ABSTRACT
Pain and anxiety are two common and undertreated non-motor symptoms in Parkinson's disease (PD), which affect the life quality of PD patients, and the underlying mechanisms remain unclear. As an important subtype of adenylyl cyclases (ACs), adenylyl cyclase subtype 1 (AC1) is critical for the induction of cortical long-term potentiation (LTP) and injury induced synaptic potentiation in the cortical areas including anterior cingulate cortex (ACC) and insular cortex (IC). Genetic deletion of AC1 or pharmacological inhibition of AC1 improved chronic pain and anxiety in different animal models. In this study, we proved the motor deficit, pain and anxiety symptoms of PD in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice model. As a lead candidate AC1 inhibitor, oral administration (1 dose and seven doses) of NB001 (20 and 40 mg/kg) showed significant analgesic effect in MPTP-treated mice, and the anxiety behavior was also reduced (40 mg/kg). By using genetic knockout mice, we found that AC1 knockout mice showed reduced pain and anxiety symptoms after MPTP administration, but not AC8 knockout mice. In summary, genetic deletion of AC1 or pharmacological inhibition of AC1 improved pain and anxiety symptoms in PD model mice, but didn't affect motor function. These results suggest that NB001 is a potential drug for the treatment of pain and anxiety symptoms in PD patients by inhibiting AC1 target.
Subject(s)
Adenylyl Cyclases , Anxiety , Disease Models, Animal , Mice, Inbred C57BL , Parkinson Disease , Animals , Adenylyl Cyclases/metabolism , Adenylyl Cyclases/genetics , Adenylyl Cyclases/deficiency , Anxiety/drug therapy , Anxiety/etiology , Male , Parkinson Disease/drug therapy , Parkinson Disease/complications , Parkinson Disease/pathology , Adenylyl Cyclase Inhibitors/pharmacology , Adenylyl Cyclase Inhibitors/therapeutic use , Mice , Pain/drug therapy , Pain/etiology , Calcium/metabolism , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/pharmacologyABSTRACT
Aim: Despite the enormous efforts to understand Congenital hyperinsulinism (CHI), up to 50% of the patients are genetically unexplained. We aimed to functionally characterize a novel candidate gene in CHI. Patient: A 4-month-old boy presented severe hyperinsulinemic hypoglycemia. A routine CHI genetic panel was negative. Methods: A trio-based whole-exome sequencing (WES) was performed. Gene knockout in the RIN-m cell line was established by CRISPR/Cas9. Gene expression was performed using real-time PCR. Results: Hyperinsulinemic hypoglycemia with diffuse beta-cell involvement was demonstrated in the patient, who was diazoxide-responsive. By WES, compound heterozygous variants were identified in the adenylyl cyclase 7, ADCY7 gene p.(Asp439Glu) and p.(Gly1045Arg). ADCY7 is calcium-sensitive, expressed in beta-cells and converts ATP to cAMP. The variants located in the cytoplasmic domains C1 and C2 in a highly conserved and functional amino acid region. RIN-m(-/-Adcy7) cells showed a significant increase in insulin secretion reaching 54% at low, and 49% at high glucose concentrations, compared to wild-type. In genetic expression analysis Adcy7 loss of function led to a 34.1-fold to 362.8-fold increase in mRNA levels of the insulin regulator genes Ins1 and Ins2 (p ≤ 0.0002), as well as increased glucose uptake and sensing indicated by higher mRNA levels of Scl2a2 and Gck via upregulation of Pdx1, and Foxa2 leading to the activation of the glucose stimulated-insulin secretion (GSIS) pathway. Conclusion: This study identified a novel candidate gene, ADCY7, to cause CHI via activation of the GSIS pathway.
Subject(s)
Adenylyl Cyclases/genetics , Congenital Hyperinsulinism/enzymology , Congenital Hyperinsulinism/metabolism , Insulin-Secreting Cells/metabolism , Insulin/metabolism , Adenylyl Cyclases/deficiency , Amino Acid Sequence , Animals , CRISPR-Cas Systems , Cell Line , Child, Preschool , Congenital Hyperinsulinism/genetics , Gene Knockout Techniques , Glucose/metabolism , Hepatocyte Nuclear Factor 3-beta/genetics , Hepatocyte Nuclear Factor 3-beta/metabolism , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Humans , Insulin/genetics , Insulin Secretion , Male , Rats , Sequence Alignment , Trans-Activators/genetics , Trans-Activators/metabolismABSTRACT
To investigate the influence of miR-18a-3p and ADCY5 on OP and osteogenic differentiation of human Mesenchymal stem cell (hBMSCs) and its possible mechanism. Samples were collected from osteoporotic patients with or without vertebral compression fracture, and without OP volunteers. MiR-18a-3p and ADCY5 mRNA expression levels in the tissue samples and hBMSCs during osteogenic differentiation were detectedãMiR-18a-3p mimic and OE-ADCY5 were introduced into hBMSCs to research the effects of miR-18a-3p and ADCY5 on osteogenesis differentiation of hBMSCs. Dual luciferase reporter system and RNA pull-down were applied to determine whether ADCY5 was a target gene of miR-18a-3p. Compared with the control group, ADCY5 expression level was down-regulated in patients with OP-no-Frx and OP-Frx, but that of miR-18a-3p was up-regulated. In addition, ADCY5 increased during osteogenesis differentiation of hBMSCs, whereas miR-18a-3p did not. OE-ADCY5 significantly facilitated calcium deposition, ALP activity, osteoblast protein expression (OSX, ALP and EUNX2), miR-18a-3p mimic inhibited osteogenic differentiation, and partially reversed the effect of OE-ADCY5 on osteogenic differentiation. In general, miR-18a-3p targets ADCY5 to promote OP and may be involved in spinal fracturs.
