Human Truncated Tau Induces Mature Neurofibrillary Pathology in a Mouse Model of Human Tauopathy

Abstract

Alzheimer’s disease (AD) represents the most common neurodegenerative disorder. Several animal models have been developed in order to test pathophysiological mechanisms of the disease and to predict effects of pharmacological interventions. Here we examine the molecular and behavioral features of R3m/4 transgenic mice expressing human non-mutated truncated tau protein (3R tau, aa151–391) that were previously used for efficacy testing of passive tau vaccine. The mouse model reliably recapitulated crucial histopathological features of human AD, such as pre-tangles, neurofibrillary tangles, and neuropil threads. The pathology was predominantly located in the brain stem. Transgenic mice developed mature sarkosyl insoluble tau complexes consisting of mouse endogenous and human truncated and hyperphosphorylated forms of tau protein. The histopathological and biochemical features were accompanied by significant sensorimotor impairment and reduced lifespan. The sensorimotor impairment was monitored by a highly sensitive, fully-automated tool that allowed us to assess early deficit in gait and locomotion. We suggest that the novel transgenic mouse model can serve as a valuable tool for analysis of the therapeutic efficacy of tau vaccines for AD therapy.

Keywords

Alzheimer’s disease neurofibrillary degeneration tauopathies transgenic mouse truncated tau protein

INTRODUCTION

Alzheimer’s disease (AD), the most common cause of dementia, is currently a major research and societal challenge. Recent epidemiological estimates of over 24 million cases of AD worldwide, and these are expected to quadruple by 2050 [1]. AD encompasses a list of pathophysiological hallmarks, including depositions of amyloid-β, neurofibrillary tangles (NFTs) composed of post-translationally modified protein tau, synaptic and neuronal loss, microglial activation, astrogliosis, and inflammation. Although amyloid-β has been strongly implicated as the trigger factor in the disease, synaptic loss and accumulation of NTFs associated with tau protein pathology correlate most strongly with dementia [2].

There is an emerging consensus that tau protein represents a promising therapeutic target in the treatment of AD. To identify and validate potential tau disease modifying treatments, several efforts were made to create disease models mimicking the crucial aspects of tau pathology in the mouse [3]. Passive and active immunological interventions targeting tau pathology in mouse models of AD have recently attained promising results. Numerous independent studies have demonstrated the efficacy of tau-targeted immunotherapy in several transgenic mouse models. Those studies clearly showed that immunotherapy could prevent tau aggregation or clear tau aggregates and reduce tau hyperphosphorylation [4]. Most of the studies, however, performed pre-clinical therapeutic trials on mouse models expressing mutant human tau. Since tau mutations are absent in AD, these animal models do not fully recapitulate the AD tau neurodegenerative cascade.

In contrast to tau mutation, protein truncation is one of the most powerful pathological modifications of tau protein. Truncation of tau protein will not only alleviate the steric hindrance associated with protein folding but also induce structural changes, forming a misordered protein. Pathologically modified proteins can act as seeds for nucleation and take part in a self-propagating process resulting in the formation of insoluble assemblies, ultimately leading to degeneration and death of affected cells. In AD, several truncation points have been found including E391 or D421 [5].

We have previously shown that truncated tau (151–391) induced extensive neurofibrillary pathology in the brain of transgenic rats [6, 7]. The rat models demonstrated that truncated tau was an inducer of neurodegeneration. Here we generated a novel transgenic mouse line (R3m/4) expressing non-mutated truncated tau derived from human AD brains. The R3m/4 transgenic mice display early onset of AD tau pathology and sensorimotor impairment, which renders this model an ideal test system for immunotherapy targeting tau neurofibrillary lesions. We have previously demonstrated reduced levels of sarkosyl insoluble tau oligomers and fewer early and mature NFTs after tau passive vaccination in R3m/4 mice [8]. The R3m/4 transgenic mouse line thus represents a promising tool for preclinical development of tau vaccines.

