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. 2019 Oct 29;36(3):263–276. doi: 10.1007/s12264-019-00437-x

Role of Elevated Thrombospondin-1 in Kainic Acid-Induced Status Epilepticus

Yurong Zhang 1,#, Mengdi Zhang 1,#, Wei Zhu 2, Xiaohong Pan 1, Qiaoyun Wang 1, Xue Gao 1, Chaoyun Wang 1, Xiuli Zhang 1, Yuxia Liu 1, Shucui Li 1, Hongliu Sun 1,✉
PMCID: PMC7056766  PMID: 31664678

Abstract

Previous studies have suggested that thrombospondin-1 (TSP-1) regulates the transforming growth factor beta 1 (TGF-β1)/phosphorylated Smad2/3 (pSmad2/3) pathway. Moreover, TSP-1 is closely associated with epilepsy. However, the role of the TSP-1-regulated TGF-β1/pSmad2/3 pathway in seizures remains unclear. In this study, changes in this pathway were assessed following kainic acid (KA)-induced status epilepticus (SE) in rats. The results showed that increases in the TSP-1/TGF-β1/pSmad2/3 levels spatially and temporally matched the increases in glial fibrillary acidic protein (GFAP)/chondroitin sulfate (CS56) levels following KA administration. Inhibition of TSP-1 expression by small interfering RNA or inhibition of TGF-β1 activation with a Leu-Ser-Lys-Leu peptide significantly reduced the severity of KA-induced acute seizures. These anti-seizure effects were accompanied by decreased GFAP/CS56 expression and Smad2/3 phosphorylation. Moreover, inhibiting Smad2/3 phosphorylation with ponatinib or SIS3 also significantly reduced seizure severity, alongside reducing GFAP/CS56 immunoreactivity. These results suggest that the TSP-1-regulated TGF-β1/pSmad2/3 pathway plays a key role in KA-induced SE and astrogliosis, and that inhibiting this pathway may be a potential anti-seizure strategy.

Electronic supplementary material

The online version of this article (10.1007/s12264-019-00437-x) contains supplementary material, which is available to authorized users.

Keywords: Astrogliosis, Status epilepticus, Ponatinib, Thrombospondin-1

Introduction

Epilepsy is a common disease that affects approximately 0.5%–1% of the global population. Although pharmacotherapy is the most widely-used and effective treatment option, up to one-third of patients are resistant to antiepileptic drugs [1, 2]. Research on the mechanisms underlying epilepsy may identify new targets for antiepileptic drugs.

Astrocytes are associated with neuronal excitability and epileptic seizures [3–5]. Almost all prolonged seizures result in reactive gliosis or astrogliosis, characterized by severe morphological and biochemical changes in astrocytes [6]. Moreover, astrogliosis is one of the most important factors promoting neuronal hyperexcitability and epileptogenesis [7].

Astrocytes secrete thrombospondin-1 (TSP-1) and regulate its expression [8]. TSP-1 is a unique member of the TSP family in that it activates transforming growth factor beta 1 (TGF-β1) via two binding sites. TGF-β1 signaling controls many cellular responses and developmental processes in animals [9]. Previous studies have confirmed that enhanced TGF-β1 signaling contributes to epileptogenesis and epileptic seizures [10–13]. TGF-β1 further modulates cytokine responses and the secretion of other growth factors [14]. Among TGF-β1 signaling pathways, the canonical TGF-β1/Smad2/3 pathway plays an important epileptogenic role [10, 11, 15] and has therefore been identified as a therapeutic target for treating astrogliosis [6]. The Smad family includes the main downstream messenger molecules that transmit TGF-β1 signals from cell membrane receptors to the nucleus [16]. Smad proteins are thought to play an important role in regulating intracellular responses to TGF-β1 through the TGF-β1-regulated phosphorylation of Smad2 and Smad3 [17]. Phosphorylated Smad2 (pSmad2) and pSmad3 further combine with co-Smads before entering the nucleus and forming transcription complexes that complete the intracellular signal transduction process [17].

Consequently, we hypothesized that the TSP-1/TGF-β1/pSmad2/3 pathway participates in and contributes to both seizures and astrogliosis. We therefore investigated changes in the TSP-1/TGF-β1/pSmad2/3 pathway in a kainic acid (KA)-induced rat model of status epilepticus (SE). To this end, we used drugs and small interfering RNA (siRNA) to manipulate pathway activity in order to elucidate the role of the pathway in KA-induced SE.

