RESUMO
BACKGROUND: Alemtuzumab efficacy and safety was demonstrated in CARE-MS I and extension studies (CAMMS03409; TOPAZ). OBJECTIVE: Evaluate serum neurofilament light chain (sNfL) in CARE-MS I patients and highly active disease (HAD) subgroup, over 7 and 2 years for alemtuzumab and subcutaneous interferon beta-1a (SC IFNB-1a), respectively. METHODS: Patients received SC IFNB-1a 44 µg 3×/week or alemtuzumab 12 mg/day at baseline and month 12, with further as-needed 3-day courses. sNfL was measured using single-molecule array (Simoa™). HAD definition was ⩾2 relapses in year before randomization and ⩾1 baseline gadolinium-enhancing lesion. RESULTS: Baseline median sNfL levels were similar in alemtuzumab (n = 354) and SC IFNB-1a-treated (n = 159) patients (31.7 vs 31.4 pg/mL), but decreased with alemtuzumab versus SC IFNB-1a until year 2 (Y2; 13.2 vs 18.7 pg/mL; p < 0.0001); 12.7 pg/mL for alemtuzumab at Y7. Alemtuzumab-treated patients had sNfL at/below healthy control median at Y2 (72% vs 47%; p < 0.0001); 73% for alemtuzumab at Y7. HAD patients (n = 102) had higher baseline sNfL (49.4 pg/mL) versus overall population; alemtuzumab HAD patients attained similar levels (Y2, 12.8 pg/mL; Y7, 12.7 pg/mL; 75% were at/below control median at Y7). CONCLUSION: Alemtuzumab was superior to SC IFNB-1a in reducing sNfL, with levels in alemtuzumab patients remaining stable through Y7. CLINICALTRIALS.GOV IDENTIFIER: NCT00530348, NCT00930553, NCT02255656.
Assuntos
Filamentos Intermediários , Esclerose Múltipla Recidivante-Remitente , Alemtuzumab/efeitos adversos , Humanos , Interferon beta-1a/uso terapêutico , Esclerose Múltipla Recidivante-Remitente/tratamento farmacológico , Proteínas de NeurofilamentosRESUMO
BACKGROUND: Acute neurological insults caused by infection, systemic inflammation, ischemia, or traumatic injury are often associated with breakdown of the blood-brain barrier (BBB) followed by infiltration of peripheral immune cells, cytotoxic proteins, and water. BBB breakdown and extravasation of these peripheral components into the brain parenchyma result in inflammation, oxidative stress, edema, excitotoxicity, and neurodegeneration. These downstream consequences of BBB dysfunction can drive pathophysiological processes and play a substantial role in the morbidity and mortality of acute and chronic neurological insults, and contribute to long-term sequelae. Preserving or rescuing BBB integrity and homeostasis therefore represents a translational research area of high therapeutic potential. METHODS: Induction of general and localized BBB disruption in mice was carried out using systemic administration of LPS and focal photothrombotic ischemic insult, respectively, in the presence and absence of the monoacylglycerol lipase (MAGL) inhibitor, CPD-4645. The effects of CPD-4645 treatment were assessed by gene expression analysis performed on neurovascular-enriched brain fractions, cytokine and inflammatory mediator measurement, and functional assessment of BBB permeability. The mechanism of action of CPD-4645 was studied pharmacologically using inverse agonists/antagonists of the cannabinoid receptors CB1 and CB2. RESULTS: Here, we demonstrate that the neurovasculature exhibits a unique transcriptional signature following inflammatory insults, and pharmacological inhibition of MAGL using a newly characterized inhibitor rescues the transcriptional profile of brain vasculature and restores its functional homeostasis. This pronounced effect of MAGL inhibition on blood-brain barrier permeability is evident following both systemic inflammatory and localized ischemic insults. Mechanistically, the protective effects of the MAGL inhibitor are partially mediated by cannabinoid receptor signaling in the ischemic brain insult. CONCLUSIONS: Our results support considering MAGL inhibitors as potential therapeutics for BBB dysfunction and cerebral edema associated with inflammatory brain insults.
