Passive Immunotherapy Targeting Tau Oligomeric Strains Reverses Tauopathy Phenotypes in Aged Human-Tau Mice in a Mouse Model-Specific Manner

Abstract

Background:

Tau oligomers are one of the most toxic species, displaying prion-like strains which have different conformations resulting in different tauopathies. Passive immunotherapy targeting different tau species is a promising therapeutic approach. Age is one of the greatest risk factors; however, most immunotherapy studies are done in young to middle-aged mice tauopathy models, which is not representative of the many clinical trials done with older humans with established tauopathies.

Objective:

We utilized two different clones of tau oligomer monoclonal antibodies (TOMAs) in aged Htau and JNPL3 mouse models to investigate the potential of passive immunotherapy.

Methods:

Aged mice received a single intravenous injection of 120 μg/animal of either TOMA1, TOMA3 clones or a non-specific IgG. Their cognitive functions were assessed one-week post-injection using Y-maze and novel object recognition tests. Brain tissues were analyzed using biochemical and immunological assays.

Results:

TOMA 1 and 3 rescues cognitive phenotypes in aged animals in a mouse model-specific manner, indicative by a reduction in tau oligomers levels. The TOMAs were shown to have strong reactivity with different tau oligomeric species in the different mouse models in vitro and ex vivo.

Conclusion:

This is the first study testing tau passive immunotherapy in aged animals and supports our previous reports on of the role of oligomeric tau in disease progression further validating the potential of TOMAs to rescue the late-stage disease pathology and phenotype. Moreover, this study suggests that multiple tau oligomeric strains exist in aged animals; therefore, it is of great importance to further characterize these strains.

Keywords

Aged mouse models brain-derived tau oligomers tau immunotherapy tau oligomers tau oligomers strains tauopathies TOMA clones

INTRODUCTION

Age is the number one risk factor for neurodegenerative diseases. As aging occurs, proteostasis becomes less efficient leading to protein misfolding and accumulation in the brain [1, 2]. Sporadic forms of tauopathies such as Alzheimer’s disease (AD), Parkinson’s disease, Pick’s disease, frontotemporal dementia, corticobasal degeneration, and progressive supranuclear palsy are characterized by the presence of aggregated species of the microtubule associated protein, tau [3, 4]. The main drivers of neurodegeneration in these diseases are thought to be the loss-of-function and toxic gain-of-function of the tau protein, which leads to subsequent spreading of tau aggregates [5].

Normally, tau stabilizes microtubules and facilitates intracellular trafficking of compounds and organelles [6]. Hyperphosphorylation of tau leads to its detachment from the microtubule followed by its misfolding and aggregation into oligomers, protofibrils, and ultimately neurofibrillary tangles (NFTs) [7]. Despite previous indications that large insoluble NFTs were the drivers of neurodegeneration, NFTs alone have been shown to be non-toxic to neurons. On the other hand, it is well established that soluble tau oligomers, the building blocks of NFTs, are the most toxic and pathologically relevant species [8]. The onset and progression of pathology correlates with both the presence and spread of tau oligomers [9]. Tau oligomers are observed both intracellularly and extracellularly. Moreover, they have been shown to spread in a neuronal network-dependent fashion which further suggests their significance in the spread of tau pathology [10, 11]. In addition, the presence of tau oligomers correlates with disease staging and the severity of dementia making them very compelling therapeutic targets [12, 13]. Reduction of tau oligomers coincides with a reduction of disease-related pathology and a significant improvement in the memory and cognitive functions in different animal models [7 , 14–20]. However, at the translational level, clinical trials targeting tau oligomers are still in their infancy.

Passive immunotherapy targeting different tau species is a very promising and advantageous approach over several other therapeutic techniques [21]. It allows for the targeting of both intracellular and extracellular tau species and limits the spread of tau from neuron to neuron. These characteristics make it a promising approach for developing therapeutic interventions for AD and other neurodegenerative tauopathies [22]. Despite their potential, the success of clinical studies testing passive immunotherapy has been hindered due to two major challenges: the old age of the subjects and the difficulty of targeting specific disease-relevant tau species [7]. In this study, we utilize two novel tau oligomer monoclonal antibodies (TOMAs) to perform passive immunotherapy in two human-tau mouse models, accounting for age as a major risk factor.

Two complementary human-tau expressing mouse models were utilized: Aged Htau mice, expressing all six isoforms of human tau, and JNPL3 mice, expressing P301L mutant tau. Mice received a single 120 μg intravenous dose of one of the two different TOMA clones or a control non-specific IgG. This is the first study to assess the efficacy of tau-targeted passive immunotherapy in aged tauopathy models with two different TOMA clones. Results from this study support our previous reports of the role of tau oligomers in disease progression and validate the potential of TOMAs in reversing disease course, even at older ages [12 , 23]. Moreover, this study suggests that targeting only one tau oligomeric strain may not be sufficient to improve or reverse all tauopathy-related phenotypes in aged animals; therefore, it is of great importance to characterize tau oligomeric strains in aged mouse models as well as in human tissues and evaluate the efficacy of antibodies targeted against them.

METHODS

Animals

Both male and female homozygous P301L (JNPL3) (Taconic farms) and Htau (The Jackson Laboratory) mice were used. The JNPL3 mouse model [24] expresses a frontotemporal dementia-associated human mutant tau transgene (P301L) and has been used previously for tau immunotherapy [14 , 26]. Htau mice express six isoforms of human tau, but do not express mouse tau, and have been used for immunotherapy in the past [14]. Mice were housed in the UTMB animal care facility and maintained according to USDA standards (12 h light/dark cycle, food and water ad libitum), in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health).

Immunotherapy with TOMAs

14-16-month-old JNPL3 and 20-24-month-old Htau mice (n = 10 animals/group) received intravenous injections of 120 μl per animal of 1 mg/ml TOMA1, TOMA3 or a non-specific IgG (Rhodamine antibody, Genetex cat. GTX29093) as a control. Briefly, mice were placed in a restrainer (Braintree Scientific) and an inch of the tail was shaved. Next, a total of 120 μg was injected into the lateral tail vein. The mice were then returned to their home cages and kept under observation.

Behavioral tests

In order to examine the effect of passive immunotherapy with TOMAs on cognitive phenotypes, the spatial and recognition memory functions of JNPL3 and Htau mice were assessed by Y-maze and Novel Object recognition (NOR) tests [27, 28]. The Y-maze and NOR tests were performed four and seven days after immunization, respectively.

Y-maze test

This test is widely used to assess spatial learning and memory in mice and rats and is based on the rodents’ innate explorative nature to alternate arms for exploring new environments. This test has been used previously in our lab to detect working memory impairment in JNPL3 mice [14]. The mice were placed in a symmetrical Y-shaped maze with identical interiors to prevent any intra-maze cues. The arms of the maze were 15” long, 3” wide, and 5” high (San Diego Instruments), allowing the mouse to see distal spatial landmarks. The floors and walls were beige, non-reflective to eliminate glare, and made with plastic odor-resistant material. The arms were randomly designated A, B, or C. Y-maze is often preferred over the T-maze due to the equidistant arms with milder turns allowing for faster learning. Black and white pattern cues were placed at the walls next to each arm. Red light was used in the testing area to reduce anxiety in the mice. After each test, the apparatus was thoroughly cleaned using 70% ethanol and allowed to dry prior to placement of a new mouse, to prevent any influence from previous test. The entire Y-maze was elevated to a height of 50 cm from the floor (Tabletop). All the tests for the mice were conducted once and by the same experimenter. Each mouse was placed in an arm facing the center (arm A, start arm) and allowed to freely explore all three arms of the maze for 6 min. The number of arms entered and the sequence of entries were recorded. A correct alternation occurred when the animal moved from the arm in which it began to the other two arms without retracing its steps (i.e., ABC or ACB). Spontaneous alternation, expressed as a percentage, was calculated as the number of consecutive entries into all three different arms (correct choices) divided by the number of possible alternations (total number of arm entries subtracted by two) and multiplied by 100. A high spontaneous alternation rate is indicative of sustained working memory as it suggests the animals memorized their previous choice of arms. Mice with less than 5 total arm entries were excluded from the analysis.

