Hydrogen sulfide and polysulfides induce GABA/glutamate/D-serine release, facilitate hippocampal LTP, and regulate behavioral hyperactivity.

Hydrogen sulfide (H2S) and polysulfides (H2Sn, n ≥ 2) are signaling molecules produced by 3-mercaptopyruvate sulfurtransferase (3MST) that play various physiological roles, including the induction of hippocampal long-term potentiation (LTP), a synaptic model of memory formation, by enhancing N-methyl-D-aspartate (NMDA) receptor activity. However, the presynaptic action of H2S/H2Sn on neurotransmitter release, regulation of LTP induction, and animal behavior are poorly understood. Here, we showed that H2S/H2S2 applied to the rat hippocampus by in vivo microdialysis induces the release of GABA, glutamate, and D-serine, a co-agonist of NMDA receptors. Animals with genetically knocked-out 3MST and the target of H2S2, transient receptor potential ankyrin 1 (TRPA1) channels, revealed that H2S/H2S2, 3MST, and TRPA1 activation play a critical role in LTP induction, and the lack of 3MST causes behavioral hypersensitivity to NMDA receptor antagonism, as in schizophrenia. H2S/H2Sn, 3MST, and TRPA1 channels have therapeutic potential for psychiatric diseases and cognitive deficits.

3',4'-Dihydroxyflavonol Attenuates Lipopolysaccharide-Induced Neuroinflammatory Responses of Microglial Cells by Suppressing AKT-mTOR and NF-κB Pathways.

Microglia-related neuroinflammation contributes to the pathogenesis of a variety of neurodegenerative disorders such as Alzheimer's disease. The synthetic flavonoid, 3',4'-dihydroxyflavonol (3,3',4'-trihydroxyflavone), has been shown to protect brain or myocardial ischemia reperfusion-induced cell death and prevent the aggregation of amyloid-ß protein, a process that causes progressive neurodegeneration in Alzheimer's disease. Here, we explored the anti-neuroinflammatory ability of 3',4'-dihydroxyflavonol in lipopolysaccharide (LPS)-activated MG6 microglial cells. 3',4'-Dihydroxyflavonol attenuated LPS-induced tumor necrosis factor-α and nitric oxide secretion in MG6 cells. LPS-induced phosphorylation of mammalian target of rapamycin (mTOR), nuclear factor-κB (NF-κB), and protein kinase B (AKT) (which are all associated with the neuroinflammatory response in microglia) were attenuated by 3',4'-dihydroxyflavonol treatment. Treatment with the mTOR inhibitor, rapamycin, NF-κB inhibitor, caffeic acid phenethyl ester, or AKT inhibitor, LY294002, also attenuated LPS-induced tumor necrosis factor-α and nitric oxide secretion in MG6 cells. LY294002 treatment attenuated LPS-induced phosphorylation of mTOR and NF-κB in MG6 cells. Hence, our study suggests that 3',4'-dihydroxyflavonol can attenuate the neuroinflammatory response of microglial cells by suppressing the AKT-mTOR and NF-κB pathways.

3',4',7-Trihydroxyflavone Downregulates NO Production in LPS- or IFN-γ-Activated MG6 Microglial Cells by Attenuating the JNK-STAT1 Pathway.

Neuroinflammation induced by activated microglia is a key feature of neurodegenerative diseases such as Alzheimer's disease. The natural flavonoid 3',4',7-trihydroxyflavone protects nerve cells from oxidative stress-mediated apoptosis and inhibits the aggregation of amyloid ß protein in vitro. However, little is known about its effects on microglial activation. In this study, we investigated the effects of 3',4',7-trihydroxyflavone on lipopolysaccharide (LPS)- or interferon-γ (IFN-γ)-induced neuroinflammatory responses in MG6 microglial cells. 3',4',7-Trihydroxyflavone inhibited LPS- or IFN-γ-mediated nitric oxide (NO) generation and the upregulation of inducible NO synthase (iNOS) in MG6 cells. 3',4',7-Trihydroxyflavone also suppressed LPS- or IFN-γ-mediated phosphorylation of signal transducer and activator of transcription 1 (STAT1), which is crucial for iNOS expression. LPS stimulation induced rapid phosphorylation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and extracellular signal-regulated kinase (ERK) in MG6 cells. 3',4',7-Trihydroxyflavone significantly inhibited the LPS-mediated phosphorylation of JNK, but not that of ERK and p38 MAPK. The inhibitory effect of 3',4',7-trihydroxyflavone on NO generation was mimicked by pharmacological inhibition of the JNK signaling pathway with SP600125. Furthermore, SP600125 significantly inhibited LPS- or IFN-γ-mediated phosphorylation of STAT1 in MG6 cells. These results suggest that 3',4',7-trihydroxyflavone exerts anti-neuroinflammatory effects via inhibition of the JNK-STAT1 pathway in microglia.