Subject(s)
Adenylyl Cyclases/deficiency , Adenylyl Cyclases/genetics , MicroRNAs/genetics , Osteogenesis/genetics , Osteoporosis/genetics , Osteoporosis/pathology , Spinal Fractures/genetics , Base Sequence , Calcium/metabolism , Genes, Reporter , Humans , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Osteoblasts/cytology , Osteoblasts/metabolism , Spinal Fractures/pathologyABSTRACT
Motile cilia are hairlike structures that line the respiratory and reproductive tracts and the middle ear and generate fluid flow in these organs via synchronized beating. Cilium growth is a highly regulated process that is assumed to be important for flow generation. Recently, Kif19a, a kinesin residing at the cilia tip, was identified to be essential for ciliary length control through its microtubule depolymerization function. However, there is a lack of information on the nature of proteins and the integrated signaling mechanism regulating growth of motile cilia. Here, we report that adenylate cyclase 6 (AC6), a highly abundant AC isoform in airway epithelial cells, inhibits degradation of Kif19a by inhibiting autophagy, a cellular recycling mechanism for damaged proteins and organelles. Using epithelium-specific knockout mice of AC6, we demonstrated that AC6 knockout airway epithelial cells have longer cilia compared with the WT cells because of decreased Kif19a protein levels in the cilia. We demonstrated in vitro that AC6 inhibits AMP-activated kinase (AMPK), an important modulator of cellular energy-conserving mechanisms, and uncouples its binding with ciliary kinesin Kif19a. In the absence of AC6, activation of AMPK mobilizes Kif19a into autophagosomes for degradation in airway epithelial cells. Lower Kif19a levels upon pharmacological activation of AMPK in airway epithelial cells correlated with elongated cilia and vice versa. In all, the AC6-AMPK pathway, which is tunable to cellular cues, could potentially serve as one of the crucial ciliary growth checkpoints and could be channeled to develop therapeutic interventions for cilia-associated disorders.
Subject(s)
Adenylyl Cyclases/metabolism , Cilia/physiology , Kinesins/metabolism , AMP-Activated Protein Kinases/antagonists & inhibitors , AMP-Activated Protein Kinases/metabolism , Adenylyl Cyclases/chemistry , Adenylyl Cyclases/deficiency , Adenylyl Cyclases/genetics , Animals , Autophagosomes/metabolism , Autophagy/drug effects , Autophagy-Related Protein 5/antagonists & inhibitors , Autophagy-Related Protein 5/genetics , Autophagy-Related Protein 5/metabolism , Chloroquine/pharmacology , Cilia/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Kinesins/antagonists & inhibitors , Kinesins/genetics , Male , Mice , Mice, Knockout , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Signal Transduction , Trachea/cytology , Trachea/metabolismABSTRACT
INTRODUCTION: Dyskinesia of the ADCY5 mutation is a rare movement-onset disorder in childhood. It is characterized by isolated chorea movements or associated with myoclonus and dystonia affecting the limbs, neck and face. The low number of patients and families still does not allow an adequate genotype-phenotype relationship. AIMS: The case of a child with movement disorders of early onset is presented in a family with three generations of affected members. An updated review of the casuistry and management of this rare disease is made. CASE REPORT: A 6-year-old boy referred for language delay and hyperactivity. After six months of follow-up he begins to show chorea movements of predominantly facial and limb roots, especially when waking up. At one year of follow-up, generalized chorea at rest with orofacial involvement and awkward gait begins to show. His family history includes his mother, grandfather, maternal uncle and cousin, who were diagnosed with Meige's syndrome (oromandibular dystonia and periorbital muscles) with choreiform-like movement disorders without affiliation since childhood. The brain study by MRI showed no alterations. A clinical exome targeting movement disorders was performed that discovered the pathogenic mutation in the ADCY5 gene causing autosomal familial dyskinesia. CONCLUSION: The c.1126G>A p.A376T mutation shows a natural history with a non-progressive clinical phenotype in three generations of affected members, with childhood debut and response to guanfacine treatment.
TITLE: Discinesia asociada a ADCY5 en la infancia: a propósito de una familia y revisión actualizada.Introducción. La discinesia de la mutación ADCY5 es un raro trastorno del movimiento de inicio en la infancia. Se caracteriza por movimientos coreicos aislados o asociados a mioclonías y distonías que afectan a las extremidades, el cuello y la cara. El escaso número de pacientes y familias no permite aún una adecuada relación genotipo-fenotipo. Objetivos. Presentar el caso de un niño con trastornos del movimiento de inicio precoz en el seno de una familia con tres generaciones de afectados, y realizar una revisión actualizada de la casuística y el tratamiento de esta rara enfermedad. Caso clínico. Varón de 6 años, remitido por retraso del lenguaje e hiperactividad. Tras seis meses de seguimiento, comenzó a presentar movimientos coreicos de predominio facial y de la raíz de los miembros, especialmente al despertar. Al año de seguimiento, se evidenció corea generalizado en reposo con afectación orofacial y torpeza en la marcha. Como antecedentes familiares destacaban su madre, abuelo, tío y prima maternos, que fueron diagnosticados de síndrome de Meige (distonía oromandibular y músculos periorbitarios) con trastornos del movimiento de tipo coreiforme sin filiar desde la infancia. El estudio cerebral por resonancia magnética no presentó alteraciones. Se realizó un exoma clínico dirigido a trastornos del movimiento que descubrió la mutación patógena en el gen ADCY5 causante de la discinesia familiar autosómica. Conclusión. La mutación c.1126G>A p.A376T muestra una historia natural con un fenotipo clínico no progresivo en tres generaciones de afectados, con inicio en la infancia y respuesta al tratamiento con guanfacina.