Methods

Animals and Experimental Groups

Male Sprague-Dawley rats (280 g–320 g) were obtained from Jinan Jinfeng Experimental Animal Co. Ltd. (Certificate No. SCXK2014-0006; China). All procedures were approved by the Animal Ethics Committee of Binzhou Medical University (Approval No. 2015005) and conducted in complete compliance with Chinese law and the US National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 80-23, revised 1996). Water and food were provided ad libitum. All experiments were performed between 09:00 and 17:00.

Rats were assigned to the KA-treated group (n = 336) or the saline-treated control group (n = 36). The KA-treated rats were further assigned to groups receiving siRNA (n = 36) or negative control RNA (n = 36), those receiving Leu-Ser-Lys-Leu (LSKL; Sigma-Aldrich, St. Louis, MO; n = 36) or saline (n = 36), those receiving SIS3 (Sigma-Aldrich; n = 42) or saline (n = 42), and those receiving ponatinib (MedChem Express, Princeton, NJ; n = 36) or saline (n = 36).

Surgical Implantation of Electrodes and Cannulae

As we previously described [18], after intraperitoneal administration of sodium pentobarbital (50 mg/kg; CAS, 57-33-0, Xiya Reagent, Chengdu, China), each rat was secured in a stereotaxic apparatus (Stoelting, Wood Dale, IL), and an electrode was implanted into the right cortex at a point 3.2 mm posterior and 3.0 mm lateral to bregma and at a depth of 1.8 mm. The electrode consisted of two twisted-pair stainless-steel wires 0.2 mm in diameter (A.M. Systems, Carlsborg, WA) and ~ 3 cm long, soldered to a miniature socket to record electroencephalograms (EEGs) using a PowerLab device (AD Instruments, Bella Vista, New South Wales, Australia). Each rat also had a cannula (Reward Biotech, Shenzhen, China) implanted into the left ventricle at a point 1.8 mm posterior and 1 mm lateral to bregma and at a depth of 3.6 mm. The implanted electrode and cannula were fixed to the skull with dental cement. Each rat was allowed a week-long postoperative recovery period.

Drug Interventions and siRNA

For intraventricular injections, drugs were infused into the left ventricle over a 10-min period through a needle (Reward Biotech, China) inserted 0.2 mm beyond the end of the guide cannula. The needle was left in place for 5 min before being slowly retracted.

SE was induced by a single intraventricular KA injection (1.25 mg/mL, 0.65 μL per rat; Sigma-Aldrich, St. Louis, MO) via the implanted cannula. Following KA administration, almost all rats showed immediate and continuous Racine’s stage 4 or 5 seizures [19]. Sixty minutes later, the seizures were terminated with an intraperitoneal diazepam injection (1 mg/mL solution at 2 mg/kg; Sigma-Aldrich). Seven rats were excluded from our study as they did not develop SE following KA administration.

To target TSP-1, siRNAs were designed, synthesized, and assessed by Tuoran Biological Technology (Shanghai, China). The siRNA sequences were as follows: TSP-1 siRNA 5′-GCCAGUAUGUUUACAACGUdTdT-3′ and 5′-ACGUUGUAAACAUACUGGCdTdT-3′; negative control siRNA 5′-UUCUCCGAACGUGUCACGUTT-3′ and 5′- ACGUGACACGUUCGGAGAATT-3′.

Twenty-four hours prior to KA injection, the siRNA group received a 4-μL (0.5 μg/μL) intraventricular injection of anti-TSP-1 siRNA, while the control group received 4 μL (0.5 μg/μL) of negative control RNA. The siRNA or negative control RNA injection was then repeated every 48 h. Smad3 phosphorylation was altered in both the hippocampus and cortex, so 4 h before KA injections, the SIS3 group received intraventricular SIS3 (0.085 µg/µL solution at 0.85 μg per 300 g) to selectively inhibit TGF-β1-regulated Smad3 phosphorylation [20], while the associated control group received the same volume of saline. Thirty minutes prior to the KA injections, the ponatinib group received an intraperitoneal ponatinib injection (1 mg/mL solution at 0.002 mg/g) to inhibit Smad2/3 phosphorylation [21], while the control group received the same volume of saline. The LSKL group received an intraventricular injection of 10 µL (0.2 mg) LSKL, while the associated control group received the same volume of saline. SIS3, ponatinib, LSKL, or saline was administered daily for 7 days.

