Leptomycin B

Leptomycin B attenuates neuronal death via PKA- and PP2B-mediated ERK1/2 activation in the rat hippocampus following status epilepticus
Su-Ji Min, Hye-Won Hyun, Tae-Cheon Kang ⇑
Keywords: Calcineurin H-89
Cyclosporin A Epilepsy Seizure U0126

A B S T R A C T

Leptomycin B (LMB), originally developed as an anti-fungal agent, has potent neuroprotective properties against status epilepticus (SE, a prolonged seizure activity). However, the pharmacological profiles and mechanisms of LMB for neuroprotection remain elusive. In the present study, we found that LMB increased phosphorylation levels of protein kinase A (PKA) catalytic subunits, protein phosphatase 2B (PP2B, calcineurin) and extracellular signal–regulated kinase 1/2 (ERK1/2) under normal condition, and abolished SE-induced neuronal death. Co-treatment of H-89 (a PKA inhibitor) with LMB could not affect the seizure latency and its severity in response to pilocarpine. However, H-89 co-treatment abrogated the protective effect of LMB on SE-induced neuronal damage. Cyclosporin A (CsA, a PP2B inhibitor) co- treatment effectively prevented SE-induced neuronal death without altered seizure susceptibility in response to pilocarpine more than LMB alone. H-89 co-treatment inhibited LMB-mediated ERK1/2 phos- phorylation, but CsA enhanced it. U0126 (an ERK1/2 inhibitor) co-treatment abolished the protective effect of LMB on SE-induced neuronal death without alterations in PKA and PP2B phosphorylations. To the best of our knowledge, the present data demonstrate a previously unreported potential neuroprotec- tive role of LMB against SE via PKA- and PP2B-mediated ERK1/2 activation.
© 2017 Elsevier B.V. All rights reserved.

1. Introduction

Leptomycin B (LMB), originally developed as an anti-fungal agent, has potent anti-inflammatory and anti-tumor properties. In addition, LMB inhibits chromosome region maintenance 1 (CRM1)/exportin 1 nuclear export carrier protein (Loewe et al., 2002; Lu et al., 2012). Recently, we have reported that LMB atten- uates neuronal death induced by status epilepticus (SE, prolonged seizure activity, Hyun et al., 2016). SE-induced neuronal death is closely related to dysfunction of mitochondrial dynamics (fusion and fission). Indeed, impaired dynamin-related protein-1 (DRP1)- mediated mitochondrial fission results in programmed necrosis in CA1 neurons following SE (Kim et al., 2014; Ko et al., 2015; Hyun et al., 2016). In this process, translocation of high mobility group box 1 (HMGB1) into mitochondria plays an important role in the facilitation of SE-induced programmed necrosis, which is abrogated by LMB (Hyun et al., 2016).
HMGB1 is a nuclear protein to regulate gene transcription, and is transported to the cytoplasm by CRM1/exportin 1 during necro-

⇑ Corresponding author at: Department of Anatomy and Neurobiology, College of Medicine, Hallym University, Chunchon 24252, South Korea.
E-mail address: tckang@hallym.ac.kr (T.-C. Kang).

sis (Scaffidi et al., 2002; Faraco et al., 2007). When HMGB1 is released into the extracellular space from damaged or dying cells during necrosis (Qiu et al., 2008), it induces potentially inflamma- tory responses via toll-like receptor 4 (TLR4) or receptor for advanced glycan endproducts (RAGE) (Abraham et al., 2000; Scaffidi et al., 2002; Maroso et al., 2010). Interestingly, exogenous HMGB1 enters the mitochondria and leads to the formation of giant mitochondria independently of HMGB1 receptors (Gdynia et al., 2010). However, we have reported that inhibition of nuclear HMGB1 export by LMB cannot ameliorate the impaired mitochon- drial dynamics in CA1 neurons following SE, although LMB abol- ishes SE-induced neuronal death (Hyun et al., 2016). This discrepancy suggests the additional signaling pathways of neu- ronal damage affected by LMB, which remain elusive, independent of mitochondrial elongation. Thus, these findings raise the question concerning the pharmacological properties of LMB against SE- induced neuronal death.
On the other hand, LMB inhibits the nuclear export of cAMP- dependent protein kinase inhibitor-a (PKI), which inhibits the activity of protein kinase A (PKA) catalytic subunits (Chen et al., 2005). Furthermore, PKA inhibits CRM1/exportin-dependent nuclear export (Nishiyama et al., 2007). Therefore, it is likely that LMB may affect PKA activity. With regard to these connections, it

http://dx.doi.org/10.1016/j.brainres.2017.06.002 0006-8993/© 2017 Elsevier B.V. All rights reserved.

S.-J. Min et al. / Brain Research 1670 (2017) 14–23 15

is worthy of note that LMB would affect PKA-mediated signaling pathway and neuronal viability in response to SE. We report herein that LMB enhanced phosphorylations of PKA, protein phosphatase 2B (PP2B, calcineurin) and extracellular signal-regulated kinase 1/2 (ERK1/2) under normal condition, and ameliorated SE-induced neuronal death. Furthermore, this effect of LMB was reversed by H-89 (a PKA inhibitor) and U0126 (an ERK1/2 inhibitor). Cyclos- porin A (CsA, a PP2B inhibitor) enhanced the LMB-mediated ERK1/2 phosphorylation without changed PKA phosphorylation, and increased the neuroprotective effect of LMB on SE-induced neuronal death. These findings suggest that LMB may protect neu- rons from SE via PKA- and PP2B-mediated ERK1/2 activations.

