Phosphorylation Of cAMP Response Element-binding Protein Research Articles

Effects of tianeptine on symptoms of fibromyalgia via BDNF signaling in a fibromyalgia animal model.

Previous reports have suggested that physical and psychological stresses may trigger fibromyalgia (FM). Stress is an important risk factor in the development of depression and memory impairments. Antidepressants have been used to prevent stress-induced abnormal pain sensation. Among various antidepressants, tianeptine has been reported to be able to prevent neurodegeneration due to chronic stress and reverse decreases in hippocampal volume. To assess the possible effect of tianeptine on FM symptoms, we constructed a FM animal model induced by restraint stress with intermittent cold stress. All mice underwent nociceptive assays using electronic von Frey anesthesiometer and Hargreaves equipment. To assess the relationship between tianeptine and expression levels of brain-derived neurotrophic factor (BDNF), cAMP response element-binding protein (CREB), and phosphorylated cAMP response element-binding protein (p-CREB), western blotting and immunohistochemistry analyses were performed. In behavioral analysis, nociception tests showed that pain threshold was significantly decreased in the FM group compared to that in the control group. Western blot and immunohistochemical analyses of medial prefrontal cortex (mPFC) and hippocampus showed downregulation of BDNF and p-CREB proteins in the FM group compared to the control group. However, tianeptine recovered these changes in behavioral tests and protein level. Therefore, this FM animal model might be useful for investigating mechanisms linking BDNF-CREB pathway and pain. Our results suggest that tianeptine might potentially have therapeutic efficacy for FM.

Scopoletin Stimulates Melanogenesis via cAMP/PKA Pathway and Partially p38 Activation.

Scopoletin was recently shown to stimulate melanogenesis through cAMP-response element-binding protein (CREB) phosphorylation. In this study, we investigated the molecular events of melanogenesis-induced by scopoletin. After exposure to scopoletin, the protein levels of tyrosinase and tyrosianse related protein-1 (TRP-1) were significantly increased in B16F10 cells. The mRNA levels of tyrosinase and microphthalmia-associated transcription factor (MITF) were also enhanced by scopoletin. cAMP production and phosphorylation of p38 mitogen-activated protein kinase (MAPK) were increased by scopoletin treatment. Scopoletin-mediated increase of intracellular melanin and tyrosinase expression were significantly attenuated by protein kinase A (PKA) inhibitors (H-89 and KT5720), while a protein kinase C (PKC) inhibitor (Ro-32-0432) had no effect and a p38 MAPK inhibitor (SB203580) partially blocked the scopoletin-induced intracellular melanin and tyrosinase expression. Moreover, scopoletin synergistically with cell-permeable cAMP analog (dibutyryl cAMP) significantly induced tyrosinase activity and melanin content in B16F10 cells. The silencing of p38 MAPK by small interfering RNA (siRNA) decreased the scopoletin-induced tyrosinase expression in B16F10 cells. These results suggest that scopoletin could induce melanin synthesis through the cAMP/PKA pathway and partially p38 MAPK activation in B16F10 cells.

Treadmill Exercise Improves Memory Function Depending on Circadian Rhythm Changes in Mice.

PurposeExercise enhances memory function by increasing neurogenesis in the hippocampus, and circadian rhythms modulate synaptic plasticity in the hippocampus. The circadian rhythm-dependent effects of treadmill exercise on memory function in relation with neurogenesis were investigated using mice.MethodsThe step-down avoidance test was used to evaluate short-term memory, the 8-arm maze test was used to test spatial learning ability, and 5-bromo-2’-deoxyuridine immunofluorescence was used to assess neurogenesis. Western blotting was also performed to assess levels of synaptic plasticity-associated proteins, such as brain-derived neurotrophic factor, tyrosine kinase receptor B, phosphorylated cAMP response element-binding protein, early growth response protein 1, postsynaptic density protein 95, and growth-associated protein 43. The mice in the treadmill exercise at zeitgeber 1 group started exercising 1 hour after sunrise, the mice in the treadmill exercise at zeitgeber 6 group started exercising 6 hours after sunrise, and the mice in the treadmill exercise at zeitgeber 13 group started exercising 1 hour after sunset. The mice in the exercise groups were forced to run on a motorized treadmill for 30 minutes once a day for 7 weeks.ResultsTreadmill exercise improved short-term memory and spatial learning ability, and increased hippocampal neurogenesis and the expression of synaptic plasticity-associated proteins. These effects of treadmill exercise were stronger in mice that exercised during the day or in the evening than in mice that exercised at dawn.ConclusionsTreadmill exercise improved memory function by increasing neurogenesis and the expression of synaptic plasticity-associated proteins. These results suggest that the memory-enhancing effect of treadmill exercise may depend on circadian rhythm changes.

