AMPA Receptor Antagonists Facilitate NEDD4-2-Mediated GRIA1 Ubiquitination by Regulating PP2B-ERK1/2-SGK1 Pathway in Chronic Epilepsy Rats

The neural precursor cell expressed by developmentally downregulated gene 4-2 (NEDD4-2) is a ubiquitin E3 ligase that has a high affinity toward binding and ubiquitinating glutamate ionotropic receptor α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type subunit 1 (GRIA1, also referred to GluR1 or GluA1). Since dysregulation of GRIA1 surface expression is relevant to the responsiveness to AMPA receptor (AMPAR) antagonists (perampanel and GYKI 52466) in chronic epilepsy rats, it is likely that NEDD4-2 may be involved in the pathogenesis of intractable epilepsy. However, the role of NEDD4-2-mediated GRIA1 ubiquitination in refractory seizures to AMPAR antagonists is still unknown. In the present study, both AMPAR antagonists recovered the impaired GRIA1 ubiquitination by regulating protein phosphatase 2B (PP2B)-extracellular signal-regulated kinase 1/2 (ERK1/2)-serum and glucocorticoid-regulated kinase 1 (SGK1)-NEDD4-2 signaling pathway in responders (whose seizure activities are responsive to AMPAR), but not non-responders (whose seizure activities were uncontrolled by AMPAR antagonists). In addition, cyclosporin A (CsA, a PP2B inhibitor) co-treatment improved the effects of AMPAR antagonists in non-responders, independent of AKT signaling pathway. Therefore, our findings suggest that dysregulation of PP2B-ERK1/2-SGK1-NEDD4-2-mediated GRIA1 ubiquitination may be responsible for refractory seizures and that this pathway may be a potential therapeutic target for improving the treatment of intractable epilepsy in response to AMPAR antagonists.


Introduction
Epilepsy is a brain function disorder characterized by recurrent and unprovoked seizures. The prevalence of epilepsy in the general population is approximately 0.6−0.8% [1]. Initial/acute prolonged seizure (status epilepticus, SE), trauma, stroke or infections are postulated as precipitating factors of epilepsy [2]. Mesial temporal lobe epilepsy (MTLE) is the most common form of epilepsy and a medically intractable syndrome that is partially or totally uncontrolled by conventional anti-epileptic drug (AED) treatments [3]. Although disturbances in glutamatergic/GABAergic transmissions and the related signaling pathways are associated with the pathogenesis of MTLE in humans, the underlying mechanisms of MTLE remain largely unclear.
The pilocarpine model (including LiCI-pilocarpine model) serves as a reliable animal model of intractable epilepsy. The profiles of spontaneous recurrent seizures in this model resemble those of human MTLE. This model shows limbic seizures that become secondarily generalized, evolving to SE, which lasts for several hours (acute period). The SE is followed by a latent "seizure-free" period (about 15−30 days) and by a chronic period Biomedicines 2021, 9, 1069 2 of 22 characterized by the presence of spontaneous recurrent seizures. The lesions of mesial temporal structures, including a well-known hippocampal sclerosis, in this model are also similar to those of human MTLE patients [4,5]. Therefore, the pilocarpine model provides the opportunity to investigate the pathogenesis of MTLE.
On the other hand, multifactorial events are involved in the underlying mechanisms of pharmacoresistant epilepsy: (1) a reduced yield of AED concentration in the brain by hyper-activation of drug efflux transporter or sustained inflammatory conditions [23,24], (2) dysfunctions of ion/neurotransmitter channels of transporters [25], and (3) abnormal neural networks [25]. Interestingly, GRIA1 surface expression is higher in the hippocampus of chronic epilepsy rats than that of normal rats, which is attenuated by AMPAR antagonists (such as perampanel and GYKI 52466) in responders whose seizure activities are reduced by them [4,10,26]. Since ubiquitination of GRIA1 is linked to AMPAR surface expression and trafficking [27][28][29], it is postulated that AMPAR antagonists may modulate NEDD4-2 activity that is required for limiting GluA1 surface expression and functionality of AMPAR in the epileptic hippocampus. However, little data are available to describe whether NEDD4-2-mediated GRIA1 ubiquitination is changed, and this alteration is relevant to the generation of refractory seizures to AMPAR antagonists in a chronic epilepsy model. In the present study, therefore, we investigated the effects of AMPAR antagonists on NEDD4-2mediated GRIA1 regulation in responders and non-responders (whose seizure activities were uncontrolled by AMPAR antagonists) of a LiCl-pilocarpine epilepsy rat model to elucidate the role of NEDD4-2 in MTLE.
Here, we demonstrate that the anti-convulsive effects of AMPAR antagonists are closely related to the regulation of GRIA1 ubiquitination via protein phosphatase 2B (PP2B)extracellular signal-regulated kinase 1/2 (ERK1/2)-serum and glucocorticoid-regulated kinase 1 (SGK1)-NEDD4-2 signaling pathway. In addition, impairment of this signaling pathway resulted in refractory seizures to AMPAR antagonists, which was improved by cyclosporin A (CsA, a PP2B inhibitor) co-treatment. Therefore, our findings suggest that the PP2B-ERK1/2-SGK1-NEDD4-2 pathway may be a potential therapeutic strategy to improve the treatment of intractable MTLE in response to AMPAR antagonists.