Novel Object Recognition test

This test is based on the inherent tendency of rodents to preferentially explore novel objects and environments over familiar ones. This task has been used previously in our laboratory to detect memory impairment in Htau mice [14]. Four days post-injection, animals were tested in the NOR task. Four square test boxes (40×40×35 cm) were used for the test, with a counterbalanced design of the placement of familiar and novel objects between the boxes. The first day was a habituation stage where mice were allowed to freely explore empty boxes, as open field arenas, for 15 min, with a camera positioned centrally above. 24 h after the habituation stage (second day), mice were placed in the boxes for the training stage with two identical, black-painted wooden cubes, and allowed to explore for 15 min. 24 h after the training stage (third day), mice were placed again in the boxes for the testing stage. The boxes contained one familiar object, which was previously explored in the training phase (black-painted wooden cube), and one novel object differing in color and shape, allowing for the mice to distinguish it from the familiar object, but sharing a common size and volume (white-painted wooden sphere). During the test, the mice were allowed to explore for 15 min. After each trial, the apparatus was thoroughly cleaned using 70% ethanol and allowed to dry prior to placement of a new mouse. All trials were recorded, and the time spent exploring each object was measured using ANY-Maze software. Object exploration was defined by head orientation within 2 cm of the object or physical contact with the object. The discrimination index was calculated as the time spent exploring the novel object divided by the total time spent exploring both objects and the quotient multiplied by 100. Object exploration data were analyzed using Graph Pad Prism 7.0 software, by one-way analysis of variance (ANOVA) followed by Bonferroni’s post-hoc multiple comparisons test.

Tissue harvesting

Brain tissues were collected 10 days post-injection. Mice were deeply anesthetized followed by transcardial perfusion with 1X PBS prior to decapitation. Animals were anesthetized according to IACUC-approved procedures for animal sacrifice. After sacrifice, brains were divided down the midline, where one hemisphere was snap-frozen and stored at –80°C until processed for biochemical analysis. The other hemisphere was embedded in OCT and stored at –80°C until sectioned for immunohistochemical analysis.

Frozen brain hemispheres, extracted from JNPL3 and Htau mice, were homogenized using a 1:3 ratio of 1X PBS with protease (Pierce, Rockford, IL) and phosphatase inhibitor cocktails (PhosStop, Roche, Risch-Rotkreuz, Switzerland). Samples were then centrifuged at 10,000× g for 10 min at 4°C. The supernatants were aliquoted and stored at –80°C as PBS-soluble fractions until use (28).

Protein assay

Total protein concentrations were measured using Micro BCA protein assay kit with bovine serum albumin as a protein standard (ThermoScientific, Waltham, MA).

Western blot analysis

20 μg each of brain homogenate PBS-soluble fractions were resolved in a pre-cast NuPAGE 4–12% Bis-Tris Gel for SDS-PAGE (Invitrogen, Waltham, MA) at 80 volts for the first 10 min then 110 volts in MES-SDS running buffer and subsequently electro-transferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA). After blocking overnight at 4°C with 10% nonfat dried milk/Tris-Buffered Saline with 0.01% Tween 20 (TBS-T), membranes were probed overnight with T22 (1:100) for tau oligomers, or with Tau5 (1:10,000, Covance, Princeton, NJ), or Tau13 (1:15,000, Covance, Princeton, NJ) for 1 h at room temperature for total tau. Membranes were stripped, washed, reblocked, then reprobed for β-actin (1:1000), diluted in 5% nonfat dried milk/TBS-Low Tween. T22 immunoreactivity was detected with horseradish peroxidase (HRP)-conjugated anti-rabbit IgG secondary antibody (1:10,000, GE Healthcare, NJ). Tau5, Tau13, and actin immunoreactivity were detected with horseradish peroxidase-conjugated anti-mouse IgG secondary antibody (1:10,000, GE Healthcare, NJ). Western Bright ECL (Advansta, San Jose, CA) was used for signal generation. For protein quantification, the densitometry of each band was normalized with the corresponding actin band. Densitometric analysis was performed using ImageJ software (Fiji). All densitometry results represent the mean and standard errors of the mean.

ELISA

ELISA plates were coated with 25 μg of samples (brain homogenate PBS-soluble fractions) using 50 mM sodium bicarbonate solution, pH 9.6, overnight at 4°C. The next day, plates were washed one time with 1X TBS-Low Tween (TBST), followed by blocking for 2 h at room temperature with 10% non-fat dry milk in 1X TBST. The plates were then washed one time with 1X TBST and incubated with T22 (1:100) or Tau13 (1:10,000) diluted in 5% nonfat milk in 1X TBST overnight at 4°C for T22 or 1 h at room temperature for Tau13. After washing four times with 1X TBST, immunoreactivity was detected using 100 μl of HRP-conjugated anti-rabbit IgG (GE Healthcare, NJ) for T22 and HRP-conjugated anti-mouse IgG (GE Healthcare, NJ) for Tau13, diluted at 1:3000 in 5% nonfat milk in 1X TBST for 1 h at room temperature. Finally, plates were washed four times with 1X TBST and incubated with 100 μl of 3,3,5,5-tetramethylbenzidine substrate solution (TMB-1 component substrate, Dako, Santa Clara, CA) for up to 1 h in the dark. The reaction was stopped by adding 100 μl of 2M HCl and the optical density (OD) was measured at 450 nm in a Polar Star Omega plate reader (BMG Labtech). Each sample was measured in duplicates and results were analyzed using Graph Pad Prism 7.0 software, by one-way analysis of variance (ANOVA) followed by Bonferroni’s post-hoc multiple comparisons test.

Immunoprecipitation of brain-derived tau oligomers

The immunoprecipitation of tau oligomers from aged Htau and JNPL3 mice brains was completed as previously described [23, 30]. Thirty microliters of tosyl-activated magnetic Dynabeads (Dynal Biotech, Lafayette Hill, PA, USA) were coated with 20 μg of the polyclonal antibody, TTC18 (1.0 mg/ml and diluted in 50 μl of 0.1 M borate, pH 9.5, to a final beads concentration of 20 mg/ml. TTC18 has been characterized previously and recognizes toxic tau confirmations [29, 30]. The beads were kept overnight at 37°C. The beads were then washed (0.2 M Tris-HCl, 0.1% bovine serum albumin, pH 8.5) and incubated with 100 μl of the brain homogenates [phosphate-buffered saline (PBS) soluble fraction] and rotated for 1 h at room temperature. The beads were then washed three times with PBS and eluted using 0.1 M glycine at pH 2.8. Fractions were centrifuged in a micro centrifugal filter device. The molecular weight cut-off was 25 kDa (Millipore, Cat # 42415) at 14,000 g for 25 min at 4°C. Oligomers were re-suspended in sterile PBS and protein concentration was measured using the bicinchoninic acid protein assay (Pierce). The samples were centrifuged again in a microcentrifugal filter device (cut-off 25 kDa) at 14,000 g for 25 min at 4°C. Oligomers were stored at –80°C until use [23].

Dot blot

Dot blot assays to detect tau oligomers were performed as previously described [29]. Briefly, 0.5 and 1 μl (upper most two rows respectively) of flow through samples and 2 and 4 μl (bottom two rows respectively) of IPed samples were applied onto nitrocellulose membranes and then blocked with 10% nonfat milk in Tris-buffered saline with Tween 0.01% (TBS-T) overnight at 4°C. Membranes were then probed with either TOMA1 or TOMA3 (1:250) diluted in 5% nonfat milk for 1 h at RT. Membranes were then incubated with HRP-conjugated IgG anti-mouse (1:3000) to detect TOMA immunoreactivity. Blots were then washed three times in TBS-T before ECL plus (GE Healthcare) was used for signal detection. Quantification corresponds to lowermost row of IPed samples.