Low-level laser irradiation modulates brain-derived neurotrophic factor mRNA transcription through calcium-dependent activation of the ERK/CREB pathway.

Low-level laser (LLL) irradiation has been reported to promote neuronal differentiation, but the mechanism remains unclear. Brain-derived neurotrophic factor (BDNF) has been confirmed to be one of the most important neurotrophic factors because it is critical for the differentiation and survival of neurons during development. Thus, this study aimed to investigate the effects of LLL irradiation on Bdnf messenger RNA (mRNA) transcription and the molecular pathway involved in LLL-induced Bdnf mRNA transcription in cultured dorsal root ganglion neurons (DRGNs) using Ca2+ imaging, pharmacological detections, RNA interference, immunocytochemistry assay, Western blot, and qPCR analysis. We show here that LLL induced increases in the [Ca2+] i level, Bdnf mRNA transcription, cAMP-response element-binding protein (CREB) phosphorylation, and extracellular signal-regulated kinase (ERK) phosphorylation, mediated by Ca2+ release via inositol triphosphate receptor (IP3R)-sensitive calcium (Ca2+) stores. Blockade of Ca2+ increase suppressed Bdnf mRNA transcription, CREB phosphorylation, and ERK phosphorylation. Downregulation of phosphorylated (p)-CREB reduced Bdnf mRNA transcription triggered by LLL. Furthermore, blockade of ERK using PD98059 inhibitor reduced p-CREB and Bdnf mRNA transcription induced by LLL. Taken together, these findings establish the Ca2+-ERK-CREB cascade as a potential signaling pathway involved in LLL-induced Bdnf mRNA transcription. To our knowledge, this is the first report of the mechanisms of Ca2+-dependent Bdnf mRNA transcription triggered by LLL. These findings may help further explore the complex molecular signaling networks in LLL-triggered nerve regeneration in vivo and may also provide experimental evidence for the development of LLL for clinical applications.

Phosphodiesterase-7 inhibition affects accumbal and hypothalamic thyrotropin-releasing hormone expression, feeding and anxiety behavior of rats

Thyrotropin-releasing hormone (TRH) has anorexigenic and anxiolytic functions when injected intraventricularly. Nucleus accumbens (NAcc) is a possible brain region involved, since it expresses proTRH. TRH from hypothalamic paraventricular nucleus (PVN) has a food intake-regulating role. TRHergic pathways of NAcc and PVN are implicated in anxiety and feeding. Both behaviors depend on cAMP and phosphorylated-cAMP response element binding protein (pCREB) intracellular levels.Intracellular levels of cAMP are controlled by the degrading activity of phosphodiesterases (PDEs). Since TRH transcription is activated by pCREB, a specific inhibitor of PDE7B may regulate TRH-induced effects on anxiety and feeding.We evaluated the effectiveness of an intra-accumbal and intraperitoneal (i.p.) administration of a PDE7 inhibitor (BRL-50481) on rats’ anxiety-like behavior and food intake; also on TRH mRNA and protein expression in NAcc and PVN to define its mediating role on the PDE7 inhibitor-induced behavioral changes.Accumbal injection of 4μg/0.3μL of PDE7 inhibitor decreased rats’ anxiety. The i.p. injection of 0.2mg/kg of the inhibitor was able to increase the PVN TRH mRNA expression and to decrease feeding but did not change animals’ anxiety levels; in contrast, 2mg/kg b.w inhibitor enhanced accumbal TRH mRNA, induced anxiolysis with no change in food intake.PDE7 inhibitor induced anxiolytic and anorexigenic like behavior depending on the dose used. Results supported hypothalamic TRH mediated feeding-reduction effects, and accumbal TRH mediation of inhibitor-induced anxiolysis. Thus, an i.p dose of this inhibitor might be reducing anxiety with no change in feeding, which could be useful for obese patients.

Long-Lasting Impairment of mGluR5-Activated Intracellular Pathways in the Striatum After Withdrawal of Cocaine Self-Administration.