2. Results

2.1. LMB attenuates SE-induced neuronal death via PKA activation

Fig. 1 shows that LMB could not affect the seizure latency and its severity induced by pilocarpine (Fig. 1A–C). Consistent with our previous study (Hyun et al., 2016), LMB alleviated SE-induced neuronal death as compared to vehicle (p < 0.05; Fig. 1D–E). These findings indicate that LMB may attenuate SE-induced neuronal death without changed seizure activity. Since LMB inhibits the nuclear export of cAMP-dependent protein kinase inhibitor-a (PKI), which inhibits the activity of PKA catalytic subunits (Chen et al., 2005), we investigated whether LMB affects PKA activity under physiological condition and post-SE condition. LMB increased the phospho (p)-PKA catalytic subunit (T197) level to 2.78-fold of vehicle-treated control animals (p < 0.05; Fig. 2A-B). Following SE, pPKA catalytic subunit level in LMB-treated animals was 2.64-fold of vehicle-treated control ani- mals (p < 0.05; Fig. 2A-B). However, vehicle did not affect pPKA level (Fig. 2A-B). Consistent with a previous study (Chen et al., 2005), these findings indicate that LMB may increase PKA activity in control and post-SE animals. Next, we co-applied H-89 with LMB to investigate the role of LMB-mediated PKA activation in SE- induced neuronal death. Co-treatment of H-89 with LMB could not affect the seizure latency and its severity in response to pilo- carpine (Fig. 2C–E). However, H-89 co-treatment abolished the protective effect of LMB on SE-induced neuronal damage (Fig. 2F–G). These findings indicate that LMB-mediated PKA activa- tion may protect neurons from SE insult without changed seizure activity. 2.2. LMB increases PP2B serine-197 phosphorylation independent of PKA activity It is well known that SE-induced neuronal death is regulated by various protein phosphatase activities (Zeng et al., 2007; Shin et al., 2012). Furthermore, PKA-mediated phosphorylation affects the Fig. 1. The effect of LMB on neuronal death and seizure activity induced by pilocarpine. (A–C) The effect of LMB on seizure susceptibility induced by pilocarpine. LMB does not affect the seizure susceptibility and its severity in response to pilocarpine. (A) Representative EEG traces in response to pilocarpine. (B) Representative frequency-power spectral temporal maps in response to pilocarpine. (C) Quantification of effect of LMB on SE induction, latency and total EEG power (mean ± S.E.M.; n = 7, respectively). (D–E) The effect of LMB on SE-induced neuronal death. LMB effectively attenuates SE-induced neuronal death. (D) Representative FJB staining in the CA1 region 3 days after SE. Bar = 50 lm. (E) Quantification of effect of LMB on SE-induced neuronal death (mean ± S.E.M.; *p < 0.05 vs. vehicle; n = 7, respectively). 16 S.-J. Min et al. / Brain Research 1670 (2017) 14–23 Fig. 2. Role of PKA in neuronal death and seizure activity induced by pilocarpine. (A-B) The effect of LMB on PKA-cat phosphorylation. LMB significantly increases PKA phosphorylation in the hippocampus. (A) Western blot of PKA-cat phosphorylation. (B) Quantification of PKA-cat phosphorylation (mean ± S.E.M.; #p < 0.05 vs. vehicle; n = 7, respectively). (C-E) The effect of H-89 co-treatment with LMB on seizure susceptibility induced by pilocarpine. H-89 co-treatment does not affect the seizure susceptibility and its severity in response to pilocarpine. (C) Representative EEG traces in response to pilocarpine. (D) Representative frequency-power spectral temporal maps in response to pilocarpine. (E) Quantification of effect of H-89 co-treatment on SE induction, latency and total EEG power (mean ± S.E.M.; n = 7, respectively). (F-G) The effect of H-89 co- treatment on SE-induced neuronal death. H-89 co-treatment exacerbates SE-induced neuronal death more than LMB. (F) Representative FJB staining in the CA1 region 3 days after SE. Bar = 50 lm. (G) Quantification of effect of H-89 co-treatment on SE-induced neuronal death (mean ± S.E.M.; *p < 0.05 vs. LMB; n = 7, respectively). protein phosphatase activities (Yan and Surmeier, 1997; Flores- Hernandez et al., 2000; Ahn et al., 2007; Kim et al., 2015). There- fore, we tested whether LMB also affects protein phosphatase phosphorylation. LMB did not change the phosphorylation of pro- tein phosphatase (PP) 1 and PP2A, but LMB significantly increased PP2B phosphorylation at serine (S) 197 site (p < 0.05 vs. vehicle; Fig. 3A). Following SE, PP2B S197 phosphorylation was reduced to 0.7-fold of control level in vehicle-treated animals (p < 0.05 vs. control animals; Fig. 3A), but it was unaltered in LMB-treated ani- mals (Fig. 3A). Since the phosphorylation of this site inhibits PP2B activity (Hashimoto et al., 1988; MacDonnell et al., 2009), these findings indicate that LMB-mediated PKA activation might inhibit PP2B activity. To elucidate this hypothesis, we co-applied H-89 with LMB. Consistent with the pharmacological properties of H- 89 (Cauthron et al., 1998), H-89 decreased PKA phosphorylation (p < 0.05 vs. vehicle). Furthermore, H-89 co-treatment with LMB abolished the LMB-induced PKA phosphorylation (p < 0.05 vs. vehi- cle). However, it did not affect LMB-mediated PP2B phosphoryla- tion (Fig. 3B). Next, we co-applied CsA with LMB to evaluate the effect of LMB-mediated PP2B inhibition on SE-induced neuronal damage. CsA did not affect the levels of PP2B expression and its phosphorylation (Fig. 3C). These findings are agreement with pre- vious studies demonstrating that CsA inhibits PP2B activity by ster- ically hindering the access of substrates to the catalytic site and the S.