Hetong decoction relieves loperamide-induced constipation in rats by regulating expression of aquaporins.

OBJECTIVE: To investigate whether Hetong decoction (, HTT) alleviates constipation via regulating AQPs expression. METHODS: Constipation in rats was induced by loperamide, and rats were randomly assigned into model (saline), HHT-low (95 g/kg), HTT-medium (190 g/kg), HTT-high (380 g/kg) and positive control (mosapride) groups. Then the defecation function, the concentration of serum arginine vasopressin (AVP) and cyclic adenosine monophosphate (cAMP), and the expression of AQP3 and AQP8 in colon tissues were assessed. NCM460 colon cells with AQP3 and AQP8 knockdown or overexpression were exposed to serum from rats that received low or high dose of HTT, followed by detection of AQP3 and AQP8 expression. RESULTS: The model group showed lower fecal weight and water content, weaker intestinal transit, higher serum concentration of AVP and cAMP, increased proximal and distal AQP8 expression, increased proximal but decreased distal AQP3 expression. However, these trends were reversed in both the HTT group (low, medium and high dose) and the positive control group. In NCM460 cells, HTT dose-dependently stabilized AQP3 and AQP8 expression under AQP3/8 plasmid interference or overexpression. CONCLUSIONS: HTT relieves constipation in rats through regulating AQP3 and AQP8 expression.

The psychosis risk factor RBM12 encodes a novel repressor of GPCR/cAMP signal transduction.

RBM12 is a high-penetrance risk factor for familial schizophrenia and psychosis, yet its precise cellular functions and the pathways to which it belongs are not known. We utilize two complementary models, HEK293 cells and human iPSC-derived neurons, and delineate RBM12 as a novel repressor of the G protein-coupled receptor/cAMP/PKA (GPCR/cAMP/PKA) signaling axis. We establish that loss of RBM12 leads to hyperactive cAMP production and increased PKA activity as well as altered neuronal transcriptional responses to GPCR stimulation. Notably, the cAMP and transcriptional signaling steps are subject to discrete RBM12-dependent regulation. We further demonstrate that the two RBM12 truncating variants linked to familial psychosis impact this interplay, as the mutants fail to rescue GPCR/cAMP signaling hyperactivity in cells depleted of RBM12. Lastly, we present a mechanism underlying the impaired signaling phenotypes. In agreement with its activity as an RNA-binding protein, loss of RBM12 leads to altered gene expression, including that of multiple effectors of established significance within the receptor pathway. Specifically, the abundance of adenylyl cyclases, phosphodiesterase isoforms, and PKA regulatory and catalytic subunits is impacted by RBM12 depletion. We note that these expression changes are fully consistent with the entire gamut of hyperactive signaling outputs. In summary, the current study identifies a previously unappreciated role for RBM12 in the context of the GPCR-cAMP pathway that could be explored further as a tentative molecular mechanism underlying the functions of this factor in neuronal physiology and pathophysiology.

CREB-associated glycosylation and function in human disease.

The shortcomings of mRNA sequencing in explaining biological functions have resulted in proteomics gradually becoming a hotspot for research. However, the function of proteins becomes complicated as a result of post-translational modifications (PTMs) such as phosphorylation, glycosylation, acetylation, etc. Post-translational modifications do not change the physicochemical properties such as charge and solubility of the proteins, but they can have significant consequences on disease initiation in living organisms. The cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB) is an important transcription regulator in eukaryotic cells. It is involved in the development of neurodegenerative diseases, diabetic complications, tumorigenesis, and neurogenesis. Previously, researchers have paid much more attention to the phosphorylation modification of CREB. However, it seems that the functional regulation-mediated glycosylation modification of CREB was just beginning to be understood. In this review, the current studies and most updated insights on how the glycosylation modification of CREB affects targeted gene expression and disease development will be comprehensively discussed. We hope to further evaluate the role of CREB glycosylation on the regulation of gene function.

PaOctß2R: Identification and Functional Characterization of an Octopamine Receptor Activating Adenylyl Cyclase Activity in the American Cockroach Periplaneta americana.