Subject(s)
Adenylyl Cyclases/deficiency , Movement Disorders/genetics , Adenylyl Cyclases/genetics , Adenylyl Cyclases/physiology , Amino Acid Substitution , Attention Deficit Disorder with Hyperactivity/drug therapy , Attention Deficit Disorder with Hyperactivity/genetics , Child , Developmental Disabilities/genetics , Drug Resistance , Female , Guanfacine/therapeutic use , Humans , Language Development Disorders/genetics , Levetiracetam/adverse effects , Male , Meige Syndrome/genetics , Mutation, Missense , Pedigree , Point MutationABSTRACT
The subunits KCNQ1 and KCNE1 generate the slowly activating, delayed rectifier potassium current, IKs, that responds to sympathetic stimulation and is critical for human cardiac repolarization. The A-kinase anchoring protein Yotiao facilitates macromolecular complex formation between IKs and protein kinase A (PKA) to regulate phosphorylation of KCNQ1 and IKs currents following beta-adrenergic stimulation. We have previously shown that adenylyl cyclase Type 9 (AC9) is associated with a KCNQ1-Yotiao-PKA complex and facilitates isoproterenol-stimulated phosphorylation of KCNQ1 in an immortalized cell line. However, requirement for AC9 in sympathetic control of IKs in the heart was unknown. Using a transgenic mouse strain expressing the KCNQ1-KCNE1 subunits of IKs, we show that AC9 is the only adenylyl cyclase (AC) isoform associated with the KCNQ1-KCNE1-Yotiao complex in the heart. Deletion of AC9 resulted in the loss of isoproterenol-stimulated KCNQ1 phosphorylation in vivo, even though AC9 represents less than 3% of total cardiac AC activity. Importantly, a significant reduction of isoproterenol-stimulated IKs currents was also observed in adult cardiomyocytes from IKs-expressing AC9KO mice. AC9 and Yotiao co-localize with N-cadherin, a marker of intercalated disks and cell-cell junctions, in neonatal and adult cardiomyocytes, respectively. In conclusion, AC9 is necessary for sympathetic regulation of PKA phosphorylation of KCNQ1 in vivo and for functional regulation of IKs in adult cardiomyocytes.
Subject(s)
Adenylyl Cyclases/metabolism , Isoproterenol/pharmacology , KCNQ1 Potassium Channel/metabolism , Myocytes, Cardiac/drug effects , Adenylyl Cyclases/deficiency , Animals , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Myocytes, Cardiac/metabolism , Phosphorylation/drug effectsABSTRACT
The neuropeptide of calcitonin gene-related peptide (CGRP) plays critical roles in chronic pain, especially in migraine. Immunohistochemistry and in situ hybridization studies have shown that CGRP and its receptors are expressed in cortical areas including pain perception-related prefrontal anterior cingulate cortex. However, less information is available for the functional roles of CGRP in cortical regions such as the anterior cingulate cortex (ACC). Recent studies have consistently demonstrated that long-term potentiation is a key cellular mechanism for chronic pain in the ACC. In the present study, we used 64-electrode array field recording system to investigate the effect of CGRP on excitatory transmission in the ACC. We found that CGRP induced potentiation of synaptic transmission in a dose-dependently manner (1, 10, 50, and 100 nM). CGRP also recruited inactive circuit in the ACC. An application of the calcitonin receptor-like receptor antagonist CGRP8-37 blocked CGRP-induced chemical long-term potentiation and the recruitment of inactive channels. CGRP-induced long-term potentiation was also blocked by N-methyl-D-aspartate (NMDA) receptor antagonist AP-5. Consistently, the application of CGRP increased NMDA receptor-mediated excitatory postsynaptic currents. Finally, we found that CGRP-induced long-term potentiation required the activation of calcium-stimulated adenylyl cyclase subtype 1 (AC1) and protein kinase A. Genetic deletion of AC1 using AC1-/- mice, an AC1 inhibitor NB001 or a protein kinase A inhibitor KT5720, all reduced or blocked CGRP-induced potentiation. Our results provide direct evidence that CGRP may contribute to synaptic potentiation in important physiological and pathological conditions in the ACC, an AC1 inhibitor NB001 may be beneficial for the treatment of chronic headache.
Subject(s)
Calcitonin Gene-Related Peptide/pharmacology , Excitatory Postsynaptic Potentials/drug effects , Gyrus Cinguli/drug effects , Nerve Net/drug effects , 2-Amino-5-phosphonovalerate/pharmacology , Adenylyl Cyclases/deficiency , Adenylyl Cyclases/genetics , Animals , Calcitonin Gene-Related Peptide/metabolism , Carbazoles/pharmacology , Electric Stimulation , Enzyme Inhibitors/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Gene Expression Regulation/drug effects , Gene Expression Regulation/genetics , Gyrus Cinguli/metabolism , In Vitro Techniques , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Patch-Clamp Techniques , Pyrroles/pharmacology , Receptors, Calcitonin Gene-Related Peptide/metabolism , Signal Transduction/drug effectsABSTRACT
BACKGROUND: Cholera toxin (CT)-induced diarrhea is mediated by cyclic adenosine monophosphate (cAMP)-mediated active Cl- secretion via the cystic fibrosis transmembrane conductance regulator (CFTR). Although the constitutive activation of adenylyl cyclase (AC) in response to CT is due to adenosine diphosphate ribosylation of the small G protein α-subunit activating CFTR with consequent secretory diarrhea, the AC isoform(s) involved remain unknown. METHODS: We generated intestinal epithelial cell-specific adenylyl cyclase 6 (AC6) knockout mice to study its role in CT-induced diarrhea. RESULTS: AC6 messenger RNA levels were the highest of all 9 membrane-bound AC isoforms in mouse intestinal epithelial cells. Intestinal epithelial-specific AC6 knockout mice (AC6loxloxVillinCre) had undetectable AC6 levels in small intestinal and colonic epithelial cells. No significant differences in fluid and food intake, plasma electrolytes, intestinal/colon anatomy and morphology, or fecal water content were observed between genotypes. Nevertheless, CT-induced fluid accumulation in vivo was completely absent in AC6loxloxVillinCre mice, associated with a lack of forskolin- and CT-induced changes in the short-circuit current (ISC) of the intestinal mucosa, impaired cAMP generation in acutely isolated small intestinal epithelial cells, and significantly impaired apical CFTR levels in response to forskolin. CONCLUSIONS: AC6 is a novel target for the treatment of CT-induced diarrhea.
