta }}_{\mathrm{age}}=0.168$ (95% CrI: $-0.139$; $0.472$), with an 85.5% probability of a positive age effect. In the cerebellum, the age effect was slightly stronger: ${\widehat{\beta }}_{\mathrm{age}}=0.194$ (95% CrI: $-0.105$; $0.491$), with an 89.5% probability of a positive effect.

Although the 95% credible intervals include zero, reflecting the small sample size, the consistently high probabilities (>85%) of positive age effects provide substantial evidence of age-related tau accumulation. Effect sizes on the logit scale correspond to substantial increases in the proportion of AT100 across age categories, with adults showing markedly higher expression than juveniles in both brain regions.

3.4.3. Sex Effects on AT100 Expression

Sex effects were minimal and centered near zero in both brain regions. In the cerebral cortex, ${\widehat{\beta }}_{\mathrm{sex}}=0.028$ (95% CrI: $-0.346$; $0.401$), and in the cerebellum, ${\widehat{\beta }}_{\mathrm{sex}}=-0.005$ (95% CrI: $-0.364$; $0.357$). These results provide strong evidence against sexual dimorphism in AT100 expression, consistent with the hypothesis that tau accumulation follows similar patterns in male and female cetaceans. Age and sex effects in both brain regions, along with their 80% and 95% credible intervals, are shown comparatively in Figure 7.

3.5. Regional Comparison

Comparison between the cerebral cortex and cerebellum revealed remarkably similar patterns of AT100 expression. Although mean expression was slightly higher in the cerebral cortex (57.3%) compared to the cerebellum (49.0%), age-related trends were nearly identical, and the strong inter-regional correlation ($r=0.932$) suggests coordinated tau phosphorylation mechanisms operating across different brain regions. Both regions displayed comparable age effect sizes and a similar absence of sex effects, indicating that AT100 expression patterns are consistent across distinct neural tissues in cetaceans. The distribution of values by age category and sex in both regions is shown in Figure 8.

3.6. Sensitivity Analysis for Prior Specification

To assess the robustness of the Bayesian results to prior choice, a sensitivity analysis was conducted comparing three specifications for the prior of ${\beta }_{\mathrm{age}}$: the Original prior used in the main analysis [$\mathcal{N}(+0.10,0.20)$], an Agnostic prior centered at zero with greater diffusivity [$\mathcal{N}(0.00,0.50)$], and a Skeptical prior of equal precision to the original but with no directional bias [$\mathcal{N}(0.00,0.20)$]. Figure 9 shows the posterior distributions of ${\beta }_{\mathrm{age}}$ under each scenario for both brain regions. All three posteriors exhibited substantial overlap and consistently pointed toward positive values, with probabilities $P({\beta }_{\mathrm{age}}>0)$ ranging from 76.2% (Skeptical, cortex) to 92.1% (Agnostic, cerebellum). This stability indicates that the direction of the age effect is supported by the data and is not an artifact of the informative prior, although the estimated magnitude shows some sensitivity to prior specification, particularly in the cortex under the Skeptical prior.

Figure 10 presents model predictions back-transformed to percentage points (pp) for direct interpretation of the age effect. Under the Agnostic prior—the most neutral—the predicted difference in AT100 expression between a juvenile and an adult is $+9.5$ pp in the cerebral cortex and $+13.9$ pp in the cerebellum. Using the Original prior, these differences are $+7.4$ pp and $+10.4$ pp, respectively. The 95% credible interval bands are wide, reflecting the uncertainty inherent to the small sample size ($n=12$), but in all scenarios, the predicted interval for adults lies clearly above that of juveniles, reinforcing the qualitative consistency of the observed age effect.

4. Discussion

The present study demonstrates that adult cetaceans exhibit a higher proportion of neurons with nuclear immunoreactivity for phosphorylated tau (p-tau) at the AT100 epitope in both the frontal cortex and cerebellum compared with subadult and juvenile individuals. Our Bayesian analysis, yielding posterior probabilities of 85.5% and 89.5% for positive age effects in the cortex and cerebellum respectively, provides robust evidence of an age-associated increase in tau phosphorylation, even within the constraints of a limited sample size ($n=12$) [34,35].

The pattern of AT100 immunolabeling observed in the frontal cortex is consistent with findings reported in humans and other mammals [28,29]. Similarly, age-related increases in hyperphosphorylated tau and changes in its subcellular localization have been described in non-human primates such as Callithrix jacchus and in murine models [25,30,42]. The presence of comparable patterns across phylogenetically distant mammalian lineages suggests that tau phosphorylation may be linked to conserved biological processes. In this context, the AT100 epitope (Thr212/Ser214), associated with conformational changes in tau [21], may represent a relevant indicator of age-related neuronal modifications, although its role as a biomarker should be interpreted with caution.

The progressive increase in phosphorylated tau with age is likely driven by multiple interacting mechanisms. Imbalances between kinase activity (including GSK-3 and CDK5) and phosphatase function (such as PP2A), together with reduced efficiency of proteasomal and autophagic degradation systems and increased oxidative stress, have been implicated in tau dysregulation [21,32]. These processes may not only promote hyperphosphorylation but also facilitate the redistribution of tau from axonal compartments to the soma and nucleus. Although these mechanisms were not directly assessed in the present study, they provide a biologically plausible framework for interpreting the observed patterns. Nuclear localization of tau has been proposed to play a protective role in maintaining DNA integrity and regulating chromatin organization under conditions of cellular stress [15,17,43], although this function may vary depending on the physiological or pathological context [20]. In the specific case of cetaceans, this nuclear migration may be fundamentally linked to the extreme physiological demands of their environment. Unlike terrestrial mammals, cetaceans are frequently exposed to ischemia-reperfusion events during diving, which trigger significant production of reactive oxygen species (ROS). This suggests that the nuclear translocation of p-tau could be a specialized response to an oxidative stress environment that is inherently more severe than that of terrestrial mammals, acting as a “nuclear shield” to protect the genome against hypoxia-induced damage throughout their long lifespans [18,19].

