RESUMO
Many physiologic effects of l-glutamate, the major excitatory neurotransmitter in the mammalian central nervous system, are mediated via signaling by ionotropic glutamate receptors (iGluRs). These ligand-gated ion channels are critical to brain function and are centrally implicated in numerous psychiatric and neurologic disorders. There are different classes of iGluRs with a variety of receptor subtypes in each class that play distinct roles in neuronal functions. The diversity in iGluR subtypes, with their unique functional properties and physiologic roles, has motivated a large number of studies. Our understanding of receptor subtypes has advanced considerably since the first iGluR subunit gene was cloned in 1989, and the research focus has expanded to encompass facets of biology that have been recently discovered and to exploit experimental paradigms made possible by technological advances. Here, we review insights from more than 3 decades of iGluR studies with an emphasis on the progress that has occurred in the past decade. We cover structure, function, pharmacology, roles in neurophysiology, and therapeutic implications for all classes of receptors assembled from the subunits encoded by the 18 ionotropic glutamate receptor genes. SIGNIFICANCE STATEMENT: Glutamate receptors play important roles in virtually all aspects of brain function and are either involved in mediating some clinical features of neurological disease or represent a therapeutic target for treatment. Therefore, understanding the structure, function, and pharmacology of this class of receptors will advance our understanding of many aspects of brain function at molecular, cellular, and system levels and provide new opportunities to treat patients.
Assuntos
Receptores de Glutamato , Receptores Ionotrópicos de Glutamato , Animais , Sistema Nervoso Central , Ácido Glutâmico , Humanos , Neurotransmissores , Receptores Ionotrópicos de Glutamato/genéticaRESUMO
BACKGROUND: Tourette syndrome is a neurodevelopmental movement disorder involving basal ganglia dysfunction. PDE10A inhibitors modulate signaling in the striatal basal ganglia nuclei and are thus of interest as potential therapeutics in treating Tourette syndrome and other movement disorders. METHODS: The preclinical pharmacology and toxicology, human safety and tolerability, and human PET striatal enzyme occupancy data for the PDE10A inhibitor EM-221 are presented. RESULTS: EM-221 inhibited PDE10A with an in vitro IC50 of 9 pM and was >100,000 selective vs. other PDEs and other CNS receptors and enzymes. In rats, at doses of 0.05-0.50 mg/kg, EM-221 reduced hyperlocomotion and the disruption of prepulse inhibition induced by MK-801, attenuated conditioned avoidance, and facilitated novel object recognition, consistent with PDE10A's inhibition. EM-221 displayed no genotoxicity and was well tolerated up to 300 mg/kg in rats and 100 mg/kg in dogs. In single- and multiple-day ascending dose studies in healthy human volunteers, EM-221 was well tolerated up to 10 mg, with a maximum tolerated dose of 15 mg. PET imaging indicated that a PDE10A enzyme occupancy of up to 92.8% was achieved with a ~24 h half-life. CONCLUSIONS: The preclinical and clinical data presented here support the study of EM-221 in phase 2 trials of Tourette syndrome and other movement disorders.
Assuntos
Diester Fosfórico Hidrolases , Síndrome de Tourette , Adulto , Animais , Cães , Feminino , Humanos , Masculino , Ratos , Transtornos dos Movimentos/tratamento farmacológico , Inibidores de Fosfodiesterase/farmacologia , Inibidores de Fosfodiesterase/uso terapêutico , Diester Fosfórico Hidrolases/metabolismo , Tomografia por Emissão de Pósitrons , Ratos Sprague-Dawley , Síndrome de Tourette/tratamento farmacológico , HaplorrinosRESUMO
Over the past decade, human genome-wide association and expression studies have strongly implicated dysregulation of the innate immune system in the pathogenesis of Alzheimer's disease (AD). Single cell mRNA sequencing studies have identified innate immune cell subtypes that are minimally present in normal healthy brain, but whose numbers greatly increase in association with AD pathology. These AD pathology-associated immune cells are putatively the locus for the immune-related AD risk. While the prevailing view is that these immune cells arise from transformation of resident brain microglia, studies across several decades and using multiple techniques and strategies suggest instead that the pathology-associated immune cells are bone-marrow derived hematopoietic cells that are recruited into brain. We critically review this translational literature, emphasizing the strengths and limitations of techniques used to address recruitment and the experimental designs employed. We conclude that the aggregate evidence points toward recruitment into brain of innate immune cells of the myeloid dendritic cell lineage. Recruitment of dendritic cells and their role in AD pathogenesis has broad implications for our understanding of the etiology and pathobiology of AD that impact the strategies to develop new, immune system-targeted therapeutics for this devastating disease.
