The genus Daphne: A review of its traditional uses, phytochemistry and pharmacology.

The genus Daphne belongs to the Thymeleaceae family and contains over 90 species that are distributed in Asia, Europe and parts of North Africa. The species of the genus Daphne are used in the traditional medicine of China, Tibet, Korea, and the Middle East for the treatment of various conditions. A broad range of studies has shown the significant biological potential of these species as sources of biologically and pharmacologically active compounds. Daphne species are a source of several classes of valuable phytochemicals such as coumarins, flavonoids, lignans, steroids and different classes of terpenes. The phytochemical diversity of this genus is demonstrated by over 350 secondary metabolites isolated from various species. The genus possesses a broad spectrum of biological activities including antibacterial, antifungal, antioxidant, analgesic, anti-inflammatory, cytotoxic, antiviral, abortive and haemostatic effects. A variety of bioactive secondary metabolites found in this genus may have potential use in pharmaceutical, cosmetic and food industries. Thus, species belonging to the genus Daphne can be considered an important source both for the treatment of various disorders, due to the presence of a plethora of bioactive constituents with potent bioactivities, and as possible leads in the discovery and synthesis of new medications.

Astrocyte-shed extracellular vesicles regulate the peripheral leukocyte response to inflammatory brain lesions.

Brain injury induces a peripheral acute cytokine response that directs the transmigration of leukocytes into the brain. Because this brain-to-peripheral immune communication affects patient recovery, understanding its regulation is important. Using a mouse model of inflammatory brain injury, we set out to find a soluble mediator for this phenomenon. We found that extracellular vesicles (EVs) shed from astrocytes in response to intracerebral injection of interleukin-1ß (IL-1ß) rapidly entered into peripheral circulation and promoted the transmigration of leukocytes through modulation of the peripheral acute cytokine response. Bioinformatic analysis of the protein and microRNA cargo of EVs identified peroxisome proliferator-activated receptor α (PPARα) as a primary molecular target of astrocyte-shed EVs. We confirmed in mice that astrocytic EVs promoted the transmigration of leukocytes into the brain by inhibiting PPARα, resulting in the increase of nuclear factor κB (NF-κB) activity that triggered the production of cytokines in liver. These findings expand our understanding of the mechanisms regulating communication between the brain and peripheral immune system and identify astrocytic EVs as a molecular regulator of the immunological response to inflammatory brain damage.

Measurement of Inositol Triphosphate Levels from Rat Hippocampal Slices.

Inositol triphosphate (IP3) is an important second messenger that participates in signal transduction pathways in diverse cell types including hippocampal neurons. Stimulation of phospholipase C in response to various stimuli (hormones, growth factors, neurotransmitters, neurotrophins, neuromodulators, odorants, light, etc) results in hydrolysis of phosphatidylinositol 4, 5-bisphosphate (PIP2), a phospholipid that is located in the plasma membrane, and leads to the production of IP3 and diacylglycerol. Binding of IP3 to the IP3 receptor (IP3R) induces Ca2+ release from intracellular stores and enables the initiation of intracellular Ca2+-dependent signaling. Here we describe a procedure for the measurement of cellular IP3 levels in tissue homogenates prepared from rat hippocampal slices.

Sex differences in cerebellar synaptic transmission and sex-specific responses to autism-linked Gabrb3 mutations in mice.

Neurons of the cerebellar nuclei (CbN) transmit cerebellar signals to premotor areas. The cerebellum expresses several autism-linked genes, including GABRB3, which encodes GABAA receptor ß3 subunits and is among the maternal alleles deleted in Angelman syndrome. We tested how this Gabrb3 m-/p+ mutation affects CbN physiology in mice, separating responses of males and females. Wild-type mice showed sex differences in synaptic excitation, inhibition, and intrinsic properties. Relative to females, CbN cells of males had smaller synaptically evoked mGluR1/5-dependent currents, slower Purkinje-mediated IPSCs, and lower spontaneous firing rates, but rotarod performances were indistinguishable. In mutant CbN cells, IPSC kinetics were unchanged, but mutant males, unlike females, showed enlarged mGluR1/5 responses and accelerated spontaneous firing. These changes appear compensatory, since mutant males but not females performed indistinguishably from wild-type siblings on the rotarod task. Thus, sex differences in cerebellar physiology produce similar behavioral output, but provide distinct baselines for responses to mutations.

