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1.
Bioconjug Chem ; 32(9): 1979-1983, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34448572

RESUMO

Adenosine receptors (ARs) play many important roles in physiology and have been recognized as potential targets for pain relief. Here, we introduce three photoswitchable adenosine derivatives that function as light-dependent agonists for ARs and confer optical control to these G protein-coupled receptors. One of our compounds, AzoAdenosine-3, was evaluated in the classical formalin model of pain. The molecule, active in the dark, was not metabolized by adenosine deaminase and effectively reduced pain perception in a light-dependent manner. These antinociceptive effects suggested a major role for A1R and A3R in peripheral-mediated pain sensitization, whereas an average adenosine-mediated antinociceptive effect will be facilitated by A2AR and A2BR. Our results demonstrate that a photoswitchable adenosine derivative can be used to map the contribution of ARs mediating analgesia in vivo.


Assuntos
Adenosina , Receptor A1 de Adenosina
2.
Pharmacol Res ; 170: 105731, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34157422

RESUMO

Psoriasis is a chronic and relapsing inflammatory skin disease lacking a cure that affects approximately 2% of the population. Defective keratinocyte proliferation and differentiation, and aberrant immune responses are major factors in its pathogenesis. Available treatments for moderate to severe psoriasis are directed to immune system causing systemic immunosuppression over time, and thus concomitant serious side effects (i.e. infections and cancer) may appear. In recent years, the Gi protein-coupled A3 receptor (A3R) for adenosine has been suggested as a novel and very promising therapeutic target for psoriasis. Accordingly, selective, and high affinity A3R agonists are known to induce robust anti-inflammatory effects in animal models of autoimmune inflammatory diseases. Here, we demonstrated the efficacy of a selective A3R agonist, namely MRS5698, in preventing the psoriatic-like phenotype in the IL-23 mouse model of psoriasis. Subsequently, we photocaged this molecule with a coumarin moiety to yield the first photosensitive A3R agonist, MRS7344, which in photopharmacological experiments prevented the psoriatic-like phenotype in the IL-23 animal model. Thus, we have demonstrated the feasibility of using a non-invasive, site-specific, light-directed approach to psoriasis treatment.


Assuntos
Agonistas do Receptor A3 de Adenosina/farmacologia , Adenosina/análogos & derivados , Fotoquimioterapia , Psoríase/prevenção & controle , Receptor A3 de Adenosina/efeitos dos fármacos , Pele/efeitos dos fármacos , Adenosina/farmacologia , Animais , Modelos Animais de Doenças , Interleucina-23 , Ligantes , Psoríase/imunologia , Psoríase/metabolismo , Psoríase/patologia , Receptor A3 de Adenosina/metabolismo , Transdução de Sinais , Pele/imunologia , Pele/metabolismo , Pele/patologia
3.
NPJ Parkinsons Dis ; 10(1): 172, 2024 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-39256360

RESUMO

The orphan G protein-coupled receptor 37 (GPR37), widely associated with Parkinson's disease (PD), undergoes proteolytic processing under physiological conditions. The N-terminus domain is proteolyzed by a disintegrin and metalloproteinase 10 (ADAM-10), which generates various membrane receptor forms and ectodomain shedding (ecto-GPR37) in the extracellular environment. We investigated the processing and density of GPR37 in several neurodegenerative conditions, including Lewy body disease (LBD), multiple system atrophy (MSA), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), and Alzheimer's disease (AD). The presence of ecto-GPR37 peptides in the cerebrospinal fluid (CSF) of PD, MSA, CBD and PSP patients was assessed through an in-house nanoluciferase-based immunoassay. This study identified increased receptor processing in early-stage LBD within the PFC and striatum, key brain areas in neurodegeneration. In MSA only the 52 kDa form of GPR37 appeared in the striatum. This form was also significantly elevated in the striatum of AD necropsies. On the contrary, GPR37 processing remained unchanged in the brains of CBD and PSP patients. Furthermore, while CSF ecto-GPR37 increased in PD patients, its levels remained unchanged in MSA, CBD, and PSP subjects. Importantly, patients with PD with rapid progression of the disease did not have elevated ecto-GPR37 in the CSF, while those with slow progression showed a significant increase, suggesting a possible prognostic use of ecto-GPR37 in PD. This research underscores the distinctive processing and density patterns of GPR37 in neurodegenerative diseases, providing crucial insights into its potential role as an indicator of PD progression rates.