RESUMO
N-methyl-D-aspartate (NMDA) receptors mediate a slow component of excitatory synaptic transmission, are widely distributed throughout the central nervous system, and regulate synaptic plasticity. NMDA receptor modulators have long been considered as potential treatments for psychiatric disorders including depression and schizophrenia, neurodevelopmental disorders such as Rett Syndrome, and neurodegenerative conditions such as Alzheimer's disease. New interest in NMDA receptors as therapeutic targets has been spurred by the findings that certain inhibitors of NMDA receptors produce surprisingly rapid and robust antidepressant activity by a novel mechanism, the induction of changes in the brain that well outlast the presence of drug in the body. These findings are driving research into an entirely new paradigm for using NMDA receptor antagonists in a host of related conditions. At the same time positive allosteric modulators of NMDA receptors are being pursued for enhancing synaptic function in diseases that feature NMDA receptor hypofunction. While there is great promise, developing the therapeutic potential of NMDA receptor modulators must also navigate the potential significant risks posed by the use of such agents. We review here the emerging pharmacology of agents that target different NMDA receptor subtypes, offering new avenues for capturing the therapeutic potential of targeting this important receptor class.
Assuntos
Psiquiatria , Esquizofrenia , Humanos , Receptores de N-Metil-D-Aspartato/metabolismo , Sistema Nervoso Central , Encéfalo/metabolismoRESUMO
α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) positive allosteric modulation (i.e., "potentiation") has been proposed to overcome cognitive impairments in schizophrenia, but AMPAR overstimulation can be excitotoxic. Thus, it is critical to define carefully a potentiator's mechanism-based therapeutic index (TI) and to determine confidently its translatability from rodents to higher-order species. Accordingly, the novel AMPAR potentiator N-{(3R,4S)-3-[4-(5-cyano-2-thienyl)phenyl]tetrahydro-2H-pyran-4-yl}propane-2-sulfonamide (PF-4778574) was characterized in a series of in vitro assays and single-dose animal studies evaluating AMPAR-mediated activities related to cognition and safety to afford an unbound brain compound concentration (Cb,u)-normalized interspecies exposure-response relationship. Because it is unknown which AMPAR subtype(s) may be selectively potentiated for an optimal TI, PF-4778574 binding affinity and functional potency were determined in rodent tissues expected to express a native mixture of AMPAR subunits and their associated proteins to afford composite pharmacological values. Functional activity was also quantified in recombinant cell lines stably expressing human GluA2 flip or flop homotetramers. Procognitive effects of PF-4778574 were evaluated in both rat electrophysiological and nonhuman primate (nhp) behavioral models of pharmacologically induced N-methyl-d-aspartate receptor hypofunction. Safety studies assessed cerebellum-based AMPAR activation (mouse) and motor coordination disruptions (mouse, dog, and nhp), as well as convulsion (mouse, rat, and dog). The resulting empirically derived exposure-response continuum for PF-4778574 defines a single-dose-based TI of 8- to 16-fold for self-limiting tremor, a readily monitorable clinical adverse event. Importantly, the Cb,u mediating each physiological effect were highly consistent across species, with efficacy and convulsion occurring at just fractions of the in vitro-derived pharmacological values.
Assuntos
Agonistas de Aminoácidos Excitatórios/farmacologia , Receptores de AMPA/agonistas , Receptores de AMPA/fisiologia , Tiofenos/farmacologia , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Animais , Células Cultivadas , Cães , Agonistas de Aminoácidos Excitatórios/uso terapêutico , Feminino , Células HEK293 , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Desempenho Psicomotor/efeitos dos fármacos , Desempenho Psicomotor/fisiologia , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Convulsões/fisiopatologia , Convulsões/prevenção & controle , Tiofenos/uso terapêutico , Resultado do TratamentoRESUMO
The mammalian ionotropic glutamate receptor family encodes 18 gene products that coassemble to form ligand-gated ion channels containing an agonist recognition site, a transmembrane ion permeation pathway, and gating elements that couple agonist-induced conformational changes to the opening or closing of the permeation pore. Glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system and are localized on neuronal and non-neuronal cells. These receptors regulate a broad spectrum of processes in the brain, spinal cord, retina, and peripheral nervous system. Glutamate receptors are postulated to play important roles in numerous neurological diseases and have attracted intense scrutiny. The description of glutamate receptor structure, including its transmembrane elements, reveals a complex assembly of multiple semiautonomous extracellular domains linked to a pore-forming element with striking resemblance to an inverted potassium channel. In this review we discuss International Union of Basic and Clinical Pharmacology glutamate receptor nomenclature, structure, assembly, accessory subunits, interacting proteins, gene expression and translation, post-translational modifications, agonist and antagonist pharmacology, allosteric modulation, mechanisms of gating and permeation, roles in normal physiological function, as well as the potential therapeutic use of pharmacological agents acting at glutamate receptors.
