RESUMO
Membrane adenylyl cyclase AC8 is regulated by G proteins and calmodulin (CaM), mediating the crosstalk between the cAMP pathway and Ca2+ signalling. Despite the importance of AC8 in physiology, the structural basis of its regulation by G proteins and CaM is not well defined. Here, we report the 3.5 Å resolution cryo-EM structure of the bovine AC8 bound to the stimulatory Gαs protein in the presence of Ca2+/CaM. The structure reveals the architecture of the ordered AC8 domains bound to Gαs and the small molecule activator forskolin. The extracellular surface of AC8 features a negatively charged pocket, a potential site for unknown interactors. Despite the well-resolved forskolin density, the captured state of AC8 does not favour tight nucleotide binding. The structural proteomics approaches, limited proteolysis and crosslinking mass spectrometry (LiP-MS and XL-MS), allowed us to identify the contact sites between AC8 and its regulators, CaM, Gαs, and Gßγ, as well as to infer the conformational changes induced by these interactions. Our results provide a framework for understanding the role of flexible regions in the mechanism of AC regulation.
Assuntos
Adenilil Ciclases , Calmodulina , Animais , Bovinos , Adenilil Ciclases/química , Adenilil Ciclases/metabolismo , Colforsina/farmacologia , Microscopia Crioeletrônica , Proteômica , Proteínas de Ligação ao GTP/metabolismoRESUMO
Activation of the cAMP pathway is one of the common mechanisms underlying long-term potentiation (LTP). In the Drosophila mushroom body, simultaneous activation of odour-coding Kenyon cells (KCs) and reinforcement-coding dopaminergic neurons activates adenylyl cyclase in KC presynaptic terminals, which is believed to trigger synaptic plasticity underlying olfactory associative learning. However, learning induces long-term depression (LTD) at these synapses, contradicting the universal role of cAMP as a facilitator of transmission. Here, we developed a system to electrophysiologically monitor both short-term and long-term synaptic plasticity at KC output synapses and demonstrated that they are indeed an exception in which activation of the cAMP-protein kinase A pathway induces LTD. Contrary to the prevailing model, our cAMP imaging found no evidence for synergistic action of dopamine and KC activity on cAMP synthesis. Furthermore, we found that forskolin-induced cAMP increase alone was insufficient for plasticity induction; it additionally required simultaneous KC activation to replicate the presynaptic LTD induced by pairing with dopamine. On the other hand, activation of the cGMP pathway paired with KC activation induced slowly developing LTP, proving antagonistic actions of the two second-messenger pathways predicted by behavioural study. Finally, KC subtype-specific interrogation of synapses revealed that different KC subtypes exhibit distinct plasticity duration even among synapses on the same postsynaptic neuron. Thus, our work not only revises the role of cAMP in synaptic plasticity by uncovering the unexpected convergence point of the cAMP pathway and neuronal activity, but also establishes the methods to address physiological mechanisms of synaptic plasticity in this important model. KEY POINTS: Although presynaptic cAMP increase generally facilitates synapses, olfactory associative learning in Drosophila, which depends on dopamine and cAMP signalling genes, induces long-term depression (LTD) at the mushroom body output synapses. By combining electrophysiology, pharmacology and optogenetics, we directly demonstrate that these synapses are an exception where activation of the cAMP-protein kinase A pathway leads to presynaptic LTD. Dopamine- or forskolin-induced cAMP increase alone is not sufficient for LTD induction; neuronal activity, which has been believed to trigger cAMP synthesis in synergy with dopamine input, is required in the downstream pathway of cAMP. In contrast to cAMP, activation of the cGMP pathway paired with neuronal activity induces presynaptic long-term potentiation, which explains behaviourally observed opposing actions of transmitters co-released by dopaminergic neurons. Our work not only revises the role of cAMP in synaptic plasticity, but also provides essential methods to address physiological mechanisms of synaptic plasticity in this important model system.
