PDI augments kainic acid-induced seizure activity and neuronal death by inhibiting PP2A-GluA2-PICK1-mediated AMPA receptor internalization in the mouse hippocampus.

Protein disulfide isomerase (PDI) is a redox-active enzyme and also serves as a nitric oxide donor causing S-nitrosylation of cysteine residues in various proteins. Although PDI knockdown reduces α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR)-mediated neuronal activity, the underlying mechanisms are largely unknown. In the present study, we found that under physiological condition PDI knockdown increased CaMKII activity (phosphorylation) in the mouse hippocampus. However, PDI siRNA inhibited protein phosphatase (PP) 2A-mediated GluA2 S880 dephosphorylation by increasing PP2A oxidation, independent of S-nitrosylation. PDI siRNA also enhanced glutamate ionotropic receptor AMPA type subunit 1 (GluA1) S831 and GluA2 S880, but not GluA1 S845 and GluA2 Y869/Y873/Y876 phosphorylations, concomitant with the enhanced protein interacting with C kinase 1 (PICK1)-mediated AMPAR internalization. Furthermore, PDI knockdown attenuated seizure activity and neuronal damage in response to kainic acid (a non-desensitizing agonist of AMPAR). Therefore, these findings suggest that PDI may regulate surface AMPAR expression through PP2A-GluA2-PICK1 signaling pathway, and that PDI may be one of the therapeutic targets for epilepsy via AMPAR internalization without altering basal neurotransmission.

Distinct Roles of CK2- and AKT-Mediated NF-κB Phosphorylations in Clasmatodendrosis (Autophagic Astroglial Death) within the Hippocampus of Chronic Epilepsy Rats.

The downregulation of glutathione peroxidase-1 (GPx1) plays a role in clasmatodendrosis (an autophagic astroglial death) in the hippocampus of chronic epilepsy rats. Furthermore, N-acetylcysteine (NAC, a GSH precursor) restores GPx1 expression in clasmatodendritic astrocytes and alleviates this autophagic astroglial death, independent of nuclear factor erythroid-2-related factor 2 (Nrf2) activity. However, the regulatory signal pathways of these phenomena have not been fully explored. In the present study, NAC attenuated clasmatodendrosis by alleviating GPx1 downregulation, casein kinase 2 (CK2)-mediated nuclear factor-κB (NF-κB) serine (S) 529 and AKT-mediated NF-κB S536 phosphorylations. 2-[4,5,6,7-Tetrabromo-2-(dimethylamino)-1H-benzo[d]imidazole-1-yl]acetic acid (TMCB; a selective CK2 inhibitor) relieved clasmatodendritic degeneration and GPx1 downregulation concomitant with the decreased NF-κB S529 and AKT S473 phosphorylations. In contrast, AKT inhibition by 3-chloroacetyl-indole (3CAI) ameliorated clasmatodendrosis and NF-κB S536 phosphorylation, while it did not affect GPx1 downregulation and CK2 tyrosine (Y) 255 and NF-κB S529 phosphorylations. Therefore, these findings suggest that seizure-induced oxidative stress may diminish GPx1 expression by increasing CK2-mediated NF-κB S529 phosphorylation, which would subsequently enhance AKT-mediated NF-κB S536 phosphorylation leading to autophagic astroglial degeneration.

Epigallocatechin-3-Gallate Attenuates Leukocyte Infiltration in 67-kDa Laminin Receptor-Dependent and -Independent Pathways in the Rat Frontoparietal Cortex following Status Epilepticus.