Assuntos
Canais Iônicos/fisiologia , Receptores de Glutamato/fisiologia , Expressão Gênica , Humanos , Canais Iônicos/química , Canais Iônicos/genética , Processamento de Proteína Pós-Traducional , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologia , Receptores de Glutamato/química , Receptores de Glutamato/genética , Transmissão Sináptica , Terminologia como AssuntoRESUMO
Cyclic nucleotides are critical regulators of synaptic plasticity and participate in requisite signaling cascades implicated across multiple neurotransmitter systems. Phosphodiesterase 9A (PDE9A) is a high-affinity, cGMP-specific enzyme widely expressed in the rodent central nervous system. In the current study, we observed neuronal staining with antibodies raised against PDE9A protein in human cortex, cerebellum, and subiculum. We have also developed several potent, selective, and brain-penetrant PDE9A inhibitors and used them to probe the function of PDE9A in vivo. Administration of these compounds to animals led to dose-dependent accumulation of cGMP in brain tissue and cerebrospinal fluid, producing a range of biological effects that implied functional significance for PDE9A-regulated cGMP in dopaminergic, cholinergic, and serotonergic neurotransmission and were consistent with the widespread distribution of PDE9A. In vivo effects of PDE9A inhibition included reversal of the respective disruptions of working memory by ketamine, episodic and spatial memory by scopolamine, and auditory gating by amphetamine, as well as potentiation of risperidone-induced improvements in sensorimotor gating and reversal of the stereotypic scratching response to the hallucinogenic 5-hydroxytryptamine 2A agonist mescaline. The results suggested a role for PDE9A in the regulation of monoaminergic circuitry associated with sensory processing and memory. Thus, PDE9A activity regulates neuronal cGMP signaling downstream of multiple neurotransmitter systems, and inhibition of PDE9A may provide therapeutic benefits in psychiatric and neurodegenerative diseases promoted by the dysfunction of these diverse neurotransmitter systems.
Assuntos
3',5'-AMP Cíclico Fosfodiesterases/antagonistas & inibidores , 3',5'-GMP Cíclico Fosfodiesterases/metabolismo , Colinérgicos/farmacologia , GMP Cíclico/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Inibidores de Fosfodiesterase/farmacologia , 3',5'-AMP Cíclico Fosfodiesterases/genética , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Animais , Aprendizagem da Esquiva/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Feminino , Humanos , Macaca fascicularis , Masculino , Memória/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Neurotransmissores/farmacologia , Ratos , Ratos Long-Evans , Ratos Wistar , Filtro Sensorial/efeitos dos fármacos , Comportamento Estereotipado/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacosRESUMO
A promising target for memory improvement is phosphodiesterase type 5 (PDE5), which selectively hydrolyzes cyclic guanosine monophosphate (cGMP). In rodents, PDE5 inhibitors (PDE5-Is) have been shown to improve memory performance in many behavioral paradigms. However, it is questioned whether the positive effects in animal studies result from PDE5 inhibition in the central nervous system or the periphery. Therefore, we studied the effects of PDE5 inhibition on memory and determined whether compound penetration of the blood-brain barrier (BBB) is required for this activity. Two selective PDE5-Is, vardenafil and UK-343,664, were tested in the object recognition task (ORT) in both a MK-801- and scopolamine-induced memory deficit model, and a time-delay model without pharmacological intervention. Compounds were dosed 30 min before the learning trial of the task. To determine if the PDE5-Is crossed the BBB, their concentrations were determined in plasma and brain tissue collected 30 min after oral administration. Vardenafil improved object recognition memory in all three variants of the ORT. UK-343,664 was ineffective at either preventing MK-801-induced memory disruption or time-dependent memory decay. However, UK-343,664 attenuated the memory impairment of scopolamine. Vardenafil crossed the BBB whereas UK-343,664 did not. Further, co-administration of UK-343,664 and scopolamine did not alter the brain partitioning of either molecule. This suggests that the positive effect of UK-343,664 on scopolamine-induced memory decay might arise from peripheral PDE5 inhibition. The results herein suggest that there may be multiple mechanisms that mediate the efficacy of PDE5 inhibition to improve memory performance in tasks such as the ORT and that these involve PDE5 located both within and outside of the brain. To further elucidate the underlying mechanisms, the cellular and subcellular localization of PDE5 needs to be determined.