Pharmacological inhibition of STriatal-Enriched protein tyrosine Phosphatase by TC-2153 reduces hippocampal excitability and seizure propensity

SUMMARY

Objective:

STriatal-Enriched protein tyrosine Phosphatase (STEP) is a brain-specific tyrosine phosphatase. Membrane-bound STEP₆₁ is the only isoform expressed in hippocampus and cortex. Genetic deletion of STEP enhances excitatory synaptic currents and long-term potentiation in the hippocampus. However, whether STEP₆₁ affects seizure susceptibility is unclear. Here we investigated the effects of STEP inhibitor TC-2153 on seizure propensity in a murine model displaying kainic acid (KA)-induced status epilepticus and its effect on hippocampal excitability.

Methods:

Adult male and female C57BL/6J mice received intraperitoneal injection of either vehicle (2.8% DMSO in saline) or TC-2153 (10 mg/kg) and then either saline or KA (30 mg/kg) 3 hours later before being monitored for behavioral seizures. A subset of female mice was ovariectomized (OVX). Acute hippocampal slices from GCaMP6s mice were treated with either DMSO or TC-2153 (10 μM) for 1 hour, and then incubated in ACSF and potassium chloride (15 mM) for 2 min prior to live calcium imaging. Pyramidal neurons in dissociated rat hippocampal culture (DIV 8–10) were pre-treated with DMSO or TC-2153 (10 uM) for 1 hour before whole-cell patch clamp recording.

Results:

TC-2153 treatment significantly reduced KA-induced seizure severity, with greater trend seen in females. OVX abolished this TC-2153-induced decrease in seizure severity in females. TC-2153 application significantly decreased overall excitability of acute hippocampal slices from both sexes. Surprisingly, TC-2153 treatment hyperpolarized resting membrane potential and decreased firing rate, sag voltage, and hyperpolarization-induced current (I_h) of cultured hippocampal pyramidal neurons.

Significance:

This study is the first to demonstrate that pharmacological inhibition of STEP with TC-2153 decreases seizure severity and hippocampal activity in both sexes, and dampens hippocampal neuronal excitability and I_h. We propose that anti-seizure effects of TC-2153 are mediated by its unexpected action on suppressing neuronal intrinsic excitability.

INTRODUCTION

Temporal Lobe Epilepsy (TLE) is the most common form of focal-onset epilepsy in adults and accounts for 60% of epileptic patients¹. In Mesial TLE, seizures often begin in the hippocampus and progressively worsen over time. Current anti-seizure drugs are ineffective for ~75% of the patients with advanced mesial TLE, leading to severe consequences including hippocampal sclerosis, high mortality rate, cognitive decline, depression, and temporal lobe resection¹. Furthermore, dysregulation of intrinsic excitability and synaptic transmission has been widely thought to underlie hippocampal hyperactivity that drives the development of spontaneous seizures in TLE^2,3, underscoring a critical need to identify the underlying mechanisms and novel therapeutic targets.

STriatal Enriched protein tyrosine Phosphatase (STEP) is a brain-specific tyrosine (Tyr) phosphatase encoded by the PTPN5 gene⁴. Among four STEP isoforms, cytosolic STEP₄₆ and membrane-bound STEP₆₁ are catalytically active and widely expressed in the brain except the cerebellum⁴. However, only STEP₆₁ is expressed in the hippocampus and neocortex⁵ where it dephosphorylates N-Methyl-D-aspartic acid receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), the key glutamate receptors that mediate fast excitatory synaptic transmission. Specifically, STEP₆₁ dephosphorylates NMDAR subunit GluN2B at Tyr¹⁴⁷² and AMPAR subunit GluA2 at Tyr⁸⁶⁹, Tyr⁸⁷³, and Tyr⁸⁷⁶, resulting in their internalization^6–10. STEP₆₁ also dephosphorylates and inactivates protein kinases including extracellular signal-regulated kinase ½ (ERK1/2), p38, Fyn, and Pyk2⁴. Reduction of STEP increases NMDAR and AMPAR surface expression and excitatory synaptic currents^11,12, enhances long term potentiation¹³, and prevents the internalization of GluA2-containing AMPARs during metabotropic glutamate receptor-dependent long term depression in the hippocampus⁹. Activity-dependent regulation of STEP₆₁ also contributes to homeostatic stabilization of excitatory synapses by regulating Tyr phosphorylation of GluN2B and GluA2¹⁰. Thus, STEP₆₁ weakens excitatory synaptic strength.

Emerging evidence suggests that STEP may be a molecular target for seizure treatment. Deletion of STEP gene PTPN5 results in resistance to pilocarpine-induced seizures¹⁴ and diminishes audiogenic seizures in a fragile X syndrome (FXS) mouse model¹⁵. This reduction in seizure propensity is puzzling since loss of STEP would be expected to increase seizure susceptibility by potentiating excitatory synaptic strength. It is possible that genetic deletion of STEP may have compensatory effects on other related genes and/or pathways, complicating delineation of the role of STEP in seizure susceptibility. Therefore, here we tested the hypothesis that acute pharmacological inhibition of STEP increases seizure propensity.

