STK24 modulates excitatory synaptic transmission in epileptic hippocampal neurons

Abstract Introduction A large amount of literature has indicated that excitatory synaptic transmission plays a crucial role in epilepsy, but the detailed pathogenesis still needs to be clarified. Methods In the present study, we used samples from patients with temporal lobe epilepsy, pentylenetetrazole‐kindled mice, and Mg2+‐free‐induced epileptic cultured hippocampal neurons to detect the expression pattern of STK24. Then, the whole‐cell recording was carried out after STK24 overexpression in the Mg2+‐free‐induced epileptic cultured hippocampal neurons. In addition, coimmunoprecipitation was performed to detect the association between endogenous STK24 and main subunits of NMDARs and AMPARs in the hippocampus of PTZ‐kindled mice. Results Here, we reported that STK24 was specifically located in epileptic neurons of human and pentylenetetrazole‐kindled mice. Meanwhile, the expression of STK24 was significantly down‐regulated in these samples which are mentioned above. Besides, we found that the amplitude of miniature excitatory postsynaptic currents was increased in STK24 overexpressed epileptic hippocampal cultured neurons, which means the excitatory synaptic transmission was changed. Moreover, the coimmunoprecipitation, which further supported the previous experiment, indicated an association between STK24 and the subunits of the NMDA receptor. Conclusion These findings expand our understanding of how STK24 involved in the excitatory synaptic transmission in epilepsy and lay a foundation for exploring the possibility of STK24 as a drug target.

Up to date, the pathogenesis of epilepsy remains unclear and needs to be further clarified. The imbalance of excitation and inhibition of the neural circuit is considered to be one of the most important pathogenesis of epilepsy. 4 Dendritic spines, which have mainly involved in the afferent of excitatory signals, are critical for the formation of functional neural circuits. 5 Serine/threonine kinase 24 (STK24), which belongs to the sterile 20 kinase family, also known as mammalian sterile 20-like kinase 3 (MST3), is widely expressed in many tissues including the brain. 6,7 STK24 was reported to facilitate dendritic spine development and maintain the structure of excitatory synapse. 8 Further, STK24 was also involved in changing the spontaneous excitatory postsynaptic currents of the mouse cortical pyramidal neurons in upper layers II-III, which indicates its possible role in regulating cortical excitability. 9 However, it has not been studied whether STK24 modulates the excitatory synaptic transmission in epilepsy.
Here, we investigated the location and expression pattern of

| Human samples
This study protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University and complied with the Declaration of Helsinki and the ethical principles and guidelines of the National Institutes of Health. Patients were diagnosed with retractable TLE in accordance with the criteria proposed by the International League Against Epilepsy (ILAE). 10 Control brain tissues were acquired from patients who underwent brain surgery owing to severe head trauma. All control patients had no history of epilepsy, seizures, or any other central nervous system disease and did not take any antiepileptic therapy before head trauma.
Temporal cortical tissue samples (14 from refractory TLE patients, 10 from patients with severe head trauma) were obtained from the Xinqiao Hospital of Third Military Medical University and the First Affiliated Hospital of Chongqing Medical University, and the clinical data of these patients were reported in our previous research. 11,12 For the use of clinical data and brain tissue, informed consent was signed by the patients or their guardians.