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/fisiologia , Imidazóis/farmacologia , Inibidores da Fosfodiesterase 5/farmacologia , Piperazinas/farmacologia , Pirimidinonas/farmacologia , Reconhecimento Psicológico/fisiologia , Animais , Barreira Hematoencefálica , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/efeitos dos fármacos , Maleato de Dizocilpina/farmacocinética , Maleato de Dizocilpina/farmacologia , Imidazóis/farmacocinética , Masculino , Inibidores da Fosfodiesterase 5/farmacocinética , Piperazinas/farmacocinética , Pirimidinonas/farmacocinética , Ratos , Ratos Wistar , Reconhecimento Psicológico/efeitos dos fármacos , Escopolamina/farmacocinética , Escopolamina/farmacologia , Sulfonas/farmacocinética , Sulfonas/farmacologia , Triazinas/farmacocinética , Triazinas/farmacologia , Dicloridrato de VardenafilaRESUMO
We report the X-ray crystal structure of a phosphodiesterase (PDE) that includes both catalytic and regulatory domains. PDE2A (215-900) crystallized as a dimer in which each subunit had an extended organization of regulatory GAF-A and GAF-B and catalytic domains connected by long alpha-helices. The subunits cross at the GAF-B/catalytic domain linker, and each side of the dimer contains in series the GAF-A and GAF-B of one subunit and the catalytic domain of the other subunit. A dimer interface extends over the entire length of the molecule. The substrate binding pocket of each catalytic domain is occluded by the H-loop. We deduced from comparisons with structures of isolated, ligand-bound catalytic subunits that the H-loop swings out to allow substrate access. However, in dimeric PDE2A (215-900), the H-loops of the two catalytic subunits pack against each other at the dimer interface, necessitating movement of the catalytic subunits to allow for H-loop movement. Comparison of the unliganded GAF-B of PDE2A (215-900) with previous structures of isolated, cGMP-bound GAF domains indicates that cGMP binding induces a significant shift in the GAF-B/catalytic domain linker. We propose that cGMP binding to GAF-B causes movement, through this linker region, of the catalytic domains, such that the H-loops no longer pack at the dimer interface and are, instead, free to swing out to allow substrate access. This increase in substrate access is proposed as the basis for PDE2A activation by cGMP and may be a general mechanism for regulation of all PDEs.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/química , Regulação Alostérica , Animais , Biocatálise , Linhagem Celular , Cristalografia por Raios X , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Ativação Enzimática , Humanos , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de Proteína , Homologia Estrutural de ProteínaRESUMO
The phosphodiesterases (PDEs) are metal ion-dependent enzymes that regulate cellular signaling by metabolic inactivation of the ubiquitous second messengers cAMP and cGMP. In this role, the PDEs are involved in many biological and metabolic processes and are proven targets of successful drugs for the treatments of a wide range of diseases. However, because of the rapidity of the hydrolysis reaction, an experimental knowledge of the enzymatic mechanisms of the PDEs at the atomic level is still lacking. Here, we report the structures of reaction intermediates accumulated at the reaction steady state in PDE9/crystal and preserved by freeze-trapping. These structures reveal the catalytic process of a PDE and explain the substrate specificity of PDE9 in an actual reaction and the cation requirements of PDEs in general.
