Preventive effect of propolis on cognitive decline in Alzheimer's disease model mice.

Brazilian green propolis (propolis) is a chemically complex resinous substance that is a potentially viable therapeutic agent for Alzheimer's disease. Herein, propolis induced a transient increase in intracellular Ca2+ concentration ([Ca2+]i) in Neuro-2A cells; moreover, propolis-induced [Ca2+]i elevations were suppressed prior to 24-h pretreatment with amyloid-ß. To reveal the effect of [Ca2+]i elevation on impaired cognition, we performed memory-related behavioral tasks in APP-KI mice relative to WT mice at 4 and 12 months of age. Propolis, at 300-1000 mg/kg/d for 8 wk, significantly ameliorated cognitive deficits in APP-KI mice at 4 months, but not at 12 months of age. Consistent with behavioral observations, injured hippocampal long-term potentiation was markedly ameliorated in APP-KI mice at 4 months of age following repeated propolis administration. In addition, repeated administration of propolis significantly activated intracellular calcium signaling pathway in the CA1 region of APP-KI mice. These results suggest a preventive effect of propolis on cognitive decline through the activation of intracellular calcium signaling pathways in CA1 region of AD mice model.

Effect of methoxyflavones contained in Kaempferia parviflora on CRE-mediated transcription in PC12D cells.

The cAMP-response element (CRE) is critical in the formation of long-term memory. To prove the pharmacological effects of the methoxyflavones-rich residue (MRR) and its constituent methoxyflavones (1-9) extracted from the rhizomes of Kaempferia parviflora on the nervous system, we examined the effects of the MRR and methoxyflavones (1-9) on CRE-mediated transcription in PC12D cells. The MRR increased CRE-mediated transcription in PC12D cells. In addition, among methoxyflavones (1-9) isolated from MRR, compounds 1-4 increased CRE-mediated transcription. These results suggest that K. parviflora and methoxyflavone might be very useful materials for preventing and recovering from cognitive decline.

Extracellular α-synuclein enters dopaminergic cells by modulating flotillin-1-assisted dopamine transporter endocytosis.

The neuropathological hallmarks of Parkinson's disease (PD) include the appearance of α-synuclein (α-SYN)-positive Lewy bodies (LBs) and the loss of catecholaminergic neurons. Thus, a potential mechanism promoting the uptake of extracellular α-SYN may exist in susceptible neurons. Of the various differentially expressed proteins, we are interested in flotillin (FLOT)-1 because this protein is highly expressed in the brainstem catecholaminergic neurons and is strikingly up-regulated in PD brains. In this study, we found that extracellular monomeric and fibrillar α-SYN can potentiate FLOT1-dopamine transporter (DAT) binding and pre-endocytic clustering of DAT on the cell surface, thereby facilitating DAT endocytosis and down-regulating its transporter activity. Moreover, we demonstrated that α-SYN itself exploited the DAT endocytic process to enter dopaminergic neuron-like cells, and both FLOT1 and DAT were found to be the components of LBs. Altogether, these findings revealed a novel role of extracellular α-SYN on cellular trafficking of DAT and may provide a rationale for the cell type-specific, functional, and pathologic alterations in PD.-Kobayashi, J., Hasegawa, T., Sugeno, N., Yoshida, S., Akiyama, T., Fujimori, K., Hatakeyama, H., Miki, Y., Tomiyama, A., Kawata, Y., Fukuda, M., Kawahata, I., Yamakuni, T., Ezura, M., Kikuchi, A., Baba, T., Takeda, A., Kanzaki, M., Wakabayashi, K., Okano, H., Aoki, M. Extracellular α-synuclein enters dopaminergic cells by modulating flotillin-1-assisted dopamine transporter endocytosis.

A novel mechanism of depression: role for connexins.

