Data_Sheet_1_Nucleocytoplasmic p27Kip1 Export Is Required for ERK1/2-Mediated Reactive Astroglial Proliferation Following Status Epilepticus.PDF

<p>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 p27^Kip1 level. U0126, an ERK1/2 inhibitor, showed opposite effects. Leptomycin B (LMB), an inhibitor of chromosomal maintenance 1 (CRM1), attenuated nucleocytoplasmic p27^Kip1 export and astroglial proliferation, although it up-regulated ERK1/2 phosphorylation and GFAP expression. Roscovitine ameliorated the reduced nuclear p27^Kip1 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 p27^Kip1 transport in ERK1/2-mediated reactive astrogliosis independent of SE-induced neuronal death and astroglial apoptosis. Therefore, our findings suggest that nucleocytoplasmic p27^Kip1 export may be required for ERK1/2-mediated astroglial proliferation during reactive astrogliosis, and that nuclear p27^Kip1 entrapment may be a potential therapeutic strategy for anti-proliferation in reactive astrocytes.</p

SE-induced vasogenic edema formation via ETB receptor-mediated NADPH oxidase pathway.

<p>(<b>A</b>–<b>E</b>) Effects of BQ788 and apocynin on SE-induced up-regulation of 4-HNE immunoreactivity in astrocytes 12 h after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 versus non-SE induced animals; #P < 0.05 versus vehicle-treated animals; one-way ANOVA followed by Tukey’s test. (<b>F</b>–<b>J</b>) Effects of BQ788 and apocynin on dystrophin and AQP4 expression 12h after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 versus non-SE induced animals; #P < 0.05 versus vehicle-treated animals; one-way ANOVA followed by Tukey’s test. (<b>K</b>–<b>M</b>) Effect of apocynin on dystrophin and AQP4 mRNA/protein expression levels after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 versus non-SE animals; #P < 0.05 versus vehicle-treated animals; one-way ANOVA followed by Tukey’s test. (<b>N</b>–<b>Q</b>) Quantification of the attenuation of vasogenic edema formation by BQ788 and apocynin in the PC (means ± s.e.m., n = 5, respectively); *P < 0.05 versus vehicle treated animals; #P < 0.05 versus BQ788-treated animals; one-way ANOVA followed by Tukey’s test. Scale bars: <b>A-D</b>, 12.5 μm; <b>insertion</b> in <b>B</b>, 10 μm; <b>F–I</b>, 25 μm; <b>N–P</b>, 400 μm.</p

SE-induced vasogenic edema formation via the ETB receptor-mediated eNOS pathway.

<p>(<b>A</b>–<b>B</b>) Effect of BQ788 on SMI-71 expression and ETB receptor expression 12 h after SE. (<b>C</b>) Effect of BQ788 on ET_B receptor mRNA expression 12 h after SE (means ± s.e.m., n = 5, respectively); paired Student’s t-test. (<b>D</b>) Nitrate/nitrite (NO products) concentration in the PC after SE (mean ± s.d., n = 5): *P < 0.05 versus basal level; paired Student’s t-test. (<b>E</b>–<b>G</b>) Effects of BQ788 and Cav1-peptide on eNOS mRNA/protein expression 12 h after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 versus non-SE animals; #P < 0.05 versus vehicle-treated animals; P < 0.05 versus BQ788-treated animals; one-way ANOVA followed by Tukey’s test. (H–J) Effects of of BQ788 and Cav1-peptide on eNOS, SMI-71 and NT expression 12 h after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 versus non-SE animals; #P < 0.05 versus vehicle-treated animals; P < 0.05 versus BQ788-treated animals; one-way ANOVA followed by Tukey’s test. (<b>K</b>–<b>N</b>) Quantification of vasogenic edema formation 3days after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 versus vehicle-treated animals; #P < 0.05 versus BQ788-treated animals; one-way ANOVA followed by Tukey’s test. Scale bars: <b>A, B, H</b> and <b>I</b>, 25 μm; <b>K-M</b>, 400 μm.</p

Scheme depicting the role of the ET-1 in vasogenic edema formation induced by SE.

<p>Scheme depicting the role of the ET-1 in vasogenic edema formation induced by SE.</p

TNF-α/NFκB-mediated ET-1 release and expression in the PC following SE.

<p>(<b>A</b>) The extracellular ET-1 concentration in the PC after SE (mean ± s.d., n = 5): *P < 0.05 versus basal level; paired Student’s t-test. (<b>B</b>) The effect of sTNFp55R, and SN50 pretreatment on ET-1 mRNA expression 12 h after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 versus non-SE animals; #P < 0.05 versus vehicle-treated animals; one-way ANOVA followed by Tukey’s test. (<b>C</b>–<b>F</b>) ET-1 expression in neurons and endothelial cells 12 h after SE. (<b>G</b>–<b>H</b>) Effects of sTNFp55R and SN50 pretreatment on ET-1 expression and SMI-71 immunoreactivity 12 h after SE. (<b>I</b>–<b>K</b>) Quantification of ET_B receptor levels by western blotting and qRT-PCR in the PC 12 h after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 versus non-SE animals; #P < 0.05 versus vehicle-treated animals; one-way ANOVA followed by Tukey’s test. (<b>L</b>–<b>O</b>) Effect of SN50 on ETB receptor expression and SMI-71 immunoreactivity 12h after SE. Scale bars: <b>C–H</b>, <b>L-O</b>, 25 μm.</p

ETB receptor-mediated reduction of dystrophin and AQP4 expression in astrocytes.