TC-2153 (benzopentathiepin 8-(trifluoro-methyl)-1,2,3,4,5-benzopentathiepin-6-amine-hydrochloride) is a selective STEP inhibitor, which forms irreversible covalent bonds with the cysteine residues near a signature catalytic domain in STEP₄₆ and STEP₆₁¹⁶. Despite the low IC50 (24.6 nM), a higher concentration of TC-2153 is required to increase Tyr phosphorylation of STEP₆₁ substrates in primary cortical neuronal culture (1–10 μM) and the cortex in vivo (10 mg/kg) and reverse cognitive deficits in a 3xTg mouse model of Alzheimer’s Disease (AD)¹⁶ which display hippocampal hyperactivity and spontaneous seizures^17,18. With a low level of acute toxicity (LD 50 > 1000 mg/kg)¹⁹, TC-2153 can alleviate audiogenic seizures in FXS mouse model¹⁵ and block pentylenetetrazole-induced convulsions¹⁹ although the sex-dependence of anti-seizure effects of TC-2153 and the underlying mechanism were not described.

In this study, we discovered that TC-2153 dampens hippocampal activity and exerts anti-convulsant activity in both C57BL/6J male and female mice against a single systemic injection of kainic acid (KA), which induces status epilepticus (SE) arising from the hippocampus²⁰. Furthermore, TC-2153 decreases action potential (AP) firing rate, sag voltage, and hyperpolarization-induced current (I_h) in hippocampal neurons, providing novel evidence that pharmacological inhibition of STEP by TC-2153 downregulates intrinsic neuronal excitability in contrast to its well-known role in synaptic transmission.

MATERIALS AND METHODS

Kainic acid-induced seizures

All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Illinois at Urbana Champaign and conformed to the ARRIVE Guidelines. Both male and female Ptpn5 homozygous knock-out mice (Ptpn5^−/−)⁸, wild-type mice (Ptpn5 ^+/+), C57BL/6J mice (Jax.org, Stock Number: 000664) were used for seizure studies at 6–12 weeks old. C57BL/6J females received ovariectomy surgeries at 9–10 weeks old as described²¹ and were used in seizure studies at 7–10 days after surgery. Behavioral seizures were induced in mice by a single intraperitoneal (i.p.) injection of saline or KA, (30 mg/kg, Abcam)²⁰ and monitored using modified Racine scale for 2 hours (h)²². To test the effects of TC-2153, C57BL/6J mice received KA at 3 h post i.p. injections with either vehicle control (saline containing 2.8% DMSO) or TC-2153 (10 mg/kg in saline containing 2.8% DMSO, Sigma Aldrich). This treatment was previously demonstrated to increase Tyr-phosphorylation of STEP substrates in the cortex¹⁶.

Immunoblot Analysis

At 3 h post injection with either vehicle control or TC-2153, the mouse hippocampi were biochemically fractionated to the supernatant and the membrane fractions (0.5 mg/mL) as described²³. Primary dissociated hippocampal cultures were prepared from Sprague-Dawley rat embryos at embryonic day 18 and plated at 330 cells/mm² as described¹⁰. To maximally inhibit STEP, neurons at 9–10 days in vitro (DIV) were treated for 1 h with either vehicle control (0.14% DMSO) or TC-2153 (10 μM)¹⁶. Although the pharmacokinetics of TC-2153 is unknown, Tyr-phosphorylation of STEP₆₁ substrates were previously reported to increase in both neuron culture upon 10 μM TC-2153 application and forebrain tissues upon its i.p. injection at 10 mg/kg¹⁶, suggesting that a 10 μM concentration used in in vitro studies is equivalent or close to its brain concentration achieved by in vivo delivery. Samples were immunoblotted for STEP₆₁ and its substrates. Densitometric quantification was performed with ImageJ Software (National Institutes of Health)^10,23.

GCaMP6s imaging in acute hippocampal slices

Acute coronal hippocampal slices (200 μm) were prepared from Thy1-GCaMP6s mice (Jax.org, Stock Number: 024275) at 4–11 weeks old. Slices were incubated for 1 h at room temperature in basal artificial cerebrospinal fluid (ACSF) with either DMSO (0.14%) or TC-2153 (10 μM) which was previously shown to enhance hippocampal LTP²⁴ similar to STEP deletion¹³. Time lapse fluorescence images of GCaMP6s (size: 640 × 404 pixel) were acquired in ACSF for 1 minute (min) at 3.6 frame per second (sec) with 25 msec exposure time, and 3×3 binning under a Zeiss Axio Observer microscope. Slices were then incubated with potassium chloride (KCl, 15 mM) in ACSF for 2 min and imaged for another 1 min. Images from 10–60 sec after KCl exposure were analyzed for mean fluorescence intensity (F) in dentate gyrus (DG), CA1, and CA3 using ImageJ. ΔF/F= (F-F_min)/F_min was computed as described²⁵, where ΔF indicates the difference between the initial intensity in ACSF and the intensity after KCl stimulation. ΔF/F was normalized to ACSF.