The Synthetic Curcumin Derivative CNB-001 Attenuates Thrombin-Stimulated Microglial Inflammation by Inhibiting the ERK and p38 MAPK Pathways.

We have recently found that the synthetic curcumin derivative CNB-001 suppresses lipopolysaccharide (LPS)-induced nitric oxide (NO) production in cultured microglia, demonstrating that it exerts anti-neuroinflammatory effects by regulating microglial activation. To explore the molecular mechanisms underlying the anti-inflammatory effect of CNB-001, the present study investigated whether CNB-001 is also effective for microglial NO production induced by other stimulants than LPS. Treatment of primary cultured rat microglia with thrombin, a serine protease that has been proposed as a mediator of cerebrovascular injuries, caused the expression of inducible NO synthase (iNOS) and the production of NO. The thrombin-induced NO production was completely blocked by the presence of SCH-79797, a selective protease-activated receptor 1 (PAR-1) antagonist, suggesting that the effect of thrombin is mediated by PAR-1. CNB-001 (1-10 µM) attenuated the thrombin-induced iNOS expression and NO production without affecting the PAR-1 expression. In addition, thrombin treatment caused rapid phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). The changes in ERK and p38 MAPK were significantly suppressed by the presence of CNB-001. These results demonstrate that CNB-001 suppresses thrombin-stimulated microglial activation by inhibiting the ERK and p38 MAPK pathways.

Oral administration of fisetin promotes the induction of hippocampal long-term potentiation in vivo.

To explore memory enhancing effect of the flavonoid fisetin, we investigated the effect of oral administration of flavonoids on the induction of long-term potentiation (LTP) at hippocampal CA1 synapses of anesthetized rats. Among four flavonoids (fisetin, quercetin, luteolin and myricetin) tested, only fisetin significantly facilitated the induction of hippocampal LTP. The effect of oral fisetin was abolished by intracerebroventricular injection of U0126, an agent that was previously found to inhibit its effect in hippocampal slices in vitro. These results suggest that orally administered fisetin crosses the blood-brain barrier and promotes synaptic functions in the hippocampus.

CNB-001, a synthetic pyrazole derivative of curcumin, suppresses lipopolysaccharide-induced nitric oxide production through the inhibition of NF-κB and p38 MAPK pathways in microglia.

CNB-001, a pyrazole derivative of curcumin, has been found to exert neuroprotective and memory-enhancing effects that may be effective for the treatment of Alzheimer's disease. Since aberrant activation of microglia is involved in the pathogenesis of Alzheimer's disease, the present study was undertaken to investigate the effect of CNB-001 on microglia-mediated inflammatory responses. In primary cultured rat microglia, CNB-001 (1-10µM) suppressed the lipopolysaccharide (LPS)-induced nitric oxide (NO) production and expression of inducible NO synthase (iNOS), and the potency of CNB-001 was stronger than curcumin. CNB-001 also suppressed the LPS-induced nuclear translocation of nuclear factor κB (NF-κB), which is essential for the expression of iNOS. LPS treatment promoted phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK). CNB-001 significantly suppressed the LPS-induced phosphorylation of p38 MAPK, but not ERK and JNK. The suppressive effect of CNB-001 on NO production was mimicked by blockade of the p38 MAPK signaling pathway with SB203580. These results suggest that CNB-001 exerts anti-inflammatory effects through inhibition of NF-κB and p38 MAPK pathways in microglia.