Filter trap assay

The filter trap assay was performed using a Bio-Dot SF microfiltration apparatus (Bio-Rad). 1 μg of each end-product reaction was applied onto nitrocellulose membranes, previously prewetted in 1X TBS with very low Tween 0.01% (TBS-T), using a vacuum-based bio-slot apparatus. Membranes were then blocked with 10% nonfat milk in 1X TBS-T overnight at 4°C. The next day, membranes were probed with the polyclonal oligomer-specific tau antibody, T22 (1:250; in-house), monoclonal misfolded tau aggregates TTCM2 (1:1000; in-house), TOMA1 (1:250), and TOMA3 (1:250) diluted in 5% nonfat milk for 1 h at room temperature. Membranes were then incubated with HRP-conjugated IgG anti-rabbit (1:10,000) to detect T22, TTCM2, and anti-mouse (1:10,000) secondary antibody to detect TOMA1 and 3. Membranes were then washed three times in TBS-T, and ECL Plus (GE Healthcare) was used for signal detection.

Oligomer amplification

Recombinant tau 2N4R monomers, obtained by dissolving lyophilized pellets of recombinant 4R tau at 1 mg/ml concentration in PBS [31], were seeded with aged Htau and JNPL3 brain-derived tau oligomers. The oligomer-monomer mixture was made at a ratio of 1:100 (w/w) with rotation at room temperature for 48 h. Aliquots were taken and immediately used for biochemical analysis.

Immunofluorescence

Immunofluorescence was performed on frozen sections. All sections were processed simultaneously under the same conditions. In brief, 12 μm frozen sections were fixed with 4% paraformaldehyde for 10 min, followed by 5-min washes in 1X PBS three times. After blocking in 5% goat serum diluted in 1X PBS (Gibco) for 1 h, sections were incubated overnight in a humidity chamber with T22 (1:250) and AT180 (1:500) diluted in Ab diluent (Dako, Santa Clara, CA) at 4°C. The next day, sections were washed in PBS three times (10 min each) and then incubated with goat anti-mouse IgG Alexa-568 (1:500, Invitrogen, Waltham, MA) and goat anti-rabbit IgG Alexa-647 (1:500, Invitrogen, Waltham, MA) diluted in Ab diluent (Dako, Santa Clara, CA) for 1 h at room temperature then the sections were incubated for 40 min with mouse Fab (1:30) followed by three washes with PBS (10 min each). Sections were then incubated overnight in a humidity chamber with Tau13 (1:1000) at 4°C. The next day, sections were washed in PBS three times (10 min each) and then incubated with goat anti-mouse IgG Alexa-488 (1:500, Invitrogen, Waltham, MA) for 1 h at room temperature, followed by washing three times (10 min each) in PBS. Sections were then mounted using ProLong Gold Antifade Mounting Medium with DAPI (Invitrogen, Waltham, MA). Images were acquired using a Keyence BZ-800E Fluorescence Microscope (Keyence) equipped with a monochrome CCD camera using standard Chrome FITC, Far red, Texas Red, and DAPI filters. Analysis was conducted using BZ-X Analyzer Software (Keyence). Fluorescence intensity was quantified using ImageJ software and analyzed by one-way ANOVA followed by Bonferroni’s post-hoc multiple comparisons test, performed using GraphPad Prism 7.0.

Immunohistochemistry

Immunohistochemistry was performed on frozen sections. All sections were processed simultaneously under the same conditions. Briefly, 12 μm frozen sections were fixed in chilled methanol for 10 min at –20°C, followed by incubation in 0.3% H₂O₂ for 15 min to quench endogenous peroxides. Sections were then washed in 1X PBS two times (5 min each) and blocked in 5% goat serum diluted in 1X PBS (Gibco) for 1 h. Sections were incubated overnight in a humidity chamber with T22 (1:250) or AT180 (1:500) diluted in Ab diluent (Dako, Santa Clara, CA) at 4°C. The next day, sections were washed in 1X PBS three times (10 min each) and then incubated with biotinylated goat anti-mouse IgG (1:200, Vectastain ABC kit, Vector Labs, Burlingame, CA) for 1 h at room temperature. The sections were then washed four times (10 min each) in 1X PBS and incubated with avidin biotinylated horseradish peroxidase (HRP) complex (ABC solution, Vectastain ABC Kit, Vector Labs) for 1 h at room temperature, followed by washing in 1X PBS three times (10 min each). Peroxidase labeling was detected with DAB, followed by washing in 1X PBS three times (10 min each), then washing in warm water for 30 s. Sections were then incubated in Hematoxylin for 1–3 min at room temperature for counterstaining and washed again in warm water for 30 s. Sections were dehydrated in a series of 80% ethanol for 1 then 2 min, 95% ethanol for 1 then 2 min, 100% ethanol for 2 then 3 min, xylene for 3 then 4 min. Sections were mounted using Cytosol 60 Mounting Medium. Images were acquired using a Keyence BZ-800E Microscope (Keyence) equipped with a monochrome CCD camera using brightfield. Analysis was conducted using BZ-X Analyzer Software (Keyence). Staining intensity was quantified using ImageJ software and analyzed by one-way ANOVA, followed by Bonferroni’s post-hoc multiple comparisons test, performed using GraphPadPrism 7.0.

Quantitative histochemical analyses

For quantification, ImageJ was used to quantify 3 regions of interest (ROIs) in the dentate gyrus, 3 in CA3 and 3 in CA1 regions from JNPL3 and Htau mice immunized with TOMAs, ornon-specific IgG.

Electrophysiology

Acute brain slices methods are described in [32]. In brief, 5-month-old Htau mice (n = 3 per treatment) were deeply anaesthetized with isoflurane followed by transcardial perfusion with 25–30 ml of carbogen (95% O₂ and 5% CO₂ gas mixture) -N-methyl-D-gluconate-artificial cerebrospinal fluid at room temperature. Transverse brain sections of 375 μm containing Schaffer collateral synapses were generated using the Compresstome VF-300 (Precisionary Instruments, Greenville, NC, USA). Slices were recovered in NMDG-HEPES cutting solution at 32–34°C for 10 min and were then transferred to carbogen bubbling HEPES-artificial cerebrospinal fluid solution at room temperature for the rest of the experiment time. Slices were treated with oligomers or oligomers preincubated with TOMAs (50 nM final concentration) diluted in carbogen bubbling HEPES-artificial cerebrospinal fluid solution at room temperature for 1 h before recording. After treatment, the slices were briefly (5–10 min) washed by placing them in an oligomer-free recovery artificial cerebrospinal fluid before placing them on the recording stage. Two slices at a time were transferred to the recording chamber and perfused with carbogen bubbling room temperature normal artificial cerebrospinal fluid at a rate of approximately 3 ml/min for recordings. Recording electrodes were pulled from borosilicate glass capillaries using a horizontal P-97 Flaming/Brown micropipette puller (Sutter Instruments, Novato, CA). Evoked field excitatory post-synaptic potentials (fEPSPs) in the CA1 were obtained by stimulating Schaffer collateral. A stable baseline was obtained by delivering single-pulse stimulation at 20 s inter-stimulus intervals. Long-term potentiation (LTP) was induced by exposing the slices to high-frequency stimulation (HFS; 3×100 Hz, 20 s) using Digidata 1550B (Molecular Devices, Sunnyvale, CA, USA), and measured using an Axon MultiClamp 700B differential amplifier (Molecular Devices). Clampex 10.6 software (Molecular Devices) was used. All data are represented as percent change from the initial average baseline fEPSP slope, which was defined as the average slope obtained for 10 min before HFS.

Statistics

All data are presented as mean ± SEM and analyzed with GraphPad prism 7.0 (GraphPad Software Inc., La Jolla, CA, USA). One-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparisons test, was used for all biochemical and immunohistochemical analyses. Results were considered significant if the p value was less than 0.05.

