Blockade of Kv1.3 Potassium Channel Inhibits Microglia-Mediated Neuroinflammation in Epilepsy

Epilepsy is a chronic neurological disorder whose pathophysiology relates to inflammation. The potassium channel Kv1.3 in microglia has been reported as a promising therapeutic target in neurological diseases in which neuroinflammation is involved, such as multiple sclerosis (MS), Alzheimer’s disease (AD), Parkinson’s disease (PD), and middle cerebral artery occlusion/reperfusion (MCAO/R). Currently, little is known about the relationship between Kv1.3 and epilepsy. In this study, we found that Kv1.3 was upregulated in microglia in the KA-induced mouse epilepsy model. Importantly, blocking Kv1.3 with its specific small-molecule blocker 5-(4-phenoxybutoxy)psoralen (PAP-1) reduced seizure severity, prolonged seizure latency, and decreased neuronal loss. Mechanistically, we further confirmed that blockade of Kv1.3 suppressed proinflammatory microglial activation and reduced proinflammatory cytokine production by inhibiting the Ca2+/NF-κB signaling pathway. These results shed light on the critical function of microglial Kv1.3 in epilepsy and provided a potential therapeutic target.


Introduction
Epilepsy is a chronic neurological disorder characterized by spontaneous recurrent seizures. Approximately 50 million people worldwide suffer from epilepsy [1,2], which seriously affects the quality of life of patients. Although several factors (including traumatic brain injury, gene mutations, and infections) have been identified as causes of epilepsy [3][4][5], approximately 60% of epileptic disorders have unknown etiology. At present, surgery and antiepileptic drugs can prevent 70% of patients from seizures [6]. However, due to the existence of drug resistance, there are still a considerable number of patients who cannot be effectively treated. Therefore, there is an urgent need to develop novel antiepileptic drugs.
One of the vital pathophysiological hallmarks of epilepsy is neuroinflammation, which predominantly involves activated microglia and astrocytes releasing many types of inflammatory mediators [7,8]. A previous study manifested that proinflammatory cytokines increased neuronal excitability and lowered seizure threshold [9]. The concentration of proinflammatory cytokines detected in cerebral spinal fluid (CSF) was higher in epileptic patients than in healthy volunteers, further implying that the activation of cytokine cascade and associated inflammatory signals was responsible for epileptogenesis [10]. Additionally, dysregulated neuroinflammatory signals contribute to seizure generation, specifically We found that the mRNA level of Kv1.3 was increased significantly in the brains of KA-induced epileptic mice (p = 0.0156), while the mRNA levels of other potassium channels such as Kv1.1 (p = 0.2347) or Kv1.2 (p = 0.4045) were not changed ( Figure 1A). Likewise, an increase of Kv1.3 protein level was found in the brains of KA mice (p = 0.0404, Figure 1B,C), suggesting that the Kv1.3 channel was involved in the process of epilepsy. Moreover, we explored the cellular localization of Kv1.3 and confirmed that this upregulation of Kv1.3 was localized to Iba-1-positive microglia (p = 0.0037, Figure 1D-F), indicating that Kv1.3 may play a crucial role in mediating microglial activation in KA-induced epileptic mice.
Considering that neuroinflammation is critical in epilepsy, these results suggested a potential role of microglial Kv1.3 in epileptic pathogenesis and progression.