<p>(<b>A</b>–<b>C</b>) Effects of BQ788 and Cav1-peptide on dystrophin and AQP4 mRNA/protein expression levels 12 h after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 versus non-SE animals; #P < 0.05 versus vehicle-treated animals; P < 0.05 versus BQ788-treated animals; one-way ANOVA followed by Tukey’s test. (D–F) Effects of BQ788 and Cav-1 peptide on dystrophin and AQP4 expression in astrocytes 12 h after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 versus non-SE animals; #P < 0.05 versus vehicle-treated animals; P < 0.05 versus BQ788-treated animals; one-way ANOVA followed by Tukey’s test. Scale bar: <b>D</b> and <b>E</b>, 12.5 μm.</p

The roles of TNF-α in SE-induced vasogenic edema in the PC.

<p>(<b>A</b>) The extracellular TNF-α concentration after SE (mean ± s.d., n = 5): *P < 0.05 versus the basal level; paired Student’s t-test. (<b>B</b> and <b>C</b>) Quantification of western blots for TNF-α protein expression and p65-Thr435 NFκB phosphorylation 12 h after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 by Student’s t-test. (<b>D</b>–<b>I</b>) Immunofluorescence data for TNFp75R, p65-Thr435 NFκB phosphorylation and SMI-71 12 h after SE. (<b>J</b>–<b>K</b>) Effects of TNFp55R and SN50 on p65-Thr435 NF-κB phosphorylation and SMI-71 immunoreactivity. (<b>L</b>–<b>M</b>) Quantification of the fluorescence intensities of SMI-71 expression and p65-Thr435 NFκB phosphorylation 12 h after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 versus non-SE animals; #P < 0.05 versus vehicle-treated animals; one-way analysis of variance (ANOVA) followed by Tukey’s test. (<b>N</b>–<b>Q</b>) Quantification of vasogenic edema attenuation by sTNFp55R and SN50 3 days after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 versus vehicle treated animals by one-way ANOVA followed by Tukey’s test. Scale bars: <b>D–K</b>, 25 μm; <b>O-Q</b>, 400 μm.</p

ETB receptor-mediated p47phox expression in astrocytes.

<p>(<b>A</b>–<b>C</b>) Effects of BQ788 and Cav1-peptide on p47phox mRNA/protein expression level 12 h after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 versus non-SE animals; #P < 0.05 versus vehicle-treated animals; P < 0.05 versus BQ788-treated animals; one-way ANOVA followed by Tukey’s test. (D–E) Effects of BQ788 and Cav-1 peptide on p47phox expression in astrocytes 12 h after SE (means ± s.e.m., n = 5, respectively); *P < 0.05 versus non-SE animals; #P < 0.05 versus vehicle-treated animals; P < 0.05 versus BQ788-treated animals; one-way ANOVA followed by Tukey’s test (<b>E</b>). Scale bar: <b>D</b>, 25 μm.</p

Data_Sheet_1_Dysfunction of 67-kDa Laminin Receptor Disrupts BBB Integrity via Impaired Dystrophin/AQP4 Complex and p38 MAPK/VEGF Activation Following Status Epilepticus.PDF

Status epilepticus (SE, a prolonged seizure activity) impairs brain-blood barrier (BBB) integrity, which results in secondary complications following SE. The non-integrin 67-kDa laminin receptor (67-kDa LR) plays a role in cell adherence to laminin (a major glycoprotein component in basement membrane), and participates laminin-mediated signaling pathways including p38 mitogen-activated protein kinase (p38 MAPK). Thus, we investigated the role of 67-kDa LR in SE-induced vasogenic edema formation in the rat piriform cortex (PC). SE diminished 67-kDa LR expression, but increased laminin expression, in endothelial cells accompanied by the reduced SMI-71 (a rat BBB barrier marker) expression. Astroglial 67-kDa LR expression was also reduced in the PC due to massive astroglial loss. 67-kDa LR neutralization led to serum extravasation in the PC concomitant with the reduced SMI-71 expression. 67-kDa LR neutralization also decreased expressions of dystrophin and aquaporin-4 (AQP4). In addition, it increased p38 MAPK phosphorylation and expressions of vascular endothelial growth factor (VEGF), laminin and endothelial nitric oxide synthase (eNOS), which were abrogated by SB202190, a p38 MAPK inhibitor. Therefore, our findings indicate that 67-kDa LR dysfunction may disrupt dystrophin-AQP4 complex, which would evoke vasogenic edema formation and subsequent laminin over-expression via activating p38 MAPK/VEGF axis.</p

Data_Sheet_1_Blockade of AMPA Receptor Regulates Mitochondrial Dynamics by Modulating ERK1/2 and PP1/PP2A-Mediated DRP1-S616 Phosphorylations in the Normal Rat Hippocampus.PDF

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.</p