4.
Curr Protoc ; 3(6): e794, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37289022

RESUMO

G protein-coupled receptors (GPCRs) constitute the largest family of plasma membrane receptors and the main drug targets in therapeutics. GPCRs can establish direct receptor-receptor interactions (oligomerization), which can also be considered as targets for drug development (GPCR oligomer-based drugs). However, prior to designing any novel GPCR oligomer-based drug development program, demonstrating the existence of a named GPCR oligomer in native tissues is needed as part of its target engagement definition. Here, we discuss the proximity ligation in situ assay (P-LISA), an experimental approach that reveals GPCR oligomerization in native tissues. We provide a detailed step-by-step protocol to perform P-LISA experiments and visualize GPCR oligomers in brain slices. We also provide instructions for slide observation, data acquisition, and quantification. Finally, we discuss the critical aspects determining the success of the technique, namely the fixation process and the validation of the primary antibodies used. Overall, this protocol may be used to straightforwardly visualize GPCR oligomers in the brain. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Visualization of GPCR oligomers by proximity ligation in situ assay (P-LISA) Support Protocol: Slide observation, image acquisition, and quantification.


Assuntos
Encéfalo , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/metabolismo , Ligação Proteica , Proteínas de Transporte/metabolismo , Transporte Proteico
5.
Br J Pharmacol ; 180(7): 958-974, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-34363210

RESUMO

BACKGROUND AND PURPOSE: Opioid-based drugs are the gold standard medicines for pain relief. However, tolerance and several side effects (i.e. constipation and dependence) may occur upon chronic opioid administration. Photopharmacology is a promising approach to improve the benefit/risk profiles of these drugs. Thus, opioids can be locally activated with high spatiotemporal resolution, potentially minimizing systemic-mediated adverse effects. Here, we aimed at developing a morphine photo-derivative (photocaged morphine), which can be activated upon light irradiation both in vitro and in vivo. EXPERIMENTAL APPROACH: Light-dependent activity of pc-morphine was assessed in cell-based assays (intracellular calcium accumulation and electrophysiology) and in mice (formalin animal model of pain). In addition, tolerance, constipation and dependence were investigated in vivo using experimental paradigms. KEY RESULTS: In mice, pc-morphine was able to elicit antinociceptive effects, both using external light-irradiation (hind paw) and spinal cord implanted fibre-optics. In addition, remote morphine photoactivation was devoid of common systemic opioid-related undesired effects, namely, constipation, tolerance to the analgesic effects, rewarding effects and naloxone-induced withdrawal. CONCLUSION AND IMPLICATIONS: Light-dependent opioid-based drugs may allow effective analgesia without the occurrence of tolerance or the associated and severe opioid-related undesired effects. LINKED ARTICLES: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.


Assuntos
Analgesia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Camundongos , Animais , Morfina/farmacologia , Analgésicos Opioides/farmacologia , Dor/tratamento farmacológico , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/tratamento farmacológico , Constipação Intestinal/induzido quimicamente , Constipação Intestinal/tratamento farmacológico
6.
Mol Neurobiol ; 59(10): 5955-5969, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35829830

