RESUMO
AIMS: Gene therapy with cardiac phosphodiesterases (PDEs), such as phosphodiesterase 4B (PDE4B), has recently been described to effectively prevent heart failure (HF) in mice. However, exact molecular mechanisms of its beneficial effects, apart from general lowering of cardiomyocyte cyclic adenosine monophosphate (cAMP) levels, have not been elucidated. Here, we studied whether gene therapy with two types of PDEs, namely PDE2A and PDE4B, can prevent pressure-overload-induced HF in mice by acting on and restoring altered cAMP compartmentation in distinct subcellular microdomains. METHODS AND RESULTS: HF was induced by transverse aortic constriction followed by tail-vein injection of adeno-associated-virus type 9 vectors to overexpress PDE2A3, PDE4B3, or luciferase for 8 weeks. Heart morphology and function was assessed by echocardiography and histology which showed that PDE2A and especially PDE4B gene therapy could attenuate cardiac hypertrophy, fibrosis, and decline of contractile function. Live cell imaging using targeted cAMP biosensors showed that PDE overexpression restored altered cAMP compartmentation in microdomains associated with ryanodine receptor type 2 (RyR2) and caveolin-rich plasma membrane. This was accompanied by ameliorated caveolin-3 decline after PDE2A3 overexpression, reduced RyR2 phosphorylation in PDE4B3 overexpressing hearts, and antiarrhythmic effects of both PDEs measured under isoproterenol stimulation in single cells. Strong association of overexpressed PDE4B but not PDE2A with RyR2 microdomain could prevent calcium leak and arrhythmias in human-induced pluripotent stem-derived cardiomyocytes with the A2254V mutation in RyR2 causing catecholaminergic polymorphic ventricular tachycardia. CONCLUSION: Our data indicate that gene therapy with phosphodiesterases can prevent HF including associated cardiac remodelling and arrhythmias by restoring altered cAMP compartmentation in functionally relevant subcellular microdomains.
Assuntos
AMP Cíclico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2 , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4 , Modelos Animais de Doenças , Terapia Genética , Insuficiência Cardíaca , Miócitos Cardíacos , Canal de Liberação de Cálcio do Receptor de Rianodina , Animais , AMP Cíclico/metabolismo , Insuficiência Cardíaca/enzimologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/terapia , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/genética , Miócitos Cardíacos/enzimologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Humanos , Camundongos Endogâmicos C57BL , Masculino , Arritmias Cardíacas/enzimologia , Arritmias Cardíacas/genética , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Arritmias Cardíacas/prevenção & controle , Remodelação Ventricular , Células-Tronco Pluripotentes Induzidas/enzimologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Função Ventricular Esquerda , Sinalização do Cálcio , Fosforilação , Frequência CardíacaRESUMO
Phosphodiesterase 2A (Pde2A) is a dual-specific PDE that breaks down both cAMP and cGMP cyclic nucleotides. We recently highlighted a direct relationship between Pde2A impairment, a consequent increase of cAMP, and the appearance of mouse congenital heart defects (CHDs). Here we aimed to characterize the pathways involved in the development of CHDs and in their prevention by pharmacological approaches targeting cAMP and cGMP signaling. Transcriptome analysis revealed a modulation of more than 500 genes affecting biological processes involved in the immune system, cardiomyocyte development and contractility, angiogenesis, transcription, and oxidative stress in hearts from Pde2A-/- embryos. Metoprolol and H89 pharmacological administration prevented heart dilatation and hypertabeculation in Pde2A-/- embryos. Metoprolol was also able to partially impede heart septum defect and oxidative stress at tissue and molecular levels. Amelioration of cardiac defects was also observed by using the antioxidant NAC, indicating oxidative stress as one of the molecular mechanisms underpinning the CHDs. In addition, Sildenafil treatment recovered cardiac defects suggesting the requirement of cAMP/cGMP nucleotides balance for the correct heart development.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 2 , Cardiopatias Congênitas , Camundongos , Animais , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Metoprolol , Transdução de Sinais , GMP Cíclico/metabolismo , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/prevenção & controle , Estresse OxidativoRESUMO