Electrophysiology

At 1 h post treatment with DMSO (0.14%) or TC-2153 (10 μM), whole-cell patch-clamp recordings of evoked AP firing, sag voltage, and rebound potential were performed at 30–32°C from cultured hippocampal pyramidal neurons held at −60 mV in external solution containing CNQX (20 μM), DL-AP5 (100 μM) and bicuculline (20 μM) under current clamp mode using a Multiclamp 700B amplifier, Digidata1440A, and pClamp 10.6 software (Molecular Devices)²⁶. Voltage clamp recording of I_h was performed with CNQX (20 μM), DL-AP5 (50 μM), bicuculline (10 μM), and TTX (0.5 μM) as described²⁷. Clampfit 10.7 software (Molecular Devices) were used for recording analyses²⁷.

Statistical analysis

Data are reported as mean ± STDEV. Statistical analyses were performed using Origin Pro 9.5 (Origin Lab) to compare differences between means in 2 groups using the Student’s two-tailed t test, and in groups of ≥3 using post-hoc Tukey test. For non-parametric data, the Mann-Whitney U-test was used. Sex difference was analyzed by two-way ANOVA with sex as one factor and treatment as the other. The priori value (p) < 0.05 was considered statistically significant.

Detailed description of each method is provided in Supplementary Information.

RESULTS

Homozygous loss of STEP affects sensitivity to KA-induced seizure severity in age- and sex-dependent manner.

Since STEP₆₁ weakens excitatory synaptic strength^7–10,13, we hypothesized that genetic deletion of PTPN5 would increase susceptibility to acute seizures. To test this hypothesis, homozygous PTPN5^−/− mice (STEP KO) and their wild-type PTPN5^+/+ littermates (STEP WT) were treated with KA (30 mg/kg, i.p.)²⁰, and behavioral seizures were scored every 10 min for the first 2 h using a modified Racine scale (Fig. 1)²². Initially, we tested 6 to 7 week old adolescent STEP WT and KO mice (P42–55) in reference to the age range that was originally investigated by Briggs et al., which combined both sexes for analysis and showed that STEP KO mice are resistant to pilocarpine-induced seizures¹⁴. Consistent with Briggs et al., STEP KO males at this age displayed a decrease in KA-induced seizure severity at nearly every time interval (Fig. 1B), resulting in a lower cumulative seizure score compared to WT males (Fig. 1C). A similar trend was observed for STEP KO females, but did not reach statistical significance (Fig. 1B–C, Table S2). The percentage (%) of mice that reached Stage 5 (SE) and Stage 6 (death) were also decreased in KO males but not females compared to WT mice (Fig. 1D–E).

Figure 1. Homozygous loss of STEP affects sensitivity to KA-induced seizures in age- and sex-dependent manner.

(A) Experimental schematic of kainic acid (KA)-induced seizures in STEP knockout (KO) and wild-type (WT) mice. A modified Racine scale was used to score behavioral seizures. (B-D) KA-induced seizures in STEP WT and KO mice at age 6 to 7 weeks (P42-P55). (B) STEP KO males at age 6–7 weeks (n=12) show a significant decrease in seizure scores at 20–110 min after KA injection compared to WT males (n=21). STEP KO females at age 6–7 weeks (n=12) show a significant decrease in seizure scores at 30 min post KA injection compared to WT females (n=12). Mann-Whitney U-test results are shown (*p<0.05). (C) Cumulative seizure scores. Two-tailed Student t-test results are shown (*p<0.05). (D-E) Percentage (%) of mice that achieved Stage 5 (D) and Stage 6 (E). (F-I) KA induced seizures in STEP WT and KO mice at age 8 to 12 weeks (P56-P90). (F) STEP KO males at age 8–12 weeks (n=12) show a significant increase in seizure scores at 110 and 120 min compared to WT males (n=13). STEP KO females at age 8–12 weeks (n=16) show a decreased trend in seizure propensity compared to WT females (n=13), but this trend was not statistically significant. Mann-Whitney U-test results are shown (*p<0.05). (G) Cumulative seizure scores. Two-tailed Student t-test results are shown (*p<0.05). (H-I) Percentage (%) of mice that achieved Stage 5 (D) and Stage 6 (E). Data shown represent the mean ± STDEV (*p<0.05).

We next tested adult mice at 8 to 12 weeks of age (P56-P90) to avoid the adolescence period when puberty onset and maturation occurs and corticolimbic circuits are still developing^28,29. In contrast to 6 to 7 weeks old STEP KO males (Fig. 1B–D), 8 to12 weeks old STEP KO males showed similar seizure severity to WT males for the first 100 min following KA injection. However, their seizure scores at 110 and 120 min post-KA injection, % SE, and % Death were higher than WT males (Fig. 1F–I), consistent with our original hypothesis that STEP KO mice would show increased seizure susceptibility. In contrast, STEP KO females displayed a decreasing trend in seizure severity, the % SE, and % Death compared to WT females (Fig. 1F–I). Significant interaction between sex and treatment was noted (Interaction: F_{(1, 53)}=7.69, p=0.008, Table S2).