RESUMO

The adenosine A2A receptor (A2AR), dopamine D2 receptor (D2R) and metabotropic glutamate receptor type 5 (mGluR5) form A2AR-D2R-mGluR5 heteroreceptor complexes in living cells and in rat striatal neurons. In the current study, we present experimental data supporting the view that the A2AR protomer plays a major role in the inhibitory modulation of the density and the allosteric receptor-receptor interaction within the D2R-mGluR5 heteromeric component of the A2AR-D2R-mGluR5 complex in vitro and in vivo. The A2AR and mGluR5 protomers interact and modulate D2R protomer recognition and signalling upon forming a trimeric complex from these receptors. Expression of A2AR in HEK293T cells co-expressing D2R and mGluR5 resulted in a significant and marked increase in the formation of the D2R-mGluR5 heteromeric component in both bioluminescence resonance energy transfer and proximity ligation assays. A highly significant increase of the the high-affinity component of D2R (D2RKi High) values was found upon cotreatment with the mGluR5 and A2AR agonists in the cells expressing A2AR, D2R and mGluR5 with a significant effect observed also with the mGluR5 agonist alone compared to cells expressing only D2R and mGluR5. In cells co-expressing A2AR, D2R and mGluR5, stimulation of the cells with an mGluR5 agonist like or D2R antagonist fully counteracted the D2R agonist-induced inhibition of the cAMP levels which was not true in cells only expressing mGluR5 and D2R. In agreement, the mGluR5-negative allosteric modulator raseglurant significantly reduced the haloperidol-induced catalepsy in mice, and in A2AR knockout mice, the haloperidol action had almost disappeared, supporting a functional role for mGluR5 and A2AR in enhancing D2R blockade resulting in catalepsy. The results represent a relevant example of integrative activity within higher-order heteroreceptor complexes.


Assuntos
Dopamina , Doença de Parkinson , Adenosina , Animais , Catalepsia , Células HEK293 , Haloperidol , Humanos , Camundongos , Subunidades Proteicas , Ratos , Receptor A2A de Adenosina/metabolismo , Receptores de Dopamina D2/metabolismo
7.
Neuropsychopharmacology ; 46(3): 665-672, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33010795

RESUMO

According to the adenosine hypothesis of schizophrenia, the classically associated hyperdopaminergic state may be secondary to a loss of function of the adenosinergic system. Such a hypoadenosinergic state might either be due to a reduction of the extracellular levels of adenosine or alterations in the density of adenosine A2A receptors (A2ARs) or their degree of functional heteromerization with dopamine D2 receptors (D2R). In the present study, we provide preclinical and clinical evidences for this latter mechanism. Two animal models for the study of schizophrenia endophenotypes, namely the phencyclidine (PCP) mouse model and the A2AR knockout mice, were used to establish correlations between behavioural and molecular studies. In addition, a new AlphaLISA-based method was implemented to detect native A2AR-D2R heteromers in mouse and human brain. First, we observed a reduction of prepulse inhibition in A2AR knockout mice, similar to that observed in the PCP animal model of sensory gating impairment of schizophrenia, as well as a significant upregulation of striatal D2R without changes in A2AR expression in PCP-treated animals. In addition, PCP-treated animals showed a significant reduction of striatal A2AR-D2R heteromers, as demonstrated by the AlphaLISA-based method. A significant and pronounced reduction of A2AR-D2R heteromers was next demonstrated in postmortem caudate nucleus from schizophrenic subjects, even though both D2R and A2AR were upregulated. Finally, in PCP-treated animals, sub-chronic administration of haloperidol or clozapine counteracted the reduction of striatal A2AR-D2R heteromers. The degree of A2AR-D2R heteromer formation in schizophrenia might constitute a hallmark of the illness, which indeed should be further studied to establish possible correlations with chronic antipsychotic treatments.


Assuntos
Receptor A2A de Adenosina , Esquizofrenia , Adenosina , Animais , Corpo Estriado/metabolismo , Dopamina , Camundongos , Receptor A2A de Adenosina/genética , Receptor A2A de Adenosina/metabolismo , Receptores de Dopamina D2/metabolismo
8.
Methods Mol Biol ; 2040: 41-50, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31432474

RESUMO

Proximity ligation assay (PLA) is an antibody-based method that permits studying protein-protein interactions with high specificity and sensitivity. In brief, when a pair of specific antibodies is in close proximity, the complementary DNA strands they bear engage into a rolling circle amplification and generate, in situ, a single fluorescent signal, which indicates the presence of a protein-protein interaction. Proper image analysis methods are needed to provide accurate quantitative assessment of the obtained fluorescent signals, namely, PLA data. In this chapter, we outline basic aspects of image analysis (including software, data import, image processing functions, and analytical tools) that can be used to extract PLA data from confocal microscopy images using ImageJ. A step-by-step protocol to determine and quantify PLA fluorescence signals is included. Overall, the accurate capture and subsequent analysis of PLA confocal images constitutes a crucial step to properly interpret data obtained with this powerful experimental approach.