Biogenic amines constitute an important group of neuroactive substances that control and modulate various neural circuits. These small organic compounds engage members of the guanine nucleotide-binding protein coupled receptor (GPCR) superfamily to evoke specific cellular responses. In addition to dopamine- and 5-hydroxytryptamine (serotonin) receptors, arthropods express receptors that are activated exclusively by tyramine and octopamine. These phenolamines functionally substitute the noradrenergic system of vertebrates Octopamine receptors that are the focus of this study are classified as either α- or ß-adrenergic-like. Knowledge on these receptors is scarce for the American cockroach (Periplaneta americana). So far, only an α-adrenergic-like octopamine receptor that primarily causes Ca2+ release from intracellular stores has been studied from the cockroach (PaOctα1R). Here we succeeded in cloning a gene from cockroach brain tissue that encodes a ß-adrenergic-like receptor and leads to cAMP production upon activation. Notably, the receptor is 100-fold more selective for octopamine than for tyramine. A series of synthetic antagonists selectively block receptor activity with epinastine being the most potent. Bioinformatics allowed us to identify a total of 19 receptor sequences that build the framework of the biogenic amine receptor clade in the American cockroach. Phylogenetic analyses using these sequences and receptor sequences from model organisms showed that the newly cloned gene is an ß2-adrenergic-like octopamine receptor. The functional characterization of PaOctß2R and the bioinformatics data uncovered that the monoaminergic receptor family in the hemimetabolic P. americana is similarly complex as in holometabolic model insects like Drosophila melanogaster and the honeybee, Apis mellifera. Thus, investigating these receptors in detail may contribute to a better understanding of monoaminergic signaling in insect behavior and physiology.

Cloning, expression, solubilization, and purification of a functionally active recombinant cAMP-dependent protein kinase catalytic subunit-like protein PKAC1 from Trypanosoma equiperdum.

The gene encoding the cAMP-dependent protein kinase (PKA) catalytic subunit-like protein PKAC1 from the Venezuelan TeAp-N/D1 strain of Trypanosoma equiperdum was cloned, and the recombinant TeqPKAC1 protein was overexpressed in bacteria. A major polypeptide with an apparent molecular mass of ∼38 kDa was detected by SDS-polyacrylamide gel electrophoresis, and immunoblotting using antibodies against the human PKA catalytic subunit α. Unfortunately, most of the expressed TeqPKAC1 was highly insoluble. Polypeptides of 36-38 kDa and 45-50 kDa were predominantly seen by immunoblotting in the bacterial particulate and cytosolic fractions, respectively. Since the incorporation of either 4% Triton X-100 or 3% sarkosyl or a mixture of 10 mM MgCl2 and 1 mM ATP (MgATP) improved the solubilization of TeqPKAC1, we used a combination of Triton X-100, sarkosyl and MgATP to solubilize the recombinant protein. TeqPKAC1 was purified by first reconstituting a hybrid holoenzyme between the recombinant protein and a mammalian poly-His-tagged PKA regulatory subunit that was immobilized on a Ni2+-chelating affinity resin, and then by eluting TeqPKAC1 using cAMP. TeqPKAC1 was functional given that it was capable of phosphorylating PKA catalytic subunit substrates, such as kemptide (LRRASLG), histone type II-AS, and the peptide SP20 (TTYADFIASGRTGRRNSIHD), and was inhibited by the peptide IP20 (TTYADFIASGRTGRRNAIHD), which contains the inhibitory motif of the PKA-specific heat-stable inhibitor PKI-α. Optimal enzymatic activity was obtained at 37 °C and pH 8.0-9.0; and the order of effectiveness of nucleotide triphosphates and divalent cations was ATP ¼ GTP â‰… ITP and Mg2+ â‰… Mn2+ â‰… Fe2+ ¼ Ca2+ â‰… Zn2, respectively.

"ADPKD-omics": determinants of cyclic AMP levels in renal epithelial cells.