TC-2153 decreases KA-induced seizure severity in C57BL/6J mice.

To test if effects of acute pharmacological inhibition of STEP on seizure susceptibility are similar to genetic deletion of STEP, C57BL/6J mice at 8 to 12 weeks of age (P56-P90) were i.p. injected first with STEP inhibitor TC-2153 (10 mg/kg) or vehicle control and 3 h later with KA (30 mg/kg) to induce behavioral seizures (Fig. 2A). Such treatment with TC-2153 was previously shown to increase Tyr-phosphorylation of STEP₆₁ substrates in the cortex including GluN2B and ERK1/2¹⁶. Upon TC-2153 injection, males displayed lower seizure scores at 50, 90, 110, and 120 min post-KA injection (Fig. 2B), decreasing cumulative seizure score, % SE, and % Death compared to vehicle injection (Fig. 2C–E). In females, TC-2153 application induced a larger decrease in seizure scores at nearly every time intervals, except 20–40 min (Fig. 2B) and reduced cumulative seizure score, % SE, and % Death compared to vehicle controls (Fig. 2C–E). However, no sex difference was observed for the effect of TC-2153 on cumulative seizure scores (Sex: F_{(1, 48)}=1.32, p=0.26, Table S2). Importantly, TC-2153 treatment reduced the number of mice that died by KA injection from 5 to 1 in both sexes (Fig. 2E). These data indicate that TC-2153 reduced KA-induced seizure severity in C57BL/6J mice.

Figure 2. TC-2153 treatment decreases KA-induced seizure severity in adult C57BL/6J mice compared to vehicle control with greater effects seen in females.

(A) Experimental schematic of kainic acid (KA)-induced seizure severity in both male and female C57BL/6J mice at 8–12 weeks (P56-P90) and ovariectomized (OVX) C57BL/6J females at 9–10 weeks (P63-P76) at 3 h post i.p. injection with STEP inhibitor TC-2153 (10 mg/kg in saline containing 2.8% DMSO) or vehicle control (saline containing 2.8% DMSO). Behavioral seizures were monitored using modified Racine scale. (B) TC-2153 treatment in male mice (n=12) decreases severity of KA-induced seizures at 90–120 min post injection compared to vehicle treatment (n=13). TC-2153 injection in female mice (n=12) significantly decreases KA-induced seizure severity at nearly every time point compared to vehicle treatment (n=12), whereas KA-induced seizures were similar between vehicle-injected OVX females (n=6) and TC-2153-injected OVX females (n=7) for the first 2 h. Mann-Whitney U-test results are shown (*p<0.05). (C) Cumulative seizure scores. Two-tailed Student t-test results are shown (*p<0.05). (D-E) Percentage (%) of mice that achieved Stage 5 (D) and Stage 6 (E). Data shown as mean ± STDEV. Table S1 shows two‐way ANOVA test results with sex as one factor and treatment as the other.

This result was contrary to our original hypothesis that seizure susceptibility will increase by acute pharmacological inhibition of STEP. To confirm that TC-2153 was blocking STEP activity, primary rat hippocampal neuronal culture was treated with either DMSO (vehicle control) or TC-2153 (10 μM) for 1 h. Immunoblot analysis of STEP₆₁ substrates revealed that TC-2153 application significantly increased the levels of Tyr¹⁴⁷²-phosphorylated GluN2B and Tyr²⁰⁴/Tyr¹⁸⁷-phosphorylated ERK1/2 compared to DMSO, without affecting the expression of GluN2B, ERK1/2, and STEP₆₁ (Fig. S1A–B). Consistent with the previous reports in cultured cortical neurons¹⁶, these results demonstrate that TC-2153 inhibits STEP₆₁ activity in cultured hippocampal neurons.

To confirm that TC-2153 inhibits STEP₆₁ in the hippocampus in vivo, the hippocampi of C57BL/6J mice were collected at 3 h post injection with either vehicle control or TC-2153 (10 mg/kg). Unlike previous reports¹⁶, TC-2153 treatment did not alter hippocampal level of phosphorylated ERK1/2 in both sexes (Fig. S2). However, TC-2153 application significantly increased the hippocampal level of phosphorylated GluN2B in females but not males, indicative of STEP₆₁ inhibition in females (Fig. S2). It is interesting to note that the effect of TC-2153 on enhancing phosphorylated GluN2B in females but not males (Fig. S2) mirrors its greater trends on reducing KA-induced seizure severity in females (Fig. 2B–D).

Ovariectomy abolishes the TC-2153-induced seizure suppression in female C57BL/6J mice.

