cAMP Response Element-binding Protein Signaling Research Articles

SMOC1 is a glucose-responsive hepatokine and therapeutic target for glycemic control.

Intertissue communication is a fundamental feature of metabolic regulation, and the liver is central to this process. We have identified sparc-related modular calcium-binding protein 1 (SMOC1) as a glucose-responsive hepatokine and regulator of glucose homeostasis. Acute intraperitoneal administration of SMOC1 improved glycemic control and insulin sensitivity in mice without changes in insulin secretion. SMOC1 exerted its favorable glycemic effects by inhibiting adenosine 3',5'-cyclic monophosphate (cAMP)-cAMP-dependent protein kinase (PKA)-cAMP response element-binding protein (CREB) signaling in the liver, leading to decreased gluconeogenic gene expression and suppression of hepatic glucose output. Overexpression of SMOC1 in the liver or once-weekly intraperitoneal injections of a stabilized SMOC1-FC fusion protein induced durable improvements in glucose tolerance and insulin sensitivity in db/db mice, without adverse effects on adiposity, liver histopathology, or inflammation. Furthermore, circulating SMOC1 correlated with hepatic and systemic insulin sensitivity and was decreased in obese, insulin-resistant humans. Together, these findings identify SMOC1 as a potential pharmacological target for the management of glycemic control in type 2 diabetes.

The role of omega-3 fatty acids in preventing glucocorticoid-induced reduction in human hippocampal neurogenesis and increase in apoptosis

Glucocorticoids have been suggested to be involved in several neuropsychiatric disorders, including depression. One of the possible mechanisms through which glucocorticoids contribute to the development of the depressive symptomatology is via regulation of distinct neurogenic mechanisms in the brain. A preventive or protective approach for these patients might be the use of omega-3 polyunsaturated fatty acids (n-3 PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are known for they neuroprotective properties. We used the human hippocampal progenitor cell line HPC0A07/03C and pre-treated cells with either EPA or DHA, followed by treatment with the glucocorticoid cortisol either alone, or in co-treatment with the same n-3 PUFA during subsequent 3 days of proliferation and 7 days of differentiation. During proliferation, both EPA and DHA were able to prevent cortisol-induced reduction in proliferation and increase in apoptosis, when used in pre-treatment, and both pre- and co-treatment. During differentiation, EPA was able to prevent cortisol-induced reduction in neurogenesis and increase in apoptosis, when used in pre-treatment, and both pre- and co-treatment only during the proliferation stage; however, DHA required continuous treatment also during the differentiation stage to prevent cortisol-induced reduction in neurogenesis. Using transcriptomic analyses, we showed that both EPA and DHA regulated pathways involved in oxidative stress and immune response [e.g., nuclear factor (erythroid-derived 2)-like 2 (Nrf2), Signal transducer and activator of transcription 3 (STAT3), Interferon (IFN) and Interleukin (IL)-1 signaling], whereas DHA also regulated pathways involved in cell development and neuronal formation [e.g., cAMP-response element binding protein (CREB) signaling]. We provide the first evidence for treatment with both EPA and DHA to prevent cortisol-induced reduction in human hippocampal neurogenesis, and identify novel molecular mechanisms underlying these effects.

Modeling human age-associated increase in Gadd45γ expression leads to spatial recognition memory impairments in young adult mice

Aging is associated with the progressive decay of cognitive function. Hippocampus-dependent processes, such as the formation of spatial memory, are particularly vulnerable to aging. Currently, the molecular mechanisms responsible for age-dependent cognitive decline are largely unknown. Here, we investigated the expression and function of the growth arrest DNA damage gamma (Gadd45γ) during aging and cognition. We report that Gadd45γ expression is increased in the hippocampus of aged humans and that Gadd45γ overexpression in the young adult mouse hippocampus compromises cognition. Moreover, Gadd45γ overexpression in hippocampal neurons disrupted cAMP response element-binding protein signaling and the expression of well-established activity-regulated genes. This work shows that Gadd45γ expression is tightly controlled in the hippocampus and its disruption may be a mechanism contributing to age-related cognitive impairments observed in humans.

Single-Nucleus RNA Sequencing of the Hypothalamic Arcuate Nucleus of C57BL/6J Mice After Prolonged Diet-Induced Obesity.