MATERIALS AND METHODS

Preparation of transgene and generation of transgenic mice

R3m/4 transgenic mice overexpress disordered 3-repeat tau protein truncated at aa151 and aa391 and driven by mouse Thy-1 promoter. The gene construct was prepared as described earlier [7]. Briefly, a complementary DNA (cDNA) coding for human tau protein truncated at amino acid positions 151 and 391 was ligated into the mouse Thy-1 gene downstream of the “brain promoter/enhancer” sequence. The part of original Thy-1 gene sequence coding for exons II-IV together with the “thymus enhancer” sequence was replaced by the cDNA. For pronuclear microinjection, the transgenic DNA was linearized by cleavage with EcoRI and purified after separation in SeaKem GTG agarose. All prokaryotic sequences were removed prior to microinjection into 1-day old mouse C57BL/6 embryos. Founders were double screened by polymerase chain reaction using Thy-1-specific and human tau-specific primers amplifying START codon (forward: 5‘-GTGGATCTCAAGCCCTCAAG-3‘, reverse: 5‘-CCTGATTTTGGAGGT-3‘) and STOP codon (forward: 5‘-CCTGATTTTGGAGGT-3‘, reverse: 5‘-TATGCATGGAGGGAGAAG-3‘) containing sequ-ences. Transgenic line R3m/4 reliably expresses human truncated tau for several generations.

Animal husbandry

All R3m/4 transgenic mice (Tg) used in this study were hemizygous for the transgene construct. Wild type C57BL/6 (Wt) age-matched males were used as control for behavioral assessments. All mice were weaned at the age of 21 days and housed under standard laboratory conditions, in plastic cages (4 animals per cage) with ad libitum access to food and water. Animals were kept under diurnal lighting conditions (12 h light/dark cycles with light phase starting at 7:00 a.m.) and under controlled temperature (22±2°C) and humidity (55±10%). To reduce stress and discomfort of experimental animals during behavioral experiments, animals were not forced to walk back to the dark cage. All experiments were performed according to the institutional animal care guidelines, conforming to international standards and were approved by the State Veterinary and Food Committee of Slovak Republic and by Ethics Committee of Institute of Neuroimmunology (Slovak Academy of Sciences, Bratislava).

Histology and immunohistochemistry

Deeply anesthetized animals were perfused intracardially for 1 min with phosphate-buffered saline (PBS), followed by 3 min perfusion with 4% paraformaldehyde (PFA) in PBS (4% PFA, pH 7.2). The brain was post-fixed for 24 h in 4% PFA, then transferred to PBS, embedded in paraffin and serially cut using Leica RM 2255 microtome into 8μm-thick sagittal brain sections. Immunostaining was performed using the standard immunohistochemistry staining procedure. Briefly, brain sections were treated with 80% formic acid (40 s) followed by heat pretreatment for 20 min in antigen retrieval solution (Retrieval 2100, Aptum, Southampton, UK). Sections were incubated with primary antibodies (Table 1) overnight at 4°C and were immunostained using the standard avidin-biotin-peroxidase method (Vectastain ABC kit) with VIP as chromogen (VIP kit, Vector Laboratories, Burlingame, CA, USA). Gallyas silver iodide staining methods were used to demonstrate mature neurofibrillary pathology in neurons [9].

Extraction of soluble tau

Total proteins were isolated from 3 or 6.5-month old mice using buffer containing 20 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 2 mM DTT, 0.5% Triton X-100 with protease and phosphatase inhibitors. Homogenates were centrifuged at 20,000 × g for 20 min and supernatant was collected. Protein concentration was determined using Bradford reagent (Bio-Rad laboratories GmbH, Germany) and aliquots were stored at –80°C until use.

Extraction of sarkosyl insoluble tau

Sarkosyl insoluble tau was isolated as described earlier [10] with minor modifications. Approximately 0.5 g of brain stem of 6.5–7 month old R3 m/4 transgenic mice was homogenized in 10 volumes of ice-cold homogenization buffer (20 mM Tris, 0.8M NaCl, 1 mM EGTA, 1 mM EDTA, and 10% sucrose) supplemented with protease (Complete, EDTA free, Roche Diagnostics, USA) and phosphatase inhibitors (1 mM Sodium orthovanadate, 20 mM NaF) followed by centrifugation at 20,000 × g for 20 min. The supernatant was adjusted to 1% (w/v) N-lauroylsarcosine and incubated for 1 h at room temperature with stirring. After incubation supernatant was centrifuged at 100,000 × g for 1.5 h at 25°C. Pellets werere-suspended gently in 1/50 of initial homogenization volume in 1xSDS sample loading buffer and analyzed by western blotting.