Study approval

Animal handling and experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health) and according to protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Texas MedicalBranch

RESULTS

TOMA 1 and 3 rescue neuronal function and LTP impairment induced by tau oligomers ex-vivo in a mouse model-specific manner

Cognitive memory function is directly correlated with neuronal function in the hippocampus and is measured by short- and long-term potentiation, STP and LTP, respectively. Oligomers have been shown to interfere with hippocampal synaptic plasticity during the progression of AD and other neurodegenerative diseases. To assess the efficacy of TOMA1 and 3 in inhibiting tau oligomer-induced neuronal impairment at the functional level, we measured the effect of oligomers alone as well as oligomers preincubated with TOMA1 or TOMA3 on hippocampal basal neuronal transmission and LTP ex vivo. Brain-derived tau oligomers (BDTOs) from naïve (non-immunized) aged Htau or JNPL3 mice were preincubated and rotated for 2 h with the TOMAs at room temperature at a ratio of 1:4 oligomers to antibodies molar concentration. Then we treated hippocampal slices from 5-month-old Htau mice for 1 h with 50 nM final concentration of the following treatments: 1) BDTOs from naïve aged Htau or JNPL3 mice, 2) BDTOs from naïve aged Htau or JNPL3 mice preincubated with TOMA1, 3) BDTOs from naïve aged Htau mice or JNPL3 mice preincubated with TOMA3, 4) Control group slices treated with oligomer-free ACSF. Five-month-old Htau mice were used in this experiment because at this early age, the mice lack pathology, which enables us to isolate the effect of the tau oligomers from the effects of age on Htau mice and p301L mice.

Paired pulse fEPSP traces, a form of STP, were recorded from slices treated with BDTOs from naïve aged Htau mice alone or preincubated with either TOMA 1 or 3 (Fig. 1A). Statistical quantification of the paired pulse ratio of the slope of fEPSPs shows that oligomers from naïve aged Htau mice inhibited paired pulse facilitation in comparison to control treated slices, with a ratio around 1, i.e., no facilitation observed (Fig. 1B). Immunodepletion of oligomers with TOMA1 did not reverse the impairment, whereas immunodepletion with TOMA3 restored paired pulse facilitation (Fig. 1B). On the other hand, basal neuronal transmission showed no significant differences between the treatment groups (Fig. 1C).

Fig. 1

Ex vivo TOMA1 and TOMA3 rescue oligomer-induced neuronal dysfunction in a mouse model-specific manner. Hippocampal fresh brain sections from 5-month-old Htau mice were incubated for 1 h with tau oligomers IPed from either naïve aged Htau and JNPL3 mice alone (50 nM) or with tau oligomers preincubated with TOMA1 or TOMA3 (ratio 1:4, oligomers:antibody). Control sections were incubated in oligomer free-ACSF. A, D) Paired-pulse traces of fEPSPs after treatment with oligomers from Htau and JNPL3-derived tau oligomers treatments, respectively. B, E) Only TOMA3 rescued paired-pulse facilitation that was impaired by Htau BDTOs, whereas only TOMA1 rescued paired-pulse facilitation that was impaired by JNPL3 BDTOs. C, F) No significant changes in the slope of basal neuronal transmission between the groups in Htau and JNPL3 brain-derived oligomers treated sections. G, H) Both TOMA1 and 3 rescued LTP impairment induced by Htau brain-derived tau oligomers. I, J) Only TOMA1 steadily rescued and maintained LTP impairment induced by JNPL3 brain-derived tau oligomers. (One-Way ANOVA followed by Bonferroni post hoc multiple comparison test, ^***p < 0.001; ^****p < 0.0001) Data expressed as mean±SEM. N = 3 mice per treatment, n = 3 slices per treatment.

For JNPL3-derived oligomers, paired pulse fEPSP traces were recorded after treating brain slices with naïve aged JNPL3-derived tau oligomers alone or immunodepleted with either TOMA 1 or 3 (Fig. 1D). Our results show that naïve aged JNPL3-derived oligomers significantly impair paired pulse facilitation in comparison to the control group as shown in the statistical quantification of the paired pulse ratios (Fig. 1E). TOMA1, when preincubated with JNPL3 oligomers, reversed that impairment, whereas TOMA3 did not (Fig. 1E). Our data also show that aged JNPL3-derived oligomers suppressed basal neuronal transmission below control, whereas both TOMA1 and 3 increased basal neuronal transmission. This shows that both TOMA1 and 3 might be protective against oligomers from aged JNPL3 mice (Fig. 1F).

Similar to the observations in the paired pulse analysis of STP, Htau- and JNPL3-derived oligomers alone both impair LTP in comparison to control groups (Fig. 1G, 1I, respectively). While TOMA3 prevented LTP impairment induced by Htau BDTOs, TOMA1 prevented impairment induced by JNPL3 BDTOs (Fig. 1G, 1I, respectively). TOMA1 also prevented LTP impairment when preincubated with Htau BDTOs as well, but to a lesser extent than TOMA3. Analysis of the last 10 min of the LTP recording show that LTP was maintained when the Htau BDTOs were preincubated with TOMA1 and 3 (Fig. 1H). On the other hand, for JNPL3 BDTOs, LTP was only significantly maintained when these BDTOs were preincubated with TOMA1 antibodies (Fig. 1J). Results from electrophysiological analysis suggest that there could be strain differences between the oligomers derived from aged Htau and JNPL3 mice at the functional level supporting the need to carefully characterize oligomeric species in different mouse models and in humans for better immunotherapy outcomes.

Differential TOMA 1 and 3 effects on neuronal recovery might correspond to tau oligomeric strain differences in aged Htau and JNPL3 mice

The prion-like characteristic of tau protein is now very well established and accepted [33]. Several recent studies have shown that tau protein, across different aggregation states, may represent different conformational strains [34 –36]. Compelling evidence from in vivo and in vitro experiments suggests that distinct tau strains may be associated with different disorders [4 , 37]. Specifically, tau oligomers have been recently shown to form different disease relevant strains that correspond to different tauopathies [4 , 37]. Therefore, we sought to characterize tau oligomers in aged Htau and JNPL3 mice. Figure 2A shows a representation of the tau protein sequence that shows the corresponding non-continuous TOMA1 and 3 conformational epitopes. TOMA1 binds to three non-continuous sequences in the proline rich region and the beginning of the microtubule binding region (MTBR), whereas TOMA3 binds to two non-continuous sequences in the MTBR. To validate conformational differences between tau oligomers in aged Htau and JNPL3 mouse models, we immunoprecipitated tau oligomers from the aged mouse brain homogenates using TTC18 antibody (since aged mice showed significant differences in the filter trap assay). We then treated the tau oligomers with 0 and 0.5 μg/ml of Proteinase K (PK) enzyme to compare the oligomers core resistance to digestion. PK digestion is the most reliable method to detect prion strain differences and has been well established in the field of tau strains [4, 38]. PK digestion showed differential band pattern and intensity across the three mouse models (wild type, Htau, and JNPL3) supporting strain-indicating differences between oligomers derived from Htau, JNPL3, and wild type aged mouse models (Fig. 2B). Data from secondary alone controls as well as from commercial tau antibodies can be found in Supplementary Figure 3.

Fig. 2

Different tauopathy mouse models exhibit different tau oligomeric strains at old ages. Filter trap assay was performed on brain homogenates from naïve (non-immunized) aged Htau and JNPL3. A) Representative diagram of TOMA1 and TOMA3 non-continuous epitopes on tau sequence. TOMA1 binds to three sequences in proline-rich region and the beginning of the MTBR, whereas TOMA3 binds to two non-continuous sequences in the MTBR. B) Tau oligomers from naïve aged Htau, JNPL3 and wild type mice show different banding and intensity patterns after PK digestion suggesting different conformational strains. C) TOMA1 recognizes significantly higher levels of oligomers in the aged JNPL3 mice in comparison to aged Htau and wild type mice. As expected, no immunoreactivity was seen in the TauKO mice. Oligomeric levels detected by TOMA1 significantly increased with age in all mouse models. D) TOMA3-detected oligomeric levels were similar across the aged mouse models. Oligomeric levels detected by TOMA3 significantly increased with age in all mouse models. E) TTCM2 (toxic tau conformations monoclonal antibody) was similar to TOMA1 in terms of specificity towards oligomeric species in different mouse models. F) T22 tau oligomer polyclonal antibody detected oligomers in wild type mice more than in Htau and JNPL3 but none in TauKO mice. Tau oligomers from naïve aged Htau, JNPL3, and wild type brain homogenates were then immunoprecipitated using TTC18 which binds to toxic tau conformations. The input and the IPed samples were ran on a dot blot assay probing with TOMA1 and 3. G) TOMA1 was reactive to significantly higher levels of oligomers in Htau and JNPL3 mice in comparison to wild type mice. H) TOMA3 reactivity followed a higher trend of reactivity to tau oligomers from Htau mice in comparison to those from JNPL3 and wild type mice. Following dot blot, the same IPed samples were exposed to proteinase K digestion (0.5 μg/ml) and were run on a western blot probing with Tau5 (1:8000) looking for conformational strain differences. (One-Way ANOVA followed by Bonferroni post hoc multiple comparison test, ^****p < 0.0001, ^***p < 0.001, ^**p < 0.01; ^*p < 0.05) Data expressed as mean±SEM. N = 3 mice per treatment.