-J. Min et al. / Brain Research 1670 (2017) 14–23 17 Fig. 3. The effect of LMB on PP phosphorylations by pilocarpine. (A) The effect of LMB on PP phosphorylations. LMB significantly increases only PP2B phosphorylation in the hippocampus. In addition, SE reduces PP2B phosphorylation, but LMB attenuated it. (Left panel) Western blot of PP phosphorylations. (Right panel) Quantification of PP phosphorylations (mean ± S.E.M.; *, #p < 0.05 vs. control (non-SE) and vehicle, respectively; n = 7, respectively). (B) The effect of H-89 on PKA-cat and PP2B phosphorylations. H-89 treatment reduces PKA-cat phosphorylation, but not PP2B phosphorylation. (Left panel) Western blot of PKA-cat and PP2B phosphorylations. (Right panel) Quantification of PKA-cat and PP2B phosphorylations (mean ± S.E.M.; *, #p < 0.05 vs. control (non-SE) and vehicle, respectively; n = 7, respectively). (C) The effect of CsA on PP2B and PKA-cat phosphorylations. CsA treatment does not affect PP2B and PKA-cat phosphorylations. (Left panel) Western blot of PP2B and PKA-cat phosphorylation. (Right panel) Quantification of PP2B and PKA-cat phosphorylation (mean ± S.E.M.; *,#p < 0.05 vs. control (non-SE) and vehicle, respectively; n = 7, respectively). formation of complex with cyclophilin, but not its phosphorylation or expression (Liu et al., 1991; Shiraishi et al., 2001). In addition, CsA did not alter PKA phosphorylation (Fig. 3C). As compared to LMB alone, however, CsA co-treatment effectively attenuated SE- induced neuronal death without altered seizure susceptibility in response to pilocarpine (p < 0.05 vs. LMB; Fig. 4A–E). Therefore, our findings indicate that LMB may increase PP2B S197 phosphory- lation independent of PKA activity, and this LMB-mediated PP2B inhibition may attenuate SE-induced neuronal death. 2.3. LMB-mediated PKA activation and PP2B inhibition elevate ERK1/2 phosphorylation CRM1 inhibition elicits persistent ERK1/2 hyperactivation (Pathria et al., 2012), and PP2B inhibition leads to ERK1/2 phospho- rylation and its activation (Choe et al., 2005; Gabryel et al., 2006). Interestingly, PKA activates ERK1/2 pathway (Zanassi et al., 2001), which plays an important role in neuroprotection against SE (Choi et al., 2007). Therefore, it is likely that the neuroprotective effect of LMB may be relevant to ERK1/2 signaling pathway. To clarify this hypothesis, we tested the effect of LMB-mediated PKA activation or PP2B inhibition on ERK1/2 phosphorylation. LMB significantly increased pERK1/2 level in control and post-SE animals (p < 0.05 vs. vehicle; Fig. 5A). In addition, H-89 reduced ERK1/2 phosphory- lation (p < 0.05 vs. vehicle; Fig. 5B), but CsA increased it (p < 0.05 vs. vehicle; Fig. 5C). Similarly, H-89 co-treatment inhibited LMB- mediated ERK1/2 phosphorylation (p < 0.05 vs. LMB; Fig. 5B), but CsA co-treatment enhanced it (p < 0.05 vs. LMB; Fig. 5C). We fur- ther investigated the direct effect of ERK1/2 activation in SE- induced neuronal death by U0126 treatment. Consistent with a 18 S.-J. Min et al. / Brain Research 1670 (2017) 14–23 Fig. 4. Role of PP2B in neuronal death and seizure activity induced by pilocarpine. (A–C) The effect of CsA co-treatment with LMB on seizure susceptibility induced by pilocarpine. CsA co-treatment does not affect the seizure susceptibility and its severity in response to pilocarpine. (A) Representative EEG traces in response to pilocarpine. (B) Representative frequency-power spectral temporal maps in response to pilocarpine. (C) Quantification of effect of CsA co-treatment on SE induction, latency and total EEG power (mean ± S.E.M.; n = 7, respectively). (D-E) The effect of CsA co-treatment on SE-induced neuronal death. CsA co-treatment diminishes SE-induced neuronal death more than LMB. (F) Representative FJB staining in the CA1 region 3 days after SE. Bar = 50 lm. (G) Quantification of effect of CsA co-treatment on SE-induced neuronal death (mean ± S.E.M.; *p < 0.05 vs. LMB; n = 7, respectively). previous study (Zhu et al., 2013), U0126 reduced ERK1/2 phospho- rylation (p < 0.05 vs. vehicle; Fig. 6A-B). U0126 co-treatment with LMB attenuated LMB-mediated ERK1/2 