The molecular mechanisms by which lymphatic vessels induce cell contact inhibition are not understood. Here, we identify the cGMP-dependent phosphodiesterase 2A (PDE2A) as a selective regulator of lymphatic but not of blood endothelial contact inhibition. Conditional deletion of Pde2a in mouse embryos reveals severe lymphatic dysplasia, whereas blood vessel architecture remains unaltered. In the absence of PDE2A, human lymphatic endothelial cells fail to induce mature junctions and cell cycle arrest, whereas cGMP levels, but not cAMP levels, are increased. Loss of PDE2A-mediated cGMP hydrolysis leads to the activation of p38 signaling and downregulation of NOTCH signaling. However, DLL4-induced NOTCH activation restores junctional maturation and contact inhibition in PDE2A-deficient human lymphatic endothelial cells. In postnatal mouse mesenteries, PDE2A is specifically enriched in collecting lymphatic valves, and loss of Pde2a results in the formation of abnormal valves. Our data demonstrate that PDE2A selectively finetunes a crosstalk of cGMP, p38, and NOTCH signaling during lymphatic vessel maturation.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 2 , Vasos Linfáticos , Animais , Humanos , Camundongos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Regulação para Baixo , Células Endoteliais/metabolismo , Vasos Linfáticos/metabolismo , Transdução de SinaisRESUMO
Intellectual developmental disorder with paroxysmal dyskinesia or seizures (IDDPADS, OMIM#619150) is an ultra-rare childhood-onset autosomal recessive movement disorder manifesting paroxysmal dyskinesia, global developmental delay, impaired cognition, progressive psychomotor deterioration and/or drug-refractory seizures. We investigated three consanguineous Pakistani families with six affected individuals presenting overlapping phenotypes partially consistent with the reported characteristics of IDDPADS. Whole exome sequencing identified a novel missense variant in Phosphodiesterase 2A (PDE2A): NM_002599.4: c.1514T > C p.(Phe505Ser) that segregated with the disease status of individuals in these families. Retrospectively, we performed haplotype analysis that revealed a 3.16 Mb shared haplotype at 11q13.4 among three families suggesting a founder effect in this region. Moreover, we also observed abnormal mitochondrial morphology in patient fibroblasts compared to controls. Belonging to diverse age groups (13 years-60 years), patients presented paroxysmal dyskinesia, developmental delay, cognitive abnormalities, speech impairment, and drug-refractory seizures with variable onset of disease (as early as 3 months of age to 7 years). Together with the previous reports, we observed that intellectual disability, progressive psychomotor deterioration, and drug-refractory seizures are consistent outcomes of the disease. However, permanent choreodystonia showed variability. We also noticed that the later onset of paroxysmal dyskinesia manifests severe attacks in terms of duration. Being the first report from Pakistan, we add to the clinical and mutation spectrum of PDE2A-related recessive disease raising the total number of patients from six to 12 and variants from five to six. Together, with our findings, the role of PDE2A is strengthened in critical physio-neurological processes.
Assuntos
Coreia , Deficiência Intelectual , Humanos , Deficiência Intelectual/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/genética , Coreia/genética , Estudos Retrospectivos , Linhagem , Mutação/genética , Consanguinidade , ConvulsõesRESUMO
Cyclic nucleotide phosphodiesterases 2A (PDE2A) and PDE3A play an important role in the regulation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP)-to-cAMP crosstalk. Each of these PDEs has up to three distinct isoforms. However, their specific contributions to cAMP dynamics are difficult to explore because it has been challenging to generate isoform-specific knock-out mice or cells using conventional methods. Here, we studied whether the CRISPR/Cas9 approach for precise genome editing can be used to knock out Pde2a and Pde3a genes and their distinct isoforms using adenoviral gene transfer in neonatal and adult rat cardiomyocytes. Cas9 and several specific gRNA constructs were cloned and introduced into adenoviral vectors. Primary adult and neonatal rat ventricular cardiomyocytes were transduced with different amounts of Cas9 adenovirus in combination with PDE2A or PDE3A gRNA constructs and cultured for up to 6 (adult) or 14 (neonatal) days to analyze PDE expression and live cell cAMP dynamics. A decline in mRNA expression for PDE2A (~80%) and PDE3A (~45%) was detected as soon as 3 days post transduction, with both PDEs being reduced at the protein level by >50-60% in neonatal cardiomyocytes (after 14 days) and >95% in adult cardiomyocytes (after 6 days). This correlated with the abrogated effects of selective PDE inhibitors in the live cell imaging experiments based on using cAMP biosensor measurements. Reverse transcription PCR analysis revealed that only the PDE2A2 isoform was expressed in neonatal myocytes, while adult cardiomyocytes expressed all three PDE2A isoforms (A1, A2, and A3) which contributed to the regulation of cAMP dynamics as detected by live cell imaging. In conclusion, CRISPR/Cas9 is an effective tool for the in vitro knock-out of PDEs and their specific isoforms in primary somatic cells. This novel approach suggests distinct regulation of live cell cAMP dynamics by various PDE2A and PDE3A isoforms in neonatal vs. adult cardiomyocytes.