| PTZ-kindled mouse model
The animal experiments in our research were approved by the ethics committee of Chongqing Medical University. All animal studies were conducted abiding by the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the rules of the Animal Ethical Committee of Chongqing Medical University. Healthy male C57BL/6 mice (20-25 g) were provided by the Experimental Animal Center of Chongqing Medical University. All of the mice were kept in a specific pathogen-free animal facility under standard conditions at 21-22°C with 50-60% humidity in a 12 h/12 h light/dark cycle and were freely eating and drinking. Every effort had been made to minimize the suffering of the animals and their quantity.
The mice in the PTZ-kindled model were intraperitoneally injected with a daily subconvulsive dose of PTZ (35 mg/kg, Sigma-Aldrich, USA) between 9:00 AM and 11:00 AM. 13,14 The rest mice were assigned as controls and were injected with the corresponding dose of saline solution. The intensity of seizure was evaluated on the basis of Racine's scale evaluation 13 : 1: mouse and facial movements; 2: head nodding; 3: forelimb clonus; 4: rearing; and 5: rearing and falling. Mice were deemed fully kindled when exhibiting seizure attacks (score 4 or 5) after each PTZ injection for at least three consecutive days. The mice were anaesthetized with pentobarbital (6 mg/100 g) before sacrificed.
Thereafter, half of the serum-free medium was exchanged with an equivalent volume of fresh serum-free medium every three days.
Neurons were plated in 6-well plates with coverglasses at a density of 2 million cells/well for electrophysiology and 5 million cells/6 cm plates for Western blot analysis.
Flag-tagged mouse STK24 plasmids with pcDNA mammalian expression vector were constructed and purchased from youBio, China. DIV (day in vitro) 10, neurons were transfected with Venus and Flag-tagged empty vector pcDNA3.1 or Venus together with STK24 using a calcium phosphate method, respectively. 16 The amount of total cDNA was balanced by adding pcDNA3.1 in control.
During whole-cell recordings, Venus was observed as an indicator of cell selection and the mEPSCs and mIPSCs were recorded to measure the postsynaptic currents.

| Mg 2+ -free-induced spontaneous recurrent epileptiform discharge model of hippocampal neurons (SREDs)
Mg 2+ -free-treated neuronal model, which can simulate spontaneous recurrent epileptiform discharges, was set up to research epilepsy according to the previous study. 17 At DIV10 (for Western blot) or 16 (for whole-cell recordings), the serum-free medium was exchanged with Mg 2+ -free medium (in mM) (145 NaCl, 2.5 KCl, 10 HEPES, 2 CaCl2, 10 glucose, and 0.002 glycine, pH 7.2-7.4, 280-320 mOsm) or nonmagnesium-free medium (non-MGF, supplemented with 1 mM MgCl 2 ) for three hours. Three hours later, the Mg 2+ -free medium or non-Mg 2+ -free medium was discarded and the serum-free culture medium was added in. Within 12-24 hours, normal Mg2+ concentration in the culture has been restored and SREDs are typically observed and continue throughout the cell culture period. Then, neurons were harvested for Western blot (DIV 10) and whole-cell patch-clamp recordings (DIV 16).

| Protein extraction and Western blot
Brain tissues of human and mouse were homogenized to extract total proteins via total protein extraction kit (Sangon, China). The concen-
On the next day, the sections were incubated with a mixture of corresponding secondary antibodies at room temperature

| Coimmunoprecipitation
Coimmunoprecipitation was conducted in the light of the manufacturer's manual of protein A/G magnetic beads (HY-K0202, MedChem Express, USA). In short, 40 μl of protein A/G magnetic beads was added into an eppendorf tube and washed with 400 ul binding/wash buffer four times. Next, the beads were incubated with 3-4 μg primary antibody as previously mentioned. Rabbit monoclonal IgG (ab172730, Abcam, USA) was also used as a negative control. Subsequently, the supernatant was discarded and the magnetic beads were washed with binding/wash buffer. Then, the magnetic beads were incubated with protein lysates which obtained from hippocampi of PTZ-kindled mice. After washing, the supernatant was removed, and 1× SDS loading buffer was added. After denaturing by metal bath, the supernatants were collected for Western blot. Uncropped images of Western blots after coimmunoprecipitation are shown in Figure S1.

| Whole-cell recordings
The whole-cell recording was carried out using previously established procedures. 17 Briefly, DIV16-17, a coverslip of neurons, was transferred to a chamber with an extracellular solution on the in-

| Data analysis
Shapiro-Wilk test was used to verify the normal distribution, and Levene's test was used to analyze the homogeneity of variance.
Fisher's exact test was used to compare the difference in sex distribution between TLE patients and control patients. For the two independent samples with normal distribution, if the variances are homogeneous, unpaired Student's t test is adopted to compare the difference between two groups; otherwise, Welch's t test is adopted. The data were expressed as means ± SEM. P-value < 0.05 was considered to be statistically significant.