To further characterize the oligomers in terms of their reaction with TOMA1 and TOMA3 antibodies, we used filter trap assay. We compared TOMA1 and 3, TTCM2, and T22 reactivity in brain homogenates taken from naïve young (4–7 months) and aged (15–18 months) wild type C57BL/6, Htau, JNPL3, and TauKO mice. No significant differences were found in the four antibodies’ reactivity in the young groups across the mice strains. However, across the aged groups, TOMA1 detected higher levels of oligomers in JNPL3 mice in comparison to both Htau and wild type groups, however TOMA3 reactivity did not significantly differ across aged groups (Fig. 2C, D). TTCM2 showed a similar trend to that of TOMA1 (Fig. 2E). Finally, T22 detected higher tau oligomeric levels in wild type mice in comparison to Htau and JNPL3 mice (Fig. 2F). Additionally, we performed a dot blot on the immunoprecipitated oligomers (used for PK above) using TOMA1 and 3. Similar to our previous results, dot blots on immunoprecipitated tau oligomers showed higher TOMA1 reactivity in JNPL3 mice, whereas TOMA3 was highly reactive to oligomers from Htau mice (Fig. 2G, H). Results were expressed as integrated density of IP normalized to FT. These results suggest that Htau and JNPL3 mouse models develop different conformations of tau oligomers leading to different pathological phenotypes and distinct antibodyreactivity.

TOMA3 improves cognitive function in aged Htau mice whereas TOMA1 improves cognitive function in aged JNPL3 mice

To investigate if the ex vivo results above are reproduced in vivo, we immunized aged Htau and JNPL3 mice with TOMA1 or TOMA3, as displayed in the schematic in Fig. 3A, and ran behavioral paradigms to assess the efficacy of these antibodies in reversing memory deficits. We have previously evaluated the efficacy of TOMA1 in mitigating or preventing cognitive deficits in several young/middle aged AD mouse models such as Htau, JNPL3, and Tg2576. Tau oligomers showed conformational differences in the aged Htau and JNPL3 mice (Figs. 1 and 2). Therefore, the same aged human-tau mouse models (Htau and JNPL3) were utilized for behavioral experiments. Each animal was immunized intravenously (i.v.) with a single dose of 120 μg of TOMA1, 3, or non-specific IgG as a control. Cognitive performance was evaluated 4 days to one-week post-injection using the Y-maze and NOR tasks, respectively. Results in Fig. 3B show that intravenous TOMA3 treatment significantly improved the working memory in aged Htau mice whereas TOMA1 improved the working memory in aged JNPL3 mice. There were no significant differences in total arm entries in the Y-maze between the groups, indicating that all animals had the ability to complete the task similarly(Fig. 3C).

Fig. 3

Passive immunotherapy using TOMA3 and TOMA1 reverses memory deficit in aged Htau and JNPL3 mouse models respectively. A) Behavioral paradigm followed in this study: Aged Htau (18–23 months) and JNPL3 (14–16 months) mice were immunized intravenously with TOMA1, TOMA3, and IgG control, 120 μg/mouse, then assessed behaviorally for cognitive deficits. B) TOMA3 significantly increased % arm alternation of aged Htau mice whereas TOMA1 significantly increased that of aged JNPL3 mice in comparison to control IgG-immunized mice. TOMA1 did not increase % alternation in aged Htau, neither did TOMA3 in aged JNPL3 mice in comparison to controls. C) Total number of entries to the Y-maze arms did not significantly differ between the groups. D) NOR discrimination index increased in aged Htau mice only with TOMA3 immunization and in JNPL3 only with TOMA1 immunization in comparison to IgG. E) Number of entries to novel/familiar object zones did not significantly change with TOMA1 and 3 treatments in comparison to IgG treatment across both groups. Due to old age, we lost a few mice from some groups as the experiments proceeded. Also, some mice were removed from the experiment due to not qualifying due to an experimental error or for being an outlier. Remaining N used for analysis for each group is shown as the individual data points presented in the bar graph. (One-Way ANOVA followed by Bonferroni post hoc multiple comparison test, ^**p < 0.01; ^*p < 0.05) Data expressed as mean±SEM. N = 10 mice per treatment as a start.

Similarly, TOMA clone-dependent cognitive improvement was observed in the NOR task. Htau mice treated with TOMA3 exhibited significantly higher discrimination index in comparison to Htau mice treated with TOMA1 and IgG as indicated by their preference to explore the novel object over the familiar object. On the other hand, only TOMA1 improved discrimination index in JNPL3 mice in comparison to control IgG-treated group (Fig. 3D). As expected, mice receiving IgG from both mouse models showed no preference for the novel object over the familiar object due to their severe memory impairment at this old age range. The number of entries (novel versus familiar) did not reach significance with the antibody treatments in both mouse models (Fig. 3E).

Cognitive improvement by TOMA3 and TOMA1 single dose coincides with reduction of tau oligomers in the hippocampal regions of Htau and JNPL3 mice, respectively

Following behavioral testing, mice were sacrificed, and brains were collected and divided into two hemispheres for biochemical and immunohistochemical analyses to characterize tau pathology in the hippocampus.

Immunohistochemistry of Htau brains with T22 (1:250) revealed a trend of reduction in the CA1, CA3, and dentate gyrus (DG) regions in the TOMA3-treated groups compared to the IgG-and TOMA1-treated groups (Fig. 4A). On the other hand, a significant reduction in hippocampal T22 staining intensity in JNPL3 mice was observed in the TOMA1-treated groups compared to the TOMA3 and IgG-treated groups (Fig. 4B).

Fig. 4

Passive immunotherapy by TOMA3 and TOMA1 reduces tau oligomer levels in a mouse model-dependent manner. A) Immunohistochemistry with T22 of hippocampal frozen sections of aged Htau mice showed a trend of Tau oligomers reduction in CA1, CA3, and DG regions after TOMA3 immunization but not after TOMA1 immunization. B) Tau oligomers levels were significantly decreased by TOMA1 immunization in the CA1, CA3, and DG regions in aged JNPL3 mice. TOMA3 reduced tau oligomers level only in the DG of aged JNPL3 mice. Three ROIs from each mouse were quantified and averaged. N = 3 mice per treatment were imaged and quantified. (One-Way ANOVA followed by Bonferroni post hoc multiple comparison test, ^*p < 0.05; ^**p < 0.01; ^***p < 0.001) Data expressed as mean±SEM.

Keeping in mind that these mice are aged with advanced cognitive impairment and tau pathology, when staining for tau oligomers using T22 (a polyclonal tau oligomer-specific antibody), significant reduction by specific tau oligomers (either TOMA1 or TOMA3) might not be easily observed due to the possible existence of multiple and most likely different tau oligomeric species hence the trend in reduction in the Htau group, especially that they were older than JNPL3 mice when immunized.