Status epilepticus (SE) evokes leukocyte infiltration in the frontoparietal cortex (FPC) without the blood-brain barrier disruption. Monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-2 (MIP-2) regulate leukocyte recruitments into the brain parenchyma. Epigallocatechin-3-gallate (EGCG) is an antioxidant and a ligand for non-integrin 67-kDa laminin receptor (67LR). However, it is unknown whether EGCG and/or 67LR affect SE-induced leukocyte infiltrations in the FPC. In the present study, SE infiltrated myeloperoxidase (MPO)-positive neutrophils, as well as cluster of differentiation 68 (CD68)-positive monocytes in the FPC are investigated. Following SE, MCP-1 was upregulated in microglia, which was abrogated by EGCG treatment. The C-C motif chemokine receptor 2 (CCR2, MCP-1 receptor) and MIP-2 expressions were increased in astrocytes, which were attenuated by MCP-1 neutralization and EGCG treatment. SE reduced 67LR expression in astrocytes, but not endothelial cells. Under physiological conditions, 67LR neutralization did not lead to MCP-1 induction in microglia. However, it induced MIP-2 expression and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in astrocytes and leukocyte infiltration in the FPC. Co-treatment of EGCG or U0126 (an ERK1/2 inhibitor) attenuated these events induced by 67LR neutralization. These findings indicate that the EGCG may ameliorate leukocyte infiltration in the FPC by inhibiting microglial MCP-1 induction independent of 67LR, as well as 67LR-ERK1/2-MIP-2 signaling pathway in astrocytes.

PLPP/CIN-mediated NF2 S10 dephosphorylation distinctly regulates kainate-induced seizure susceptibility and neuronal death through PAK1-NF-κB-COX-2-PTGES2 signaling pathway.

BACKGROUND: Pyridoxal-5'-phosphate phosphatase/chronophin (PLPP/CIN) selectively dephosphorylates serine (S) 10 site on neurofibromin 2 (NF2, also known as merlin (moesin-ezrin-radixin-like protein) or schwannomin). p21-activated kinase 1 (PAK1) is a serine/threonine protein kinase, which is involved in synaptic activity and plasticity in neurons. NF2 and PAK1 reciprocally regulate each other in a positive feedback manner. Thus, the aim of the present study is to investigate the effects of PLPP/CIN-mediated NF2 S10 dephosphorylation on PAK1-related signaling pathways under physiological and neuroinflammatory conditions, which are largely unknown. METHODS: After kainate (KA) injection in wild-type, PLPP/CIN-/- and PLPP/CINTg mice, seizure susceptibility, PAK1 S204 autophosphorylation, nuclear factor-κB (NF-κB) p65 S276 phosphorylation, cyclooxygenase-2 (COX-2) upregulation, prostaglandin E synthase 2 (PTGES2) induction and neuronal damage were measured. The effects of 1,1'-dithiodi-2-naphthtol (IPA-3, a selective inhibitor of PAK1) pretreatment on these responses to KA were also validated. RESULTS: PLPP/CIN overexpression increased PAK1 S204 autophosphorylation concomitant with the enhanced NF2 S10 dephosphorylation in hippocampal neurons under physiological condition. Following KA treatment, PLPP/CIN overexpression delayed the seizure on-set and accelerated PAK1 S204 phosphorylation, NF-κB p65 S276 phosphorylation, COX-2 upregulation and PTGES2 induction, which were ameliorated by PLPP/CIN deletion or IPA-3. Furthermore, IPA-3 pretreatment shortened the latency of seizure on-set without affecting seizure severity (intensity) and ameliorated CA3 neuronal death induced by KA. CONCLUSIONS: These findings indicate that PLPP/CIN may regulate seizure susceptibility (the latency of seizure on-set) and CA3 neuronal death in response to KA through NF2-PAK1-NF-κB-COX-2-PTGES2 signaling pathway.

Deregulation of Astroglial TASK-1 K+ Channel Decreases the Responsiveness to Perampanel-Induced AMPA Receptor Inhibition in Chronic Epilepsy Rats.