Assuntos
Ácidos Araquidônicos/antagonistas & inibidores , Ácidos Araquidônicos/metabolismo , Barreira Hematoencefálica/metabolismo , Lesões Encefálicas/tratamento farmacológico , Lesões Encefálicas/metabolismo , Permeabilidade Capilar/fisiologia , Endocanabinoides/antagonistas & inibidores , Endocanabinoides/metabolismo , Glicerídeos/antagonistas & inibidores , Glicerídeos/metabolismo , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Lesões Encefálicas/induzido quimicamente , Permeabilidade Capilar/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Hidrólise/efeitos dos fármacos , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monoacilglicerol Lipases/antagonistas & inibidores , Monoacilglicerol Lipases/metabolismoRESUMO
OBJECTIVE: Status epilepticus (SE) is a life-threatening and commonly drug-refractory condition. Novel therapies are needed to rapidly terminate seizures to prevent mortality and morbidity. Monoacylglycerol lipase (MAGL) is the key enzyme responsible for the hydrolysis of the endocannabinoid 2-arachidonoylglycerol (2-AG) and a major contributor to the brain pool of arachidonic acid (AA). Inhibiting of monoacylglycerol lipase modulates synaptic activity and neuroinflammation, 2 mediators of excessive neuronal activation underlying seizures. We studied the effect of a potent and selective irreversible MAGL inhibitor, CPD-4645, on SE that was refractory to diazepam, its neuropathologic sequelae, and the mechanism underlying the drug's effects. METHODS: Diazepam-resistant SE was induced in adult mice fed with standard or ketogenic diet or in cannabinoid receptor type 1 (CB1) receptor knock-out mice. CPD-4645 (10 mg/kg, subcutaneously) or vehicle was dosed 1 and 7 h after status epilepticus onset in video-electroencephalography (EEG) recorded mice. At the end of SE, mice were examined in the novel object recognition test followed by neuronal cellloss analysis. RESULTS: CPD-4645 maximal plasma and brain concentrations were attained 0.5 h postinjection (half-life = 3.7 h) and elevated brain 2-AG levels by approximately 4-fold. CPD-4645 administered to standard diet-fed mice progressively reduced spike frequency during 3 h postinjection, thereby shortening SE duration by 47%. The drug immediately abrogated SE in ketogenic diet-fed mice. CPD-4645 rescued neuronal cell loss and cognitive deficit and reduced interleukin (IL)-1ß and cyclooxygenase 2 (COX-2) brain expression resulting from SE. The CPD-4645 effect on SE was similar in mice lacking CB1 receptors. SIGNIFICANCE: MAGL represents a novel therapeutic target for treating status epilepticus and improving its sequelae. CPD-4645 therapeutic effects appear to be predominantly mediated by modulation of neuroinflammation.
Assuntos
Carbamatos/uso terapêutico , Monoacilglicerol Lipases/antagonistas & inibidores , Piperidinas/uso terapêutico , Estado Epiléptico , Sulfonamidas/uso terapêutico , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Ondas Encefálicas/efeitos dos fármacos , Ondas Encefálicas/fisiologia , Carbamatos/química , Carbamatos/farmacocinética , Transtornos Cognitivos/tratamento farmacológico , Transtornos Cognitivos/etiologia , Diazepam/efeitos adversos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Epilepsia Resistente a Medicamentos/induzido quimicamente , Epilepsia Resistente a Medicamentos/enzimologia , Epilepsia Resistente a Medicamentos/terapia , Eletroencefalografia , Agonistas de Aminoácidos Excitatórios/toxicidade , Fluoresceínas/metabolismo , Ácido Caínico/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monoacilglicerol Lipases/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Piperidinas/química , Piperidinas/farmacologia , Distribuição Aleatória , Receptor CB1 de Canabinoide/deficiência , Receptor CB1 de Canabinoide/genética , Reconhecimento Psicológico/efeitos dos fármacos , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/complicações , Estado Epiléptico/enzimologia , Estado Epiléptico/terapia , Sulfonamidas/química , Sulfonamidas/farmacocinética , Fatores de TempoRESUMO