To further investigate different changes in pathological tau (other than tau oligomers) following TOMA injections, immunohistochemistry, targeting late stage phospho-tau pathology, using AT180 phospho-tau monoclonal antibody, in the hippocampal regions of both Htau and JNPL3 strain groups, was performed. Figure 5A shows a clear trend of increase in Htau phospho-tau pathology following antibody treatment. On the other hand, in the JNPL3 mouse model, AT180-imunoreactivesignal was significantly increased after TOMA1 treatment in the whole hippocampus and reduced by TOMA3 treatment in the CA1 and DG as shown in Fig. 5B.

Fig. 5

Late phosphorylated tau pathology in aged Htau and JNPL3 mice. Frozen aged Htau and JNPL3 hippocampal sections were stained with AT180 antibody using immunohistochemistry. A) Number of AT180 positive cells did not significantly change in the aged Htau hippocampus after TOMA1 and TOMA3 treatments in comparison to the control treated group. However, a trend of increase of AT180 positive cells was observed in CA3, DG, and the whole hippocampus combined after TOMA1 and 3 immunization. B) Number of AT180 positive cells significantly increased in the whole hippocampus and CA3 regions of aged JNPL3 mice with TOMA1 treatment in comparison to control IgG treated group. (One-Way ANOVA followed by Bonferroni post hoc multiple comparison test, ^**p < 0.01; ^*p < 0.05) Data expressed as mean±SEM.

It is established that cells such as microglia are involved in hyperphosphorylated tau clearance but not in oligomeric tau clearance after immunization with tau monoclonal antibodies [16, 39]. This non-consistent trend of change in phospho-tau levels at Thr231 could correspond to neuronal and non-neuronal tau, such as tau engulfed by microglia. Further investigation is warranted to determine the exact reason behind the increase in Th231 phospho-tau after TOMA1 and 3 treatments in aged Htau and JNPL3 mouse models.

In summary, memory improvement coincides with changes in the presence tau oligomers after TOMA 1 and TOMA 3 immunotherapy. Biochemical analysis of tau species in treated animals and controls was performed using PBS-soluble fractions of brain homogenates. Western blot analysis of the brain homogenates of Htau mice showed a reduction in tau oligomers only in TOMA3-treated groups in comparison to IgG-treated groups, supporting data from the immunohistochemical staining. ELISA analysis of PBS-soluble fractions using T22 was not sensitive enough to detect differences between the TOMA and the IgG-treated groups in Htau brain homogenates (Supplementary Figure 1).

JNPL3 mice brain homogenates, on the other hand, showed a significant decrease in high molecular weight tau oligomers using T22 antibody in TOMA1-treated groups in comparison to both TOMA3 and IgG-treated groups. This decrease is also supported by immunohistochemical analysis. ELISA analysis was also not-sensitive enough to detect those differences between the groups of JNPL3 mice (Supplementary Figure 2, *p<0.05). Keeping in mind that these mice were aged, biochemical analysis using denaturing western blots and probing with polyclonal T22 and Tau13 antibodies might not be sensitive enough to show differences between the groups.

Significant decrease in colocalization between tau oligomers and AT180 phospho-tau pathology after immunotherapy might correspond to perinuclear and extracellular tau oligomer clearance by TOMA1 and 3

To acquire additional insight into the changes in tau pathology following immunotherapy, the colocalization between phospho-tau pathology (AT180) and tau oligomers in the hippocampus and the para-hippocampal gyrus (PHG) was measured. Figure 6A shows a significant decrease in the colocalization of AT180 tau pathology and tau oligomers in the TOMA1- and TOMA3-treated aged Htau mice in the CA1 and CA3 regions in comparison to IgG-treated mice. TOMA1-treated mice showed a similar decrease in the PHG. On the other hand, in Fig. 6B, only TOMA1-treated aged JNPL3 mice showed a decrease in colocalization between phospho-tau and tau oligomers in the three regions. The colocalization between phospho-tau and tau oligomers are shown to be mostly paranuclear or extracellular in the IgG treated mice. The decrease in colocalization after immunotherapy indicates tau oligomers clearance by the TOMAs.

Fig. 6

Co-immunofluorescence of phospho-tau and tau oligomers in TOMA1 and TOMA3 immunized aged Htau and JNPL3 mice reveals extracellular and perinuclear tau oligomer clearance. Co-immunofluorescence with T22 (red), and AT180 (magenta) in CA1, CA3, and para hippocampal gyrus (PHG) regions of immunized aged Htau (A) and JNPL3 mice (B). Results show that perinuclear and extracellular colocalization significantly decreases after immunization with TOMA1 in both Htau and JNPL3 mice; whereas TOMA3 reduces colocalization only in Htau mice. DAPI (grey) represents nuclei. Three ROIs were taken from each of the 3 sections per mouse. N = 3 mice per treatment. (One-Way ANOVA followed by Bonferroni post hoc multiple comparison test, ^**p < 0.01; ^*p < 0.05) Data expressed as mean±SEM.

DISCUSSION

Tau oligomers in neurodegenerative diseases have become an attractive target in the tauopathy field. Recent mouse models and clinical studies have explored many passive immunotherapy approaches to target tau, but these studies mostly use antibodies against phospho-tau. Tau hyperphosphorylation is believed to be one of the major causes of tau pathology [7 , 40–42]. However, it should be noted that tau proteins undergo other changes besides or due to hyperphosphorylation such as conformational aggregation and pathogenic oligomerization. These changes play a crucial role in antibody epitope determination and ultimately the response to immunotherapy [8, 43]. In addition, the changes in tau oligomer levels are more reflective of the changes in memory function than the changes in phospho-tau levels, as shown by the results.

More importantly, most studies are being performed on young to middle-aged mice, which are not representative of the AD human population; AD disease pathology changes significantly with age and disease onset starts late in non-familial AD cases [44]. Several clinical tau-targeted immunotherapy trials are ongoing. However, promising trends have emerged only from studies done on prodromal or MCI cases and not from studies done on older populations. Three challenges have limited the success of these clinical trials: 1) the difficulty of preventing cognitive impairment in older populations in comparison to younger populations; 2) antibody non-specificity to the main pathological tau species in the studied populations; and 3) the need for further tau pathology characterization (Active Tau Vaccine: Hints of Slowing Neurodegeneration; ALZFORUM 2020).

To address the first challenge, in this study we sought to evaluate tau-targeted immunotherapy in two aged human tau mouse models using two in-house monoclonal oligomer-specific tau antibodies; TOMA 1 and 3. TOMA1 was previously evaluated in young JNPL3 [16], as well as in Htau mice [14]. Those studies showed that TOMA1 was effective in reversing the behavioral deficits as well as the pathology in young JNPL3 and Htau animals.

In this study we followed a similar approach, we chose to use two cognitive tests to evaluate more than one aspect of memory deficit: Y-maze for working memory, and NOR for object discrimination. We also selected tests that are minimally invasive and less stressful due to old age of the mice. We showed that although therapeutic benefit would be expected to be less in older age, these expectations were consistent with our data from younger mice. TOMA1 significantly improved cognitive impairment and reduced oligomeric levels but not late phospho-tau pathology in aged JNPL3 mice. However, TOMA3 significantly reversed memory impairment in Htau mice with matching trends of reduced tau oligomeric pathology and no consistent change in late phospho-tau pathology. These results strongly indicate that the observed cognitive improvements correspond to the reduction of the targeted oligomeric species. These results also suggest that targeting tau oligomers at young and old age differs due to possible changes in antibody efficacy towards progressive tau pathology.

To address the second challenge, we characterized BDTOs from naïve aged Htau and JNPL3 to investigate TOMA clones’ specificity to different tau species in different mouse models. Our results indicated that tau BDTOs from Htau and JNPL3 mouse models correspond to different conformations/strains that might be pathologically relevant to the cognitive impairment observed at the aged stage, shown by PK digestion assay. These results are supported by ex vivo functional characterization of the efficacy of TOMA 1 and 3 to reverse STP and LTP which directly correlates with memory function in vivo. These results emphasize the need to characterize pathologically relevant tau species in different tauopathies as well as in young versus old human populations.