phosphorylation (p < 0.05 vs. LMB; Fig. 6A-B). Furthermore, U0126 co-treatment abolished the neuroprotective effect of LMB against SE without alterations in seizure activity, and PKA and PP2B phosphorylations (Fig. 6A- G). These findings indicate that LMB may mitigate SE-induced neu- ronal death via ERK1/2 activation, which is regulated by PKA and PP2B. 2.4. Post-SE treatment of LMB and CsA attenuate SE-induced neuronal death To confirm neuroprotective role of LMB, we applied each com- pound 1 day after SE. Compared to vehicle, both LMB and CsA effectively alleviated SE-induced neuronal death (p < 0.05 vs. vehi- cle; Fig. 7A-B). However, neither H-89 nor U0126 affected neuronal loss induced by SE. These findings indicate that post-SE treatment of LMB may also be effective to prevent SE-induced neuronal death. 3. Discussion Although neuronal death is one of the precipitating events induced by SE, the molecular mechanisms are still unknown. Recently, we have reported that dysfunction of mitochondrial fis- sion play an important role in SE-induced neuronal death (Kim et al., 2014). Interestingly, LMB delays SE-induced neuronal death by inhibiting nuclear HMGB1 release, but cannot recover the abnormal mitochondrial elongation (Hyun et al., 2016). Similarly, LMB protects neuronal damage from kainite-induced seizures and transient ischemia through preventing translocation of diacyl- glycerol kinase f (DGKf) from nucleus to cytoplasm (Okada et al., 2012). Since LMB inhibits CRM1/exportin 1 (Loewe et al., 2002; Lu et al., 2012), it is plausible that LMB-mediated inhibition of nuclear exports may ameliorate SE-induced neuronal death. How- ever, LMB also affects many signal transductions unrelated to nucleocytoplasmic shuttle. For example, LMB inhibits nuclear fac- tor (NF)-jB-dependent signal transduction by the inhibition of IjB (Rodriguez et al., 1999). In the present study, LMB elevated PKA phosphorylation under physiological condition. With respect to the inhibitory effect of LMB on PKI (Chen et al., 2005), it is likely that LMB may activate PKA by inhibition of nuclear PKI export. Fur- thermore, PKA itself inhibits CRM1/exportin-dependent nuclear export. This is because H-89 facilitates nucleocytoplasmic shut- tling of Id1 that is suppressed by LMB (Nishiyama et al., 2007). Thus, it is presumable that LMB would inhibit CRM1 via PKA acti- vation. Interestingly, the present study shows that LMB-mediated PKA phosphorylation may play a neuroprotective role via ERK1/2 activation. PKA-mediated mechanism is important for ERK1/2 sig- S.-J. Min et al. / Brain Research 1670 (2017) 14–23 19 Fig. 5. The effect of LMB on ERK1/2 phosphorylation by pilocarpine. (A) The effect of LMB on ERK1/2 phosphorylation. LMB significantly increases ERK1/2 phosphorylation in the hippocampus. In addition, SE reduces ERK1/2 phosphorylation, but LMB attenuated it. (Left panel) Western blot of ERK1/2 phosphorylation. (Right panel) Quantification of ERK1/2 phosphorylation (mean ± S.E.M.; *,#p < 0.05 vs. control (non-SE) and vehicle, respectively; n = 7, respectively). (B) The effect of H-89 on PKA-cat and ERK1/2 phosphorylations. H-89 treatment abolishes both PKA-cat and ERK1/2 phosphorylations. (Left panel) Western blot of PKA-cat and ERK1/2 phosphorylations. (Right panel) Quantification of PKA-cat and ERK1/2 phosphorylations (mean ± S.E.M.; *,#,$p < 0.05 vs. control (non-SE), vehicle and LMB, respectively; n = 7, respectively). (C) The effect of CsA on PP2B and ERK1/2 phosphorylations. CsA enhances ERK1/2 phosphorylation, but it cannot affect PP2B phosphorylation. (Left panel) Western blot of PP2B and ERK1/2 phosphorylation. (Right panel) Quantification of PP2B and ERK1/2 phosphorylations (mean ± S.E.M.; *,#,$p < 0.05 vs. control (non-SE), vehicle and LMB, respectively; n = 7, respectively). naling pathway (Roberson et al., 1999; Grewal et al., 2000), which is involve in neuroprotective responses to SE insults (Choi et al., 2007). In addition, CRM1 inhibition results in persistent ERK1/2 hyperactivation (Pathria et al., 2012). In the present study, inhibition of PKA by H-89 abrogated the neuroprotective effect of LMB against SE, accompanied by reduced ERK1/2 phosphorylation. Furthermore, U0126 co-treatment with LMB aggravated SE- induced neuronal death, as compared to LMB alone, although U0126 did not affect PKA phosphorylation. Therefore, our findings indicate that PKA may be one of the upstream regulators for ERK1/2 activation in response to CRM1 inhibition by LMB, and that PKA-dependent ERK1/2 phosphorylation may be a key set of sig- naling events that protect neurons against SE insults. In the present study, LMB increased PP2B phosphorylation level representing the reduction of its phosphatase activity (Hashimoto et al., 1988; MacDonnell et al., 2009). This LMB-mediated PP2B phosphorylation is accompanied by enhanced