Kv1.3 Potassium Channel in Microglia Is Upregulated in KA-Induced Epileptic Mice
Since Kv1.3 expression has been reported to increase in microglia in some neurological diseases such as AD and PD [25,27], we investigated whether the expression of Kv1.3 was changed in brains of KA-induced epileptic mice compared with the Ctrl group. We found that the mRNA level of Kv1.3 was increased significantly in the brains of KA-induced epileptic mice (p = 0.0156), while the mRNA levels of other potassium channels such as Kv1.1 (p = 0.2347) or Kv1.2 (p = 0.4045) were not changed ( Figure 1A). Likewise, an increase of Kv1.3 protein level was found in the brains of KA mice (p = 0.0404, Figure 1B,C), suggesting that the Kv1.3 channel was involved in the process of epilepsy. Moreover, we explored the cellular localization of Kv1.3 and confirmed that this upregulation of Kv1.3 was localized to Iba-1-positive microglia (p = 0.0037, Figure 1D-F), indicating that Kv1.3 may play a crucial role in mediating microglial activation in KA-induced epileptic mice.
Considering that neuroinflammation is critical in epilepsy, these results suggested a potential role of microglial Kv1.3 in epileptic pathogenesis and progression. channels were detected in the brain of mice in Ctrl (n = 4 to 5) and KA (n = 9) groups; statistics by unpaired t-test. (B,C) Western blot analysis of Kv1.3 in the brain of mice in Ctrl and KA groups (n = 3); statistics by unpaired t-test. (D-F) Representative confocal images of Iba-1/Kv1.3/DAPI staining in the hippocampus of the two groups and colocalization analysis of Iba-1 and Kv1.3 (n = 3); statistics by unpaired t-test. Scale bar = 10 μm. Data are depicted as the mean ± SEM. * p < 0.05, ** p < 0.01.

Blockade of Kv1.3 Attenuates KA-Induced Epilepsy
To investigate the specific effect of microglial Kv1.3 on epilepsy, we assessed seizure severity and latency by observing neurobehaviors after treatment of PAP-1, a specific blocker of the Kv1.3 channel (Figure 2A). Then, we performed HE, Nissl, and NeuN staining to detect neuropathic changes after seizures.
The behaviors of the mice were observed and recorded for 2 h after KA injection. The results showed that mice of the KA+PAP-1 group had lower seizure severity (3.889 ± 0.2606 vs. 5.300 ± 0.2603, p = 0.0019) and longer seizure latency (42.38 ± 3.500 vs. 31.05 ± 3.730 min, p = 0.0418), compared with KA mice ( Figure 2B,C). Next, we investigated mRNA levels of potassium channels were detected in the brain of mice in Ctrl (n = 4 to 5) and KA (n = 9) groups; statistics by unpaired t-test. (B,C) Western blot analysis of Kv1.3 in the brain of mice in Ctrl and KA groups (n = 3); statistics by unpaired t-test. (D-F) Representative confocal images of Iba-1/Kv1.3/DAPI staining in the hippocampus of the two groups and colocalization analysis of Iba-1 and Kv1.3 (n = 3); statistics by unpaired t-test. Scale bar = 10 µm. Data are depicted as the mean ± SEM. * p < 0.05, ** p < 0.01.

Blockade of Kv1.3 Attenuates KA-Induced Epilepsy
To investigate the specific effect of microglial Kv1.3 on epilepsy, we assessed seizure severity and latency by observing neurobehaviors after treatment of PAP-1, a specific blocker of the Kv1.3 channel (Figure 2A). Then, we performed HE, Nissl, and NeuN staining to detect neuropathic changes after seizures.
The behaviors of the mice were observed and recorded for 2 h after KA injection. The results showed that mice of the KA+PAP-1 group had lower seizure severity (3.889 ± 0.2606 vs. 5.300 ± 0.2603, p = 0.0019) and longer seizure latency (42.38 ± 3.500 vs. 31.05 ± 3.730 min, p = 0.0418), compared with KA mice ( Figure 2B,C). Next, we investigated neuronal loss in the CA3 region in the hippocampus, which is susceptible to KA. Compared with the Ctrl group, many neurons exhibited cytoplasmic shrinkage, triangulated pyknotic nuclei, and darker Nissl staining in the KA group (HE, p = 0.0041; Nissl, p < 0.0001). In contrast, most neurons showed standard cell shape in the KA+PAP-1 group, and the number of neurons with darker blue staining decreased (HE, p = 0.0489; Nissl, p = 0.0007, Figure 2D-G). In addition, KA-induced neuronal loss was relieved by PAP-1 treatment (KA vs. Ctrl, p < 0.0001; KA+PAP-1 vs. KA, p < 0.0001, Figure 2H,I).
Taken together, the Kv1.3 blocker PAP-1 dramatically ameliorated KA-induced hippocampal neuronal damage and epileptic seizures. neuronal loss in the CA3 region in the hippocampus, which is susceptible to KA. Compared with the Ctrl group, many neurons exhibited cytoplasmic shrinkage, triangulated pyknotic nuclei, and darker Nissl staining in the KA group (HE, p = 0.0041; Nissl, p < 0.0001). In contrast, most neurons showed standard cell shape in the KA+PAP-1 group, and the number of neurons with darker blue staining decreased (HE, p = 0.0489; Nissl, p = 0.0007, Figure 2D-G). In addition, KA-induced neuronal loss was relieved by PAP-1 treatment (KA vs. Ctrl, p < 0.0001; KA+PAP-1 vs. KA, p < 0.0001, Figure 2H,I).

