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N-Methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) activations induce fast and transient mitochondrial fragmentation under pathophysiological conditions. However, it is still unknown whether NMDAR or AMPAR activity contributes to mitochondrial dynamics under physiological conditions. In the present study, MK801 (a non-competitive NMDAR antagonist) did not affect mitochondrial length in hippocampal neurons as well as phosphorylation levels of dynamin-related protein 1 (DRP1)-serine (S) 616, extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK) and AMPAR. In contrast, perampanel (a non-competitive AMPAR antagonist) elongated mitochondrial length in neurons concomitant with diminishing phosphorylations of DRP1-S616, ERK1/2, and JNK, but not p38 MAPK. Perampanel also reduced protein phosphatase (PP) 1, PP2A and PP2B phosphorylations, indicating activations of these PPs which were unaffected by MK801. U0126 (an ERK1/2 inhibitor) elongated mitochondrial length, accompanied by the reduced DRP1-S616 phosphorylation. SP600125 (a JNK inhibitor) did not influence mitochondrial length and DRP1 phosphorylations. Okadaic acid (a PP1/PP2A inhibitor) reduced mitochondrial length with the up-regulated DRP1-S616 phosphorylation, while CsA (a PP2B inhibitor) increased it with the elevated DRP1-S637 phosphorylation. Co-treatment of okadaic acid or CsA with perampanel attenuated the reductions in DRP1-S616 and -S637 phosphorylation without changing DRP1 expression level, respectively. GYKI 52466 (another non-competitive AMPAR antagonist) showed the similar effects of perampanel on phosphorylations of DRP1, ERK1/2, JNK, PPs, and GluR1 AMPAR subunits. Taken together, our findings suggest that a blockade of AMPAR may regulate the cooperation of ERK1/2- and PP1/PP2A for the modulation of DRP1 phosphorylations, which facilitate mitochondrial fusion.

To elucidate the pharmacological properties of perampanel [2-(2-oxo-1-phenyl-5-pyridin-2-yl-1,2-dihydropyridin-3-yl)benzonitrile, a novel non-competitive antagonist of AMPA receptor], we investigated its effects on the up-stream regulatory pathways of GluA1 phosphorylation including protein kinase C (PKC), Ca2+-calmodulin-dependent protein kinase II (CAMKII), protein kinase A (PKA), extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), protein phosphatase (PP) 1, PP2A, and PP2B in normal and pilocarpine-induced epileptic rat model using Western blot analysis. In normal animals, perampanel affected GluA1 expression/phosphorylation, PKC, CAMKII, PKA, ERK1/2, JNK, and PPs activities. In epileptic rats, perampanel effectively inhibited spontaneous seizure activities. Perampanel enhanced phospho (p)-GluA1-S831 and -S845 ratios (phosphoprotein/total protein), while it reduced GluA1 expression. Perampanel also increased pCAMKII and pPKA ratios, which phosphorylate GluA1-S831 and -S845 site, respectively. Perampanel elevated pJNK and pPP2B ratios, which phosphorylates and dephosphorylates both GluA1-S831 and -S845 sits. Perampanel also increased pERK1/2 ratio in epileptic animals, while U0126 (an ERK1/2 inhibitor) did not affect pGluA1 ratios. Perampanel did not influence PKC, PP1, and PP2A expression levels and their phosphorylation ratios. In addition, perampanel did not have a detrimental impact on cognitive abilities of epileptic and normal rats in Morris water maze test. These findings suggest that perampanel may regulate AMPA receptor functionality via not only blockade of AMPA receptor but also the regulations of multiple molecules (CAMKII, PKA, JNK, and pPP2B)-mediated GluA1 phosphorylations without negative effects on cognition, although the effects of perampanel on PKC, PP1, and PP2A activities were different between normal and epileptic rats.

Redox modulation and S-nitrosylation of cysteine residues are the post-translational modifications of N-methyl-D-aspartate receptor (NMDAR) to regulate its functionality. Recently, we have reported that protein disulfide isomerase (PDI) reduces disulfide bond (S-S) to free thiol (-SH) on NMDAR. Since PDI is a modulator of S-nitrosylation on various proteins, it is noteworthy whether PDI affects S-nitrosylation of NMDAR in acute seizure and chronic epilepsy models. In the present study, we found that acute seizures in response to pilocarpine and spontaneous seizures in chronic epilepsy rats led to the reduction in S-nitrosylated thiol (SNO-thiol)-to-total thiol ratio on NMDAR, while they elevated nitric oxide (NO) level and S-nitrosylation on NMDAR. N-nitro-L-arginine methyl ester (L-NAME, a non-selective NOS inhibitor) did not affect seizure activities in both models, although it decreased SNO-thiol levels on NMDAR. However, PDI knockdown effectively inhibited pilocarpine-induced acute seizures and spontaneous seizures in chronic epilepsy rats, accompanied by increasing the SNO-thiol-to-total thiol ratio on NMDAR due to diminishing the amounts of total thiols on GluN1 and GluN2A. Therefore, these findings indicate that PDI may not be a NO donor or a denitrosylase for NMDAR, and that PDI knockdown may inhibit seizure activity by the S-nitrosylation-independent thiolation on NMDAR.

