Antiepileptic Drugs Modulate Alzheimer-Related Tau Aggregation in a Neuronal Activity-Independent Manner

The number of older adults with dementia and epilepsy is rapidly growing due to aging population [1, 2]. Patients with dementia are at a several times higher risk of developing epilepsy than those with normal cognitive function [2, 3]. Recent findings show that epilepsy accelerates cognitive impairment in patients with dementia [2, 3]. Epilepsy in older patients is commonly nonconvulsive and often overlooked because the epilepsy-induced symptoms are similar to memory impairment due to dementia [2]. As such, comorbid epilepsy in patients with dementia is often underdiagnosed and not treated with appropriate antiepileptic agents in routine clinical practice.

Alzheimer's disease (AD) is associated with a particularly high risk for epilepsy; thus, the pathological relationship between the two conditions has attracted attention [3]. Cross-sectional studies have reported that approximately 50% of AD patients have comorbid epilepsy [4] and the incidence of epilepsy is significantly higher in AD patients than in patients with other causes of dementia [5]. AD itself is a significant predictor of developing epilepsy, and advanced age is related to a higher incidence rate of epilepsy in patients with AD [6]. Epilepsy reportedly accelerates the progression of cognitive impairment in AD patients [7]. Epileptic seizures start occurring around 3–4 years after the onset of AD [6], with temporal lobe epilepsy being the most common type [2, 3, 8]. Notably, a study using intracranial electrodes to record the electrical activity of the temporal lobe in AD patients has reported frequent silent hippocampal seizures, which were not clinically manifested as convulsive seizures [8]. Transgenic mice with AD-like pathologies have been shown to have increased susceptibility to epileptic seizures, suggesting an interplay between AD pathologies and epilepsy [9, 10].

The specific mechanism underlying the pathological association between AD and epilepsy remains largely unknown. Aggregates of β-amyloid and tau accumulate in the AD brain and are used as basis for AD pathological diagnosis and important targets for the development of disease-modifying therapy [11, 12]. Particularly, the amounts of intraneuronal tau aggregates known as neurofibrillary tangles correlate with cognitive impairment severity in AD patients, suggesting direct links between pathological tau with neuronal dysfunction [13‒17].

Recent basic studies have shown neuronal activity-dependent extracellular release of tau [18] and cellular uptake of tau followed by intracellular formation of new aggregates [17, 19, 20]. Based on these release/uptake pathways, the hypothesis of tau propagation has been proposed, which explains the progression of tau pathology in the brain as being mediated by cell-to-cell transmission of tau [15‒17, 20‒23]. In vivo experiments in an AD mouse model have also shown that neuronal activity can affect tau propagation, supporting the link between epileptic seizures and tau pathology development [24].

Notably, some antiepileptic agents reportedly have a protective effect on cognitive function [25]. However, the mechanism by which epileptic agents modify cognitive function in patients with dementia and, particularly, their effects on brain neuropathology remain to be elucidated. Understanding the effects of antiepileptic drugs on AD neuropathology is expected to be helpful for treatment optimization for older patients with dementia and comorbid epilepsy and the identification of a target for the development of a novel therapeutic approach to AD.

In this study, we used multiple in vitro assay systems to evaluate the effects of antiepileptic agents on AD tau pathology. First, a tau-biosensor cell [26]-based screening assay was used to evaluate the effects of seven antiepileptic agents on intracellular tau aggregation. A drug that impacts intracellular tau aggregation in a cell-based assay is assumed to exert its effect by: (i) altering the intracellular environment and subsequently affecting the tau aggregation rate or (ii) binding itself to tau and directly affecting its aggregation. Therefore, the antiepileptic agents found to inhibit (phenobarbital [PB]) and promote (sodium valproate [VAP]) intracellular tau aggregation in tau-biosensor cells were tested in a thioflavin T (ThT)-based, cell-free tau aggregation assay to verify their direct effects on tau aggregation.

The detailed descriptions of the materials and methods are provided in the online supplementary material.

The antiepileptic drugs used in this study were summarized in Table 1.