Generation of Chronic Epilepsy Rats
Animals were intraperitoneally (i.p.) given LiCl (127 mg/kg) 24 h before pilocarpine treatment. On the next day, animals were treated with pilocarpine (30 mg/kg, i.p.) 20 min after atropine methylbromide (5 mg/kg i.p.). Two hours after SE on-set, animals were administered diazepam (Valium; Hoffman la Roche, Neuilly sur-Seine, France; 10 mg/kg, i.p.) as needed. Control animals received saline in place of pilocarpine. Animals were video-monitored 8 h a day for general behavior and occurrence of spontaneous seizures by four weeks after SE (Figure 1). We classified chronic epilepsy rats that showed behavioral seizures with seizure score ≥ 3 more than once.

Generation of Chronic Epilepsy Rats
Animals were intraperitoneally (i.p.) given LiCl (127 mg/kg) 24 h before pilocarpine treatment. On the next day, animals were treated with pilocarpine (30 mg/kg, i.p.) 20 min after atropine methylbromide (5 mg/kg i.p.). Two hours after SE on-set, animals were administered diazepam (Valium; Hoffman la Roche, Neuilly sur-Seine, France; 10 mg/kg, i.p.) as needed. Control animals received saline in place of pilocarpine. Animals were video-monitored 8 h a day for general behavior and occurrence of spontaneous seizures by four weeks after SE (Figure 1). We classified chronic epilepsy rats that showed behavioral seizures with seizure score ≥ 3 more than once.

Surgery
Control and epilepsy rats were implanted with monopolar stainless steel electrodes (Plastics One, Roanoke, VA, USA) in the right hippocampus (stereotaxic coordinates was −3.8 mm posterior; 2.0 mm lateral; −2.6 mm depth to bregma) under isoflurane anesthesia (3% induction, 1.5−2% for surgery, and 1.5% maintenance in a 65:35 mixture of N2O:O2). Some animals were also implanted with a brain infusion kit 1 (Alzet, Cupertino, CA, USA) to infuse with vehicle or cyclosporin A (CsA, a PP2B inhibitor, 250 μM) into the right lateral ventricle (1 mm posterior; 1.5 mm lateral; −3.5 mm depth to the bregma, see below). The CsA concentration did not affect spontaneous seizure activities in chronic epilepsy rats [30]. Throughout surgery, the core temperature of each rat was maintained at 37-38 °C. Electrodes were secured to the exposed skull with dental acrylic.