Assuntos
Bioensaio , Processamento de Imagem Assistida por Computador/métodos , Mapeamento de Interação de Proteínas/métodos , Receptores Acoplados a Proteínas G/metabolismo , Animais , Encéfalo/citologia , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Núcleo Celular/metabolismo , Camundongos , Camundongos Knockout , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Neurônios/metabolismo , Mapas de Interação de Proteínas , Software
9.
Methods Mol Biol ; 1947: 351-359, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30969427

RESUMO

Optopharmacology is a very promising approach based on the use of light-deliverable drugs, which allows manipulating physiological processes with high spatiotemporal resolution. Light-dependent drugs (i.e. caged-compounds) targeting G protein-coupled receptors (GPCRs) have been developed to provide great pharmacological precision on the control of pain. Metabotropic glutamate type 5 (mGlu5) receptors are widely expressed through the pain neuraxis and play a key role in pain transmission. In line with this, selective mGlu5 receptor negative allosteric modulators (NAMs) have consistently shown analgesic activity in experimental animal models of inflammatory pain. Accordingly, we synthesized a light-deliverable drug (i.e. caged compound) using the chemical structure of raseglurant, a mGlu5 receptor NAM, as a molecular scaffold. And thereafter, we evaluated the analgesic activity of the caged compound in formalin-injected (hind paw) mice upon light irradiation (405 nm). Of note, light was both delivered at the peripheral (i.e. hind paw) and central level (i.e. thalamus), by means of brain-implanted fiber-optics. The novel light-deliverable drug, JF-NP-26, showed antinociceptive activity upon violet light irradiation either of the hind paw or the thalamus, demonstrating the ability of precisely activating, in time and space, the caged compound. Here, we describe in detail the protocol used to perform a reliable and reproducible formalin nociception test in mice using an optopharmacology approach (i.e. light-deliverable compounds).


Assuntos
Encéfalo/efeitos dos fármacos , Luz , Medição da Dor/métodos , Dor/tratamento farmacológico , Fármacos Fotossensibilizantes/farmacologia , Receptor de Glutamato Metabotrópico 5/metabolismo , Regulação Alostérica , Animais , Encéfalo/metabolismo , Masculino , Camundongos , Dor/metabolismo
10.
Mol Neurobiol ; 56(8): 5382-5391, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30610611

RESUMO

At present, clinical interest in the plant-derived cannabinoid compound cannabidiol (CBD) is rising exponentially, since it displays multiple therapeutic properties. In addition, CBD can counteract the undesirable effects of the psychoactive cannabinoid Δ9-tetrahydrocannabinol (Δ9-THC) that hinder clinical development of cannabis-based therapies. Despite this attention, the mechanisms of CBD action and its interaction with Δ9-THC are still not completely elucidated. Here, by combining in vivo and complementary molecular techniques, we demonstrate for the first time that CBD blunts the Δ9-THC-induced cognitive impairment in an adenosine A2A receptor (A2AR)-dependent manner. Furthermore, we reveal the existence of A2AR and cannabinoid CB1 receptor (CB1R) heteromers at the presynaptic level in CA1 neurons in the hippocampus. Interestingly, our findings support a brain region-dependent A2AR-CB1R functional interplay; indeed, CBD was not capable of modifying motor functions presumably regulated by striatal A2AR/CB1R complexes, nor anxiety responses related to other brain regions. Overall, these data provide new evidence regarding the mechanisms of action of CBD and the nature of A2AR-CB1R interactions in the brain.


Assuntos
Canabidiol/uso terapêutico , Disfunção Cognitiva/induzido quimicamente , Disfunção Cognitiva/tratamento farmacológico , Dronabinol/efeitos adversos , Hipocampo/metabolismo , Multimerização Proteica , Receptor A2A de Adenosina/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Animais , Canabidiol/farmacologia , Disfunção Cognitiva/fisiopatologia , Hipocampo/fisiopatologia , Hipocampo/ultraestrutura , Locomoção/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Multimerização Proteica/efeitos dos fármacos
11.
Front Pharmacol ; 9: 1200, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30405415