EKR1/2 phosphorylation, but not PKA phosphorylation. Furthermore CsA co-treatment with LMB increased ERK1/2 phosphorylation and attenuated SE-induced neuronal death more than LMB alone. How- ever, U0126 did not affect PP2B phosphorylation. Since PP2B antagonizes ERK1/2 phosphorylation and its activity (Choe et al., 2005; Gabryel et al., 2006), these findings indicate that PP2B may be another upstream regulator for LMB-mediated ERK1/2 activa- tion, which attenuates SE-induced neuronal death. How does LMB increase PP2B phosphorylation? It is difficult to clarify the direct mechanism of LMB for regulating PP2B phosphorylation. However, nuclear imports of various molecules, such as nuclear factor of activated T cells (NFAT), transducers of regulated CREB and eukaryotic initiation factor 6, are regulated by PP2B- mediated dephosphorylation (Hogan et al., 2003; Bittinger et al., 2004; Biswas et al., 2011). However, PP2B inhibits the CRM1- mediated nuclear export of NFAT4 not by virtue of its phosphatase activity, but by competing with CRM1 for binding to NFAT4 (Zhu and McKeon, 1999). Therefore, it cannot be excluded that LMB would regulate PP2B activity or its binding ability, which would 20 S.-J. Min et al. / Brain Research 1670 (2017) 14–23 Fig. 6. Role of ERK1/2 in neuronal death and seizure activity induced by pilocarpine. (A-B) The effect of U0126 on ERK1/2, PKA-cat and PP2B phosphorylations. U0126 reduces ERK1/2 phosphorylation, while it does not affect PKA-cat and PP2B phosphorylations. (A) Western blot of ERK1/2, PKA-cat and PP2B phosphorylations. (B) Quantification of ERK1/2, PKA-cat and PP2B phosphorylations (mean ± S.E.M.; *,#p < 0.05 vs. control (non-SE) and vehicle, respectively; n = 7, respectively). (C–E) The effect of U0126 co- treatment with LMB on seizure susceptibility induced by pilocarpine. U0126 co-treatment does not affect the seizure susceptibility and its severity in response to pilocarpine. (C) Representative EEG traces in response to pilocarpine. (D) Representative frequency-power spectral temporal maps in response to pilocarpine. (E) Quantification of effect of U0126 co-treatment on SE induction, latency and total EEG power (mean ± S.E.M.; n = 7, respectively). (F-G) The effect of U0126 co-treatment on SE-induced neuronal death. U0126 co-treatment aggravates SE-induced neuronal death more than LMB. (F) Representative FJB staining in the CA1 region 3 days after SE. Bar = 50 lm. (G) Quantification of effect of U0126 co-treatment on SE-induced neuronal death (mean ± S.E.M.; *p < 0.05 vs. LMB; n = 7, respectively). be required for a balance between PP2B-related nuclear import and CRM1-mediated export. To elucidate the molecular mechanism of effect of LMB on PP2B functionality would be a subject of further investigation. In conclusion, we found that that LMB attenuated SE-induced neuronal death via ERK1/2 activation, which might be mediated by PKA- and PP2B-dependent mechanism (Fig. 8). Furthermore, our findings suggest that LMB may have multi-pharmacological targets beyond the inhibition of nucleocytoplasmic trafficking machinery. To the best of our knowledge, the present data demon- strate a previously unreported potential neuroprotective role of LMB, and propose a mechanism for neuronal viability and a groundwork for the development and application of CRM1 inhibi- tors against neuronal death. S.-J. Min et al. / Brain Research 1670 (2017) 14–23 21 Fig. 7. Effect of each compound on SE-induced neuronal death 1 day after SE. Both LMB and CsA attenuates SE-induced neuronal death. However, H-89 and U0126 do not affect SE-induced neuronal death. (A) Representative FJB staining in the CA1 region 3 days after SE. Bar = 50 lm. (B) Quantification of effect of each compound on SE-induced neuronal death (mean ± S.E.M.; #,$p < 0.05 vs. vehicle and LMB, respectively; n = 7, respectively). Fig. 8. Scheme of the effect of LMB on SE-induced neuronal death. LMB activates PKA, which increases ERK1/2 phosphorylation (activity). LMB also inhibits PP2B activity that dephosphorylates (inactivates) ERK1/2. Subsequently, enhanced ERK1/ 2 activity by these distinct pathways attenuates SE-induced neuronal death. 4. Experimental procedures 4.1. Experimental animals and chemicals Adult male Sprague-Dawley (SD) rats (weight 320–370 g, Dae- han Biolink, South Korea) were used in the study. Animals were kept under controlled environmental conditions (23–25 °C, 12 h light/dark cycle) with free access to water and standard laboratory food. All animal experiments were approved by the Institutional Animal Care and Use Committee of the Hallym University (Chun- cheon, Korea). All reagents were obtained from Sigma-Aldrich (USA), unless otherwise noted. 