The regulation of cyclic adenosine monophosphate (cAMP) levels in kidney epithelial cells is important in at least 2 groups of disorders, namely water balance disorders and autosomal dominant polycystic kidney disease. Focusing on the latter, we review genes that code for proteins that are determinants of cAMP levels in cells. We identify which of these determinants are expressed in the 14 kidney tubule segments using recently published RNA-sequencing and protein mass spectrometry data ("autosomal dominant polycystic kidney disease-omics"). This includes G protein-coupled receptors, adenylyl cyclases, cyclic nucleotide phosphodiesterases, cAMP transporters, cAMP-binding proteins, regulator of G protein-signaling proteins, G protein-coupled receptor kinases, arrestins, calcium transporters, and calcium-binding proteins. In addition, compartmentalized cAMP signaling in the primary cilium is discussed, and a specialized database of the proteome of the primary cilium of cultured "IMCD3" cells is provided as an online resource (https://esbl.nhlbi.nih.gov/Databases/CiliumProteome/). Overall, this article provides a general resource in the form of a curated list of proteins likely to play roles in determination of cAMP levels in kidney epithelial cells and, therefore, likely to be determinants of progression of autosomal dominant polycystic kidney disease.

Senkyunolide H protects PC12 cells from OGD/R-induced injury via cAMP-PI3K/AKT signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Senkyunolide H (SNH) is a bioactive phthalide isolated from Ligusticum chuanxiong Hort rhizome and was reported to have multiple pharmacological effects. AIM OF THE STUDY: The study was performed to verify the potency of SNH protecting PC12 cells from oxygen glucose deprivation/reperfusion (OGD/R)-induced injury and to elucidate the underlying mechanisms. MATERIALS AND METHODS: OGD/R model was established in PC12 cells and the cell viability was measured by MTT assay. The cell morphology was observed using scanning electron microscope (SEM). The potential targets of SNH and related targets of OGD/R were screened, and a merged protein-protein interaction (PPI) network of SNH and OGD/R was constructed based on the network pharmacology analysis. Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used for pathway analysis. Intracellular cAMP level and the protein expression levels were measured to elucidate the underlying mechanisms. RESULTS: SNH pretreatment protected PC12 cells against OGD/R-induced cell death. SNH also significantly protected the cell protrusion. A merged PPI network was constructed and the shared candidate targets significantly enriched in cAMP signaling pathway. The level of intracellular cAMP and the protein level of p-CREB, p-AKT, p-PDK1 and PKA protein were up-regulated after the treatment of SNH compared with OGD/R modeling. CONCLUSIONS: The present study indicated that SNH protected PC12 cells from OGD/R-induced injury via cAMP-PI3K/AKT signaling pathway.

Crosstalk between protein kinase A and the HOG pathway under impaired biosynthesis of complex sphingolipids in budding yeast.

Complex sphingolipids are important components of the lipid bilayer of budding yeast Saccharomyces cerevisiae, and a defect of the biosynthesis causes widespread cellular dysfunction. In this study, we found that mutations causing upregulation of the cAMP/protein kinase A (PKA) pathway cause hypersensitivity to the defect of complex sphingolipid biosynthesis caused by repression of AUR1 encoding inositol phosphorylceramide synthase, whereas loss of PKA confers resistance to the defect. Loss of PDE2 encoding cAMP phosphodiesterase or PKA did not affect the reduction in complex sphingolipid levels and ceramide accumulation caused by AUR1 repression, suggesting that the change in sensitivity to the AUR1 repression due to the mutation of the cAMP/PKA pathway is not caused by exacerbation or suppression of the abnormal metabolism of sphingolipids. We also identified PBS2 encoding MAPKK in the high-osmolarity glycerol (HOG) pathway as a multicopy suppressor gene that rescues the hypersensitivity to AUR1 repression caused by deletion of IRA2, which causes hyperactivation of the cAMP/PKA pathway. Since the HOG pathway has been identified as one of the rescue systems against the growth defect caused by the impaired biosynthesis of complex sphingolipids, it was assumed that PKA affects activation of the HOG pathway under AUR1-repressive conditions. Under AUR1-repressive conditions, hyperactivation of PKA suppressed the phosphorylation of Hog1, MAPK in the HOG pathway, and transcriptional activation downstream of the HOG pathway. These findings suggested that PKA is possibly involved in the avoidance of excessive activation of the HOG pathway under impaired biosynthesis of complex sphingolipids.