RESUMO

Adenosine A2A receptor (A2AR) antagonists have emerged as complementary non-dopaminergic drugs to alleviate Parkinson's disease (PD) symptomatology. Here, we characterize a novel non-xhantine non-furan A2AR antagonist, PBF509, as a potential pro-dopaminergic drug for PD management. First, PBF509 was shown to be a highly potent ligand at the human A2AR, since it antagonized A2AR agonist-mediated cAMP accumulation and impedance responses with KB values of 72.8 ± 17.4 and 8.2 ± 4.2 nM, respectively. Notably, these results validated our new A2AR-based label-free assay as a robust and sensitive approach to characterize A2AR ligands. Next, we evaluated the efficacy of PBF509 reversing motor impairments in several rat models of movement disorders, including catalepsy, tremor, and hemiparkinsonism. Thus, PBF509 (orally) antagonized haloperidol-mediated catalepsy, reduced pilocarpine-induced tremulous jaw movements and potentiated the number of contralateral rotations induced by L-3,4-dihydroxyphenylalanine (L-DOPA) in unilaterally 6-OHDA-lesioned rats. Moreover, PBF509 (3 mg/kg) inhibited L-DOPA-induced dyskinesia (LID), showing not only its efficacy on reversing parkinsonian motor impairments but also acting as antidyskinetic agent. Overall, here we describe a new orally selective A2AR antagonist with potential utility for PD treatment, and for some of the side effects associated to the current pharmacotherapy (i.e., dyskinesia).

12.
Mol Neurobiol ; 55(6): 4952-4958, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28779351

RESUMO

Dopamine D2 receptor (D2R) activation triggers both G protein- and ß-arrestin-dependent signaling. Biased D2R ligands activating ß-arrestin pathway have been proposed as potential antipsychotics. The ability of D2R to heteromerize with adenosine A2A receptor (A2AR) has been associated to D2R agonist-induced ß-arrestin recruitment. Accordingly, here we aimed to demonstrate the A2AR dependence of D2R/ß-arrestin signaling. By combining bioluminescence resonance energy transfer (BRET) between ß-arrestin-2 tagged with yellow fluorescent protein and bimolecular luminescence complementation (BiLC) of D2R/A2AR homomers and heteromers, we demonstrated that the D2R agonists quinpirole and UNC9994 could promote ß-arrestin-2 recruitment only when A2AR/D2R heteromers were expressed. Subsequently, the role of A2AR in the antipsychotic-like activity of UNC9994 was assessed in wild-type and A2AR-/- mice administered with phencyclidine (PCP) or amphetamine (AMPH). Interestingly, while UNC9994 reduced hyperlocomotion in wild-type animals treated either with PCP or AMPH, in A2AR-/- mice, it failed to reduce PCP-induced hyperlocomotion or produced only a moderate reduction of AMPH-mediated hyperlocomotion. Overall, the results presented here reinforce the notion that D2R/A2AR heteromerization facilitates D2R ß-arrestin recruitment, and furthermore, reveal a pivotal role for A2AR in the antipsychotic-like activity of the ß-arrestin-biased D2R ligand, UNC9994.


Assuntos
Antipsicóticos/farmacologia , Atividade Motora/efeitos dos fármacos , Receptor A2A de Adenosina/metabolismo , Receptores de Dopamina D2/agonistas , Transdução de Sinais/efeitos dos fármacos , Adenosina/análogos & derivados , Adenosina/farmacologia , Anfetamina/farmacologia , Animais , Dimerização , Dopaminérgicos/farmacologia , Agonistas de Dopamina/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Camundongos , Camundongos Knockout , Fenciclidina/farmacologia , Fenetilaminas/farmacologia , Quimpirol/farmacologia , Receptor A2A de Adenosina/genética
13.
J Control Release ; 283: 135-142, 2018 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-29859955