Western blotting

Proteins were separated using 12% SDS-PAGE gels and transferred to nitrocellulose membrane as described previously [10]. Membranes were incubated with primary antibodies diluted in 5% non-fat dry milk or 5% BSA for 1 h or overnight at 4°C or according to manufacturer’s instructions. Blots were developed using enhanced chemiluminiscence detection system (SuperSignal West Pico chemiluminiscence substrate, Thermo Scientific, USA). All western blots were repeated at least twice for consistency; however, only one of each pair was used for further analysis. For phospho-tau analysis, a cocktail of phospho-tau antibodies (pS199, pT205, pT212, S396, and pS404) was used.

Behavioral assays

Four days prior to the first testing session, all animals were handled daily to minimize stress and maximize validity of acquired data. Testing was performed between 1:00–4:00 p.m. on 3 consecutive days in 4 sessions (at 3, 4, 5, and 6 months of age of experimental animals). Each animal was required to finish 4 correct runs during each session. Animals unable to complete four consecutive runs were excluded from measurement. Only three 6-month-old transgenic animals performed the required number of runs and we excluded data measured at 6 months from further analysis. Experimenters were blind to animal genotype.

Sensorimotor impairment assessment

Gait of unforced walking rats was analyzed with CatWalk XT (Noldus, Wageningen, Netherlands). The system consists of a corridor that directs the movement of animals to a straight line, a glass walkway with illuminated footprint technology, a cage at the end of the walkway, high speed color camera, and software for recording and automated analysis of the locomotor ability of rodents [11, 12]. Animals were required to cross the walkway without interruptions and at constant speed (total duration of the run in range 1–5 s, speed variation ≤30%). At least four correct crossings of the walkway were acquired for each animal [13]. All data were recorded with a pixel threshold value ≥25 (arbitrary units; range 0–255) in a completely dark room.

For further analysis we selected general run parameters (run duration, body speed variation, number of steps, and step sequence regularity) and gait parameters (step cycle, duty cycle, swing speed, print position, contact area, and intensity).

Statistical analysis

Statistical analysis was performed using GraphPad Prism software (GraphPad Software, CA, USA) and using custom-written scripts in Matlab (Mathworks, Natick, MA, USA).

Analysis of total tau and phospho-tau levels was performed using non-parametric Mann-Whitney U test. Survival time curves were analyzed using the Kaplan-Meier method. Changes in gait parameters between R3m/4 and wild type mice were evaluated using repeated-measures ANOVA with Greenhouse-Geisser bound correction. When the change in a gait parameter between the two groups was significant, values at different time points were evaluated further using two-sample t tests. Obtained p-values were corrected for multiple comparisons using Benjamini-Hochberg procedure to keep the false discovery rate <0.05. All data are expressed as mean±SEM, unless stated otherwise.

RESULTS

Expression of human truncated tau protein reduced lifespan of transgenic mice

The ratio of human truncated tau levels to endogenous tau protein levels from 3 month-old transgenic R3m/4 mice (n = 3; males) was analyzed in 3 different brain regions in order to characterize regional expression of human truncated tau (Fig. 1A). The expression levels of human truncated tau protein differed in various brain areas. There was twofold increase in truncated human tau levels compared to endogenous mouse tau levels in the brain stem (2.4±0.114; mean±SEM) and cortex (1.9±0.05; mean±SEM) and only mild overexpression of truncated tau in the hippocampus (1.18±0.08; mean±SEM; Fig. 1B).

Next, we examined the effect of the expression of human truncated tau protein on lifespan of R3m/4 transgenic mice (Fig. 1C). Interestingly, median survival time of transgenic males (n = 11, 244±20.87 days) and females (n = 17, 224±13.69 days) was significantly shorter compared to wild type animals (median survival = 827 days in males, n = 15, and 818 days in females, n = 15). The lifespan of R3m/4 transgenic mice was thus reduced to approximately one third of the lifespan of wild type mice.

Expression of human truncated tau protein did not dysregulate endogenous total and phospho-tau protein levels in transgenic mouse model

We investigated whether the expression of the human truncated tau protein in mouse brain might alter the endogenous tau proteome. We analyzed total and phosphorylated tau protein from three different brain regions: the hippocampus, the cerebral cortex and the brainstem from wild type and transgenic mice (Wt, n = 3; and Tg, n = 3 mice; age 6.5 months, males). Pan-tau antibody DC25 detected tau protein in all brain regions (Fig. 2). We observed no significant changes in the levels of endogenous tau protein in any of the brain regions examined in transgenic mice when compared to wild type animals. To investigate phospho-tau protein changes, we performed western blotting using a cocktail of anti-phospho-tau antibodies pS199, pT205, pT212, pS396, and pS404 (Fig. 3). We found only a moderate increase in phospho-tau levels in the protein extracts from R3m/4 mice; however, the differences were not significant.