Assuntos
Sistemas CRISPR-Cas , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2 , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3 , Miócitos Cardíacos , Animais , Camundongos , Ratos , Sistemas CRISPR-Cas/genética , AMP Cíclico/metabolismo , Dietilestilbestrol , Miócitos Cardíacos/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Isoformas de Proteínas/metabolismoRESUMO
In Neurospora crassa, caffeine and other methylxanthines are known to inhibit phosphodiesterase (PDE) activity, leading to augmented cAMP levels. In this organism, it has also been shown that the addition of these drugs significantly lengthens the circadian period, as seen by conidiation rhythms. Utilizing in vivo bioluminescence reporters, pharmacological inhibitors, and cAMP analogs, we revisited the effect of methylxanthines and the role of cAMP signaling in the Neurospora clockworks. We observed that caffeine, like all tested methylxanthines, led to significant period lengthening, visualized with both core-clock transcriptional and translational reporters. Remarkably, this phenotype is still observed when phosphodiesterase (PDE) activity is genetically or chemically (via 3-isobutyl-1-methylxanthine) abrogated. Likewise, methylxanthines still exert a period effect in several cAMP signaling pathway mutants, including adenylate cyclase (cr-1) and protein kinase A (PKA) (Δpkac-1) mutants, suggesting that these drugs lead to circadian phenotypes through mechanisms different from the canonical PDE-cAMP-PKA signaling axis. Thus, this study highlights the strong impact of methylxanthines on circadian period in Neurospora, albeit the exact mechanisms somehow remain elusive. IMPORTANCE Evidence from diverse organisms show that caffeine causes changes in the circadian clock, causing period lengthening. The fungus Neurospora crassa is no exception; here, several methylxanthines such as caffeine, theophylline, and aminophylline cause period lengthening in a concentration-dependent manner. Although methylxanthines are expected to inhibit phosphodiesterase activity, we were able to show by genetic and pharmacological means that these drugs exert their effects through a different mechanism. Moreover, our results indicate that increases in cAMP levels and changes in PKA activity do not impact the circadian period and therefore are not part of underlying effects of methylxanthine. These results set the stage for future analyses dissecting the molecular mechanisms by which these drugs dramatically modify the circadian period.
Assuntos
Cafeína , Neurospora crassa , Neurospora crassa/efeitos dos fármacos , Neurospora crassa/fisiologia , Ritmo Circadiano/efeitos dos fármacos , AMP Cíclico/metabolismo , Cafeína/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/antagonistas & inibidores , 1-Metil-3-Isobutilxantina , Proteínas Quinases/metabolismo , Transdução de SinaisRESUMO
BACKGROUND: Post-traumatic stress disorder (PTSD) is the prevalent psychiatric disorder that induces alcohol use disorders (AUD) such as abnormal alcohol intake and anxiety. However, little is known about whether phosphodiesterase 2 (PDE2)-cAMP/cGMP signaling is involved in PTSD-induced AUD. METHODS: The present study used single-prolonged stress (SPS) to mimic PTSD that induced increases in ethanol intake and preference (2-bottle choice test) and anxiety-like behavior (elevated-plus maze test and novelty suppressed feeding test). PDE2 inhibitor Bay 60-7550 (Bay) was administered to the mice and protein kinase A (PKA) inhibitor H89 and PKG inhibitor KT5823 were micro-injected into dorsolateral striatum (DLS) and central amygdala (CA) of mice to determine whether the effects of Bay on anxiety-like behavior in SPS mice are brain region dependent. RESULTS: PDE2 inhibitor Bay rescued SPS-induced decreases in open arm entries and open arm time exposure in elevated-plus maze test and reversed increased latency to feed in the novelty suppressed feeding test. Moreover, SPS-induced ethanol use disorder was reversed by Bay as evidenced by decreased ethanol intake and preference without changing total fluid intake in the SPS mice after treatment with Bay. However, Bay did not change the ethanol metabolism or sucrose or quinine intake and preference. The locomotor activity was not affected after treatment with Bay. Interestingly, microinjection of PKA or PKG inhibitor H89 or KT5823 into DLS prevented the effects of Bay on alcohol intake and preference and cAMP-response element binding proteins phosphorylation and brain derived neurotrophic factor expression in DLS but not on the anxiety-like behavior in SPS mice. Microinjection of these inhibitors into CA prevented Bay-induced anxiolytic-like effects and cAMP-response element binding proteins phosphorylation and brain derived neurotrophic factor levels in CA but did not affect ethanol intake in SPS mice, indicating that the effects of Bay on different behaviors are brain region dependent. CONCLUSIONS: These findings support the hypothesis that PDE2-cAMP/cGMP signaling may differentially mediate PTSD-induced AUD and anxiety-like behavior.