RESUMO

G protein-coupled adenosine receptors are promising therapeutic targets for a wide range of neuropathological conditions, including Parkinson's disease (PD). However, the ubiquity of adenosine receptors and the ultimate lack of selectivity of certain adenosine-based drugs have frequently diminished their therapeutic use. Photopharmacology is a novel approach that allows the spatiotemporal control of receptor function, thus circumventing some of these limitations. Here, we aimed to develop a light-sensitive caged adenosine A2A receptor (A2AR) antagonist to photocontrol movement disorders. We synthesized MRS7145 by blocking with coumarin the 5-amino position of the selective A2AR antagonist SCH442416, which could be photoreleased upon violet light illumination (405 nm). First, the light-dependent pharmacological profile of MRS7145 was determined in A2AR-expressing cells. Upon photoactivation, MRS7145 precluded A2AR ligand binding and agonist-induced cAMP accumulation. Next, the ability of MRS7145 to block A2AR in a light-dependent manner was assessed in vivo. To this end, A2AR antagonist-mediated locomotor activity potentiation was evaluated in brain (striatum) fiber-optic implanted mice. Upon irradiation (405 nm) of the dorsal striatum, MRS7145 induced significant hyperlocomotion and counteracted haloperidol-induced catalepsy and pilocarpine-induced tremor. Finally, its efficacy in reversing motor impairment was evaluated in a PD animal model, namely the hemiparkinsonian 6-hydroxydopamine (6-OHDA)-lesioned mouse. Photo-activated MRS7145 was able to potentiate the number of contralateral rotations induced by L-3,4-dihydroxyphenylalanine (l-DOPA). Overall, MRS7145 is a new light-operated A2AR antagonist with potential utility to manage movement disorders, including PD.


Assuntos
Antagonistas do Receptor A2 de Adenosina/administração & dosagem , Antagonistas do Receptor A2 de Adenosina/efeitos da radiação , Luz , Transtornos dos Movimentos/tratamento farmacológico , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Modelos Animais de Doenças , Células HEK293 , Humanos , Locomoção/efeitos dos fármacos , Camundongos , Transtornos dos Movimentos/metabolismo , Transtornos dos Movimentos/fisiopatologia , Fibras Ópticas , Receptor A2A de Adenosina/metabolismo
14.
Bio Protoc ; 7(23): e2628, 2017 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-34595296

RESUMO

Pain research is mostly based on experimental assays that use animal models, which may allow deciphering the physiopathology of this condition and to propel drug discovery. The formalin nociception test is considered one of the most predictive approaches to study acute pain in rodents. This test permits monitoring pain-related responses (i.e., itch) caused by a subcutaneous injection of an inflammatory agent, namely 2.5% formalin solution, in the hind paw. After the injection, two distinct time periods or phases of licking/biting behaviour occur, which are separated by a quiescent period. Importantly, these phases differ in duration and underlying mechanisms. Hence, the initial acute phase (phase I), commonly recorded for 5 min just after formalin administration, reflects acute peripheral pain, probably due to direct activation of nociceptors through TRPA1 channels. On the other hand, the phase II, which starts after the quiescent period (5-15 min) and is commonly recorded for 15-30 min, is due to the ongoing inflammatory input and central nociceptive sensitization. Here, we describe in detail the protocol used to perform a reliable and reproducible formalin test in mice.

15.
Neuropharmacology ; 126: 48-57, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28844595

RESUMO

The study of psychiatric disorders usually focuses on emotional symptoms assessment. However, cognitive deficiencies frequently constitute the core symptoms, are often poorly controlled and handicap individual's quality of life. Adenosine receptors, through the control of both dopamine and glutamate systems, have been implicated in the pathophysiology of several psychiatric disorders such as schizophrenia and attention deficit/hyperactivity disorder. Indeed, clinical data indicate that poorly responsive schizophrenia patients treated with adenosine adjuvants show improved treatment outcomes. The A2A adenosine receptor subtype (A2AR) is highly expressed in brain areas controlling cognition and motivational responses including the striatum, hippocampus and cerebral cortex. Accordingly, we study the role of A2AR in the regulation of cognitive processes based on a complete cognitive behavioural analysis coupled with the assessment of neurogenesis and sub-synaptic protein expression in adult and middle-aged A2AR constitutional knockout mice and wild-type littermates. Our results show overall cognitive impairments in A2AR knockout mice associated with a decrease in new-born hippocampal neuron proliferation and concomitant changes in synaptic protein expression, in both the prefrontal cortex and the hippocampus. These results suggest a deficient adenosine signalling in cognitive processes, thus providing new opportunities for the therapeutic management of cognitive deficits associated with psychiatric disorders.