Sarkosyl insoluble tau was composed of human truncated tau and mouse endogenous tau

Sarkosyl insoluble tau protein is the main feature of human tauopathies, including AD. Therefore, we examined sarkosyl insoluble protein from the brain stem of transgenic R3m/4 mice (n = 4, 7 months, males). As a control, wild type mice were used (n = 3, 7 months, males). Sarkosyl insoluble fractions were immuno-labeled with pan tau mAb DC25. In the brainstem of all transgenic mice, abundant insoluble tau was found. In contrast, brain extracts from wild type mice did not display any traces of insoluble tau species (Fig. 4A). To monitor the level of phosphorylation of insoluble tau, phospho-tau antibody pT212 was used (Fig. 4B). The ratio of the total tau to phospho-tau pT212 was 2:1 (Fig. 4D). DC39C antibody detected only endogenous mouse insoluble tau and stained the A68 triplet (Fig. 4C). In this case, the ratio of the total tau to endogenous tau was 20:1 (Fig. 4D).

Transgenic mouse model developed progressive neurofibrillary degeneration in brain stem

Abnormally phosphorylated tau was found in the somatodendritic compartment of pyramidal neurons in the cortex (Fig. 5A), as well as in the mossy fibers of hippocampus (Fig. 5B). The classical neurofibrillary pathology was distributed almost exclusively in the brain stem (Fig. 5C). Mature neurofibrillary lesions represented the main histopathological feature of transgenic mouse line R3m/4. NFTs were distributed mainly in the brain stem and occasionally in the cortex (n = 3 mice, 6 months, males). Some NFTs were positive for Gallyas silver staining, suggesting the presence of mature NFTs (Fig. 6A). Moreover, NFTs were detected using a panel of antibodies which recognize phospho-tau species, endogenous mouse tau, or conformationally modified tau (Table 2, Fig. 6B-H). Antibodies AT180 (phospho Ser231/Thr235), pT212, and pS214 labeled pre-tangles, tangles, and neuropil threads mostly in the brain stem. In addition to tau pathology, antibody DC11 (recognizing conformationally modified tau) also detected mossy fibers in the hippocampus. Interestingly, antibody PHF1 (recognizing endogenous mouse tau only) exclusively recognized tangles but not pre-tangles and neuropil threads. The overall appearance of NFTs closely resembled the neurofibrillary pathology described in human patients with AD (Fig. 6G, H).

Transgenic mice displayed mild sensorimotor impairment in pre-symptomatic stage and rapid decline in phenotype in pre-terminal stage of disease

Health status and weight of experimental animals were checked regularly in two-week intervals from weaning to natural death/euthanasia. Assessment of sensorimotor impairment was performed in one-month intervals between 3 and 6 months of age (four time points). While all animals (Tg males, n = 12; Wt males, n = 11) were able to pass the walkway correctly at the age of 3–4 months, at month 5 one transgenic animal was already not able to pass the walkway. By month 6, most of the tested transgenic animals developed paresis or total paralysis of hind limbs, had progressively atrophied skeletal muscles and were not able to pass the walkway.

Across the population of tested animals, the gait of transgenic mice was indistinguishable from control mice during the first two months of testing (month 3 and 4, Fig. 7). At month 5, however, transgenic animals moved significantly slower across the walkway when compared to control animals (Fig. 7A). Although the movement of transgenic animals was slower, the variability was not significant (Fig. 7B). Rather, this slower movement was accompanied by shorter, irregular steps, as demonstrated by the overall increase in the number of steps necessary to walk across the walkway (Fig. 7C) and lower regularity of each step sequence (Fig. 7D).

Individual steps of 5-month-old transgenic mice were slower (longer step cycle duration, Fig. 7E), with paws spending more time in contact with the walkway (duty cycle, Fig. 7F) and slower swing speed (Fig. 7G). However, paw print position, i.e., the distance between the position of the hind limb paw and the position of the previously placed front paw at the same side of the body and in the same step cycle, remained unchanged (Fig. 7H). Other paw parameters, such as contact area of the paw and intensity of the contact area decreased slightly, most likely as a result of locomotor discomfort (Fig. 7I, J).