Phosphodiesterase 8A Regulates CFTR Activity in Airway Epithelial Cells.

BACKGROUND/AIMS: Cystic fibrosis transmembrane conductance regulator (CFTR), the anion channel that is defective in cystic fibrosis (CF), is phosphorylated and activated by cAMP-dependent protein kinase (PKA). cAMP levels are downregulated by a large family of phosphodiesterases that have variable expression in different cell types. We have previously observed high levels of PDE8A expression in well-differentiated primary human bronchial epithelial (pHBE) cells and thus aimed to assess whether it played a role in cAMP-dependent regulation of CFTR activity. METHODS: We assessed the effect of the selective PDE8 inhibitor PF-04957325 (PF) on intracellular cAMP levels ([cAMP]i) in well differentiated pHBE cells from non-CF or CF donors and also in CFBE41o- cells that stably express wild-type CFTR (CFBE41o- WT) using ELISA and FRET-FLIM microscopy. CFTR channel function was also measured using electrophysiological recordings from pHBE and CFBE41o- WT cells mounted in Ussing Chambers. RESULTS: PDE8 inhibition elevated [cAMP]i in well-differentiated pHBE cells and stimulated wild-type CFTR-dependent ion transport under basal conditions or after cells had been pre-stimulated with physiological cAMP-elevating agents. The response to PDE8 inhibition was larger than to PDE3 or PDE5 inhibition but smaller and synergistic with that elicited by PDE4 inhibition. CRISPR Cas9-mediated knockdown of PDE8A enhanced CFTR gene and protein expression yet reduced the effect of PDE8 inhibition. Acute pharmacological inhibition PDE8 increased CFTR activity in CF pHBE cells (F508del/F508del and F508del/R117H-5T) treated with clinically-approved CFTR modulators. CONCLUSION: These results provide the first evidence that PDE8A regulates CFTR and identifies PDE8A as a potential target for adjunct therapies to treat CF.

Resveratrol Treatment in Human Parkin-Mutant Fibroblasts Modulates cAMP and Calcium Homeostasis Regulating the Expression of Mitochondria-Associated Membranes Resident Proteins.

Parkin plays an important role in ensuring efficient mitochondrial function and calcium homeostasis. Parkin-mutant human fibroblasts, with defective oxidative phosphorylation activity, showed high basal cAMP level likely ascribed to increased activity/expression of soluble adenylyl cyclase and/or low expression/activity of the phosphodiesterase isoform 4 and to a higher Ca2+ level. Overall, these findings support the existence, in parkin-mutant fibroblasts, of an abnormal Ca2+ and cAMP homeostasis in mitochondria. In our previous studies resveratrol treatment of parkin-mutant fibroblasts induced a partial rescue of mitochondrial functions associated with stimulation of the AMPK/SIRT1/PGC-1α pathway. In this study we provide additional evidence of the potential beneficial effects of resveratrol inducing an increase in the pre-existing high Ca2+ level and remodulation of the cAMP homeostasis in parkin-mutant fibroblasts. Consistently, we report in these fibroblasts higher expression of proteins implicated in the tethering of ER and mitochondrial contact sites along with their renormalization after resveratrol treatment. On this basis we hypothesize that resveratrol-mediated enhancement of the Ca2+ level, fine-tuned by the ER-mitochondria Ca2+ crosstalk, might modulate the pAMPK/AMPK pathway in parkin-mutant fibroblasts.

Enhanced ATP and antioxidant levels for cAMP biosynthesis by Arthrobacter sp. CCTCC 2013431 with polyphosphate addition.

