Prenatal exposure to valproic acid increases the neural progenitor cell pool and induces macrocephaly in rat brain via a mechanism involving the GSK-3ß/ß-catenin pathway.

Autism is a spectrum of neurodevelopmental disorders characterized by social isolation and lack of interaction. Anatomically, autism patients often show macrocephaly and high neuronal density. To investigate the mechanism underlying the higher neuronal populations seen in ASD, we subcutaneously injected VPA (400 mg/kg) into pregnant Sprague-Dawley rats on E12, an animal model often used in ASD study. Alternatively, cultured rat neural progenitor cells were treated with VPA. Until E18, VPA induced NPC proliferation and delayed neurogenesis in fetal brain, but the subsequent differentiation of NPCs to neurons increased brain neuronal density afterward. Similar findings were observed with NPCs treated with VPA in vitro. At a molecular level, VPA enhanced Wnt1 expression and activated the GSK-3ß/ß-catenin pathway. Furthermore, inhibition of this pathway attenuated the effects of VPA. The findings of this study suggest that an altered developmental process underlies the macrocephaly and abnormal brain structure observed in the autistic brain.

Positive feedback regulation of Akt-FMRP pathway protects neurons from cell death.

J. Neurochem. (2012) 123, 226-238. ABSTRACT: Fragile X syndrome (FXS), the most common single genetic cause of mental retardation and autistic spectrum disease, occurs when FMR1 gene is mutated. FMR1 encodes fragile X mental retardation protein (FMRP) which regulates translation of mRNAs playing important roles in the development of neurons as well as formation and maintenance of synapses. To examine whether FMRP regulates cell viability, we induced apoptosis in rat primary cortical neurons with glutamate in vitro and with middle cerebral artery occlusion (MCAO) in striatal neurons in vivo. Both conditions elicited a rapid, but transient FMRP expression in neurons. This up-regulated FMRP expression was abolished by pre-treatment with PI3K and Protein Kinase B (Akt) inhibitors: LY294002, Akt inhibitor IV, and VIII. Reduced FMRP expression in vitro or in vivo using small hairpin Fmr1 virus exacerbated cell death by glutamate or MCAO, presumably via hypophosphorylation of Akt and reduced expression of B-cell lymphoma-extra large (Bcl-xL). However, over-expression of FMRP using enhanced green fluorescent protein (eGFP)-FMRP constructs alleviated cell death, increased Akt activity, and enhanced Bcl-xL production. The pro-survival role of Akt-dependent up-regulation of FMRP in glutamate-stimulated cultured neuron as well as in ischemic brain may have a clinical importance in FXS as well as in neurodegenerative disorders and traumatic brain injury.

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-3ß 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.

Cognitive dysfunctions induced by a cholinergic blockade and Aß 25-35 peptide are attenuated by salvianolic acid B.

Alzheimer's disease (AD) is a neurodegenerative disorder associated with progressive cognitive and memory loss and neuronal cell death. Current therapeutic strategies for AD are very limited; thus, traditional herbal medicines or their active constituents receive much attention. The aim of this study was to investigate the cognitive enhancing effects of salvianolic acid B (SalB) isolated from Salvia miltiorrhiza and its ameliorating effects on various drug-induced amnesic models using the passive avoidance, Y-maze, and Morris water maze tasks. Drug-induced amnesia was induced by administering scopolamine, diazepam, muscimol, or amyloid-ß (Aß)(25-35) peptide. SalB (10 mg/kg, p.o.) was found to significantly reverse the cognitive impairments induced by scopolamine (1 mg/kg, i.p.) or Aß(25-35) (10 nmol/5 µl, i.c.v.) injection. This ameliorating effect of SalB was antagonized by the GABA(A) receptor agonists, muscimol or diazepam, respectively. In addition, SalB alone was capable of improving cognitive performances. Furthermore, SalB (100 µM) was found to inhibit GABA-induced outward Cl(-) currents in single hippocampal CA1 neuron. These results suggest that the observed ameliorations of cholinergic dysfunction- or Aß(25-35)-induced memory impairment by SalB were mediated, in part, via the GABAergic neurotransmitter system after a single administration.