Sex Differences in Molecular Signaling at Inhibitory Synapses in the Hippocampus.

The possibility that mechanisms of synaptic modulation differ between males and females has far-reaching implications for understanding brain disorders that vary between the sexes. We found recently that 17ß-estradiol (E2) acutely suppresses GABAergic inhibition in the hippocampus of female rats through a sex-specific estrogen receptor α (ERα), mGluR, and endocannabinoid-dependent mechanism. Here, we define the intracellular signaling that links ERα, mGluRs, and endocannabinoids in females and identify where in this pathway males and females differ. Using a combination of whole-cell patch-clamp recording and biochemical analyses in hippocampal slices from young adult rats, we show that E2 acutely suppresses inhibition in females through mGluR1 stimulation of phospholipase C, leading to inositol triphosphate (IP3) generation, activation of the IP3 receptor (IP3R), and postsynaptic endocannabinoid release, likely of anandamide. Analysis of sex differences in this pathway showed that E2 stimulates a much greater increase in IP3 levels in females than males, whereas the group I mGluR agonist DHPG increases IP3 levels equivalently in each sex. Coimmunoprecipitation showed that ERα-mGluR1 and mGluR1-IP3R complexes exist in both sexes but are regulated by E2 only in females. Independently of E2, a fatty acid amide hydrolase inhibitor, which blocks breakdown of anandamide, suppressed >50% of inhibitory synapses in females with no effect in males, indicating tonic endocannabinoid release in females that is absent in males. Together, these studies demonstrate sex differences in both E2-dependent and E2-independent regulation of the endocannabinoid system and suggest that manipulation of endocannabinoids in vivo could affect physiological and behavioral responses differently in each sex. SIGNIFICANCE STATEMENT: Many brain disorders vary between the sexes, yet the degree to which this variation arises from differential experience versus intrinsic biological sex differences is unclear. In this study, we demonstrate intrinsic sex differences in molecular regulation of a key neuromodulatory system, the endocannabinoid system, in the hippocampus. Endocannabinoids are involved in diverse aspects of physiology and behavior that involve the hippocampus, including cognitive and motivational state, responses to stress, and neurological disorders such as epilepsy. Our finding that molecular regulation of the endocannabinoid system differs between the sexes suggests mechanisms through which experiences or therapeutics that engage endocannabinoids could affect males and females differently.

Quantitative analysis of long-form aromatase mRNA in the male and female rat brain.

In vitro studies show that estrogens acutely modulate synaptic function in both sexes. These acute effects may be mediated in vivo by estrogens synthesized within the brain, which could fluctuate more rapidly than circulating estrogens. For this to be the case, brain regions that respond acutely to estrogens should be capable of synthesizing them. To investigate this question, we used quantitative real-time PCR to measure expression of mRNA for the estrogen-synthesizing enzyme, aromatase, in different brain regions of male and female rats. Importantly, because brain aromatase exists in two forms, a long form with aromatase activity and a short form with unknown function, we targeted a sequence found exclusively in long-form aromatase. With this approach, we found highest expression of aromatase mRNA in the amygdala followed closely by the bed nucleus of the stria terminalis (BNST) and preoptic area (POA); we found moderate levels of aromatase mRNA in the dorsal hippocampus and cingulate cortex; and aromatase mRNA was detectable in brainstem and cerebellum, but levels were very low. In the amygdala, gonadal/hormonal status regulated aromatase expression in both sexes; in the BNST and POA, castration of males down-regulated aromatase, whereas there was no effect of estradiol in ovariectomized females. In the dorsal hippocampus and cingulate cortex, there were no differences in aromatase levels between males and females or effects of gonadal/hormonal status. These findings demonstrate that long-form aromatase is expressed in brain regions that respond acutely to estrogens, such as the dorsal hippocampus, and that gonadal/hormonal regulation of aromatase differs among different brain regions.