Prolonged obesity is associated with blunted feeding and thermogenic autonomic responses to leptin, but cardiovascular responses to leptin are maintained. This state of selective leptin resistance is, therefore, proposed to contribute to the pathogenesis and maintenance of obesity-associated hypertension. Cells of the arcuate nucleus of the hypothalamus detect leptin, and although the cellular and molecular mechanisms remain unclear, altered arcuate nucleus biology is hypothesized to contribute to selective leptin resistance. Male C57BL/6J mice were fed a high-fat diet (HFD) or chow from 8 to 18 weeks of age, as this paradigm models selective leptin resistance. Nuclei were then isolated from arcuate nucleus for single-nucleus RNA sequencing. HFD caused expected gains in adiposity and circulating leptin. Twenty-three unique cell-type clusters were identified, and Ingenuity Pathway Analysis was used to explore changes in gene expression patterns due to chronic HFD within each cluster. Notably, gene expression signatures related to leptin signaling exhibited suppression predominantly in neurons identified as the Agouti-related peptide (Agrp) subtype. Ingenuity Pathway Analysis results were also consistent with alterations in CREB (cAMP response element-binding protein) signaling in Agrp neurons after HFD, and reduced phosphorylated CREB was confirmed in arcuate nucleus after prolonged HFD by capillary electrophoresis-based Western blotting. These findings support the concept that prolonged HFD-induced obesity is associated with selective changes in Agrp neuron biology, possibly secondary to altered CREB signaling.

Strength exercise suppresses STZ-induced spatial memory impairment and modulates BDNF/ERK-CAMKII/CREB signalling pathway in the hippocampus of mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that has generated scientific interest because of its prevalence in the population. Studies indicate that physical exercise promotes neuroplasticity and improves cognitive function in animal models and in human beings. The aim of the present study was to investigate the effects of strength exercise on the hippocampal protein contents and memory performance in mice subjected to a model of sporadic AD induced by streptozotocin (STZ). Swiss mice received two injections of STZ (3 mg/kg, intracerebroventricular). After 21 days, they began physical training using a ladde. Mice performed this protocol for 4 weeks. After the last exercise training session, mice performed the Morris Water Maze test. The samples of hippocampus were excised and used to determine protein contents of brain-derived neurotrophic factor (BDNF), extracellular signal-regulated kinase-Ca2+ (ERK), calmodulin-dependent protein kinase (CAMKII) and cAMP-response element-binding protein (CREB) signalling pathway. Strength exercise was effective against the decrease in the time spent and distance travelled in the target quadrant by STZ-injected mice. Strength exercise was also effective against the reduction of mature BDNF, tropomyosin receptor kinase B and neuronal nuclear antigen (NeuN) hippocampal protein levels in STZ mice. The decrease in the hippocampal ratio of pERK/ERK, pCAMKII/CAMKII and pCREB/CREB induced by STZ was reversed by strength exercise. Strength exercise decreased Bax/Bcl2 ratio in the hippocampus of STZ-injected mice. The present study demonstrates that strength exercise modulated the hippocampal BDNF/ERK-CAMKII/CREB signalling pathway and suppressed STZ-induced spatial memory impairment in mice.

Activation of α7 nAChR by PNU-282987 improves synaptic and cognitive functions through restoring the expression of synaptic-associated proteins and the CaM-CaMKII-CREB signaling pathway.

Ligands of nicotinic acetylcholine receptors (nAChRs) are widely considered as potential therapeutic agents. The present study used primary hippocampus cells and APPswe/PSEN1dE9 double-transgenic mice models to study the possible therapeutic effect and underlying mechanism of the specific activation of α7 nAChR by PNU-282987 in the pathogenesis of Alzheimer’s disease. The results indicated that activation of α7 nAChR attenuated the Aβ-induced cell apoptosis, decreased the deposition of Aβ, increased the expression of synaptic-associated proteins, and maintained synaptic morphology. Furthermore, in the APP/PS1_DT mice model, activation of α7 nAChR attenuated Aβ-induced synaptic loss, reduced the deposition of Aβ in the hippocampus, maintained the integral structure of hippocampus-derived synapse, and activated the calmodulin (CaM)-calmodulin-dependent protein kinase II (CaMKII)-cAMP response element-binding protein signaling pathway by upregulation of its key signaling proteins. In addition, activation of α7 nAChR improved the learning and memory abilities of the APP/PS1_DT mice. Collectively, the activation of α7 nAChR by PNU-282987 attenuated the toxic effect of Aβ in vivo and in vitro, which including reduced deposition of Aβ in the hippocampus, maintained synaptic morphology by partially reversing the expression levels of synaptic-associated proteins, activation of the Ca2+ signaling pathway, and improvement of the cognitive abilities of APP/PS1_DT mice.