Subject(s)
Adenylyl Cyclases/metabolism , Cholera Toxin/toxicity , Cholera/physiopathology , Diarrhea/physiopathology , Epithelial Cells/enzymology , Epithelial Cells/metabolism , Adenylyl Cyclases/deficiency , Animals , Colforsin/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Epithelial Cells/drug effects , Male , Mice, Inbred C57BL , Mice, KnockoutABSTRACT
BACKGROUND: Pharmacogenomic studies have shown that ADCY9 genotype determines the effects of the CETP (cholesteryl ester transfer protein) inhibitor dalcetrapib on cardiovascular events and atherosclerosis imaging. The underlying mechanisms responsible for the interactions between ADCY9 and CETP activity have not yet been determined. METHODS: Adcy9-inactivated ( Adcy9Gt/Gt) and wild-type (WT) mice, that were or not transgenic for the CETP gene (CETPtg Adcy9Gt/Gt and CETPtg Adcy9WT), were submitted to an atherogenic protocol (injection of an AAV8 [adeno-associated virus serotype 8] expressing a PCSK9 [proprotein convertase subtilisin/kexin type 9] gain-of-function variant and 0.75% cholesterol diet for 16 weeks). Atherosclerosis, vasorelaxation, telemetry, and adipose tissue magnetic resonance imaging were evaluated. RESULTS: Adcy9Gt/Gt mice had a 65% reduction in aortic atherosclerosis compared to WT ( P<0.01). CD68 (cluster of differentiation 68)-positive macrophage accumulation and proliferation in plaques were reduced in Adcy9Gt/Gt mice compared to WT animals ( P<0.05 for both). Femoral artery endothelial-dependent vasorelaxation was improved in Adcy9Gt/Gt mice (versus WT, P<0.01). Selective pharmacological blockade showed that the nitric oxide, cyclooxygenase, and endothelial-dependent hyperpolarization pathways were all responsible for the improvement of vasodilatation in Adcy9Gt/Gt ( P<0.01 for all). Aortic endothelium from Adcy9Gt/Gt mice allowed significantly less adhesion of splenocytes compared to WT ( P<0.05). Adcy9Gt/Gt mice gained more weight than WT with the atherogenic diet; this was associated with an increase in whole body adipose tissue volume ( P<0.01 for both). Feed efficiency was increased in Adcy9Gt/Gt compared to WT mice ( P<0.01), which was accompanied by prolonged cardiac RR interval ( P<0.05) and improved nocturnal heart rate variability ( P=0.0572). Adcy9 inactivation-induced effects on atherosclerosis, endothelial function, weight gain, adipose tissue volume, and feed efficiency were lost in CETPtg Adcy9Gt/Gt mice ( P>0.05 versus CETPtg Adcy9WT). CONCLUSIONS: Adcy9 inactivation protects against atherosclerosis, but only in the absence of CETP activity. This atheroprotection may be explained by decreased macrophage accumulation and proliferation in the arterial wall, and improved endothelial function and autonomic tone.
Subject(s)
Adenylyl Cyclases/deficiency , Aorta/enzymology , Aortic Diseases/prevention & control , Atherosclerosis/prevention & control , Cholesterol Ester Transfer Proteins/deficiency , Plaque, Atherosclerotic , Adenylyl Cyclases/genetics , Adiposity , Animals , Aorta/pathology , Aorta/physiopathology , Aortic Diseases/enzymology , Aortic Diseases/genetics , Aortic Diseases/pathology , Atherosclerosis/enzymology , Atherosclerosis/genetics , Atherosclerosis/pathology , Autonomic Nervous System/physiopathology , Biological Factors/metabolism , Cell Proliferation , Cholesterol Ester Transfer Proteins/genetics , Diet, High-Fat , Disease Models, Animal , Endothelial Cells/enzymology , Endothelial Cells/pathology , Lipids/blood , Lipolysis , Macrophages/enzymology , Macrophages/pathology , Male , Mice, Inbred C57BL , Mice, Knockout , Nitric Oxide/metabolism , Proprotein Convertase 9/genetics , Prostaglandin-Endoperoxide Synthases/metabolism , Signal Transduction , Vasodilation , Weight GainABSTRACT
Cyclic AMP promotes cyst growth in polycystic kidney disease (PKD) by stimulating cell proliferation and fluid secretion. Previously, we showed that the primary cilium of renal epithelial cells contains a cAMP regulatory complex comprising adenylyl cyclases 5 and 6 (AC5/6), polycystin-2, A-kinase anchoring protein 150, protein kinase A, and phosphodiesterase 4C. In Kif3a mutant cells that lack primary cilia, the formation of this regulatory complex is disrupted and cAMP levels are increased. Inhibition of AC5 reduces cAMP levels in Kif3a mutant cells, suggesting that AC5 may mediate the increase in cAMP in PKD. Here, we examined the role of AC5 in an orthologous mouse model of PKD caused by kidney-specific ablation of Pkd2. Knockdown of AC5 with siRNA attenuated the increase in cAMP levels in Pkd2-deficient renal epithelial cells. Levels of cAMP and AC5 mRNA transcripts were elevated in the kidneys of mice with collecting duct-specific ablation of Pkd2. Compared with Pkd2 single mutant mice, AC5/Pkd2 double mutant mice had less kidney enlargement, lower cyst index, reduced kidney injury, and improved kidney function. Importantly, cAMP levels and cAMP-dependent signaling were reduced in the kidneys of AC5/Pkd2 double mutant compared to the kidneys of Pkd2 single mutant mice. Additionally, we localized endogenous AC5 in the primary cilium of renal epithelial cells and showed that ablation of AC5 reduced ciliary elongation in the kidneys of Pkd2 mutant mice. Thus, AC5 contributes importantly to increased renal cAMP levels and cyst growth in Pkd2 mutant mice, and inhibition of AC5 may be beneficial in the treatment of PKD.