Activity-dependent Wnt 7 dendritic targeting in hippocampal neurons: plasticity- and tagging-related retrograde signaling mechanism?

Wnt proteins have emerged as transmembrane signaling molecules that regulate learning and memory as well as synaptic plasticity at central synapses (Inestrosa and Arenas (2010) Nat Rev Neurosci 11:77-86; Maguschak and Ressler (2011) J Neurosci 31:13057-13067; Tabatadze et al. (2012) Hippocampus 22: 1228-1241; Fortress et al. (2013) J Neurosci 33:12619-12626). For example, there is both a training-selective and Wnt isoform-specific increase in Wnt 7 levels in hippocampus seven days after spatial learning in rats (Tabatadze et al. (2012) Hippocampus 22: 1228-1241). Despite growing interest in Wnt signaling pathways in the adult brain, intracellular distribution and release of Wnt molecules from synaptic compartments as well as their influence on synaptic strength and connectivity remain less well understood. As a first step in such an analysis, we show here that Wnt 7 levels in primary hippocampal cells are elevated by potassium or glutamate activation in a time-dependent manner. Subsequent Wnt 7 elevation in dendrites suggests selective somato-dendritic trafficking followed by transport from dendrites to their spines. Wnt 7 elevation is also TTX-reversible, establishing that its elevation is indeed an activity-dependent process. A second stimulation given 6 h after the first significantly reduces Wnt 7 levels in dendrites 3 h later as compared to non-stimulated controls suggesting activity-dependent Wnt 7 release from dendrites and spines. In a related experiment designed to mimic the release of Wnt 7, exogenous recombinant Wnt 7 increased the number of active zones in presynaptic terminals as indexed by bassoon. This suggests the formation of new presynaptic release sites and/or presynaptic terminals. Wnt signaling inhibitor sFRP-1 completely blocked this Wnt 7-induced elevation of bassoon cluster number and cluster area. We suggest that Wnt 7 is a plasticity-related protein involved in the regulation of presynaptic plasticity via a retrograde signaling mechanism as previously proposed (Routtenberg (1999) Trends in Neuroscience 22:255-256). These findings provide support for this proposal, which offers a new perspective on the synaptic tagging mechanism (Redondo and Morris (2011) Nat Rev Neurosci 12:17-30).

Estradiol regulates large dense core vesicles in the hippocampus of adult female rats.

Previous work has shown that the steroid hormone estradiol facilitates the release of anticonvulsant neuropeptides from inhibitory neurons in the hippocampus to suppress seizures. Because neuropeptides are packaged in large dense core vesicles, estradiol may facilitate neuropeptide release through regulation of dense core vesicles. In the current study, we used serial section electron microscopy in the hippocampal CA1 region of adult female rats to test three hypotheses about estradiol regulation of dense core vesicles: (1) Estradiol increases the number of dense core vesicles in axonal boutons, (2) Estradiol increases the size of dense core vesicles in axonal boutons, (3) Estradiol shifts the location of dense core vesicles toward the periphery of axonal boutons, potentially lowering the threshold for neuropeptide release during seizures. We found that estradiol increases the number and size of dense core vesicles in inhibitory axonal boutons, consistent with increased neuropeptide content, but does not shift the location of dense core vesicles closer to the bouton periphery. These effects were specific to large dense core vesicles (>80 nm) in inhibitory boutons. Estradiol had no effects on small dense core vesicles or dense core vesicles in excitatory boutons. Our results indicate that estradiol suppresses seizures at least in part by increasing the potentially releasable pool of neuropeptides in the hippocampus, and that estradiol facilitation of neuropeptide release involves a mechanism other than mobilization of dense core vesicles toward sites of release.

Distribution and posttranslational modification of synaptic ERα in the adult female rat hippocampus.