To investigate whether ovarian hormones were implicated in the TC-2153-induced reduction in seizure severity seen in females, female C57BL6/J mice received ovariectomy (OVX), which eliminates bulk circulation of ovarian-derived hormones from the system³⁰. Under DMSO injection, OVX females reached SE more quickly than ovary-intact females following KA injection (Fig. S3). Remarkably, TC-2153 injection no longer decreased KA-induced seizure severity, cumulative seizure scores, and % Death in OVX mice compared to DMSO injection (Fig. 2B–C, E). Furthermore, similar numbers of OVX females reached SE regardless of treatment (vehicle: 6 out of 6 mice, TC-2153: 6 out of 7 mice) (Fig. 2D). These data indicate that OVX abolishes the TC-2153-induced suppression of seizure severity seen in intact females.

TC-2153 treatment reduces the excitability of acute hippocampal slices.

To investigate if TC-2153 affects hippocampal excitability, calcium imaging was performed on acute hippocampal slices prepared from mice containing genetically encoded calcium indicator GCaMP6s²⁵. Acute slices were treated with either DMSO or TC-2153 (10 μM) in ACSF for 1 h prior to imaging of GCaMP6s (Fig. 3A–C). Under DMSO application, KCl-mediated depolarization significantly increased calcium signals in the somatic and dendritic layers in the CA1, CA3, and DG regions from both sexes (Fig. 3C–D, Table S3). In contrast, TC-2153 treatment significantly reduced the KCl-evoked calcium signals in every region (Fig. 3C–D, Table S3), indicating that TC-2153 decreases the excitability of the hippocampal slices.

Figure 3. TC-2153 treatment reduces the excitability of acute hippocampal slices.

(A) Experimental schematic of calcium imaging on coronal acute hippocampal slices prepared from Thy1-GCaMP6s mice at P28–90. The slices were incubated with DMSO (0.14%) or TC-2153 (10 μM) for 1 h, and subjected to GCaMP6s imaging first in ACSF and then after 2 min of 15 mM KCl application. (B) Manual tracing of hippocampal CA1, CA3, and DG regions for analysis. (C) Representative GCaMP6s fluorescence images of slices. Raw pixel intensity is shown. (D) Quantification of GCaMP6s fluorescence (ΔF/F) in DG, CA1, and CA3 regions normalized to ACSF. The total number of slices imaged: DMSO-treated slices (n=23 including 10 from 4 males, 13 from 5 females); TC-2153-treated slices (n=16 including 7 from 4 males and 9 from 5 females). Compared to DMSO treatment, TC-2153 treatment reduces GCaMP6s signals in DG, CA1, and CA3 regions of hippocampal slices prepared from both males and females. The total number of analyzed slices from males: DG (10 DMSO, 7 TC-2153), CA1 (10 DMSO, 7 TC-2153), and CA3 (8 DMSO, 6 TC-2153). The total number of analyzed slices from females: DG (11 DMSO, 9 TC-2153), CA1 (11 DMSO, 9 TC-2153), CA3 (8 DMSO, 7 TC-2153). Data shown as mean ± STDEV. Post-hoc Tukey test results are shown for ACSF vs. KCl (^#p<0.05, ^###p<0.005) and for DMSO+KCl vs. TC+KCl (*p<0.05, ***p < 0.005). Table S2 shows two‐way ANOVA test results with sex as one factor and treatment as the other.

TC-2153 treatment hyperpolarizes resting membrane potential and decreases intrinsic excitability in cultured hippocampal neurons.

The inhibitory action of TC-2153 on the excitability of the hippocampal slices was contrary to the well-known role of STEP₆₁ in weakening excitatory synaptic strength in the hippocampus. Therefore, we hypothesized that TC-2153 may regulate intrinsic excitability of hippocampal pyramidal cells. To test this, we performed whole-cell patch-clamp recording of cultured hippocampal neurons (DIV 8–10) after pre-treating with either DMSO or TC-2153 (10 μM) for 1 h (Fig. 4A–C). TC-2153 application hyperpolarized resting membrane potentials (RMP) and decreased the input resistance (R_in), but did not affect membrane capacitance of recorded neurons (Fig. 4D). Current clamp recording in the presence of synaptic transmission blockers revealed that TC-2153 application reduced instantaneous firing rates and the number of APs at 20 to 200 pA injections compared to DMSO or no treatment (Fig. 4A–C). TC-2153 treatment also increased average rheobase current, interspike interval (ISI), AP rise time, AP decay time, and AP half width, while decreasing fast after-hyperpolarization (fAHP) amplitude at 100 pA injection (Table 1). These data indicate that TC-2153 decreases hippocampal neuronal excitability.

Figure 4. TC-2153 hyperpolarizes RMP and reduces AP firing and input resistance in cultured hippocampal neurons.