EEG Recordings and Seizure Severity Classification

After KA treatment, each rat was placed in a Plexiglas arena (50 cm × 30 cm × 30 cm). Their behaviors were observed and EEGs recorded during the following 60 min, and the power spectrum was analyzed using a Powerlab system (AD Instruments, Bella Vista, New South Wales, Australia). Seizure severity was classified according to modified Racine’s stages [19]: stage 1, non-selective chewing; stage 2, head nodding; stage 3, unilateral forelimb clonus; stage 4, “wet dog” shakes with bilateral forelimb clonus and rearing; and stage 5, falling. We also recorded generalized seizure durations (GSDs), seizure durations, and seizure latencies to evaluate the severity of seizures.

Immunohistochemistry

At 24 h, 3 days, and 7 days following KA administration, 6 rats from each group were anesthetized with intraperitoneal sodium pentobarbital (50 mg/kg; CAS, 57-33-0, Xiya Reagent, Chengdu, China). The chest was opened to expose the heart, a needle was inserted into the left ventricle, the right auricle opened, and 250 mL saline rapidly injected followed by 250 mL of 4% paraformaldehyde (PFA). Each rat was decapitated and the brain placed in 4% PFA and left for at least 24 h. After soaking in 30% sucrose, the brain was cut at 10 μm on a cryostatic microtome (CM3050 S, Leica, Weztlar, Germany).

For double immunofluorescent staining of glial fibrillary acidic protein (GFAP) and chondroitin sulfate (CS56) or TSP-1, the sections were first washed three times with 0.01 mol/L phosphate-buffered saline (PBS) for 15 min, and then blocked with 10% bovine serum albumin in PBS for 2 h at room temperature. Then the sections were incubated overnight at 4°C in antibody dilution buffer containing rabbit anti-GFAP immunoglobulin G (IgG; 1:100; BM0055, Boster, Wuhan, China), mouse anti-CS56 IgG (1:100; ab11570, EMD Millipore, Billerica, MA), or rabbit anti-TSP-1 IgG (1:100; ab85762, Abcam, Cambridge, UK). After 3 washes in PBS for 15 min each, the sections were sequentially incubated at 37°C for 2 h in antibody dilution buffer with fluorescein isothiocyanate-conjugated (1:200; A22110, EMD Millipore) and Cy3-conjugated (1:200; A0516, Beyotime Institute of Biotechnology, Shanghai, China) secondary antibodies. After three washes in PBS for 15 min on a shaking table, the sections were coverslipped.

Fluorescence intensities corresponding to GFAP and either CS56 or TSP-1 levels were assessed by laser confocal microscopy (LSM880, Zeiss, Oberkochen, Germany). Six stained slices from each rat were obtained and un-collapsed confocal images from the same brain region were analyzed. The fluorescence intensities in different brain regions were comparable because the slices were stained according to the same protocol and the same exposure intensity and time were used for image acquisition. Fluorescence intensities were quantified using ImageJ 1.37 software (National Institutes of Health, Bethesda, MD) as previously described [18].

Western Blotting

At 24 h, 3 days, and 7 days following KA administration, we decapitated six rats from each group and immediately removed their brains. The hippocampus and cortex were isolated and proteins were extracted. The protein concentrations were determined using a bicinchoninic acid assay kit (P0011, Beyotime Institute of Biotechnology) and emission at 562 nm was measured with a microplate reader (ELx800, BioTek Instruments, Winooski, VT). The proteins were separated by electrophoresis on 12% sodium dodecyl sulfate polyacrylamide gels and electroblotted to a polyvinylidene difluoride membrane. The membrane was then blocked with 5% fat-free milk for 3 h at room temperature.

The blocked membranes were incubated with rabbit anti-GFAP IgG (1:1000; BM0055, Boster), mouse anti-CS56 IgG (1:1000; ab11570, EMD Millipore) or rabbit anti-TSP-1 (1:100; ab85762, Abcam), mouse monoclonal anti-TGF-β1 (1:800; ab64751, Abcam), rabbit anti-Smad2/3/pSmad2/3 monoclonal antibodies (1:2000; ab47083 and ab40854, Abcam; Ser_465/467, Cell Signaling Technology, Boston, MA, USA; ab52903, Abcam), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:3000; AB-P-R001, Zhejiang Kangcheng Biotech, Jiaxing, China) at room temperature for 2 h and then at 4°C overnight. We visualized immunoreactive bands after 2-h exposure to horseradish peroxidase-conjugated IgG secondary antibodies. We acquired images with the Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE) and analyzed them with the accompanying software. TGF-β1, Smad2/3, and pSmad2/3 expression intensities were expressed as ratios relative to the GAPDH expression intensities and normalized to the control group values.