Subject(s)
Adenylyl Cyclases/deficiency , Adenylyl Cyclases/metabolism , Cyclic AMP/metabolism , Epithelial Cells/enzymology , Kidney/enzymology , Polycystic Kidney, Autosomal Dominant/enzymology , Animals , Cilia/enzymology , Cilia/pathology , Disease Models, Animal , Disease Progression , Down-Regulation , Epithelial Cells/pathology , Female , Kidney/pathology , Kidney/physiopathology , Male , Mice, Inbred C57BL , Mice, Knockout , Polycystic Kidney, Autosomal Dominant/genetics , Polycystic Kidney, Autosomal Dominant/pathology , Polycystic Kidney, Autosomal Dominant/prevention & control , RNA Interference , Second Messenger Systems , TRPP Cation Channels/deficiency , TRPP Cation Channels/geneticsABSTRACT
Neuroadaptive responses to chronic ethanol, such as behavioral sensitization, are associated with N-methyl-D-aspartate receptor (NMDAR) recruitment. Ethanol enhances GluN2B-containing NMDAR function and phosphorylation (Tyr-1472) of the GluN2B-NMDAR subunit in the dorsal medial striatum (DMS) through a protein kinase A (PKA)-dependent pathway. Ethanol-induced phosphorylation of PKA substrates is partially mediated by calcium-stimulated adenylyl cyclase 1 (AC1), which is enriched in the dorsal striatum. As such, AC1 is poised as an upstream modulator of ethanol-induced DMS neuroadaptations that promote drug responding, and thus represents a therapeutic target. Our hypothesis is that loss of AC1 activity will prevent ethanol-induced locomotor sensitization and associated DMS GluN2B-NMDAR adaptations. We evaluated AC1's contribution to ethanol-evoked locomotor responses and DMS GluN2B-NMDAR phosphorylation and function using AC1 knockout (AC1KO) mice. Results were mechanistically validated with the AC1 inhibitor, NB001. Acute ethanol (2.0 g/kg) locomotor responses in AC1KO and wild-type (WT) mice pretreated with NB001 (10 mg/kg) were comparable to WT ethanol controls. However, repeated ethanol treatment (10 days, 2.5 g/kg) failed to produce sensitization in AC1KO or NB001 pretreated mice, as observed in WT ethanol controls, following challenge exposure (2.0 g/kg). Repeated exposure to ethanol in the sensitization procedure significantly increased pTyr-1472 GluN2B levels and GluN2B-containing NMDAR transmission in the DMS of WT mice. Loss of AC1 signaling impaired ethanol-induced increases in DMS pGluN2B levels and NMDAR-mediated transmission. Together, these data support a critical and specific role for AC1 in striatal signaling that mediates ethanol-induced behavioral sensitization, and identify GluN2B-containing NMDARs as an important AC1 target.
Subject(s)
Adenylyl Cyclases/deficiency , Corpus Striatum/metabolism , Ethanol/administration & dosage , Locomotion/physiology , Receptors, N-Methyl-D-Aspartate/metabolism , Animals , Corpus Striatum/drug effects , Excitatory Postsynaptic Potentials/drug effects , Excitatory Postsynaptic Potentials/physiology , Locomotion/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation/drug effects , Phosphorylation/physiologyABSTRACT
Adenylate cyclase 3 (AC3) is an important component of the cyclic adenosine 3',5'-monophosphate (cAMP) signaling pathway and converts adenosine triphosphate into cAMP. Male mice with AC3 deletion (AC3-/-) are sterile. However, the mechanical mechanism remains unclear. By TUNEL staining, we found that cell apoptosis in the testicular tissues of AC3-/- mice increased significantly compared with that in the wild-type (AC3+/+) mice. Differentially expressed genes regulated by AC3 in the testicular tissues were identified by gene chip hybridization. We observed that the expression of 693 genes was altered in the testicular tissues of AC3-/- mice, including 330 up-regulated and 363 down-regulated gene expression with fold changes higher than 2 (≥2) as the standards. Furthermore, part of these differentially expressed genes was verified by the real-time fluorescence quantification PCR and immunofluorescent staining. The expression levels of the genes related to olfactory receptors, cell apoptosis, transcriptional activity, defensive reaction, cell adhesion, cell death, and immunoreactions were significantly altered in the testicular tissues of AC3-/- mice compared with AC3+/+ mice. In addition, the corresponding Ca2+, cAMP, and cell adhesion signaling pathways, as well as the signaling pathways related to axon guidance and cell interaction, were altered significantly in the AC3-/- mice. These data would help elucidate the general understanding of the mechanisms underlying the sterility in AC3-/- male mice.