Under pathophysiological conditions, aberrant mitochondrial dynamics lead to the different types of neuronal death: excessive mitochondrial fission provokes apoptosis and abnormal mitochondrial elongation induces necrosis. However, the underlying mechanisms how the different mitochondrial dynamics result in the distinct neuronal death patterns have been elusive. In the present study, status epilepticus (SE) evoked excessive mitochondrial fission in parvalbumin (PV) cells (one of GABAergic interneurons) and abnormal mitochondrial elongation in CA1 neurons in the rat hippocampus. These impaired mitochondrial dynamics were accompanied by mitochondrial translocations of active caspase-3 and high mobility group box 1 (HMGB1) in PV cells and CA1 neurons, respectively. WY14643 (an activator of mitochondrial fission) aggravated SE-induced PV cell loss by enhancing active caspase-3 induction and its mitochondrial translocation, which were attenuated by Mdivi-1 (an inhibitor of mitochondrial fission). Mitochondrial HMGB1 import was not observed in PV cell. In contrast to PV cells, Mdivi-1 deteriorated SE-induced CA1 neuronal death concomitant with mitochondrial HMGB1 translocation, which was abrogated by WY14643. These findings suggest that SE-induced aberrant mitochondrial dynamics may be involved in translocation of active caspase-3 and HMGB1 into mitochondria, which regulate neuronal apoptosis and necrosis, respectively.

Reactive astrogliosis is a prominent and ubiquitous reaction of astrocytes to many types of brain injury. Up-regulation of glial fibrillary acidic protein (GFAP) expression and astroglial proliferation are hallmarks of reactive astrogliosis. However, the mechanisms that regulate reactive astrogliosis remain elusive. In the present study, status epilepticus (SE, a prolonged seizure activity) led to reactive astrogliosis showing the increases in GFAP expression and the number of proliferating astrocytes with prolonged extracellular signal receptor-activated kinases 1/2 (ERK1/2) activation and reduced nuclear p27Kip1 level. U0126, an ERK1/2 inhibitor, showed opposite effects. Leptomycin B (LMB), an inhibitor of chromosomal maintenance 1 (CRM1), attenuated nucleocytoplasmic p27Kip1 export and astroglial proliferation, although it up-regulated ERK1/2 phosphorylation and GFAP expression. Roscovitine ameliorated the reduced nuclear p27Kip1 level and astroglial proliferation without changing GFAP expression and ERK1/2 phosphorylation. U0126 aggravated SE-induced astroglial apoptosis in the molecular layer of the dentate gyrus that was unaffected by LMB and roscovitine. In addition, U0126 exacerbated SE-induced neuronal death, while LMB mitigated it. Roscovitine did not affect SE-induced neuronal death. The present data elucidate for the first time the roles of nucleocytoplasmic p27Kip1 transport in ERK1/2-mediated reactive astrogliosis independent of SE-induced neuronal death and astroglial apoptosis. Therefore, our findings suggest that nucleocytoplasmic p27Kip1 export may be required for ERK1/2-mediated astroglial proliferation during reactive astrogliosis, and that nuclear p27Kip1 entrapment may be a potential therapeutic strategy for anti-proliferation in reactive astrocytes.

Recently, we have reported that transient receptor potential channel-6 (TRPC6) plays an important role in the regulation of neuronal excitability and synchronization of spiking activity in the dentate granule cells (DGC). However, the underlying mechanisms of TRPC6 in these phenomena have been still unclear. In the present study, we investigated the role of TRPC6 in subcellular localization of Kv4.3 and its relevance to neuronal excitability in the rat hippocampus. TRPC6 knockdown increased excitability and inhibitory transmission in the DGC and the CA1 neurons in response to a paired-pulse stimulus. However, TRPC6 knockdown impaired γ-aminobutyric acid (GABA)ergic inhibition in the hippocampus during and after high-frequency stimulation (HFS). TRPC6 knockdown reduced the Kv4.3 clusters in membrane fractions and its dendritic localization on DGC and GABAergic interneurons. TRPC6 knockdown also decreased extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and the efficacy of 4-aminopyridine (4-AP) in neuronal excitability. An ERK1/2 inhibitor generated multiple population spikes in response to a paired-pulse stimulus, concomitant with reduced membrane Kv4.3 translocation. A TRPC6 activator (hyperforin) reversed the effects of TRPC knockdown, except paired-pulse inhibition. These findings provide valuable clues indicating that TRPC6-mediated ERK1/2 activation may regulate subcellular Kv4.3 localization in DGC and interneurons, which is cause-effect relationship between neuronal excitability and seizure susceptibility.