Following previously described methods [15, 22, 26], the intracellular tau aggregation assay was performed with some modifications (online suppl. Fig. 1; for all online suppl. material, see www.karger.com/doi/10.1159/000529915). A previous report used flow cytometry (fluorescence-activated cell sorting [FACS]) to assess FRET signals and the number of tau-positive cells [26]. In this study, we performed high-throughput screening using high content imaging (IN Cell Analyzer 6000, GE Healthcare, Tokyo, Japan) in the 384-well format; then, the candidate drug identified to affect tau aggregation was re-evaluated using FACS.

Brain extracts from the tau-transgenic (tau-tg) AD mouse model were used to induce tau aggregation in tau-biosensor cells, as previously described [15, 21]. The tau-biosensor cells were plated in poly-d-lysine-coated 384-well plates (PhenoPlate-384, #6057500, PerkinElmer, MA, USA) at a density of 8,000 cells/well (30 μL/well). After 16 h, a mixture of lipofectamine (Lipofectamine 2000, #11668-027, Invitrogen, Massachusetts) (1.0% final volume), optiMEM (Opti-MEM, #31985-070, Gibco, Massachusetts), brain extracts (total protein 0.5 μg/well), and each antiepileptic drug dissolved in PBS (#14249-95, Nacalai Tesque Inc., Kyoto, Japan) at designated concentrations was applied to each well (30 μL/well), then incubated overnight.

Tau-biosensor cells were incubated overnight with the tau seed-containing brain extract; then, they were fixed with 2% paraformaldehyde (#163-20145, FUJIFILM Wako Chemicals, Osaka, Japan), followed by nuclear staining with Hoechst (Hoechst 33342, #H3570, Invitrogen, Massachusetts). Subsequently, images were obtained using a high content automated laser-based confocal imaging system (IN Cell Analyzer 6000) from nine distinct regions in each well. Then, the images were analyzed using the IN Cell Investigator software (#28926256, GE Healthcare, Tokyo, Japan); subsequently, the number of intracellular tau-aggregate-positive cells/well was counted and used for quantification. Finally, the total number of cells was determined based on nuclear staining with Hoechst.

While the high content imaging method is suitable for high-throughput assessments because tau aggregates are rapidly counted by image analysis, the FACS method is superior in terms of sensitivity and specificity of detection. Tau-biosensor cells were plated at a density of 30,000 cells per well (100 μL/well) in a 96-well plate (CELLSTAR 96 Well Cell Culture Plate, #655180, Greiner Bio-One, Tokyo, Japan). Sixteen hours later, the cells were treated with the tau seed-containing brain extract at a volume of 50 μL/well (total protein: 1.65 μg/well in optiMEM) and mixed with 49.0 μL optiMEM and 1.0 μL lipofectamine, followed by overnight incubation. Then, BD FACSCanto™ II (#338962, Becton, Dickinson and Company, New Jersey) was used to perform FRET flow cytometry (excited with a 450-nm laser and fluorescence captured with a 530-nm filter). We analyzed 10,000 cells/well. The percentage of FRET-positive cells (tau aggregate-positive cells) was determined using the number of these FRET-positive cells divided by the total number of cells. Data were analyzed using the BD FACSDiva™ software.

The ThT assay was used for cell-free tau aggregation, as previously described [27], with some modifications (online suppl. Fig. 2). Aggregation-prone recombinant tau (Active Human Recombinant Tau Protein monomer [K18], #SPR-328C, Funakoshi, Tokyo, Japan) was dissolved in PBS to a final concentration of 20.0 μm; then, ThT (#202-01002, FUJIFILM, Tokyo, Japan), heparin (#H3393-50KU, Sigma-Aldrich, Tokyo, Japan), recombinant tau, and each epileptic drug were mixed in a 0.5-mL tube (Microresico tube, #92016, Richell, Toyama, Japan). The mixture was applied to a 384-well plate at designated antiepileptic drug concentrations with 50 μM ThT, 10 uM heparin, and 100 μM tau in each well. We used the tau aggregation inhibitor KT-430 (#119-01141, FUJIFILM, Tokyo, Japan) as a positive control for halting tau aggregation. After the mixture had been incubated at 37°C, luminescence was measured using a fluorescence microplate reader (SH-9000Lab, Corona Electric Co., Ltd., Ibaraki, Japan) at 0, 4, 18, 24, and 48 h after initiating incubation.