Assuntos
Alcoolismo , Ansiolíticos , Transtornos de Estresse Pós-Traumáticos , Animais , Camundongos , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Transtornos de Estresse Pós-Traumáticos/tratamento farmacológico , Fator Neurotrófico Derivado do Encéfalo , Diester Fosfórico Hidrolases , GMP Cíclico/metabolismo , Consumo de Bebidas Alcoólicas/tratamento farmacológico , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Etanol , Modelos Animais de DoençasRESUMO
Phosphodiesterase (PDE) inhibitors have been safely and effectively used in the clinic and increase the concentration of intracellular cyclic nucleotides (cAMP/cGMP). These molecules activate downstream mediators, including the cAMP response element-binding protein (CREB), which controls neuronal excitability and growth responses. CREB gain of function enhances learning and allocates neurons into memory engrams. CREB also controls recovery after stroke. PDE inhibitors are linked to recovery from neural damage and to stroke recovery in specific sites within the brain. PDE2A is enriched in cortex. In the present study, we use a mouse cortical stroke model in young adult and aged male mice to test the effect of PDE2A inhibition on functional recovery, and on downstream mechanisms of axonal sprouting, tissue repair, and the functional connectivity of neurons in recovering cortex. Stroke causes deficits in use of the contralateral forelimb, loss of axonal projections in cortex adjacent to the infarct, and functional disconnection of neuronal networks. PDE2A inhibition enhances functional recovery, increases axonal projections in peri-infarct cortex, and, through two-photon in vivo imaging, enhances the functional connectivity of motor system excitatory neurons. PDE2A inhibition after stroke does not have an effect on other aspects of tissue repair, such as angiogenesis, gliogenesis, neurogenesis, and inflammatory responses. These data suggest that PDE2A inhibition is an effective therapeutic approach for stroke recovery in the rodent and that it simultaneously enhances connectivity in peri-infarct neuronal populations.SIGNIFICANCE STATEMENT Inhibition of PDE2A enhances motor recovery, axonal projections, and functional connectivity of neurons in peri-infarct tissue. This represents an avenue for a pharmacological therapy for stroke recovery.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 2 , Acidente Vascular Cerebral , Animais , Masculino , Camundongos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Infarto , Neurônios Motores , Neurogênese , Inibidores de Fosfodiesterase/farmacologia , Recuperação de Função Fisiológica/fisiologia , Acidente Vascular Cerebral/tratamento farmacológico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/antagonistas & inibidoresRESUMO
BACKGROUND: Aldosterone is a critical pathological driver for cardiac and renal diseases. We recently discovered that mutant atrial natriuretic peptide (MANP), a novel atrial natriuretic peptide (ANP) analog, possessed more potent aldosterone inhibitory action than ANP in vivo. MANP and natriuretic peptide (NP)-augmenting therapy sacubitril/valsartan are under investigations for human hypertension treatment. Understanding the elusive mechanism of aldosterone inhibition by NPs remains to be a priority. Conflicting results were reported on the roles of the pGC-A (particulate guanylyl cyclase A receptor) and NP clearance receptor in aldosterone inhibition. Furthermore, the function of PKG (protein kinase G) and PDEs (phosphodiesterases) on aldosterone regulation are not clear. METHODS: In the present study, we investigated the molecular mechanism of aldosterone regulation in a human adrenocortical cell line H295R and in mice. RESULTS: We first provided evidence to show that pGC-A, not NP clearance receptor, mediates aldosterone inhibition. Next, we confirmed that MANP inhibits aldosterone via PDE2 (phosphodiesterase 2) not PKG, with specific agonists, antagonists, siRNA silencing, and fluorescence resonance energy transfer experiments. Further, the inhibitory effect is mediated by a reduction of intracellular Ca2+ levels. We then illustrated that MANP directly reduces aldosterone synthase CYP11B2 (cytochrome p450 family 11 subfamily b member 2) expression via PDE2. Last, in PDE2 knockout mice, consistent with in vitro findings, embryonic adrenal CYP11B2 is markedly increased. CONCLUSIONS: Our results innovatively explore and expand the NP/pGC-A/3',5', cyclic guanosine monophosphate (cGMP)/PDE2 pathway for aldosterone inhibition by MANP in vitro and in vivo. In addition, our data also support the development of MANP as a novel ANP analog drug for aldosterone excess treatment.
