RESUMO
Cannabinoid receptor 1 (CB(1) receptor) controls several neuronal functions, including neurotransmitter release, synaptic plasticity, gene expression and neuronal viability. Downregulation of CB(1) expression in the basal ganglia of patients with Huntington's disease (HD) and animal models represents one of the earliest molecular events induced by mutant huntingtin (mHtt). This early disruption of neuronal CB(1) signaling is thought to contribute to HD symptoms and neurodegeneration. Here we determined whether CB(1) downregulation measured in patients with HD and mouse models was ubiquitous or restricted to specific striatal neuronal subpopulations. Using unbiased semi-quantitative immunohistochemistry, we confirmed previous studies showing that CB(1) expression is downregulated in medium spiny neurons of the indirect pathway, and found that CB(1) is also downregulated in neuropeptide Y (NPY)/neuronal nitric oxide synthase (nNOS)-expressing interneurons while remaining unchanged in parvalbumin- and calretinin-expressing interneurons. CB(1) downregulation in striatal NPY/nNOS-expressing interneurons occurs in R6/2 mice, Hdh(Q150/Q150) mice and the caudate nucleus of patients with HD. In R6/2 mice, CB(1) downregulation in NPY/nNOS-expressing interneurons correlates with diffuse expression of mHtt in the soma. This downregulation also occludes the ability of cannabinoid agonists to activate the pro-survival signaling molecule cAMP response element-binding protein in NPY/nNOS-expressing interneurons. Loss of CB(1) signaling in NPY/nNOS-expressing interneurons could contribute to the impairment of basal ganglia functions linked to HD.
Assuntos
Gânglios da Base/metabolismo , Regulação para Baixo , Doença de Huntington/metabolismo , Interneurônios/metabolismo , Neuropeptídeo Y/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Adulto , Idoso , Animais , Gânglios da Base/citologia , Calbindina 2 , Agonistas de Receptores de Canabinoides/farmacologia , Estudos de Casos e Controles , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Feminino , Expressão Gênica , Humanos , Proteína Huntingtina , Interneurônios/classificação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/genética , Neuropeptídeo Y/genética , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Proteínas Nucleares/genética , Parvalbuminas/genética , Parvalbuminas/metabolismo , Receptor CB1 de Canabinoide/agonistas , Receptor CB1 de Canabinoide/genética , Proteína G de Ligação ao Cálcio S100/genética , Proteína G de Ligação ao Cálcio S100/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismoRESUMO
Agonists at cannabinoid receptors, such as the phytocannabinoid Δ(9)-tetrahydrocannabinol, exert a remarkable array of therapeutic effects but are also associated with undesirable psychoactive side effects. Conversely, targeting enzymes that hydrolyze endocannabinoids (eCBs) allows for more precise fine-tuning of cannabinoid receptor signaling, thus providing therapeutic relief with reduced side effects. Here, we report the development and characterization of an inhibitor of eCB hydrolysis, UCM710, which augments both N-arachidonoylethanolamine and 2-arachidonoylglycerol levels in neurons. This compound displays a unique pharmacological profile in that it inhibits fatty acid amide hydrolase and α/ß-hydrolase domain 6 but not monoacylglycerol lipase. Thus, UCM710 represents a novel tool to delineate the therapeutic potential of compounds that manipulate a subset of enzymes that control eCB signaling.
Assuntos
Amidoidrolases/antagonistas & inibidores , Ácidos Araquidônicos/metabolismo , Moduladores de Receptores de Canabinoides/metabolismo , Endocanabinoides , Inibidores Enzimáticos/farmacologia , Glicerídeos/metabolismo , Monoacilglicerol Lipases/antagonistas & inibidores , Proteínas do Tecido Nervoso/antagonistas & inibidores , Neurônios/metabolismo , Amidoidrolases/metabolismo , Animais , Células COS , Chlorocebus aethiops , Camundongos , Monoacilglicerol Lipases/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Alcamidas Poli-Insaturadas , Receptores de CanabinoidesRESUMO
Large-scale proteomic and functional analysis of isolated pseudopodia revealed the Lim, actin, and SH3 domain protein (Lasp-1) as a novel protein necessary for cell migration, but not adhesion to, the extracellular matrix (ECM). Lasp-1 is a ubiquitously expressed actin-binding protein with a unique domain configuration containing SH3 and LIM domains, and is overexpressed in 8-12% of human breast cancers. We find that stimulation of nonmotile and quiescent cells with growth factors or ECM proteins facilitates Lasp-1 relocalization from the cell periphery to the leading edge of the pseudopodium, where it associates with nascent focal complexes and areas of actin polymerization. Interestingly, although Lasp-1 dynamics in migratory cells occur independently of c-Abl kinase activity and tyrosine phosphorylation, c-Abl activation by apoptotic agents specifically promotes phosphorylation of Lasp-1 at tyrosine 171, which is associated with the loss of Lasp-1 localization to focal adhesions and induction of cell death. Thus, Lasp-1 is a dynamic focal adhesion protein necessary for cell migration and survival in response to growth factors and ECM proteins.