Cyclin B1 expression regulated by cytoplasmic polyadenylation element binding protein in astrocytes.

Astrocytes are the most abundant cells in the brain, playing vital roles in neuronal survival, growth, and function. Understanding the mechanism(s) regulating astrocyte proliferation will have important implications in brain development, response to injury, and tumorigenesis. Cyclin B1 is well known to be a critical regulator of mitotic entry via its interaction with cyclin-dependent kinase 1. In rat astrocytes, we now show that the mRNA binding protein cytoplasmic polyadenylation element binding protein 1 (CPEB1) is associated with cyclin B1 mRNA and that this interaction is enriched at the centrosome. In addition, if growth-arrested astrocytes are stimulated to divide, CPEB1 is phosphorylated and cyclin B1 mRNA is polyadenylated, both hallmarks of CPEB1 activation, resulting in an increase in cyclin B1 protein. CPEB1 binding to mRNA initially inhibits translation; therefore, removing CPEB1 from mRNA should result in an increase in translation due to derepression. Indeed, when we either knocked down CPEB1 protein with siRNA or sequestered it from endogenous mRNA by expressing RNA containing multiple CPEB1 binding sites, cyclin B1 protein was increased and cell proliferation was stimulated. Our data suggest a mechanism wherein CPEB1 is bound and represses cyclin B1 mRNA translation until a signal to proliferate phosphorylates CPEB1, resulting in an increase in cyclin B1 protein and progression into mitosis. Our results demonstrate for the first time a role for CPEB1 in regulating cell proliferation in the brain.

Valproic acid inhibits neural progenitor cell death by activation of NF-κB signaling pathway and up-regulation of Bcl-XL.

BACKGROUND: At the beginning of neurogenesis, massive brain cell death occurs and more than 50% of cells are eliminated by apoptosis along with neuronal differentiation. However, few studies were conducted so far regarding the regulation of neural progenitor cells (NPCs) death during development. Because of the physiological role of cell death during development, aberration of normal apoptotic cell death is detrimental to normal organogenesis.Apoptosis occurs in not only neuron but also in NPCs and neuroblast. When growth and survival signals such as EGF or LIF are removed, apoptosis is activated as well as the induction of differentiation. To investigate the regulation of cell death during developmental stage, it is essential to investigate the regulation of apoptosis of NPCs. METHODS: Neural progenitor cells were cultured from E14 embryonic brains of Sprague-Dawley rats. For in vivo VPA animal model, pregnant rats were treated with VPA (400 mg/kg S.C.) diluted with normal saline at E12. To analyze the cell death, we performed PI staining and PARP and caspase-3 cleavage assay. Expression level of proteins was investigated by Western blot and immunocytochemical assays. The level of mRNA expression was investigated by RT-PCR. Interaction of Bcl-XL gene promoter and NF-κB p65 was investigated by ChIP assay. RESULTS: In this study, FACS analysis, PI staining and PARP and caspase-3 cleavage assay showed that VPA protects cultured NPCs from cell death after growth factor withdrawal both in basal and staurosporine- or hydrogen peroxide-stimulated conditions. The protective effect of prenatally injected VPA was also observed in E16 embryonic brain. Treatment of VPA decreased the level of IκBα and increased the nuclear translocation of NF-κB, which subsequently enhanced expression of anti-apoptotic protein Bcl-XL. CONCLUSION: To the best of our knowledge, this is the first report to indicate the reduced death of NPCs by VPA at developmentally critical periods through the degradation of IκBα and the activation of NF-κB signaling. The reduced NPCs death might underlie the neurodevelopmental defects collectively called fetal valproate syndrome, which shows symptoms such as mental retardation and autism-like behavior.

Activation of adenosine A2A receptor up-regulates BDNF expression in rat primary cortical neurons.