3,3',4',5'-Tetrahydroxyflavone induces formation of large aggregates of amyloid ß protein.

Amyloid ß protein (Aß) self-assembles into insoluble fibrils, and forms the senile plaques associated with Alzheimer's disease. 3,3',4',5'-Tetrahydroxyflavone, a synthetic analogue of the natural flavonoid fisetin, has been found to potently inhibit Aß fibril formation. In the present study, we investigated how inhibition of Aß fibril formation by this flavonoid affects Aß conformation and neurotoxicity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of Aß1-42 (20 µM) incubated with or without 3,3',4',5'-tetrahydroxyflavone demonstrated that 3,3',4',5'-tetrahydroxyflavone (100 µM) rapidly caused formation of atypical Aß conformers, which appeared as a very broad, smear-like band in the high molecular weight region and were distinguishable from soluble Aß oligomers or mature Aß fibrils. Transmission electron microscopy (TEM) revealed that large spherical Aß aggregates were preferentially formed in the presence of 3,3',4',5'-tetrahydroxyflavone. The SDS-resistant, smear-like band on SDS-PAGE and the large spherical aggregates in TEM both disappeared after heat treatment (100°C, 10 min). Furthermore, a neurotoxicity assay with cultured rat hippocampal neurons demonstrated that Aß incubated with 3,3',4',5'-tetrahydroxyflavone was significantly less toxic than Aß incubated without the flavonoid. These results suggest that the newly synthesized fisetin analogue 3,3',4',5'-tetrahydroxyflavone directly produces atypical, large Aß aggregates and reduces Aß toxicity.

Extract from Nandina domestica inhibits lipopolysaccharide-induced cyclooxygenase-2 expression in human pulmonary epithelial A549 cells.

Extract from fruits of Nandina domestica THUNBERG (NDE) has been used to improve cough and breathing difficulty in Japan for many years. To explore whether NDE may alleviate respiratory inflammation, we investigated its effect on expression of cyclooxygenase-2 (COX-2) and production of prostaglandin E2 (PGE2) in human pulmonary epithelial A549 cells in culture. Treatment with lipopolysaccharide (LPS; 6 µg/mL) resulted in an increase of COX-2 expression and PGE2 production in A549 cells. Both the LPS-induced COX-2 expression and PGE2 production were significantly inhibited by NDE (1-10 µg/mL) in a concentration-dependent manner. NDE did not affect COX-1 expression nor COX activity. These results suggest that NDE downregulates LPS-induced COX-2 expression and inhibits PGE2 production in pulmonary epithelial cells. Furthermore, higenamine and nantenine, two major constituents responsible for tracheal relaxing effect of NDE, did not mimic the inhibitory effect of NDE on LPS-induced COX-2 expression in A549 cells. To identify active constituent(s) of NDE responsible for the anti-inflammatory effect, NDE was introduced in a polyaromatic absorbent resin column and stepwise eluted to yield water fraction, 20% methanol fraction, 40% methanol fraction, 99.8% methanol fraction, and 99.5% acetone fraction. However, none of these five fractions alone inhibited LPS-induced COX-2 expression. On the other hand, exclusion of water fraction from NDE abolished the inhibitory effect of NDE on LPS-induced COX-2 expression. These results suggest that constituent(s) present in water fraction is required but not sufficient for the anti-inflammatory activity of NDE, which may result from interactions among multiple constituents.

3,3',4',5,5'-Pentahydroxyflavone is a potent inhibitor of amyloid ß fibril formation.

The natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) is neurotrophic and prevents fibril formation of amyloid ß protein (Aß). It is a promising lead compound for the development of therapeutic drugs for Alzheimer's disease. To find even more effective drugs based on the structure of fisetin, we synthesized a series of fisetin analogues lacking the 7-hydroxyl group and compared their effects on Aß fibril formation determined by the thioflavin T fluorescence assay. 3,3',4'-Trihydroxyflavone and 3',4'-dihydroxyflavone inhibited Aß fibril formation more potently than fisetin or 3',4',7-trihydroxyflavone, suggesting that the 7-hydroxy group is not necessary for anti-amyloidogenic activity. 3,3',4',5'-Tetrahydroxyflavone and 3',4',5'-trihydroxyflavone inhibited Aß fibril formation far more potently than 3,3',4'-trihydroxyflavone and 3',4'-dihydroxyflavone, suggesting that 3',4',5'-trihydroxyl group of the B ring is crucial for the anti-amyloidogenic activity of flavonoids. Based on the structure-activity relationship, we synthesized 3,3',4',5,5'-pentahydroxyflavone, and confirmed that this compound is the most potent inhibitor of Aß fibril formation among fisetin analogues that have been tested. Cytotoxicity assay using rat hippocampal neuron cultures demonstrated that Aß preincubated with 3,3',4',5,5'-pentahydroxyflavone was significantly less toxic than Aß preincubated with vehicle. 3,3',4',5,5'-Pentahydroxyflavone could be a new therapeutic drug candidate for the treatment of Alzheimer's disease.

A novel neurotrophic drug for cognitive enhancement and Alzheimer's disease.

Currently, the major drug discovery paradigm for neurodegenerative diseases is based upon high affinity ligands for single disease-specific targets. For Alzheimer's disease (AD), the focus is the amyloid beta peptide (Aß) that mediates familial Alzheimer's disease pathology. However, given that age is the greatest risk factor for AD, we explored an alternative drug discovery scheme that is based upon efficacy in multiple cell culture models of age-associated pathologies rather than exclusively amyloid metabolism. Using this approach, we identified an exceptionally potent, orally active, neurotrophic molecule that facilitates memory in normal rodents, and prevents the loss of synaptic proteins and cognitive decline in a transgenic AD mouse model.

Biphasic tracheal relaxation induced by higenamine and nantenine from Nandina domestica Thunberg.

We compared the effects of the extract from fruits of Nandina domestica Thunberg (NDE) and its constituents, higenamine and nantenine, on contractile responses in isolated guinea-pig trachea. NDE (1 mg/ml) caused biphasic relaxation of the trachea precontracted with high-K(+) stimulation: the fast component was blocked by propranolol and mimicked by higenamine; and the slow was resistant to propranolol and mimicked by nantenine. Ca(2+)-induced contraction under high-K(+) stimulation was antagonized by nantenine or NDE + propranolol. These results suggest that NDE relaxes the trachea quickly through ß-adrenoceptor stimulation by higenamine and slowly through Ca(2+) antagonism by nantenine.

Biphasic Tracheal Relaxation Induced by Higenamine and Nantenine From Nandina domestica THUNBERG.

We compared the effects of the extract from fruits of Nandina domestica THUNBERG (NDE) and its constituents, higenamine and nantenine, on contractile responses in isolated guineapig trachea. NDE (1 mg/ml) caused biphasic relaxation of the trachea precontracted with high-K+ stimulation: the fast component was blocked by propranolol and mimicked by higenamine; and the slow was resistant to propranolol and mimicked by nantenine. Ca2+-induced contraction under high-K+ stimulation was antagonized by nantenine or NDE + propranolol. These results suggest that NDE relaxes the trachea quickly through ß-adrenoceptor stimulation by higenamine and slowly through Ca2+ antagonism by nantenine.

A pyrazole derivative of curcumin enhances memory.

Reduced cognitive function is associated with Alzheimer's and Parkinson's diseases as well as brain trauma and ischemia. However, there are few compounds that enhance memory and are also orally active. We recently synthesized a pyrazole derivative of curcumin called CNB-001 that enhances the activity of Ca(2+)/calmodulin dependent protein kinase II (CaMKII). Since CaMKII plays a central role in long-term potentiation (LTP) and memory, it was asked if CNB-001 can facilitate the induction of LTP in rat hippocampal slices and enhance memory in a rat object recognition test. It is shown that CNB-001 enhances both LTP and memory.

Beta2-adrenoceptor-mediated tracheal relaxation induced by higenamine from Nandina domestica Thunberg.