Major depressive disorder (MDD) is a chronic and debilitating illness that affects over 350 million people worldwide; however, current treatments have failed to cure or prevent the progress of depression. Increasing evidence suggests a crucial role for connexins in MDD. In this review, we have summarised recent accomplishments regarding the role of connexins, gap junctions, and hemichannels in the aetiology of MDD, and discussed the limitations of current research. A blockage of gap junctions or hemichannels induces depressive behaviour. Possible underlying mechanisms include the regulation of neurosecretory functions and synaptic activity by gap junctions and hemichannels. Gap junctions are functionally inhibited under stress conditions. Conversely, hemichannel permeability is increased. Antidepressants inhibit hemichannel permeability; however, they have contrasting effects on the function of gap junctions under normal conditions and can protect them against stress. In conclusion, the blockage of hemichannels concurrent with improvements in gap junction functionality might be potential targets for depression treatment.

Imidacloprid, a neonicotinoid insecticide, facilitates tyrosine hydroxylase transcription and phenylethanolamine N-methyltransferase mRNA expression to enhance catecholamine synthesis and its nicotine-evoked elevation in PC12D cells.

Imidacloprid is a neonicotinoid insecticide acting as an agonist of nicotinic acetylcholine receptors (nAChRs) in the target insects. However, questions about the safety to mammals, including human have emerged. Overactivation of mammalian peripheral catecholaminergic systems leads to onset of tachycardia, hypertension, vomiting, etc., which have been observed in acutely imidacloprid-poisoned patients as well. Physiological activation of the nAChRs is known to drive catecholamine biosynthesis and secretion in mammalian adrenal chromaffin cells. Yet, the impacts of imidacloprid on the catecholaminergic function of the chromaffin cells remain to be evaluated. In this study using PC12D cells, a catecholaminergic cell line derived from the medulla chromaffin-cell tumors of rat adrenal gland, we examined whether imidacloprid itself could impact the catecholamine-synthesizing ability. Imidacloprid alone did facilitate tyrosine hydroxylase (TH) transcription via activation of α3ß4 nAChR and the α7 subunit-comprising receptor. The insecticide showed the TH transcription-facilitating ability at the concentrations of 3 and 30 µM, at which acetylcholine is known to produce physiological responses, including catecholamine secretion through the nAChRs in adrenal chromaffin cells. The insecticide-facilitated TH transcription was also dependent on PKA- and RhoA-mediated signaling pathways. The insecticide coincidentally raised levels of TH and phenylethanolamine N-methyltransferase (PNMT) mRNA, and as a consequence, increased catecholamine production, although the efficacy of the neonicotinoid was lesser than that of nicotine, indicating its partial agonist-like action. Intriguingly, in cultured rat adrenal chromaffin cells, imidacloprid did increase levels of TH and PNMT protein. When the chromaffin cells were treated with nicotine in the presence of the insecticide, nicotine-elevated adrenaline production was enhanced due to facilitation of nicotine-increased TH and PNMT protein expression, and simultaneous enhancement of nicotine-elevated adrenaline secretion also took place. These findings thus suggest that imidacloprid may facilitate the physiological functions of adrenal glands in mammals.

Ginsenoside Rg1 alleviates corticosterone-induced dysfunction of gap junctions in astrocytes.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginsenoside Rg1 (Rg1), one of the major bioactive ingredients of Panax ginseng C. A. Mey, has neuroprotective effects in animal models of depression, but the mechanism underlying these effects is still largely unknown AIM OF THE STUDY: Gap junction intercellular communication (GJIC) dysfunction is a potentially novel pathogenic mechanism for depression. Thus, we investigated that whether antidepressant-like effects of Rg1 were related to GJIC. MATERIALS AND METHODS: Primary rat prefrontal cortical and hippocampal astrocytes cultures were treated with 50µM CORT for 24h to induce gap junction damage. Rg1 (0.1, 1, or 10µM) or fluoxetine (1µM) was added 1h prior to CORT treatment. A scrape loading and dye transfer assay was performed to identify the functional capacity of gap junctions. Western blot was used to detect the expression and phosphorylation of connexin43 (Cx43), the major component of gap junctions. RESULTS: Treatment of primary astrocytes with CORT for 24h inhibited GJIC, decreased total Cx43 expression, and increased the phosphorylation of Cx43 at serine368 in a dose-dependent manner. Pre-treatment with 1µM and 10µM Rg1 significantly improved GJIC in CORT-treated astrocytes from the prefrontal cortex and hippocampus, respectively, and this was accompanied by upregulation of Cx43 expression and downregulation of Cx43 phosphorylation. CONCLUSION: These findings provide the first evidence indicating that Rg1 can alleviate CORT-induced gap junction dysfunction, which may have clinical significance in the treatment of depression.