Subject(s)
Adenylyl Cyclases/deficiency , Testis/metabolism , Adenylyl Cyclases/genetics , Animals , Apoptosis/genetics , Apoptosis/physiology , Epigenesis, Genetic , Gene Expression , Gene Expression Profiling , Infertility, Male/genetics , Infertility, Male/metabolism , Infertility, Male/pathology , Male , Mice , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Signal Transduction/genetics , Testis/pathologyABSTRACT
The fungal pathogen Candida albicans can transition from budding to hyphal growth, which promotes biofilm formation and invasive growth into tissues. Stimulation of adenylyl cyclase to form cAMP induces hyphal morphogenesis. The failure of cells lacking adenylyl cyclase (cyr1Δ) to form hyphae has suggested that cAMP signaling is essential for hyphal growth. However, cyr1Δ mutants also grow slowly and have defects in morphogenesis, making it unclear whether hyphal inducers must stimulate cAMP, or if normal basal levels of cAMP are required to maintain cellular health needed for hyphal growth. Interestingly, supplementation of cyr1Δ cells with low levels of cAMP enabled them to form hyphae in response to the inducer N-acetylglucosamine (GlcNAc), suggesting that a basal level of cAMP is sufficient for stimulation. Furthermore, we isolated faster-growing cyr1Δ pseudorevertant strains that can be induced to form hyphae even though they lack cAMP. The pseudorevertant strains were not induced by CO2 , consistent with reports that CO2 directly stimulates adenylyl cyclase. Mutational analysis showed that induction of hyphae in a pseudorevertant strain was independent of RAS1, but was dependent on the EFG1 transcription factor that acts downstream of protein kinase A. Thus, cAMP-independent signals contribute to the induction of hyphal responses.
Subject(s)
Candida albicans/growth & development , Candida albicans/metabolism , Cyclic AMP/metabolism , Hyphae/growth & development , Signal Transduction , Acetylglucosamine/pharmacology , Adenylyl Cyclases/deficiency , Adenylyl Cyclases/genetics , Candida albicans/drug effects , Candida albicans/genetics , Cyclic AMP/pharmacology , Cyclic AMP-Dependent Protein Kinases/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Fungal Proteins/genetics , Gene Expression Regulation, Fungal , Hyphae/drug effects , Hyphae/genetics , Hyphae/physiology , Signal Transduction/genetics , Transcription Factors/geneticsABSTRACT
BACKGROUND & AIMS: Genetic defects in polycystin-1 or -2 (PC1 or PC2) cause polycystic liver disease associated with autosomal dominant polycystic kidney disease (PLD-ADPKD). Progressive cyst growth is sustained by a cAMP-dependent Ras/ERK/HIFα pathway, leading to increased vascular endothelial growth factor A (VEGF-A) signaling. In PC2-defective cholangiocytes, cAMP production in response to [Ca2+]ER depletion is increased, while store-operated Ca2+ entry (SOCE), intracellular and endoplasmic reticulum [Ca2+]ER levels are reduced. We investigated whether the adenylyl cyclases, AC5 and AC6, which can be inhibited by Ca2+, are activated by the ER chaperone STIM1. This would result in cAMP/PKA-dependent Ras/ERK/HIFα pathway activation in PC2-defective cells, in response to [Ca2+]ER depletion. METHODS: PC2/AC6 double conditional knockout (KO) mice were generated (Pkd2/AC6 KO) and compared to Pkd2 KO mice. The AC5 inhibitor SQ22,536 or AC5 siRNA were used in isolated cholangiocytes while the inhibitor was used in biliary organoid and animals; liver tissues were harvested for histochemical analysis. RESULTS: When comparing Pkd2/AC6 KO to Pkd2 KO mice, no decrease in liver cyst size was found, and cellular cAMP after [Ca2+]ER depletion only decreased by 12%. Conversely, in PC2-defective cells, inhibition of AC5 significantly reduced cAMP production, pERK1/2 expression and VEGF-A secretion. AC5 inhibitors significantly reduced growth of biliary organoids derived from Pkd2 KO and Pkd2/AC6 KO mice. In vivo treatment with SQ22,536 significantly reduced liver cystic area and cell proliferation in PC2-defective mice. After [Ca2+]ER depletion in PC2-defective cells, STIM1 interacts with AC5 but not with Orai1, the Ca2+ channel that mediates SOCE. CONCLUSION: [Ca2+]ER depletion in PC2-defective cells activates AC5 and results in stimulation of cAMP/ERK1-2 signaling, VEGF production and cyst growth. This mechanism may represent a novel therapeutic target. LAY SUMMARY: Polycystic liver diseases are characterized by progressive cyst growth until their complications mandate surgery or liver transplantation. In this manuscript, we demonstrate that inhibiting cell proliferation, which is induced by increased levels of cAMP, may represent a novel therapeutic target to slow the progression of the disease.
Subject(s)
Adenylyl Cyclases/metabolism , Calcium/metabolism , Cyclic AMP/metabolism , Cysts/genetics , Cysts/metabolism , Liver Diseases/genetics , Liver Diseases/metabolism , Adenylyl Cyclase Inhibitors/pharmacology , Adenylyl Cyclases/deficiency , Adenylyl Cyclases/genetics , Animals , Cell Proliferation , Cysts/pathology , Disease Models, Animal , Homeostasis , Humans , Liver Diseases/pathology , MAP Kinase Signaling System , Mice , Mice, Knockout , Polycystic Kidney, Autosomal Dominant/genetics , Polycystic Kidney, Autosomal Dominant/metabolism , Polycystic Kidney, Autosomal Dominant/pathology , RNA Interference , Signal Transduction , Stromal Interaction Molecule 1/metabolism , TRPP Cation Channels/deficiency , TRPP Cation Channels/genetics , TRPP Cation Channels/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
An emerging secondary messenger c-di-AMP plays an important role in bacterial physiology. It was reported by Cheng et al. that inactivation of a gene coding for diadenylate cyclase (DAC), a c-di-AMP producing enzyme, resulted in enhanced synthesis of extracellular polysaccharides (EPS) by a cariogenic bacterium, Streptococcus mutans (Cheng et al., 2016). We constructed a similar mutant and observed a completely different effect, the DAC deficiency resulted in a decrease in the production of EPS. Our studies provided the following compelling evidence, (1) the DAC mutant we constructed can be readily complemented for the production of EPS, while the mutant from the Cheng group cannot; (2) Our mutant exhibits the regular pattern of key enzymes that produce EPS, glucosyltransferases (Gtfs), while Cheng et al. reported an irregular pattern, which was inconsistent with their earlier studies. (3) We demonstrated that the response of the DAC mutant to oxidative stress is independent of GtfB, the key enzyme producing EPS, while the Cheng report suggests that overproduction of EPS is a responsive mechanism for the DAC mutant to adapt to the oxidative stress. Therefore, the validity of the relationship between DAC and EPS reported by Cheng et al. warrants further investigation and clarification.