Moreover, it has been implicated that AD-related pathological tau is truncated at the N-terminal and/or at the C-terminal rendering the antibodies targeting tau terminals ineffective in immunotherapy clinical trials [7]. Herein, it becomes worthy to mention that the TOMA clones used in this study only bind to non-continuous epitopes in the central regions of the tau protein (proline rich region and the MTBR), which could account for their effectiveness even in the advanced age of the utilized mouse models. These results might also imply the importance of using a combination of tau antibodies as opposed to antibody mono-immunotherapy to improve tau-targeted immunotherapy clinical trial outcomes. Finally, this is the first study to assess the efficacy of passive immunotherapy targeting specific tau oligomers in aged tauopathy mouse models. Results from this study provide evidence for effective immunotherapy in older populations as well as validate the efficacy of TOMA1 and TOMA3 as immunotherapy tools in mice and potentially in humans.

One of the limitations of this study is that we have not investigated if the TOMAs injected intravenously into aged animals would pass through the blood-brain barrier (BBB). However, we would expect that a significant amount of the injected TOMAs would cross the BBB considering it may be compromised in aged animals [7 , 46]. We have previously shown that a fraction of the intravenously injected TOMA1 crosses the BBB and binds to tau oligomers in the brain and spinal cord by in vivo imaging of 8-month-old JNPL3 mice and using intravenous injection of biotinylated TOMA1 [16]. Moreover, the effect that reducing tau oligomers has on amyloid-β depositions requires further investigation; however, it can be speculated from previous studies that the reduction of tau oligomers also reduces amyloid-β load [15]. In addition, astrocytes can be considered the guarding cells of the BBB. Astrocyte mediated degeneration may impact neuronal loss and disease progression, suggesting further research is needed to characterize the effects of passive immunotherapy in astrocytes. While passive immunotherapy is now highly considered for the treatment of neurodegenerative diseases, a major potential side effect is hypersensitivity. This could potentially be addressed by administering the appropriate dosage.

We also previously analyzed the mechanism of TOMA1-induced tau oligomer clearance and showed that the TOMA1-mediated clearance of tau oligomers appears to occur peripherally while extracellular tau oligomers are cleared, preventing their spreading between neurons and reducing toxicity [15, 47]. In this study, this mechanism is shown be very plausible due to the significant reduction of extracellular and perinuclear colocalization between tau oligomers and phospho-tau. This mechanism has been suggested before in several studies showing a shift in the perinuclear and intracellular tau oligomeric load after tau-targeted immunotherapy [7 , 16]. Those studies suggest that the extracellular tau oligomer-antibody complex is engulfed by microglia for degradation [39], which may explain changes in tau levels in the microglia shown in that study. In addition to this mechanism, another possible mechanism of clearance involving microglia is direct microglia receptor-mediated clearance [7, 39]. Further investigation is needed to determine which of these mechanisms apply for older animals with progressed pathology and to investigate the corresponding mechanisms underlying the observed cognitive improvement. For instance, Bi et al. observed astrocyte activation after active immunization only in older mice (24 months). This astrocyte activation may be responsible for the reduced tau pathology in older mice. In addition, after the noted increase in phospho-Th231 tau levels in some of the mice, experiments are ongoing to examine the inflammatory response after TOMA1 and TOMA3 immunization. These studies will highlight the importance of studying older mice as the cellular responses might differ with aging [48].

In conclusion, results of this study emphasize the need to characterize pathologically relevant tau species in different tauopathies as well as in young versus old human populations. These results also suggest the importance of using a combination of tau antibodies as opposed to antibody monotherapy for better tau-targeted immunotherapy clinical trial outcomes. Finally, this is the first study to assess the efficacy of passive immunotherapy targeting specific tau oligomers in aged tauopathy mouse models. These results give hope for usage of immunotherapy in older populations as well as validate the efficacy of TOMA1 and 3 as immunotherapy tools in mice and hopefully in humans.

Footnotes

ACKNOWLEDGMENTS

We thank Dr. Limon, Dr. Taglialatela, and Dr. Guptarak for allowing space and time to generate electrophysiology data using their field recording systems.

Authors’ disclosures available online ().

The supplementary material is available in the electronic version of this article: .

References

Sin

, Nollen

(2015) Regulation of protein homeostasis in neurodegenerative diseases: The role of coding and non-coding genes. Cell Mol Life Sci 72, 4027–4047.

Hafycz

, Naidoo

(2019) Sleep, aging, and cellular health: Aged-related changes in sleep and protein homeostasis converge in neurodegenerative diseases. Front Aging Neurosci 11, 140.

Morris

, Maeda

, Vossel

, Mucke

(2011) The many faces of tau. Neuron 70, 410–426.

Lo Cascio

, Garcia

, Montalbano

, Puangmalai

, McAllen

, Pace

, Palumbo Piccionello

, Kayed

(2020) Modulating disease-relevant tau oligomeric strains by small molecules. J Biol Chem 295, 14807–14825.

Winklhofer

, Tatzelt

, Haass

(2008) The two faces of protein misfolding: Gain- and loss-of-function in neurodegenerative diseases. EMBO J 27, 336–349.

Barbier

, Zejneli

, Martinho

, Lasorsa

, Belle

, Smet-Nocca

, Tsvetkov

, Devred

, Landrieu

(2019) Role of tau as a microtubule-associated protein: Structural and functional asects.Front Aging Neurosci 11, 204.

Bittar

, Bhatt

, Kayed

(2020) Advances and considerations in AD tau-targeted immunotherapy. Neurobiol Dis 134, 104707.

Niewiadomska

, Niewiadomski

, Steczkowska

, Gasiorowska

(2021) Tau oligomers neurotoxicity. Life (Basel) 11, 28.

Cardenas-Aguayo Mdel

, Gomez-Virgilio

, DeRosa

, Meraz-Rios

(2014) The role of tau oligomers in the onset of Alzheimer’s disease neuropathology. ACS Chem Neurosci 5, 1178–1191.

10.

Mudher

, Colin

, Dujardin

, Medina

, Dewachter

, Alavi Naini

, Mandelkow

, Buee

, Goedert

, Brion

(2017) What is the evidence that tau pathology spreads through prion-like propagation? Acta Neuropathol Commun 5, 99.

11.

Brunello

, Merezhko

, Uronen

, Huttunen

(2020) Mechanisms of secretion and spreading of pathological tau protein. Cell Mol Life Sci 77, 1721–1744.

12.

Lasagna-Reeves

, Castillo-Carranza

, Sengupta

, Sarmiento

, Troncoso

, Jackson

, Kayed

(2012) Identification of oligomers at early stages of tau aggregation in Alzheimer’s disease. FASEB J 26, 1946–1959.

13.

Guzman-Martinez

, Farias

, Maccioni

(2013) Tau oligomers as potential targets for Alzheimer’s diagnosis and novel drugs. Front Neurol 4, 167.

14.

Castillo-Carranza

, Gerson

, Sengupta

, Guerrero-Munoz

, Lasagna-Reeves

, Kayed

(2014) Specific targeting of tau oligomers in Htau mice prevents cognitive impairment and tau toxicity following injection with brain-derived tau oligomeric seeds, J Alzheimers Dis 40 (Suppl 1), S97–S111.

15.

Castillo-Carranza

, Guerrero-Munoz

, Sengupta

, Hernandez

, Barrett

, Dineley

, Kayed

(2015) Tau immunotherapy modulates both pathological tau and upstream amyloid pathology in an Alzheimer’s disease mouse model. J Neurosci 35, 4857–4868.

16.

Castillo-Carranza

, Sengupta

, Guerrero-Munoz

, Lasagna-Reeves

, Gerson

, Singh

, Estes

, Barrett

, Dineley

, Jackson

, Kayed

(2014) Passive immunization with tau oligomer monoclonal antibody reverses tauopathy phenotypes without affecting hyperphosphorylated neurofibrillary tangles. J Neurosci 34, 4260–4272.

17.

Gerson

, Farmer

, Henson

, Castillo-Carranza

, Carretero Murillo

, Sengupta

, Barrett

, Kayed

(2018) Tau oligomers mediate alpha-synuclein toxicity and can be targeted by immunotherapy. Mol Neurodegener 13, 13.