4.2. Intracerebroventricular drug infusion Under Isoflurane anesthesia (1–2% in O2 and N2O), animals were stereotaxically implanted a brain infusion kit 1 (Alzet, USA) into the right lateral ventricle (1 mm posterior; 1.5 mm lateral; 3.5 mm depth; flat skull position with bregma as reference). Thereafter, an infusion kit was connected to an osmotic pump (1007D, Alzet, USA) containing: (1) vehicle; (2) H-89 (10 lM); (3) LMB (30 mg/ml); (4) LMB (30 mg/ml) + H-89 (10 lM); (5) CsA (250 lM); (6) LMB (30 mg/ml) + CsA (250 lM); (7) U0126 (25 lM); and (8) LMB (30 mg/ml) + U0126 (25 lM). In pilot study, each compound treatment did not affect seizure threshold in response to pilocarpine. Some animals were also implanted a depth electrode into the left dorsal hippocampus ( 3.8 mm poste- rior; 2.0 mm lateral; 2.6 mm depth). Three days after surgery, animals were used for SE induction. Other animals were given each compound by the same methods 1 day after SE. 4.3. Seizure induction SE was induced by a single dose (30 mg/kg) of pilocarpine in rats pretreated (24 h before pilocarpine injection) with 127 mg/kg lithium chloride, as previously described (Kim and Kang, 2015; Hyun et al., 2016). Before pilocarpine injection, animals were given 22 S.-J. Min et al. / Brain Research 1670 (2017) 14–23 atropine methylbromide (5 mg/kg i.p.) to block the peripheral effect of pilocarpine. Two hours after SE, animals received diazepam (10 mg/kg, i.p.) to terminate SE. As controls, rats were treated with saline instead of pilocarpine. Electrode-implanted rats were injected with pilocarpine after baseline recording for 30 min. EEG signals were acquired using LabChart Pro v7 (AD Instruments, NSW, Australia), and latency or seizure onset and total power were measured from each animal (Kim and Kang, 2015; Min and Kang, 2016). 4.4. Tissue processing and FJB staining Three days after SE, tissue processing and Fluoro-Jade B (FJB) staining were performed as previously described (Ko et al., 2015; Hyun et al., 2016). Briefly, animals were deeply anesthetized with urethane anesthesia (1.5 g/kg, i.p.) and perfused with phosphate- buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). The brains were removed, and cryoprotected by infiltration with 30% sucrose overnight. Thereafter, the tissues were sectioned with a cryostat at 30㎛ and consecutive sections were collected in six-well plates contain- ing PBS. For FJB staining, sections were immersed in a solution con- taining 1% sodium hydroxide in 80% ethanol, 70% ethanol and distilled water. The slides were then incubated in a solution of potassium permanganate and subsequently in 0.001% FJB (Histo- Chem Inc. Jefferson, AR, USA). Two different investigators per- formed cell counts with optical dissector methods (Kim et al., 2014; Ko et al., 2015; Hyun et al., 2016). For western blot, tissues were homogenized and the protein concentration in the super- natant was determined using a Micro BCA Protein Assay Kit (Pierce Chemical, Rockford, IL, USA). 4.5. Western blot Western blot was performed by the standard protocol. Briefly, aliquots were loaded into a polyacrylamide gel. After electrophore- sis, gels were transferred to nitrocellulose transfer membranes. Membranes were incubated with primary antibodies including: rab- bit anti-phospho (p)-PKA-cat (1:1000, Assay Biotech, USA), PKA-cat (1:1000, Biovision, USA), pPP2B (1:1000, Badrilla, UK), PP2B (1:1000, Millipore, USA), pERK1/2 (1:1000, Millipore, USA) and ERK1/2 (1:1000, Biorbyt, USA). Thereafter, membranes were reacted with a HRP-conjugated secondary antibody and ECL kit (GE Healthcare, Piscataway, NJ, USA). The bands were detected and quantified on ImageQuant LAS4000 system (GE Healthcare, Piscataway, NJ, USA). The rabbit anti-b-actin primary antibody (1:5000) was used as inter- nal reference. Intensity measurements were represented as the mean gray-scale value and normalized against b-actin. 4.6. Statistical analysis A single data point for each animal was used for analysis. Parameters were tested for the normality and equality of variance. Thereafter, data were analyzed by Student t-test or one-way anal- ysis of variance (ANOVA) coupled with Bonferroni’s post hoc test for multiple comparison. Values are presented as mean ± SEM. Dif- ferences were considered as significant for p < 0.05. Conflicts of interests The authors have declared that no conflict of interest exists. Acknowledgments This work was supported by the National Research Foundation of Korea (NRF) grants (No. 2009-0093812 and 2015R1A2A2A01003539) and Hallym University (HRF-201610- 010). References Abraham, E., Arcaroli, J., Carmody, A., Wang, H., Tracey, K.J., 2000. HMG-1 as a mediator of acute lung inflammation. J. Immunol. 