Introduction

Microtubule-associated Tau protein is a key neuronal element with several different functions in the central nervous system. One of the first known functions of this protein was the stabilization of cellular microtubules through tubulin interaction and, therefore, its main role is the regulation of the cellular cytoskeleton and axonal transport. However, over the years, new functions have been described for this protein. According to recent studies, Tau could be involved in synaptic plasticity processes, cellular transcriptome regulation, or even the maintenance of DNA and RNA integrity1,2.

Although Tau is mostly expressed in the nervous system (mainly in neurons, but also in oligodendrocytes and astrocytes), it has also been found in peripheral nerves, renal tubules and other tissues and cells3,4. The interaction of Tau with tubulin, actin, or any other protein is affected by post-translational modifications. In an aqueous solution, Tau behaves as a protein with no defined secondary or tertiary structure (intrinsically disordered protein), but after modifications to certain residues, the protein may acquire alpha helix or beta sheet structures. This may have an impact on Tau localization, protein–protein interaction, or even undesirable self-aggregation. Among the possible modifications, phosphorylation is probably the one that has been most widely studied, but acetylation, deamidation, methylation, O-Glycosylation, ubiquitination, SUMOylation, oxidation, and nitration have also been proposed as key regulatory processes5.

Abnormal pTau (hyperphosphorylation) has been associated with pathology. pTau is the main component of the paired helical filaments present in the brain of Alzheimer’s disease (AD) cases and is a pre-step in the formation of NFTs, which—Together with beta-amyloid plaques— are the major neuropathological hallmarks of this disease. The mechanism of Tau aggregation leading to NFT formation remains unsolved and controversial. Currently, there are no effective treatments or neuronal regenerative drugs available to cure AD6. The lack of efficacy of the tested drugs could be explained by them being administered at too late a stage of the disease. Thus, early clinical diagnosis has recently become a well-studied field that could help to improve results after treatment. Nowadays, several research articles indicate that pTau biomarkers are suitable for evaluating the preclinical stage of AD7. Plasma pTau at threonine 181, in combination with neurofilament light and glial fibrillary acidic protein, is a promising, simple and accessible biomarkers for the screening and diagnosis of AD and age-related cognitive decline8,9. In addition, it has been described that plasma pTau181 can be used to differentiate AD from other neurodegenerative disorders10.

However, Tau phosphorylation is not always indicative of a pathological event, nor does it necessarily lead to Tau aggregation11. Natural processes, such as hibernation, elevate brain phosphorylation levels without causing apparent neurological damage12. Animals undergoing hibernation follow several metabolic adaptations and brain plastic changes, including generalized Tau phosphorylation throughout the brain (except in regions where Tau is not expressed, such as the cerebellum). It is striking that this phosphorylation disappears very rapidly when the animal wakes up, and several investigations point to a neuronal protective mechanism, deactivating certain brain functions, possibly to avoid energy expenditure or to prevent neuronal damage13. Hibernation is, therefore, an extraordinary model to study the phosphorylation/dephosphorylation events of Tau protein, since (1) it is a natural non-pathological process, (2) it does not involve the generation of transgenic animals, eliminating possible adverse effects due to genetic modification, and (3) the fast pTau clearance in the brain can indicate there is a mechanism to successfully eliminate a potential neuronal toxic element.

In the present study, we have analyzed the values of pTau181 in the serum of Syrian hamsters during hibernation and after awakening. The optical biosensing system based on the Interferometric optical detection method, which is extensively reported in the scientific literature14,15,16,17,18 was used to measure the concentration of pTau181 protein. This in vitro detection system, whose measurement signal is the Increased Relative in Optical Power (IROP%)19,20, has been employed previously for the detection of several markers in serum and saliva samples21,22. Silicon dioxide nanoparticles (SiO2 NPs) were used as a refractometric amplification method for higher sensitivity23, making it possible to enhance the limit of dejection and eliminating the inherent matrix effect of serum samples by performing a competitive assay—For example, for measuring total Tau protein in serum24. Other applications have also been previously described25,26. This detection system has been validated on multiple occasions with the gold standard ELISA technique, demonstrating even lower detection limits27. The detection limit of 10 pg/mL has been reached24 and can be lower and thus compared with techniques such as Single Molecule Array (SiMOA). In addition, the advantages of IODM should be highlighted, since it is a multiplexed system, which uses low sample volumes and allows the experiment to be monitored at all stages.