Tandem of P domains in a weak inwardly rectifying K+ channel (TWIK)-related acid sensitive K+-1 channel (TASK-1) is activated under extracellular alkaline conditions (pH 7.2-8.2), which are upregulated in astrocytes (particularly in the CA1 region) of the hippocampi of patients with temporal lobe epilepsy and chronic epilepsy rats. Perampanel (PER) is a non-competitive α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) antagonist used for the treatment of focal seizures and primary generalized tonic-clonic seizures. Since AMPAR activation leads to extracellular alkaline shifts, it is likely that the responsiveness to PER in the epileptic hippocampus may be relevant to astroglial TASK-1 regulation, which has been unreported. In the present study, we found that PER ameliorated astroglial TASK-1 upregulation in responders (whose seizure activities were responsive to PER), but not non-responders (whose seizure activities were not responsive to PER), in chronic epilepsy rats. ML365 (a selective TASK-1 inhibitor) diminished astroglial TASK-1 expression and seizure duration in non-responders to PER. ML365 co-treatment with PER decreased spontaneous seizure activities in non-responders to PER. These findings suggest that deregulation of astroglial TASK-1 upregulation may participate in the responsiveness to PER, and that this may be a potential target to improve the efficacies of PER.

CDDO-Me Abrogates Aberrant Mitochondrial Elongation in Clasmatodendritic Degeneration by Regulating NF-κB-PDI-Mediated S-Nitrosylation of DRP1.

Clasmatodendrosis is a kind of astroglial degeneration pattern which facilitates excessive autophagy. Although abnormal mitochondrial elongation is relevant to this astroglial degeneration, the underlying mechanisms of aberrant mitochondrial dynamics are still incompletely understood. Protein disulfide isomerase (PDI) is an oxidoreductase in the endoplasmic reticulum (ER). Since PDI expression is downregulated in clasmatodendritic astrocytes, PDI may be involved in aberrant mitochondrial elongation in clasmatodendritic astrocytes. In the present study, 26% of CA1 astrocytes showed clasmatodendritic degeneration in chronic epilepsy rats. 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me; bardoxolone methyl or RTA 402) and SN50 (a nuclear factor-κB (NF-κB) inhibitor) ameliorated the fraction of clasmatodendritic astrocytes to 6.8 and 8.1% in CA1 astrocytes, accompanied by the decreases in lysosomal-associated membrane protein 1 (LAMP1) expression and microtubule-associated protein 1A/1B light-chain 3 (LC3)-II/LC3-I ratio, indicating the reduced autophagy flux. Furthermore, CDDO-Me and SN50 reduced NF-κB S529 fluorescent intensity to 0.6- and 0.57-fold of vehicle-treated animal level, respectively. CDDO-Me and SN50 facilitated mitochondrial fission in CA1 astrocytes, independent of dynamin-related protein 1 (DRP1) S616 phosphorylation. In chronic epilepsy rats, total PDI protein, S-nitrosylated PDI (SNO-PDI), and SNO-DRP1 levels were 0.35-, 0.34- and 0.45-fold of control level, respectively, in the CA1 region and increased CDDO-Me and SN50. Furthermore, PDI knockdown resulted in mitochondrial elongation in intact CA1 astrocytes under physiological condition, while it did not evoke clasmatodendrosis. Therefore, our findings suggest that NF-κB-mediated PDI inhibition may play an important role in clasmatodendrosis via aberrant mitochondrial elongation.

PLPP/CIN inhibits dopamine D1 receptor-mediated seizure activity via DARPP-32 serine 97 dephosphorylation in the mouse hippocampus.

Dopamine plays a central role in the regulation of psychomotor functions in the brain. Furthermore, the dopaminergic system is involved in the ictogenesis in human patients and animal models of epilepsy. Dopamine and cAMP-regulated phosphoprotein, 32 kDa (DARPP-32) plays an important role in the regulation of interactions between dopamine and glutamate receptors in neurons. Indeed, SKF 83822 (a specific D1 receptor agonist) facilitates DARPP-32-mediated protein phosphatase 1 (PP1) inhibition leading to the increase in phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR), which potentiates channel activities and currents and thereby generates seizure activity. In the present study, we found that pyridoxal-5'-phosphate phosphatase/chronophin (PLPP/CIN), a selective phosphatase for serine (S) residues, attenuated seizure susceptibility in response to SKF 83822 by dephosphorylating DARPP-32 S97 site. Similarly, inhibition of DARPP-32 S97 phosphorylation by 2-[4,5,6,7-Tetrabromo-2-(dimethylamino)-1H-benzo[d]imidazole-1-yl]acetic acid (TMCB; a selective casein kinase 2 inhibitor) attenuated SKF 83822-induced seizure activity. These inhibitory effects of PLPP/CIN and TMCB were relevant to the regulations of DARPP-32-PP1-AMPAR signaling pathway. Therefore, our findings suggest that PLPP/CIN may be a modulator in dopaminergic neurotransmission as well as glutamatergic systems, and that the PLPP/CIN-mediated DARPP-32 regulation may be one of the potential therapeutic targets for medication of seizure or epilepsy induced by D1 receptor hyperactivation.