Background:Cocaine addiction continues to be a major heath concern, and despite public health intervention there is a lack of efficient pharmacological treatment options. A newly identified potential target are the group I metabotropic glutamate receptors, with allosteric modulators showing particular promise.Methods:We evaluated the capacity of group I metabotropic glutamate receptors to induce functional responses in ex vivo striatal slices from rats with (1) acute cocaine self-administration, (2) chronic cocaine self-administration, and (3) 60 days cocaine self-administration withdrawal by Western blot and extracellular recordings of synaptic transmission.Results:We found that striatal group I metabotropic glutamate receptors are the principal mediator of the mGluR1/5 agonist (RS)-3,5-dihydroxyphenylglycine-induced cAMP responsive-element binding protein phosphorylation. Both acute and chronic cocaine self-administration blunted group I metabotropic glutamate receptor effects on cAMP responsive-element binding protein phosphorylation in the striatum, which correlated with the capacity to induce long-term depression, an effect that was maintained 60 days after chronic cocaine self-administration withdrawal. In the nucleus accumbens, the principal brain region mediating the rewarding effects of drugs, chronic cocaine self-administration blunted group I metabotropic glutamate receptor stimulation of extracellular signal-regulated protein kinases 1/2 and cAMP responsive-element binding protein. Interestingly, the group I metabotropic glutamate receptor antagonist/inverse-agonist, 2-methyl-6-(phenylethynyl)pyridine hydrochloride, led to a specific increase in cAMP responsive-element binding protein phosphorylation after chronic cocaine self-administration, specifically in the nucleus accumbens, but not in the striatum.Conclusions:Prolonged cocaine self-administration, through withdrawal, leads to a blunting of group I metabotropic glutamate receptor responses in the striatum. In addition, specifically in the accumbens, group I metabotropic glutamate receptor signaling to cAMP responsive-element binding protein shifts from an agonist-induced to an antagonist-induced cAMP responsive-element binding protein phosphorylation.

MPTP Impairs Dopamine D1 Receptor-Mediated Survival of Newborn Neurons in Ventral Hippocampus to Cause Depressive-Like Behaviors in Adult Mice.

Parkinson’s disease (PD) is characterized by motor symptoms with depression. We evaluated the influence of dopaminergic depletion on hippocampal neurogenesis process to explore mechanisms of depression production. Five consecutive days of 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injection in mice (MPTP-mice) reduced dopaminergic fibers in hippocampal dentate gyrus (DG). MPTP-mice exhibited depressive-like behaviors later for 2–3 weeks. BrdU was injected 4 h after last-injection of MPTP. BrdU-positive (BrdU+) cells in dorsal (d-DG) and ventral (v-DG) DG were examined on day 1 (D1), 7 (D7), 14 (D14) and 21 (D21) after BrdU injection. Fewer D7-, D14- and D21-BrdU+ cells or BrdU+/NeuN+ cells, but not D1-BrdU+ cells, were found in v-DG of MPTP-mice than in controls. However, the number of BrdU+ cells in d-DG did not differ between the both. Loss of doublecortin-positive (DCX+) cells was observed in v-DG of MPTP-mice. Protein kinase A (PKA) and Ca2+/cAMP-response element binding protein (CREB) phosphorylation were reduced in v-DG of MPTP-mice, which were reversed by D1-like receptor (D1R) agonist SKF38393, but not D2R agonist quinpirole. The treatment of MPTP-mice with SKF38393 on days 2–7 after BrdU-injection reduced the loss of D7- and D21-BrdU+ cells in v-DG and improved the depressive-like behaviors; these changes were sensitive to PKA inhibitor H89. Moreover, the v-DG injection of SKF38393 in MPTP-mice could reduce the loss of D21-BrdU+ cells and relieve the depressive-like behaviors. In control mice, the blockade of D1R by SCH23390 caused the reduction of D21-BrdU+ cells in v-DG and the depressive-like behaviors. Our results indicate that MPTP-reduced dopaminergic depletion impairs the D1R-mediated early survival of newborn neurons in v-DG, producing depressive-like behaviors.