Prominent cerebral amyloid angiopathy is often observed in the brains of elderly individuals and is almost universally found in patients with Alzheimer's disease. Cerebral amyloid angiopathy is characterized by accumulation of the shorter amyloid-ß isoform(s) (predominantly amyloid-ß40) in the walls of leptomeningeal and cortical arterioles and is likely a contributory factor to vascular dysfunction leading to stroke and dementia in the elderly. We used transgenic mice with prominent cerebral amyloid angiopathy to investigate the ability of ponezumab, an anti-amyloid-ß40 selective antibody, to attenuate amyloid-ß accrual in cerebral vessels and to acutely restore vascular reactivity. Chronic administration of ponezumab to transgenic mice led to a significant reduction in amyloid and amyloid-ß accumulation both in leptomeningeal and brain vessels when measured by intravital multiphoton imaging and immunohistochemistry. By enriching for cerebral vascular elements, we also measured a significant reduction in the levels of soluble amyloid-ß biochemically. We hypothesized that the reduction in vascular amyloid-ß40 after ponezumab administration may reflect the ability of ponezumab to mobilize an interstitial fluid pool of amyloid-ß40 in brain. Acutely, ponezumab triggered a significant and transient increase in interstitial fluid amyloid-ß40 levels in old plaque-bearing transgenic mice but not in young animals. We also measured a beneficial effect on vascular reactivity following acute administration of ponezumab, even in vessels where there was a severe cerebral amyloid angiopathy burden. Taken together, the beneficial effects ponezumab administration has on reducing the rate of cerebral amyloid angiopathy deposition and restoring cerebral vascular health favours a mechanism that involves rapid removal and/or neutralization of amyloid-ß species that may otherwise be detrimental to normal vessel function.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Anticorpos Monoclonais Humanizados/administração & dosagem , Angiopatia Amiloide Cerebral/tratamento farmacológico , Angiopatia Amiloide Cerebral/metabolismo , Endotélio Vascular/metabolismo , Imunização Passiva/métodos , Peptídeos beta-Amiloides/imunologia , Animais , Angiopatia Amiloide Cerebral/imunologia , Circulação Cerebrovascular/efeitos dos fármacos , Circulação Cerebrovascular/fisiologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/imunologia , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Placa Amiloide/tratamento farmacológico , Placa Amiloide/imunologia , Placa Amiloide/metabolismoRESUMO
Hepcidin is a key regulator of systemic iron homeostasis. Hepcidin deficiency induces iron overload, whereas hepcidin excess induces anemia. Mutations in the gene encoding hemojuvelin (HFE2, also known as HJV) cause severe iron overload and correlate with low hepcidin levels, suggesting that hemojuvelin positively regulates hepcidin expression. Hemojuvelin is a member of the repulsive guidance molecule (RGM) family, which also includes the bone morphogenetic protein (BMP) coreceptors RGMA and DRAGON (RGMB). Here, we report that hemojuvelin is a BMP coreceptor and that hemojuvelin mutants associated with hemochromatosis have impaired BMP signaling ability. Furthermore, BMP upregulates hepatocyte hepcidin expression, a process enhanced by hemojuvelin and blunted in Hfe2-/- hepatocytes. Our data suggest a mechanism by which HFE2 mutations cause hemochromatosis: hemojuvelin dysfunction decreases BMP signaling, thereby lowering hepcidin expression.
Assuntos
Peptídeos Catiônicos Antimicrobianos/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana/fisiologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Sequência de Aminoácidos , Animais , Proteína Morfogenética Óssea 2 , Células CHO , Cricetinae , Proteínas Ligadas por GPI , Proteína da Hemocromatose , Hepcidinas , Humanos , Fígado/citologia , Fígado/metabolismo , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação , Reação em Cadeia da PolimeraseRESUMO
Repulsive guidance molecule (RGM) family members RGMa, RGMb/Dragon, and RGMc/hemojuvelin were found recently to act as bone morphogenetic protein (BMP) coreceptors that enhance BMP signaling activity. Although our previous studies have shown that hemojuvelin regulates hepcidin expression and iron metabolism through the BMP pathway, the role of the BMP signaling mediated by Dragon remains largely unknown. We have shown previously that Dragon is expressed in neural cells, germ cells, and renal epithelial cells. In this study, we demonstrate that Dragon is highly expressed in macrophages. Studies with RAW264.7 and J774 macrophage cell lines reveal that Dragon negatively regulates IL-6 expression in a BMP ligand-dependent manner via the p38 MAPK and Erk1/2 pathways but not the Smad1/5/8 pathway. We also generated Dragon knockout mice and found that IL-6 is upregulated in macrophages and dendritic cells derived from whole lung tissue of these mice compared with that in respective cells derived from wild-type littermates. These results indicate that Dragon is an important negative regulator of IL-6 expression in immune cells and that Dragon-deficient mice may be a useful model for studying immune and inflammatory disorders.