At month 6, transgenic mice developed atrophy of skeletal muscles, accompanied by shaking-like movements and paresis or total paralysis of hind limbs. Hind limb extension reflex response during tail suspension progressively deteriorated (Fig. 8B, C) when compared to wild type mice (Fig. 8A). Clasping reflex of the hind limbs appeared in the terminal stage of the disease (Fig. 8D).

DISCUSSION

Epidemiological studies that demonstrate growing number of AD patients sound the alarm bells over our knowledge of tau pathology and current state of tau-targeting therapeutic approaches to the disease. Indeed, considerable efforts have been undertaken to generate an appropriate animal model mirroring the crucial aspects of AD-like tau pathology that would be suitable for testing immunological therapeutic interventions [14].

Several animal models have been widely used for preclinical efficacy studies. P301L and P301S mutant tau models became the most commonly used in the preclinical drug development. For instance, P301L mutant tau transgenic mouse model with progressively increased phosphorylation of tau at multiple sites was used for active immunization with phosphorylated tau epitope Tau379–408 (phospho-Ser396/404). Administration of this immunogen led to antibody response, decreased level of insoluble tau forms and deceleration of motor impairment characteristic of untreated P301L mice [15]. In another active immunization study, P301L mice were immunized with twelve amino acid peptide of the human tau (aa395–406) comprising phosphorylated Ser396/Ser404 of the PHF-1 antibody epitope. The treatment reduced neurofibrillary pathology and decreased tau phosphorylation [16]. A single dose of passive immunization of P301L transgenic mice with oligomer-specific monoclonal antibody (TOMA) designed to intervene with tau oligomers that occur in the brain before NFTs formation was sufficient to alter behavioral deficits in mice and led to striking reduction of tau oligomers but not phosphorylated NFTs or monomeric tau [17].

Another commonly utilized transgenic tau model strain P301S expresses 4R0N isoform of human tau with the P301S mutation and accumulates detectable levels of hyperphosphorylated tau in both soluble and insoluble fractions [18]. P301S mice were used to demonstrate efficacy of two peptide vaccines composed of double phosphorylated tau epitopes (pSer202/pThr205, pThr212/pSer214, and pThr231/pSer235) that was associated with behavioral benefits and prolongation of lifespan of transgenic mice [19]. Furthermore, P301S mice were used also as a preclinical model of passive immunotherapy with MC1 (a conformation-dependent antibody that recognizes an early pathological tau conformation) and PHF1 (pSer396/Ser404). The therapy significantly reduced levels of tau pathology after administration of both PHF1 and MC1, however, improvement of sensorimotor deficit of P301S mutant mice was observed only in the case of MC1 administration [20].

Other preclinical studies used THY-Tau22 mutant mouse model that expresses transgene containing the cDNA of the 412 amino acid isoform of human 4-repeat tau mutated at sites G272 and P301S. These transgenic mice develop hippocampal NFT-like inclusions responsible for cognitive impairment and express phosphorylation of tau in several AD-relevant tau epitopes. When used as a model for testing active tau immunotherapy, application of Y10A peptide targeting phospho-Ser422 epitope improved cognitive deficits and reduced the load of NFTs and insoluble tau complexes in THY-Tau22 mice [21].

Occasionally, other models such as triple transgenic mice harboring APP (SWE), PS2/PS19, and tau (P301L) transgenes [22 –24] or combined genomic wild type tau/mutant tau models [25, 26] were used for testing of either active or passive immunization.

Most of the preclinical trials performed so far on AD immunotherapy used mouse models expressing mutant human tau. Although tau mutation is the driving force in the pathogenesis of FTDP-17, there is no evidence of tau mutation in AD. To provide a mouse model suitable for preclinical testing of therapeutic approaches in AD, we generated transgenic mouse line R3m/4 expressing non-mutated truncated tau derived from human AD.