Phosphodiesterase 10A inhibition attenuates sleep deprivation-induced deficits in long-term fear memory

Sleep, particularly rapid eye movement (REM) sleep, is implicated in the consolidation of emotional memories. In the present study, we investigated the protective effects of a phosphodiesterase 10A (PDE10A) inhibitor MP-10 on deficits in long-term fear memory induced by REM sleep deprivation (REM-SD). REM-SD caused deficits in long-term fear memory, however, MP-10 administration ameliorated the deleterious effects of REM-SD on long term fear memory. Brain-derived neurotropic factor (BDNF) and phosphorylated cAMP response element-binding protein (pCREB) were altered in specific brain regions associated with learning and memory in REM-SD rats. Accordingly, REM-SD caused a significant decrease of pCREB in hippocampus and striatum and a significant decrease of BDNF in the hippocampus, striatum and amygdala, however, MP-10 reversed the effects of REM-SD in a dose-dependent manner. Our findings suggest that REM-SD disrupts the consolidation of long-term fear memory and that administration of MP-10 protects the REM-SD-induced deficits in fear memory, which may be due to the MP-10-induced expression of BDNF in the hippocampus, striatum and amygdala, and phosphorylation of CREB in the hippocampus and striatum.

Physical interaction between neurofibromin and serotonin 5-HT6 receptor promotes receptor constitutive activity

Active G protein-coupled receptor (GPCR) conformations not only are promoted by agonists but also occur in their absence, leading to constitutive activity. Association of GPCRs with intracellular protein partners might be one of the mechanisms underlying GPCR constitutive activity. Here, we show that serotonin 5 hydroxytryptamine 6 (5-HT6) receptor constitutively activates the Gs/adenylyl cyclase pathway in various cell types, including neurons. Constitutive activity is strongly reduced by silencing expression of the Ras-GTPase activating protein (Ras-GAP) neurofibromin, a 5-HT6 receptor partner. Neurofibromin is a multidomain protein encoded by the NF1 gene, the mutation of which causes Neurofibromatosis type 1 (NF1), a genetic disorder characterized by multiple benign and malignant nervous system tumors and cognitive deficits. Disrupting association of 5-HT6 receptor with neurofibromin Pleckstrin Homology (PH) domain also inhibits receptor constitutive activity, and PH domain expression rescues 5-HT6 receptor-operated cAMP signaling in neurofibromin-deficient cells. Furthermore, PH domains carrying mutations identified in NF1 patients that prevent interaction with the 5-HT6 receptor fail to rescue receptor constitutive activity in neurofibromin-depleted cells. Further supporting a role of neurofibromin in agonist-independent Gs signaling elicited by native receptors, the phosphorylation of cAMP-responsive element-binding protein (CREB) is strongly decreased in prefrontal cortex of Nf1+/- mice compared with WT mice. Moreover, systemic administration of a 5-HT6 receptor inverse agonist reduces CREB phosphorylation in prefrontal cortex of WT mice but not Nf1+/- mice. Collectively, these findings suggest that disrupting 5-HT6 receptor-neurofibromin interaction prevents agonist-independent 5-HT6 receptor-operated cAMP signaling in prefrontal cortex, an effect that might underlie neuronal abnormalities in NF1 patients.

Exendin-4 antagonizes Aβ1-42-induced attenuation of spatial learning and memory ability.

β-amyloid protein (Aβ) accumulation in cerebral centers involved in cognition and memory is a pivotal pathological feature of Alzheimer's disease (AD). The onset process of type 2 diabetes mellitus (T2DM) has a number of similarities compared with AD. Thus, it is hypothesized that the pharmacological therapy employed for the treatment of T2DM may help to prevent and ameliorate the symptoms of AD. This study demonstrated that Exendin-4, which is a glucagon-like peptide-1 analogue which is used as a therapeutic drug for T2DM, markedly antagonized Aβ fragment-induced attenuation of spatial learning and memory ability, as indicated by a Morris water maze experiment. In addition, we investigated the potential underlying electrophysiological and molecular mechanisms. The results indicate that Exendin-4 rescued long-term potentiation from Aβ1-42-induced damage in the rat hippocampal CA1 region in vivo, and antagonized Aβ1-42-induced reduction of cyclic adenosine monophosphate and phosphorylated-cAMP response element-binding protein in rat hippocampal tissue using ELISA and western blot analysis, respectively. Thus, the results of the present study provide theoretical support for the application of Exendin-4 for improving AD.