Flow Cytometry

Three days after KA administration, we anesthetized and decapitated 6 rats from each group and immediately removed their brains. The hippocampus and cortex were separated and immediately immersed in PBS. Single-cell suspensions were prepared by filtration. Repetitive centrifugation was used to wash the suspension with PBS. Cell solutions with densities of 5–10 × 106 cells/mL were fixed in 4% PFA, and 200-µL volumes of cell suspension, each containing 1–5 × 105 cells, were incubated with 5% bovine serum albumin on ice for 10 min. Monoclonal rabbit anti-Smad3 antibody (1:100; ab40854, Abcam) was added to each tube, and the tubes were incubated for 20 min at 4 °C. The cells were washed three times by centrifugation. Fluorescein isothiocyanate-conjugated secondary antibodies (1:150; A22110, Beyotime, Shanghai, China) were added to each tube, and the tubes were again incubated for 15 min. The cells were again washed three times by centrifugation, and 500–550-nm fluorescence intensities produced by the samples were analyzed with a flow cytometer (BD FACSVerse, Becton Dickinson, Franklin Lakes, NJ).

Statistical Analysis

All values are presented as the mean ± SEM. We performed statistical analyses in SPSS 13.0 for Windows (IBM, Amund, NY). One-way analysis of variance was used to compare seizure latencies, cumulative seizure durations, and GSDs. The nonparametric Mann–Whitney U test was used to compare cumulative times in each seizure stage and changes in protein expression levels. We defined statistical significance as P < 0.05.

Results

KA Administration Upregulates Hippocampal and Cortical Expression of GFAP, CS56, and TSP-1

We evaluated the immunoreactivity of GFAP, an astrocyte-specific marker [22, 23], and CS56, secreted by astrocytes and accordingly used as another astrocytic marker [24]. At all time points from 24 h onwards, hippocampal and cortical GFAP immunoreactivity levels in the KA-treated group were significantly higher than in the control group (P < 0.001; Fig. 1A, D, G, J, L, M). Similarly, hippocampal and cortical CS56 immunoreactivity levels were also higher in the KA-treated group than those in the control group (P < 0.001; Fig. 1B, E, H, K, L, N). Western blotting yielded similar results (P < 0.001; Fig. 1L–N).

Fig. 1.

Fig. 1

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KA-induced upregulation of GFAP/CS56 expression in rat brain. A–I Representative images of immunoreactivity for GFAP and CS56 in the right dentate gyrus in controls and after KA treatment (scale bar, 100 μm; n = 6/group). J, K Quantified GFAP and CS56 immunoreactivity levels in the hippocampus and cortex of controls and KA-treated rats. L–N Western blots (L) and statistics for GFAP (M) and CS56 (N) immunoreactivity. Data are shown as the mean ± SEM. ***P < 0.001 vs. controls. Hip, hippocampus; Cor, cortex.

Further, TSP-1 immunoreactivity was detected. The immunohistochemistry results indicated that TSP-1 immunoreactivity was elevated in the hippocampus (P = 0.048; Fig. 2A, C, I) and cortex (P = 0.02; Fig. 2J) 30 min after KA administration. Western blotting revealed similar increases in hippocampal and cortical TSP-1 immunoreactivity 30 min after KA administration (Fig. S1A, B). Western blotting further confirmed that TGF-β1 immunoreactivity was significantly elevated from 24 h onwards in the hippocampus (P < 0.001; Fig. 2K, M) and cortex (P < 0.001; Fig. 2M) in the KA-treated group.

Fig. 2.

Fig. 2

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KA-induced upregulation of TSP-1, TGF-β1, Smad2/3, and pSmad2/3. A–H Representative images of immunoreactivity for TSP-1 and GFAP in CA2 in controls and after KA treatment (scale bar, 150 μm; n = 6/group). I, J Quantified TSP-1 immunoreactivity in the hippocampus and cortex in controls and KA-treated rats. K–Q Western blots (K) and statistics for hippocampal and cortical expression of TSP-1 (L), TGF-β1 (M), Smad2/3 (N, P), and pSmad2/3 (O, Q) (n = 6/group). Data are shown as the mean ± SEM. *P < 0.05, ***P < 0.001 vs. controls. Hip, hippocampus.

We further found that KA significantly increased Smad2/3 protein levels in the hippocampus (Smad2, P < 0.001; Smad3, P < 0.001) and cortex (Smad2, P < 0.001; Smad3, P < 0.001) from 24 h onwards (Fig. 2K, N, P). KA administration also promoted Smad2/3 phosphorylation, as evidenced by increased pSmad2/3 levels in the hippocampus (pSmad2, P < 0.001; pSmad3, P < 0.001) and cortex (pSmad2, P < 0.001; pSmad3, P < 0.001) from 24 h onwards (Fig. 2K, O, Q).