Fermented Citrus reticulata (ponkan) fruit squeezed draff that contains a large amount of 4'-demethylnobiletin prevents MK801-induced memory impairment.

A previous study reported biotransformation of a citrus peel polymethoxyflavone, nobiletin, by Aspergillus enabling production of 4'-demethylnobiletin, and the product's antimutagenic activity. However, the effects of fermented citrus peel on the basal forebrain-hippocampal system remain unidentified. Citrus reticulata (ponkan) fruit squeezed draffs are generated as mass waste in beverage factories. In this study using PC12D cells and cultured central nervous system neurons, we therefore examined whether Aspergillus kawachii-fermented citrus fruit squeezed draff could affect cAMP response element (CRE)- and choline acetyltransferase gene (ChAT) promoter region-mediated transcriptional activities relevant to memory formation and cholinergic function. Our current fermentation yielded approximately 80% nobiletin bioconversion, and a sample of hot-water extract of the fermented fruit squeezed draff was stronger than that of the unfermented one in facilitating CRE-mediated transcription in cultured hippocampal neurons as well as in PC12D cells. A sample of 0-80% ethanol-eluted fraction of Diaion HP-20 column-adsorbed components of the preparation obtained by the fermentation concentration-dependently and more strongly facilitated CRE-mediated transcription than did the fraction of the unfermented one in both cell culture systems. In a separate study, this polymethoxyflavone-rich fraction of the fermented fruit squeezed draff showed a potent ability to facilitate CRE-mediated and ChAT transcription in a co-culture of hippocampal neurons and basal forebrain neurons. Repeated oral gavage of mice with the fermented fraction sample prevented MK801-impaired memory formation in mice. These findings suggest that the 4'-demethylnobiletin-rich fraction prepared from the Aspergillus-fermented ponkan squeezed draff has a potential anti-dementia effect.

Tyrosine hydroxylase gene expression is facilitated by alcohol followed by the degradation of the protein by ubiquitin proteasome system.

OBJECTIVES: Alcohol intake induces brief periods of euphoria; however, its continuous consumption can lead the development of alcohol tolerance. The euphoria, an intense feeling of wellbeing, is deeply associated with dopamine. Dopamine biosynthesis is strictly regulated by tyrosine hydroxylase (TH), a rate-limiting enzyme of dopamine. The aim of this study was to examine the transient or chronic effects of ethanol treatment on TH protein level in vitro. METHODS: Cultured primary mesencephalic neurons were prepared and exposed to 100 mM ethanol for 48 hours or 168 hours. TH and cAMP-responsive element (CRE)-mediated transcriptional activity was measured by reporter gene assay using pTH9.0kb-Luc and pCRE-Luc reporter plasmid. TH protein expression and TH phosphorylation was analyzed by Western blot analysis. Dopamine content was measured by high-performance liquid chromatography (HPLC). RESULTS: Ethanol treatment for 48 hours facilitates TH transcriptional activity and TH protein expression in a cAMP-dependent protein kinase A (PKA) and MAPK/Erk kinase (MEK)-dependent manner in cultured mesencephalic neurons. Ethanol also facilitated TH phosphorylation, which resulted in the elevation of dopamine content. On the other hand, treatment with ethanol for 168 hours did not show significant elevation of TH gene expression and dopamine biosynthesis. Intriguingly, simultaneous treatment with MG-132, a 26S proteasomal inhibitor, recovered the ethanol-induced increase of TH protein expression and dopamine biosynthesis. CONCLUSION: Transient ethanol-treatment facilitates TH gene expression and its phosphorylation in a PKA- and MEK-dependent manner to elevate dopamine biosynthesis, whereas continuous exposure to ethanol abolishes its potent effects on the dopaminergic function to reduce dopamine content. This reduction seems to originate from the decrease of TH protein level by degradation of the protein. Our current data may contribute to the better understanding of alcohol tolerance associated with degradation of TH protein to reduce total-TH level and dopamine biosynthesis.