EPA-enriched ethanolamine plasmalogen and EPA-enriched phosphatidylethanolamine enhance BDNF/TrkB/CREB signaling and inhibit neuronal apoptosis in vitro and in vivo.

Our previous study showed that EPA-enriched ethanolamine plasmalogen (EPA-pPE) exerted more significant effects than EPA-enriched phosphatidylethanolamine (EPA-PE) in improving learning and memory deficit. However, the results of the mechanism study were not consistent with the improved cognitive function, which suggested that other signaling pathways might be involved. In the present study, primary cultured hippocampal neurons and cognitive deficiency rats were used to compare the effects of EPA-pPE and EPA-PE on brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB)/cAMP response element-binding protein (CREB) signaling and neuronal apoptosis. The in vitro experiment showed that both EPA-pPE and EPA-PE could relieve cell death and improve the cellular morphology of neurons via upregulating anti-apoptotic proteins and downregulating pro-apoptotic proteins. The in vivo experiment showed that EPA-pPE exerted more significant effects than EPA-PE in improving the number of neuronal Nissl bodies, increasing the branching of dendrites and dendritic spine density in cortical neurons, as well as improving the expression of synaptic vesicle-related proteins synaptophysin (SYN) and PSD95 via BDNF/TrkB/CREB signaling. These results indicated that EPA-pPE exerted neuroprotection at least partly through inhibiting neuronal apoptosis and enhancing the BDNF/TrkB/CREB pathway, which suggests that EPA-enriched plasmalogen can be explored as a potential therapeutic agent in long-term Alzheimer's disease therapy.

Astaxanthin Inhibits Mitochondrial Permeability Transition Pore Opening in Rat Heart Mitochondria

The mitochondrion is the main organelle of oxidative stress in cells. Increased permeability of the inner mitochondrial membrane is a key phenomenon in cell death. Changes in membrane permeability result from the opening of the mitochondrial permeability transition pore (mPTP), a large-conductance channel that forms after the overload of mitochondria with Ca2+ or in response to oxidative stress. The ketocarotenoid astaxanthin (AST) is a potent antioxidant that is capable of maintaining the integrity of mitochondria by preventing oxidative stress. In the present work, the effect of AST on the functioning of mPTP was studied. It was found that AST was able to inhibit the opening of mPTP, slowing down the swelling of mitochondria by both direct addition to mitochondria and administration. AST treatment changed the level of mPTP regulatory proteins in isolated rat heart mitochondria. Consequently, AST can protect mitochondria from changes in the induced permeability of the inner membrane. AST inhibited serine/threonine protein kinase B (Akt)/cAMP-responsive element-binding protein (CREB) signaling pathways in mitochondria, which led to the prevention of mPTP opening. Since AST improves the resistance of rat heart mitochondria to Ca2+-dependent stress, it can be assumed that after further studies, this antioxidant will be considered an effective tool for improving the functioning of the heart muscle in general under normal and medical conditions.

Hippocampus-specific regulation of long non-coding RNA and mRNA expression in germ-free mice.

Gut microbiota can affect multiple brain functions and cause behavioral alterations through the microbiota-gut-brain axis. In our previous study, we found that the absence of gut microbiota can influence the expression of microRNAs and mRNAs in the hippocampal region of the germ-free (GF) mice. Long non-coding RNAs (lncRNAs) are increasingly being recognized as an important functional transcriptional regulator in the brain. In the present study, we aim to identify possible biological pathways and functional networks for lncRNA-associated transcript of the gut microbiota in relation to the brain function. The profiles of lncRNA and mRNA from specific pathogen-free (SPF), colonized GF (CGF), and GF mice were generated using the Agilent Mouse LncRNA Array v2.0. Differentially expressed (DE) lncRNAs and mRNAs were identified, and lncRNA target genes were also predicted. Ingenuity pathway analysis (IPA) was performed to analyze related signaling pathways and biological functions associated with these dysregulated mRNAs and target genes. Validation with quantitative real-time PCR was performed on several key genes. Compared with SPF mice a total of 2230 DE lncRNAs were found in GF mice. Among these, 1355 were upregulated and 875 were downregulated. After comparing the target genes of DE lncRNAs with mRNA datasets, 669 overlapping genes were identified. IPA core analyses revealed that most of these genes were highly associated with cardiac hypertrophy, nuclear factors of activated T cells (NFAT) gonadotropin-releasing hormone (GnRH), calcium, and cAMP-response element-binding protein (CREB) signaling pathways. Additionally, mRNA expression levels of APP, CASP9, IGFBP2, PTGDS, and TGFBR2 genes that are involved in central nervous system functions were significantly changed in the GF mouse hippocampus. Through this study, for the first time, we describe the effect of gut microbiota on the hippocampal lncRNA regulation. This will help in enhancing the overall knowledge about microbiota-gut-brain axis.