Whole-cell current-clamp recording of hippocampal pyramidal neurons in dissociated culture (DIV 8–10) was performed in current clamp mode after 1 h treatment with TC-2153 (10 μM) or DMSO (0.14%). (A-C) TC-2153 reduces AP firing in cultured hippocampal neurons. Spike trains were evoked in pyramidal neurons in the presence of synaptic transmission blockers by delivering constant somatic current pulses of 500 ms duration in the range 0–200 pA with a step interval of 10 s at a holding potential of −60 mV. (A) Representative traces of APs at 100 pA injection. (B) Average instantaneous AP firing rate. (C) Average number of APs. The number of recorded neurons: untreated (gray circle, n=12), DMSO (black square, n=15), or TC-2153 (red triangle, n=18). (D) TC-2153 reduces hyperpolarizes RMP and decreases input resistance in cultured hippocampal neurons. Average resting membrane potential, capacitance, and input resistance in the recorded neurons: untreated (n=24), DMSO (n=28), or TC-2153 (n=36). Data shown as mean ± STDEV. Post-hoc Tukey test results are shown for TC-2153 vs. untreated or DMSO (***p < 0.005).

Table 1.

AP properties of cultured hippocampal pyramidal neurons treated with DMSO or TC-2153 in Figure 5.

	n	Rheobase (pA)	AP latency (ms)	VT (mV)	ISI (ms)	AP height (mV)	AP rise time (ms)	AP decay time (ms)	AP HW (ms)	fAHP (mV)
Untreated	12	34.0 ± 13.46	16.9 ± 6.01	−36.5 ± 1.46	33.6 ± 4.11	53.5 ± 7.36	0.83 ± 0.15	2.73 ± 0.30	1.83 ± 0.33	21.5 ± 4.79
DMSO	15	33.2 ± 16.60	15.4 ± 3.08	−36.5 ± 2.35	32.1 ± 4.63	49.7 ± 7.12	0.83 ± 0.15	2.86 ± 0.35	1.91 ± 0.27	20.1 ± 2.29
TC-2153	18	63.2 ± 26.26*^	27.1 ± 24.50	−35.3 ± 4.22	48.1 ± 21.03*^	50.5 ± 12.35	1.18 ± 0.45*^	3.81 ± 1.46*^	2.58 ± 1.12*^	16.4 ± 4.90*^

n, number; average rheobase current (the minimal current that elicited at least one spike). AP properties were measured from the first action potential evoked by a current step to 100 pA at a holding potential of −60 mV. AP latency was measured from the start time of current step to the peak of the first AP. V_T, voltage threshold for AP; Inter-spike interval (ISI); AP rise time, 10–90% rise time of AP; AP decay time, 10–90% decay time of AP; AP half-width (HW); fast afterhyperpolarization (fAHP). Each value represents the mean ± STDEV. Post-hoc Tukey test results are shown for TC-2153 vs. untreated (*p<0.05) and TC-2153 vs. DMSO (^p<0.05).

^*p<0.05 for untreated vs. TC 2153.

^{^}p<0.05 for DMSO vs. TC 2153.

TC-2153 treatment decreases sag voltage and I_h in cultured hippocampal neurons.

To test if TC-2153 regulates intrinsic membrane properties of hippocampal pyramidal neurons upon membrane hyperpolarization, we measured the amplitude of voltage sag and rebound potential. We found that TC-2153 treatment significantly reduced voltage sag by 75.2% and rebound potentials by 51.6% at −200 to −40 pA current injections compared to DMSO or no treatment (Fig. 5A–C).

Figure 5. TC-2153 treatment reduces I_h in cultured hippocampal neurons.

Whole-cell current clamp recording of cultured hippocampal neurons (DIV 8–10) was performed after 1 h treatment with TC 2153 (10 μM) or DMSO (0.14%). (A-C) TC-2153 reduces voltage sag and rebound voltage in cultured hippocampal neurons. (A) Representative responses to hyperpolarizing current steps from −200 to 0 pA in 20 pA increments in current clamp mode. The amount of voltage sag (blue lines) and rebound potential (red lines) was determined as difference between the maximum and steady-state voltage during the hyperpolarizing current injection. (B) Average sag voltage. (C) Average rebound voltage. The number of recorded neurons: untreated (gray circle, n=12), DMSO (black square, n=13), or TC-2153 (red triangle, n=18). (D-F) In voltage clamp mode, I_h was evoked by applying voltage steps from the holding potential of −60 mV to −120 mV in 5 mV decrements. (D) Representative traces of I_h. (E) I_h density at all voltage steps. (F) Normalized conductance (G/G_max) at all voltage steps. The number of recorded neurons: untreated (n=9), DMSO (n=12), TC-2153 (n=15). Data shown as mean ± STDEV. Post-hoc Tukey test results are shown for TC-2153 vs. untreated or DMSO (***p < 0.005).