Assuntos
Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Catálise , Cristalografia por Raios X , Nucleotídeos de Guanina/química , Nucleotídeos de Guanina/metabolismo , Humanos , Hidrólise , Cinética , Modelos Moleculares , Mutação/genética , Diester Fosfórico Hidrolases/genética , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
PDE10A, a phosphodiesterase that inactivates both cAMP and cGMP, is a unique signaling molecule in being highly and nearly exclusively expressed in striatal medium spiny neurons. These neurons dynamically integrate cortical information with dopamine-signaled value to mediate action selection among available behavioral options. Medium spiny neurons are components of either the direct or indirect striatal output pathways. Selective activation of indirect pathway medium spiny neurons by dopamine D2 receptor antagonists is putatively a key element in the mechanism of their antipsychotic efficacy. While PDE10A is expressed in all medium spiny neurons, studies in rodents indicated that PDE10A inhibition has behavioral effects in several key assays that phenocopy dopamine D2 receptor inhibition. This finding gave rise to the hypothesis that PDE10A inhibition also preferentially activates indirect pathway medium spiny neurons, a hypothesis that is consistent with electrophysiological, neurochemical, and molecular effects of PDE10A inhibitors. These data underwrote industry-wide efforts to investigate and develop PDE10A inhibitors as novel antipsychotics. Disappointingly, PDE10A inhibitors from 3 companies failed to evidence antipsychotic activity in patients with schizophrenia to the same extent as standard-of-care D2 antagonists. Given the notable similarities between PDE10A inhibitors and D2 antagonists, gaining an understanding of why only the latter class is antipsychotic affords a unique window into the basis for this therapeutic efficacy. With this in mind, we review the data on PDE10A inhibition as a step toward back-translating the limited antipsychotic efficacy of PDE10A inhibitors, hopefully to inform new efforts to develop better therapeutics to treat psychosis and schizophrenia.
RESUMO
NMDA receptors containing GluN2D subunits are expressed in the subthalamic nucleus and external globus pallidus, key nuclei of the indirect and hyperdirect pathways of the basal ganglia. This circuitry integrates cortical input with dopaminergic signaling to select advantageous behaviors among available choices. In the experiments described here, we characterized the effects of PTC-174, a novel positive allosteric modulator (PAM) of GluN2D subunit-containing NMDA receptors, on response control regulated by this circuitry. The indirect pathway suppresses less advantageous behavioral choices, a manifestation of which is suppression of locomotor activity in rats. Systemic administration of PTC-174 produced a dose-dependent reduction in activity in rats placed in a novel open field or administered the stimulants MK-801 or amphetamine. The hyperdirect pathway controls release of decisions from the basal ganglia to the cortex to optimize choice processing. Such response control was modeled in rats as premature responding in the 5-choice serial reaction time (5-CSRT) task. PTC-174 produced a dose-dependent reduction in premature responding in this task. These data suggest that potentiation of GluN2D receptor activity by PTC-174 facilitates the complex basal ganglia information processing that underlies response control. The behavioral effects occurred at estimated free PTC-174 brain concentrations predicted to induce 10-50% increases in GluN2D activity. The present findings suggest the potential of GluN2D PAMs to modulate basal ganglia function and to treat neurological disorders related to dysfunctional response control.
Assuntos
Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Regulação Alostérica , Anfetamina/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Maleato de Dizocilpina/farmacologia , Locomoção/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
NMDA receptors are ionotropic glutamate receptors that mediate excitatory neurotransmission. The diverse functions of these receptors are tuned by deploying different combinations of GluN1 and GluN2 subunits (GluN2A-D) to form either diheteromeric NMDA receptors, which contain two GluN1 and two identical GluN2 subunits, or triheteromeric NMDA receptors, which contain two GluN1 and two distinct GluN2 subunits. Here, we characterize PTC-174, a novel positive allosteric modulator (PAM) of receptors containing GluN2C or GluN2D subunits. PTC-174 potentiates maximal current amplitudes by 1.8-fold for diheteromeric GluN1/2B receptors and by > 10-fold for GluN1/2C and GluN1/2D receptors. PTC-174 also potentiates responses from triheteromeric GluN1/2B/2D and GluN1/2A/2C receptors by 4.5-fold and 1.7-fold, respectively. By contrast, PTC-174 produces partial inhibition of responses from diheteromeric GluN1/2A and triheteromeric GluN1/2A/2B receptors. PTC-174 increases potencies of co-agonists glutamate and glycine by 2- to 5-fold at GluN1/2C and GluN1/2D receptors, and NMDA receptor activation facilitates allosteric modulation by PTC-174. At native NMDA receptors in GluN2D-expressing subthalamic nucleus neurons, PTC-174 increases the amplitude of responses to NMDA application and slows the decay of excitatory postsynaptic currents (EPSCs) evoked by internal capsule stimulation. Furthermore, PTC-174 increases the amplitude and slows the decay of EPSCs in hippocampal interneurons, but has not effect on the amplitudes of NMDA receptor-mediated EPSCs in hippocampal CA1 pyramidal neurons. Thus, PTC-174 provides a useful new pharmacological tool to investigate the molecular pharmacology and physiology of GluN2C- and GluN2D-containing NMDA receptors.