Assuntos
AMP Cíclico , Corpos Pedunculados , Plasticidade Neuronal , Animais , Corpos Pedunculados/fisiologia , AMP Cíclico/metabolismo , Plasticidade Neuronal/fisiologia , Dopamina , Potenciação de Longa Duração/fisiologia , Drosophila melanogaster/fisiologia , GMP Cíclico/metabolismo , Sinapses/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismoRESUMO
Recent studies using different experimental approaches demonstrate that silent synapses may exist in the adult cortex including the sensory cortex and anterior cingulate cortex (ACC). The postsynaptic form of long-term potentiation (LTP) in the ACC recruits some of these silent synapses and the activity of calcium-stimulated adenylyl cyclases (ACs) is required for such recruitment. It is unknown if the chemical activation of ACs may recruit silent synapses. In this study, we found that activation of ACs contributed to synaptic potentiation in the ACC of adult mice. Forskolin, a selective activator of ACs, recruited silent responses in the ACC of adult mice. The recruitment was long-lasting. Interestingly, the effect of forskolin was not universal, some silent synapses did not undergo potentiation or recruitment. These findings suggest that these adult cortical synapses are not homogenous. The application of a selective calcium-permeable AMPA receptor inhibitor 1-naphthyl acetyl spermine (NASPM) reversed the potentiation and the recruitment of silent responses, indicating that the AMPA receptor is required. Our results strongly suggest that the AC-dependent postsynaptic AMPA receptor contributes to the recruitment of silent responses at cortical LTP.
Assuntos
Adenilil Ciclases , Colforsina , Giro do Cíngulo , Potenciação de Longa Duração , Animais , Camundongos , Giro do Cíngulo/efeitos dos fármacos , Giro do Cíngulo/metabolismo , Colforsina/farmacologia , Potenciação de Longa Duração/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Masculino , Receptores de AMPA/metabolismo , Camundongos Endogâmicos C57BL , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Cálcio/metabolismoRESUMO
Dopamine D2 receptor (D2R) is expressed in striatopallidal neurons and decreases forskolin-stimulated cyclic adenine monophosphate (cAMP) accumulation and gamma-aminobutyric acid (GABA) release. Dopamine D3 receptor (D3R) mRNA is expressed in a population of striatal D2R-expressing neurons. Also, D3R protein and binding have been reported in the neuropil of globus pallidus. We explore whether D2R and D3R colocalize in striatopallidal terminals and whether D3R modulates the D2R effect on forskolin-stimulated [3H]cAMP accumulation in pallidal synaptosomes and high K+ stimulated-[3H]GABA release in pallidal slices. Previous reports in heterologous systems indicate that calmodulin (CaM) and CaMKII modulate D2R and D3R functions; thus, we study whether this system regulates its functional interaction. D2R immunoprecipitates with CaM, and pretreatment with ophiobolin A or depolarization of synaptosomes with 15 mM of K+ decreases it. Both treatments increase the D2R inhibition of forskolin-stimulated [3H]cAMP accumulation when activated with quinpirole, indicating a negative modulation of CaM on D2R function. Quinpirole also activates D3R, potentiating D2R inhibition of cAMP accumulation in the ophiobolin A-treated synaptosomes. D2R and D3R immunoprecipitate in pallidal synaptosomes and decrease after the kainic acid striatal lesion, indicating the striatal origin of the presynaptic receptors. CaM-kinase II alfa (CaMKIIα) immunoprecipitates with D3R and increases after high K+ depolarization. In the presence of KN62, a CaMKIIα blocker, D3R potentiates D2R effects on cAMP accumulation in depolarized synaptosomes and GABA release in pallidal slices, indicating D3R function regulation by CaMKIIα. Our data indicate that D3R potentiates the D2R effect on cAMP accumulation and GABA release at pallidal terminals, an interaction regulated by the CaM-CaMKIIα system.
Assuntos
Calmodulina , Receptores de Dopamina D3 , Sesterterpenos , Receptores de Dopamina D3/metabolismo , Quimpirol/farmacologia , Calmodulina/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Colforsina , Receptores de Dopamina D2/metabolismo , Ácido gama-Aminobutírico/metabolismoRESUMO
The skeletal transformations of diterpenoid forskolin were achieved by employing an oxidative rearrangement strategy. A library of 36 forskolin analogues with structural diversity was effectively generated. Computational analysis shows that 12 CTD compounds with unique scaffolds and ring systems were produced during the course of this work.