Auricular electroacupuncture improves depression possibly by promoting hippocampal Raf/ERK/RSK/CREB signaling in chronic unpredictable mild stress induced depression rats

To observe the effect of auricular electroacupuncture (EA) on intracellular Raf/ extracellular signal-regulated kinase (ERK)/ ribosomal S6 kinase (RSK)/ cAMP response element-binding protein (CREB) signal pathway in the hippocampus of depression model rats, so as to explore its anti-depressive mechanism. A total of 60 male SD rats were randomly divided into control, model, auricular EA, PD98059(ERK inhibitor), DMSO (Dimethylsulfoxide), PD98059+EA groups (n=10 in each group). The rats in the control group were fed with normal diet without any treatment. The depression model was induced by chronic unpredictable mild stress (CUMS) for consecutive 21 days. EA (20 Hz, 2 mA) was applied to bilateral auricular "Xin"(Heart) and "Shenmen" for 30 min, once daily for 28 days. Rats of the PD98059, DMSO and PD98059+EA groups received intracerebroventricular injection of PD98059(100 µmol/L), DMSO and PD98059 (dissolved by DMSO) solutions (5 µL/d), respectively, once daily for 28 days. Sucrose preference test (sucrose consumption) was conducted at the baseline, before and after the intervention. The expression of hippocampal Raf, phosphorylated (p)-Raf, ERK, p-ERK, RSK, CREB and p-CREB proteins were detected by Western blot after EA intervention. Following modeling, the sucrose consumption volume, and the expression levels of hippocampal Raf, p-Raf, ERK, p-ERK, RSK, CREB, p-CREB proteins were significantly lower in the model group than those in the control group (P<0.01). Following the treatment, the sucrose consumption and the expression levels of Raf, p-Raf, ERK, p-ERK, RSK, CREB, p-CREB in the auricular EA group and those of p-Raf, ERK and CREB in the PD98059+EA group were obviously increased in comparison with the model group (P<0.05, P<0.01), the expression level of p-ERK in the PD98059 group was obviously decreased in comparison with the model group (P<0.05), suggesting an improvement of depression after auricular EA. Compared with auricular EA group, the expression level of p-ERK, p-CREB and RSK in the PD98059+EA group were significantly decreased (P<0.05). EA of auricular "Xin" and "Shenmen" is able to improve depression in depression rats, which is probably related to its effect in promoting activities of hippocampal Raf/ERK/RSK/CREB signaling.

Abstract WP564: Deep Immune Profiling of the Post-Stroke Peripheral Immune Response Reveals Tri-phasic Response and Correlations With Long-Term Cognitive Outcomes