Hyperpolarization-activated cyclic nucleotide-gated ion (HCN) channels produce a slowly depolarizing non-selective inward cationic current called I_h, which mediates voltage sag and rebound potentials and regulates RMP and excitability of the hippocampal pyramidal neurons³¹. Among HCN1–4 subunits, HCN1 and HCN2 subunits are predominantly expressed in the hippocampal and cortical excitatory neurons³². To test if TC-2153 modulates I_h, we first confirmed that cultured hippocampal neurons expressed HCN1 and HCN2 subunits (Fig. S1C–D). Voltage-clamp recording upon membrane hyperpolarization revealed that TC-2153-treated neurons displayed smaller I_h density (pA/pF) at −70 mV to −120 mV compared to DMSO-treated and untreated neurons (Fig. 5D–E). The normalized conductance (G/Gmax) and V_1/2 calculated from G/Gmax showed a depolarizing shift in voltage dependence (Fig. 5F, Table S4). Consistent with decreased sag voltage and rebound potential (Fig. 5A–C), these data indicate that TC-2153 down-regulates I_h.

DISCUSSION

The role of STEP₆₁ in weakening excitatory synaptic strength in the hippocampus and cortex has been well-established^7–10,13, but its effect on seizure susceptibility and regulation of hippocampal excitability remains elusive. In this study, we provide evidence that acute pharmacological inhibition of STEP with TC-2153 decreases KA-induced seizure severity and hippocampal excitability. Our study has also revealed a previously unknown actions of TC-2153 in modulating intrinsic membrane properties.

Genetic ablation of STEP exerts age- and sex-dependent effects on seizure severity

Since STEP₆₁ weakens excitatory synaptic strength in the hippocampus, seizure susceptibility should increase in STEP KO mice which deletes all STEP isoforms⁸. However, Briggs et al. combined both sexes for analyses and showed that STEP KO mice at 6–8 weeks of age are resistant to pilocarpine-induced seizures¹⁴. When we separated the sexes in our analysis for KA-induced seizure severity, there was a decrease in STEP KO males at 6–7 weeks of age but an increase at 8–12 weeks of age (Fig. 1). STEP KO females at both age groups showed a decreasing trend in seizure severity (Fig. 1). Our study thus demonstrates that genetic ablation of STEP, especially catalytic STEP₄₆ and STEP₆₁, most likely affects seizure severity in an age- and sex-dependent manner.

In rodents at 6 to 8 weeks of age, sexual maturation occurs in association with significant changes in sex hormone-dependent synapse formation and circuit maturation in the brain, particularly in the hippocampus and cortex^28,29. The 8^th week of age marks the end of the puberty and adolescence period²⁸. These critical changes in hippocampal circuitry and synapse formation due to sex hormone shifts may influence sensitivity to pilocarpine and KA which have different mechanisms for inducing limbic seizures^20,33 and may explain the age-dependent switch in seizure susceptibility of STEP KO mice.

Anti-seizure effect of the STEP inhibitor TC-2153.

TC-2153 inhibits two catalytically active STEP isoforms, STEP₄₆ and STEP₆₁⁴. Both isoforms are expressed in the striatum and amygdala⁵ although only STEP₆₁ is expressed in the hippocampus and neocortex⁵. In both male and female mice, TC-2153 treatment decreased hippocampal excitability (Fig. 3) and the severity of KA-induced seizures (Fig. 2) that arise mostly from the hippocampus where KA subtype glutamate receptors are highly expressed especially in the CA3 region compared to other brain regions including amygdala, striatum, and cortex²⁰. Therefore, the majority of anti-seizure effect of TC-2153 is likely mediated by STEP₆₁ inhibition in the hippocampus, although we cannot exclude the possible contributions of inhibiting both STEP₄₆ and STEP₆₁ in other brain regions. While our studies with TC-2153 pretreatment demonstrate its proof-of-concept anticonvulsant efficacy, investigating its pharmacokinetics and anti-seizure effects during SE and TLE will be critical to assess its clinically relevant efficacy.

Compared to males, females displayed a greater trend in anti-seizure effect of TC-2153, which was abolished by OVX (Fig. 2). Greater anti-seizure potency in females than males has also been reported for neurosteroids in various acute seizure models³⁴. The possible involvement of ovarian-derived hormones³⁰ is interesting because prevalence, frequency, and semiology of focal seizures and TLE have been reported to differ by sex in both clinical patient populations and preclinical animal models due to their neurobiological actions³⁵. Estrogen exacerbates seizures in women with epilepsy³⁶ and both estrogen and testosterone increase seizure susceptibility in rodent KA models^37,38. In contrast, seizure frequency in mice and women with epilepsy is reduced by high progesterone level^36,39.