Mogrol Derived from Siraitia grosvenorii Mogrosides Suppresses 3T3-L1 Adipocyte Differentiation by Reducing cAMP-Response Element-Binding Protein Phosphorylation and Increasing AMP-Activated Protein Kinase Phosphorylation

This study investigated the effects of mogrol, an aglycone of mogrosides from Siraitia grosvenorii, on adipogenesis in 3T3-L1 preadipocytes. Mogrol, but not mogrosides, suppressed triglyceride accumulation by affecting early (days 0–2) and late (days 4–8), but not middle (days 2–4), differentiation stages. At the late stage, mogrol increased AMP-activated protein kinase (AMPK) phosphorylation and reduced glycerol-3-phosphate dehydrogenase activity. At the early stage, mogrol promoted AMPK phosphorylation, inhibited the induction of CCAAT/enhancer-binding protein β (C/EBPβ; a master regulator of adipogenesis), and reduced 3T3-L1 cell contents (e.g., clonal expansion). In addition, mogrol, but not the AMPK activator AICAR, suppressed the phosphorylation and activity of the cAMP response element-binding protein (CREB), which regulates C/EBPβ expression. These results indicated that mogrol suppressed adipogenesis by reducing CREB activation in the initial stage of cell differentiation and by activating AMPK signaling in both the early and late stages of this process.

Period1 gates the circadian modulation of memory-relevant signaling in mouse hippocampus by regulating the nuclear shuttling of the CREB kinase pP90RSK.

Memory performance varies over a 24-h day/night cycle. While the detailed underlying mechanisms are yet unknown, recent evidence suggests that in the mouse hippocampus, rhythmic phosphorylation of mitogen-activated protein kinase (MAPK) and cyclic adenosine monophosphate response element-binding protein (CREB) are central to the circadian (~24h) regulation of learning and memory. We recently identified the clock protein PERIOD1 (PER1) as a vehicle that translates information encoding time of day to hippocampal plasticity. We here elaborate how PER1 may gate the sensitivity of memory-relevant hippocampal signaling pathways. We found that in wild-type mice (WT), spatial learning triggers CREB phosphorylation only during the daytime, and that this effect depends on the presence of PER1. The time-of-day-dependent induction of CREB phosphorylation can be reproduced pharmacologically in acute hippocampal slices prepared from WT mice, but is absent in preparations made from Per1-knockout (Per1(-/-) ) mice. We showed that the PER1-dependent CREB phosphorylation is regulated downstream of MAPK. Stimulation of WT hippocampal neurons triggered the co-translocation of PER1 and the CREB kinase pP90RSK (pMAPK-activated ribosomal S6kinase) into the nucleus. In hippocampal neurons from Per1(-/-) mice, however, pP90RSK remained perinuclear. A co-immunoprecipitation assay confirmed a high-affinity interaction between PER1 and pP90RSK. Knocking down endogenous PER1 in hippocampal cells inhibited adenylyl cyclase-dependent CREB activation. Taken together, the PER1-dependent modulation of cytoplasmic-to-nuclear signaling in the murine hippocampus provides a molecular explanation for how the circadian system potentially shapes a temporal framework for daytime-dependent memory performance, and adds a novel facet to the versatility of the clock gene protein PER1. We provide evidence that the circadian clock gene Period1 (Per1) regulates CREB phosphorylation in the mouse hippocampus, sculpturing time-of-day-dependent memory formation. This molecular mechanism constitutes the functional link between circadian rhythms and learning efficiency. In hippocampal neurons of wild-type mice, pP90RSK translocates into the nucleus upon stimulation with forskolin (left), whereas in Period1-knockout (Per1(-/-) ) mice (right) the kinase is trapped at the nuclear periphery, unable to efficiently phosphorylate nuclear CREB. Consequently, the presence of PER1 in hippocampal neurons is a prerequisite for the time-of-day-dependent phosphorylation of CREB, as it regulates the shuttling of pP90RSK into the nucleus. Representative immunofluorescence images show a temporal difference in phosphorylated cAMP response element-binding protein (pCREB; green color) levels in all regions of the dorsal hippocampus between a wild-type C3H mouse (WT; left) and a Period1-knockout (Per1(-/-) ; right) mouse. Images were taken 2h after lights on, thus, when fluctuating levels of pCREB peak in WT mouse hippocampus. Insets show a representative hippocampal neuron, in response to activating cAMP signaling, stained for the neuronal marker NeuN (red), the nuclear marker DAPI (blue) and the activated CREB kinase pP90RSK (green). The image was taken 2h after light onset (at the peak of the endogenous CREB phosphorylation that fluctuates with time of day). Magnification: 100X, inset 400X. Read the Editorial Highlight for this article on page 650. Cover image for this issue: doi: 10.1111/jnc.13332.

North American ginseng (Panax quinquefolius) suppresses β-adrenergic-dependent signalling, hypertrophy, and cardiac dysfunction.