Introduction: Stroke produces profound local and systemic immune responses that engage all major innate and adaptive immune compartments. The aim of this study was to characterize the systemic immune response to stroke and to determine if it contributes to long-term cognitive disability. Methods: We included 24 consecutive subjects with ischemic stroke and excluded patients for autoimmune disorders, use of immunosuppressant drugs, or life expectancy &lt;90 days. Blood samples were collected for up to 9 timepoints after stroke (days 1, 2, 3, 5, 7, 14, 30, 90, and 365). Change in cognitive function between days 90 and 365 was assessed using the Montreal Cognitive Assessment (MoCA). Control samples were from a cohort of 24 sex- and age-matched patients prior to hip replacement surgery. We used mass cytometry to acquire 240 immune features from each sample, representing 20 immune cell subtypes, their frequency, cell surface markers, and activation states. Elastic Net (EN) regularized regression modeling was used to characterize phases of the immune response and to correlate stages of the immune response with change in cognitive function. Results: The EN model identified three distinct phases of the systemic immune response to ischemic stroke: The acute phase (day 2) was characterized by increased STAT3 (signal transducer and activator of transcription 3) signaling responses in innate immune cell types. The intermediate phase (day 5) was characterized by increased CREB (cAMP response element-binding protein) signaling responses in adaptive immune cell types. The late phase (day 90) was characterized by persistent elevation of neutrophils and IgM+ B cells. By day 365 there was a return to baseline immune responses, comparable to the controls. A decline in MoCA scores between day 90 and day 365 after stroke correlated with a stronger inflammatory response in the acute phase (r = -0.692, Bonferroni-corrected p = 0.04). Conclusions: The results demonstrate three distinct phases of the peripheral immune response that occur after stroke, spanning from days 2 to day 90. The acute phase immune response predicts post-stroke cognitive decline, suggesting that therapies aimed at optimizing this response could lead to preservation of cognitive functioning post-stroke.

Kaempferide prevents cognitive decline via attenuation of oxidative stress and enhancement of brain-derived neurotrophic factor/tropomyosin receptor kinase B/cAMP response element-binding signaling pathway.

Kaempferide (KF) is a compound of flavonoids from Alpinae oxyphylla Miq, and the herb itself is used as a classical tonic agent. This paper aims to investigate the effects of KF on cognitive function impairment and neurodegeneration in the mouse model of Alzheimer's disease induced by intracerebroventricular (ICV) injection of Aβ1-42 . The mice were treated with KF at doses of 0.02 and 0.2mg/kg/day (ICV) for five consecutive days after Aβ1-42 exposures. The behavioral test results showed that KF could prevent cognitive decline in mice induced by Aβ1-42 as assessed by the locomotor activity test, Y-maze test, and Morris water maze test. Furthermore, the activities of superoxide dismutase and malondialdehyde in the hippocampus and cerebral cortex were elevated by KF administration. Results of hippocampus slices showed that neurons were integrated and regularly arranged in the groups, which were administered along with KF. In addition, we found KF could boost brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB)/cAMP response element-binding (CREB) protein signal in the hippocampus. All results illustrated that KF could exert neuroprotective effects at least partly through alleviating oxidative stress and enhancing the BDNF/TrkB/CREB pathway in Aβ1-42 -induced mice.

Serum orexin-A levels are associated with disease progression and motor impairment in multiple sclerosis

Diencephalon is frequently affected in multiple sclerosis (MS), and lesions of this region are associated with increased disability. Orexin-A and melatonin, two foremost mediators of diencephalon, modulate cognitive and motor functions through several pathways including the brain-derived neurotrophic factor (BDNF)-cAMP response element-binding protein (CREB) signaling pathway. In this pilot study, our aim was to investigate the prognostic value of these factors in progression of cognitive and physical disability. Levels of BDNF, melatonin, CREB, and orexin-A were determined by ELISA in sera of 25 relapsing remitting MS (RRMS) patients, 15 secondary progressive MS (SPMS) patients, and 20 healthy controls. Cognitive and motor functions were assessed by a neuropsychological test battery, timed 25-ft walk (T25-FW) and 9-hole peg (9-HP) tests. MS patients had significantly lower serum levels of orexin-A and BDNF than healthy controls, and SPMS patients had significantly lower levels of melatonin and orexin-A than RRMS patients. Serum orexin-A levels were negatively correlated with 9-HP, T25-FW test scores, and progression index in RRMS patients. BDNF, CREB, and melatonin levels did not show any significant correlation with clinical features including EDSS and cognitive/motor performance of the patients. Our results suggest that orexin-A levels are decreased in parallel to disease progression and motor system deterioration in the earlier stages of the disease. Thus, orexin-A might be used as a potential biomarker of physical disability.

Identification of Anti-Melanogenesis Constituents from Morus alba L. Leaves.