Sp1-Mediated Prdx6 Upregulation Leads to Clasmatodendrosis by Increasing Its aiPLA2 Activity in the CA1 Astrocytes in Chronic Epilepsy Rats.

Clasmatodendrosis is an autophagic astroglial degeneration (a non-apoptotic (type II) programmed cell death) whose underlying mechanisms are fully understood. Peroxiredoxin-6 (Prdx6), the "non-selenium glutathione peroxidase (NSGPx)", is the only member of the 1-cysteine peroxiredoxin family. Unlike the other Prdx family, Prdx6 has multiple functions as glutathione peroxidase (GPx) and acidic calcium-independent phospholipase (aiPLA2). The present study shows that Prdx6 was upregulated in CA1 astrocytes in chronic epilepsy rats. 2-Cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me) and N-acetylcysteine (NAC, a precursor of glutathione) ameliorated clasmatodendrosis accompanied by reduced Prdx6 level in CA1 astrocytes. Specificity protein 1 (Sp1) expression was upregulated in CA1 astrocyte, which was inhibited by mithramycin A (MMA). MMA alleviated clasmatodendrosis and Prdx6 upregulation. Sp1 expression was also downregulated by CDDO-Me and NAC. Furthermore, 1-hexadecyl-3-(trifluoroethgl)-sn-glycerol-2 phosphomethanol (MJ33, a selective inhibitor of aiPLA2 activity of Prdx6) attenuated clasmatodendrosis without affecting Prdx6 expression. All chemicals shortened spontaneous seizure duration but not seizure frequency and behavioral seizure severity in chronic epilepsy rats. Therefore, our findings suggest that Sp1 activation may upregulate Prdx6, whose aiPLA2 activity would dominate over GPx activity in CA1 astrocytes and may lead to prolonged seizure activity due to autophagic astroglial degeneration.

PLPP/CIN-mediated DARPP-32 serine 97 dephosphorylation delays the seizure onset in response to kainic acid in the mouse hippocampus.

Dopamine and cAMP-regulated phosphoprotein, 32 kDa (DARPP-32)-mediated protein phosphatase 1 (PP1) inhibition leads to the increase in phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR), which potentiates channel activity and current and thereby may facilitate seizure activity. In the present study, we found that pyridoxal-5'-phosphate phosphatase/chronophin (PLPP/CIN) transiently dephosphorylated DARPP-32 serine (S) 97 site in the early time window, and casein kinase 2 (CK2) subsequently phosphorylated this site in the later time points after kainic acid (KA) injection, which increased the latency of seizure onset in response to KA, but exacerbated the intensity (severity), duration and progression of seizures. TMCB (a CK2 inhibitor) delayed the seizure onset in response to KA, concomitant with the reduced DARPP-32 S97 phosphorylation. Therefore, our findings suggest that PLPP/CIN may play an important role in the latency of seizure onset via DARPP-32-PP1-AMPAR signaling pathway, and may be one of the potential therapeutic targets for medication of seizure or epilepsy.

2,4-Diacetylphloroglucinol Reduces Beta-Amyloid Production and Secretion by Regulating ADAM10 and Intracellular Trafficking in Cellular and Animal Models of Alzheimer's Disease.

