Sung Hoon Lee

Oroxylin A increases BDNF production by activation of MAPK-CREB pathway in rat primary cortical neuronal culture.

Oroxylin A (5,7-dihydroxy-6-methoxyfavone) is a flavonoid compound originated from the root of Scutellaria baicalensis Georgi. Our previous reports suggested that oroxylin A improves memory function in rat, at least in part, by its antagonistic effects on GABA(A) receptor. In addition, oroxylin A protects neurons from ischemic damage by mechanisms currently not clear. In this study we determined whether oroxylin A modulates the level of brain derived neurotrophic factor (BDNF) in primary rat cortical neuronal culture, which is well known for its role on neuronal survival, neurogenesis, differentiation of neurons and synapses and learning and memory. Treatment of oroxylin A for 3-48h increased BDNF expression which was analyzed by ELISA assay and Western blot analysis. Oroxylin A induced slow but sustained increases in intracellular calcium level and activated ERK1/2 mitogen activated protein kinase (MAPK). In addition, oroxylin A phosphorylated cyclic AMP response element binding protein (CREB) at Ser 133 in concentration and time dependent manner. Pretreatment with the MAPK inhibitor PD98059 (10μM) attenuated phosphorylation of ERK1/2 and CREB as well as BDNF production, which suggests that oroxylin A regulates BDNF production by activating MAPK-CREB pathway. GABA(A) antagonist bicuculline mimicked the effects of oroxylin A on BDNF production as well as MAPK-CREB pathway. Increase in intracellular Ca(2+) concentration, phosphorylation of ERK1/2 and CREB, and BDNF expression by oroxylin A was blocked by NMDA receptor inhibitor MK-801 (10μM) as well as tetrodotoxin (TTX, 0.5 and 1μM). The results from the present study suggest that the calcium and p-CREB dependent induction of BDNF expression, possibly via activation of synaptic NMDA receptor through the blockade of GABA(A) activity in cortical neuronal circuitry, might be responsible for the neuroprotective or memory enhancing effects of oroxylin A.

Biphasic regulation of tissue plasminogen activator activity in ischemic rat brain and in cultured neural cells: essential role of astrocyte-derived plasminogen activator inhibitor-1.

In brain, the serine protease tissue plasminogen activator (tPA) and its endogenous inhibitor plasminogen activator inhibitor-1 (PAI-1) have been implicated in the regulation of various neurophysiological and pathological responses. In this study, we investigated the differential role of neurons and astrocytes in the regulation of tPA/PAI-1 activity in ischemic brain. The activity of tPA peaked transiently and then decreased in cortex and striatum along with delayed induction of PAI-1 in the inflammatory stage after MCAO/reperfusion injury. In cultured primary cells, glutamate stimulation increased tPA activity in neurons but not in other cells such as microglia and astrocytes. With LPS stimulation, a model of neuroinflammatory insults, robust PAI-1 induction was observed in astrocytes but not in neurons and microglia. The upregulation of PAI-1 by LPS in astrocytes was also verified by RT-PCR analysis as well as PAI-1 promoter reporter assay. Lastly, we checked the effects of hypoxia on tPA/PAI-1 activity. Hypoxia increased tPA release from neurons without effects on microglia, while the activity of tPA in astrocyte was decreased consistent with increased PAI-1 activity in astrocyte. Taken together, the results from the present study suggest that neurons are the major source of tPA and that the glutamate-induced stimulated release is mainly governed by neurons in the acute phase. In contrast, the massive up-regulation of PAI-1 in astrocytes during subchronic and chronic inflammatory conditions, leads to decreased tPA activity in the later stages of MCAO. Differential regulation of tPA and PAI-1 in neurons, astrocytes and microglia suggest more attention is required to understand the role of local tPA activity in the vicinity of individual cell types.

Valproic acid induces astrocyte-dependent neurite outgrowth from cultured rat primary cortical neuron via modulation of tPA/PAI-1 activity.