As a member of neurotrophin family, brain derived neurotrophic factor (BDNF) plays critical roles in neuronal development, differentiation, synaptogenesis, and neural protection from the harmful stimuli. There have been reported that adenosine A2(A) receptor subtype is widely distributed in the brain regions, such as hippocampus, striatum, and cortex. Adenosine A2(A) receptor is colocalized with BDNF in brain regions and the functional interaction between A2(A) receptor stimulation and BDNF action has been suggested. In this study, we investigated the possibility that the activation of A2(A) receptor modulates BDNF production in rat primary cortical neuron. CGS21680, an adenosine A2(A) receptor agonist, induced BDNF expression and release. An antagonist against A2(A) receptor, ZM241385, prevented CGS21680-induced increase in BDNF production. A2(A) receptor stimulation induced the activation of Akt-GSK-3ß signaling pathway and the blockade of the signaling pathway with specific inhibitors abolished the increase in BDNF production, possibly via modulation of ERK1/2-CREB pathway. The physiological roles of A2(A) receptor-induced BDNF production was demonstrated by the protection of neurons from the excitotoxicity and increased neurite extension as well as synapse formation from immature and mature neurons. Taken together, activation of A2(A) receptor regulates BDNF production in rat cortical neuron, which provides neuro-protective action.

Role of oxidative stress in epileptic seizures.

Oxidative stress resulting from excessive free-radical release is likely implicated in the initiation and progression of epilepsy. Therefore, antioxidant therapies aimed at reducing oxidative stress have received considerable attention in epilepsy treatment. However, much evidence suggests that oxidative stress does not always have the same pattern in all seizures models. Thus, this review provides an overview aimed at achieving a better understanding of this issue. We summarize work regarding seizure models (i.e., genetic rat models, kainic acid, pilocarpine, pentylenetetrazol, and trimethyltin), oxidative stress as an etiologic factor in epileptic seizures (i.e., impairment of antioxidant systems, mitochondrial dysfunction, involvement of redox-active metals, arachidonic acid pathway activation, and aging), and antioxidant strategies for seizure treatment. Combined, this review highlights pharmacological mechanisms associated with oxidative stress in epileptic seizures and the potential for neuroprotection in epilepsy that targets oxidative stress and is supported by effective antioxidant treatment.

Neuroprotective effect of forsythiaside against transient cerebral global ischemia in gerbil.

Forsythiaside, a phenylethanoside, has been reported to have anti-oxidative activity and memory ameliorating effects against a scopolamine-induced memory deficit model. The aim of this study was to determine whether forsythiaside has neuroprotective activity on transient cerebral global ischemia in gerbil. Transient cerebral ischemia was induced by bilateral common carotid artery occlusion for 5 min and followed by reperfusion for 7 days. Oral administration of forsythiaside was conducted immediately after reperfusion and once a day over the next 7 days. The forsythiaside administration significantly increased the number of viable neurons detected by neuronal nuclei immunostaining and decreased degenerating neuronal cells detected by Fluoro-Jade B staining in the hippocampal CA1 region, at the 7th day post-ischemia (P<0.05). Forsythiaside also significantly decreased the number of ionized calcium-binding adaptor molecule-1-detected activated microglia and glial fibrillary acidic protein-detected astrocytes, both of which were increased after ischemic insults, and decreased interleukin-1ß and tumor necrosis factor-α expression levels, which were also increased after the insults (P<0.05). In addition, forsythiaside significantly improved ischemia-induced cognitive impairments in the Y-maze task (P<0.05). These results suggest that forsythiaside exhibits neuroprotective properties, which are, in part, mediated by its anti-inflammatory activities supported by forsythiaside-induced reductions of activated glial cells and expression levels of interleukin-1ß and tumor necrosis factor-α.

Convulsion-related activities of Scutellaria flavones are related to the 5,7-dihydroxyl structures.