Acute 17ß-estradiol (E2) signaling in the brain is mediated by extranuclear estrogen receptors. Here we used biochemical methods to investigate the distribution, posttranslational modification, and E2 regulation of estrogen receptor-α (ERα) in synaptosomal fractions isolated by differential centrifugation from the adult female rat hippocampus. We find that ERα is concentrated presynaptically and is highly enriched with synaptic vesicles. Immunoisolation of vesicles using vesicle subtype-specific markers showed that ERα is associated with both glutamate and γ-aminobutyric acid-containing neurotransmitter vesicles as well as with some large dense core vesicles. Experiments using broad spectrum and residue-specific phosphatases indicated that a portion of ERα in synaptosomal fractions is phosphorylated at serine/threonine residues leading to a mobility shift in SDS-PAGE and creating a double band on Western blots. The phosphorylated form of ERα runs in the upper of the two bands and is particularly concentrated with synaptic vesicles. Finally, we used E2 with or without the acyl protein thioesterase 1 inhibitor, Palmostatin B, to show that 20 min of E2 treatment of hippocampal slices depletes ERα from the synaptosomal membrane by depalmitoylation. We found no evidence that E2 regulates phosphorylation of synaptosomal ERα on this time scale. These studies begin to fill the gap between detailed molecular characterization of extranuclear ERα in previous in vitro studies and acute E2 modulation of hippocampal synapses in the adult brain.

Delayed yet persistent hippocampal molecular and structural alteration after learning: new players in the debate on time-dependent long-lasting memory processes.

Two articles in this issue concern the presynaptic structural remodeling and the molecular events that are important novel mechanisms underlying long-term memory storage processes.

Wnt transmembrane signaling and long-term spatial memory.

Transmembrane signaling mechanisms are critical for regulating the plasticity of neuronal connections underlying the establishment of long-lasting memory (e.g., Linden and Routtenberg (1989) Brain Res Rev 14:279-296; Sossin (1996) Trends Neurosci 19:215-218; Mayr and Montminy (2001) Nat Rev Mol Cell Biol 2:599-609; Chen et al. (2011) Nature 469:491-497). One signaling mechanism that has received surprisingly little attention in this regard is the well-known Wnt transmembrane signaling pathway even though this pathway in the adult plays a significant role, for example, in postsynaptic dendritic spine morphogenesis and presynaptic terminal neurotransmitter release (Inestrosa and Arenas (2010) Nat Rev Neurosci 11:77-86). The present report now provides the first evidence of Wnt signaling in spatial information storage processes. Importantly, this Wnt participation is specific and selective. Thus, spatial, but not cued, learning in a water maze selectively elevates the levels in hippocampus of Wnt 7 and Wnt 5a, but not the Wnt 3 isoform, indicating behavioral selectivity and isoform specificity. Wnt 7 elevation is subfield-specific: granule cells show an increase with no detectable change in CA3 neurons. Wnt 7 elevation is temporally specific: increased Wnt signaling is not observed during training, but is seen 7 days and, unexpectedly, 30 days later. If the Wnt elevation after learning is activity-dependent, then it may be possible to model this effect in primary hippocampal neurons in culture. Here, we evaluate the consequence of potassium or glutamate depolarization on Wnt signaling. This represents, to our knowledge, the first demonstration of an activation-dependent elevation of Wnt levels and surprisingly an increased number of Wnt-stained puncta in neurites suggestive of trafficking from the cell body to neuronal processes, probably dendrites. It is proposed that Wnt signaling pathways regulate long-term information storage in a behavioral-, cellular-, and isoform-specific manner.

Inhibition of neutral sphingomyelinase-2 perturbs brain sphingolipid balance and spatial memory in mice.