Tissue plasminogen activator (tPA) is expressed in several regions of brain and plays regulatory roles such as neurite outgrowth, synaptic plasticity and long term potentiation. The activity of tPA is regulated by an endogenous inhibitor plasminogen activator inhibitor‐1 (PAI‐1), which is expressed mainly in astrocytes. Valproic acid (VPA), a histone deacetylase inhibitor that is used for the treatment of epilepsy and bipolar disorders, promotes neurite extension, neuronal growth and has neuroprotective effect in neurodegenerative diseases. In this study, we examined whether the neurite extension effects of VPA is mediated by modulating tPA/PAI‐1 system. VPA dose‐dependently increased tPA activity and decreased PAI‐1 activity in rat primary astrocytes but not in neurons. PAI‐1 protein level secreted into the culture medium but not tPA per se was decreased by VPA. In co‐culture system or in neuronal culture stimulated with astrocyte conditioned media but not in pure neuronal cell culture, VPA induced neurite outgrowth via increased tPA activity due to the decreased PAI‐1 activity in astrocytes. The decrease in PAI‐1 activity and increased neurite extension was regulated via JNK mediated post‐transcriptional pathway. The essential role of tPA/PAI‐1 system in the regulation of VPA‐mediated neurite extension was further demonstrated by experiments using astrocyte conditioned media obtained from tPA or PAI‐1 knockout mice. Regulation of PAI‐1 activity in astrocyte by VPA may affect both physiological and pathological processes in brain by upregulating tPA activity. GLIA 2013

The Role of TLR4 and Fyn Interaction on Lipopolysaccharide-Stimulated PAI-1 Expression in Astrocytes

Plasminogen activator inhibitor-1 (PAI-1) is an endogenous inhibitor of tissue plasminogen activator (tPA) that acts as a neuromodulator in various neurophysiological and pathological conditions. Several researchers including us reported the induction of PAI-1 during inflammatory condition; however, the mechanism regulating PAI-1 induction is not yet clear. In this study, we investigated the role of non-receptor tyrosine kinase Fyn in the regulation of lipopolysaccharide (LPS)-induced upregulation of PAI-1 in rat primary astrocyte. The activation of toll-like receptor 4 (TLR4) signaling, induced by its ligand LPS, stimulated a physical interaction between TLR4 and Fyn along with phosphorylation of tyrosine residue in both molecules as determined by co-immunoprecipitation experiments. Immunofluorescence staining also showed increased co-localization of TLR4-Fyn on cultured rat primary astrocytes after LPS treatment. The increased TRLR4-Fyn interaction induced expression of PAI-1 through the activation of PI3k/Akt/NFĸB pathway. Treatment with Src kinase inhibitor (PP2) or transfection of Fyn small interfering RNA (siRNA) into cultured rat primary astrocytes inhibited phosphorylation of tyrosine residue of TLR4 and blocked the interaction between TLR4 and Fyn resulting to the inhibition of LPS-induced expression of PAI-1. The activation of PI3K/Akt/NFĸB signaling cascades was also inhibited by Fyn knockdown in rat primary astrocytes. The induction of PAI-1 in rat primary astrocytes, which resulted in downregulation of tPA activity in culture supernatants, inhibited neurite outgrowth in cultured rat primary cortical neuron. The inhibition of neurite extension was prevented by PP2 or Fyn siRNA treatment in rat primary astrocytes. These results suggest the critical physiological role of TRL4-Fyn interaction in the modulation of PAI-1-tPA axis in astrocytes during neuroinflammatory responses such as ischemia/reperfusion injuries.

Urokinase-type plasminogen activator induces BV-2 microglial cell migration through activation of matrix metalloproteinase-9.

In response to brain injury, microglia migrate and accumulate in the affected sites, which is an important step in the regulation of inflammation and neuronal degeneration/regeneration. In this study, we investigated the effect of urokinase-type plasminogen activator (uPA) on the BV-2 microglial cell migration. At resting state, BV-2 microglial cells secreted uPA and the release of uPA was increased by ATP, a chemoattractant released from injured neuron. The migration of BV-2 cell was significantly induced by uPA and inhibited by uPA inhibitors. In this condition, uPA increased the activity of matrix metalloproteinase (MMP-9) and the inhibition of MMP activity with pharmacological inhibitors against either uPA (amiloride) or MMP (phenanthrolene and SB-3CT) effectively prevented BV2 cell migration. Interestingly, the level of MMP-9 protein and mRNA in the cell were not changed by uPA. These results suggest that the increase of MMP-9 activity by uPA is regulated at the post-translational level, possibly via increased activation of the enzyme. Unlike the uPA inhibitor, plasmin inhibitor PAI-1 only partially inhibited uPA-induced cell migration and MMP-9 activation. The incubation of recombinant MMP-9 with uPA resulted in the activation of MMP-9. These results suggest that uPA plays a critical role in BV-2 microglial cell migration by activating pro-MMP-9, in part by its direct action on MMP-9 and also in part by the activation of plasminogen/plasmin cascade.