Blockade of Kv1.3 Inhibits the Activation of Microglia in KA-Induced Epileptic Mice
After we found that Kv1.3 blockade attenuated KA-induced epilepsy in mice, we explored how Kv1.3 blocker PAP-1 mitigated seizures and alleviated neuronal damage. Neuroinflammation promotes the occurrence of convulsions and neuronal damage after seizures, which is accompanied by the release of proinflammatory cytokines, such as IL-1β, IL-6, and TNF-α. These proinflammatory cytokines are released primarily by activated glia. Given the indispensable role of microglia in epilepsy, we examined the proliferation and inflammatory activation of microglia and related proinflammatory cytokine production with the treatment of Kv1.3 blocker PAP-1 in KA-induced epileptic mice.
These findings demonstrated that blockade of Kv1.3 dramatically reduced microglial activation and proinflammatory cytokine production in vivo and attenuated epilepsy as a consequence.

Blockade of Kv1.3 Suppresses Proinflammatory Activation of Microglia In Vitro
To further verify the anti-inflammatory effect of Kv1.3 blockade on microglia, we cultured BV2 microglia and primary microglia with LPS stimulation in vitro. We first examined the change of Kv1.3 expression in LPS-stimulated microglia and then investigated microglial activation and proinflammatory cytokine production to assess the effects of Kv1.3 blockade on reducing microglial inflammation.
These findings suggested that blockade of Kv1.3 directly reduced microglial activation and proinflammatory cytokine production in vitro.

Blockade of Kv1.3 Attenuates Microglial Activation through the Ca 2+ /NF-κB Signaling Pathway
After we found that blockade of Kv1.3 could suppress proinflammatory microglia in vivo and in vitro, we then investigated the underlying molecular mechanism of the anti-inflammatory effect of Kv1.3 blocker PAP-1 in epilepsy. Considering that the NF-κB signaling pathway is associated strongly with inflammation, we examined whether it is upregulated in epilepsy and verified the inhibitory effect of PAP-1 on it.
Collectively, these results suggested that Kv1.3 blockade may suppress microglial activation and proinflammatory cytokine production by inhibiting NF-κB pathway.
Recent studies have suggested that blocking Kv1.3 disrupted Ca 2+ influx [34], and calcium signaling participated in the inflammation of microglia as a critical secondary messenger [35]. Thus, we speculated that calcium signaling might be involved in inhibiting microglial activation and proinflammatory cytokine production induced by PAP-1 and acted upstream of NF-κB signaling pathway. We measured the concentration of intracellular calcium in BV2 microglia treated with PAP-1 and LPS and then detected the expression of the NF-κB signaling pathway and proinflammatory cytokines after chelating cytosolic calcium.

Discussion
Epilepsy is a complex multifactorial disease, and it is difficult to perform an effective treatment because of pharmacoresistance [6,36,37]. In recent years, the research of epilepsy has been focused on discovering new therapeutic targets. Here, we tried to illustrate