Materials and methods

Animals

Animal housing and maintenance protocols followed the guidelines of the Council of Europe Convention ETS123, revised as indicated in the Directive 86/609/EEC. Animal experiments were performed under protocols approved by the Institutional Animal Experiment Ethics Committee (Comité de Ética de Experimentación Animal de la Universidad CEU San Pablo, CEBA-CEU, Madrid, Spain; PROEX_203-4-21 and PROEX 165.6/21) and following ARRIVE guidelines.

All animals were anesthetized by intraperitoneal injection of sodium pentobarbital (200 mg/kg) and subsequently perfused intracardially with 0.9% saline solution followed by 4% paraformaldehyde in phosphate buffer (PB; 0.1M, pH 7.4).

Syrian hamster

In the present study, we used 16 male 3-month-old Syrian hamsters (Mesocricetus auratus) that were purchased from Janvier Labs (Le Genest-Saint-Isle, France). All experimental procedures were carried out at the animal facility of Centro de Tecnología Biomédica (CTB) from the Universidad Politécnica of Madrid (UPM), whose registration number is ES280790002070.

The animals had free access to food and water and were kept at 23 °C with an 8:16-h light/dark cycle. After the acclimatization period (4 weeks), 11 out of the 16 animals were transferred to a special chamber to artificially induce hibernation and obtain torpor and arousal experimental groups (controlling the temperature and illumination, while also allowing the hamsters to be monitored by measuring their locomotor activity). To induce a torpor state, temperature and light were gradually decreased until total darkness and a temperature of 4 °C were reached. Torpor was considered to have started after 24 h of inactivity. We considered animals to be torpid only when they had completed three full bouts of torpor (3–4 days of inactivity) before they were sacrificed by a lethal intraperitoneal injection of sodium pentobarbital (Dolethal. Vetoquinol; 200 mg/kg). We sacrificed the torpid animals on day 2 after torpor started, to make sure the animals were in deep torpor. The arousal experimental group was obtained by removing the torpid animals from the hibernation chamber and inducing their awakening 1 h before they were sacrificed. To ensure the animals were in deep torpor, their temperatures were checked using an infrared thermometer before blood collection and perfusion (n = 6; temperature 5–7.5 ºC) or arousal (n = 5; temperature: 32–35 °C). Control animals (n = 5) were housed, until sacrifice, under normal temperature conditions (not transferred to the hibernation chamber) for the same time as the torpor and arousal groups. See Ref.28 for further details.

P301 transgenic mice

In this study, we also used adult P301 transgenic mice as a control for detecting pTau181. The animals were obtained from The Jackson Laboratory (B6; C3-Tg, Prnp-MAPT*P301S, PS19Vle/J-Strain #008169) and express the human Tau protein P301S mutation (1N4R isoform), controlled by the mouse prion protein promoter. All experimental procedures were carried out at the animal facility of the San Pablo CEU University (SVA-CEU.USP, ES 280220000015). All animals were kept on a 12-h light–dark schedule with access to food and water ad libitum.

All animals were sacrificed by a lethal intraperitoneal injection of sodium pentobarbital (Dolethal. Vetoquinol 570,681.8; 200 mg/kg). Serum samples were obtained by drawing blood directly from the heart of the anesthetized animal and the volume obtained was transferred to an Eppendorf tube. Sampling took place in the morning, between 10 and 12 pm. After 4 h at room temperature, samples were centrifuged for 10 min at 2000g and the supernatant was collected for serum assays with NPs. Finally, animals were perfused intracardially with a saline solution followed by 4% paraformaldehyde (Electron Microscopy Sciences; 15714) in phosphate buffer (PB; 0.1M, pH 7.4). The brain of each animal was removed and post-fixed by immersion in the same fixative for 24 h at 4 °C. After rinsing in PB, the brains were cut in the coronal plane using a vibratome (Leica VT2100S). Serial sections (50 mm thick) were cryoprotected in 30% sucrose solution in 0.1 M PB and stored in ethylene glycol/glycerol at – 20°C until they were used for immunohistochemistry assays as described in Ref.28.