Inhibiting TSP-1 Expression with siRNA Attenuates KA-Evoked Seizures Accompanied by Reduced GFAP/CS56 Expression

Rats that received anti-TSP-1 siRNA in addition to KA spent more time in stages 0–3 (53.7 ± 0.8 min vs. 12.0 ± 0.9 min; P < 0.001) and less time in stage 4 (4.0 ± 0.5 min vs. 15.6 ± 0.7 min; P < 0.001) and stage 5 (2.3 ± 0.4 min vs. 32.4 ± 1.4 min; P < 0.001) than rats that received negative control RNA in addition to KA (Fig. 3A). The siRNA group also showed reduced GSDs (6.3 ± 0.8 min vs. 48.0 ± 0.9 min; P < 0.001; Fig. 3B) and seizure durations (9.8 ± 0.7 min vs. 51.5 ± 0.8 min; P < 0.001; Fig. 3C) as well as increased latencies (51.2 ± 0.9 min vs. 2.8 ± 0.1 min; P < 0.001; Fig. 3D). Representative EEGs and EEG power spectra are shown in Fig. 3N.

Fig. 3.

Fig. 3

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Effects of inhibiting TSP-1 expression with siRNA in KA model rats. A–D siRNA treatment altered the cumulative time in every stage (A), shortened the GSDs (B) and seizure durations (C), and increased the latencies (D) (n = 10/group). E–M siRNA treatment reduced the KA-induced increases in immunoreactivity for GFAP (E, F, H), CS56 (E, G, I), and TSP-1 (E, J-M) in the hippocampus (n = 6/group; scale bars for F–I, 150 μm; J–M, 100 μm). N Representative EEGs (upper) and EEG power spectra (lower) in a control (left) and a siRNA-treated rat (right). Data are shown as the mean ± SEM. ***P < 0.001 vs. controls.

Immunohistochemical and western blotting experiments revealed that hippocampal (P < 0.001, Fig. 3J, L, E; P < 0.001, Fig. 4A, B) and cortical (P < 0.001; Fig. S2A) TSP-1 immunoreactivity was significantly lower in the siRNA group than in the negative control RNA group. The siRNA group also exhibited reduced hippocampal (P < 0.001 and 0.001; Fig. 3E, F–I) and cortical (P < 0.001 and 0.001; Fig. S2A) immunoreactivity for GFAP and CS56.

Fig. 4.

Fig. 4

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Changes in TSP-1, TGF-β1, Smad2/3, and pSmad2/3 levels after anti-TSP-1 siRNA treatment. Western blots (A) and statistics (B–G) for TSP-1, TGF-β1, Smad2/3, and pSmad2/3 (n = 6/group). The data are shown as the mean ± SEM. ***P < 0.001 vs. controls.

Western blotting showed that hippocampal (P < 0.001; Fig. 4A, C) and cortical (P < 0.001; Fig. S2C) TGF-β1 expression levels were significantly greater in the siRNA group than in the control group from 24 h onwards. We found similar increases in Smad2 (Fig. 4A, D; Fig. S2D) and Smad3 expression (Fig. 4A, F; Fig. S2F) in the siRNA group from 24 h onwards, while the pSmad2/3 levels were significantly decreased (Fig. 4A, E, G; Fig. S2E, G).

These results indicated that inhibiting TSP-1 expression attenuates KA-induced seizures, along with reduced Smad2/3 phosphorylation and GFAP and CS56 expression.

Inhibiting TGF-β1 Activation with LSKL Attenuates KA-Evoked Seizures Accompanied by Reduced GFAP/CS56 Expression

LSKL is a competitive antagonist that inhibits TGF-β1 activation by specifically blocking the binding of TSP-1 to TGF-β1. Our behavioral results showed that administering LSKL before KA significantly prolonged the time spent in stages 0–3 (52.0 ± 0.8 min vs. 15.0 ± 0.8 min; P < 0.001) and shortened the time spent in stage 4 (4.3 ± 0.6 min vs. 19.1 ± 0.8 min; P < 0.001) and stage 5 (3.7 ± 0.5 min vs. 25.9 ± 1.5 min; P < 0.001) (Fig. 5A). Moreover, LSKL treatment significantly reduced GSDs (8.0 ± 0.8 min vs. 45.0 ± 0.8 min; P < 0.001; Fig. 5B) and seizure durations (10.9 ± 0.7 min vs. 46.2 ± 0.6 min; P < 0.001; Fig. 5C), and increased the latencies (35.3 ± 1.8 min vs. 3.0 ± 0.1 min; P < 0.001; Fig. 5D). Representative EEGs and their power spectra are shown in Fig. 5N.