Here we performed molecular and behavioral analyses of the R3m/4 transgenic mouse model. Transgenic mice expressed human truncated tau in various areas of the brain such as the isocortex, the hippocampus, and the brainstem. Tau was abnormally phosphorylated and distributed in the somatodendritic compartment of cortical pyramidal cells and in axonal fibres of hippocampal neurons. Expression of truncated tau did not dysregulate the level of endogenous tau or modified phosphorylation of endogenous tau. The mice developed mature neurofibrillary degeneration that progressed through several histologically defined maturation stages, from accumulation of pre-tangles to mature NFTs. NFTs were identified with antibodies specific for hyperphosphorylated forms of tau protein and antibodies recognizing an abnormal tau conformation such as DC11, used to detect pathologic tau in the human brain. Previously we have shown that human truncated tau developed extensive tau neurofibrillary pathology in the brain of two independent transgenic rat lines [6, 7].

Sarkosyl insoluble tau is considered to be an important feature of mature neurofibrillary degeneration. In two transgenic rat models expressing human truncated tau we have previously shown that sarkosyl insoluble tau underwent several maturation stages beginning with an early one band stage, followed by stage of shifted monomer, and maturing as stage of the tau ladder. The stage of extending phosphorylation-shifting monomer stage-was reported in several mutant mouse models of tauopathies [27, 28]. However, the mature stage of the tau ladder encompassing both exogenous human tau and endogenous mouse tau was described only in a handful of transgenic mice [29, 30]. Here, we described a transgenic mouse line which develops the final stage of sarkosyl insoluble tau suggesting that the truncated tau is the driving force of the full neurofibrillary cascade.

Most of the neurofibrillary pathology identified in the brains of R3m/4 mice was localized in the brain stem. Such localization likely contributed to reduced lifespan of R3m/4 mice and, given the role of brainstem in regulation of motor function, led to specific motor disturbances. Therefore, we focused our behavioral investigations of R3m/4 model on the expected sensorimotor impairment. We evaluated the impact of transgene expression on motor function using the CatWalk system, a highly sensitive fully automated system that quantifies gait disturbances of freely walking rodents.

Sensorimotor tests, such as Rota-rod, static rods or balance beam have been used for screening animals in most of immunotherapeutic studies performed in tau transgenic mice [15 , 31]. The limitations of these methods were described elsewhere [32]. Briefly, these tests were designed to measure motor coordination and balance, thus offering only partial information about functioning of the complex sensorimotor system. On the other hand, the automated quantitative analysis system used in this study provides additional and more detailed information about general and specific gait parameters. R3m/4 transgenic mice displayed changes in motor function that were manifested by significantly slower motion and shortened steps with irregular step pattern.

The JNPL3 transgenic mouse strain is currently one of the most commonly used animal models for testing the efficacy of tau-targeted immunotherapy at the behavioral level. The behavioral phenotype of these transgenic mice, however, can be variable. While the initial reports describing heterozygous P301L mice in F1 generation showed sensorimotor impairments presented by clasping response during tail elevation and hunched postures with clenched paws while sitting that typically started at 6.5 months of age and were followed by hind limb paralysis at 8–10 months of age, a later study reported no generalized sensorimotor impairment within 8.5 months of age [33]. These findings suggest that JNPL3 mice do not have stable phenotype and the phenotypic variability could be explained by heterogeneous genetic background (C57BL/6, DBA/2, SW). On the contrary, R3m/4 transgenic line was developed on a genetically homogeneous background (C57Bl6) and it has been stable for more than 10generations.

Finally, R3m/4 transgenic mouse line successfully recapitulates the features of AD-like tau pathology observed in our previously described SHR72 transgenic rat line [6, 34]. R3m/4 mice displayed similar onset, localization and progress of neurofibrillary pathology, including the development of mature NFTs that we also observed in transgenic rats. Additionally, the similar onset and progress of behavioral phenotype in both transgenic mice and rats suggests that the truncated tau is sufficient to induce full tau neurodegenerative cascade in two different species. In conclusion, R3m/4 transgenic mouse line expressing human non-mutated truncated tau (3R tau 151–391) opens new possibilities for a broad range of applications, from characterization of the molecular mechanisms of tau neurodegeneration to the determination of efficacies of various therapeutic approaches.

Footnotes

ACKNOWLEDGMENTS

The work was supported by Axon Neuroscience R&D Services SE, Bratislava, Slovak Republic.

Author is an employee of Axon Neuroscience R&D Services SE that develops tau-based immunotherapy.

Author is an employee of Axon Neuroscience SE that develops tau-based immunotherapy.

Authors’ disclosures available online ().

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