Subject(s)
Adenylyl Cyclases/deficiency , Bacterial Proteins/genetics , Extracellular Matrix/metabolism , Polysaccharides/biosynthesis , Streptococcus mutans/enzymology , Adenylyl Cyclases/genetics , Bacterial Proteins/metabolism , Dinucleoside Phosphates/metabolism , Extracellular Matrix/genetics , Mutation , Streptococcus mutans/genetics , Streptococcus mutans/metabolismABSTRACT
Brain-derived neurotrophic factor (BDNF) mediates neuron growth and is regulated by adenylyl cyclases (ACs). Mice lacking AC1/8 (DKO) have a basal reduction in the dendritic complexity of medium spiny neurons in the caudate putamen and demonstrate increased neurotoxicity in the striatum following acute neonatal ethanol exposure compared to wild type (WT) controls, suggesting a compromise in BDNF regulation under varying conditions. Although neonatal ethanol exposure can negatively impact BDNF expression, little is known about the effect on BDNF receptor activation and its downstream signaling, including Akt activation, an established neuroprotective pathway. Therefore, here we determined the effects of AC1/8 deletion and neonatal ethanol administration on BDNF and proBDNF protein expression, and activation of tropomyosin-related kinase B (TrkB), Akt, ERK1/2, and PLCγ. WT and DKO mice were treated with a single dose of 2.5 g/kg ethanol or saline at postnatal days 5-7 to model late-gestational alcohol exposure. Striatal and cortical tissues were analyzed using a BDNF enzyme-linked immunosorbent assay or immunoblotting for proBDNF, phosphorylated and total TrkB, Akt, ERK1/2, and PLCɣ1. Neither postnatal ethanol exposure nor AC1/8 deletion affected total BDNF protein expression at any time point in either region examined. Neonatal ethanol increased the expression of proBDNF protein in the striatum of WT mice 6, 24, and 48 h after exposure, with DKO mice demonstrating a reduction in proBDNF expression 6 h after exposure. Six and 24 h after ethanol administration, phosphorylation of full-length TrkB in the striatum was significantly reduced in WT mice, but was significantly increased in DKO mice only at 24 h. Interestingly, 48 h after ethanol, both WT and DKO mice demonstrated a reduction in phosphorylated full-length TrkB. In addition, Akt and PLCɣ1 phosphorylation was also decreased in ethanol-treated DKO mice 48 h after injection. These data demonstrate dysregulation of a potential survival pathway in the AC1/8 knockout mice following early-life ethanol exposure.
Subject(s)
Adenylyl Cyclases/metabolism , Brain-Derived Neurotrophic Factor/metabolism , Fetal Alcohol Spectrum Disorders/metabolism , Membrane Glycoproteins/metabolism , Protein Precursors/metabolism , Protein-Tyrosine Kinases/metabolism , Adenylyl Cyclases/deficiency , Animals , Ethanol/toxicity , Fetal Alcohol Spectrum Disorders/etiology , Fetal Alcohol Spectrum Disorders/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Phosphorylation/drug effects , Phosphorylation/physiology , Receptor, trkB , Signal Transduction/drug effects , Signal Transduction/physiologyABSTRACT
We recently reported that olfactory sensory neurons in the dorsal zone of the mouse olfactory epithelium exhibit drastic location-dependent differences in cilia length. Furthermore, genetic ablation of type III adenylyl cyclase (ACIII), a key olfactory signaling protein and ubiquitous marker for primary cilia, disrupts the cilia length pattern and results in considerably shorter cilia, independent of odor-induced activity. Given the significant impact of ACIII on cilia length in the dorsal zone, we sought to further investigate the relationship between cilia length and ACIII level in various regions throughout the mouse olfactory epithelium. We employed whole-mount immunohistochemical staining to examine olfactory cilia morphology in phosphodiesterase (PDE) 1C-/-;PDE4A-/- (simplified as PDEs-/- hereafter) and ACIII-/- mice in which ACIII levels are reduced and ablated, respectively. As expected, PDEs-/- animals exhibit dramatically shorter cilia in the dorsal zone (i.e., where the cilia pattern is found), similar to our previous observation in ACIII-/- mice. Remarkably, in a region not included in our previous study, ACIII-/- animals (but not PDEs-/- mice) have dramatically elongated, comet-shaped cilia, as opposed to characteristic star-shaped olfactory cilia. Here, we reveal that genetic ablation of ACIII has drastic, location-dependent effects on cilia architecture in the mouse nose. These results add a new dimension to our current understanding of olfactory cilia structure and regional organization of the olfactory epithelium. Together, these findings have significant implications for both cilia and sensory biology.
