2-Cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me) is a triterpenoid analogue of oleanolic acid that exhibits promising anti-cancer, anti-inflammatory, antioxidant and neuroprotective activities. In addition, CDDO-Me affects cellular differentiation and cell cycle arrest, and irreversibly inhibits Lon protease-1 (LONP1). In the present study, we evaluate the effects of CDDO-Me on mitochondrial dynamics and its downstream effectors in order to understand the underlying mechanism of the neuronal death following status epilepticus (SE, a prolonged seizure activity). CDDO-Me increased dynamin-related proteins 1 (DRP1)-serine 616 phosphorylation via activating extracellular-signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK), but not protein kinase A (PKA) or protein phosphatases (PPs). In addition, CDDO-Me facilitated DRP1-mediated mitochondrial fissions, which selectively attenuated SE-induced CA1 neuronal death. Unlike CDDO-Me, LONP1 knockdown led to SE-induced massive degeneration of dentate granule cells, CA1 neurons and hilus interneurons without altering the expression and phosphorylation of DRP1, ERK1/2, JNK and PP2B. LONP1 knockdown could not inhibit SE-induced mitochondrial elongation in CA1 neurons. Co-treatment of CDDO-Me with LONP1 siRNA ameliorated only CA1 neuronal death, concomitant with abrogation of mitochondrial elongation induced by SE. Thus, our findings suggest that CDDO-Me may selectively attenuate SE-induced CA1 neuronal death by rescuing the abnormal mitochondrial machinery, independent of LONP1 activity.

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.

Under physiological conditions, microglia are unique immune cells resident in the brain that is isolated from the systemic immune system by brain-blood barrier. Following status epilepticus (SE, a prolonged seizure activity), microglia are rapidly activated and blood-derived monocytes that infiltrate the brain; therefore, the regulations of microglia activation and monocyte infiltration are one of the primary therapeutic strategies for inhibition of undesirable consequences from SE. Roscovitine, a potent (but not selective) cyclin-dependent kinase 5 (CDK5) inhibitor, has been found to exert anti-inflammatory and microglia-inhibiting actions in several in vivo models, although the underlying mechanisms have not been clarified. In the present study, roscovitine attenuated SE-induces monocyte infiltration without vasogenic edema formation in the frontoparietal cortex (FPC), accompanied by reducing expressions of monocyte chemotactic protein-1 (MCP-1) and lysosome-associated membrane protein 1 (LAMP1) in resident microglia, while it did not affect microglia transformation to amoeboid form. Furthermore, roscovitine ameliorated the up-regulation of p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation, but not nuclear factor-κB-S276 phosphorylation. Similar to roscovitine, SB202190, a p38 MAPK inhibitor, mitigated monocyte infiltration and microglial expressions of MCP-1 and LAMP1 in the FPC following SE. Therefore, these findings suggest for the first time that roscovitine may inhibit SE-induced neuroinflammation via regulating p38 MAPK-mediated microglial responses.

2-Cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me) is a triterpenoid analogue of oleanolic acid that has anti-inflammatory, antioxidant, and neuroprotective activities. In the present study, we evaluate the effects of CDDO-Me on serum extravasation and astroglial death in the rat piriform cortex (PC) induced by status epilepticus (a prolonged seizure activity, SE) in order to propose an underlying pharmacological mechanism of CDDO-Me and its availability for treatment of vasogenic edema. CDDO-Me effectively mitigated serum extravasation and a massive astroglial loss in the PC following SE. CDDO-Me abrogated tumor necrosis factor-α (TNF-α) synthesis in activated microglia by inhibiting nuclear factor-κB (NF-κB) p65 serine 276 phosphorylation. CDDO-Me also abolished NF-κB threonine 435 phosphorylation in endothelial cells and TNF-α-mediated-phosphatidylinositol-3-kinase (PI3K)/AKT/endothelial nitric oxide synthase (eNOS) signaling cascades, which trigger vasogenic edema following SE. Furthermore, CDDO-Me increased astroglial viability via the up-regulation of nuclear factor-erythroid 2-related factor 2 (Nrf2) expression. Therefore, our findings suggest that CDDO-Me may ameliorate SE-induced vasogenic edema formation by regulating NF-κB p65 phosphorylations in microglia as well as endothelial cells and enhancing Nrf2 expression in astrocytes, respectively.

Transient receptor potential canonical channel-6 (TRPC6) is one of the Ca2+-permeable non-selective cation channels. TRPC6 is mainly expressed in dentate granule cell (DGC), which is one of the most resistant neuronal populations to various harmful stresses. Although TRPC6 knockdown evokes the massive DGC degeneration induced by status epilepticus (a prolonged seizure activity, SE), the molecular mechanisms underlying the role of TRPC6 in DGC viability in response to SE are still unclear. In the present study, hyperforin (a TRPC6 activator) facilitated mitochondrial fission in DGC concomitant with increases in Lon protease-1 (LONP1, a mitochondrial protease) expression and extracellular-signal-regulated kinase 1/2 (ERK1/2) phosphorylation under physiological conditions, which were abrogated by U0126 (an ERK1/2 inhibitor) co-treatment. TRPC6 knockdown showed the opposite effects on LONP1 expression, ERK1/2 activity, and mitochondrial dynamics. In addition, TRPC6 siRNA and U0126 evoked the massive DGC degeneration accompanied by mitochondrial elongation following SE, independent of seizure severity. However, LONP1 siRNA exacerbated SE-induced DGC death without affecting mitochondrial length. These findings indicate that TRPC6-ERK1/2 activation may increase DGC invulnerability to SE by regulating LONP1 expression as well as mitochondrial dynamics. Therefore, TRPC6-ERK1/2-LONP1 signaling pathway will be an interesting and important therapeutic target for neuroprotection from various neurological diseases.