Fig. 5.

Fig. 5

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Effects of LSKL treatment in the KA-induced SE model. A–D LSKL altered the cumulative times for each stage (A), shortened the GSDs (B) and seizure durations (C), and increased the latencies (D) (n = 10 group). E–I LSKL reduced the KA-induced upregulation of immunoreactivity for GFAP and CS56 in the hippocampus 3 days after KA treatment (n = 6/group; scale bars, 100 μm). J–M The immunoreactivity of TSP-1 in the hippocampus 3 days after KA treatment (scale bars, 50 μm; n = 6/group). N Representative EEGs and EEG power spectra in saline- and LSKL-treated groups. Data are shown as the mean ± SEM. ***P < 0.001 vs. controls.

LSKL treatment also significantly reduced the hippocampal (P < 0.001 and 0.001; Fig. 5F–I, E) and cortical (P < 0.001 and 0.001; Fig. S3A) levels of GFAP and CS56. No effects on TSP-1 immunoreactivity were found (Figs. 5J–M, E; 6A, B; S3A).

We also found that from 24 h onwards, LSKL significantly increased the hippocampal and cortical expression of TGF-β1 (hippocampus, P < 0.001; Fig. 6A, C; cortex, P < 0.001; Fig. S3C), and Smad2/3 (P < 0.001 and 0.001; Figs. 6A, D, F; S3D, F). However, the phosphorylation levels of Smad2/3 were significantly decreased from 24 h onwards (P < 0.001 and 0.001; Figs. 6A, E, G; S3E, G).

Fig. 6.

Fig. 6

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LSKL-induced changes in hippocampal levels of TSP-1, TGF-β1, Smad2/3, and pSmad2/3. Western blots (A) and statistics (B–G) for TSP-1 (B), TGF-β1 (C), Smad2/3 (D, F), and pSmad2/3 (E, G) in hippocampus (n = 6/group). The data are shown as the mean ± SEM. ***P < 0.001 vs. controls.

These results indicated that inhibiting TGF-β1 activation with LSKL attenuates KA-evoked seizures, and reduces the Smad2/3 phosphorylation and the protein levels of GFAP and CS56. These effects are similar to those obtained by inhibiting TSP-1 expression by siRNA interference.

SIS3 and Ponatinib Attenuate KA-Evoked Seizures While Reducing GFAP/CS56 Expression

As with LSKL and siRNA treatment, SIS3 (a specific inhibitor of Smad3 phosphorylation) in addition to KA prolonged the time spent in Racine’s stages 0–3 (52.1 ± 0.6 min vs. 14.1 ± 0.8 min; P < 0.001), and reduced the time spent in stage 4 (6.4 ± 0.5 min vs. 18.5 ± 0.9 min; P < 0.001) and stage 5 (1.5 ± 0.2 min vs. 25.4 ± 1.1 min; P < 0.001) (Fig. 7A). Meanwhile, the seizure durations (10.8 ± 0.6 min vs. 48.0 ± 0.7 min; P < 0.001; Fig. 7C) and GSDs (7.9 ± 0.6 min vs. 43.9 ± 0.7 min; P < 0.001; Fig. 7B) were shortened and the latencies were increased (48.7 ± 0.8 min vs. 3.0 ± 0.1 min; P < 0.001; Fig. 7D). Representative EEGs and their power spectra are shown in Fig. S4D. SIS3 treatment also significantly reduced the hippocampal and cortical expression of GFAP and CS56 (P < 0.001 and 0.001; Fig. 7 E–L).

Fig. 7.

Fig. 7

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Effects of SIS3 in the KA-induced SE model. A–D SIS3 altered the cumulative times in each stage (A), shortened GSDs (B) and seizure durations (C), and increased the latencies (D, n = 10/group). E–L Representative images (E–J) and statistics (K, L) for the immunoreactivity for GFAP and CS56 in controls and after SIS3 treatment (scale bar, 100 μm; n = 6/group). M, N Western blots and statistics showing SIS3-induced changes in the levels of Smad2/3 and pSmad2/3. O Flow cytometry-based quantification of hippocampal Smad3 (3 days, n = 6/group). The data are shown as the mean ± SEM. **P < 0.01, ***P < 0.001 vs. controls. Hip, hippocampus.