The fruit of Nandina domestica Thunberg (ND, Berberidaceae) has been used to improve cough and breathing difficulties in Japan for many years, but very little is known about the constituent of ND responsible for this effect. We have recently reported that the crude extract from ND (NDE) inhibits histamine- and serotonin-induced contraction of isolated guinea pig trachea, and the inhibitory activity was not explained by nantenine, a well-known alkaloid isolated from ND. To explore other constituent(s) of NDE with tracheal smooth muscle relaxant activity, we fractionated NDE and assessed the pharmacological effects of the fractions using isolated guinea pig tracheal ring preparations. NDE was introduced into a polyaromatic absorbent resin column and stepwise eluted to yield five fractions, among which only the 40 % methanol fraction was active in relaxing tracheal smooth muscle precontracted with histamine. Further separation of the 40 % methanol fraction with high-performance liquid chromatography yielded multiple subfractions, one of which was remarkably active in relaxing histamine-precontracted trachea. Chemical analysis with a time-of-flight mass spectrometer and nuclear magnetic resonance spectrometer identified the constituent of the most active subfraction as higenamine, a benzyltetrahydroisoquinoline alkaloid. The potency and efficacy of the active constituent from NDE in relaxing trachea were almost equivalent to synthetic higenamine. In addition, the effect of the active constituent from NDE was competitively inhibited by the selective beta (2)-adrenoceptor antagonist ICI 118,551. These results indicate that the major constituent responsible for the effect of NDE is higenamine, which probably causes the tracheal relaxation through stimulation of beta (2) adrenoceptors.

Modulatory role of dopamine D2 receptors and fundamental role of L-type Ca2+ channels in the induction of long-term potentiation in the basolateral amygdala-dentate gyrus pathway of anesthetized rats.

We have previously found that the induction of long-term potentiation in the synaptic pathway from the basolateral amygdala to the dentate gyrus (BLA-DG LTP) is regulated by L-type Ca(2+) channels, dopamine D(2) receptors and GABAergic inhibition. In the present study, we investigated possible relations among the three mechanisms by using anesthetized rats. Blockade of GABAergic inhibition with picrotoxin abolished both the inhibitory effect of the dopamine D(2) receptor antagonist chlorpromazine and the promoting effect of the dopamine D(2) receptor agonist quinpirole on the induction of BLA-DG LTP. However, the inhibitory effect of the L-type Ca(2+) channel blocker verapamil on BLA-DG LTP was not affected by picrotoxin. These results suggest that the role of dopamine D(2) receptors in the induction of BLA-DG LTP is modulatory and depends on GABAergic inhibition, whereas the role of L-type Ca(2+) channels is fundamental.

Basolateral amygdala D1- and D2-dopaminergic system promotes the formation of long-term potentiation in the dentate gyrus of anesthetized rats.

We have previously found that the induction of hippocampal long-term potentiation (LTP) is modulated by neuron activities in the basolateral amygdala (BLA). However, little is known about what neurotransmitter system in the BLA contributes to modulation of hippocampal LTP. In the present study, we investigated possible involvement of BLA dopaminergic system in the induction of LTP at the perforant path (PP)-dentate gyrus (DG) granule cell synapses of anesthetized rats. The induction of PP-DG LTP was significantly attenuated by intra-BLA injection of the D(1) receptor antagonist SCH23390 (2 or 6 nmol) or the D(2) receptor antagonists, chlorpromazine (30 or 100 nmol) or haloperidol (4.4 or 13.3 nmol). The effects of SCH23390 and haloperidol were abolished by concomitant intra-BLA injection of the D(1) receptor agonist SKF38393 (17 nmol) and the D(2) receptor agonist quinpirole (3 nmol), respectively. Furthermore, lesioning with 6-hydroxydopamine of the ventral tegmental area, the origin of the dopaminergic system projecting to the BLA, resulted in attenuated PP-DG LTP, which was restored by intra-BLA injection of SKF38393 or quipirole. These results suggest that the induction of PP-DG LTP is promoted by the BLA dopaminergic system via both D(1) and D(2) receptors.

Stimulation of basolateral amygdaloid serotonin 5-HT(2C) receptors promotes the induction of long-term potentiation in the dentate gyrus of anesthetized rats.