There is currently no effective treatment against Alzheimer's disease (AD), although many strategies have been applied to reduce beta-amyloid (Aß) levels. Here, we investigated 2,4-diacetylphloroglucinol (DAPG) effects on Aß levels and mechanisms of action. DAPG was the most effective phloroglucinol derivative for reducing Aß levels, without being toxic, in various models including HEK293 cells overexpressing Swedish mutant amyloid precursor protein (APP) (293sw), primary astrocytes isolated from APPsw/PS1dE9 transgenic mice, and after intrahippocampal injection of DAPG in APPsw/PS1dE9 transgenic mice. DAPG-mediated Aß reduction was associated with increased soluble APPα (sAPPα) levels mediated by a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) but not ADAM17. ADAM10 inhibition in DAPG-treated cells prevented the effects on sAPPα but only partly on intracellular and secreted Aß. To identify regulators of sAPPα and Aß secretion, various inhibitors of intracellular trafficking were administered with DAPG. Brefeldin A (BFA) reversed DAPG-mediated changes in Aß secretion in 293sw cells, whereas golgicide A (GCA) and BFA were effective in primary astrocytes, indicating a cell type-specific regulation of the trafficking. Moreover, GCA or BFA effects on sAPPα, but not Aß, levels in primary astrocytes resembled those of ADAM10 inhibition, indicating at least partly independent trafficking pathways for sAPPα and Aß. In conclusion, DAPG might be a promising drug candidate against AD regulating ADAM10 and intracellular trafficking, but optimizing DAPG ability to cross the BBB will be needed.

CDDO-Me Attenuates CA1 Neuronal Death by Facilitating RalBP1-Mediated Mitochondrial Fission and 4-HNE Efflux in the Rat Hippocampus Following Status Epilepticus.

Ras-related protein Ral-A (RalA)-binding protein 1 (RalBP1, also known as Ral-interacting protein of 76 kDa (RLIP76) or Ral-interacting protein 1 (RLIP1 or RIP1)) is involved in the efflux of 4-hydroxynonenal (4-HNE, an end product of lipid peroxidation), as well as mitochondrial fission. In the present study, we found that 2-cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me) attenuated CA1 neuronal death and aberrant mitochondrial elongations in these neurons coupled with enhanced RalBP1 expression and reduced 4-HNE levels following status epilepticus (SE). RalBP1 knockdown did not affect mitochondrial dynamics and CA1 neuronal death under physiological and post-SE conditions. Following SE, however, cotreatment of RalBP1 siRNA diminished the effect of CDDO-Me on 4-HNE levels, mitochondrial hyperfusion in CA1 neurons, and CA1 neuronal death. These findings indicate that CDDO-Me may ameliorate CA1 neuronal death by facilitating RalBP1-mediated 4-HNE efflux and mitochondrial fission following SE. Therefore, our findings suggest that increased RalBP1 expression/activity may be one of the considerable targets to protect neurons from SE.

CDDO-Me Attenuates Clasmatodendrosis in CA1 Astrocyte by Inhibiting HSP25-AKT Mediated DRP1-S637 Phosphorylation in Chronic Epilepsy Rats.

Clasmatodendrosis is one of the irreversible astroglial degeneration, which is involved in seizure duration and its progression in the epileptic hippocampus. Although sustained heat shock protein 25 (HSP25) induction leads to this autophagic astroglial death, dysregulation of mitochondrial dynamics (aberrant mitochondrial elongation) is also involved in the pathogenesis in clasmatodendrosis. However, the underlying molecular mechanisms of accumulation of elongated mitochondria in clasmatodendritic astrocytes are elusive. In the present study, we found that clasmatodendritic astrocytes showed up-regulations of HSP25 expression, AKT serine (S) 473 and dynamin-related protein 1 (DRP1) S637 phosphorylations in the hippocampus of chronic epilepsy rats. 2-Cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me; bardoxolone methyl or RTA 402) abrogated abnormal mitochondrial elongation by reducing HSP25 upregulation, AKT S473- and DRP1 S637 phosphorylations. Furthermore, HSP25 siRNA and 3-chloroacetyl-indole (3CAI, an AKT inhibitor) abolished AKT-DRP1-mediated mitochondrial elongation and attenuated clasmatodendrosis in CA1 astrocytes. These findings indicate that HSP25-AKT-mediated DRP1 S637 hyper-phosphorylation may lead to aberrant mitochondrial elongation, which may result in autophagic astroglial degeneration. Therefore, our findings suggest that the dysregulation of HSP25-AKT-DRP1-mediated mitochondrial dynamics may play an important role in clasmatodendrosis, which would have implications for the development of novel therapies against various neurological diseases related to astroglial degeneration.