There is increasing evidence for a beneficial effect of ginseng on cardiac pathology. Here, we determined whether North American ginseng can modulate the deleterious effects of the β-adrenoceptor agonist isoproterenol on cardiac hypertrophy and function using in vitro and in vivo approaches. Isoproterenol was administered for 2 weeks at either 25 mg/kg per day or 50 mg/kg per day (ISO25 or ISO50) via a subcutaneously implanted osmotic mini-pump to either control rats or those receiving ginseng (0.9 g/L in the drinking water ad libitum). Isoproterenol produced time- and dose-dependent left ventricular dysfunction, although these effects were attenuated by ginseng. Improved cardiac functions were associated with reduced heart masses, as well as prevention in the upregulation of the hypertrophy-related fetal gene expression. Lung masses were similarly attenuated, suggesting reduced pulmonary congestion. In in vitro studies, ginseng (10 μg/mL) completely suppressed the hypertrophic response to 1 μmol/L isoproterenol in terms of myocyte surface area, as well as reduction in the upregulation of fetal gene expression. These effects were associated with attenuation in both protein kinase A and cAMP response element-binding protein phosphorylation. Ginseng attenuates adverse cardiac adrenergic responses and, therefore, may be an effective therapy to reduce hypertrophy and heart failure associated with excessive catecholamine production.

New insights into selective PDE4D inhibitors: 3-(Cyclopentyloxy)-4-methoxybenzaldehyde O-(2-(2,6-dimethylmorpholino)-2-oxoethyl) oxime (GEBR-7b) structural development and promising activities to restore memory impairment

Phosphodiesterase type 4D (PDE4D) has been indicated as a promising target for treating neurodegenerative pathologies such as Alzheimer's Disease (AD). By preventing cAMP hydrolysis, PDE4 inhibitors (PDE4Is) increase the cAMP response element-binding protein (CREB) phosphorylation, synaptic plasticity and long-term memory formation. Pharmacological and behavioral studies on our hit GEBR-7b demonstrated that selective PDE4DIs could improve memory without causing emesis and sedation. The hit development led to new molecule series, herein reported, characterized by a catechol structure bonded to five member heterocycles. Molecular modeling studies highlighted the pivotal role of a polar alkyl chain in conferring selective enzyme interaction. Compound 8a showed PDE4D3 selective inhibition and was able to increase intracellular cAMP levels in neuronal cells, as well as in the hippocampus of freely moving rats. Furthermore, 8a was able to readily cross the blood-brain barrier and enhanced memory performance in mice without causing any emetic-like behavior. These data support the view that PDE4D is an adequate molecular target to restore memory deficits in different neuropathologies, including AD, and also indicate compound 8a as a promising candidate for further preclinical development.

Oleanolic acid ameliorates cognitive dysfunction caused by cholinergic blockade via TrkB-dependent BDNF signaling

Oleanolic acid is a naturally occurring triterpenoid and is widely present in food and medicinal plants. To examine the effect of oleanolic acid on memory deficits, we employed a cholinergic blockade-induced cognitive deficit mouse model. A single administration of oleanolic acid significantly increased the latency on the passive avoidance task and affected the alternation behavior on the Y-maze task and the exploration time on the novel object recognition task, indicating that oleanolic acid reverses the cognitive impairment induced by scopolamine. In accordance with previous reports, oleanolic acid enhanced extracellular-signal-regulated kinase 1/2 (ERK1/2) and cAMP response element-binding protein (CREB) phosphorylation and brain-derived neurotrophic factor (BDNF) expression in the hippocampus. Interestingly, ameliorating effect of oleanolic acid on scopolamine-induced memory impairment was abolished by N2-(2-{[(2-oxoazepan-3-yl)amino]carbonyl}phenyl)benzo[b]thiophene-2-carboxamide (ANA-12), a potent and specific inhibitor of tropomyosin receptor kinase B (TrkB), in the passive avoidance task. Similarly, oleanolic acid significantly evoked long-term potentiation in a dose-dependent manner, which was diminished by ANA-12 treatment as shown in the electrophysiology study. Together, these results imply that oleanolic acid ameliorates scopolamine-induced memory impairment by modulating the BDNF-ERK1/2-CREB pathway through TrkB activation in mice, suggesting that oleanolic acid would be a potential therapeutic agent for the treatment of cognitive deficits.

Indication of nerve growth factor binding components from herbal extracts by HerboChip: a platform for drug screening on a chip.