We screened the major bioactive flavones isolated from Scutellaria baicalensis (baicalin, baicalein and oroxylin A) for their convulsion related activities. In electrogenic response score system and the pentylenetetrazole seizure model, baicalein but not oroxylin A and baicalin exhibited anticonvulsant effects. In vitro studies also revealed that baicalein induced intracellular Cl(-) influx, whereas oroxylin A blocked muscimol- and baicalein-induced intracellular Cl(-) influx. The anticonvulsant effect of baicalein was inhibited by flumazenil, a benzodiazepine(BZD) receptor antagonist. Therefore, anticonvulsive effect of baicalein was mediated by the BZD binding site of GABA(A) receptor. The 5, 7-dihydroxyl group is present in the structure of the three flavones. It is postulated that this group played a key role in inducing convulsion-related activities.

Cellular stress-induced up-regulation of FMRP promotes cell survival by modulating PI3K-Akt phosphorylation cascades.

BACKGROUND: Fragile X syndrome (FXS), the most commonly inherited mental retardation and single gene cause of autistic spectrum disorder, occurs when the Fmr1 gene is mutated. The product of Fmr1, fragile X linked mental retardation protein (FMRP) is widely expressed in HeLa cells, however the roles of FMRP within HeLa cells were not elucidated, yet. Interacting with a diverse range of mRNAs related to cellular survival regulatory signals, understanding the functions of FMRP in cellular context would provide better insights into the role of this interesting protein in FXS. Using HeLa cells treated with etoposide as a model, we tried to determine whether FMRP could play a role in cell survival. METHODS: Apoptotic cell death was induced by etoposide treatment on Hela cells. After we transiently modulated FMRP expression (silencing or enhancing) by using molecular biotechnological methods such as small hairpin RNA virus-induced knock down and overexpression using transfection with FMRP expression vectors, cellular viability was measured using propidium iodide staining, TUNEL staining, and FACS analysis along with the level of activation of PI3K-Akt pathway by Western blot. Expression level of FMRP and apoptotic regulator BcL-xL was analyzed by Western blot, RT-PCR and immunocytochemistry. RESULTS: An increased FMRP expression was measured in etoposide-treated HeLa cells, which was induced by PI3K-Akt activation. Without FMRP expression, cellular defence mechanism via PI3K-Akt-Bcl-xL was weakened and resulted in an augmented cell death by etoposide. In addition, FMRP over-expression lead to the activation of PI3K-Akt signalling pathway as well as increased FMRP and BcL-xL expression, which culminates with the increased cell survival in etoposide-treated HeLa cells. CONCLUSIONS: Taken together, these results suggest that FMRP expression is an essential part of cellular survival mechanisms through the modulation of PI3K, Akt, and Bcl-xL signal pathways.

The critical period of valproate exposure to induce autistic symptoms in Sprague-Dawley rats.

Prenatal exposure to valproic acid (VPA) induces neural tube defects and impairment in social behaviors related to autistic spectrum disorder in newborns, which make it a useful animal model of autism. In this study, we compared the effects of different time window of prenatal valproic acid exposure for inducing the altered social behaviors relevant to autism from embryonic day 7 to embryonic day 15 in Sprague-Dawley rats to determine the critical periods for the impairment. Compared to E7, E9.5 and E15 exposure, VPA exposure at E12 showed most significant changes in behaviors over control animals with reduced sociability and social preference. E9.5 exposure to valproic acid showed strong reproductive toxicity including decrease in the number of live birth. In general, exposure at E15 showed only marginal effects on reproduction and social behaviors. Finally, VPA-exposed rats at E12 were more sensitive to electric shock than VPA-exposed rats at any other periods. These results suggested that E12 is the critical period in rats when valproate exposure has prominent effects for inducing the altered social behavior similar to human autistic behavior.

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.

Prenatal exposure of ethanol induces increased glutamatergic neuronal differentiation of neural progenitor cells.