Glutathione Regulates GPx1 Expression during CA1 Neuronal Death and Clasmatodendrosis in the Rat Hippocampus following Status Epilepticus.

Glutathione peroxidase-1 (GPx1) catalyze the reduction of H2O2 by using glutathione (GSH) as a cofactor. However, the profiles of altered GPx1 expression in response to status epilepticus (SE) have not been fully explored. In the present study, GPx1 expression was transiently decreased in dentate granule cells, while it was temporarily enhanced and subsequently reduced in CA1 neurons following SE. GPx1 expression was also transiently declined in CA1 astrocytes (within the stratum radiatum) following SE. However, it was elevated in reactive CA1 astrocytes, but not in clasmatodendritic CA1 astrocytes, in chronic epilepsy rats. Under physiological condition, L-buthionine sulfoximine (BSO, an inducer of GSH depletion) increased GPx1 expression in CA1 neurons but decreased it in CA1 astrocytes. However, N-acetylcysteine (NAC, an inducer of GSH synthesis) did not influence GPx1 expression in these cell populations. Following SE, BSO aggravated CA1 neuronal death, concomitant with reduced GPx1 expression. Further. BSO also lowered GPx1 expression in CA1 astrocytes. NAC effectively prevented neuronal death and GPx1 downregulation in CA1 neurons, and restored GPx1 expression to the control level in CA1 astrocytes. In chronic epilepsy rats, BSO reduced GPx1 intensity and exacerbated clasmatodendritic degeneration in CA1 astrocytes. In contrast, NAC restored GPx1 expression in clasmatodendritic astrocytes and ameliorated this autophagic astroglial death. To the best of our knowledge, our findings report, for the first time, the spatiotemporal profiles of altered GPx1 expression in the rat hippocampus following SE, and suggest GSH-mediated GPx1 regulation, which may affect SE-induced neuronal death and autophagic astroglial degeneration.

P2X7 Receptor Augments LPS-Induced Nitrosative Stress by Regulating Nrf2 and GSH Levels in the Mouse Hippocampus.

P2X7 receptor (P2X7R) regulates inducible nitric oxide synthase (iNOS) expression/activity in response to various harmful insults. Since P2X7R deletion paradoxically decreases the basal glutathione (GSH) level in the mouse hippocampus, it is likely that P2X7R may increase the demand for GSH for the maintenance of the intracellular redox state or affect other antioxidant defense systems. Therefore, the present study was designed to elucidate whether P2X7R affects nuclear factor-erythroid 2-related factor 2 (Nrf2) activity/expression and GSH synthesis under nitrosative stress in response to lipopolysaccharide (LPS)-induced neuroinflammation. In the present study, P2X7R deletion attenuated iNOS upregulation and Nrf2 degradation induced by LPS. Compatible with iNOS induction, P2X7R deletion decreased S-nitrosylated (SNO)-cysteine production under physiological and post-LPS treated conditions. P2X7R deletion also ameliorated the decreases in GSH, glutathione synthetase, GS and ASCT2 levels concomitant with the reduced S-nitrosylations of GS and ASCT2 following LPS treatment. Furthermore, LPS upregulated cystine:glutamate transporter (xCT) and glutaminase in P2X7R+/+ mice, which were abrogated by P2X7R deletion. LPS did not affect GCLC level in both P2X7R+/+ and P2X7R-/- mice. Therefore, our findings indicate that P2X7R may augment LPS-induced neuroinflammation by leading to Nrf2 degradation, aberrant glutamate-glutamine cycle and impaired cystine/cysteine uptake, which would inhibit GSH biosynthesis. Therefore, we suggest that the targeting of P2X7R, which would exert nitrosative stress with iNOS in a positive feedback manner, may be one of the important therapeutic strategies of nitrosative stress under pathophysiological conditions.

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.