OBJECTIVES: When citrate and pyruvate were utilized to strengthen ATP generation for high cAMP production, oxidative stress became more severe in cells resulting in lower cell viability and cAMP formation at the late fermentation phase. To further improve cAMP biosynthesis, the effects of polyphosphate on cAMP fermentation performance together with intracellular ATP and oxidation levels were investigated under high oxidative stress condition and then high efficient cAMP fermentation process based on polyphosphate and salvage synthesis was developed and studied. RESULTS: With 2 g/L-broth sodium hexametaphosphate added at 24 h was determined as the optimal condition for cAMP production by Arthrobacter sp. CCTCC 2013431 in shake flasks. Under high oxidative stress condition caused by adding 15 mg/L-broth menadione, cAMP contents and cell viability were improved greatly due to hexametaphosphate addition and also exceeded those of control (without hexametaphosphate and menadione added) when fermentations were conducted in a 7 L bioreactor. Meanwhile, ATP levels and antioxidant capacity were improved obviously by hexametaphosphate as well. Moreover, a fermentation process with hexametaphosphate and hypoxanthine coupling added was developed by which cAMP concentration reached 7.25 g/L with an increment of 87.1% when compared with only hypoxanthine added batch and the high ROS contents generated from salvage synthesis were reduced significantly. CONCLUSION: Polyphosphate could improve intracellular ATP levels and antioxidant capacity significantly under high oxidative stress condition resulting in enhanced cell viability and cAMP fermentation production no matter by de novo synthesis or salvage synthesis.

ß-Agonist exposure preferentially impacts lung macrophage cyclic AMP-related gene expression in asthma and asthma COPD overlap syndrome.

Bronchoalveolar lavage (BAL) samples from Severe Asthma Research Program (SARP) patients display suppression of a module of genes involved in cAMP-signaling pathways (BALcAMP) correlating with severity, therapy, and macrophage constituency. We sought to establish if gene expression changes were specific to macrophages and compared gene expression trends from multiple sources. Datasets included single-cell RNA sequencing (scRNA-seq) from lung specimens including a fatal exacerbation of severe Asthma COPD Overlap Syndrome (ACOS) after intense therapy and controls without lung disease, bulk RNA sequencing from cultured macrophage (THP-1) cells after acute or prolonged ß-agonist exposure, SARP datasets, and data from the Immune Modulators of Severe Asthma (IMSA) cohort. THP monocytes suppressed BALcAMP network gene expression after prolonged relative to acute ß-agonist exposure, corroborating SARP observations. scRNA-seq from healthy and diseased lung tissue revealed 13 cell populations enriched for macrophages. In severe ACOS, BALcAMP gene network expression scores were decreased in many cell populations, most significantly for macrophage populations (P < 3.9e-111). Natural killer (NK) cells and type II alveolar epithelial cells displayed less robust network suppression (P < 9.2e-8). Alveolar macrophages displayed the most numerous individual genes affected and the highest amplitude of modulation. Key BALcAMP genes demonstrate significantly decreased expression in severe asthmatics in the IMSA cohort. We conclude that suppression of the BALcAMP gene module identified from SARP BAL samples is validated in the IMSA patient cohort with physiological parallels observed in a monocytic cell line and in a severe ACOS patient sample with effects preferentially localizing to macrophages.

Axonal CB1 Receptors Mediate Inhibitory Bouton Formation via cAMP Increase and PKA.