Assuntos
Disfunção Cognitiva/fisiopatologia , Hipocampo/metabolismo , Córtex Pré-Frontal/metabolismo , Receptor A2A de Adenosina/fisiologia , Sinapses/metabolismo , Animais , Aprendizagem da Esquiva , Calcineurina/metabolismo , Proliferação de Células , Disfunção Cognitiva/metabolismo , Aprendizagem , Masculino , Memória de Curto Prazo , Camundongos Knockout , Neurogênese , Neurônios/metabolismo , Receptor A2A de Adenosina/genética , Memória Espacial , Sinapsinas/metabolismo
16.
J Vis Exp ; (123)2017 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-28570537

RESUMO

Assessing the synaptic protein composition and function constitutes an important challenge in neuroscience. However, it is not easy to evaluate neurotransmission that occurs within synapses because it is highly regulated by dynamic protein-protein interactions and phosphorylation events. Accordingly, when any method is used to study synaptic transmission, a major goal is to preserve these transient physiological modifications. Here, we present a brain membrane fractionation protocol that represents a robust procedure to isolate proteins belonging to different synaptic compartments. In other words, the protocol describes a biochemical methodology to carry out protein enrichment from presynaptic, postsynaptic, and extrasynaptic compartments. First, synaptosomes, or synaptic terminals, are obtained from neurons that contain all synaptic compartments by means of a discontinuous sucrose gradient. Of note, the quality of this initial synaptic membrane preparation is critical. Subsequently, the isolation of the different subsynaptic compartments is achieved with light solubilization using mild detergents at differential pH conditions. This allows for separation by gradient and isopycnic centrifugations. Finally, protein enrichment at the different subsynaptic compartments (i.e., pre-, post- and extrasynaptic membrane fractions) is validated by means of immunoblot analysis using well-characterized synaptic protein markers (i.e., SNAP-25, PSD-95, and synaptophysin, respectively), thus enabling a direct assessment of the synaptic distribution of any particular neuronal protein.


Assuntos
Encéfalo/metabolismo , Proteínas de Membrana/isolamento & purificação , Sinapses/química , Sinaptossomos/química , Animais , Detergentes/química , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Transporte Proteico , Sinapses/metabolismo , Sinaptossomos/metabolismo
17.
Front Pharmacol ; 8: 700, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29046640

RESUMO

Guanosine (GUO) is a guanine-based purine nucleoside with important trophic functions and promising neuroprotective properties. Although the neuroprotective effects of GUO have been corroborated in cellular models of Parkinson's disease (PD), its efficacy as an antiparkinsonian agent has not been fully explored in PD animal models. Accordingly, we evaluated the effectiveness of GUO in reversing motor impairments in several rodent movement disorder models, including catalepsy, tremor, and hemiparkinsonism. Our results showed that orally administered GUO antagonized reserpine-mediated catalepsy, reduced reserpine-induced tremulous jaw movements, and potentiated the number of contralateral rotations induced by L-3,4-dihydroxyphenylalanine in unilaterally 6-hydroxidopamine-lesioned rats. In addition, at 5 and 7.5 mg/kg, GUO inhibited L-DOPA-induced dyskinesia in rats chronically treated with a pro-dopaminergic agent. Overall, we describe the therapeutic potential of GUO, which may be effective not only for reversing parkinsonian motor impairments but also for reducing dyskinesia induced by treatment for PD.

18.
Sci Rep ; 7(1): 9452, 2017 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-28842709

RESUMO

G protein-coupled receptor 37 (GPR37) is an orphan receptor associated to Parkinson's disease (PD) neuropathology. Here, we identified GPR37 as an inhibitor of adenosine A2A receptor (A2AR) cell surface expression and function in vivo. In addition, we showed that GPR37 and A2AR do oligomerize in the striatum. Thus, a close proximity of GPR37 and A2AR at the postsynaptic level of striatal synapses was observed by double-labelling post-embedding immunogold detection. Indeed, the direct receptor-receptor interaction was further substantiated by proximity ligation in situ assay. Interestingly, GPR37 deletion promoted striatal A2AR cell surface expression that correlated well with an increased A2AR agonist-mediated cAMP accumulation, both in primary striatal neurons and nerve terminals. Furthermore, GPR37-/- mice showed enhanced A2AR agonist-induced catalepsy and an increased response to A2AR antagonist-mediated locomotor activity. Overall, these results revealed a key role for GPR37 controlling A2AR biology in the striatum, which may be relevant for PD management.