Assuntos
Diterpenos , Terpenos , Terpenos/química , Colforsina/química , Diterpenos/química , Extratos Vegetais , Estresse OxidativoRESUMO
Motile cilia in the ependymal cells that line the brain ventricles play pivotal roles in cerebrospinal fluid (CSF) flow in well-defined directions. However, the substances and pathways which regulate their beating have not been well studied. Here, we used primary cultured cells derived from neonatal mouse brain that possess motile cilia and found that adenosine (ADO) stimulates ciliary beating by increasing the ciliary beat frequency (CBF) in a concentration-dependent manner, with the ED50 value being 5 µM. Ciliary beating stimulated by ADO was inhibited by A2B receptor (A2BR) antagonist MRS1754 without any inhibition by antagonists of other ADO receptor subtypes. The expression of A2BR on the cilia was also confirmed by immunofluorescence. The values of CBF were also increased by forskolin, which is an activator of adenylate cyclase, whereas they were not further increased by the addition of ADO. Furthermore, ciliary beating was not stimulated by ADO in the presence of a protein kinase A (PKA) inhibitors. These results altogether suggest that ADO stimulates ciliary beating through A2BR on the cilia, and activation of PKA.
Assuntos
Adenosina , Animais Recém-Nascidos , Encéfalo , Cílios , Proteínas Quinases Dependentes de AMP Cíclico , Receptor A2B de Adenosina , Animais , Cílios/efeitos dos fármacos , Cílios/metabolismo , Cílios/fisiologia , Receptor A2B de Adenosina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Adenosina/farmacologia , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Camundongos , Células Cultivadas , Transdução de Sinais/efeitos dos fármacos , Antagonistas do Receptor A2 de Adenosina/farmacologia , Colforsina/farmacologia , Epêndima/metabolismo , Epêndima/citologiaRESUMO
Platelets are terminally differentiated anucleated cells, but they still have cell-like functions and can even produce progeny platelets. However, the mechanism of platelet sprouting has not been elucidated so far. Here, we show that when platelet-rich plasma(PRP) was cultured at 37°C, platelets showed a spore phenomenon. The number of platelets increased when given a specific shear force. It is found that AMP-related signaling pathways, such as PKA and AMPK are activated in platelets in the spore state. Meanwhile, the mRNA expression levels of genes, such as CNN3, CAPZB, DBNL, KRT19, and ESPN related to PLS1 skeleton proteins also changed. Moreover, when we use the AMPK activator AICAR(AI) to treat washed platelets, cultured platelets can still appear spore phenomenon. We further demonstrate that washed platelets treated with Forskolin, an activator of PKA, not only platelet sprouting after culture but also the AMPK is activated. Taken together, these data demonstrate that AMPK plays a key role in the process of platelet budding and proliferation, suggesting a novel strategy to solve the problem of clinical platelet shortage.
What is new? In this study, we showed that when platelet-rich plasma(PRP) was cultured at 37°C, platelets showed spore phenomenon and increased.It was found that AMP-related signaling pathways, such as PKA and AMPK were activated in platelets in the spore state.In addition, we found that PKA acts as an upstream kinase of AMPK.In the process of platelet sprouting and proliferation, the mRNA expression levels of skeleton protein PLS1 and its related genes, such as CNN3, CAPZB, DBNL, KRT19, andESPN also changed.What is the impact? Our study proposes a new strategy to solve the problem of clinical platelet shortage.