Experience-dependent formation and removal of inhibitory synapses are essential throughout life. For instance, GABAergic synapses are removed to facilitate learning, and strong excitatory activity is accompanied by the formation of inhibitory synapses to maintain coordination between excitation and inhibition. We recently discovered that active dendrites trigger the growth of inhibitory synapses via CB1 receptor-mediated endocannabinoid signaling, but the underlying mechanism remained unclear. Using two-photon microscopy to monitor the formation of individual inhibitory boutons in hippocampal organotypic slices from mice (both sexes), we found that CB1 receptor activation mediated the formation of inhibitory boutons and promoted their subsequent stabilization. Inhibitory bouton formation did not require neuronal activity and was independent of Gi/o-protein signaling, but was directly induced by elevating cAMP levels using forskolin and by activating Gs-proteins using DREADDs. Blocking PKA activity prevented CB1 receptor-mediated inhibitory bouton formation. Our findings reveal that axonal CB1 receptors signal via unconventional downstream pathways and that inhibitory bouton formation is triggered by an increase in axonal cAMP levels. Our results demonstrate an unexpected role for axonal CB1 receptors in axon-specific, and context-dependent, inhibitory synapse formation.SIGNIFICANCE STATEMENT Coordination between excitation and inhibition is required for proper brain function throughout life. It was previously shown that new inhibitory synapses can be formed in response to strong excitation to maintain this coordination, and this was mediated by endocannabinoid signaling via CB1 receptors. As activation of CB1 receptors generally results in the suppression of synaptic transmission, it remained unclear how CB1 receptors can mediate the formation of inhibitory synapses. Here we show that CB1 receptors on inhibitory axons signal via unconventional intracellular pathways and that inhibitory bouton formation is triggered by an increase in axonal cAMP levels and requires PKA activity. Our findings point to a central role for axonal cAMP signaling in activity-dependent inhibitory synapse formation.

Knockdown of a ß-Adrenergic-Like Octopamine Receptor Affects Locomotion and Reproduction of Tribolium castaneum.

The neurohormone octopamine regulates many crucial physiological processes in insects and exerts its activity via typical G-protein coupled receptors. The roles of octopamine receptors in regulating behavior and physiology in Coleoptera (beetles) need better understanding. We used the red flour beetle, Tribolium castaneum, as a model species to study the contribution of the octopamine receptor to behavior and physiology. We cloned the cDNA of a ß-adrenergic-like octopamine receptor (TcOctß2R). This was heterologously expressed in human embryonic kidney (HEK) 293 cells and was demonstrated to be functional using an in vitro cyclic AMP assay. In an RNAi assay, injection of dsRNA demonstrated that TcOctß2R modulates beetle locomotion, mating duration, and fertility. These data present some roles of the octopaminergic signaling system in T. castaneum. Our findings will also help to elucidate the potential functions of individual octopamine receptors in other insects.

Cellular Mechanisms of the Anti-Arrhythmic Effect of Cardiac PDE2 Overexpression.

BACKGROUND: Phosphodiesterases (PDE) critically regulate myocardial cAMP and cGMP levels. PDE2 is stimulated by cGMP to hydrolyze cAMP, mediating a negative crosstalk between both pathways. PDE2 upregulation in heart failure contributes to desensitization to ß-adrenergic overstimulation. After isoprenaline (ISO) injections, PDE2 overexpressing mice (PDE2 OE) were protected against ventricular arrhythmia. Here, we investigate the mechanisms underlying the effects of PDE2 OE on susceptibility to arrhythmias. METHODS: Cellular arrhythmia, ion currents, and Ca2+-sparks were assessed in ventricular cardiomyocytes from PDE2 OE and WT littermates. RESULTS: Under basal conditions, action potential (AP) morphology were similar in PDE2 OE and WT. ISO stimulation significantly increased the incidence of afterdepolarizations and spontaneous APs in WT, which was markedly reduced in PDE2 OE. The ISO-induced increase in ICaL seen in WT was prevented in PDE2 OE. Moreover, the ISO-induced, Epac- and CaMKII-dependent increase in INaL and Ca2+-spark frequency was blunted in PDE2 OE, while the effect of direct Epac activation was similar in both groups. Finally, PDE2 inhibition facilitated arrhythmic events in ex vivo perfused WT hearts after reperfusion injury. CONCLUSION: Higher PDE2 abundance protects against ISO-induced cardiac arrhythmia by preventing the Epac- and CaMKII-mediated increases of cellular triggers. Thus, activating myocardial PDE2 may represent a novel intracellular anti-arrhythmic therapeutic strategy in HF.

Impacts of OrX and cAMP-insensitive Orco to the insect olfactory heteromer activity.