Tau-tg PS19 mice were purchased from Charles River Laboratories, Japan, Inc. (Kanagawa, Japan). Tau-tg PS19 mice overexpress the P301S-mutant form of the human microtubule-associated protein tau and develop AD-like tau pathology in the brain [28]. 12-month-old female tau-tg and non-tg (wild-type) littermates were used (see online suppl. material).

All data were expressed as mean ± standard error of the mean, and statistical analysis was performed using Statcel 4 (OMS Publishing Inc., Tokorozawa, Japan). Comparison of mean values among three or more groups was performed using one-way analysis of variance, followed by the Dunnett or Tukey-Kramer test. p values of < 0.05 were considered significant.

Seven antiepileptic agents (VAP, levetiracetam, PB, gabapentin [GBP], piracetam [PIR], ethosuximide, and acetazolamide) were screened for their effects on intracellular tau aggregation using a tau-biosensor cell assay (Fig. 1a, b). The previously reported method of using tau-biosensor cells with FACS measurement was improved to allow for high-throughput screening assays using the high content imaging technology, which allowed evaluation of multiple antiepileptic agents at multiple concentrations. With the improved method, cells containing tau aggregates could be rapidly counted by image processing in a 384-well format (Fig. 1b).

Various antiepileptic agents and tau seeds were added to the medium in which tau-biosensor cells were cultured, and tau aggregate-positive cells were counted using a high content imaging technology 24 h later to evaluate the effects of the antiepileptic agents on intracellular tau aggregation (Fig. 1a, b). The tau seed used in this experiment was pathological tau (high-molecular-weight phosphorylated tau [15, 21, 22]) derived from brain tissue extracts of AD mice (12-month-old tau-tg PS19). Various antiepileptic agents were tested in 100–1,000-fold dilutions of the maximum concentrations determined for the individual antiepileptic agents based on medium solubility. The cells treated with tau seeds and vehicle (PBS) were only used as the positive control group for inducing intracellular tau aggregates, and the cells treated with tau from wild-type mouse brain tissue extract were used as the negative control group.

The effects of the individual antiepileptic drugs on intracellular tau aggregation differed greatly and were dependent on drug concentrations (Fig. 1a). In the phenobarbital group, tau aggregation was inhibited in a concentration-dependent manner, and statistically significant decreases in the number of tau aggregates were observed at 3–10 mm (Fig. 1a, p < 0.05). Meanwhile, the number of tau aggregates increased in a concentration-dependent manner in the sodium valproate, gabapentin, and piracetam groups, and the increases observed at 1–10 (VAP), 5–15 (GBP), and 100 mm (PIR) were significant (Fig. 1a, p < 0.05). The number of tau aggregates did not change significantly in the levetiracetam, ethosuximide, and acetazolamide groups (Fig. 1a). Figure 1b shows representative images of the intracellular tau aggregates used in each treatment group at different concentrations. A lactate dehydrogenase-based cytotoxicity assay confirmed that the antiepileptic drugs did not have a significant cytotoxic effect even at their highest concentrations (online suppl. Fig. 3).

Next, we used the FACS method to confirm the inhibitory effect of PB on intracellular tau aggregation observed in the tau-biosensor cell assay (Fig. 2). The FACS-based measurement confirmed significant decreases in the number of tau aggregate-positive cells in a concentration-dependent manner in the phenobarbital group (3–10 mm) (Fig. 2). We also evaluated the effects of three drugs that increase tau aggregates: VAP, GBP, and PIR (online suppl. Fig. 4). These drugs also increased the number of tau aggregate-positive cells in a concentration-dependent manner in FACS-based assays.