Mice genotyping

Transgenic and non-transgenic offspring were identified by DNA extraction from the animal tail and, subsequently, standard PCR was carried out using previously described human Tau P301S transgene-specific primers29.

Immunocytochemistry

For immunofluorescence experiments, free-floating sections were first rinsed 3 times (10 min per rinse) in 0.1 M PB. As paraformaldehyde fixation may mask antigenic sites, the sections were then incubated with sodium citrate buffer (10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0) for 15 min at 95 °C. After washing with 0.1 M PB, samples were then preincubated for 1 h at room temperature with PB 0.25% Triton-X and 3% normal goat serum (Vector Laboratories, Burlingame, CA, USA). Thereafter, the sections were incubated for 48 h at 4 °C in the same stock solution containing the rabbit monoclonal to pTau181 antibody (Abcam, Cambridge, UK, EPR23506-107, 1:2000). After PB rinses, the sections were incubated for 1 h in biotinylated goat αRabbit-IgG (1:200; Vector Laboratories). After several washes in PB buffer, the sections were incubated for 1 h at room temperature with avidin–biotin peroxidase complex (Immuno Pure ABC, Pierce, Rockford, IL; diluted 1:125). Peroxidase activity was revealed with 0.01% hydrogen peroxide, using the chromogen 3,3′ diaminobenzidine tetrahydrochloride (Sigma-Aldrich, St Louis. MO; 0.05%). After staining, the sections were dehydrated, cleared with xylene, and cover-slipped with DEPEX (VWR, Rannor, Pennsylvania).

SiO2 NPs biofunctionalization with αpTau181

80 nm in diameter SiO2 NPs (Superior Silica, Chandler, AZ, USA) in ethanol were used for the biofunctionalization protocol. First, three washes were performed with 1 mL H2OmQ to remove all ethanol by centrifugation at 7500rpm for 20 min. In the last wash, the NPs were resuspended in 5 mM sodium carbonate buffer (Na2HCO3) (Sigma-Aldrich) and carboxyethylsilanetriol 25% silane in water (Fluorochem, UK) was added and kept under agitation for 1 h at room temperature. The same wash was then performed with H2OmQ, and in the final wash, they were resuspended in 2-Morpholinoethanesulphonic acid (MES) buffer at pH 3.5 for the addition of Ethyl(dimethylaminopropyl) carbodiimide (EDC) and N-Hydroxysuccinimid (NHS) (Sigma-Aldrich). The reaction was allowed to stand for 1 h at room temperature with agitation. After the washes, G-protein (Sigma-Aldrich) was added (500 µg/mL in H2OmQ) and left overnight at 4°C with agitation. The next day, 0.1 M diethanolamine (Sigma-Aldrich; D8885) was added as a blocker and allowed to react for 1 h at room temperature with agitation. The washes were repeated and αpTau181 antibody (Abcam, Cambridge, UK, EPR23506-107) was added (50 µg/mL); this incubation was left overnight at 4°C. Final washes were performed and the NPs were resuspended in PBS (Sigma-Aldrich) and stored at 4 °C24.

At each step of the biofunctionalization process, portions of NPs were separated to measure the size and concentration of the NPs using Dynamic Light Scattering (DLS) (Malvern Panalytical, UK).

αpTau181 NP concentration curve

KITs from 16 Biophotonic Sensing Cells (BICELLs) were immobilized with pTau181 protein (Abcam) (50 µL/mL) for 3 h in a 37°C humidity chamber. Tau protein (50 µg/mL) was also incubated as a control (Sigma-Aldrich; APREST88626). The KITs were washed with 20 mL H2OmQ and shaken in H2OmQ for 45 s. After drying with compressed air, the immobilization values were read.