BackgroundHerboChip is an array of different fractions deriving from herbal extracts. This study aimed to identify effective components from Chinese medicine (CM) that interact with nerve growth factor (NGF) as a target using HerboChip.MethodsFifty types of CM that are traditionally used as remedies for emotion imbalance were selected and extracted with 50 % ethanol. Biotinylated-NGF was hybridized with over 300 chips coated with different HPLC-separated fractions from CM extracts and straptavidin-Cy5 was used to identify the NGF-bound fractions.ResultsOver 300 chips were screened within a week, and 17 positive hits were identified. The interaction of the identified herbal extracts with NGF was confirmed in cultured PC12 cells. Co-application of NGF and herbal extract interfered with NGF-induced expression of neurofilaments, including NF68 and NF200 in cell cultures. Western blot analysis comparing the intensity of phosphorylated cAMP response element-binding protein (CREB) over total CREB showed NGF-induced CREB phosphorylation was modulated by the identified herbal extracts. Five CM herbs showed activating activities on the NGF response and nine CM herbs showed inhibiting activities.ConclusionThe current result supported the applicability of HerboChip for screening NGF binding components from herbal extracts.Electronic supplementary materialThe online version of this article (doi:10.1186/s13020-016-0107-8) contains supplementary material, which is available to authorized users.

Caffeine exposure alters adenosine system and neurochemical markers during retinal development.

Evidence points to beneficial properties of caffeine in the adult central nervous system, but teratogenic effects have also been reported. Caffeine exerts most of its effects by antagonizing adenosine receptors, especially A1 and A2A subtypes. In this study, we evaluated the role of caffeine on the expression of components of the adenosinergic system in the developing avian retina and the impact of caffeine exposure upon specific markers for classical neurotransmitter systems. Caffeine exposure (5-30mg/kg by in ovo injection) to 14-day-old chick embryos increased the expression of A1 receptors and concomitantly decreased A2A adenosine receptors expression after 48h. Accordingly, caffeine (30mg/kg) increased [(3) H]-8-cyclopentyl-1,3-dipropylxanthine (A1 antagonist) binding and reduced [(3) H]-ZM241385 (A2A antagonist) binding. The caffeine time-response curve demonstrated a reduction in A1 receptors 6h after injection, but an increase after 18 and 24h. In contrast, caffeine exposure increased the expression of A2A receptors from 18 and 24h. Kinetic assays of [(3) H]-S-(4-nitrobenzyl)-6-thioinosine binding to the equilibrative adenosine transporter ENT1 revealed an increase in Bmax with no changes in Kd , an effect accompanied by an increase in adenosine uptake. Immunohistochemical analysis showed a decrease in retinal content of tyrosine hydroxylase, calbindin and choline acetyltransferase, but not Brn3a, after 48h of caffeine injection. Furthermore, retinas exposed to caffeine had increased levels of phosphorylated extracellular signal-regulated kinase and cAMP-response element binding protein. Overall, we show an invivo regulation of the adenosine system, extracellular signal-regulated kinase and cAMP-response element binding protein function and protein expression of specific neurotransmitter systems by caffeine in the developing retina. The beneficial or maleficent effects of caffeine have been demonstrated by the work of different studies. It is known that during animal development, caffeine can exert harmful effects, impairing the correct formation of CNS structures. In this study, we demonstrated cellular and tissue effects of caffeine's administration on developing chick embryo retinas. Those effects include modulation of adenosine receptors (A1 , A2 ) content, increasing in cAMP response element-binding protein (pCREB) and extracellular signal-regulated kinase phosphorylation (pERK), augment of adenosine equilibrative transporter content/activity, and a reduction of some specific cell subpopulations. ENT1, Equilibrative nucleoside transporter 1.

The Steroid Hormone 20-Hydroxyecdysone Enhances Gene Transcription through the cAMP Response Element-binding Protein (CREB) Signaling Pathway

Animal steroid hormones regulate gene transcription through genomic pathways by binding to nuclear receptors. These steroid hormones also rapidly increase intracellular calcium and cyclic adenosine monophosphate (cAMP) levels and activate the protein kinase C (PKC) and protein kinase A (PKA) nongenomic pathways. However, the function and mechanism of the nongenomic pathways of the steroid hormones are unclear, and the relationship between the PKC and PKA pathways is also unclear. We propose that the steroid hormone 20-hydroxyecdysone (20E) activates the PKA pathway to enhance 20E-induced gene transcription in the lepidopteran insect Helicoverpa armigera The expression of the catalytic subunit 1 of PKA (PKAC1) increased during metamorphosis, and PKAC1 knockdown blocked pupation and repressed 20E-responsive gene expression. 20E regulated PKAC1 phosphorylation at threonine 200 and nuclear translocation through an ecdysone-responsive G-protein-coupled receptor 2. PKAC1 induced cAMP response element-binding protein (CREB) phosphorylation at serine 143, which bound to the cAMP response element on DNA to enhance 20E-responsive gene transcription. Through ecdysone-responsive G-protein-coupled receptor 2, 20E increased cAMP levels, which induced CREB PKA phosphorylation and 20E-responsive gene expression. This study demonstrates that the PKA/CREB pathway tightly and critically regulates 20E-induced gene transcription as well as its relationship with the 20E-induced PKC pathway.