Assuntos
Proteínas Quinases Ativadas por AMP , Plaquetas , Humanos , Plaquetas/citologia , Plaquetas/metabolismo , Diferenciação Celular , Colforsina , Técnicas de CulturaRESUMO
Three types of adipocytes, white, brown, and beige, regulate the systemic energy balance through the storage and expenditure of chemical energy. In addition, adipocytes produce various bioactive molecules known as adipokines. In contrast to white adipocyte-derived molecules, less information is available on the adipokines produced by brown adipocytes (batokine). This study explored the regulatory expression of interleukin (IL)-6 in cell culture studies. Norepinephrine or a nonselective ß-adrenergic receptor agonist increased the expression of IL-6 in primary brown adipocytes and HB2 brown adipocytes. Treatment with forskolin (Fsk), an activator of the cAMP-dependent protein kinase (PKA) pathway (downstream signaling of the ß-adrenergic receptor), efficiently stimulated IL-6 expression in brown adipocytes and myotubes. Phosphorylated CREB and phosphorylated p38 MAP kinase levels were increased in Fsk-treated brown adipocytes within 5 min. In contrast, a long-term (â¼60 min and â¼4 h) treatment with Fsk was required for increase in STAT3 phosphorylation and C/EBPß expression, respectively. The PKA, p38 MAP kinase, STAT3, and C/EBPß pathways are required for the maximal IL-6 expression induced by Fsk, which were verified by use of various inhibitors of these signal pathways. Vitamin C enhanced Fsk-induced IL-6 expression through the extracellular signal-regulated kinase activity. The present study provides basic information on the regulatory expression of IL-6 in activated brown adipocytes.
Assuntos
Adipócitos Marrons , Proteína Quinase 14 Ativada por Mitógeno , Animais , Camundongos , Adipócitos Brancos , Adipocinas , Colforsina/farmacologia , Interleucina-6RESUMO
We analyzed the effects of olfactory receptors (ORs) on transient receptor potential vanilloid 1 (TRPV1) activation using HEK293T cells co-expressing TRPV1 and OR51E1. We demonstrate here that the effect of OR51E1 on TRPV1 activation varies depending on the two TRPV1 ligands: capsaicin and eugenol. Notably, both of these ligands are vanilloid analogs. OR51E1 enhanced the response of TRPV1 to capsaicin but diminished that to eugenol. OR51E2 also showed similar effects. Based on the susceptibility to the OR's modulatory effects, various TRPV1 ligands could be classified into capsaicin and eugenol types. Activation of OR51E1 enhanced cAMP production. In addition, forskolin exhibited almost identical effects as ORs on TRPV1 responses to both types of ligands. These results suggest that OR51E1-induced cAMP elevation leads to a modification of TRPV1, presumably phosphorylation of TRPV1, which amplifies the susceptibility of TRPV1 to the two types of ligands differently.
Assuntos
Capsaicina , AMP Cíclico , Eugenol , Receptores Odorantes , Canais de Cátion TRPV , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/genética , Humanos , Eugenol/farmacologia , Células HEK293 , Capsaicina/farmacologia , AMP Cíclico/metabolismo , Receptores Odorantes/metabolismo , Receptores Odorantes/genética , Ligantes , Fosforilação/efeitos dos fármacos , Colforsina/farmacologiaRESUMO
The measles vaccine virus strain (MV-Edm) serves as a potential platform for the development of effective oncolytic vectors. Nevertheless, despite promising pre-clinical data, our comprehension of the factors influencing the efficacy of MV-Edm infection and intratumoral spread, as well as the interactions between oncolytic viruses and specific chemotherapeutics associated with viral infection, remains limited. Therefore, we investigated the potency of Forskolin in enhancing the antitumor effect of oncolytic MV-Edm by promoting the Rab27a-dependent vesicular transport system. After infecting cells with MV-Edm, we observed an increased accumulation of cytoplasmic vesicles. Our study demonstrated that MV-Edm infection and spread in tumors, which are indispensable processes for viral oncolysis, depend on the vesicular transport system of tumor cells. Although tumor cells displayed a responsive mechanism to restrain the MV-Edm spread by down-regulating the expression of Rab27a, a key member of the vesicle transport system, over-expression of Rab27a promoted the oncolytic efficacy of MV-Edm towards A549 tumor cells. Additionally, we found that Forskolin, a Rab27a agonist, was capable of promoting the oncolytic effect of MV-Edm in vitro. Our study revealed that the vesicle transporter Rab27a could facilitate the secretion of MV-Edm and the generation of syncytial bodies in MV-Edm infected cells during the MV-Edm-mediated oncolysis pathway. The results of the study demonstrate that a combination of Forskolin and MV-Edm exerts a synergistic anti-tumor effect in vitro, leading to elevated oncolysis. This finding holds promise for the clinical treatment of patients with tumors.