BACKGROUND: Prenatal ethanol exposure during pregnancy induces a spectrum of mental and physical disorders called fetal alcohol spectrum disorder (FASD). The central nervous system is the main organ influenced by FASD, and neurological symptoms include mental retardation, learning abnormalities, hyperactivity and seizure susceptibility in childhood along with the microcephaly. In this study, we examined whether ethanol exposure adversely affects the proliferation of NPC and de-regulates the normal ratio between glutamatergic and GABAergic neuronal differentiation using primary neural progenitor culture (NPC) and in vivo FASD models. METHODS: Neural progenitor cells were cultured from E14 embryo brain of Sprague-Dawley rat. Pregnant mice and rats were treated with ethanol (2 or 4 g/kg/day) diluted with normal saline from E7 to E16 for in vivo FASD animal models. Expression level of proteins was investigated by western blot analysis and immunocytochemical assays. MTT was used for cell viability. Proliferative activity of NPCs was identified by BrdU incorporation, immunocytochemistry and FACS analysis. RESULTS: Reduced proliferation of NPCs by ethanol was demonstrated using BrdU incorporation, immunocytochemistry and FACS analysis. In addition, ethanol induced the imbalance between glutamatergic and GABAergic neuronal differentiation via transient increase in the expression of Pax6, Ngn2 and NeuroD with concomitant decrease in the expression of Mash1. Similar pattern of expression of those transcription factors was observed using an in vivo model of FASD as well as the increased expression of PSD-95 and decreased expression of GAD67. CONCLUSIONS: These results suggest that ethanol induces hyper-differentiation of glutamatergic neuron through Pax6 pathway, which may underlie the hyper-excitability phenotype such as hyperactivity or seizure susceptibility in FASD patients.

The effects of daidzin and its aglycon, daidzein, on the scopolamine-induced memory impairment in male mice.

In this study, the effect of daidzin or daidzein isolated from Pueraria lobata on the memory impairments induced by scopolamine was assessed in male mice using the passive avoidance and the Morris water maze tasks. Administration of daidzin (5 mg/kg) or daidzein (5 mg/kg) significantly reversed the scopolamine (1 mg/kg)-induced cognitive impairments in male mice as evidenced by the passive avoidance test (p < 0.05) and on the Morris water maze test (p < 0.05). Moreover, the ameliorating effects of daidzin or daidzein were antagonized by tamoxifen (1 mg/kg), the nonspecific estrogen receptor antagonist. These results indicate that daidzin or daidzein may be useful in cognitive impairment induced by cholinergic dysfunction, and this beneficial effect is mediated, in part, via estrogen receptor.

Mismatch between changes in baicalein-induced memory-related biochemical parameters and behavioral consequences in mouse.

Baicalein is one of the major flavonoids originally isolated from the roots of Scutellaria baicalensis Georgi (Labiatae). Reports on baicalein-induced changes in memory-related biochemical parameters including extracellular signal-regulated kinases (ERK), Akt, cAMP response element-binding protein (CREB), and brain-derived neurotrophic factor (BDNF) have been scarce, and the action of baicalein is controversial. Baicalein promotes phosphorylation of ERK under normal conditions; on the other hand, it inhibits phosphorylation of ERK extracellularly under oxidative conditions. In the present study, we observed that baicalein (20mg/kg, p.o.) as compared to vehicle significantly increased the expression of phosphorylated ERK (pERK), phosphorylated CREB (pCREB), and BDNF but did not increase phosphorylated Akt expression in the hippocampus of naive mice. Baicalein also significantly increased the expression of pERK and BDNF in the cortex of naive mice. However, baicalein had no effect on memory acquisition in the step-through passive avoidance task. On the contrary, baicalein (20mg/kg, p.o.) co-injected with flumazenil (10mg/kg, i.p.) significantly increased the retention latency in the passive avoidance task in comparison to the flumazenil-treated group, baicalein-treated group, and vehicle-treated control group. In addition, the number of platform crossings in the Morris water maze test during the probe trial session was significantly increased by co-administration of baicalein with flumazenil. Furthermore, the expressions level of BDNF was significantly increased in the baicalein with flumazenil-treated group compared to the baicalein- or flumazenil-treated groups in the hippocampus after an acquisition trial. These results suggest that the reasons why baicalein does not exert cognitive enhancement although it enhances the expression levels of pERK, pCREB, and BDNF are, in part, derived from its GABA(A) receptor agonistic property which is antagonized by flumazenil.

The ameliorating effect of the extract of the flower of Prunella vulgaris var. lilacina on drug-induced memory impairments in mice.