Assuntos
Aldosterona , Fator Natriurético Atrial , Aldosterona/farmacologia , Aminobutiratos , Animais , Fator Natriurético Atrial/farmacologia , Compostos de Bifenilo , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2 , Citocromo P-450 CYP11B2/genética , Humanos , Camundongos , Camundongos Knockout , Peptídeos NatriuréticosRESUMO
Our previous study suggested that inhibition of Phosphodiesterase 2 ameliorates memory loss upon exposure to oxidative stress. While whether memory enhancing effects of PDE2 inhibition on Alzheimer's disease mouse model are involved in antioxidant defense and neuronal remodeling, are largely unexplored. The present study addressed whether and how PDE2 inhibitor Bay 60-7550 rescued Aß oligomers (Aßo)-induced neuronal damage and memory impairment. The results suggested that exposure of primary cortical neurons to Aßo induced neuronal cells damage and increased PDE2 expression, which were paralleled to an increase in the oxidative parameter malondialdehyde (MDA) level and cellular apoptosis. However, this Aßo-induced oxidative damage was blocked by pre-treatment with protein kinase A or G (PKA or PKG) inhibitor, suggesting the involvement of cAMP/cGMP signaling. Moreover, microinjection of Aßo into the prefrontal cortex of mice increased the MDA level; while Bay 60-7550 reversed this effect and increased antioxidant and anti-apoptotic factors, i.e. increased trolox-equivalent-antioxidant capacity and Bcl-2/Bax ratio. Bay 60-7550 also rescued Aßo-induced synaptic atrophy and memory deficits, as evidenced by the increased synaptic proteins' levels and spine density in the prefrontal cortex, and improved cognitive behaviors by decreased working memory errors in the eight-arm maze and increased discrimination index in the novel object recognition test. These findings suggest that inhibition of PDE2 contributes to antioxidant defense and neuronal remodeling by regulation of cAMP/cGMP signaling, which provide a theoretical basis for the future use of PDE2 inhibitors as the anti-AD drugs.
Assuntos
Doença de Alzheimer , Inibidores de Fosfodiesterase , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Antioxidantes/farmacologia , GMP Cíclico/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2 , Hipocampo , Transtornos da Memória/tratamento farmacológico , Camundongos , Camundongos Endogâmicos ICR , Neurônios , Fragmentos de Peptídeos , Inibidores de Fosfodiesterase/farmacologia , Inibidores de Fosfodiesterase/uso terapêuticoRESUMO
BACKGROUND: Disruption of cyclic nucleotide signaling in sympathetic postganglionic neurons contributes to impaired intracellular calcium handling (Ca2+) and the development of dysautonomia during the early stages of hypertension, although how this occurs is poorly understood. Emerging evidence supports the uncoupling of signalosomes in distinct cellular compartments involving cyclic nucleotide-sensitive PDEs (phosphodiesterases), which may underpin the autonomic phenotype in stellate neurons. METHODS: Using a combination of single-cell RNA sequencing together with Forster resonance energy transfer-based sensors to monitor cyclic adenosine 3',5'-monophosphate, PKA (protein kinase A)-dependent phosphorylation and cGMP (cyclic guanosine 3',5'-monophosphate), we tested the hypothesis that dysregulation occurs in a sub-family of PDEs in the cytosol and outer mitochondrial membrane of neurons from the stellate ganglion. RESULTS: PDE2A, 6D, 7A, 9A genes were highly expressed in young Wistar neurons and also conserved in neurons from spontaneously hypertensive rats (SHRs). In stellate neurons from prehypertensive SHRs, we found the levels of cyclic adenosine 3',5'-monophosphate and cGMP at the outer mitochondrial membrane were decreased compared with normal neurons. The reduced cyclic adenosine 3',5'-monophosphate response was due to the hydrolytic activity of overexpressed PDE2A2 located at the mitochondria. Normal cyclic adenosine 3',5'-monophosphate levels were re-established by inhibition of PDE2A. There was also a greater PKA-dependent phosphorylation in the cytosol and at the outer mitochondrial membrane in spontaneously hypertensive rat neurons, where this response was regulated by protein phosphatases. The cGMP response was only restored by inhibition of PDE6. CONCLUSIONS: When taken together, these results suggest that site-specific inhibition of PDE2A and PDE6D at the outer mitochondrial membrane may provide a therapeutic target to ameliorate cardiac sympathetic impairment during the onset of hypertension.