Surgery
Control and epilepsy rats were implanted with monopolar stainless steel electrodes (Plastics One, Roanoke, VA, USA) in the right hippocampus (stereotaxic coordinates was −3.8 mm posterior; 2.0 mm lateral; −2.6 mm depth to bregma) under isoflurane anesthesia (3% induction, 1.5−2% for surgery, and 1.5% maintenance in a 65:35 mixture of N 2 O:O 2 ). Some animals were also implanted with a brain infusion kit 1 (Alzet, Cupertino, CA, USA) to infuse with vehicle or cyclosporin A (CsA, a PP2B inhibitor, 250 µM) into the right lateral ventricle (1 mm posterior; 1.5 mm lateral; −3.5 mm depth to the bregma, see below). The CsA concentration did not affect spontaneous seizure activities in chronic epilepsy rats [30]. Throughout surgery, the core temperature of each rat was maintained at 37-38 • C. Electrodes were secured to the exposed skull with dental acrylic.
2.4. Drug Trials, EEG Analysis and Quantification of Behavioral Seizure Activity 2.4.1. Experiment I Figure 1 illustrates the design of the drug trial methodology, which was a modified protocol based on previous studies [10,26,30,31]. After baseline seizure activity was determined over three days, perampanel (8 mg/kg, i.p, Eisai Korea Inc., Seoul, Korea), GYKI 52466 (10 mg/kg, i.p.) or saline (vehicle) was daily administered at 6:00 PM over a oneweek period [4,30]. Electroencephalographic (EEG) signals were detected with a DAM 80 differential amplifier (0.1-3000 Hz bandpass; World Precision Instruments, Sarasota, FL, USA) 2 h a day at the same time over a one-week period. The data were digitized (1000 Hz) and analyzed using LabChart Pro v7 (ADInstruments, Bella Vista, New South Wales, Australia). Behavioral seizure severity was evaluated according to Racine's scale [32]: 1, immobility, eye closure, twitching of vibrissae, sniffing, facial clonus; 2, head nodding associated with more severe facial clonus; 3, clonus of one forelimb; 4, rearing, often accompanied by bilateral forelimb clonus; and 5, rearing with loss of balance and falling accompanied by generalized clonic seizures. After recording (18 h after the last drug treatment), animals were used for Western blot.

Experiment II
Some non-responders in experiment I were given saline (i.p.) over a seven-day period. Thereafter, perampanel or GYKI 52466 was daily administered by the aforementioned method. Non-responders were also connected with Alzet 1007D osmotic pump (Alzet, Cupertino, CA, USA) containing vehicle or CsA (250 µM). The pump was placed in a subcutaneous pocket in the dorsal region. After recording (18 h after the last drug treatment), animals were used for Western blot.

Western Blot
Animals were sacrificed by decapitation, and their hippocampi were obtained and homogenized in lysis buffer containing protease inhibitor cocktail (Roche Applied Sciences, Branford, CT, USA) and phosphatase inhibitor cocktail (PhosSTOP ® , Roche Applied Science, Branford, CT, USA). Thereafter, total protein concentration was calibrated using a Micro BCA Protein Assay Kit (Pierce Chemical, Rockford, IL, USA). Western blot was performed by the standard protocol: Sample proteins (10 µg) were separated on a Bis-Tris sodium dodecyl sulfate-poly-acrylamide gel (SDS-PAGE) and transferred to membranes. Membranes were incubated with 2% bovine serum albumin (BSA) in Tris-buffered saline (TBS; in mM 10 Tris, 150 NaCl, pH 7.5, and 0.05% Tween 20), and then reacted with primary antibodies (Table 1) overnight at 4 • C. After washing, membranes were incubated in a solution containing horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at room temperature. Immunoblots were detected and quantified using an ImageQuant LAS4000 system (GE Healthcare Korea, Seoul, Korea). Optical densities of proteins were calculated with the corresponding amount of β-actin.

Data Analysis
The Shapiro-Wilk W-test was used to evaluate the normality values. Mann-Whitney U-test, Wilcoxon signed rank test, Student's t-test, and paired Student's t-test were applied to determine statistical significance of data. Comparisons among groups were also performed using repeated measures ANOVA, Friedman test and one-way ANOVA followed by Bonferroni's post hoc comparisons. A p-value less than 0.05 was considered to be significant.