Assuntos
Regulação da Expressão Gênica/imunologia , Interleucina-6/antagonistas & inibidores , Interleucina-6/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Animais , Moléculas de Adesão Celular Neuronais , Linhagem Celular , Modelos Animais de Doenças , Regulação para Baixo/imunologia , Proteínas Ligadas por GPI , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/fisiologia , Interleucina-6/biossíntese , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos/patologia , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/fisiologia , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
Repulsive guidance molecule b (RGMb) is a bone morphogenetic protein (BMP) coreceptor and sensitizer of BMP signaling, highly expressed in adult dorsal root ganglion (DRG) sensory neurons. We used a murine RGMb knock-out to gain insight into the physiological role of RGMb in the DRG, and address whether RGMb-mediated modulation of BMP signaling influences sensory axon regeneration. No evidence for altered development of the PNS and CNS was detected in RGMb(-/-) mice. However, both cultured neonatal whole DRG explants and dissociated DRG neurons from RGMb(-/-) mice exhibited significantly fewer and shorter neurites than those from wild-type littermates, a phenomenon that could be fully rescued by BMP-2. Moreover, Noggin, an endogenous BMP signaling antagonist, inhibited neurite outgrowth in wild-type DRG explants from naive as well as nerve injury-preconditioned mice. Noggin is downregulated in the DRG after nerve injury, and its expression is highly correlated and inversely associated with the known regeneration-associated genes, which are induced in the DRG by peripheral axonal injury. We show that diminished BMP signaling in vivo, achieved either through RGMb deletion or BMP inhibition with Noggin, retarded early axonal regeneration after sciatic nerve crush injury. Our data suggest a positive modulatory contribution of RGMb and BMP signaling to neurite extension in vitro and early axonal regrowth after nerve injury in vivo and a negative effect of Noggin.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/metabolismo , Regeneração Nervosa/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Neuritos/metabolismo , Transdução de Sinais/fisiologia , Animais , Moléculas de Adesão Celular Neuronais , Células Cultivadas , Proteínas Ligadas por GPI , Gânglios Espinais/metabolismo , Camundongos , Camundongos Knockout , Compressão Nervosa , Neurônios/metabolismo , Fosforilação/fisiologia , Nervo Isquiático/fisiologiaRESUMO
A cardinal feature of peripheral inflammation is pain. The most common way of managing inflammatory pain is to use nonsteroidal antiinflammatory agents (NSAIDs) that reduce prostanoid production, for example, selective inhibitors of COX2. Prostaglandins produced after induction of COX2 in immune cells in inflamed tissue contribute both to the inflammation itself and to pain hypersensitivity, acting on peripheral terminals of nociceptors. COX2 is also induced after peripheral inflammation in neurons in the CNS, where it aids in developing a central component of inflammatory pain hypersensitivity by increasing neuronal excitation and reducing inhibition. We engineered mice with conditional deletion of Cox2 in neurons and glial cells to determine the relative contribution of peripheral and central COX2 to inflammatory pain hypersensitivity. In these mice, basal nociceptive pain was unchanged, as was the extent of peripheral inflammation, inflammatory thermal pain hypersensitivity, and fever induced by lipopolysaccharide. By contrast, peripheral inflammation-induced COX2 expression in the spinal cord was reduced, and mechanical hypersensitivity after both peripheral soft tissue and periarticular inflammation was abolished. Mechanical pain is a major symptom of most inflammatory conditions, such as postoperative pain and arthritis, and induction of COX2 in neural cells in the CNS seems to contribute to this.