Prunella vulgaris var. lilacina is widely distributed in Korea, Japan, China, and Europe, and its flowers are used to treat inflammation in traditional Chinese medicine. In the present study, we studied the effects of the ethanolic extract of the flower of P. vulgaris var. lilacina (EEPV) on drug-induced learning and memory impairment using the passive avoidance, the Y-maze, and the Morris water maze tasks in mice. EEPV (25 or 50 mg/kg, p.o.) significantly ameliorated scopolamine-induced cognitive impairments in the passive avoidance and Y-maze tasks (P<0.05). In the Morris water maze task, EEPV (25 mg/kg, p.o.) significantly shortened escape latencies in training-trials. Furthermore, swimming times within the target zone during the probe-trial were significantly increased as compared with scopolamine-treated mice (P<0.05). In addition, the reduced latency induced by MK-801 treatment in the passive avoidance task was ameliorated by EEPV (25 mg/kg, p.o.) (P<0.05). Additionally, the ameliorating effect of EEPV on scopolamine-induced memory dysfunction was antagonized by a sub-effective dose of MK-801. These results suggest that EEPV would be useful for treating cognitive impairments induced by cholinergic dysfunction, and that it exerts its effects via NMDA receptor signaling.

ATP induced microglial cell migration through non-transcriptional activation of matrix metalloproteinase-9.

In response to brain insults, microglia, the resident inflammatory cells in CNS, migrate into injured sites to initiate inflammatory responses in brain. ATP, released from apoptotic or necrotic cells induce chemoattractive responses but the mechanism is not clear yet. In this study, we investigated whether ATP modulates microglial migration by regulating the activity of matrix metalloproteinases (MMPs). ATP induced rapid microglial migration and increased the activity of MMP-9 in the culture supernatants (secreted compartments) in a concentration-dependent manner. The increased activity of secreted MMP-9 is due to the increased protein secretion, but not by the increased MMP-9 mRNA and protein expression. Inhibition of MMP-9 activity by treatment with specific inhibitors including GM6001 and SB-3CT prevented ATP-induced microglial migration. ATP-induced microglial migration was also inhibited by P2Y receptor antagonists including clopidogrel as well as PI3K inhibitor such as wortmanin. Taken together, ATP non-transcriptionally increased MMP-9 activity by activation of P2Y and PI3K. The results from the present investigation may provide further insights into the regulation of the activity of MMP-9 during microglial migration, which may play essential role in the regulation of inflammatory responses in pathological situations such as neurodegenerative disorders.

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.

Activation of microglial cells via protease-activated receptor 2 mediates neuronal cell death in cultured rat primary neuron.

The role of protease-activated receptor (PARs) in the regulation of microglial activation process is increasingly evident. In the present study, we have investigated the role of PAR-2, which can be activated by trypsin-like proteases, in microglial activation and neuronal cell death. In cultured rat primary microglia, activation of PAR-2 induced nitrite production by PKC- and MAPKs-dependent mechanism. Among the three members of MAPK pathway, ERK and JNK but not p38 mediated PAR-2-induced microglial activation. The down-stream regulator of PAR-2-PKC-MAPK pathway-induced microglial activation was NF-kappaB pathway. Besides nitrite, PAR-2 activation increased production of a variety of inflammatory mediators such as ROS and pro-inflammatory cytokines including TNF-alpha and IL-1beta. The addition of culture spent media from PAR-2 activated microglia induced neuronal cell death in primary rat cortical neuron cultures with apoptotic features such as increased number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive neurons, dissipation of mitochondrial membrane potential, increased expression of pro-apoptotic Bax, decreased expression of anti-apoptotic Bcl-2, Bcl-X(L), and activation of caspase-3 in neurons. Interestingly, the increased production of cytoactive molecules as well as the neuronal cell death was normalized by PAR-2 or trypsin inhibitor or an NO synthase inhibitor, N(G)-nitro-l-arginine-methyl ester. Taken together, these results suggest that overt PAR-2 activation may induce microglial activation, which contributes to neuronal cell death.