Discussion
Epilepsy is a complex multifactorial disease, and it is difficult to perform an effective treatment because of pharmacoresistance [6,36,37]. In recent years, the research of epilepsy has been focused on discovering new therapeutic targets. Here, we tried to illustrate the role of the microglial Kv1.3 channel in epilepsy. We demonstrated that microglial Kv1.3 was upregulated in KA-induced epileptic mice, and pharmacological blockade of the Kv1.3 channel inhibited the neuroinflammation and seizures via the Ca 2+ /NF-κB signaling pathway. Our results indicated that Kv1.3 played an essential role in microglial activation during epilepsy.
In recent studies, the potassium channels have attracted more attention as causes of epilepsy [19,20,38]. The conclusion that mutations of K + channels can result in severe epilepsy is further supported by evidence from clinical research. Pathogenic mutations of Kv2.1 and Kv3.2 channels are accepted to be linked with epileptic encephalopathies, the rare form of epilepsy [39,40]. Kv7.3 channel mutations have been implicated in resulting in BFNE (benign familial neonatal epilepsy), which is characterized by developmental delay and intellectual disability [41]. It has been reported that mutations of Kv1.1 and Kv1.2 channels can lead to episodic ataxia with generalized/focal seizures [42,43]. However, little is known about the association between Kv1.3 and epilepsy. In this study, we found that microglial Kv1.3 was upregulated in KA-induced epileptic mice, suggesting a possible role of Kv1.3 in epileptic pathogenesis and progression. Therefore, we used a small-molecule inhibitor PAP-1, which has been widely used as a specific Kv1.3 blocker in diseases, to investigate the role of Kv1.3 [25,27,28]. Our results showed that PAP-1 attenuated KAinduced epilepsy. However, the role of the Kv1.3 channel in epilepsy has not been reported in any genetic mouse models of epilepsy, and we believed that Kv1.3 might be involved in those models and should be further investigated in future.
An earlier study reported that Kv1.3 blocker Psora-4 could not obviously affect the action potential (AP) properties (including AP peak value, AHP, and AP frequency) of neurons compared with the control [44]. Kv1.3 immunoreactivity in astrocytes was reported to be unaltered after seizures in gerbils [45]. Therefore, we focused on the function of Kv1.3 in microglia. Kv1.3 has been found to contribute to the migration of the BV2 cell line and primary microglia [46]. Moreover, the Kv1.3 channel in LPS-activated microglia contributed strongly to postnatal hippocampal neuronal death [47]. Notably, much effort has focused on inflammation mediated by Kv1.3. Following differentiation with LPS, microglia exhibited high Kv1.3 current density, and the expression of Kv1.3 was upregulated [48]. Moreover, blockade of the microglial Kv1.3 channel reduced the release of IL-6 from brain slices of mice [49]. Similarly, we showed that PAP-1 reduced microglial activation and proinflammatory cytokine production, such as IL-1β, IL-6, and TNF-α. In our work, the uniqueness of the mRNA expression of IL-6 in the brain might be related to the diversity of cell types and complex transcription mechanism. The importance of microglial Kv1.3 has gained more attention in multiple neurological diseases in which neuroinflammation is involved. PD is characterized mainly by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) region of the brain. Chronic administration of PAP-1 protected multiple animal models of PD from the loss of dopaminergic neurons and reduced neuroinflammation, resulting in lower motor deficits [25]. Likewise, PAP-1 attenuated AβO-induced microglial activation and microglial neurotoxicity. Blockade of the Kv1.3 channel had a neuroprotective effect on Alzheimer's transgenic model via inhibiting microglial activation and enhancing the microglial amyloid-β clearance capacity, thereby improving behavioral deficits [27]. In addition, treatment with PAP-1 intraperitoneally polarized microglia towards the M2 phenotype and inhibited NLRP3 inflammasome activation in ischemic stroke [28]. Given the critical role of inflammation in epilepsy, we hypothesized that blocking Kv1.3 might attenuate KA-induced epilepsy. As expected, we found that Kv1.3 blockade with PAP-1 reduced seizure severity, extended seizure latency, and relieved neuronal damage. Furthermore, PAP-1 inhibited microglial activation and proinflammatory cytokine production in KA-induced epileptic mouse models. Activated neuroinflammatory microglia induced A1 neurotoxic astrocytes through secreting IL-1α, TNF, and C1q [50]. It was reported that A1 astrocytes promoted the progression of epilepsy [51]. Whether blockade of Kv1.3 regulated the phenotypic transition of astrocytes via microglia is an interesting research direction.
NF-κB signaling pathway is one of the classic pathways associated with inflammation. Nuclear Factor Kappa B (NF-κB) is a widely expressed transcription factor. Evidence suggests that NF-κB participates in multiple activities, such as immune responses, DNA transcription, and cancer [52][53][54]. The activation of the NF-κB signaling pathway induced by LPS involves several steps. Upon stimulation, TRAF6 is upregulated as an adaptor protein of TLR4/MyD88 and leads to the phosphorylation of IKK, degradation of IκB, and release of p65. The released p65 translocates to the nucleus and binds to specific sequences, resulting in numerous proinflammatory cytokines production [55]. Strong evidence suggests that NF-κB may play a key role in modulating seizure susceptibility via inflammation. The inactive NF-κB signaling pathway induced by the kappa opioid receptor (KOR) participated in suppressing neuronal injury and regulating microglial M2 polarization in epileptic rats [31]. Likewise, our laboratory also verified that sitagliptin (a DPP4 inhibitor) reduced the KA-induced activation of the NF-κB signaling pathway and suppressed the inflammatory response mediated by microglia in epilepsy [56]. Here, we found that PAP-1 inhibited the NF-κB signaling pathway, resulting in attenuation of both microglial activation and epilepsy severity.
In this work, we demonstrated that Kv1.3, playing an essential role in neuroinflammation, was upregulated in KA-induced mouse epilepsy models. Noteworthy, the blockade of Kv1.3 attenuated KA-induced epilepsy and inhibited microglial activation and the release of proinflammatory cytokines (such as IL-1β, IL-6, and TNF-α) through the Ca 2+ /NF-κB signaling pathway. We believe our findings provided a novel strategy for the potential therapy of pharmacoresistant epilepsy. Although we found that Ca 2+ was involved in the downregulation of the NF-κB signaling pathway, further studies are still required to fully understand the interaction between Ca 2+ and NF-κB signaling pathway in microglia.