The sphingolipid ceramide is a bioactive signaling lipid that is thought to play important roles in modulating synaptic activity, in part by regulating the function of excitatory postsynaptic receptors. However, the molecular mechanisms by which ceramide exerts its effects on synaptic activity remain largely unknown. We recently demonstrated that a rapid generation of ceramide by neutral sphingomyelinase-2 (nSMase2; also known as "sphingomyelin phosphodiesterase-3") played a key role in modulating excitatory postsynaptic currents by controlling the insertion and clustering of NMDA receptors (Wheeler et al. [2009] J. Neurochem. 109:1237-1249). We now demonstrate that nSMase2 plays a role in memory. Inhibition of nSMase2 impaired spatial and episodic-like memory in mice. At the molecular level, inhibition of nSMase2 decreased ceramide, increased PSD-95, increased the number of AMPA receptors, and altered the subunit composition of NMDA receptors. Our study identifies nSMase2 as an important component for efficient memory formation and underscores the importance of ceramide in regulating synaptic events related to learning and memory.

New triterpenoid saponins from Leontice smirnowii.

Three new triterpene saponins, leonticins I (1), J (2) and L (3) were isolated from the tubers of Leontice smirnowii. On the basis of spectroscopic methods, including 2D NMR experiments (DEPT, gs-COSY, gs-HMQC, gs-HMBC and gs-HSQC-TOCSY), mass spectrometry (HR-ESI-MS) and chemical degradation, the structures of the new compounds were elucidated as 3-O-ß-D-glucopyranosyl-(1 → 3)-[ß-D-xylopyranosyl-1 → 2)]-α-L-arabinopyranosyl-28-O-[α-L-rhamnopyranosyl-(1 → 4)-ß-D-glucopyranosyl-(1 → 6)-ß-D-glucopyranosyl]-3ß-hydroxy-30-norolean-12,20(29)-dien-28-oic acid (1), 3-O-[ß-D-xylopyranosyl-(1 → 3)-ß-D-galactopyranosyl-(1 → 4)-ß-D-glucopyranosyl-(1 → 3)-α-L-arabinopyranosyl]-28-O-[α-L-rhamnopyranosyl-(1 → 4)-ß-D-glucopyranosyl-(1 → 6)-ß-D-glucopyranosyl]-3ß-hydroxy-30-norolean-12,20(29)-dien-28-oic acid (2) and 3-O-[ß-D-xylopyranosyl-(1 → 3)-ß-D-galactopyranosyl-(1 → 4)-ß-D-glucopyranosyl-(1 → 3)]-[ß-D-xylopyranosyl-(1 → 2)]-α-L-arabinopyranosyl]-28-O-[α-L-rhamnopyranosyl-(1 → 4)-ß-D-glucopyranosyl-(1 → 6)-ß-D-glucopyranosyl]-3ß-hydroxy-30-norolean-12,20(29)-dien-28-oic acid (3), respectively. The aglycone 3ß-hydroxy-30-norolean-12,20(29)-dien-28-oic acid was observed for the first time in Leontice species.

Apoptosis and cytolysis induced by giganteosides and hederacolchisides in HL-60 cells.

UNLABELLED: The viability, cytolysis and apoptosis-mediated cellular death induced by giganteosides D and E (Gig-D and Gig-E) and hederacolchisides A and A1 (Hcol-A and Hcol-A1) were analysed in HL-60 cells. MATERIALS AND METHODS: The end-point metabolic (WST1) and lactate dehydrogenase (LDH) assays were used. Cell cycle analysis and apoptosis were measured by flow cytometry, DNA laddering and caspase-3 analyses. RESULTS: the HL-60 cell line was more sensitive to Hcol-A1 and Gig-D (IC50 3-5 microM) than to Gig-E and Hcol-A (IC50 8-13 microM; WST1 assay). This was related to LDH release. The induction of apoptosis could be detected without caspase-3 activation after 24 h of treatment. DNA fragmentation could be detected only with Gig-D. With Hcol-A1 and Gig-D, an accumulation of cells in the S-phase and an increase of cells in sub-G1 peak were observed. By the annexinV-fluorescein isothiocyanate (FITC)/7-amino-actinomycin D (AAD) assay, the majority of cells were in late apoptosis with Gig-D, and in necrosis with Hcol-A1. CONCLUSION: Hcol-A1 is more cytotoxic than Gig-D, followed by Gig-E and finally Hcol-A. This is related to a membrane permeabilization effect, leading to cytolysis.