Assuntos
Terapia Viral Oncolítica , Vírus Oncolíticos , Humanos , Linhagem Celular Tumoral , Colforsina/farmacologia , Vírus do Sarampo/genética , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos/genéticaRESUMO
p.Asn1303Lys (N1303K) is a common missense variant of the CFTR gene, causing cystic fibrosis (CF). In this study, we initially evaluated the influence of CFTR modulators on the restoration of N1303K-CFTR function using intestinal organoids derived from four CF patients expressing the N1303K variant. The forskolin-induced swelling assay in organoids offered valuable insights about the beneficial effects of VX-770 + VX-661 + VX-445 (Elexacaftor + Tezacaftor + Ivacaftor, ETI) on N1303K-CFTR function restoration and about discouraging the prescription of VX-770 + VX-809 (Ivacaftor + Lumacaftor) or VX-770 + VX-661 (Ivacaftor + Tezacaftor) therapy for N1303K/class I patients. Then, a comprehensive assessment was conducted on an example of one patient with the N1303K/class I genotype to examine the ETI effect on the restoration of N1303K-CFTR function using in vitro the patient's intestinal organoids, ex vivo the intestinal current measurements (ICM) method and assessment of the clinical status before and after targeted therapy. All obtained results are consistent with each other and have proven the effectiveness of ETI for the N1303K variant. ETI produced a significant positive effect on forskolin-induced swelling in N1303K/class I organoids indicating functional improvement of the CFTR protein; ICM demonstrated that ETI therapy restored CFTR function in the intestinal epithelium after three months of treatment, and the patient improved his clinical status and lung function, increased his body mass index (BMI) and reduced the lung pathogenic flora diversity, surprisingly without improving the sweat test results.
Assuntos
Aminofenóis , Aminopiridinas , Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Quinolonas , Humanos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Colforsina/uso terapêutico , Mutação , Fibrose Cística/genética , Benzodioxóis/farmacologiaRESUMO
Coleus forskohlii (Willd.) Briq. is a medicinal herb of the Lamiaceae family. It is native to India and widely present in the tropical and sub-tropical regions of Egypt, China, Ethiopia, and Pakistan. The roots of C. forskohlii are edible, rich with pharmaceutically bioactive compounds, and traditionally reported to treat a variety of diseases, including inflammation, respiratory disorders, obesity, and viral ailments. Notably, the emergence of viral diseases is expected to quickly spread; consequently, these data impose a need for various approaches to develop broad active therapeutics for utilization in the management of future viral infectious outbreaks. In this study, the naturally occurring labdane diterpenoid derivative, Forskolin, was obtained from Coleus forskohlii. Additionally, we evaluated the antiviral potential of Forskolin towards three viruses, namely the herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), hepatitis A virus (HAV), and coxsackievirus B4 (COX-B4). We observed that Forskolin displayed antiviral activity against HAV, COX-B4, HSV-1, and HSV-2 with IC50 values of 62.9, 73.1, 99.0, and 106.0 µg/mL, respectively. Furthermore, we explored the Forskolin's potential antiviral target using PharmMapper, a pharmacophore-based virtual screening platform. Forskolin's modeled structure was analyzed to identify potential protein targets linked to its antiviral activity, with results ranked based on Fit scores. Cathepsin L (PDB ID: 3BC3) emerged as a top-scoring hit, prompting further exploration through molecular docking and MD simulations. Our analysis revealed that Forskolin's binding mode within Cathepsin L's active site, characterized by stable hydrogen bonding and hydrophobic interactions, mirrors that of a co-crystallized inhibitor. These findings, supported by consistent RMSD profiles and similar binding free energies, suggest Forskolin's potential in inhibiting Cathepsin L, highlighting its promise as an antiviral agent.