The individual parts of Morus alba L. including root bark, branches, leaves, and fruits are used as a cosmetic ingredient in many Asian countries. This study identified several anti-melanogenesis constituents in a 70% ethanol extract of M. alba leaves. The ethyl acetate fraction of the initial ethanol extract decreased the activity of tyrosinase, a key enzyme in the synthetic pathway of melanin. Twelve compounds were isolated from this fraction and their structures were identified based on spectroscopic spectra. Then, the authors investigated the anti-melanogenesis effects of the isolated compounds in B16-F10 mouse melanoma cells. Compounds 3 and 8 significantly inhibited not only melanin production but also intracellular tyrosinase activity in alpha-melanocyte-stimulating-hormone (α-MSH)-induced B16-F10 cells in a dose-dependent manner. These same compounds also inhibited melanogenesis-related protein expression such as microphthalmia-associated transcription factor (MITF), tyrosinase, and tyrosinase-related protein-1 (TRP-1). Compound 3 modulated the cAMP-responsive element-binding protein (CREB) and p38 signaling pathways in α-MSH-activated B16-F10 melanoma cells, which resulted in the anti-melanogenesis effects. These results suggest that compound 3, isolated from M. alba leaves, could be used to inhibit melanin production via the regulation of melanogenesis-related protein expression.

Early-Life Neuronal-Specific Iron Deficiency Alters the Adult Mouse Hippocampal Transcriptome

Iron deficiency (ID) compromises the developing nervous system, including the hippocampus, resulting in later-life deficits despite iron repletion. The iron-dependent molecular changes driving these lasting deficits, and the effect of early iron repletion, are incompletely understood. Previous studies have utilized dietary models of maternal-fetal ID anemia (IDA) to address these questions; however, concurrent anemia prevents delineation of the specific role of iron. The aim of the study was to isolate the effects of developmental ID on adult hippocampal gene expression and to determine if iron repletion reverses these effects in a mouse model of nonanemic hippocampal neuronal ID. Nonanemic, hippocampus-specific neuronal ID was generated by using a Tet-OFF dominant negative transferrin receptor (DN-TFR1) mouse model that impairs cellular iron uptake. Hippocampal ID was reversed with doxycycline at postnatal day 21 (P21) in a subset of mice to create 2 experimental groups, chronically iron-deficient and formerly iron-deficient mice, which were compared with their respective doxycycline-treated and untreated iron-sufficient controls. RNA from adult male hippocampi was sequenced. Paired-end reads were analyzed for differential expression. Differentially expressed genes were analyzed in Ingenuity Pathway Analysis. A total of 346 genes were differentially expressed in adult, chronically iron-deficient hippocampi compared with controls. ID dysregulated genes in critical neurodevelopmental pathways, including axonal guidance, CDK5, Ephrin receptor, Rac, and Neurotrophin/Trk signaling. Iron repletion at P21 normalized adult hippocampal expression of 198 genes; however, genes involved in cAMP response element-binding protein (CREB) signaling, neurocognition, and neurologic disease remained dysregulated in adulthood. Chronic ID during development, independent of anemia, alters the adult mouse hippocampal transcriptome. Restoring iron status during a known critical period of hippocampal neurodevelopment incompletely normalized these changes, suggesting a need for additional studies to identify the most effective timeline for iron therapy, and adjunctive treatments that can fully restore ID-induced molecular changes, particularly in human populations in whom chronic ID is endemic.

Pulsed electromagnetic fields promote bone formation by activating the sAC-cAMP-PKA-CREB signaling pathway.

The application of pulsed electromagnetic fields (PEMFs) in the prevention and treatment of osteoporosis has long been an area of interest. However, the clinical application of PEMFs remains limited because of the poor understanding of the PEMF action mechanism. Here, we report that PEMFs promote bone formation by activating soluble adenylyl cyclase (sAC), cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), and cAMP response element-binding protein (CREB) signaling pathways. First, it was found that 50 Hz 0.6 millitesla (mT) PEMFs promoted osteogenic differentiation of rat calvarial osteoblasts (ROBs), and that PEMFs activated cAMP-PKA-CREB signaling by increasing intracellular cAMP levels, facilitating phosphorylation of PKA and CREB, and inducing nuclear translocation of phosphorylated (p)-CREB. Blocking the signaling by adenylate cyclase (AC) and PKA inhibitors both abolished the osteogenic effect of PEMFs. Second, expression of sAC isoform was found to be increased significantly by PEMF treatment. Blocking sAC using sAC-specific inhibitor KH7 dramatically inhibited the osteogenic differentiation of ROBs. Finally, the peak bone mass of growing rats was significantly increased after 2 months of PEMF treatment with 90 min/day. The serum cAMP content, p-PKA, and p-CREB as well as the sAC protein expression levels were all increased significantly in femurs of treated rats. The current study indicated that PEMFs promote bone formation in vitro and in vivo by activating sAC-cAMP-PKA-CREB signaling pathway of osteoblasts directly or indirectly.