AMPAR Antagonists Facilitates NEDD4-2-Mediated GRIA1 Ubiquitination by Enhancing NEDD4-2 S448 Phosphorylation in Responders
NEDD4-2 plays an important role in the regulation of seizure susceptibility, and its phosphorylation level is closely related to the maintenance of its stability, which modulates AMPAR functionality [17,33]. Thus, we explored whether AMPAR antagonists affect NEDD4-2 protein expression and its phosphorylation levels.
Consistent with our previous study [30], the present study showed that PP2A protein level was 0.5 times the control level in the epileptic hippocampus (t (12) = 16.3, p < 0.001 vs. control animals, Student t-test; Figure 6A,B and Figure S4). PP2A phosphorylation level and its ratio were 0.28 (t (12) = 26, p < 0.001 vs. control animals, Student t-test) and 0.58 times (t (12) = 8, p < 0.001 vs. control animals, Student t-test) the control level in epilepsy rats, respectively ( Figure 6A,C,D and Figure S4). Similarly, PP2B protein level was reduced to 0.49 times the control level in the epileptic hippocampus (t (12) = 12.6, p < 0.001 vs. control animals, Student t-test; Figure 6A,E and Figure S4). PP2B phosphorylation level and its ratio were decreased to 0.26 (t (12) = 30.3, p < 0.001 vs. control animals, Student t-test) and 0.55 times (t (12) = 12.9, p < 0.001 vs. control animals, Student t-test) the control level in epilepsy rats, respectively (Figure 6A,F,G and Figure S4). Since phosphorylation negatively regulates PP2A and PP2B activities [45,46], these findings indicate that PP2A and PP2B activities may be enhanced in the epileptic hippocampus as compensatory responses to downregulation of their protein levels.

Co-Treatment of PP2B Inhibitor Increases the Efficacies of AMPAR Antagonists in Non-Responders
To confirm the role of the PP2B-ERK1/2-SGK1-NEDD4-2 signaling pathway in GRIA1 ubiquitination and refractory seizures to AMPAR antagonists, cyclosporin A (CsA, a PP2B inhibitor) was co-treated with perampanel or GYKI 52466 in non-responders. In non-responders to perampanel, total seizure frequency was 12.4 ± 3.1,

Co-Treatment of PP2B Inhibitor Increases the Efficacies of AMPAR Antagonists in Non-Responders
To confirm the role of the PP2B-ERK1/2-SGK1-NEDD4-2 signaling pathway in GRIA1 ubiquitination and refractory seizures to AMPAR antagonists, cyclosporin A (CsA, a PP2B inhibitor) was co-treated with perampanel or GYKI 52466 in non-responders. In nonresponders to perampanel, total seizure frequency was 12.4 ± 3.1, total seizure duration was 946.4 ± 153.1 s, and average seizure severity was 4 ± 0.4 over a one-week period (n = 5, Figure 7A-C). In non-responders to GYKI 52466, total seizure frequency was 12 ± 2.7, total seizure duration was 908.8 ± 146.9 s, and average seizure severity was 3.8 ± 0.3 over a one-week period (n = 5, Figure 7A-C).