The biofunctionalized NPs were then incubated at a concentration of 1 × 1010 NPs/µL and a dilution of 1:10 on the KIT coated with pTau181. They were kept in a humid chamber at 37 °C for 2 h. The NPS were then washed with 20 mL H2OmQ, kept in shaking PBS for 10 min and rinsed with 40 mL H2OmQ. Finally, after drying, ΔIROP (%) values were read. All points were measured in four replicates.

Competitive assay on hamster serum samples

KITs from 65-BICELLs were immobilized with pTau181 protein (50 µg/mL; 1.5 µL/cell) for 3 h at 37 °C in a humid chamber. Tau was immobilized (50 µg/mL) as a control protein.

NPs biofunctionalized with αpTau181 antibody at a concentration of 1 × 108 NPs/µL in PBS were doped with different concentrations of pTau181 protein (from 100 µg/mL to 0.00001 µg/mL in 1:10 dilutions) for 1 h at room temperature with shaking. After washes, they were incubated in the KIT (1.5 µL/cell) for 2 h in a humidity chamber at 37 °C. Finally, they were washed with 20 mL H2OmQ, kept in PBS with agitation for 10 min, and then rinsed with 40 mL H2OmQ.

These NPs were incubated with the hamster serum samples (1:10 dilution) for 1 h at room temperature with agitation. We used 16 different serum samples: 5 samples from control hamsters (non-hibernating), 5 samples from arousal hamsters, and 6 samples from torpor hamsters. In addition, as a positive control, the biofunctionalized NPs were incubated with 4 samples from transgenic P301S mice. The KITs were washed and incubated in the same manner as the concentration standard. All measurements were performed in five replicates (Fig. 4). One-way ANOVA statistical analysis was performed with a Tukey’s comparison and a Levene’s test of homogeneity of variance with a significance level of p < 0.01.

Results

Since blood pTauT181 detection is considered as a suitable biomarker to evaluate the preclinical stage of AD, we used a biosensing system, based on the interferometric optical detection method, to monitor the Tau phosphorylation at T181 in the bloodstream during the hibernation of the Syrian hamster. This experimental model has been extensively used to study Tau phosphorylation in the brain30,31,32.

First, the NPs undergo a biofunctionalization process by modifying their surface and adding the antibody of interest (αpTau181). After this process, the NPs are incubated with the serum samples, so the protein present in the sample will bind to the NPs. After separating this fraction of NPs, they are incubated on the surface of the biosensor previously coated with the pTau181 protein. In this way, the free antibodies of the NPs will bind with the protein immobilized on the biosensor, obtaining a higher IROP (%) signal when the pTau181 protein in the sample is less concentrated. See a schematic representation of this process in Fig. 1.

Fig. 1
figure 1

Schematic representation of the steps followed to perform the competitive assay for pTau181 detection. Created with BioRender.com.

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SiO2 NPs biofunctionalization with αpTau181

80 nm SiO2 NPs were biofunctionalized with the αpTau181 antibody using a silanization protocol and G-protein. During this process, each step was verified by DLS to measure the concentration and size of the NPs.

Table 1 shows the NP size and how it increases as we progress in the biofunctionalization protocol (Fig. 2). Also shown are the concentration of the NPs after each step and the final working concentration of the NPs once they are biofunctionalized.

Table 1 NPs concentrations and size during the biofunctionalization process.
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Fig. 2
figure 2

DLS measurements of the hydrodynamic diameter of the NPs during the biofunctionalization protocol.

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αpTau181 NPs concentration curve

Following the biofunctionalization protocol, the NPs were incubated on the biofunctionalized KITs with pTau181 protein and Tau at a concentration of 5.42 × 109 and 1:10 and 1:100 dilutions (1.5 µL/cell).