A particularly notable finding was the strong correlation between AT100 immunoreactivity in the frontal cortex and cerebellum ($r=0.932$). This association suggests that tau phosphorylation may be regulated through coordinated, organism-level mechanisms rather than strictly region-specific processes. Such coordination may reflect shared regulatory pathways, common responses to age-related stressors, or synchronized developmental and aging programs affecting tau metabolism across distinct brain regions. From an evolutionary perspective, this type of systemic regulation could confer adaptive advantages by ensuring consistent neuroprotective responses in functionally interconnected neural circuits.

In the cerebellum, phosphorylated tau was predominantly localized in granule cells and, to a lesser extent, in Purkinje neurons, a pattern consistent with previous observations in other mammals [33]. Granule cells, as the principal excitatory interneurons of the cerebellum, play a central role in transmitting sensorimotor information and modulating Purkinje cell activity, which constitutes the primary output of the cerebellar cortex [34,35,36]. This distribution suggests that tau phosphorylation may influence cerebellar circuits involved in motor integration and coordination.

Furthermore, we observed that two subadult individuals of D. delphis exhibited nuclear AT100 immunoreactivity levels comparable to those observed in adults. Moreover, one of these individuals presented cerebral necrosis, with particularly elevated levels of nuclear labeling in both the cortex and cerebellum. Necrotic processes are associated with energy failure, ionic imbalance, and activation of stress-sensitive kinases, all of which can promote tau redistribution from axonal compartments to the soma and nucleus [20,32]. This finding suggests that increased nuclear tau may reflect not only physiological aging but also acute responses to neuronal injury. However, the potential contribution of infectious or concomitant pathologies cannot be excluded, as agents such as morbillivirus, Brucella ceti, and Toxoplasma gondii have been associated with central nervous system lesions in marine mammals [45,46,47,48].

Regarding biological traits, no significant differences were observed between males and females in AT100 immunolabeling patterns. This lack of variation suggests that tau regulation and function may be conserved across sexes, consistent with its fundamental role in neuronal homeostasis [20,21]. Our findings complement those reported by Orekhova et al. [38], who investigated $\beta$-amyloid and phosphorylated tau in Mediterranean cetaceans using the AT180 and AT8 epitopes. While their study focused on pathological tau aggregates potentially associated with neurodegeneration, the AT100 pattern observed in our samples appears more consistent with physiological aging processes. These differences between epitopes (AT100 versus AT180 and AT8) may explain such discrepancies, highlighting the importance of epitope-specific approaches when interpreting tau pathology across different species.

The Bayesian analytical framework was appropriate for this study given the small sample size and the proportional nature of the outcome variables. The use of Beta regression ensured appropriate modelling of bounded percentage data, and sensitivity analyses across different prior specifications consistently supported the direction of the age effect. Nevertheless, it is critical to emphasize that this work represents an exploratory or pilot study. While our Bayesian analysis suggests biological consistency across the individuals analyzed—showing no evidence of notable differences between the species included (D. delphis, P. phocoena, T. truncatus, S. coeruleoalba)—the limited sample size ($n=12$) precludes definitive generalizations about all cetaceans. Several limitations should therefore be acknowledged, including the small sample size, the cross-sectional design, and the multispecies composition of the dataset. The absence of species as a covariate represents a potential source of confounding. However, a post-hoc inspection of residuals from the main models revealed no systematic clustering by species, and the two non-Delphinus adults (T. truncatus, S. coeruleoalba) fell within the range predicted for their age class, suggesting that species-level differences, if present, are smaller than age-related variance in this dataset. Formal multi-species modelling remains a priority for future studies with adequate per-species sample sizes.

5. Conclusions

This pioneering study provides the first description of the nuclear distribution of the AT100 phosphorylated tau epitope in the cetacean brain, further distinguished as the first work to analyze this protein within the cerebellum of these species. Our results demonstrate that the increase in nuclear AT100 immunoreactivity is significantly correlated with age in both the frontal cortex and the cerebellum. This suggests a coordinated, systemic regulatory mechanism within the central nervous system as the animals mature.

The high posterior probability obtained through Bayesian analysis confirms that the nuclear translocation of tau is a robust biological event in cetaceans stranded along the Galician coast. We propose that this phenomenon acts as an adaptive “nuclear shield”[17,43], intended to preserve genomic integrity against chronic oxidative stress and intermittent hypoxia—factors inherent to the extreme diving physiology of these marine mammals. Furthermore, the observation of elevated AT100 levels in a single individual presenting cerebral necrosis suggests that this protein may also serve as a sensitive marker of acute neuronal distress in the presence of severe injury.

Despite the exploratory nature of this pilot study and the inherent limitations of the sample size, the consistency of the patterns observed across the different species analyzed highlights the importance of this neuroprotective mechanism. This work not only fills a gap in the comparative neuroanatomy literature but also establishes a fundamental starting point for future research on brain aging and neuronal resilience strategies in marine mammals.