As the tau-biosensor cell-based assays showed that PB inhibited intracellular tau aggregation, we then used a cell-free assay to evaluate the direct effects of PB on tau aggregation. The inhibitory effect of PB on tau aggregation was measured quantitatively using the ThT-based tau aggregation assay [29, 30] (Fig. 3). High aggregation-prone recombinant tau (Tau K18) [31] was used as seed tau in this experiment. Tau K18 was incubated with the vehicle (PBS) or PB (10, 100 mm), and the fluorescence intensity was measured over time (0–48 h) (Fig. 3a). The known tau aggregation inhibitor KT-430 (100 μm) was used as a positive control for halting tau aggregation. In the vehicle (PBS) group, the measurement showed time-dependent increases in the ThT fluorescence intensity, representing a progression of tau aggregation (Fig. 3a). The KT-430 group showed near complete inhibition of tau aggregation (Fig. 3b, p < 0.01). In the phenobarbital group, tau aggregation was inhibited in a concentration-dependent manner, and the ThT fluorescence intensity after 24 h was significantly lower than that in the vehicle group (Fig. 3b, p < 0.01). Particularly, the aggregation-inhibiting effect of PB at a high concentration (100 mm) was comparable to that of the tau aggregation inhibitor KT-430. These results confirmed that PB also inhibited tau aggregation in the cell-free experimental system, suggesting the possibility that PB acts directly on tau. We also examined the effect of VAP on tau aggregation using a ThT assay, as it increased intracellular tau aggregation in the tau-biosensor cell assay at a relatively low concentration (1 mm). VAP was not found to have a significant effect on tau aggregation in the cell-free ThT assay, even at high concentrations (online suppl. Fig. 5).

We evaluated the effects of multiple antiepileptic drugs using tau aggregation assay systems to determine the effects of antiepileptic agents on tau aggregation. Our results demonstrated the possibility that individual antiepileptic agents have different effects on tau aggregation. Among the seven antiepileptic agents evaluated in this study, PB inhibited tau aggregation in multiple in vitro assay systems.

The tau-biosensor cell-based intracellular tau aggregation assay used in this study was based on non-neuronal HEK293T cells [26], which shows no neuron-like electrical activity. Using the non-neuronal cell-based assay in this study allows the evaluation of neuronal activity-independent factors in terms of the effects of various antiepileptic agents on intracellular tau aggregation. Excessive electrical activity of neurons during epileptic seizures may increase tau aggregates in the brain, and overexcitement reduction with antiepileptic agents may decelerate tau pathology progression [18, 24, 32]. We showed that PB inhibited intracellular tau aggregation even in the assay system based on tau-biosensor cells devoid of neuron-like electrical activity, indicating that PB inhibits tau aggregation in a neuronal activity-independent manner. In fact, PB also inhibited tau aggregation significantly in the ThT-based, cell-free tau aggregation assay (Fig. 3). This implies that PB has a certain biochemical property that interacts with tau and directly interferes with its aggregation, although there is no apparent similarity between the structures of KT-430 (a tau-aggregation inhibitor) and PB. The detailed mechanism behind PB's inhibition of tau aggregation should be explored in future studies.

In this study, seven antiepileptic agents commercially available as laboratory reagents were evaluated in terms of tau aggregation effects. The drugs were selected from main classes based on mode of action, including gamma-aminobutyric acid enhancers, carbonic anhydrase inhibitors, sodium channel blockers, calcium channel blockers, presynaptic machinery modulators, acetylcholine transmission modulators, and carbonic anhydrase inhibitors (Table 1). Although many antiepileptic agents are used clinically, only seven antiepileptic agents were selected for evaluation based on solubility in the vehicle (PBS) in vitro assays. Tau aggregation rate is known to be affected by artificial cofactors and the biochemical properties of the vehicle [33]. Therefore, to avoid the potential effects of additive reagents, such as ethanol or dimethyl sulfoxide, on tau aggregation, we focused on antiepileptic drugs that are highly solubilized in PBS. The drug concentrations used in this study were determined based on the maximum concentration that remained soluble in the PBS.