Assuntos
Movimento Celular/genética , Adesões Focais/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinases/metabolismo , Pseudópodes/metabolismo , Actinas/biossíntese , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma/genética , Carcinoma/metabolismo , Sobrevivência Celular/genética , Proteínas do Citoesqueleto , Proteínas da Matriz Extracelular/metabolismo , Proteínas da Matriz Extracelular/farmacologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Adesões Focais/genética , Substâncias de Crescimento/metabolismo , Substâncias de Crescimento/farmacologia , Proteínas de Homeodomínio/genética , Proteínas com Domínio LIM , Camundongos , Células NIH 3T3 , Metástase Neoplásica/genética , Proteínas de Neoplasias/genética , Fosforilação , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Pseudópodes/genéticaRESUMO
This study aims to understand the roles of hydrogen and carbon monoxide during the desulfurization process in a coal gasification system that H2S of the syngas was removed by Fe2O3/SiO2 sorbents. The Fe2O3/SiO2 sorbents were prepared by incipient wetness impregnation. Through the breakthrough experiments and Fourier transform infrared spectroscopy analyses, the overall desulfurization mechanism of the Fe2O3/SiO2 sorbents was proposed in this study. The results show that the major reaction route is that Fe2O3 reacts with H2S to form FeS, and the existence of CO and H2 in the simulated gas significantly affects equilibrium concentrations of H2S and COS. The formation of COS occurs when the feeding gas is blended with CO and H2S, or CO2 and H2S. The pathways in the formation of products from the desulfurization process by the reaction of Fe2O3 with H2S have been successfully established.
Assuntos
Monóxido de Carbono/análise , Carvão Mineral/análise , Compostos Férricos/química , Gases/química , Sulfeto de Hidrogênio/isolamento & purificação , Hidrogênio/análise , Dióxido de Silício/química , AdsorçãoRESUMO
GEANT4 Application for Tomographic Emission (GATE) is a powerful Monte Carlo simulator that combines the advantages of the general-purpose GEANT4 simulation code and the specific software tool implementations dedicated to emission tomography. However, the detailed physical modelling of GEANT4 is highly computationally demanding, especially when tracking particles through voxelized phantoms. To circumvent the relatively slow simulation of voxelized phantoms in GATE, another efficient Monte Carlo code can be used to simulate photon interactions and transport inside a voxelized phantom. The simulation system for emission tomography (SimSET), a dedicated Monte Carlo code for PET/SPECT systems, is well-known for its efficiency in simulation of voxel-based objects. An efficient Monte Carlo workflow integrating GATE and SimSET for simulating pinhole SPECT has been proposed to improve voxelized phantom simulation. Although the workflow achieves a desirable increase in speed, it sacrifices the ability to simulate decaying radioactive sources such as non-pure positron emitters or multiple emission isotopes with complex decay schemes and lacks the modelling of time-dependent processes due to the inherent limitations of the SimSET photon history generator (PHG). Moreover, a large volume of disk storage is needed to store the huge temporal photon history file produced by SimSET that must be transported to GATE. In this work, we developed a multiple photon emission history generator (MPHG) based on SimSET/PHG to support a majority of the medically important positron emitters. We incorporated the new generator codes inside GATE to improve the simulation efficiency of voxelized phantoms in GATE, while eliminating the need for the temporal photon history file. The validation of this new code based on a MicroPET R4 system was conducted for (124)I and (18)F with mouse-like and rat-like phantoms. Comparison of GATE/MPHG with GATE/GEANT4 indicated there is a slight difference in energy spectra for energy below 50 keV due to the lack of x-ray simulation from (124)I decay in the new code. The spatial resolution, scatter fraction and count rate performance are in good agreement between the two codes. For the case studies of (18)F-NaF ((124)I-IAZG) using MOBY phantom with 1 × 1 × 1 mm(3) voxel sizes, the results show that GATE/MPHG can achieve acceleration factors of approximately 3.1 × (4.5 ×), 6.5 × (10.7 ×) and 9.5 × (31.0 ×) compared with GATE using the regular navigation method, the compressed voxel method and the parameterized tracking technique, respectively. In conclusion, the implementation of MPHG in GATE allows for improved efficiency of voxelized phantom simulations and is suitable for studying clinical and preclinical imaging.