| Clinical characteristics of TLE and control subjects
Fourteen TLE patients (eight males and six females) were selected in our research, and the mean age was 22.36 ± 2.00 years (range   Figure  S2, **P < .01) and anterior temporal cortex (F) (n = 6 in the control group and n = 6 in the epileptic group, technical replicates four times, **P < .01) from 12 to 36 years) (

| Localization of STK24 in TLE patients and PTZkindled mice
Immunofluorescence labeling was performed as previously described. In the anterior temporal cortex from TLE patients, STK24 (red) was mainly located in the cytoplasm of the neuron and coexpressed with the neuronal marker MAP2 (purple), but not with the astrocyte marker GFAP (green) (Figure 1A). At the same time, STK24 was also observed in the cytoplasm of the neuron but not in the astrocyte in the hippocampus ( Figure 1B) and anterior temporal cortex ( Figure 1C) of PTZ-kindled mice. In summary, consistent with earlier reports, STK24 is still located in neurons specifically and mainly presented in the cytoplasm of the neuron.

| Decreased STK24 expression in TLE patients and PTZ-kindled mice
STK24 expression in the anterior temporal cortex from TLE patients and control patients was detected by Western blot. As shown in Figure 2A and B, STK24 expression was significantly decreased in TLE patients compared with that in control patients

| SREDs induces a significant decrease of STK24 expression
Owing to complex and compensatory environmental changes in regulating metabolic homeostasis of the living animal, it is difficult to evaluate the electrophysiological function at the single neuron and molecular levels in vivo studies. 20 Hence, in our present study, a SREDs cell model of cultured hippocampal neurons was set up as previously reported, 17,21 and the whole-cell recordings were con- 0.57 ± 0.06, five independent primary cultures, technical repeated three times) (**P < .01).

| STK24 affects the excitatory synaptic transmission
To examine the role of STK24 on neuronal excitability in epilepsy, we measured miniature excitatory and inhibitory postsynaptic currents Meanwhile, no significant difference in the cumulative distribution of mEPSCs frequency and mean mEPSCs frequency was found between the STK24 overexpressed group and the control group

| D ISCUSS I ON
Excitatory synaptic transmission, which is modulated mainly by the synaptic vesicle formation, release, and recycling, postsynaptic receptors, and their regulators, 23 plays a critical role in many neurological diseases, including epilepsy. 23,24 Regulating excitatory synaptic transmission is the key step to control the development of epilepsy. [23][24][25] STK24 has multiple functions in synaptic activity, including promoting dendritic spine and excitatory synapse development 6,8,9,[26][27][28] and changing neuronal excitability. 9 According to the fundamental characteristics of STK24, we hypothesize that STK24 may involve in epilepsy.
STK24 mRNA was found widely expressed in different rat brain regions and especially abundantly expressed in the cerebral cortex and hippocampus. 29 Besides, STK24 was identified expressed in neurons of the adult rat hippocampus and the neuronal layers of the occipital cortex. 6 For now, our study demonstrated that STK24 mainly expressed in the neuronal cytoplasm rather than astrocytes in the temporal cortex of TLE patients and the temporal cortex and hippocampus of PTZ-kindled mice. These results suggested that STK24 specifically located in epileptic neurons, which indicates that STK24 probably has some effects on neurons in epilepsy. Subsequently, the Western blot indicated that the expression level of STK24 was significantly down-regulated in TLE patients and PTZ-kindled mice.
These results indicate that STK24 may have a potential regulatory effect on epilepsy.
In order to figure out whether STK24 has a regulatory effect on epilepsy, the whole-cell recordings were performed to detect the  35 In TLE patients, NMDARS were mainly increased in dentate granule cells 36,37 ; meanwhile, in epileptic animal models, the increase of NMDARs could also be detected. 38 In conclusion, the experimental evidence suggests that the expression level of STK24 was decreased in epileptic patients, PTZkindled mice, and SRED cell model, and overexpression of STK24 in SRED cell model increased the excitatory postsynaptic transmission. All these evidence give us a hint that the decreased expression of STK24 may have an inhibitory effect on epileptogenesis via decreasing the excitatory postsynaptic transmission. However, this is a hypothesis, and further study will be needed to find out whether intervene the expression of STK24 could affect epileptogenesis.
Meanwhile, although we have preliminarily confirmed the possible interactions between STK24 and NMDARs, further elucidation of the detailed mechanism of STK24 on the NMDAR is still needed.

ACK N OWLED G M ENTS
This work was supported by grants from the National Science

CO N FLI C T S O F I NTE R E S T
There is no conflict of interest.