165, 2950–2954. Ahn, J.H., McAvoy, T., Rakhilin, S.V., Nishi, A., Greengard, P., Nairn, A.C., 2007. Protein kinase A activates protein phosphatase 2A by phosphorylation of the B56delta subunit. Proc. Natl. Acad. Sci. U.S.A. 104, 2979–2984. Biswas, A., Mukherjee, S., Das, S., Shields, D., Chow, C.W., Maitra, U., 2011. Opposing action of casein kinase 1 and calcineurin in nucleo-cytoplasmic shuttling of mammalian translation initiation factor eIF6. J. Biol. Chem. 286, 3129–3138. Bittinger, M.A., McWhinnie, E., Meltzer, J., Iourgenko, V., Latario, B., Liu, X., Chen, C. H., Song, C., Garza, D., Labow, M., 2004. Activation of cAMP response element- mediated gene expression by regulated nuclear transport of TORC proteins. Curr. Biol. 14, 2156–2161. Cauthron, R.D., Carter, K.B., Liauw, S., Steinberg, R.A., 1998. Physiological phosphorylation of protein kinase A at Thr-197 is by a protein kinase A kinase. Mol. Cell. Biol. 18, 1416–1423. Chen, X., Dai, J.C., Orellana, S.A., Greenfield, E.M., 2005. Endogenous protein kinase inhibitor gamma terminates immediate-early gene expression induced by cAMP-dependent protein kinase (PKA) signaling: termination depends on PKA inactivation rather than PKA export from the nucleus. J. Biol. Chem. 280, 2700– 2707. Choe, E.S., Shin, E.H., Wang, J.Q., 2005. Inhibition of protein phosphatase 2B upregulates serine phosphorylation of N-methyl-D-aspartate receptor NR1 subunits in striatal neurons in vivo. Neurosci. Lett. 384, 38–43. Choi, Y.S., Lin, S.L., Lee, B., Kurup, P., Cho, H.Y., Naegele, J.R., Lombroso, P.J., Obrietan, K., 2007. Status epilepticus-induced somatostatinergic hilar interneuron degeneration is regulated by striatal enriched protein tyrosine phosphatase. J. Neurosci. 27, 2999–3009. Faraco, G., Fossati, S., Bianchi, M.E., Patrone, M., Pedrazzi, M., Sparatore, B., Moroni, F., Chiarugi, A., 2007. High mobility group box 1 protein is released by neural cells upon different stresses and worsens ischemic neurodegeneration in vitro and in vivo. J. Neurochem. 103, 590–603. Flores-Hernandez, J., Hernandez, S., Snyder, G.L., Yan, Z., Fienberg, A.A., Moss, S.J., Greengard, P., Surmeier, D.J., 2000. D(1) dopamine receptor activation reduces GABA(A) receptor currents in neostriatal neurons through a PKA/DARPP-32/PP1 signaling cascade. J. Neurophysiol. 83, 2996–3004. Gabryel, B., Pudelko, A., Adamczyk, J., Fischer, I., Malecki, A., 2006. Calcineurin and Erk1/2-signaling pathways are involved in the antiapoptotic effect of cyclosporine A on astrocytes exposed to simulated ischemia in vitro. Naunyn. Schmiedebergs. Arch. Pharmacol. 374, 127–139. Gdynia, G., Keith, M., Kopitz, J., Bergmann, M., Fassl, A., Weber, A.N., George, J., Kees, T., Zentgraf, H.W., Wiestler, O.D., Schirmacher, P., Roth, W., 2010. Danger signaling protein HMGB1 induces a distinct form of cell death accompanied by formation of giant mitochondria. Cancer Res. 70, 8558–8568. Grewal, S.S., Horgan, A.M., York, R.D., Withers, G.S., Banker, G.A., Stork, P.J., 2000. Neuronal calcium activates a Rap1 and B-Raf signaling pathway via the cyclic adenosine monophosphate-dependent protein kinase. J. Biol. Chem. 275, 3722– 3728. Hashimoto, Y., King, M.M., Soderling, T.R., 1988. Regulatory interactions of calmodulin-binding proteins: phosphorylation of calcineurin by autophosphorylated Ca2+/calmodulin-dependent protein kinase II. Proc. Natl. Acad. Sci. U.S.A. 85, 7001–7005. Hogan, P.G., Chen, L., Nardone, J., Rao, A., 2003. Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev. 17, 2205–2232. Hyun, H.W., Ko, A.R., Kang, T.C., 2016. Mitochondrial translocation of high mobility group box 1 facilitates LIM kinase 2-mediated programmed necrotic neuronal death. Front. Cell. Neurosci. 10, 99. Kim, Y.J., Kang, T.C., 2015. The role of TRPC6 in seizure susceptibility and seizure- related neuronal damage in the rat dentate gyrus. Neuroscience 307, 215–230. Kim, J.E., Ryu, H.J., Kim, M.J., Kang, T.C., 2014. LIM kinase-2 induces programmed necrotic neuronal death via dysfunction of DRP1-mediated mitochondrial fission. Cell Death Differ. 21, 1036–1049. Kim, S.S., Lee, E.H., Lee, K., Jo, S.H., Seo, S.R., 2015. PKA regulates calcineurin function through the phosphorylation of RCAN1: identification of a novel phosphorylation site. Biochem. Biophys. Res. Commun. 459, 604–609. Ko, A.R., Hyun, H.W., Min, S.J., Kim, J.E., Kang, T.C., 2015. Endothelin-1 induces LIMK2-mediated programmed necrotic neuronal death independent of NOS activity. Mol. Brain. 8, 58. Liu, J., Farmer Jr., J.D., Lane, W.S., Friedman, J., Weissman, I., Schreiber, S.L., 1991. Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 66, 807–815. Loewe, R., Holnthoner, W., Gröger, M., Pillinger, M., Gruber, F., Mechtcheriakova, D., Hofer, E., Wolff, K., Petzelbauer, P., 2002. Dimethylfumarate inhibits TNF- induced nuclear entry of NF-kappa B/p65 in human endothelial cells. J. Immunol. 168, 4781–4787. S.-J. Min et al. / Brain Research 1670 (2017) 14–23 23 Lu, C., Shao, C., Cobos, E., Singh, K.P., Gao, W., 2012. Chemotherapeutic sensitization of leptomycin B resistant lung cancer cells by pretreatment with doxorubicin. PLoS One 7, e32895.