Assuntos
Herpesvirus Humano 1 , Colforsina/farmacologia , Colforsina/química , Catepsina L , Simulação de Acoplamento Molecular , Herpesvirus Humano 1/metabolismo , Antivirais/farmacologia , Antivirais/químicaRESUMO
Semi-anadromous animals experience salinity fluctuations during their life-span period. Alterations of environmental conditions induce stress response where catecholamines (CA) play a central role. Physiological stress and changes in external and internal osmolarity are frequently associated with increased production of reactive oxygen species (ROS). In this work, we studied the involvement of the cAMP/PKA pathway in mediating catecholamine-dependent effects on osmoregulatory responses, intracellular production of ROS, and mitochondrial membrane potential of the river lamprey (Lampetra fluviatilis, Linnaeus, 1758) red blood cells (RBCs). We also investigated the role of hypoosmotic shock in the process of ROS production and mitochondrial respiration of RBCs. For this, osmotic stability and the dynamics of the regulatory volume decrease (RVD) following hypoosmotic swelling, intracellular ROS levels, and changes in mitochondrial membrane potential were assessed in RBCs treated with epinephrine (Epi, 25 µM) and forskolin (Forsk, 20 µM). Epi and Forsk markedly reduced the osmotic stability of the lamprey RBCs whereas did not affect the dynamics of the RVD response in a hypoosmotic environment. Activation of PKA with Epi and Forsk increased ROS levels and decreased mitochondrial membrane potential of the lamprey RBCs. In contrast, upon hypoosmotic shock enhanced ROS production in RBCs was accompanied by increased mitochondrial membrane potential. Overall, a decrease in RBC osmotic stability and the enhancement of ROS formation induced by ß-adrenergic stimulation raises concerns about stress-associated changes in RBC functions in agnathans. Increased ROS production in RBCs under hypoosmotic shock indicates that a decrease in blood osmolarity may be associated with oxidative damage of RBCs during lamprey migration.
Assuntos
Epinefrina , Eritrócitos , Lampreias , Potencial da Membrana Mitocondrial , Pressão Osmótica , Espécies Reativas de Oxigênio , Animais , Eritrócitos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Pressão Osmótica/efeitos dos fármacos , Lampreias/fisiologia , Epinefrina/farmacologia , Colforsina/farmacologia , Osmorregulação/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismoRESUMO
Rhythmic regulations are virtually described in all physiological processes, including central nervous system development and immunologic responses. Zika virus (ZIKV), a neurotropic arbovirus, has been recently linked to a series of birth defects and neurodevelopmental disorders. Given the well-characterized role of the intrinsic cellular circadian clock within neurogenesis, cellular metabolism, migration, and differentiation among other processes, this study aimed to characterize the influence of ZIKV infection in the circadian clock expression in human neuronal cells. For this, in vitro models of human-induced neuroprogenitor cells (hiNPCs) and neuroblastoma cell line SH-SY5Y, cultured as monolayer and neurospheres, were infected by ZIKV, followed by RNA-Seq and RT-qPCR investigation, respectively. Targeted circadian clock components presented mRNA oscillations only after exogenous synchronizing stimuli (Forskolin) in SH-SY5Y monolayer culture. Interestingly, when these cells were grown as 3D-arranged neurospheres, an intrinsic oscillatory expression pattern was observed for some core clock components without any exogenous stimulation. The ZIKV infection significantly disturbed the mRNA expression pattern of core clock components in both neuroblastoma cell culture models, which was also observed in hiNPCs infected with different strains of ZIKV. The ZIKV-mediated desynchronization of the circadian clock expression in human cells might further contribute to the virus impairment of neuronal metabolism and function observed in adults and ZIKV-induced congenital syndrome. In vitro models of Zika virus (ZIKV) neuronal infection. Human neuroprogenitor cells were cultured as monolayer and neurospheres and infected by ZIKV. Monolayer-cultured cells received forskolin (FSK) as a coupling factor for the circadian clock rhythmicity, while 3D-arranged neurospheres showed an intrinsic oscillatory pattern in the circadian clock expression. The ZIKV infection affected the mRNA expression pattern of core clock components in both cell culture models. The ZIKV-mediated desynchronization of the circadian clock machinery might contribute to the impairment of neuronal metabolism and function observed in both adults (e.g., Guillain-Barré syndrome) and ZIKV-induced congenital syndrome (microcephaly). The graphical abstract has been created with Canva at the canva.com website.