Discussion
Although the pathogenesis of MTLE has been studied for decades, the underlying mechanisms of intractable MTLE are still elusive. Recently, the dysregulation of ubiquitination has been considered as a potential factor for the generation of refractory epilepsy, since ubiquitination is involved in the modulation of synaptic function [47,48]. Ubiquitination is a posttranslational modification that degrades proteins through a sequential reaction by the ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2) and ubiquitin ligase enzyme (E3) [47,48]. Among E3 ubiquitin ligases, NEDD4-2 has been focused on for its dysregulation of cellular trafficking/endocytosis and lysosomal degradation of ion channels and transporters in MTLE [17,34]. Under physiological conditions, NEDD4-2 contributes to the elevation of spontaneous neuronal activity, particularly spontaneous spike frequency, when the AMPAR is activated [11]. Indeed, NEDD4-2 andi mice (in whom one of the major forms of NEDD4-2 in the brain is selectively deficient) are less sensitive to AMPAR activation but very sensitive to AMPAR inhibition. Briefly, the direct AMPAR stimulation by AMPA treatment elevates the synchrony of neuronal activity in wild-type mice more than NEDD4-2 andi mice. In contrast, NEDD4-2 andi mice are very sensitive to 2,3dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX, an AMPAR antagonist) with regard to spontaneous spike frequency, although average spontaneous spike amplitude and electrode burst activity do not differ after AMPA or NBQX treatment for either genotype [11]. Paradoxically, NEDD4-2 andi mice show a higher seizure susceptibility than kainic acid, which is recovered by the genetically reducing GRIA1 level [11]. Furthermore, kainic acid-induced seizure activity increases NEDD4-2 ubiquitination without altered GRIA1 surface expression [17]. In addition, the upregulated NEDD4-2 ubiquitination increases the latency of seizure onset and seizure progression in response to kainic acid, accompanied by reduced GRIA1 ubiquitination [17]. Therefore, it is likely that the dysregulated GRIA1/AMPAR-mediated intracellular signaling pathway, rather than the direct AMPAR functionality, may contribute to the dysfunction of NEDD4-2-mediated GRIA1 ubiquitination in the epileptic hippocampus. In the present study, NEDD4-2 protein level was lower in the epileptic hippocampus than that in the normal one, concomitant with decreased phosphorylation levels. AMPAR antagonists effectively increased NEDD4-2 protein and its S448 phosphorylation level in responders, but not in non-responders. Furthermore, AMPAR inhibitions decreased NEDD4-2 ubiquitination in responders. Since NEDD4-2 itself is a substrate of the ubiquitin-proteasome system, which is negatively regulated by phosphorylation [17,49], AMPAR antagonists may increase NEDD4-2 protein level by inhibiting its ubiquitination. Compatible with NEDD4-2 protein level, furthermore, GRIA1 ubiquitination was reduced in epilepsy rats as compared to control animals, which was restored by both AMPAR antagonists in responders. Although it could not be excluded that the higher clearance of AMPAR antagonists by multidrug efflux systems or the lower affinities of AMPAR antagonists on GRIA1 due to allosteric changes of AMPAR [23][24][25] would decrease the efficacies of AMPAR antagonists in non-responders, our findings indicate that maladaptive regulation of intracellular signaling pathways for NEDD4-2-mediated GRIA1 ubiquitination may be one of the important factors in pharmacoresistant seizures to AMPAR antagonists.
In the present study, PP2A and PP2B expressions in chronic epilepsy rats were lower than those in control animals. Furthermore, their phosphorylation ratios were also reduced in epilepsy animals. Since phosphorylation inhibits protein phosphatase activities [45,46], it is likely that the reduced phosphorylation ratios of PP2A and PP2B may be compensatory responses for maintenance of their activities against downregulation of expressions. Consistent with our previous study [30], furthermore, both AMPAR antagonists elevated PP2B phosphorylation in responders, indicating the decreased PP2B phosphatase activity. Considering that PP2A and PP2B deactivate ERK1/2 kinase activity [42,43], these findings indicate that AMPAR antagonists may enhance ERK1/2 phosphorylation by in-hibiting PP2B activity. Indeed, CsA co-treatment improved the anti-epileptic effects of AMPAR antagonists in non-responders, concomitant with the increases in ERK1/2 and SGK1 S78 phosphorylation, NEDD4-2 protein level, NEDD4-2 S448 phosphorylation and ubiquitination of NEDD4-2 and GRIA1. Taken together, these findings indicate that dysregulation of PP2B-ERK1/2-SGK1 signaling pathway may play an important role in the generation of refractory seizures to AMPAR antagonists via impaired NEDD4-2-mediated GRIA1 ubiquitination.