Ginsenoside Rg1-induced antidepressant effects involve the protection of astrocyte gap junctions within the prefrontal cortex.

Ginsenoside Rg1 (Rg1) exhibits antidepressant-like activity by increasing neurogenesis and dendritic spine density without discernible side effects. However, the molecular mechanisms underlying Rg1 antidepressant activity remain poorly understood. As the dysfunction of gap junctions between astrocytes in the prefrontal cortex (PFC) is implicated in major depression disorder, the aim of this study was to investigate the effects of Rg1 on astrocyte gap junctions in the PFC. Rats exposed to chronic unpredictable stress (CUS) were administered Rg1 (5, 10, and 20mg/kg) for 28days and analyzed for depressive symptoms using the sucrose preference and forced swimming tests. Functional and morphological changes of gap junction channels in the PFC were evaluated using dye transfer and electron microscopy, respectively. The expression of connexin 43 (Cx43) was analyzed by western blotting. Rg1 markedly alleviated depression-like behavior in rats. Long-term Rg1 treatment of CUS-exposed rats also significantly prevented the decrease in dye diffusion and improved the ultrastructure of astrocyte gap junctions in the PFC, indicating beneficial effects on the functional activity of gap junction channels in the brain. In addition, Rg1 upregulated Cx43 expression in the PFC reduced by CUS exposure, which significantly correlated with its antidepressant-like effects. The results demonstrate that Rg1-induced antidepressant effects are might be mediated, in part, by protecting astrocyte gap junctions within the prefrontal cortex.

Dopamine or biopterin deficiency potentiates phosphorylation at (40)Ser and ubiquitination of tyrosine hydroxylase to be degraded by the ubiquitin proteasome system.

The protein amount of tyrosine hydroxylase (TH), that is the rate-limiting enzyme for the biosynthesis of dopamine (DA), should be tightly regulated, whereas its degradation pathway is largely unknown. In this study, we analyzed how the TH protein is chemically modified and subsequently degraded under deficiencies of DA and tetrahydrobiopterin (BH4), a cofactor for TH, by using pharmacological agents in PC12D cells and cultured mesencephalic neurons. When inhibition of DA- or BH4-synthesizing enzymes greatly reduced the DA contents in PC12D cells, a marked and persistent increase in phosphorylated TH at (40)Ser (p40-TH) was concomitantly observed. This phosphorylation was mediated by D2 dopamine auto-receptor and cAMP-dependent protein kinase (PKA). Our immunoprecipitation experiments showed that the increase in the p40-TH level was accompanied with its poly-ubiquitination. Treatment of PC12D cells with cycloheximide showed that total-TH protein level was reduced by the DA- or BH4-depletion. Notably, this reduction in the total-TH protein level was sensitive not only to a 26S proteasomal inhibitor, MG-132, but also to a PKA inhibitor, H-89. These data demonstrated that DA deficiency should induce compensatory activation of TH via phosphorylation at (40)Ser through D2-autoreceptor and PKA-mediated pathways, which in turn give a rise to its degradation through an ubiquitin-proteasome pathway, resulting in a negative spiral of DA production when DA deficiency persists.

SLC10A4 is a protease-activated transporter that transports bile acids.