Assuntos
Relógios Circadianos , Neuroblastoma , Infecção por Zika virus , Zika virus , Adulto , Humanos , Zika virus/genética , Colforsina , RNA Mensageiro/genéticaRESUMO
OBJECTIVES: The aim of the study was to culture vital salivary gland organoids obtained through labial or parotid biopsy of primary Sjögren's syndrome (pSS) patients in order to evaluate their morphological and functional features in basal condition and after stimulation with Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) activator forskolin and phosphodiesterase 4 (PDE4) inhibitor apremilast, their in vitro regenerative capacity and the immune-histological resemblance with original tissue. METHODS: Salivary gland tissues from five pSS patients were processed to obtain vital organoids; swelling assay and cell proliferation tests were performed after forskolin and apremilast application. Immunochemistry evaluation on original salivary gland tissue and corresponding organoids was performed, and secretomics analysis was conducted to assess their functional status. REULTS: After application of forskolin and apremilast, we observed organoid swelling after 30 minutes, compatible with a positive functional status and enhancement of saliva production. In 3 cases, apremilast induced organoid proliferation. All cases were positive for cytokeratin 14 (CK14) and most for cytokeratin 5 (CK5). All the cases were positive for amylase; its secretion, and thus functional status of organoids, was confirmed by its high concentration in the culture medium. A focal ductal differentiation was found in some cases, highlighted by epithelial membrane antigen (EMA) positivity. The more differentiated EMA positive areas were negative for the staminal marker CK14, showing a sort of "complementary staining". CONCLUSIONS: Our data highlighted that differentiated cells and vital functional organoids that recapitulate the development of original salivary glands can be obtained from pSS epithelium. For the first time, the direct stimulating effect of PDE4 inhibitor apremilast on pSS human salivary gland organoids is reported, opening new perspectives on targeting oral dryness with drugs that combine secretagogue and immunomodulatory effects.
Assuntos
Inibidores da Fosfodiesterase 4 , Síndrome de Sjogren , Humanos , Inibidores da Fosfodiesterase 4/farmacologia , Secretagogos , Colforsina , Glândulas Salivares , Organoides/metabolismo , Organoides/patologiaRESUMO
Purpose: Intravenous regional anesthesia (IVRA) using lidocaine provides effective localized analgesia but its duration is limited. The mechanism by which dexmedetomidine enhances lidocaine IVRA is unclear but may involve modulation of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Materials and Methods: Lidocaine IVRA with varying dexmedetomidine concentrations was performed in the tails of Sprague-Dawley rats. Tail-flick and tail-clamping tests assessed IVRA analgesia and anesthesia efficacy and duration. Contributions of α2 adrenergic receptors and HCN channels were evaluated by incorporating an α adrenergic receptor antagonist, the HCN channel inhibitor ZD7288, and the HCN channel agonist forskolin. Furthermore, whole-cell patch clamp electrophysiology quantified the effects of dexmedetomidine on HCN channels mediating hyperpolarization-activated cation current (Ih) in isolated dorsal root ganglion neurons. Results: Dexmedetomidine dose-dependently extended lidocaine IVRA duration and analgesia, unaffected by α2 receptor blockade. The HCN channel inhibitor ZD7288 also prolonged lidocaine IVRA effects, while the HCN channel activator forskolin shortened effects. In dorsal root ganglion neurons, dexmedetomidine concentration-dependently inhibited Ih amplitude and shifted the voltage-dependence of HCN channel activation. Conclusion: Dexmedetomidine prolongs lidocaine IVRA duration by directly inhibiting HCN channel activity, independent of α2 adrenergic receptor activation. This HCN channel inhibition represents a novel mechanism underlying the anesthetic and analgesic adjuvant effects of dexmedetomidine in IVRA.