PB inhibited tau aggregation, whereas VAP, GBP, and PIR promoted tau aggregation in both high content image- and FACS-based analyses using tau-biosensor cells (Fig. 1; online suppl. Fig. 4). VAP increased tau aggregation in tau-biosensor cells at a relatively low concentration (1 mm); however, it was not found to have a significant effect in the cell-free ThT assay (online suppl. Fig. 5). This inconsistency suggests that VAP impacts tau aggregation by altering the intracellular environment rather than the tau aggregation process. Moving forward, individual antiepileptic agents should be evaluated in cell-free tau aggregation and in vivo efficacy assays to analyze effect differences in detail. To the best of our knowledge, no study has reported on antiepileptic medication history and intracerebral tau pathology assessments (e.g., histopathological analysis in autopsied brains, tau PET imaging, and measurement of in CSF). Clinical analysis using neuropathological biomarkers is a future research topic.

The concentrations of the antiepileptic drugs that affected tau aggregation in this study were higher than their therapeutic reference ranges. PB inhibited tau aggregation in the cell-based assay at a concentration of 3–10 mm (Fig. 1), which was above the toxic plasma level of the drug (0.017 mm) [34]. VAP increased tau aggregation in the cell-based assay at a concentration of 1 mm (Fig. 1), which was near the therapeutic reference level (0.3–0.6 mm) [35]. Note that these drugs did not exhibit a cytotoxic effect in the cell-based experiments, even when at the highest concentrations (online suppl. Fig. 3). In vivo efficacy and toxicity in relation to the impact on tau aggregation should be examined in future studies.

Tau aggregates in the AD brain comprise a mixture of three- and four-repeat tau isoforms [36]. The tau seed-containing brain extract used in the cell-based tau aggregation assay was derived from a tau-tg mouse model (PS19) that overexpresses four-repeat tau with disease-associated P301S mutation, producing only four-repeat tau aggregates. This animal model also lacks another cardinal feature of the AD brain – beta-amyloid plaques – and this absence may affect the development of tau aggregates [37]. Therefore, the findings of this study are not entirely translatable to the pathogenesis of AD, which is a significant limitation. Future experiments using human patient-derived brain extract are certain to provide more relevant findings that can be more readily translated to clinical practice.

In conclusion, some antiepileptic agents including PB have the potential to modify the development of tau pathology in a neuronal activity-independent manner. Antiepileptic drugs may alter the trajectory of tau aggregation in patients with concomitant AD and epilepsy. This finding is likely to provide an important insight into the optimization of antiepileptic drug therapy in elderly patients with dementia and comorbid epilepsy.

This study was supported by Center for Medical Research and Education, Graduate School of Medicine, Osaka University.

The animal experiments were carried out in accordance with the Guidelines for the Care and Use of Laboratory Animals at the Osaka University School of Medicine and the National Institute of Health Guidelines for the Care and Use of Laboratory Animals and approved by the Animal Experiments Committee of Osaka University, Osaka, Japan (the decision reference number 29-046-015). This study protocol was reviewed and approved by the Osaka University Living Modified Organisms (LMO) Research Safety Committee, approval number 04232.

The authors declare no competing interest.

This work was supported by JSPS KAKENHI Grant No. 17H05080 (grant-in-aid for young scientists [A]) and 21H02828 (grant-in-aid for scientific research [B]) (S.T.) and research grants from Cell Science Research Foundation (S.T.) and the Japan Epilepsy Research Foundation (S.T.).

Yuki Ito, Shuko Takeda, Sayaka Moroi, Tsuneo Nakajima, Akane Oyama, Kunihiro Miki, Nanami Sugihara, Yoichi Takami, Yasushi Takeya, Munehisa Shimamura, Hiromi Rakugi, and Ryuichi Morishita designed the research; Yuki Ito, Shuko Takeda, Sayaka Moroi, Tsuneo Nakajima, Akane Oyama, Kunihiro Miki, and Nanami Sugihara performed the research; Yuki Ito, Shuko Takeda, Sayaka Moroi, Yoichi Takami, Yasushi Takeya, Munehisa Shimamura, Hiromi Rakugi, and Ryuichi Morishita analyzed the data; and Yuki Ito, Shuko Takeda, Sayaka Moroi, and Ryuichi Morishita wrote the paper.

All data generated or analyzed during this study are included in this article and its supplementary material files. Further inquiries can be directed to the corresponding author.