Assuntos
Fótons , Software , Tomografia Computadorizada por Raios X/métodos , Animais , Elétrons , Camundongos , Imagens de Fantasmas , Ratos , Sensibilidade e Especificidade , Tomografia Computadorizada por Raios X/instrumentaçãoRESUMO
High-throughput screening (HTS) of chemical libraries is often used for the unbiased identification of compounds interacting with G protein-coupled receptors (GPCRs), the largest family of therapeutic targets. However, current HTS methods require removing GPCRs from their native environment, which modifies their pharmacodynamic properties and biases the screen toward false positive hits. Here, we developed and validated a molecular imaging (MI) agent, NIR-mbc94, which emits near infrared (NIR) light and selectively binds to endogenously expressed cannabinoid CB(2) receptors, a recognized target for treating autoimmune diseases, chronic pain and cancer. The precision and ease of this assay allows for the HTS of compounds interacting with CB(2) receptors expressed in their native environment.
Assuntos
Corantes Fluorescentes/química , Norbornanos/farmacologia , Pirazóis/farmacologia , Receptor CB2 de Canabinoide/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Agonismo Inverso de Drogas , Ensaios de Triagem em Larga Escala , Camundongos , Norbornanos/química , Ligação Proteica , Pirazóis/química , Receptor CB2 de Canabinoide/metabolismoRESUMO
The endocannabinoid 2-arachidonoylglycerol (2-AG) regulates neurotransmission and neuroinflammation by activating CB1 cannabinoid receptors on neurons and CB2 cannabinoid receptors on microglia. Enzymes that hydrolyze 2-AG, such as monoacylglycerol lipase, regulate the accumulation and efficacy of 2-AG at cannabinoid receptors. We found that the recently described serine hydrolase alpha-beta-hydrolase domain 6 (ABHD6) also controls the accumulation and efficacy of 2-AG at cannabinoid receptors. In cells from the BV-2 microglia cell line, ABHD6 knockdown reduced hydrolysis of 2-AG and increased the efficacy with which 2-AG can stimulate CB2-mediated cell migration. ABHD6 was expressed by neurons in primary culture and its inhibition led to activity-dependent accumulation of 2-AG. In adult mouse cortex, ABHD6 was located postsynaptically and its selective inhibition allowed the induction of CB1-dependent long-term depression by otherwise subthreshold stimulation. Our results indicate that ABHD6 is a rate-limiting step of 2-AG signaling and is therefore a bona fide member of the endocannabinoid signaling system.
Assuntos
Ácidos Araquidônicos/metabolismo , Encéfalo/metabolismo , Glicerídeos/metabolismo , Monoacilglicerol Lipases/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Transdução de Sinais/fisiologia , Animais , Células COS , Linhagem Celular , Movimento Celular , Chlorocebus aethiops , Endocanabinoides , Potenciais Pós-Sinápticos Excitadores/fisiologia , Técnicas de Silenciamento de Genes , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Microscopia Eletrônica de Transmissão , Neurônios/metabolismo , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , TransfecçãoRESUMO
We examined how lipopolysaccharide (LPS) and interferon gamma (IFN-gamma), known to differentially activate microglia, affect the expression of G protein-coupled receptor 55 (GPR55), a novel cannabinoid receptor. We found that GPR55 mRNA is significantly expressed in both primary mouse microglia and the BV-2 mouse microglial cell line, and that LPS down-regulates this message. Conversely, IFN-gamma slightly decreases GPR55 mRNA in primary microglia, while it upregulates this message in BV-2 cells. Moreover, the GPR55 agonist, lysophosphatidylinositol, increases ERK phosphorylation in BV-2 stimulated with IFN-gamma, in correlation with the increased amount of GPR55 mRNA. Remarkably, these stimuli-induced changes in GPR55 expression are similar to those observed with CB(2)-R, suggesting that both receptors might be involved in neuroinflammation and that their expression is concomitantly controlled by the state of microglial activation.