SLC10A4 belongs to the sodium bile acid cotransporter family, but has no transport activity for bile acids. We performed multiple amino acid alignments and examined the relationships between the SLC10 proteins. The extracellular N-terminus of SLC10A4 was predicted to be relatively longer at the amino acid level than those of SLC10A1, SLC10A2 and SLC10A6. We examined the relationship between the N-terminus and transport activity of SLC10A4. Rat Slc10a4 is predominantly expressed in rat cholinergic neurons; therefore, TE671 cells expressing the acetylcholine receptor and acetylcholinesterase were used. After thrombin treatment, western blotting and immunofluorescence staining demonstrated that the N-terminus of SLC10A4 might be cleaved. Substrates were added to the cells, and their uptake was quantified by liquid chromatography tandem mass spectrometry. Lithocholic acid (LCA) and taurocholic acid (TCA) uptake and cell death effects of LCA were increased by thrombin treatment. After RNA interference treatment for SLC10A4, bile acid uptake was also quantified. In consequence, increases in the LCA and TCA uptake did not occur. Therefore, SLC10A4 may have low activity but becomes activated by proteases, including thrombin, following cleavage. We have demonstrated that SLC10A4 appears to be a protease-activated transporter and transports bile acids.

Potent activity of nobiletin-rich Citrus reticulata peel extract to facilitate cAMP/PKA/ERK/CREB signaling associated with learning and memory in cultured hippocampal neurons: identification of the substances responsible for the pharmacological action.

cAMP/PKA/ERK/CREB signaling linked to CRE-mediated transcription is crucial for learning and memory. We originally found nobiletin as a natural compound that stimulates this intracellular signaling and exhibits anti-dementia action in animals. Citrus reticulata or C. unshiu peels are employed as "chinpi" and include a small amount of nobiletin. We here provide the first evidence for beneficial pharmacological actions on the cAMP/PKA/ERK/CREB cascade of extracts from nobiletin-rich C.reticulata peels designated as Nchinpi, the nobiletin content of which was 0.83 ± 0.13% of the dry weight or 16-fold higher than that of standard chinpi extracts. Nchinpi extracts potently facilitated CRE-mediated transcription in cultured hippocampal neurons, whereas the standard chinpi extracts showed no such activity. Also, the Nchinpi extract, but not the standard chinpi extract, stimulated PKA/ERK/CREB signaling. Interestingly, treatment with the Nchinpi extract at the concentration corresponding to approximately 5 µM nobiletin more potently facilitated CRE-mediated transcriptional activity than did 30 µM nobiletin alone. Consistently, sinensetin, tangeretin, 6-demethoxynobiletin, and 6-demethoxytangeretin were also identified as bioactive substances in Nchinpi that facilitated the CRE-mediated transcription. Purified sinensetin enhanced the transcription to a greater degree than nobiletin. Furthermore, samples reconstituted with the four purified compounds and nobiletin in the ratio of each constituent's content in the extract showed activity almost equal to that of the Nchinpi extract to stimulate CRE-mediated transcription. These findings suggest that above four compounds and nobiletin in the Nchinpi extract mainly cooperated to facilitate potently CRE-mediated transcription linked to the upstream cAMP/PKA/ERK/CREB pathway in hippocampal neurons.

Nobiletin induces inhibitions of Ras activity and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling to suppress cell proliferation in C6 rat glioma cells.

Ras, a small G-protein, physiologically directs cell proliferation and cell cycle via regulation of mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling cascade. Dysregulation of Ras/MEK/ERK signaling has been reported to cause tumorigenesis and gliomas. Nobiletin, a citrus flavonoid, has been shown to have anti-tumor cells action. However, it remains elusive whether nobiletin could affect Ras activity. In this study, we provide the first evidence that nobiletin suppresses the proliferation by inhibiting Ras activity in C6 glioma cells, a rat glioma cell line. First, Ras pull-down assay showed that nobiletin inhibits Ras activity in a concentration-dependent manner in C6 cells. Second, farnesyltransferase inhibitor I, a Ras inhibitor, and U0126, a MEK inhibitor, induced an inhibition of the cell proliferation in C6 cells, while the cell proliferation was inhibited by nobiletin as well. Third, western blotting revealed that nobiletin showed inhibitory effects on MEK and ERK phopsphorylation levels in a concentration-dependent manner. Finally, such an inhibitory effect on the level of ERK phosphorylation by nobiletin was appreciably prevented by Gö6976, a selective inhibitor of conventional protein kinase Cs (PKCs) showing Ca(2+)-sensitivity, while GF109203X, a general inhibitor for PKCs, and BAPTA, a cell-permeable Ca(2+) chelator, to a lesser extent, suppressed a reduction of the phosphorylation. These findings suggest that the proliferation of C6 cells is Ras- and MEK/ERK signaling-dependent, and that nobiletin suppresses the cell proliferation by inhibiting Ras activity and MEK/ERK signaling cascade probably via a Ca(2+)-sensitive PKC-dependent mechanism. Thus, the natural compound has potential to be a therapeutic agent for glioma.