Inhibition of Acid Sensing Ion Channel 3 Aggravates Seizures by Regulating NMDAR Function.

The existing data about whether acid sensing ion channels (ASICs) are proconvulsant or anticonvulsant are controversial. Particularly, acid sensing ion channel 3 (ASIC3) is the most sensitive to extracellular pH and has the characteristic ability to generate a biphasic current, but few studies have focused on the role of ASIC3 in seizure. Here we found ASIC3 expression was increased in the hippocampus of pilocarpine induced seizure rats, as well as in hippocampal neuronal cultures undergoing epileptiform discharge elicited by Mg2+-free media. Furthermore, ASIC3 blockade by the selective inhibitor APETx2 shortened seizure onset latency and increased seizure severity compared with the control in the pilocarpine induced seizure model. Incubation with APETx2 enhanced the excitability of primary cultured hippocampal neurons in Mg2+-free media. Notably, the aggravated seizure was associated with upregulation of the N-methyl-D-aspartate subtype of glutamate receptors (NMDARs), increased NMDAR mediated excitatory neurotransmission and subsequent activation of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) and cAMP-response element binding protein (CREB) signaling pathway. Moreover, co-immunoprecipitation confirmed the interaction between ASIC3 and NMDAR subunits, and NMDARs blockade prevented the aggravated seizure caused by ASIC3 inhibition. Taken together, our findings suggest that ASIC3 inhibition aggravates seizure and potentiates seizure induced hyperexcitability at least partly by the NMDAR/CaMKII/CREB signaling pathway, which implies that ASIC3 agonists may be a promising approach for seizure treatment.

Opiate-associated contextual memory formation and retrieval are differentially modulated by dopamine D1 and D2 signaling in hippocampal-prefrontal connectivity.

Contextual memory driven by abused drugs such as opiates has a central role in maintenance and relapse of drug-taking behaviors. Although dopamine (DA) signaling favors memory storage and retrieval via regulation of hippocampal-prefrontal connectivity, its role in modulating opiate-associated contextual memory is largely unknown. Here, we report roles of DA signaling within the hippocampal-prefrontal circuit for opiate-related memories. Combining-conditioned place preference (CPP) with molecular analyses, we investigated the DA D1 receptor (D1R) and extracellular signal-regulated kinase (ERK)-cAMP-response element binding protein (CREB) signaling, as well as DA D2 receptor (D2R) and protein kinase B (PKB or Akt)/glycogen synthase kinase 3 (GSK3) signaling in the ventral hippocampus (vHip) and medial prefrontal cortex (mPFC) during the formation of opiate-related associative memories. Morphine-CPP acquisition increased the activity of the D1R-ERK-CREB pathway in both the vHip and mPFC. Morphine-CPP reinstatement was associated with the D2R-mediated hyperactive GSK3 via Akt inhibition in the vHip and PFC. Furthermore, integrated D1R-ERK-CREB and D2R-Akt-GSK3 pathways in the vHip-mPFC circuit are required for the acquisition and retrieval of the morphine contextual memory, respectively. Moreover, blockage of D1R or D2R signaling could alleviate normal Hip-dependent spatial memory. These results suggest that D1R and D2R signaling are differentially involved in the acquisition and retrieval of morphine contextual memory, and DA signaling in the vHip-mPFC connection contributes to morphine-associated and normal memory, largely depending on opiate exposure states.

Granulosa cells from human primordial and primary follicles show differential global gene expression profiles.