Assuntos
Microglia/metabolismo , Receptores de Canabinoides/genética , Receptores de Canabinoides/metabolismo , Animais , Sequência de Bases , Agonistas de Receptores de Canabinoides , Linhagem Celular , Células Cultivadas , Primers do DNA/genética , Expressão Gênica/efeitos dos fármacos , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Lisofosfolipídeos/farmacologia , Camundongos , Microglia/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas RecombinantesRESUMO
PK 11195 and DAA1106 bind with high-affinity to the translocator protein (TSPO, formerly known as the peripheral benzodiazepine receptor). TSPO expression in glial cells increases in response to cytokines and pathological stimuli. Accordingly, [(11)C]-PK 11195 and [(11)C]-DAA1106 are recognized molecular imaging (MI) agents capable of monitoring changes in TSPO expression occurring in vivo and in response to various neuropathologies.Here we tested the pharmacological characteristics and TSPO-monitoring potential of two novel MI agents: NIR-conPK and NIR-6T. NIR-conPK is an analogue of PK 11195 conjugated to the near-infrared (NIR) emitting fluorophore: IRDye 800CW. NIR-6T is a DAA1106 analogue also conjugated to IRDye 800CW.We found that NIR-6T competed for [(3)H]-PK 11195 binding in astrocytoma cell homogenates with nanomolar affinity, but did not exhibit specific binding in intact astrocytoma cells in culture, indicating that NIR-6T is unlikely to constitute a useful MI agent for monitoring TSPO expression in intact cells. Conversely, we found that NIR-conPK did not compete for [(3)H]-PK 11195 binding in astrocytoma cell homogenate, but exhibited specific binding in intact astrocytoma cells in culture with nanomolar affinity, suggesting that NIR-conPK binds to a protein distinct, but related to, TSPO. Accordingly, treating intact astrocytoma cells and microglia in culture with cytokines led to significant changes in the amount of NIR-conPK specific binding without corresponding change in TSPO expression. Remarkably, the cytokine-induced changes in the protein targeted by NIR-conPK in intact microglia were selective, since IFN-gamma (but not TNFalpha and TGFbeta) increased the amount of NIR-conPK specific binding in these cells.Together these results suggest that NIR-conPK binds to a protein that is related to TSPO, and expressed by astrocytomas and microglia. Our results also suggest that the expression of this protein is increased by specific cytokines, and thus allows for the monitoring of a particular subtype of microglia activation.
Assuntos
Astrocitoma/metabolismo , Corantes Fluorescentes/metabolismo , Indóis/metabolismo , Microglia/metabolismo , Receptores de GABA-A/metabolismo , Animais , Extratos Celulares , Quimiocinas/metabolismo , Regulação da Expressão Gênica , Cinética , Camundongos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de GABA-A/genéticaRESUMO
The Vibrio harveyi rpoS gene which encodes an alternative sigma factor (sigma(s) or sigma(38)), has been cloned and characterized. The predicted protein sequence is closely related to RpoS proteins in other bacteria with up to 86% sequence identity. A rpoS null mutant of V. harveyi was constructed and the phenotype studied. Comparison of the properties of the V. harveyi wild type and rpoS deletion mutant showed that rpoS affected the ability of the cells to survive only under specific types of environmental stresses. The rpoS null mutant had a lower survival rate compared to the wild type parental strain at high concentrations of ethanol and in the stationary phase. In contrast to other bacteria, deletion of rpoS in V. harveyi did not affect the resistance of the cells to high osmolarity or hydrogen peroxide, suggesting the existence of alternative systems in V. harveyi responsible for resistance to these stresses. RpoS appears not to be involved in the control of luminescence in V. harveyi even though it is implicated in regulation of other acyl-homoserine dependent quorum sensing systems.