Western blotting experiments also showed that SIS3 significantly reduced hippocampal (P < 0.001; Fig. 7M, N) and cortical (P < 0.001; Fig. S4A) pSmad3 levels and increased hippocampal (P < 0.01 and 0.001; Fig. 7M, N) and cortical (P < 0.001; Fig. S4A) Smad3 expression from 24 h onwards. It had no significant effect on Smad2 expression or pSmad2 levels (Fig. S4B, C). Increased Smad3 immunoreactivity following SIS3 treatment was confirmed by flow cytometry (Fig. 7O).

We further found that administering the tyrosine kinase inhibitor ponatinib in addition to KA significantly prolonged the time spent in Racine’s stages 0–3 (50.2 ± 0.8 min vs. 14.8 ± 1.0 min; P < 0.001), reduced the time spent in stage 4 (5.0 ± 0.4 min vs. 19.7 ± 1.3 min; P < 0.001) and stage 5 (4.8 ± 0.5 min vs. 25.5 ± 1.6 min; P < 0.001) (Fig. 8A), and shortened seizure durations (13.1 ± 0.7 min vs. 53.4 ± 0.6 min; P < 0.001; Fig. 8C) and GSDs (9.8 ± 0.7 min vs. 45.2 ± 1.0 min; P < 0.001; Fig. 8B) in addition to increasing the latencies (41.4 ± 0.5 min vs. 2.9 ± 0.1 min; P < 0.001; Fig. 8D). Ponatinib significantly reduced the hippocampal and cortical expression of GFAP and CS56 (P < 0.001; Fig. 8E–L). It also significantly reduced the Smad2/3 and pSmad2/3 levels from 24 h onwards (P < 0.001; Fig. 8M–O; Fig. S5A, B).

Fig. 8.

Fig. 8

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Effects of ponatinib in the KA-induced SE model. A–D Ponatinib altered the cumulative times in each stage (A), shortened GSDs (B) and seizure durations (C), and increased the latencies (D) (n = 10/group). E–L Representative images (E–J) and statistics (K, L) of immunoreactivity for GFAP and CS56 in the hippocampus of controls and after KA and ponatinib treatment (scale bar, 100 μm; n = 6/group). M–O Western blots (M) and statistics (N, O) showing ponatinib-induced changes in the levels of Smad2/3 and pSmad2/3. The data are shown as the mean ± SEM. **P < 0.01, ***P < 0.001 vs. controls. Hip, hippocampus; Pon, ponatinib.

Discussion

We hypothesized that seizures are mediated by the TSP-1/TGF-β1/pSmad2/3 pathway. Accordingly, as predicted, we found previously unreported increases in TSP-1 expression, TGF-β1 expression, and pSmad2/3 levels in a KA-induced rat model of SE. Inhibiting TSP-1 expression with siRNA and inhibiting TGF-β1 activation with LSKL significantly attenuated seizure severity, along with reducing GFAP/CS56 and Smad2/3 phosphorylation in this model. Inhibiting Smad3 phosphorylation with SIS3 similarly reduced seizure severity and GFAP/CS56 levels. Moreover, ponatinib significantly reduced seizure severity, Smad2/3 phosphorylation, and GFAP/CS56 levels. Since the upregulation in the TSP-1/TGF-β1/pSmad2/3 pathway was consistent with the KA-induced SE and GFAP/CS56 levels, these results support our initial hypothesis.

TSP-1 is secreted by astrocytes and promotes synaptogenesis, neuronal migration, and axonal growth [25–29]. Moreover, TSP-1 interacts with the α2δ-1 subunit of the gabapentin receptor to stimulate the formation of excitatory synapses [30]. Consistent with these reports, we found dynamically increased TSP-1 levels in the brains of rats that experienced KA-induced SE and amygdaloid-kindling [18]. Together, these results suggest a contribution of TSP-1 in the early phase of seizures and epilepsy development, such as via the formation of an excitatory seizure network. In contrast, however, a reduction in TSP-1 expression has been reported in the ventrobasal thalamus in a rat model of spontaneous absence epilepsy and in patients with generalized epilepsy [31]. Increased sensitivity to pentylenetetrazol-induced kindling has been reported in mice lacking TSP-1 [32]. Previous reports have suggested that TSP-1 levels are closely linked to epilepsy development and seizures, with both elevated and reduced TSP-1 levels aggravating seizures. Further, these results indicate that TSP-1 plays different roles in different phases of epilepsy and seizures. For example, in the early phases of epilepsy, levels of TSP-1, which plays a vital role in synaptogenesis, are increased and may be responsible for synaptogenesis and the development of an excitatory epileptic network [18]. However, kindled animals and patients with generalized epilepsy display significantly reduced concentrations of PSD-95 [33], in addition to neuronal injury and even neuronal loss [33, 34]. The reduced number of synapses [35] may be partly due to the reduced TSP-1 levels.