Assuntos
Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Agonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Glicina/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Interneurônios/efeitos dos fármacos , Interneurônios/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia , Núcleo Subtalâmico/efeitos dos fármacos , Núcleo Subtalâmico/fisiologia , XenopusRESUMO
AMPA-type glutamate receptors are specifically inhibited by the noncompetitive antagonists GYKI-53655 and CP-465,022, which act through sites and mechanisms that are not understood. Using receptor mutagenesis, we found that these antagonists bind at the interface between the S1 and S2 glutamate binding core and channel transmembrane domains, specifically interacting with S1-M1 and S2-M4 linkers, thereby disrupting the transduction of agonist binding into channel opening. We also found that the antagonists' affinity is higher for agonist-unbound receptors than for activated nondesensitized receptors, further depending on the level of S1 and S2 domain closure. These results provide evidence for substantial conformational changes in the S1-M1 and S2-M4 linkers following agonist binding and channel opening, offering a conceptual frame to account for noncompetitive antagonism of AMPA receptors.
Assuntos
Benzodiazepinas/farmacologia , Potenciais da Membrana/fisiologia , Quinazolinas/farmacologia , Receptores de AMPA/antagonistas & inibidores , Receptores de AMPA/fisiologia , Animais , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Western Blotting/métodos , Encéfalo/efeitos dos fármacos , Linhagem Celular , Clonagem Molecular/métodos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Modelos Neurológicos , Mutagênese/fisiologia , Técnicas de Patch-Clamp/métodos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Conformação Proteica , Quinazolinas/farmacocinética , Ratos , Receptores de AMPA/química , Transfecção/métodos , Trítio/farmacocinética , Xenopus laevisRESUMO
Decreased activity of cAMP responsive element-binding protein (CREB) is thought to contribute to the death of striatal medium spiny neurons in Huntington's disease (HD). Therefore, therapies that increase levels of activated CREB, may be effective in fighting neurodegeneration in HD. In this study, we sought to determine whether the phosphodiesterase type 10 (PDE10A) inhibitor TP10 exerts a neuroprotective effect in an excitotoxic model of HD. Rats were surgically administered with quinolinic acid into striatum and subsequently treated with TP10 daily for two or eight weeks. After 2 weeks of TP10 treatment, striatal lesion size was 52% smaller and the surviving cell number was several times higher than in the vehicle-treated group. These beneficial effects of TP10 were maintained through 8 weeks. TP10 treatment also increased significantly the levels of activated CREB in the striatal spiny neurons, which is hypothesized to be a contributing mechanism for the neuroprotective effect. Our findings suggest PDE10A inhibition as a novel neuroprotective approach to the treatment of HD and confirm the importance of phosphodiesterase inhibition in fighting the disease.
Assuntos
Doença de Huntington/induzido quimicamente , Doença de Huntington/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Diester Fosfórico Hidrolases/metabolismo , Receptores de Complemento/uso terapêutico , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/patologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos/uso terapêutico , Doença de Huntington/patologia , Masculino , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Fosforilação , Ácido Quinolínico , Ratos , Ratos WistarRESUMO
The cyclic nucleotide phosphodiesterase 10A (PDE10A) is highly expressed in striatal medium-sized spiny projection neurons (MSNs), apparently playing a critical role in the regulation of both cGMP and cAMP signaling cascades. Genetic disruption or pharmacological inhibition of PDE10A reverses behavioral abnormalities associated with subcortical hyperdopaminergia. Here, we investigate the effect of PDE10A inhibition on the activity of MSNs using single-unit extracellular recordings performed in the dorsal striatum of anesthetized rats. Antidromic stimulation of the substantia nigra pars reticulata was used to identify striatonigral (SNr+) MSNs. Intrastriatal infusion of the selective PDE10A inhibitors papaverine or TP-10 [2-{4-[-pyridin-4-yl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-3-yl]-phenoxymethyl}-quinoline succinic acid] by reverse microdialysis did not affect spontaneous firing but robustly increased measures of cortically evoked spike activity in a stimulus intensity-dependent manner. Systemic administration of TP-10 also increased cortically evoked spike activity in a stimulus intensity- and dose-dependent manner. A robust increase in cortically evoked activity was apparent in SNr- MSNs (primarily striatopallidal). It is interesting that TP-10 administration did not affect cortically evoked activity in SNr+ MSNs. However, TP-10 administration increased the incidence of antidromically activated (i.e., SNr+) MSNs. These findings indicate that inhibition of striatal PDE10A activity increases the responsiveness of MSNs to depolarizing stimuli. Furthermore, given the lack of effect of TP-10 on SNr+ MSNs, we speculate that PDE10A inhibition may have a greater facilitatory effect on corticostriatal synaptic activity in striatopallidal MSNs. These data support further investigation of selective targeting of PDE signaling pathways in MSN subpopulations because this may represent a promising novel approach for treating brain disorders involving dysfunctional glutamatergic and dopaminergic neurotransmission.