Inhibition of AKT/GSK3ß/CREB Pathway Improves the Responsiveness to AMPA Receptor Antagonists by Regulating GRIA1 Surface Expression in Chronic Epilepsy Rats.

α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) has been reported as one of the targets for treatment of epilepsy. Although maladaptive regulation of surface expression of glutamate ionotropic receptor AMPA type subunit 1 (GRIA1) subunit is relevant to the responsiveness to AMPAR antagonists (perampanel and GYKI 52466) in LiCl-pilocarpine-induced chronic epilepsy rats, the underlying mechanisms of refractory seizures to AMPAR antagonists have yet been unclear. In the present study, we found that both AMPAR antagonists restored the up-regulations of GRIA1 surface expression and Src family-mediated glycogen synthase kinase 3ß (GSK3ß)-Ca2+/cAMP response element-binding protein (CREB) phosphorylations to control levels in responders (whose seizure activities were responsive to AMPAR) but not non-responders (whose seizure activities were uncontrolled by AMPAR antagonists). In addition, 3-chloroacetyl indole (3CAI, an AKT inhibitor) co-treatment attenuated spontaneous seizure activities in non-responders, accompanied by reductions in AKT/GSK3ß/CREB phosphorylations and GRIA1 surface expression. Although AMPAR antagonists reduced GRIA2 tyrosine (Y) phosphorylations in responders, they did not affect GRIA2 surface expression and protein interacting with C kinase 1 (PICK1) protein level in both responders and non-responders. Therefore, our findings suggest that dysregulation of AKT/GSK3ß/CREB-mediated GRIA1 surface expression may be responsible for refractory seizures in non-responders, and that this pathway may be a potential target to improve the responsiveness to AMPAR antagonists.

Regional specific activations of ERK1/2 and CDK5 differently regulate astroglial responses to ER stress in the rat hippocampus following status epilepticus.

Endoplasmic reticulum (ER) triggers the regional specific astroglial responses to status epilepticus (SE, a prolonged seizure activity). However, the epiphenomena/downstream effecters for ER stress and the mechanism of ER stress signaling in astroglial apoptosis have not been fully understood. In the present study, tunicamycin-induced ER stress resulted in reactive astrogliosis-like events showing astroglial hypertrophy with the elevated extracellular signal-activated protein kinase 1/2 (ERK1/2) and cyclin-dependent kinase 5 (CDK5) phosphorylations in the CA1 region of the rat hippocampus. However, tunicamycin increased CDK5, but not ERK1/2, phosphorylation in the molecular layer of the dentate gyrus. Roscovitine (a CDK5 inhibitor) suppressed the effect of tunicamycin in the molecular layer of the dentate gyrus and the CA1 region, while U0126 (an ERK1/2 inhibitor) reversed it in the CA1 region. Salubrinal (an ER stress inhibitor) abrogated activations of ERK1/2 and CDK5, and attenuated reactive astrogliosis in the CA1 region and astroglial apoptosis in the molecular layer of the dentate gyrus following status epilepticus (SE, a prolonged seizure activity). These findings indicate that ER stress may induce reactive astrogliosis via ERK1/2-mediated CDK5 activation in the CA1 region. In the molecular layer of the dentate gyrus, however, ER stress may participate in astroglial apoptosis through ERK1/2-independent CDK5 activation following SE.

PLPP/CIN-mediated NF2-serine 10 dephosphorylation regulates F-actin stability and Mdm2 degradation in an activity-dependent manner.

Neurofibromin 2 (NF2, also known as merlin) is a tumor suppressor protein encoded by the neurofibromatosis type 2 gene NF2. NF2 is also an actin-binding protein that functions in an intrinsic signaling network critical for actin dynamics. Although protein kinase A (PKA)-mediated NF2-serin (S) 10 phosphorylation stabilizes filamentous actin (F-actin), the underlying mechanisms of NF2-S10 dephosphorylation and the role of NF2 in seizures have been elusive. Here, we demonstrate that pyridoxal-5'-phosphate phosphatase/chronophin (PLPP/CIN) dephosphorylated NF2-S10 site as well as cofilin-S3 site. In addition, NF2-S10 dephosphorylation reversely regulated murine double minute-2 (Mdm2) and postsynaptic density 95 (PSD95) degradations in an activity-dependent manner, which increased seizure intensity and its progression in response to kainic acid (KA). In addition, NF2 knockdown facilitated seizure intensity and its progress through F-actin instability independent of cofilin-mediated actin dynamics. Therefore, we suggest that PLPP/CIN may be a potential therapeutic target for epileptogenesis and NF2-associated diseases.