MacDonnell, S.M., Weisser-Thomas, J., Kubo, H., Hanscome, M., Liu, Q., Jaleel, N., Berretta, R., Chen, X., Brown, J.H., Sabri, A.K., Molkentin, J.D., Houser, S.R., 2009. CaMKII negatively regulates calcineurin-NFAT signaling in cardiac myocytes. Circ. Res. 105, 316–325.
Maroso, M., Balosso, S., Ravizza, T., Liu, J., Aronica, E., Iyer, A.M., Rossetti, C., Molteni, M., Casalgrandi, M., Manfredi, A.A., Bianchi, M.E., Vezzani, A., 2010. Toll-like receptor 4 and high-mobility group box-1 are involved in ictogenesis and can be targeted to reduce seizures. Nat. Med. 16, 413–419.
Min, S.J., Kang, T.C., 2016. Positive feedback role of TRPC3 in TNF-a-mediated
vasogenic edema formation induced by status epilepticus independent of ETB receptor activation. Neuroscience 337, 37–47.
Nishiyama, K., Takaji, K., Uchijima, Y., Kurihara, Y., Asano, T., Yoshimura, M., Ogawa, H., Kurihara, H., 2007. Protein kinase A-regulated nucleocytoplasmic shuttling of Id1 during angiogenesis. J. Biol. Chem. 282, 17200–17209.
Okada, M., Hozumi, Y., Tanaka, T., Suzuki, Y., Yanagida, M., Araki, Y., Evangelisti, C., Yagisawa, H., Topham, M.K., Martelli, A.M., Goto, K., 2012. DGKf is degraded through the cytoplasmic ubiquitin-proteasome system under excitotoxic conditions, which causes neuronal apoptosis because of aberrant cell cycle reentry. Cell Signal. 24, 1573–1582.
Pathria, G., Wagner, C., Wagner, S.N., 2012. Inhibition of CRM1-mediated nucleocytoplasmic transport: triggering human melanoma cell apoptosis by perturbing multiple cellular pathways. J. Invest. Dermatol. 132, 2780–2790.
Qiu, J., Nishimura, M., Wang, Y., Sims, J.R., Qiu, S., Savitz, S.I., Salomone, S., Moskowitz, M.A., 2008. Early release of HMGB-1 from neurons after the onset of brain ischemia. J. Cereb. Blood Flow. Metab. 28, 927–938.
Roberson, E.D., English, J.D., Adams, J.P., Selcher, J.C., Kondratick, C., Sweatt, J.D., 1999. The mitogen-activated protein kinase cascade couples PKA and PKC to

cAMP response element binding protein phosphorylation in area CA1 of hippocampus. J. Neurosci. 19, 4337–4348.
Rodriguez, M.S., Thompson, J., Hay, R.T., Dargemont, C., 1999. Nuclear retention of IkappaBalpha protects it from signal-induced degradation and inhibits nuclear factor kappaB transcriptional activation. J. Biol. Chem. 274, 9108–9115.
Scaffidi, P., Misteli, T., Bianchi, M.E., 2002. Release of chromatin protein HMGB1 by necrotic cells triggers inflammation. Nature 418, 191–195.
Shin, H.J., Jeon, B.T., Kim, J., Jeong, E.A., Kim, M.J., Lee, D.H., Kim, H.J., Kang, S.S., Cho,
G.J., Choi, W.S., Roh, G.S., 2012. Effect of the calcineurin inhibitor FK506 on K+- Cl-cotransporter 2 expression in the mouse hippocampus after kainic acid- induced status epilepticus. J. Neural. Transm. 119, 669–677.
Shiraishi, S., Yanagita, T., Kobayashi, H., Uezono, Y., Yokoo, H., Minami, S.I., Takasaki, M., Wada, A., 2001. Up-regulation of cell surface sodium channels by cyclosporin A, FK506, and rapamycin in adrenal chromaffin cells. J. Pharmacol. Exp. Ther. 297, 657–665.
Yan, Z., Surmeier, D.J., 1997. D5 dopamine receptors enhance Zn2+-sensitive GABA
(A) currents in striatal cholinergic interneurons through a PKA/PP1 cascade. Neuron 19, 1115–1126.
Zanassi, P., Paolillo, M., Feliciello, A., Avvedimento, E.V., Gallo, V., Schinelli, S., 2001. CAMP-dependent protein kinase induces cAMP-response element-binding protein phosphorylation via an intracellular calcium release/ERK-dependent pathway in striatal neurons. J. Biol. Chem. 276, 11487–11495.
Zeng, L.H., Xu, L., Rensing, N.R., Sinatra, P.M., Rothman, S.M., Wong, M., 2007. Kainate seizures cause acute dendritic injury and actin depolymerization in vivo. J. Neurosci. 27, 11604–11613.
Zhu, J., McKeon, F., 1999. NF-AT activation requires suppression of Crm1-dependent export by calcineurin. Nature 398, 256–260.
Zhu, Y.M., Wang, C.C., Chen, L., Qian, L.B., Ma, L.L., Yu, J., Zhu, M.H., Wen, C.Y., Yu, L.
N., Yan, M., 2013. Both PI3K/Akt and ERK1/2 pathways participate in the protection by dexmedetomidine against transient focal cerebral ischemia/ reperfusion injury in rats. Brain Res. 1494, 1–8.