18.

Colin

, Dujardin

, Schraen-Maschke

, Meno-Tetang

, Duyckaerts

, Courade

, Buee

(2020) From the prion-like propagation hypothesis to therapeutic strategies of anti-tau immunotherapy. Acta Neuropathol 139, 3–25.

19.

Sigurdsson

(2016) Tau immunotherapy. Neurodegener Dis 16, 34–38.

20.

Bittar

, Sengupta

, Kayed

(2018) Prospects for strain-specific immunotherapy in Alzheimer’s disease and tauopathies. NPJ Vaccines 3, 9.

21.

Novak

, Kontsekova

, Zilka

, Novak

(2018) Ten years of tau-targeted immunotherapy: The path walked and the roads ahead. Front Neurosci 12, 798.

22.

Sigurdsson

(2018) Tau immunotherapies for Alzheimer’s disease and related tauopathies: Progress and potential pitfalls, J Alzheimers Dis 64, S555–S565.

23.

Lasagna-Reeves

, Castillo-Carranza

, Sengupta

, Guerrero-Munoz

, Kiritoshi

, Neugebauer

, Jackson

, Kayed

(2012) Alzheimer brain-derived tau oligomers propagate pathology from endogenous tau. Sci Rep 2, 700.

24.

Lewis

, McGowan

, Rockwood

, Melrose

, Nacharaju

, Van Slegtenhorst

, Gwinn-Hardy

, Paul Murphy

, Baker

, Yu

, Duff

, Hardy

, Corral

, Lin

, Yen

, Dickson

, Davies

, Hutton

(2000) Neurofibrillary tangles, amyotrophy and progressive motor disturbance in mice expressing mutant (P301L) tau protein. Nat Genet 25, 402–405.

25.

Asuni

, Boutajangout

, Quartermain

, Sigurdsson

(2007) Immunotherapy targeting pathological tau conformers in a tangle mouse model reduces brain pathology with associated functional improvements. J Neurosci 27, 9115–9129.

26.

Vander Zanden

, Chi

(2020) Passive immunotherapies targeting amyloid beta and tau oligomers in Alzheimer’s disease. J Pharm Sci 109, 68–73.

27.

Morgan

, Munireddy

, Alamed

, DeLeon

, Diamond

, Bickford

, Hutton

, Lewis

, McGowan

, Gordon

(2008) Apparent behavioral benefits of tau overexpression in P301L tau transgenic mice. J Alzheimers Dis 15, 605–614.

28.

Holcomb

, Gordon

, Jantzen

, Hsiao

, Duff

, Morgan

(1999) Behavioral changes in transgenic mice expressing both amyloid precursor protein and presenilin-1 mutations: Lack of association with amyloid deposits. Behav Genet 29, 177–185.

29.

Lo Cascio

, Puangmalai

, Ellsworth

, Bucchieri

, Pace

, Palumbo Piccionello

, Kayed

(2019) Toxic tau oligomers modulated by novel curcumin derivatives. Sci Rep 9, 19011.

30.

Puangmalai

, Sengupta

, Bhatt

, Gaikwad

, Montalbano

, Bhuyan

, Garcia

, McAllen

, Sonawane

, Jerez

, Zhao

, Kayed

(2022) Lysine 63-linked ubiquitination of tau oligomers contributes to the pathogenesis of Alzheimer’s disease. J Biol Chem 298, 101766.

31.

Lasagna-Reeves

, Castillo-Carranza

, Sengupta

, Clos

, Jackson

, Kayed

(2011) Tau oligomers impair memory and induce synaptic and mitochondrial dysfunction in wild-type mice. Mol Neurodegener 6, 39.

32.

Bittar

, Bhatt

, Hasan

, Montalbano

, Puangmalai

, McAllen

, Ellsworth

, Carretero Murillo

, Taglialatela

, Lucke-Wold

, Logsdon

, Rosen

, Turner

, Kayed

(2019) Neurotoxic tauoligomers after single versus repetitive mild traumatic braininjury. Brain Commun 1, fcz004.

33.

Frost

, Diamond

(2010) Prion-like mechanisms in neurodegenerative diseases. Nat Rev Neurosci 11, 155–159.

34.

Holmes

, Diamond

(2014) Prion-like properties of tau protein: The imortance of extracellular tau as a therapeutic target. J Biol Chem 289, 19855–19861 .

35.

Karikari

, Nagel

, Grainger

, Clarke-Bland

, Crowe

, Hill

, Moffat

(2019) Distinct conformations, aggregation and cellular internalization of different tau strains. Front Cell Neurosci 13, 296.

36.

Sanders

, Kaufman

, DeVos

, Sharma

, Mirbaha

, Li

, Barker

, Foley

, Thorpe

, Serpell

, Miller

, Grinberg

, Seeley

, Diamond

(2014) Distinct tau prion strains propagate in cells and mice and define different tauopathies. Neuron 82, 1271–1288.

37.

Gerson

, Mudher

, Kayed

(2016) Potential mechanisms and implications for the formation of tau oligomeric strains. Crit Rev Biochem Mol Biol 51, 482–496.

38.

Falcon

, Cavallini

, Angers

, Glover

, Murray

, Barnham

, Jackson

, O’Neill

, Isaacs

, Hutton

, Szekeres

, Goedert

, Bose

(2015) Conformation determines the seeding potencies of native and recombinant tau aggregates. J Biol Chem 290, 1049–1065.

39.

Luo

, Liu

, Hu

, Hanna

, Caravaca

, Paul

(2015) Microglial internalization and degradation of pathological tau is enhanced by an anti-tau monoclonal antibody. Sci Rep 5, 11161.

40.

Gibbons

, Kim

, Wu

, Riddle

, Leight

, Changolkar

, Xu

, Meymand

, O’Reilly

, Zhang

, Brunden

, Trojanowski

, Lee

VMY

(2020) Conformation-selective tau monoclonal antibodies inhibit tau pathology in primary neurons and a mouse model of Alzheimer’s disease. Mol Neurodegener 15, 64.

41.

Boutajangout

, Ingadottir

, Davies

, Sigurdsson

(2011) Passive immunization targeting pathological phospho-tau protein in a mouse model reduces functional decline and clears tau aggregates from the brain. J Neurochem 118, 658–667.

42.

Plotkin

, Cashman

(2020) Passive immunotherapies targeting Abeta and tau in Alzheimer’s disease. Neurobiol Dis 144, 105010.

43.

Martinisi

, Flach

, Sprenger

, Frank

, Tolnay

, Winkler

(2021) Severe oligomeric tau toxicity can be reversed without long-term sequelae. Brain 144, 963–974.

44.

Neddens

, Temmel

, Flunkert

, Kerschbaumer

, Hoeller

, Loeffler

, Niederkofler

, Daum

, Attems

, Hutter-Paier

(2018) Phosphorylation of different tau sites during progression of Alzheimer’s disease. Acta Neuropathol Commun 6, 52.

45.

Kayed

(2010) Anti-tau oligomers passive vaccination for the treatment of Alzheimer disease. Hum Vaccin 6, 931–935.

46.

Corsetti

, Borreca

, Latina

, Giacovazzo

, Pignataro

, Krashia

, Natale

, Cocco

, Rinaudo

, Malerba

, Florio

, Ciarapica

, Coccurello

, D’Amelio

, Ammassari-Teule

, Grassi

, Calissano

, Amadoro

(2020) Passive immunotherapy for N-truncated tau ameliorates the cognitive deficits in two mouse Alzheimer’s disease models. Brain Commun 2, fcaa039.

47.

Congdon

, Gu

, Sait

, Sigurdsson

(2013) Antibody uptake into neurons occurs primarily via clathrin-dependent Fcgamma receptor endocytosis and is a prerequisite for acute tau protein clearance. J Biol Chem 288, 35452–35465.

48.

, Ittner

, Ke

, Gotz

, Ittner

(2011) Tau-targeted immunization impedes progression of neurofibrillary histopathology in aged P301L tau transgenic mice. PLoS One 6, e26860.