Insect odorant receptors (ORs) have been suggested to function as ligand-gated cation channels, with OrX/Orco heteromers combining ionotropic and metabotropic activity. The latter is mediated by different G proteins and results in Orco self-activation by cyclic nucleotide binding. In this contribution, we co-express the odor-specific subunits DmOr49b and DmOr59b with either wild-type Orco or an Orco-PKC mutant lacking cAMP activation heterologously in mammalian cells. We show that the characteristics of heteromers strongly depend on both the OrX type and the coreceptor variant. Thus, methyl acetate-sensitive Or59b/Orco demonstrated 25-fold faster response kinetics over o-cresol-specific Or49b/Orco, while the latter required a 10-100 times lower ligand concentration to evoke a similar electrical response. Compared to wild-type Orco, Orco-PKC decreased odorant sensitivity in both heteromers, and blocked an outward current rectification intrinsic to the Or49b/Orco pair. Our observations thus provide an insight into insect OrX/Orco functioning, highlighting their natural and artificial tuning features and laying the groundwork for their application in chemogenetics, drug screening, and repellent design.

ADAM9 enhances Th17 cell differentiation and autoimmunity by activating TGF-ß1.

The a disintegrin and metalloproteinase (ADAM) family of proteinases alter the extracellular environment and are involved in the development of T cells and autoimmunity. The role of ADAM family members in Th17 cell differentiation is unknown. We identified ADAM9 to be specifically expressed and to promote Th17 differentiation. Mechanistically, we found that ADAM9 cleaved the latency-associated peptide to produce bioactive transforming growth factor ß1, which promoted SMAD2/3 phosphorylation and activation. A transcription factor inducible cAMP early repressor was found to bind directly to the ADAM9 promoter and to promote its transcription. Adam9-deficient mice displayed mitigated experimental autoimmune encephalomyelitis, and transfer of Adam9-deficient myelin oligodendrocyte globulin-specific T cells into Rag1-/- mice failed to induce disease. At the translational level, an increased abundance of ADAM9 levels was observed in CD4+ T cells from patients with systemic lupus erythematosus, and ADAM9 gene deletion in lupus primary CD4+ T cells clearly attenuated their ability to differentiate into Th17 cells. These findings revealed that ADAM9 as a proteinase provides Th17 cells with an ability to activate transforming growth factor ß1 and accelerates its differentiation, resulting in aberrant autoimmunity.

Transcriptional signatures in prefrontal cortex confer vulnerability versus resilience to food and cocaine addiction-like behavior.

Addiction is a chronic relapsing brain disease characterized by compulsive reward-seeking despite harmful consequences. The mechanisms underlying addiction are orchestrated by transcriptional reprogramming in the reward system of vulnerable subjects. This study aims at revealing gene expression alterations across different types of addiction. We analyzed publicly available transcriptome datasets of the prefrontal cortex (PFC) from a palatable food and a cocaine addiction study. We found 56 common genes upregulated in the PFC of addicted mice in these two studies, whereas most of the differentially expressed genes were exclusively linked to either palatable food or cocaine addiction. Gene ontology analysis of shared genes revealed that these genes contribute to learning and memory, dopaminergic synaptic transmission, and histone phosphorylation. Network analysis of shared genes revealed a protein-protein interaction node among the G protein-coupled receptors (Drd2, Drd1, Adora2a, Gpr6, Gpr88) and downstream targets of the cAMP signaling pathway (Ppp1rb1, Rgs9, Pde10a) as a core network in addiction. Upon extending the analysis to a cell-type specific level, some of these common molecular players were selectively expressed in excitatory neurons, oligodendrocytes, and endothelial cells. Overall, computational analysis of publicly available whole transcriptome datasets provides new insights into the molecular basis of addiction-like behaviors in PFC.

GLP-1 improves the neuronal supportive ability of astrocytes in Alzheimer's disease by regulating mitochondrial dysfunction via the cAMP/PKA pathway.