Mice
Eight-week-old male C57BL/6J mice (20 ± 2 g body weight) were purchased from Wuhan University Center for Animal Experiment/ABSL-3 Laboratory. The animals were housed at 20 ± 2 • C with 60 ± 5% humidity and a 12 h light/12 h dark cycle. For the experiment duration, all mice had ad libitum access to standard mouse chow and water. All protocols involving mouse models were approved by the Institutional Animal Care and Use Committee of Wuhan University.

Mouse Model and Drug Administration
On the third day of PAP-1 (MedChemExpress, New Jersey, NJ, USA) or saline treatment, the seizure model was induced by intraperitoneal (i.p.) injection of KA (Sigma, St. Louis, MO, USA). Mice were randomly divided into three groups and were treated as follows: mice in KA + PAP-1 group (n = 9) received PAP-1 (20 mg/kg/d i.p.) for 3 days before KA injection (30 mg/kg/d i.p.), the KA group (n = 10) received an equal volume of saline for 3 days before KA injection (30 mg/kg/d i.p.), and mice in the Ctrl group (n = 8) received an equal volume of saline for 3 days.

Tissue Collection
Twenty-four hours after KA injection, the mice were anesthetized using isoflurane and then intracardially perfused with saline. The mouse brain was rapidly removed and cut sagittally; one half was stored at −80 • C, and the other half was fixed in 4% paraformaldehyde and processed for paraffin embedding or frozen sections.

Microglia Culture and Drug Treatment
As previously described, primary microglia were separated from primary mixed glial cultures prepared from newborn C57BL/6J mice [58]. The BV2 microglia were purchased from the China Center for Type Culture Collection (Wuhan, China). Both BV2 cells and primary microglia were cultured in a humidified 5% CO 2 incubator at 37 • C. Primary microglia were cultured in DMEM-F12 media (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Gibco, Melbourne, Australia) and a penicillin-streptomycin solution (Biosharp, Hefei, China). BV2 microglia were cultured in DMEM media (Gibco, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (BI, Herzliya, Israel) and a penicillin-streptomycin solution (Biosharp, Hefei, China).
Primary microglia were pretreated with 10 µM PAP-1 for 1 h and then stimulated with 100 ng/mL LPS (Sigma, St. Louis, MO, USA) for 12 h. BV2 microglia were pretreated with 10 µM PAP-1 for 1 h and then stimulated with 1 µg/mL LPS for 12 h. Both cell supernatants and lysates were collected. LPS stimulation was extended to 24 h for ELISA. To observe p65 nuclear translocation after LPS stimulation, cells were fixed with 4% paraformaldehyde 45 min after exposure to LPS.