Assuntos
Doença de Parkinson/metabolismo , Receptor A2A de Adenosina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sinapses/metabolismo , Córtex Visual/metabolismo , Animais , Catalepsia/genética , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Humanos , Locomoção/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doença de Parkinson/genética , Multimerização Proteica , Receptores Acoplados a Proteínas G/genética
19.
Sci Rep ; 7(1): 1857, 2017 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-28500295

RESUMO

Tardive dyskinesia (TD) is a serious motor side effect that may appear after long-term treatment with neuroleptics and mostly mediated by dopamine D2 receptors (D2Rs). Striatal D2R functioning may be finely regulated by either adenosine A2A receptor (A2AR) or angiotensin receptor type 1 (AT1R) through putative receptor heteromers. Here, we examined whether A2AR and AT1R may oligomerize in the striatum to synergistically modulate dopaminergic transmission. First, by using bioluminescence resonance energy transfer, we demonstrated a physical AT1R-A2AR interaction in cultured cells. Interestingly, by protein-protein docking and molecular dynamics simulations, we described that a stable heterotetrameric interaction may exist between AT1R and A2AR bound to antagonists (i.e. losartan and istradefylline, respectively). Accordingly, we subsequently ascertained the existence of AT1R/A2AR heteromers in the striatum by proximity ligation in situ assay. Finally, we took advantage of a TD animal model, namely the reserpine-induced vacuous chewing movement (VCM), to evaluate a novel multimodal pharmacological TD treatment approach based on targeting the AT1R/A2AR complex. Thus, reserpinized mice were co-treated with sub-effective losartan and istradefylline doses, which prompted a synergistic reduction in VCM. Overall, our results demonstrated the existence of striatal AT1R/A2AR oligomers with potential usefulness for the therapeutic management of TD.


Assuntos
Multimerização Proteica , Receptor A2A de Adenosina/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Antagonistas do Receptor A2 de Adenosina/química , Antagonistas do Receptor A2 de Adenosina/farmacologia , Bloqueadores do Receptor Tipo 1 de Angiotensina II/química , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Células Cultivadas , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Receptor A2A de Adenosina/química , Receptor Tipo 1 de Angiotensina/química , Discinesia Tardia/tratamento farmacológico , Discinesia Tardia/metabolismo
20.
Elife ; 62017 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-28395733

RESUMO

Light-operated drugs constitute a major target in drug discovery, since they may provide spatiotemporal resolution for the treatment of complex diseases (i.e. chronic pain). JF-NP-26 is an inactive photocaged derivative of the metabotropic glutamate type 5 (mGlu5) receptor negative allosteric modulator raseglurant. Violet light illumination of JF-NP-26 induces a photochemical reaction prompting the active-drug's release, which effectively controls mGlu5 receptor activity both in ectopic expressing systems and in striatal primary neurons. Systemic administration in mice followed by local light-emitting diode (LED)-based illumination, either of the thalamus or the peripheral tissues, induced JF-NP-26-mediated light-dependent analgesia both in neuropathic and in acute/tonic inflammatory pain models. These data offer the first example of optical control of analgesia in vivo using a photocaged mGlu5 receptor negative allosteric modulator. This approach shows potential for precisely targeting, in time and space, endogenous receptors, which may allow a better management of difficult-to-treat disorders.


Assuntos
Regulação Alostérica/efeitos dos fármacos , Analgésicos/metabolismo , Luz , Neurônios/efeitos dos fármacos , Dor , Fármacos Fotossensibilizantes/metabolismo , Receptor de Glutamato Metabotrópico 5/efeitos dos fármacos , Analgesia/métodos , Analgésicos/administração & dosagem , Animais , Camundongos , Fármacos Fotossensibilizantes/administração & dosagem
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