The glucagon-like peptide-1 (GLP-1) was shown to have neuroprotective effects in Alzheimer's disease (AD). However, the underlying mechanism remains elusive. Astrocytic mitochondrial abnormalities have been revealed to constitute important pathologies. In the present study, we investigated the role of astrocytic mitochondria in the neuroprotective effect of GLP-1 in AD. To this end, 6-month-old 5 × FAD mice were subcutaneously treated with liraglutide, a GLP-1 analogue (25 nmol/kg/qd) for 8 weeks. Liraglutide ameliorated mitochondrial dysfunction and prevented neuronal loss with activation of the cyclic adenosine 3',5'-monophosphate (cAMP)/phosphorylate protein kinase A (PKA) pathway in the brain of 5 × FAD mice. Next, we exposed astrocytes to ß-amyloid (Aß) in vitro and treated them with GLP-1. By activating the cAMP/PKA pathway, GLP-1 increased the phosphorylation of DRP-1 at the s637 site and mitigated mitochondrial fragmentation in Aß-treated astrocytes. GLP-1 further improved the Aß-induced energy failure, mitochondrial reactive oxygen species (ROS) overproduction, mitochondrial membrane potential (MMP) collapse, and cell toxicity in astrocytes. Moreover, GLP-1 also promoted the neuronal supportive ability of Aß-treated astrocytes via the cAMP/PKA pathway. This study revealed a new mechanism behind the neuroprotective effect of GLP-1 in AD.

Psychotropic drugs upregulate aquaporin-2 via vasopressin-2 receptor/cAMP/protein kinase A signaling in inner medullary collecting duct cells.

Psychotropic drugs may be associated with hyponatremia, but an understanding of how they induce water retention in the kidney remains elusive. Previous studies have postulated that they may increase vasopressin production in the hypothalamus without supporting evidence. In this study, we investigated the possibility of drug-induced nephrogenic syndrome of inappropriate antidiuresis using haloperidol, sertraline, and carbamazepine. Haloperidol, sertraline, or carbamazepine were treated in inner medullary collecting duct (IMCD) suspensions and primary cultured IMCD cells prepared from male Sprague-Dawley rats. The responses of intracellular cAMP production, aquaporin-2 (AQP2) protein expression and localization, vasopressin-2 receptor (V2R) and AQP2 mRNA, and cAMP-responsive element-binding protein (CREB) were tested with and without tolvaptan and the protein kinase A (PKA) inhibitors H89 and Rp-cAMPS. In IMCD suspensions, cAMP production was increased by haloperidol, sertraline, or carbamazepine and was relieved by tolvaptan cotreatment. In primary cultured IMCD cells, haloperidol, sertraline, or carbamazepine treatment increased total AQP2 and decreased phosphorylated Ser261-AQP2 protein expression. Notably, these responses were reversed by cotreatment with tolvaptan or a PKA inhibitor. AQP2 membrane trafficking was induced by haloperidol, sertraline, or carbamazepine and was also blocked by cotreatment with tolvaptan or a PKA inhibitor. Furthermore, upregulation of V2R and AQP2 mRNA and phosphorylated CREB was induced by haloperidol, sertraline, or carbamazepine and was blocked by tolvaptan cotreatment. We conclude that, in the rat IMCD, psychotropic drugs upregulate AQP2 via V2R-cAMP-PKA signaling in the absence of vasopressin stimulation. The vasopressin-like action on the kidney appears to accelerate AQP2 transcription and dephosphorylate AQP2 at Ser261.NEW & NOTEWORTHY It is unclear whether antipsychotic drugs can retain water in the kidney in the absence of vasopressin. This study demonstrates that haloperidol, sertraline, and carbamazepine can produce nephrogenic syndrome of inappropriate antidiuresis because they directly upregulate vasopressin-2 receptor and aquaporin-2 (AQP2) via cAMP/PKA signaling. We showed that, in addition to AQP2 trafficking, AQP2 protein abundance was rapidly increased by treatment with antipsychotic drugs in association with dephosphorylation of AQP2 at Ser261 and accelerated AQP2 transcription.