Four new myrsinol diterpenes from Euphorbia prolifera.

Four new myrsinol diterpenes, euphorbialoids K-N (1-4), have been isolated from the roots of Euphorbia prolifera. Their structures were elucidated on the basis of extensive 1D and 2D NMR (COSY, HMQC, HMBC, and NOESY) and mass (ESI-MS and HR-ESI-MS) spectroscopic data analyses.

Repeated treatment with nicotine induces phosphorylation of NMDA receptor NR2B subunit in the brain regions involved in behavioral sensitization.

Protein phosphorylation is an important mechanism for the post-translational modulation of N-methyl-d-aspartate (NMDA) receptor functions. In the present study, we investigated the levels of NR2B phosphorylation at Tyr1472 and Ser1303 in the nucleus accumbens, striatum, frontal cortex, and hippocampus of rats that exhibit behavioral sensitization to nicotine. Repeated treatment of rats with nicotine (0.6mg/kg, s.c., for 7 days) produced locomotor sensitization accompanied by increased NR2B phosphorylation at Tyr1472 in the nucleus accumbens and striatum, brain regions involved in behavioral sensitization. In contrast, no changes in NR2B phosphorylation were observed after a single treatment with nicotine in these brain regions. In addition, no changes in NR2B phosphorylation at Ser1303 were observed after repeated treatment with nicotine in any examined brain regions. These results suggest that repeated treatment with nicotine induces NR2B phosphorylation at Tyr1472 in the nucleus accumbens and striatum, which might contribute to the development of synaptic and behavioral plasticity in response to nicotine.

Isolation, structural elucidation, and neuroprotective effects of iridoids from Valeriana jatamansi.

Two new iridoids, jatadoids A (1) and B (2), and two known compounds (3 and 4) were isolated from Valeriana jatamansi. Their structures were elucidated on the basis of extensive spectroscopic analyses (IR, ESI-MS, HR-ESI-MS, 1D and 2D NMR). Compound 1 possessed an isovaleroxy group at the C-3 position that has previously been unreported in the class of iridoids. Four compounds were evaluated and compounds 1 and 3 showed moderate neuroprotective effects against MPP+-induced neuronal cell death in human dopaminergic neuroblastoma SH-SY5Y cells.

New myrsinol diterpenes from Euphorbia prolifera and their inhibitory activities on LPS-induced NO production.

A phytochemical investigation of the roots of Euphorbia prolifera led to the isolation of ten new myrsinol diterpenes, named euphorbialoids A-J (1-10), and two known analogues (11 and 12). Their structures were elucidated on the basis of extensive 1D and 2D NMR (COSY, HMQC, HMBC and NOESY) spectroscopic data analyzes, and the structures of 1 and 9 were confirmed by X-ray crystallography. The inhibitory activities on LPS-induced NO production of these diterpenes were evaluated and all the compounds showed inhibitory effects.

Three new iridoids from the roots of Valeriana jatamansi.

Three new iridoids, valeriandoids D-F (1-3), have been isolated from the roots of Valeriana jatamansi. Structure elucidation, especially the positions of the acyloxy groups, and complete (1)H- and (13)C-NMR assignments of these new compounds were carried out using one- and two-dimensional NMR measurements, including (1)H- and (13)C-NMR, DEPT-135, (1)H-(1)H COSY, HMQC, HMBC, and HR-ESI-MS experiments.