Assuntos
Hipertensão , Membranas Mitocondriais , Adenosina , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Membranas Mitocondriais/metabolismo , Neurônios/metabolismo , Nucleotídeos Cíclicos , Ratos , Ratos Endogâmicos SHR , Ratos WistarRESUMO
Pharmacological inhibition of phosphodiesterase 2A (PDE2A), which catalyzes the hydrolysis of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), has recently been proposed as a novel therapeutic tool for Fragile X Syndrome (FXS), the leading monogenic cause of Autism Spectrum Disorder (ASD). Here, we investigated the role of PDE2A in ASD pathogenesis using two rat models that reflect one of either the genetic or environmental factors involved in the human disease: the genetic Fmr1-Δexon 8 rat model and the environmental rat model based on prenatal exposure to valproic acid (VPA, 500 mg/kg). Prior to behavioral testing, the offspring was treated with the PDE2A inhibitor BAY607550 (0.05 mg/kg at infancy, 0.1 mg/kg at adolescence and adulthood). Socio-communicative symptoms were assessed in both models through the ultrasonic vocalization test at infancy and three-chamber test at adolescence and adulthood, while cognitive impairments were assessed by the novel object recognition test in Fmr1-Δexon 8 rats (adolescence and adulthood) and by the inhibitory avoidance test in VPA-exposed rats (adulthood). PDE2A enzymatic activity in VPA-exposed infant rats was also assessed. In line with the increased PDE2A enzymatic activity previously observed in the brain of Fmr1-KO animals, we found an altered upstream regulation of PDE2A activity in the brain of VPA-exposed rats at an early developmental age (p < 0.05). Pharmacological inhibition of PDE2A normalized the communicative (p < 0.01, p < 0.05), social (p < 0.001, p < 0.05), and cognitive impairment (p < 0.001) displayed by both Fmr1-Δexon 8 and VPA-exposed rats. Altogether, these data highlight a key role of PDE2A in brain development and point to PDE2A inhibition as a promising pharmacological approach for the deficits common to both FXS and ASD.
Assuntos
Transtorno do Espectro Autista , Síndrome do Cromossomo X Frágil , Animais , Transtorno do Espectro Autista/induzido quimicamente , Transtorno do Espectro Autista/tratamento farmacológico , Transtorno do Espectro Autista/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Feminino , Proteína do X Frágil da Deficiência Intelectual , Síndrome do Cromossomo X Frágil/genética , Gravidez , Ratos , Ácido Valproico/farmacologiaRESUMO
Phosphodiesterase 2 (PDE2A) modulates the levels of cAMP/cGMP and was recently found to be involved in mitochondria function regulation, closely related to multiple types of tumor progression. This study aimed to estimate the prognostic significance and biological effects of PDE2A on hepatocellular carcinoma (HCC). We comprehensively analyzed the PDE2A mRNA expression in HCC based on The Cancer Genome Atlas (TCGA) database and investigated the effects of PDE2A on the proliferation and metastatic capacity of HCC cells. PDE2A was downregulated in 25 cancer types, including HCC. Lower PDE2A expression was a protective factor in HCC and was negatively associated with serum AFP levels, tumor status, vascular invasion, histologic grade, and pathologic stage of HCC. Moreover, tumors with low PDE2A expression displayed a decreased immune function. Then, the ROC curve was used to assess the diagnostic ability of PDE2A in HCC (AUC = 0.823 in TCGA and AUC = 0.901 in GSE76427). Patients with low PDE2A expression exhibited worse outcomes compared with those with high PDE2A expression. Additionally, GO functional annotations demonstrated the involvement of PDE2A in the ECM organization, systems development, and ERK-related pathways, indicating that PDE2A might regulate HCC growth and metastasis. The in vitro experiments confirmed that overexpression of PDE2A inhibited proliferation, colony formation, migration, and invasion in two HCC cell lines (HLF and SNU-368), while inhibition of PDE2A has the opposite results. The mechanism of PDE2A's effect on HCC cells is attributed to the change of mitochondrial morphology and ATP content. These data demonstrated that PDE2A closely participated in the regulation of HCC proliferation and metastasis and can be used as a predictive marker candidate and a potential therapeutic target for HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/patologia , Neoplasias Hepáticas/patologia , Linhagem Celular , Sistema de Sinalização das MAP Quinases , Trifosfato de Adenosina/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismoRESUMO
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.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/genética , Proteínas Ativadoras de GTPase/genética , Hexosiltransferases/genética , Proteínas de Saccharomyces cerevisiae/genética , Esfingolipídeos/genética , Ceramidas/biossíntese , Ceramidas/genética , AMP Cíclico/genética , Regulação Fúngica da Expressão Gênica/genética , Glicerol/metabolismo , Sistema de Sinalização das MAP Quinases , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Concentração Osmolar , Saccharomyces cerevisiae/genética , Esfingolipídeos/biossíntese , Ativação Transcricional/genéticaRESUMO
Rationale: The malignant phenotypes of glioblastomas (GBMs) are primarily attributed to glioma stem cells (GSCs). Our previous study and other reports have suggested that both miR-139 and its host gene PDE2A are putative antitumor genes in various cancers. The aim of this study was to investigate the roles and mechanisms of miR-139/PDE2A in GSC modulation. Methods: Clinical samples were used to determine miR-139/PDE2A expression. Patient-derived glioma stem-like cells (PD-GSCs) were stimulated for immunofluorescent staining, sphere formation assays and orthotopic GBM xenograft models. Bioinformatic analysis and further in vitro experiments demonstrated the downstream molecular mechanisms of miR-139 and PDE2A. OX26/CTX-conjugated PEGylated liposome (OCP) was constructed to deliver miR-139 or PDE2A into glioma tissue specifically. Results: We demonstrated that miR-139 was concomitantly transcribed with its host gene PDE2A. Both PDE2A and miR-139 indicated better prognosis of gliomas and were inversely correlated with GSC stemness. PDE2A or miR-139 overexpression suppressed the stemness of PD-GSCs. FZD3 and ß-catenin, which induced Wnt/ß-catenin signaling activation, were identified as targets of miR-139 and mediated the effects of miR-139 on GSCs. Meanwhile, PDE2A suppressed Wnt/ß-catenin signaling by inhibiting cAMP accumulation and GSK-3ß phosphorylation, thereby modulating the self-renewal of PD-GSCs. Notably, Notch1, which is also a target of miR-139, suppressed PDE2A/miR-139 expression directly via downstream Hes1, indicating that miR-139 promoted its own expression by the miR-139-Notch1/Hes1 feedback circuit. Expectedly, targeted overexpression miR-139 or PDE2A in glioma with OCP system significantly repressed the stemness and decelerated glioma progression. Conclusions: Our findings elaborate on the inhibitory functions of PDE2A and miR-139 on GSC stemness and tumorigenesis, which may provide new prognostic markers and therapeutic targets for GBMs.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Glioma/metabolismo , MicroRNAs/metabolismo , Células-Tronco Neoplásicas , Via de Sinalização Wnt , Animais , AMP Cíclico/metabolismo , Glioma/patologia , Humanos , Camundongos Nus , Receptor Notch1/metabolismo , beta Catenina/metabolismoRESUMO
Streptococcus mitis is a commensal bacterial species of the oral cavity, with the potential for opportunistic pathogenesis. For successful colonization, S. mitis must be able to adhere to surfaces of the oral cavity and survive and adapt to frequently changing environmental conditions. Cyclic-di-AMP (c-di-AMP) is a nucleotide second messenger, involved in the regulation of stress responses and biofilm formation in several bacterial species. Cyclic-di-AMP is produced by diadenylate cyclases and degraded by phosphodiesterases. We have previously shown that in S. mitis, one diadenylate cyclase (CdaA) and at least two phosphodiesterases (Pde1 and Pde2) regulate the intracellular concentration of c-di-AMP. In this study, we utilized S. mitis deletion mutants of cdaA, pde1, and pde2 to analyze the role of c-di-AMP signaling in various stress responses, biofilm formation, and adhesion to eukaryotic cells. Here, we demonstrate that the Δpde1 mutant displayed a tendency toward increased susceptibility to acetic acid at pH 4.0. Deletion of cdaA increases auto-aggregation of S. mitis but reduces biofilm formation on an abiotic surface. These phenotypes are more pronounced under acidic extracellular conditions. Inactivation of pde1 or pde2 reduced the tolerance to ciprofloxacin, and UV radiation and the Δpde1 mutant was more susceptible to Triton X-100, indicating a role for c-di-AMP signaling in responses to DNA damage and cell membrane perturbation. Finally, the Δpde2 mutant displayed a tendency toward a reduced ability to adhere to oral keratinocytes. Taken together, our results indicate an important role for c-di-AMP signaling in cellular processes important for colonization of the mouth.