Oroxylin A Induces BDNF Expression on Cortical Neurons through Adenosine A2A Receptor Stimulation: A Possible Role in Neuroprotection

Oroxylin A is a flavone isolated from a medicinal herb reported to be effective in reducing the inflammatory and oxidative stresses. It also modulates the production of brain derived neurotrophic factor (BDNF) in cortical neurons by the transactivation of cAMP response element-binding protein (CREB). As a neurotrophin, BDNF plays roles in neuronal development, differentiation, synaptogenesis, and neural protection from the harmful stimuli. Adenosine A2A receptor colocalized with BDNF in brain and the functional interaction between A2A receptor stimulation and BDNF action has been suggested. In this study, we investigated the possibility that oroxylin A modulates BDNF production in cortical neuron through the regulation of A2A receptor system. As ex-pected, CGS21680 (A2A receptor agonist) induced BDNF expression and release, however, an antagonist, ZM241385, prevented oroxylin A-induced increase in BDNF production. Oroxylin A activated the PI3K-Akt-GSK-3β signaling pathway, which is inhibited by ZM241385 and the blockade of the signaling pathway abolished the increase in BDNF production. The physiological roles of oroxylin A-induced BDNF production were demonstrated by the increased neurite extension as well as synapse formation from neurons. Overall, oroxylin A might regulate BDNF production in cortical neuron through A2A receptor stimulation, which promotes cellular survival, synapse formation and neurite extension.

tPA Regulates Neurite Outgrowth by Phosphorylation of LRP5/6 in Neural Progenitor Cells

Despite the important role of tissue plasminogen activator (tPA) as a neuromodulator in neurons, microglia, and astrocytes, its role in neural progenitor cell (NPC) development is not clear yet. We identified that tPA is highly expressed in NPCs compared with neurons. Inhibition of tPA activity or expression using tPA stop, PAI-1, or tPA siRNA inhibited neurite outgrowth from NPCs, while overexpression or addition of exogenous tPA increased neurite outgrowth. The expression of Wnt and β-catenin as well as phosphorylation of LRP5 and LRP6, which has been implicated in Wnt–β-catenin signaling, was rapidly increased after tPA treatment and was decreased by tPA siRNA transfection. Knockdown of β-catenin or LRP5/6 expression by siRNA prevented tPA-induced neurite extension. NPCs obtained from tPA KO mice showed impaired neurite outgrowth compared with WT NPCs. In ischemic rat brains, axon density was higher in the brains transplanted with WT NPCs than in those with tPA KO NPCs, suggesting increased axonal sprouting by NPC-derived tPA. tPA-mediated regulation of neuronal maturation in NPCs may play an important role during development and in regenerative conditions.

Glucose deprivation reversibly down-regulates tissue plasminogen activator via proteasomal degradation in rat primary astrocytes

AIMS Tissue plasminogen activator (tPA) is an essential neuromodulator whose involvement in multiple functions such as synaptic plasticity, cytokine-like immune function and regulation of cell survival mandates rapid and tight tPA regulation in the brain. We investigated the possibility that a transient metabolic challenge induced by glucose deprivation may affect tPA activity in rat primary astrocytes, the main cell type responsible for metabolic regulation in the CNS. MAIN METHODS Rat primary astrocytes were incubated in serum-free DMEM without glucose. Casein zymography was used to determine tPA activity, and tPA mRNA was measured by RT-PCR. The signaling pathways regulating tPA activity were identified by Western blotting. KEY FINDINGS Glucose deprivation rapidly down-regulated the activity of tPA without affecting its mRNA level in rat primary astrocytes; this effect was mimicked by translational inhibitors. The down-regulation of tPA was accompanied by increased tPA degradation, which may be modulated by a proteasome-dependent degradation pathway. Glucose deprivation induced activation of PI3K-Akt-GSK3β, p38 and AMPK, and inhibition of these pathways using LY294002, SB203580 and compound C significantly inhibited glucose deprivation-induced tPA down-regulation, demonstrating the essential role of these pathways in tPA regulation in glucose-deprived astrocytes. SIGNIFICANCE Rapid and reversible regulation of tPA activity in rat primary astrocytes during metabolic crisis may minimize energy-requiring neurologic processes in stressed situations. This effect may thereby increase the opportunity to invest cellular resources in cell survival and may allow rapid re-establishment of normal cellular function after the crisis.