Subject(s)
Adenylyl Cyclases/deficiency , Adenylyl Cyclases/genetics , Cilia/metabolism , Gene Deletion , Nose/cytology , Animals , Cilia/enzymology , Mice , Nose/enzymology , Organ Specificity , Phosphoric Diester Hydrolases/metabolismABSTRACT
BACKGROUND: Although major depressive disorder (MDD) has low heritability, a genome-wide association study in humans has recently implicated type 3 adenylyl cyclase (AC3; ADCY3) in MDD. Moreover, the expression level of AC3 in blood has been considered as a MDD biomarker in humans. Nevertheless, there is a lack of supporting evidence from animal studies. METHODS: We employed multiple approaches to experimentally evaluate if AC3 is a contributing factor for major depression using mouse models lacking the Adcy3 gene. RESULTS: We found that conventional AC3 knockout (KO) mice exhibited phenotypes associated with MDD in behavioral assays. Electroencephalography/electromyography recordings indicated that AC3 KO mice have altered sleep patterns characterized by increased percentage of rapid eye movement sleep. AC3 KO mice also exhibit neuronal atrophy. Furthermore, synaptic activity at cornu ammonis 3-cornu ammonis 1 synapses was significantly lower in AC3 KO mice, and they also exhibited attenuated long-term potentiation as well as deficits in spatial navigation. To confirm that these defects are not secondary responses to anosmia or developmental defects, we generated a conditional AC3 floxed mouse strain. This enabled us to inactivate AC3 function selectively in the forebrain and to inducibly ablate it in adult mice. Both AC3 forebrain-specific and AC3 inducible knockout mice exhibited prodepression phenotypes without anosmia. CONCLUSIONS: This study demonstrates that loss of AC3 in mice leads to decreased neuronal activity, altered sleep pattern, and depression-like behaviors, providing strong evidence supporting AC3 as a contributing factor for MDD.
Subject(s)
Adenylyl Cyclases/physiology , Behavior, Animal/physiology , Depressive Disorder, Major/enzymology , Depressive Disorder, Major/physiopathology , Adenylyl Cyclases/deficiency , Animals , Disease Models, Animal , Long-Term Potentiation/physiology , Mice , Mice, Knockout , Phenotype , Sleep Wake Disorders/physiopathology , Spatial Navigation/physiologyABSTRACT
OBJECTIVE: Adenylyl cyclases (ACs) catalyze the synthesis of cAMP from ATP, and cAMP signaling affects a large number of neuronal processes. Ca(2+)-stimulated adenylyl cyclase 8 (AC8) expressed in the CNS plays a role in synaptic plasticity, drug addiction and ethanol sensitivity, and chronic pain. This study was to aim at examining the contributions of AC8 to epileptogenesis. METHODS: In this study, we observed the seizure behavior induced by kainic acid (20 mg/kg or 30 mg/kg) or pilocarpine (350 mg/kg) in AC8 KO and wild-type mice. Next we injected kainic acid or pilocarpine to induce status epilepticus (SE), and examined neuronal degeneration (by Fluoro-Jade B staining) and mossy fiber sprouting (by Timm staining) 24h and 2 weeks after SE termination in the hippocampus, respectively. Finally, 15 min after intraperitoneal injection of kainic acid (30 mg/kg), we examined phosphor-ERK1/2 in the hippocampus by Western blot and immunochemistry staining. RESULTS: We first observed that AC8 KO mutants display reduced susceptibility (including seizure latency and episodes) to two chemoconvulsants, kainic acid and pilocarpine. Moreover, we found that degenerative neurons and mossy fiber sprouting induced by chemoconvulsants were significant decreased in the hippocampus. Further, Western blot and immunochemistry analysis revealed that the MAPK signaling in the hippocampus was attenuated in kainic acid-injected AC8 KO mice. CONCLUSION: AC8 is involved in epileptogenesis, and may serve as a potential target for the treatment of epilepsy.
Subject(s)
Adenylyl Cyclases/deficiency , Convulsants/toxicity , Seizures/chemically induced , Seizures/physiopathology , Status Epilepticus/chemically induced , Status Epilepticus/physiopathology , Adenylyl Cyclases/genetics , Animals , Cell Death/drug effects , Cell Death/physiology , Disease Models, Animal , Hippocampus/drug effects , Hippocampus/pathology , Hippocampus/physiopathology , Kainic Acid/toxicity , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Male , Mice, Inbred C57BL , Mice, Knockout , Nerve Degeneration/chemically induced , Nerve Degeneration/pathology , Nerve Degeneration/physiopathology , Neurons/drug effects , Neurons/pathology , Neurons/physiology , Pilocarpine/toxicity , Seizures/pathology , Status Epilepticus/pathologyABSTRACT
Adenylyl Cyclase 3 (AC3) plays an important role in the olfactory sensation-signaling pathway in mice. AC3 deficiency leads to defects in olfaction. However, it is still unknown whether AC3 deficiency affects gene expression or olfactory signal transduction pathways within the main olfactory epithelium (MOE). In this study, gene microarrays were used to screen differentially expressed genes in MOE from AC3 knockout (AC3(-/-)) and wild-type (AC3(+/+)) mice. The differentially expressed genes identified were subjected to bioinformatic analysis and verified by qRT-PCR. Gene expression in the MOE from AC3(-/-) mice was significantly altered, compared to AC3(+/+) mice. Of the 41266 gene probes, 3379 had greater than 2-fold fold change in expression levels between AC3(-/-) and AC3(+/+) mice, accounting for 8% of the total gene probes. Of these genes, 1391 were up regulated, and 1988 were down regulated, including 425 olfactory receptor genes, 99 genes that are specifically expressed in the immature olfactory neurons, 305 genes that are specifically expressed in the mature olfactory neurons, and 155 genes that are involved in epigenetic regulation. Quantitative RT-PCR verification of the differentially expressed epigenetic regulation related genes, olfactory receptors, ion transporter related genes, neuron development and differentiation related genes, lipid metabolism and membrane protein transport etc. related genes showed that P75NTR, Hinfp, Gadd45b, and Tet3 were significantly up-regulated, while Olfr370, Olfr1414, Olfr1208, Golf, Faim2, Tsg101, Mapk10, Actl6b, H2BE, ATF5, Kirrrel2, OMP, Drd2 etc. were significantly down-regulated. In summary, AC3 may play a role in proximal olfactory signaling and play a role in the regulation of differentially expressed genes in mouse MOE.