After incubation, the KITs were washed and the ΔIROP (%) signals were read (Fig. 3), obtaining a curve in which the signal decreases as the concentration of NPs decreases. In addition, it is observed that the NPs have specificity on the surface covered with pTau181 protein since a very low background signal is obtained on the Tau protein.

Fig. 3
figure 3

Concentration curve of NPs biofunctionalized with pTau181 antibody.

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Competitive assay on hamster serum samples

After testing the functionality of the NPs, a competitive assay was performed. For this purpose, the NPs were incubated at a concentration of 1 × 109 NPs/µL with different concentrations of pTau181 protein (from 100 to 0.00001 µg/mL in 1:10 dilutions) and left at a final concentration of 1 × 108 NPs/µL. After the incubation time, they were applied to the sensor surface coated with pTau181 and left to incubate for the calibration curve.

At the same time, hamster serum samples (1:10 dilution) were incubated with the NPs at a final concentration of 1 × 108 NPs/µL and the same procedure as for the calibration curve was followed. Serum from transgenic mice were incubated as a positive control.

Once the ΔIROP (%) signals of the NPs were read, the calibration curve was obtained (Fig. 4), in which the signals obtained from the samples were extrapolated to determine the concentration of pTau181 protein in the serum of hamster and transgenic mice (Table 2).

Fig. 4
figure 4

Calibration curve of the competitive assay to determine pTau181.

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Table 2 Data of p181Tau protein concentration in hamster and in P301 mouse serum once extrapolated on the calibration curve.
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Analyzing the results, noticeable differences emerge between the control samples, exhibiting levels ranging from 3 to 16 µg/mL, and the samples obtained from torpor hamsters, which display a pTau181 concentration between 35 and 100 µg/mL. Conversely, arousal hamsters have concentrations similar to the controls. This alignment with control levels is plausible, as the animals should activate the Tau protein dephosphorylation machinery upon cessation of hibernation, a process that is inactive during hibernation.

On the other hand, the positive controls of the transgenic mice also present high concentrations of pTau181 protein (140–200 µg/mL). This would be expected, since these P301 transgenic mice express the human Tau protein P301S mutation (1N4R isoform), leading to early symptoms of human tauopathies such as cognitive decline. Additionally, they promote a progressive accumulation of filamentous Tau in association with neuron loss33,34,35.

Figure 5 shows the box plot obtained after performing the One-Way ANOVA statistical analysis with a Tukey’s comparison of means and a Levene’s test of homogeneity of variance with a significance level of p < 0.01. As shown, there are significant differences in the concentration of pTau181 protein between control and torpor hamsters, as well as between torpor and arousal hamsters.

Fig. 5
figure 5

Comparison of pTau181 protein concentration in control, torpor, and arousal hamsters. ***p < 0.01.

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Immunohistochemistry assay using αpTau 181 antibody

Previous studies evaluating the effect of hibernation on Tau protein revealed that, during the torpor state, Tau is phosphorylated at several residues. Increased labeling has been detected during torpor in western blot assays using the antibodies AT100, AT180, AT270, PHF-1, and 12E8. These antibodies recognize different phosphorylated amino acids at positions 212/214, 181, 231, 396, and 262/356, respectively. However, the presence of pTau has only been demonstrated by immunohistochemistry using the AT8 antibody, which recognizes phosphorylated Tau at the S202/T205 epitope. To further investigate the presence of pTau in the brain of hibernating Syrian hamsters, we performed immunohistochemistry assays using the same specific antibody used here for serum pTau detection (pTau181). Figure 6 shows the labeling in the neocortex and hippocampus in the Syrian hamster brain for different states: torpor, arousal, and non-hibernating (control). As expected, there is no appreciable labeling of pTau181 in control animals, whereas in the torpor state, a clear labeling is observed in hippocampal neurons (Fig. 6K) and in the apical dendrites of pyramidal neurons in the neocortex (Fig. 6J), as previously described using AT8 antibody36. Also, in line with previous western blot results12, pTau181 phosphorylation labeling was fully reversible after arousal (Fig. 6E,H). These results are in accordance with the pTau 181 serum levels found in the same animals.