Argon mediates protection by interleukin-8 suppression via a TLR2/TLR4/STAT3/NF-κB pathway in a model of apoptosis in neuroblastoma cells invitro and following ischemia-reperfusion injury in rat retina invivo.

Argon has recently come into scientific focus as a neuroprotective agent. The underlying neuroprotective mechanism remains unknown although toll-like receptors were recently suggested to play an important role. We hypothesized that TLR-associated downstream transcription factors are responsible for argon's effects, leading to anti-apoptotic and anti-inflammatory properties. Apoptosis was induced in human neuroblastoma cells. Immediately afterwards, argon treatment (75 Vol% for 2h) was initiated. Cells were analyzed, measuring mitochondrial membrane potential, reactive-oxygen-species, annexin-V/propidium iodide staining, transcription factor phosphorylation and binding activity as well as protein and mRNA expression of interleukins. Argon's invivo effects were analyzed by quantification of retinal ganglion cell density, mRNA expression, serum cytokine analysis and immunohistochemistry after retinal ischemia reperfusion injury (IRI) in rats. Argon diminished rotenone-induced kappa-light-chain-enhancer' of activated B-cells (NF-κB) and signal transducer and activator of transcription 3 (STAT3) but not STAT5 or cAMP-response element-binding protein (CREB) phosphorylation and DNA-binding activity. Argon treatment attenuated apoptosis by preservation of mitochondrial membrane potential and decline in reactive oxygen species (ROS) generation. NF-κB and STAT3 inhibition, as well as TLR2 and TLR4 inhibition reversed argon's effects on IL-8 mRNA expression. Argon attenuated rotenone-induced IL-8 protein and mRNA expression invitro. Inhibition of TLR2 and 4 attenuated argon's protective effect invivo reducing IRI driven retinal IL-8 expression. IL-8 expression was found in the retina in co-localization with Müller cells and retinal ganglion cells. Argon mediates its neuroprotective effects by TLR-mediated regulation of transcription factors NF-κB and STAT3, thus decreasing interleukin-8 expression invitro and invivo. These findings may open up new opportunities to effectively treat cerebral ischemia and reperfusion injury through the inhalation of argon. Argon exerts its protective effects invitro and in vivo via toll-like receptors TLR2 and TLR4 signaling, followed by alteration of downstream enzymes. In conclusion, argon mediates its beneficial effects by suppression of STAT3 and NF-κB phosphorylation and subsequent suppression of interleukin IL-8 protein expression. These novel findings may open up opportunities for argon as a therapeutic agent, particularly in the treatment of neuronal injury. Cover image for this issue: doi: 10.1111/jnc.13334.

Short-term environmental enrichment enhances synaptic plasticity in hippocampal slices from aged rats

Age-associated changes in cognition are mirrored by impairments in cellular models of memory and learning, such as long-term potentiation (LTP) and long-term depression (LTD). In young rodents, environmental enrichment (EE) can enhance memory, alter LTP and LTD, as well as reverse cognitive deficits induced by aging. Whether short-term EE can benefit cognition and synaptic plasticity in aged rodents is unclear. Here, we tested if short-term EE could overcome age-associated impairments in induction of LTP and LTD. LTP and LTD could not be induced in the CA1 region of hippocampal slices in control, aged rats using standard stimuli that are highly effective in young rats. However, exposure of aged littermates to EE for three weeks enabled successful induction of LTP and LTD. EE-facilitated LTP was dependent upon N-methyl-d-aspartate receptors (NMDARs). These alterations in synaptic plasticity occurred with elevated levels of phosphorylated cAMP response element-binding protein and vascular endothelial growth factor, but in the absence of changes in several other synaptic and cellular markers. Importantly, our study suggests that even a relatively short period of EE is sufficient to alter synaptic plasticity and molecular markers linked to cognitive function in aged animals.