Assuntos
Ciclo-Oxigenase 2/fisiologia , Inflamação/fisiopatologia , Neurônios/enzimologia , Dor/etiologia , Medula Espinal/enzimologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
BACKGROUND: Monoacylglycerol lipase (MAGL) is a key serine hydrolase which terminates endocannabinoid signaling and regulates arachidonic acid driven inflammatory responses within the central nervous system. To develop [11C]PF-06809247 into a clinically usable MAGL positron emission tomography (PET) radioligand, we assessed the occupancy of MAGL by an inhibitor in the non-human primate (NHP) brain. Additionally, we measured the whole-body distribution of [11C]PF-06809247 in NHP and estimated human effective radiation doses. METHODS: Seven cynomolgus monkeys were enrolled for brain PET measurements. Two PET measurements along with arterial blood sampling were performed in each NHP: one baseline and one pretreatment condition with intravenous administration of PF-06818883, a pro-drug of a selective MAGL inhibitor (total of seven doses between 0.01 and 1.27 mg/kg). Kinetic parameters K1, k2 and k3 were estimated by a two tissue compartment (2TC) model using metabolite corrected plasma radioactivity as the input function. k4 was set as 0 according to the irreversible binding of [11C]PF-06809247. Ki by 2TC and Patlak analysis were calculated as the influx constant. The target occupancy was calculated using Ki at baseline and pretreatment conditions. Two cynomolgus monkeys were enrolled for whole-body PET measurements. Estimates of the absorbed radiation dose in humans were calculated with OLINDA/EXM 1.1 using the adult male reference model. RESULTS: Radioactivity retention was decreased in all brain regions following pretreatment with PF-06818883. Occupancy was measured as 25.4-100.5% in a dose dependent manner. Whole-body PET showed high radioactivity uptake values in the liver, small intestine, kidney, and brain. The effective dose of [11C]PF-06809247 was calculated as 4.3 µSv/MBq. CONCLUSIONS: [11C]PF-06809247 is a promising PET ligand for further studies of MAGL in the human brain.
RESUMO
Endocannabinoids (eCBs) function as retrograde signaling molecules at synapses throughout the brain, regulate axonal growth and guidance during development, and drive adult neurogenesis. There remains a lack of genetic evidence as to the identity of the enzyme(s) responsible for the synthesis of eCBs in the brain. Diacylglycerol lipase-alpha (DAGLalpha) and -beta (DAGLbeta) synthesize 2-arachidonoyl-glycerol (2-AG), the most abundant eCB in the brain. However, their respective contribution to this and to eCB signaling has not been tested. In the present study, we show approximately 80% reductions in 2-AG levels in the brain and spinal cord in DAGLalpha(-/-) mice and a 50% reduction in the brain in DAGLbeta(-/-) mice. In contrast, DAGLbeta plays a more important role than DAGLalpha in regulating 2-AG levels in the liver, with a 90% reduction seen in DAGLbeta(-/-) mice. Levels of arachidonic acid decrease in parallel with 2-AG, suggesting that DAGL activity controls the steady-state levels of both lipids. In the hippocampus, the postsynaptic release of an eCB results in the transient suppression of GABA-mediated transmission at inhibitory synapses; we now show that this form of synaptic plasticity is completely lost in DAGLalpha(-/-) animals and relatively unaffected in DAGLbeta(-/-) animals. Finally, we show that the control of adult neurogenesis in the hippocampus and subventricular zone is compromised in the DAGLalpha(-/-) and/or DAGLbeta(-/-) mice. These findings provide the first evidence that DAGLalpha is the major biosynthetic enzyme for 2-AG in the nervous system and reveal an essential role for this enzyme in regulating retrograde synaptic plasticity and adult neurogenesis.