We have previously found that the induction of hippocampal long-term potentiation (LTP) is modulated by neuron activities in the basolateral amygdala (BLA). However, little is known about what neurotransmitter system in the BLA contributes to modulation of hippocampal LTP. In the present study, we investigated possible involvement of BLA serotonergic system in the induction of LTP at the perforant path (PP)-dentate gyrus (DG) granule cell synapses of anesthetized rats. The induction of PP-DG LTP was significantly inhibited by intra-BLA injection of the 5-HT(2) receptor antagonist cinanserin (25-50nmol), but not by intra-BLA injection of the 5-HT(1,7) receptor antagonist methiothepin (50nmol), the 5-HT(3) receptor antagonist ondansetron (50nmol) or the 5-HT(4) receptor antagonist RS23597-190 (100nmol). In addition, intra-BLA injection of the 5-HT(2C) receptor agonist MK212 (50nmol) facilitated the induction of PP-DG LTP. These results suggest that the induction of PP-DG LTP is promoted by activation of 5-HT(2C) receptors in the BLA.

Involvement of dopamine D2 receptors in the induction of long-term potentiation in the basolateral amygdala-dentate gyrus pathway of anesthetized rats.

We have previously found that synaptic pathway from the basolateral amygdala (BLA) to the dentate gyrus (DG) displays N-methyl-D-aspartate (NMDA) receptor-independent form of long-term potentiation (LTP), which should be a valuable model for elucidating neural mechanisms linking emotion and memory. To explore its cellular mechanisms, we investigated possible involvement of the beta-adrenergic, muscarinic cholinergic and dopaminergic systems on LTP in this pathway of anesthetized rats. The induction of BLA-DG LTP was not affected by administration of the beta-adrenoceptor antagonist propranolol (50-150nmol, i.c.v.), the muscarinic receptor antagonist scopolamine (2-6mg/kg, i.p.), the cholinesterase inhibitor physostigmine (50 nmol, i.c.v.) or the dopamine D(1) receptor antagonist SCH23390 (100nmol, i.c.v.), but significantly inhibited by the dopamine D2 receptor antagonists, chlorpromazine (15nmol, i.c.v.) and haloperidol (0.15-0.5mg/kg, i.p.), and significantly promoted by the dopamine D2 receptor agonist quinpirole (78nmol, i.c.v.). Furthermore, lesioning with 6-hydroxydopamine of the ventral tegmental area (VTA), the origin of mesolimbic dopaminergic neurons, resulted in attenuated BLA-DG LTP. These results suggest that the D2-dopaminergic system, but not the beta-adrenergic, muscarinic or D1-dopaminergic system, is involved in the induction of BLA-DG LTP. In addition, inhibition of BLA-DG LTP by haloperidol or VTA lesion was abolished by blockade of GABAergic inhibition with picrotoxin. It is probable that the D2-dopaminergic system promotes the induction of BLA-DG LTP by suppressing GABAergic inhibition.

Structural requirements for the flavonoid fisetin in inhibiting fibril formation of amyloid beta protein.

Fisetin (3,3',4',7-tetrahydroxyflavone) has been found to be neuroprotective, induce neuronal differentiation, enhance memory, and inhibit the aggregation of the amyloid beta protein (Abeta) that may cause the progressive neuronal loss in Alzheimer's disease. The diverse collection of biological activities of this compound may lead to a new type of therapeutic drug for Alzheimer's disease. As the first step to design even more effective drugs based upon the structure of fisetin, the present study investigated the structural requirements for the anti-amyloidogenic activity of fisetin by comparing the effects of several structurally related flavonoids on Abeta fibril formation in vitro. Abeta1-42 (20muM) and the flavonoids were incubated for 0-48h at 37 degrees C, and fibril formation was quantitatively determined by the thioflavin T fluorescence assay. Among ten flavonoids tested, fisetin, 3',4',7-trihydroxylflavone, 3,3',4'-trihydroxyflavone, luteolin, quercetin and myricetin inhibited Abeta fibril formation. On the other hand, 3,3',7-trihydroxyflavone, 5-deoxykaempferol, chrysin and kaempferol enhanced Abeta fibril formation. These results suggest that the 3',4'-dihydroxyl group, but not the 3- or 7-hydroxyl group, is essential for the inhibitory effect of fisetin on Abeta fibril formation.