GAP-43 gene expression regulates information storage.

Previous reports have shown that overexpression of the growth- and plasticity-associated protein GAP-43 improves memory. However, the relation between the levels of this protein to memory enhancement remains unknown. Here, we studied this issue in transgenic mice (G-Phos) overexpressing native, chick GAP-43. These G-Phos mice could be divided at the behavioral level into "spatial bright" and "spatial dull" groups based on their performance on two hidden platform water maze tasks. G-Phos dull mice showed both acquisition and retention deficits on the fixed hidden platform task, but were able to learn a visible platform task. G-Phos bright mice showed memory enhancement relative to wild type on the more difficult movable hidden platform spatial memory task. In the hippocampus, the G-Phos dull group showed a 50% greater transgenic GAP-43 protein level and a twofold elevated transgenic GAP-43 mRNA level than that measured in the G-Phos bright group. Unexpectedly, the dull group also showed an 80% reduction in hippocampal Tau1 staining. The high levels of GAP-43 seen here leading to memory impairment find its histochemical and behavioral parallel in the observation of Rekart et al. (Neuroscience 126: 579-584) who described elevated levels of GAP-43 protein in the hippocampus of Alzheimer's patients. The present data suggest that moderate overexpression of a phosphorylatable plasticity-related protein can enhance memory, while excessive overexpression may produce a "neuroplasticity burden" leading to degenerative and hypertrophic events culminating in memory dysfunction.

Antimalarial activity of alkaloids isolated from Stephania rotunda.

Stephania rotunda (Menispermaceae) is used in traditional medicine for the treatment of fever. Four major alkaloids: dehydroroemerine, tetrahydropalmatine, xylopinine, cepharanthine as well as aqueous extract (SA), dichloromethane extracts (SD1 and SD2) from this plant were tested against Plasmodium falciparum W2 in vitro. Dehydroroemerine, cepharanthine and SD1 were the most active against W2 with IC(50) of 0.36, 0.61microM and 0.7microg/mL, respectively. Their IC(50) on human monocytic THP1 cells were 10.8, 10.3microM and >250microg/mL, respectively. Cepharanthine, SD1 and SA were selected for in vivo antimalarial test against Plasmodium berghei in mice. The results of SD1 and SA at dose of 150mg/kg showed a decrease of 89 and 74% of parasitaemia by intra-peritoneal injection and 62.5 and 46.5% of parasitaemia by oral administration, respectively. The result of cepharanthine at dose of 10mg/kg showed a decrease of 47% of parasitaemia by intra-peritoneal injection and 50% of parasitaemia by oral administration. Drug interaction of chloroquine and major alkaloids indicates that cepharanthine-chloroquine and tetrahydropalmatine-xylopinine associations are synergistic. These results are in agreement with the use of this plant in the treatment of malaria. This is the first report on in vivo antimalarial investigation for Stephania rotunda.

Cytotoxic triterpenoid saponins from the roots of Cephalaria gigantea.

Three new oleanane-type saponins, giganteosides L (1), M (2) and N (3) along with eight known ones were isolated from the roots of Cephalaria gigantea. Their structures were established as 3-O-[beta-D-galactopyranosyl-(1-->2)-beta-D-glucuronopyranosyl]-28-O-[beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl]-oleanolic acid, 3-O-[beta-D-galactopyranosyl-(1-->2)-beta-D-glucuronopyranosyl]-28-O-[beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl]-hederagenin, 3-O-[alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucuronopyranosyl]-28-O-[beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl]-hederagenin, respectively, by means of spectroscopic methods (1D and 2D NMR, HR-ESI-MS). Cytotoxic activity of monodesmosides was investigated in vitro using three cancer cell lines, namely, human non pigmented melanoma MEL-5 and human leukemia HL-60. Giganteosides D (4) and E (5) showed antiproliferative effect on human cell lines with IC(50) values in the range 3.15-7.5 microM.