Assuntos
Corpo Estriado/fisiologia , Neurônios/fisiologia , Inibidores de Fosfodiesterase/farmacologia , Diester Fosfórico Hidrolases/metabolismo , Animais , Corpo Estriado/efeitos dos fármacos , Estimulação Elétrica , Eletrofisiologia/métodos , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/fisiologia , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/fisiologia , Globo Pálido/efeitos dos fármacos , Globo Pálido/fisiologia , Masculino , Microdiálise , Neurônios/efeitos dos fármacos , Papaverina/farmacologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/fisiologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Substância Negra/efeitos dos fármacos , Substância Negra/fisiologiaRESUMO
Phosphodiesterase 5A (PDE5A) inhibitors improve functional recovery after middle cerebral artery occlusion (MCA-o) in rats. We used the PDE5A inhibitor 3-(4-(2-hydroxyethyl)piperazin-1-yl)-7-(6-methoxypyridin-3-yl)-1-(2-propoxyethyl)pyrido[3,4-b]pyrazin-2(1H)-one hydrochloride (PF-5) to determine the timing, duration, and degree of inhibition that yields maximum efficacy. We also investigated the localization of PDE5A to determine the tissues and cells that would be targets for PDE5 inhibition and that may mediate efficacy. Nearly complete inhibition of PDE5A, starting 24 h after MCA-o and continued for 7 days, resulted in nearly complete recovery of sensorimotor function that was sustained for 3 months. Delaying administration until 72 h after MCA-o resulted in equivalent efficacy, whereas delaying treatment for 14 days was ineffective. Treatment for 7 days was equivalently efficacious to 28 or 84 days of treatment, whereas treatment for 1 day was less effective. In the normal forebrain, PDE5A immunoreactivity was prominent in smooth muscle of meningeal arteries and a few smaller blood vessels, with weak staining in a few widely scattered cortical neurons and glia. At 24 and 48 h after MCA-o, the number and intensity of blood vessel staining increased in the infarcted cortex and striatum. PDE5A immunoreactivity also was increased at 48 h in putative microglia in penumbra, whereas there was no change in staining of the scattered cortical neurons. Given the window for efficacy and the PDE5A distribution, we hypothesize that efficacy results from an effect on vasculature, and perhaps modulation of microglial function, both of which may facilitate recovery of neuronal function.