It is interesting that anti-seizure effect of TC-2153 was lost in OVX females, whereas the effect was present in males (Fig. 2). TC-2153 application also reduced the excitability of hippocampal slices from both male and female mice (Fig. 3) even after circulating gonadal hormones had washed away while acclimating the slices in ACSF⁴⁰, suggesting that gonadal hormone cannot fully explain the subtle sex difference in the response to TC-2153. Considering that women display higher drug concentration in blood and longer duration for drug metabolism and clearance than men³⁵, a greater trend in anti-seizure effect in females may arise from higher brain concentration of TC-2153 in females than males. Alternatively, the enhanced seizure severity in OVX females compared to naïve females (Fig. S3) may also contribute to the diminished efficacy of TC-2153 in OVX females. Future studies shall evaluate ADME properties of TC-2153 in both sexes and use gonadectomy in combination with hormone replacement to confirm whether TC-2153 decreases KA-induced seizure severity by altered pharmacokinetics of TC-2153 or suppressing proconvulsant actions of estrogen or testosterone, and/or potentiating anti-convulsant actions of progesterone.

Decrease in hippocampal intrinsic excitability as a mechanism for anti-seizure effect of TC-2153.

Our study demonstrates novel actions of STEP inhibitor TC-2153 (Fig. 4–5). TC-2153 application markedly decreases intrinsic excitability of cultured hippocampal pyramidal neurons (Fig. 4). Considering that STEP₆₁ interacts with a variety of proteins including ion channels, ion transporters, and signaling proteins important for neuronal excitability and synaptic transmission¹², our findings suggest a compelling possibility that STEP₆₁ may regulate ionic currents critical for intrinsic neuronal excitability and AP waveform, in contrast to its well-known role in weakening synaptic transmission.

Indeed, hyperpolarized RMP and decreased R_in in TC-2153-treated hippocampal neurons (Fig. 4D) suggest the opening of potassium channels. Among major potassium currents in hippocampal pyramidal neurons⁴¹, fast activating and inactivating I_A and fast activating and slowly inactivating I_D delay the onset of firing and contribute to AP repolarization and firing rate^41,42. Slowly activating and inactivating I_K mediates AP repolarization⁴², whereas slowly activating and non-inactivating I_M hyperpolarizes RMP and suppresses repetitive firing of APs without affecting their latency⁴³. Calcium-activated I_C contributes to fAHP and regulates AP repolarization, firing rate, and half-width⁴⁴. The effects of TC-2153 on ISI, AP rise and decay times, AP half width, and fAHP amplitude (Table 1) suggest that one or more of these potassium currents may be regulated by TC-2153 to control intrinsic excitability and AP waveform.

I_h as a novel target of TC-2153.

We discover that TC-2153 treatment decreases I_h which contributes to sag voltage and rebound potential evoked by membrane hyperpolarization (Fig. 5). The role of I_h in hippocampal neuronal excitability is complex, as I_h exerts both excitatory and inhibitory effects on the ability of an excitatory postsynaptic potential (EPSP) to trigger an AP³¹. In hippocampal CA1 pyramidal neurons, HCN1 and HCN2 are preferentially enriched in the distal dendrites³¹, where I_h decreases EPSP summation⁴⁵, but dendritic excitability can be enhanced or reduced by I_h^46,47. I_h can also increase AP firing rate by depolarizing RMP and decreasing R_in⁴⁸. Therefore, TC-2153-induced I_h reduction (Fig. 5) may contribute to hyperpolarized RMP and decreased excitability seen in TC-2153-treated neurons (Fig. 4).

How TC-2153 decreases I_h is unknown. Activation kinetic of HCN2 channel is regulated by Src phosphorylation of its Tyr⁴⁷⁶, whereas the receptor-like protein-tyrosine phosphatase-alpha can dephosphorylate HCN2 and decrease its surface and current expression⁴⁹, raising a possibility that STEP₆₁ may dephosphorylate HCN2, and its inhibition by TC-2153 may modify HCN2 channel function or expression. Alternatively, TC-2153 may directly bind to and decrease current expression of HCN1 and/or HCN2 channels. The mechanism underlying inhibitory actions of TC-2153 on I_h warrants future studies.

Therapeutic potential of TC-2153.

High levels of STEP₆₁ are associated with Alzheimer’s disease (AD)⁷ and FXS¹⁵. Pharmacological inhibition of STEP with TC-2153 alleviates excitatory synaptic defects and memory loss observed in AD mouse model¹⁶ and reverses behavioral and synaptic deficits in FXS mouse model¹⁵. In addition to these therapeutic potentials, our present study demonstrates that TC-2153 reduces seizure severity in both male and female mice and the activity of their hippocampi (Fig. 1–3) and dampens intrinsic excitability and I_h of hippocampal neurons (Fig. 4–5). Clinical challenges exist in the use of anti-seizure drugs that are ineffective or can differ by sex^1,50, urging a need for new therapeutic targets. Our study presents TC-2153 as an attractive therapeutic candidate for epilepsy with novel mechanistic actions.

KEY POINTS BOX.

• Administration of TC-2153 significantly reduces seizure severity in both males and females.

• Ovariectomy abolishes the TC-2153-induced decrease in seizure severity observed in females.

• TC-2153 treatment significantly decreases overall excitability of acute hippocampal slices prepared from both sexes.

• TC-2153 application decreases intrinsic excitability and hyperpolarization-induced currents of cultured hippocampal neurons.