Regulation of tissue plasminogen activator/plasminogen activator inhibitor‐1 by hydrocortisone in rat primary astrocytes

Although originally known as a plasma serine protease involved in clot dissolution, tPA and its primary inhibitor, PAI‐1, play crucial roles in synaptic reorganization and plasticity in the central nervous system. In contrast to the wide array of work conducted in neural cells, relatively little is known about the regulatory mechanism governing tPA/PAI‐1 expression in astrocytes. Glucocorticoids (GCs) such as hydrocortisone regulate the expression of tPA/PAI‐1 in various biological systems in a tissue‐specific manner. However, little is known about GC‐mediated regulation of tPA/PAI‐1 system in CNS. The aims of the present study were to investigate whether tPA/PAI‐1 expression is regulated by hydrocortisone in rat primary astrocytes. Enzyme activity of tPA was decreased in a concentration‐dependent manner by hydrocortisone treatment, and the activity of PAI‐1 was increased by hydrocortisone. Hydrocortisone did not affect the level of tPA mRNA, which suggests that transcriptional down‐regulation of tPA mRNA is not involved in the down‐regulation of tPA enzyme activity in astrocytes. However, the level of PAI‐1 mRNA and protein was increased. Both hydrocortisone and a tPA‐Stop treatment prevented glutamate‐induced neurotoxicity in rat cortical primary mixed astrocyte–neuron culture, which suggests a neurotoxic role for tPA in our culture system. Interestingly, hydrocortisone further increased LPS‐induced up‐regulation of PAI‐1 while inhibiting the up‐regulation of iNOS and COX‐2 expression. Our data show that hydrocortisone up‐regulated PAI‐1 expression along with down‐regulation of tPA activity in both normal and inflammatory conditions.

Valproic Acid Induces Telomerase Reverse Transcriptase Expression during Cortical Development

The valproic acid (VPA)-induced animal model is one of the most widely utilized environmental risk factor models of autism. Autism spectrum disorder (ASD) remains an insurmountable challenge among neurodevelopmental disorders due to its heterogeneity, unresolved pathological pathways and lack of treatment. We previously reported that VPA-exposed rats and cultured rat primary neurons have increased Pax6 expression during post-midterm embryonic development which led to the sequential upregulation of glutamatergic neuronal markers. In this study, we provide experimental evidence that telomerase reverse transcriptase (TERT), a protein component of ribonucleoproteins complex of telomerase, is involved in the abnormal components caused by VPA in addition to Pax6 and its downstream signals. In embryonic rat brains and cultured rat primary neural progenitor cells (NPCs), VPA induced the increased expression of TERT as revealed by Western blot, RT-PCR, and immunostainings. The HDAC inhibitor property of VPA is responsible for the TERT upregulation. Chromatin immunoprecipitation revealed that VPA increased the histone acetylation but blocked the HDAC1 binding to both Pax6 and Tert genes. Interestingly, the VPA-induced TERT overexpression resulted to sequential upregulations of glutamatergic markers such as Ngn2 and NeuroD1, and inter-synaptic markers such as PSD-95, α-CaMKII, vGluT1 and synaptophysin. Transfection of Tert siRNA reversed the effects of VPA in cultured NPCs confirming the direct involvement of TERT in the expression of those markers. This study suggests the involvement of TERT in the VPA-induced autistic phenotypes and has important implications for the role of TERT as a modulator of balanced neuronal development and transmission in the brain.