PP2A also leads to SGK1 inactivation [44]. In the present study, however, neither AMPAR antagonist affected PP2A expression or its phosphorylation. Recently, we have reported that AMPAR antagonists inhibit casein kinase 2 (CK2) that binds to PP2A and increases PP2A activity [53]. Thus, the possibility that AMPAR antagonists may reduce CK2-PP2A-mediated SGK1 dephosphorylation in a phosphorylation-independent manner cannot be excluded.
Under physiological conditions, AMPARs contain the GRIA2 subunit, which are permeable to Na + and K + , but not Ca 2+ [54]. In chronic epilepsy rats, membrane GRIA1/GRIA2 ratio is significantly higher than that in control animals, indicating a preponderance of GRIA2-lacking (Ca 2+ -permeable) AMPAR [4,10,26]. A higher expression of Ca 2+ -permeable AMPAR in the epileptic hippocampus results in a subsequent elevated Ca 2+ influx followed by PP2B activation [55][56][57]. AMPAR antagonists decrease GRIA1/GRIA2 ratio in responders by reducing GRIA1, but not GRIA2, surface expression [4,10,26]. The present study also demonstrates that AMPAR antagonists increased GRIA1 ubiquitination in responders, which indicates the decreased Ca 2+ -permeable AMPAR level. Furthermore, the hyper-activation of AKT-glycogen synthase kinase 3β (GSK3β)-Ca 2+ /cAMP response element-binding protein (CREB) pathway leads to increased Ca 2+ -permeable AMPAR in non-responders [20,26], suggesting that dysregulation of AKT/GSK3β/CREB-mediated GRIA1 surface expression may also be responsible for the prolonged PP2B activation. Therefore, it is plausible that the increased Ca 2+ -permeable AMPAR expression would be a fundamental reason for the lack of response to the AMPAR antagonists in the non-responders. However, the present data show that the lower efficacies of AMPAR antagonists to the PP2B-ERK1/2-SGK1-NEDD4-2-mediated GRIA1 ubiquitination and the inhibition of spontaneous seizure activities (presumably due to the higher clearance of AMPAR antagonists or the lower affinities of AMPAR antagonists on GRIA1 [23][24][25]) in non-responders were improved by CsA co-treatment. If the upregulated GRIA2-lacking AMPAR expression resulted in refractory seizures to AMPAR antagonists, CsA co-treatment would not inhibit seizure activity in non-responders, since a lack of effects of CsA on neuronal excitability and seizure activity is well known [30,58,59]. Therefore, our findings suggest that the upregulated GRIA2-lacking AMPAR expression in non-responders may not be a primary cause of intractable seizures to AMPAR antagonists (although it may be relevant to ictogenesis), but may be a consequence of the irresponsiveness to AMPAR antagonists. Moreover, PP2B inhibition may enhance the efficacies of AMPAR antagonists in non-responders by recovering the dysregulation of ERK1/2-SGK1-NEDD4-2-mediated GRIA1 ubiquitination and secondarily reducing Ca 2+ -permeable AMPAR expression.
On the other hand, deubiquitination (the removal of ubiquitin moieties) also plays a role in AMPAR-mediated neurotransmission [60,61]. Ubiquitin-specific protease (USP) 46, a deubiquitinating enzyme, inhibits GRIA1 ubiquitination, accompanied by a decreased rate in GRIA1 degradation and an increase in AMPAR synaptic accumulation [60]. With respect to this, it is likely that downregulated GRIA1 deubiquitination may be involved in the intractable seizures to AMPAR antagonists, and CsA co-treatment would also inhibit deubiquitinases of GRIA1 in non-responders via unknown mechanisms. However, retigabine, an AED and a Kv7 channel opener, alleviates the acute stress-induced GRIA1 downregulation by increasing USP2 expression [61]. Therefore, further studies are needed to elucidate the role of GRIA1 deubiqutination in intractable seizures to AMPAR antagonists.

Conclusions
The present study reveals that AMPAR antagonists ameliorated spontaneous seizure activity by affecting the PP2B-mediated ERK1/2-SGK1-NEDD4-2 signaling pathway, which is relevant to the enhanced GRIA1 ubiquitination. In addition, the dysregulation of this pathway was one of the causes of refractory seizures to AMPAR antagonists. Therefore, our findings suggest that PP2B-ERK1/2-SGK1-NEDD4-2 pathway may be one of the potential therapeutic targets for the treatment of intractable TLE.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/biomedicines9081069/s1; Figure S1: Representative full-gel images of Western blots in Figure 3; Figure S2: Representative full-gel images of Western blots in Figure 4; Figure S3: Representative full-gel images of Western blots in Figure 5; Figure S4: Representative full-gel images of Western blots in Figure 6; Figure S5: Representative full-gel images of Western blots in Figure 8.