Can novel genetic candidates involved in follicle dormancy, activation and integrity be identified from transcriptomic profiles of isolated granulosa cells from human primordial and primary follicles? The granulosa cell compartment of the human primordial and primary follicle was extensively enriched in signal transducer and activator of transcription 3 (STAT3) and cAMP-response element binding protein (CREB) signalling, and several other putative signalling pathways that may also be mediators of follicle growth and development were identified. Mechanistic target of rapamycin kinase (mTOR) signalling and the factors Forkhead Box L2 (FOXL2) and KIT proto-oncogene receptor tyrosine kinase (KITL) may be involved in defining the early steps of mammalian follicular recruitment through complex bidirectional signalling between the oocyte and granulosa cells. cAMP/protein kinase K (PKA)/CREB signalling is a feature of FSH-induced regulation of granulosa cell steroidogenesis that is essential to normal human fertility. A class comparison study was carried out on primordial follicles (n = 539 follicles) and primary follicles (n = 261) follicles) donated by three women having ovarian tissue cryopreserved before chemotherapy. RNA samples from isolates of laser capture micro-dissected oocytes and follicles from the primordial and primary stage, respectively, were sequenced on the HiSeq Illumina platform. Data mapping, quality control, filtering, FPKM (fragments per kilobase of exon per million) normalization and comparisons were performed. The granulosa cell contribution in whole follicle isolates was extracted in silico. Modelling of complex biological systems was performed using Ingenuity Pathway Analysis (IPA). For validation of transcriptomic findings, we performed quantitative RT-PCR of selected candidate genes. Furthermore, we interrogated the in situ localization of selected corresponding proteins using immunofluorescence. Our differentially expressed gene analysis revealed a number of transcripts in the granulosa cells to be significantly down- (736 genes) or up- (294 genes) regulated during the human primordial-to-primary follicle transition. The IPA analysis revealed enriched canonical signalling pathways not previously associated with granulosa cells from human primordial and primary follicles. Immunofluorescent staining of human ovarian tissue explored the intra-ovarian localization of FOG2, and FOXL2, which revealed the presence of forkhead box L2 (FOXL2) in both oocytes and granulosa cells in primary follicles, with a more enriched staining in the granulosa cells in primary follicles. Friend of GATA 2 (FOG2) stained strongly in oocytes in primordial follicles, with a shift towards granulosa cell as follicle stage advanced. http://users-birc.au.dk/biopv/published_data/ernst_et_al_GC_2017/. This is a descriptive study, and no functional assays were employed. The study was based on a limited number of patients, and it is acknowledged that natural biological variance exists in human samples. Strict filters were applied to accommodate the in silico extraction of the granulosa cell contribution. In support of this, quantitative RT-PCR was used to confirm selected candidate genes, and immunofluorescent staining was employed to interrogate the intra-ovarian distribution of selected corresponding proteins. Moreover, it is unknown whether the primordial follicles analysed represent those still in the resting pool, or those from the cohort that have entered the growing pool. We present, for the first time, a detailed description of global gene activity in the human granulosa cell compartment of primordial and primary follicles. These results may be utilized in the development of novel clinical treatment strategies aimed at improving granulosa cell function. E.H.E. was supported by the Health Faculty, Aarhus University and Kong Christian Den Tiendes Fond. K.L.H. was supported by a grant from Fondens til Lægevidenskabens Fremme and Kong Christian Den Tiendes Fond. No authors have competing interests to declare.

The Differential Effects of Repetitive Magnetic Stimulation in an In Vitro Neuronal Model of Ischemia/Reperfusion Injury.

Repetitive transcranial magnetic stimulation (rTMS) is a non-invasive therapy that has been implicated in treatment of serious neurological disorders. However, the neurobiological mechanisms underlying the effects of rTMS remain unclear. Therefore, this study examined the differential effects of repetitive magnetic stimulation (rMS) in an in vitro neuronal model of ischemia/reperfusion (I/R) injury, depending on low and high frequency. Neuro-2a cells were differentiated with retinoic acid and established for in vitro neuronal model of I/R injury under a subsequent 3 h of oxygen and glucose deprivation/reoxygenation (OGD/R) condition. After the I/R injury, the differentiated neuronal cells were stimulated with rMS on day 1 and randomly divided into three groups: OGD/R+sham, OGD/R+low-frequency, and OGD/R+high-frequency groups. High-frequency rMS increases cell proliferation through activation of extracellular signal-regulated kinases and AKT-signaling pathway and inhibits apoptosis in OGD/R-injured cells. Furthermore, high-frequency rMS increases Ca2+–calmodulin-dependent protein kinase II (CaMKII)-cAMP-response element binding protein (CREB) signaling pathway, further leading to alternation of brain-derived neurotrophic factor expression and synaptic plasticity in OGD/R injured cells. These results verified the neurobiological mechanisms of frequency-dependent rMS in I/R injury-treated neuronal cells. These mechanisms will help develop more powerful and credible rTMS stimulation treatment protocols.