Fig. 6
figure 6

Photomicrographs showing the pattern of hyperphosphorylated Tau immunostaining with pTau181 antibody in control (A,D,G), arousal (B,E,H), and torpid (C,F,I,J) Syrian hamsters. Small, squared zones in (D–F) are shown at higher magnification in (G–J). Black arrows in (I) indicate pTau181-ir bundles of apical dendrites. Black arrows in (J) indicate 181-positive dentate gyrus neurons, whereas red arrows indicate CA3 pTau181-ir neurons. Scale bar in J indicates 115 μm in (A–F), and 57 μm in (G–J). Adobe Photoshop CS4 software was used to compose figures.

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Discussion

In recent years, much progress has been made in the development of early diagnostic techniques for neurodegenerative diseases, specifically for AD. Cerebrospinal fluid (CSF) analysis and PET imaging, although highly accurate, are expensive and invasive procedures. It has been reported that the amount and phosphorylation of CSF Tau correlate with AD state and the amyloid load37. Currently, plasma and genetic markers may be combined in search of an early diagnosis, allowing a relatively easy and non-invasive approach. One of the most commonly used plasma biomarkers is phosphorylated Tau at threonine 181, which can be used to accurately differentiate AD from other non-AD neurodegenerative diseases38. This biomarker may also be used to predict pre-dementia or even the presymptomatic stage of AD39. Other biomarkers can be used together to detect AD at the earliest possible stage. Concerning this, ultrasensitive immunoassays allow the measurement of several pTau isoforms, such as pTau181, pTau217, and pTau231, which are the most promising AD biomarkers in plasma. In addition, a recent research article suggested that NfL and GFAP can also be used to improve diagnostic accuracy40. Therefore, efforts are focused on developing Alzheimer’s blood tests for primary care, providing an inexpensive and accurate means to predict the risk of developing AD.

In this study, a competitive assay was conducted to determine the concentration of pTau181 protein in serum samples from a Syrian hamster hibernation model.

The key finding is that phosphorylated Tau at threonine 181, one of the most promising AD biomarkers, is present in the serum of hibernating Syrian hamsters and fully disappears during arousal.

Unlike human AD cases, the Syrian hamster does not show any known pathologies due to elevated pTau levels in the brain or serum. This raises the question: why is Tau secreted into the bloodstream? Currently, the presence of Tau protein in the blood of AD patients can be attributed to (1) the release of brain pTau into the extracellular space upon neuronal death, (2) abnormal neuronal pTau secretion under a pathological condition, and/or (3) pTau transport from peripheral tissues to the bloodstream. Concerning the third point, Barthélemy et al. indicated that peripheral contribution has a different phosphorylation pattern compared to CSF and this may affect the search for pTau biomarkers10. Some studies have shown that truncated Tau species appear in brain lysates and CSF of AD cases41. Truncation is critical in the N-terminal region, playing a role in the progression of AD, as this may improve the misfolding/aggregation and lead to neurodegeneration41,42. Future studies are needed to determine whether Syrian hamster blood contains the same Tau truncated species, as this could explain why it does not promote any pathological event. It has been described that phosphorylated Tau can be delivered from neuronal cells transported in exosomes. This mechanism could be responsible for the slow Tau propagation or spreading to other brain regions in AD43 and might explain the pTau transport from cells to the bloodstream.

The augmented pTau levels detected here in serum are not associated with any pathological event, which opens up new questions regarding the mechanism of Tau protein diffusion in the organism. Since tau protein is predominantly found in the brain, the presence of pTau in serum most likely has this origin. If so, this would indicate transport across the blood–brain barrier during hibernation in a reversible way, which would be a surprising finding. In general, this work may help to better understand neurodegenerative diseases and improve preclinical diagnostic methods for tauopathies.

Finally, the present results may be useful for exploring immunotherapy approaches targeting pTau, which could help prevent the formation of tau tangles and offer a potential therapeutic avenue for tauopathies.