Upstream regulation of TGF-β1 activation is provided by TSP-1, an important astrocytic secretion [36, 37]. TGF-β1 is stored in the extracellular matrix bound to latency-associated peptide (LAP) [38, 39]. TSP-1 converts latent TGF-β1 to its active form by binding to LAP and thereby releases active TGF-β1, which then binds to the TGF-β1 receptor to induce signal transduction [40]. TGF-β1 exerts its downstream effects on target cells, with Smad proteins playing a pivotal role in relaying signals from cell-surface receptors to the nucleus [41, 42]. The best-described intracellular pathway for TGF-β1 is the canonical cascade, which involves the upregulation and phosphorylation of Smad2/3 [42]. TGF-β1 promotes Smad2/3 phosphorylation and TGF-β1/Smad signaling has therefore been recognized as a therapeutic target for the treatment of astrogliosis [43].

TGF-β1 is associated with astrogliosis and epilepsy [43, 44]. TGF-β1 levels and Smad2/3 phosphorylation, which are increased in activated astrocytes and microglia [6, 45, 46], are elevated in animals and patients with epilepsy [48–49]. Cacheaux et al. [10] reported that direct activation of the TGF-β1 pathway by TGF-β1 results in epileptiform activity. Importantly, TGF-β pathway blockers prevent the generation of epileptiform activity [10], and an antagonist to the TGF-β receptor prevents the development of spontaneous seizures [11]. These studies point to a contributory role of the TGF-β1 pathway to the generation of epileptic seizures.

We found that inhibiting TSP-1 activity with siRNA or inhibiting TGF-β1 activity with LSKL alleviated the seizures induced by KA, which led us to speculate that increased TSP-1 expression may be the key factor in seizure generation via TGF-β1 pathway activation. We obtained similar results with SIS3, a potent and selective inhibitor of TGF-β1-regulated Smad3 phosphorylation [51]. This indicates that increased Smad3 phosphorylation, induced by TSP-1 and TGF-β1, may be the key factor underlying KA-induced astrogliosis and seizures.

Ponatinib is a multi-target tyrosine kinase inhibitor suitable for treating cases of chronic myelogenous leukemia that are otherwise resistant to complex amino-acid kinase inhibitors [50]. Ponatinib modulates the TGF-β1/pSmad2/3 pathway [48], which contributes to seizures. In our study, ponatinib also alleviated KA-induced seizures and astrogliosis. These results point to the inhibitory effect of ponatinib, further supporting the contribution of pSmad2/3 to astrogliosis and seizures.

As reported in previous studies, we found increased hippocampal and cortical immunoreactivity for GFAP and CS56 in KA-treated rats. Hippocampal dysregulation is crucial for epileptic seizures induced by several kindling stimuli [51, 52]. Similar to the hippocampus, the cortex has been shown to be an important region for the propagation of epileptic discharges, and most interictal-like discharges originating in the cortex show secondary propagation [53–56]. Our results confirm the increased immunoreactivity of GFAP and CS56 in KA-treated rats. Moreover, this increase was attenuated by siRNA or pharmacological intervention to inhibit the TSP-1/TGF-β1/pSmad2/3 pathway. These results support the possible contribution of the TSP-1-regulated pathway directly or indirectly to KA-induced astrogliosis. Moreover, since astrogliosis contributes to both acute seizures and chronic epilepsy [6, 7], we speculate that the TSP-1/TGF-β1/pSmad2/3 pathway may also contribute to spontaneous recurrent seizures, in addition to KA-induced SE.

In conclusion, astrocytes increase TSP-1 levels in response to KA-induced SE, subsequently activating the TGF-β1/Smad2/3 pathway, perhaps in astrocytes or other cell types, thereby increasing astrogliosis. Interfering with either TSP-1 or TGF-β1 signaling reduces seizure severity and astrogliosis, indicating that inhibiting the TSP-1/TGF-β1/Smad2/3 pathway is a potential therapeutic target for seizure management.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 882 kb) (881.6KB, pdf)

Acknowledgements

This work was supported by the National Natural Science Foundation of China (81573412), the Key Research and Development Plan (2018GSF121004), and the Natural Science Foundation of Shandong Province, China (ZR2014JL055).

Conflict of interest

The authors declare that they have no conflict of interest.

Footnotes

Yurong Zhang and Mengdi Zhang have contributed equally to this work.

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