Protein disulfide isomerase-mediated S-nitrosylation facilitates surface expression of P2X7 receptor following status epilepticus.

BACKGROUND: P2X7 receptor (P2X7R) is an ATP-gated nonselective cationic channel playing important roles in a variety of physiological functions, including inflammation, and apoptotic or necrotic cell death. An extracellular domain has ten cysteine residues forming five intrasubunit disulfide bonds, which are needed for the P2X7R trafficking to the cell surface and the recognition of surface epitopes of apoptotic cells and bacteria. However, the underlying mechanisms of redox/S-nitrosylation of cysteine residues on P2X7R and its role in P2X7R-mediated post-status epilepticus (SE, a prolonged seizure activity) events remain to be answered. METHODS: Rats were given pilocarpine (380 mg/kg i.p.) to induce SE. Animals were intracerebroventricularly infused Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME, a NOS inhibitor) 3 days before SE, or protein disulfide isomerase (PDI) siRNA 1 day after SE using an osmotic pump. Thereafter, we performed Western blot, co-immunoprecipitation, membrane fraction, measurement of S-nitrosylated (SNO)-thiol and total thiol, Fluoro-Jade B staining, immunohistochemistry, and TUNEL staining. RESULTS: SE increased S-nitrosylation ratio of P2X7R and the PDI-P2X7R bindings, which were abolished by L-NAME and PDI knockdown. In addition, both L-NAME and PDI siRNA attenuated SE-induced microglial activation and astroglial apoptosis. L-NAME and PDI siRNA also ameliorated the increased P2X7R surface expression induced by SE. CONCLUSIONS: These findings suggest that PDI-mediated redox/S-nitrosylation may facilitate the trafficking of P2X7R, which promotes microglial activation and astroglial apoptosis following SE. Therefore, our findings suggest that PDI-mediated regulations of dynamic redox status and S-nitrosylation of P2X7R may be a critical mechanism in the neuroinflammation and astroglial death following SE.

Src/CK2/PTEN-Mediated GluN2B and CREB Dephosphorylations Regulate the Responsiveness to AMPA Receptor Antagonists in Chronic Epilepsy Rats.

Both α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor (AMPAR) and N-methyl-D-aspartate receptor (NMDAR) have been reported as targets for treatment of epilepsy. To investigate the roles and interactions of AMPAR and NMDAR in ictogenesis of epileptic hippocampus, we analyzed AMPAR antagonists (perampanel and GYKI 52466)-mediated phosphatase and tensin homolog deleted on chromosome 10 (PTEN) regulation and glutamate ionotropic receptor NMDA type subunit 2B (GluN2B) tyrosine (Y) 1472 phosphorylation in epilepsy rats. Both perampanel and GYKI 52466 increased PTEN expression and its activity (reduced phosphorylation), concomitant with decreased activities (phosphorylations) of Src family-casein kinase 2 (CK2) signaling pathway. Compatible with these, they also restored the upregulated GluN2B Y1472 and Ca2+/cAMP response element-binding protein (CREB) serine (S) 133 phosphorylations and surface expression of glutamate ionotropic receptor AMPA type subunit 1 (GRIA1) to basal level in the epileptic hippocampus. These effects of perampanel and GYKI 52466 are observed in responders (whose seizure activities are responsive to AMPAR antagonists), but not non-responders (whose seizure activities were uncontrolled by AMPAR antagonists). Therefore, our findings suggest that Src/CK2/PTEN-mediated GluN2B Y1472 and CREB S133 regulations may be one of the responsible signaling pathways for the generation of refractory seizures in non-responders to AMPAR antagonists.