Assuntos
Anestesia por Condução , Dexmedetomidina , Ratos , Animais , Lidocaína/farmacologia , Dexmedetomidina/farmacologia , Ratos Sprague-Dawley , Colforsina , CátionsRESUMO
Repeated sublethal hypoxia exposure induces brain inflammation and affects the initiation and progression of cognitive dysfunction. Experiments from the current study showed that hypoxic exposure downregulates PKA/CREB signaling, which is restored by forskolin (FSK), an adenylate cyclase activator, in both Neuro2a (N2a) cells and zebrafish brain. FSK significantly protected N2a cells from hypoxia-induced cell death and neurite shrinkage. Intraperitoneal administration of FSK for 5 days on zebrafish additionally led to significant recovery from hypoxia-induced social interaction impairment and learning and memory (L/M) deficit. FSK suppressed hypoxia-induced neuroinflammation, as indicated by the observed decrease in NF-κB activation and GFAP expression. We further investigated the potential effect of FSK on O-GlcNAcylation changes induced by hypoxia. Intriguingly FSK induced marked upregulation of the protein level of O-GlcNAc transferase catalyzing addition of the GlcNAc group to target proteins, accompanied by elevated O-GlcNAcylation of nucleocytoplasmic proteins. The hypoxia-induced O-GlcNAcylation decrease in the brain of zebrafish was considerably restored following FSK treatment. Based on the collective results, we propose that FSK rescues hypoxia-induced cognitive dysfunction, potentially through regulation of HBP/O-GlcNAc cycling.
Assuntos
Disfunção Cognitiva , Peixe-Zebra , Animais , Colforsina/farmacologia , Cognição , Hipóxia/complicações , Transtornos da MemóriaRESUMO
Human somatic cells can be reprogrammed into neuron cell fate through regulation of a single transcription factor or application of small molecule cocktails. Methods: Here, we report that forskolin efficiently induces the conversion of human somatic cells into induced neurons (FiNs). Results: A large population of neuron-like phenotype cells was observed as early as 24-36 h post-induction. There were >90% TUJ1-, >80% MAP2-, and >80% NEUN-positive neurons at 5 days post-induction. Multiple subtypes of neurons were present among TUJ1-positive cells, including >60% cholinergic, >20% glutamatergic, >10% GABAergic, and >5% dopaminergic neurons. FiNs exhibited typical neural electrophysiological activity in vitro and the ability to survive in vitro and in vivo more than 2 months. Mechanistically, forskolin functions in FiN reprogramming by regulating the cAMP-CREB1-JNK signals, which upregulates cAMP-CREB1 expression and downregulates JNK expression. Conclusion: Overall, our studies identify a safer and efficient single-small-molecule-driven reprogramming approach for induced neuron generation and reveal a novel regulatory mechanism of neuronal cell fate acquisition.
Assuntos
Reprogramação Celular , Fatores de Transcrição , Humanos , Colforsina/farmacologia , Diferenciação Celular/fisiologia , Fatores de Transcrição/metabolismo , Neurônios Dopaminérgicos/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP CíclicoRESUMO
BACKGROUND: Many disease-causing variants in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene remain uncharacterized and untreated. Restoring the function of the impaired CFTR protein is the goal of personalized medicine, particularly in patients carrying rare CFTR variants. In this study, functional defects related to the rare R334W variant were evaluated after treatment with CFTR modulators or Roflumilast, a phosphodiesterase-4 inhibitor (PDE4i). METHODS: Rectal organoids from subjects with R334W/2184insA and R334W/2183AA > G genotypes were used to perform the Forskolin-induced swelling (FIS) assay. Organoids were left drug-untreated or treated with modulators VX-770 (I), VX-445 (E), and VX-661 (T) mixed, and their combination (ETI). Roflumilast (R) was used alone or as a combination of I + R. RESULTS: Our data show a significant increase in FIS rate following treatment with I alone. The combined use of modulators, such as ETI, did not increase further swelling than I alone, nor in protein maturation. Treatment with R shows an increase in FIS response similar to those of I, and the combination R + I significantly increases the rescue of CFTR activity. CONCLUSIONS: Equivalent I and ETI treatment efficacy was observed for both genotypes. Furthermore, significant organoid swelling was observed with combined I + R used that supports the recently published data describing a potentiating effect of only I in patients carrying the variant R334W and, at the same time, corroborating the role of strategies that include PDE4 inhibitors further to potentiate the effect of I for this variant.