Parvalbumin (PV) is one of the calcium-binding proteins, which plays an important role in the responsiveness of inhibitory neurons to an adaptation to repetitive spikes. Furthermore, PV neurons are highly vulnerable to status epilepticus (SE, prolonged seizure activity), although the underlining mechanism remains to be clarified. In the present study, we found that p47Phox expression was transiently and selectively increased in PV neurons 6 h after SE. This up-regulated p47Phox expression was accompanied by excessive mitochondrial fission. In this time point, CDK5-tyrosine 15 and dynamin-related protein 1 (DRP1)-serine 616 phosphorylations were also increased in PV cells. Apocynin (a p47Phox inhibitor) effectively mitigated PV cell loss via inhibition of CDK5/DRP1 phosphorylations and mitochondrial fragmentation induced by SE. Roscovitine (a CDK5 inhibitor) and Mdivi-1 (a DRP1 inhibitor) attenuated SE-induced PV cell loss by inhibiting aberrant mitochondrial fission. These findings suggest that p47Phox/CDK5/DRP1 may be one of the important upstream signaling pathways in PV cell degeneration induced by SE via excessive mitochondrial fragmentation.

Reactive astrogliosis is a prominent and ubiquitous reaction of astrocytes to many types of brain injury. Up-regulation of glial fibrillary acidic protein (GFAP) expression and astroglial proliferation are hallmarks of reactive astrogliosis. However, the mechanisms that regulate reactive astrogliosis remain elusive. In the present study, status epilepticus (SE, a prolonged seizure activity) led to reactive astrogliosis showing the increases in GFAP expression and the number of proliferating astrocytes with prolonged extracellular signal receptor-activated kinases 1/2 (ERK1/2) activation and reduced nuclear p27Kip1 level. U0126, an ERK1/2 inhibitor, showed opposite effects. Leptomycin B (LMB), an inhibitor of chromosomal maintenance 1 (CRM1), attenuated nucleocytoplasmic p27Kip1 export and astroglial proliferation, although it up-regulated ERK1/2 phosphorylation and GFAP expression. Roscovitine ameliorated the reduced nuclear p27Kip1 level and astroglial proliferation without changing GFAP expression and ERK1/2 phosphorylation. U0126 aggravated SE-induced astroglial apoptosis in the molecular layer of the dentate gyrus that was unaffected by LMB and roscovitine. In addition, U0126 exacerbated SE-induced neuronal death, while LMB mitigated it. Roscovitine did not affect SE-induced neuronal death. The present data elucidate for the first time the roles of nucleocytoplasmic p27Kip1 transport in ERK1/2-mediated reactive astrogliosis independent of SE-induced neuronal death and astroglial apoptosis. Therefore, our findings suggest that nucleocytoplasmic p27Kip1 export may be required for ERK1/2- mediated astroglial proliferation during reactive astrogliosis, and that nuclear p27Kip1 entrapment may be a potential therapeutic strategy for anti-proliferation in reactive astrocytes. [ABSTRACT FROM AUTHOR]

Heat shock protein (HSP) 25 (murine/rodent 25 kDa, human 27 kDa) is one of the major astroglial HSP families, which has a potent anti-apoptotic factor contributing to a higher resistance of astrocytes to the stressful condition. However, impaired removals of HSP25 decrease astroglial viability. In the present study, we investigated whether HSP25 is involved in astroglial apoptosis or clasmatodendrosis (autophagic astroglial death) in the rat hippocampus induced by status epilepticus (SE). Following SE, HSP25 expression was transiently increased in astrocytes within the dentate gyrus (DG), while it was sustained in CA1 astrocytes until 4 weeks after SE. HSP25 knockdown exacerbated SE-induced apoptotic astroglial degeneration, but mitigated clasmatodendrosis accompanied by abrogation of endoplasmic reticulum (ER) stress without changed seizure susceptibility or severity. These findings suggest that sustained HSP25 induction itself may result in clasmatodendrosis via prolonged ER stress. To the best of our knowledge, the present study demonstrates for the first time the double-edge properties of HSP25 in astroglial death induced by SE. [ABSTRACT FROM AUTHOR]

The response and susceptibility to astroglial degenerations are relevant to the distinctive properties of astrocytes in a hemodynamic-independent manner following status epilepticus (SE). Since impaired mitochondrial fission plays an important role in mitosis, apoptosis and programmed necrosis, we investigated whether the unique pattern of mitochondrial dynamics is involved in the characteristics of astroglial death induced by SE. In the present study, SE induced astroglial apoptosis in the molecular layer of the dentate gyrus, accompanied by decreased mitochondrial length. In contrast, clasmatodendritic (autophagic) astrocytes in the CA1 region showed mitochondrial elongation induced by SE. Mdivi-1 (an inhibitor of mitochondrial fission) effectively attenuated astroglial apoptosis, but WY14643 (an enhancer of mitochondrial fission) aggravated it. In addition, Mdivi-1 accelerated clasmatodendritic changes in astrocytes. These regional specific mitochondrial dynamics in astrocytes were closely correlated with dynamin-related protein 1 (DRP1; a mitochondrial fission protein) phosphorylation, not optic atrophy 1 (OPA1; a mitochondrial fusion protein) expression. To the best of our knowledge, the present data demonstrate for the first time the novel role of DRP1-mediated mitochondrial fission in astroglial loss. Thus, the present findings suggest that the differential astroglial mitochondrial dynamics may participate in the distinct characteristics of astroglial death induced by SE. [ABSTRACT FROM AUTHOR]