Assuntos
Encéfalo/efeitos dos fármacos , Inibidores da Fosfodiesterase 5 , Inibidores de Fosfodiesterase/administração & dosagem , Inibidores de Fosfodiesterase/uso terapêutico , Acidente Vascular Cerebral/tratamento farmacológico , Animais , Comportamento Animal/efeitos dos fármacos , Encéfalo/irrigação sanguínea , Encéfalo/enzimologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 5/biossíntese , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Esquema de Medicação , Masculino , Microglia/efeitos dos fármacos , Microglia/enzimologia , Atividade Motora/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Inibidores de Fosfodiesterase/farmacologia , Ratos , Ratos Sprague-Dawley , Acidente Vascular Cerebral/enzimologia , Acidente Vascular Cerebral/fisiopatologia , Fatores de TempoRESUMO
It is well established that cAMP signaling is an important regulator of the oocyte meiotic cell cycle. Conversely, the function of cGMP during oocyte maturation is less clear. Herein, we evaluated the expression of cGMP-hydrolyzing phosphodiesterases (PDEs) in the somatic and germ cell compartments of the mouse ovarian follicle and demonstrate that PDE5 is preferentially expressed in somatic cells. Cyclic GMP is a potent inhibitor of cAMP hydrolysis from oocyte extracts, with a 50% inhibitory concentration of 97 nM. Luteinizing hormone (LH) stimulation of cultured preovulatory follicles results in a marked decrease in cGMP content, and a nadir is reached in 1.5 h; similarly, oocyte cGMP levels decrease after gonadotropin stimulation in vivo. The LH-dependent decrease in cGMP requires activation of the epidermal growth factor network. Treatment of follicles with a PDE5 inhibitor increases cGMP in the follicle well above unstimulated levels. Although LH causes a decrease in cGMP in follicles preincubated with PDE5 inhibitors, the levels of this nucleotide remain above unstimulated levels. Under these conditions of elevated cGMP, LH stimulation does not cause oocyte maturation after 5 h of incubation. Microinjection of a cGMP-specific PDE into oocytes causes meiotic maturation of wild-type oocytes, suggesting that an intraoocyte pool of cGMP is involved in the maintenance of meiotic arrest. This effect is absent in PDE3A-deficient oocytes. Taken together, these findings provide evidence that cGMP and cAMP signaling cooperate in maintaining meiotic arrest via regulation of PDE3A and that a decrease in cGMP in the somatic compartment is one of the signals contributing to meiotic maturation.
Assuntos
GMP Cíclico/fisiologia , Hormônio Luteinizante/farmacologia , Meiose/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Animais , Células Cultivadas , GMP Cíclico/metabolismo , GMP Cíclico/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/genética , Nucleotídeo Cíclico Fosfodiesterase do Tipo 3/metabolismo , Fatores de Crescimento Endotelial/metabolismo , Fatores de Crescimento Endotelial/fisiologia , Feminino , Meiose/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oócitos/metabolismo , Oócitos/fisiologia , Oogênese/efeitos dos fármacos , Oogênese/genética , Folículo Ovariano/citologia , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , Ovulação/metabolismo , Ovulação/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Phosphodiesterases (PDEs) comprise a family of enzymes that regulate the levels of cyclic nucleotides, key second messengers that mediate a diverse array of functions. PDE2A is an evolutionarily conserved cGMP-stimulated cAMP and cGMP PDE. In the present study, the regional and cellular distribution of PDE2A in tissues of rats, mice, cynomolgus monkeys, dogs, and humans was evaluated by immunohistochemistry. A polyclonal antibody directed to the C-terminal portion of PDE2A specifically detected PDE2A by Western blotting and by immunohistochemistry. The pattern of PDE2A immunoreactivity (ir) was consistent across all species. Western blot analysis demonstrated that PDE2A was most abundant in the brain relative to peripheral tissues. PDE2A ir was heterogeneously distributed within brain and was selectively expressed in particular peripheral tissues. In the brain, prominent immunoreactivity was apparent in components of the limbic system, including the isocortex, hippocampus, amygdala, habenula, basal ganglia, and interpeduncular nucleus. Cytoplasmic PDE2A staining was prominent in several peripheral tissues, including the adrenal zona glomerulosa, neurons of enteric ganglia, endothelial cells in all organs, lymphocytes of spleen and lymph nodes, and pituitary. These studies suggest that PDE2A is evolutionarily conserved across mammalian species and support the hypothesis that the enzyme plays a fundamental role in signal transduction.
Assuntos
Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/biossíntese , Animais , Western Blotting , Encéfalo/anatomia & histologia , Encéfalo/enzimologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/genética , Cães , Gânglios Espinais/anatomia & histologia , Gânglios Espinais/enzimologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Macaca fascicularis , Camundongos , Camundongos Knockout , Especificidade de Órgãos , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Medula Espinal/anatomia & histologia , Medula Espinal/enzimologiaRESUMO
Utilizing structure-based drug design, a 4-aminoimidazole heterocyclic core was synthesized as a replacement for a 2-aminothiazole due to potential metabolically mediated toxicity. The synthetic route utilized allowed for ready synthesis of 1-substituted-4-aminoimidazoles. SAR exploration resulted in the identification of a novel cis-substituted cyclobutyl group that gave improved enzyme and cellular potency against cdk5/p25 with up to 30-fold selectivity over cdk2/cyclin E.