Assuntos
Adaptação Fisiológica/fisiologia , Aderência Bacteriana/fisiologia , Biofilmes/crescimento & desenvolvimento , AMP Cíclico/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Streptococcus mitis/metabolismo , Ácido Acético/farmacologia , Linhagem Celular Tumoral , Ciprofloxacina/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 1/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Queratinócitos/microbiologia , Boca/microbiologia , Octoxinol/farmacologia , Fósforo-Oxigênio Liases/genética , Fósforo-Oxigênio Liases/metabolismo , Streptococcus mitis/crescimento & desenvolvimento , Estresse Fisiológico/fisiologiaRESUMO
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.
Assuntos
Arritmias Cardíacas/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Animais , Antiarrítmicos/farmacologia , Arritmias Cardíacas/induzido quimicamente , Arritmias Cardíacas/patologia , Cálcio/metabolismo , AMP Cíclico/genética , GMP Cíclico/genética , Regulação da Expressão Gênica/genética , Coração/fisiopatologia , Humanos , Isoproterenol/toxicidade , Camundongos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismoRESUMO
Cyclic nucleotide phosphodiesterase (PDE) enzymes catalyze the hydrolysis and inactivation of the cyclic nucleotides cyclic adenosine monophosphate and cyclic guanosine monophosphate, which act as intracellular second messengers for many signal transduction pathways in the central nervous system. Several classes of PDE enzymes with specific tissue distributions and cyclic nucleotide selectivity are highly expressed in brain regions involved in cognitive and motor functions, which are known to be implicated in neurodegenerative diseases, such as Parkinson's disease and Huntington's disease. The indication that PDEs are intimately involved in the pathophysiology of different movement disorders further stems from recent discoveries that mutations in genes encoding different PDEs, including PDE2A, PDE8B, and PDE10A, are responsible for rare forms of monogenic parkinsonism and chorea. We here aim to provide a translational overview of the preclinical and clinical data on PDEs, the role of which is emerging in the field of movement disorders, offering a novel venue for a better understanding of their pathophysiology. Modulating cyclic nucleotide signaling, by either acting on their synthesis or on their degradation, represents a promising area for development of novel therapeutic approaches. The study of PDE mutations linked to monogenic movement disorders offers the opportunity of better understanding the role of PDEs in disease pathogenesis, a necessary step to successfully benefit the treatment of both hyperkinetic and hypokinetic movement disorders. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Assuntos
Doença de Parkinson , Diester Fosfórico Hidrolases , 3',5'-AMP Cíclico Fosfodiesterases , AMP Cíclico , GMP Cíclico , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2 , Humanos , Diester Fosfórico Hidrolases/genéticaRESUMO
A focused SAR study was conducted on a series of N1-substituted pyrazolopyrimidinone PDE2 inhibitors to reveal compounds with excellent potency and selectivity. The series was derived from previously identified internal leads and designed to enhance steric interactions with key amino acids in the PDE2 binding pocket. Compound 26 was identified as a lead compound with excellent PDE2 selectivity and good physicochemical properties.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/antagonistas & inibidores , Descoberta de Drogas , Inibidores de Fosfodiesterase/farmacologia , Pirazóis/farmacologia , Pirimidinonas/farmacologia , Cristalografia por Raios X , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Inibidores de Fosfodiesterase/síntese química , Inibidores de Fosfodiesterase/química , Pirazóis/síntese química , Pirazóis/química , Pirimidinonas/síntese química , Pirimidinonas/química , Relação Estrutura-AtividadeRESUMO
The multi-target-directed-ligand (MTDL) strategy has been widely applied in the discovery of novel drugs for the treatment of Alzheimer's disease (AD) because of the multifactorial pathological mechanisms of AD. Phosphodiesterase-2 (PDE2) has been identified to be a novel and promising target for AD. However, MTDL combining with the inhibitory activity against PDE2A and other anti-AD factors such as antioxidants has not been developed yet. Herein, a novel series of PDE2 inhibitors with antioxidant capacities were designed, synthesized, and evaluated. Most compounds showed remarkable inhibitory activities against PDE2A as well as antioxidant activities. Compound 6d was selected, which showed good IC50 of 6.1 nM against PDE2A, good antioxidant activity (ORAC (Trolox) = 8.4 eq.) and no cytotoxicity to SH-SY5Y cells. Molecular docking and dynamics simulations were applied for the rational design and explanation of structure-activity relationship (SAR) of lead compounds.