Nissl and HE Staining
Paraffin sections were dewaxed with xylene and then rehydrated in an ethanol gradient. For Nissl staining, paraffin sections were stained with a 1% toluidine blue solution (Boster Biotech, Wuhan, China). For HE staining, paraffin sections were immersed in hematoxylin solution for 3 min, soaked in a hydrochloric acid alcohol solution for 5 s, and soaked in eosin solution for 2 min.

Quantitative PCR
Total RNA from the mouse brain or microglia was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Then, reverse transcription was performed using the HiScript ® III RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China) according to the manufacturer's protocol. Quantitative PCR (qPCR) was performed with ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China). The expression of target genes was normalized to GAPDH. The 2 −∆∆Ct relative quantification method was used to calculate the target genes' expression. A complete list of primer sequences is provided in Table 1.

Western Blot
Tissues and cells were lysed on ice using radioimmunoprecipitation assay (RIPA; Biosharp, Hefei, China) buffer supplemented with phenylmethanesulfonyl fluoride (PMSF; Biosharp, Hefei, China), protease inhibitors (TargetMol, Boston, MA, USA), and phosphatase inhibitors (TargetMol, Boston, MA, USA). The total protein concentration was quantified using a BCA protein assay (Beyotime Biotechnology, Shanghai, China). The detailed western blot procedure was previously described [51]. Finally, the fluorescence signal was determined by super Western blot ECL substrates (PUMOKE, Wuhan, China). Detailed information of the primary and secondary antibodies were as follows: Kv1.

Flow Cytometry
Following stimulation, microglia were double-washed with PBS. Then, cells were incubated in the Kv1.3 antibody (1:100, Alomone Labs, Jerusalem, Israel) for 2 h at 37 • C and goat anti-guinea pig IgG FITC (1:100, Bioss, Beijing, China) for 1 h at 37 • C. Then, any unbound antibody was double-washed with PBS, and the cells were suspended in 300 µL PBS and analyzed by flow cytometer (Beckman Coulter, Miami, FL, USA).

ELISA
The protein levels of IL-6 and TNF-α in the supernatants of microglia were quantified using ELISA kits (IL-6, 4A Biotech Co., Ltd., Beijing, China; TNF-α, Multisciences, Hangzhou, China). The microtiter plate was pre-coated with anti-mouse IL-6 and TNF-α monoclonal antibodies, and moderately diluted samples and standards were added, in which IL-6 and TNF-α would bind to their monoclonal antibodies. Next, biotinylated anti-mouse antibodies were added, which connected with mouse IL-6 and TNF-α bound to monoclonal antibodies to form an immune complex. Then, horseradish peroxidase-labeled avidin was added and bound explicitly to biotin. Finally, chromogenic reagent and stop solution were added, and the OD value was measured at 450 nm. The concentration of IL-6 and TNF-α were proportional to the OD450 value, and the concentrations of IL-6 and TNF-α were calculated by drawing a standard curve.

The Intracellular Concentration of Ca 2+ Determination Assay
After treatments and being washed three times with PBS, BV2 microglia were incubated with 2 µM Fluo-4 AM fluorescent probe (Beyotime Biotechnology, Shanghai, China) for 30 min. Then, any unbound fluorescent probe was washed with PBS, and the cells were suspended in 300 µL PBS for 30 min to ensure complete intracellular conversion of Fluo-4 AM into Fluo-4. Finally, the intracellular Ca 2+ level was quantified using flow cytometer.

Statistical Analysis
The data were presented as the mean ± standard error of the mean (SEM). GraphPad Prism 8.0 software was used to analyze the data and generate graphs. All of the statistical details of experiments can be found in the figure legends, including the statistical tests used, number of mice in animal experiments, and number of replicates for cell experiments. The data between two groups were analyzed using unpaired t-tests or unpaired t-tests with Welch's correction, while one-way ANOVA with Tukey's multiple comparisons tests were applied for more than two groups. Racine Scores of KA and KA+PAP-1 mice were analyzed using Mann-Whitney test. p < 0.05 was considered to suggest statistical significance.