Assuntos
Encéfalo/metabolismo , Moduladores de Receptores de Canabinoides/fisiologia , Endocanabinoides , Lipase Lipoproteica/genética , Animais , Ácidos Araquidônicos/metabolismo , Encéfalo/citologia , Glicerídeos/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Knockout , Neurogênese , Plasticidade Neuronal , Transdução de Sinais , Medula Espinal/metabolismo , Sinapses/fisiologiaRESUMO
The neuronal adhesion protein Dragon acts as a bone morphogenetic protein (BMP) coreceptor that enhances BMP signaling. Given the importance of BMP signaling in nephrogenesis and its putative role in the response to injury in the adult kidney, we studied the localization and function of Dragon in the kidney. We observed that Dragon localized predominantly to the apical surfaces of tubular epithelial cells in the thick ascending limbs, distal convoluted tubules, and collecting ducts of mice. Dragon expression was weak in the proximal tubules and glomeruli. In mouse inner medullary collecting duct (mIMCD3) cells, Dragon generated BMP signals in a ligand-dependent manner, and BMP4 is the predominant endogenous ligand for the Dragon coreceptor. In mIMCD3 cells, BMP4 normally signaled through BMPRII, but Dragon enhanced its signaling through the BMP type II receptor ActRIIA. Dragon and BMP4 increased transepithelial resistance (TER) through the Smad1/5/8 pathway. In epithelial cells isolated from the proximal tubule and intercalated cells of collecting ducts, we observed coexpression of ActRIIA, Dragon, and BMP4 but not BMPRII. Taken together, these results suggest that Dragon may enhance BMP signaling in renal tubular epithelial cells and maintain normal renal physiology.
Assuntos
Proteínas Morfogenéticas Ósseas/fisiologia , Células Epiteliais/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Moléculas de Adesão de Célula Nervosa/fisiologia , Animais , Células Cultivadas , Rim , Camundongos , Transdução de Sinais , Urotélio/citologiaRESUMO
P2X5 is a member of the P2X family of ATP-gated nonselective cation channels, which exist as trimeric assemblies. P2X5 is believed to trimerize with another member of this family, P2X1. We investigated the single-nucleotide polymorphism (SNP) at the 3' splice site of exon 10 of the human P2X5 gene. As reported previously, presence of a T at the SNP location results in inclusion of exon 10 in the mature transcript, whereas exon 10 is excluded when a G is present at this location. Our genotyping of human DNA samples reveals predominance of the G-bearing allele, which was exclusively present in DNA samples from white American, Middle Eastern, and Chinese donors. Samples from African American donors were polymorphic, with the G allele more frequent. Reverse transcription-polymerase chain reaction analysis of lymphocytes demonstrated a 100% positive correlation between genotype and P2X5 transcript. Immunostaining of P2X1/P2X5 stably coexpressing cell lines showed full-length P2X5 to be expressed at the cell surface and the exon 10-deleted isoform to be cytoplasmic. Fluorometric imaging-based pharmacological characterization indicated a ligand-dependent increase in intracellular calcium in 1321N1 astrocytoma cells transiently expressing full-length P2X5 but not the exon 10-deleted isoform. Likewise, electrophysiological analysis showed robust ATP-evoked currents when full-length but not the exon 10-deleted isoform of P2X5 was expressed. Taken together, our findings indicate that most humans express only a nonfunctional isoform of P2X5, which is in stark contrast to what is seen in other vertebrate species in which P2X5 has been studied, from which only the full-length isoform is known.
Assuntos
Éxons , Polimorfismo de Nucleotídeo Único , Isoformas de Proteínas/genética , Receptores Purinérgicos P2/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Linhagem Celular , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiologia , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2/fisiologia , Receptores Purinérgicos P2X5 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido NucleicoRESUMO
Endocannabinoids are lipid molecules that serve as natural ligands for the cannabinoid receptors CB1 and CB2. They modulate a diverse set of physiological processes such as pain, cognition, appetite, and emotional states, and their levels and functions are tightly regulated by enzymatic biosynthesis and degradation. 2-Arachidonoylglycerol (2-AG) is the most abundant endocannabinoid in the brain and is believed to be hydrolyzed primarily by the serine hydrolase monoacylglycerol lipase (MAGL). Although 2-AG binds and activates cannabinoid receptors in vitro, when administered in vivo, it induces only transient cannabimimetic effects as a result of its rapid catabolism. Here we show using a mouse model with a targeted disruption of the MAGL gene that MAGL is the major modulator of 2-AG hydrolysis in vivo. Mice lacking MAGL exhibit dramatically reduced 2-AG hydrolase activity and highly elevated 2-AG levels in the nervous system. A lack of MAGL activity and subsequent long-term elevation of 2-AG levels lead to desensitization of brain CB1 receptors with a significant reduction of cannabimimetic effects of CB1 agonists. Also consistent with CB1 desensitization, MAGL-deficient mice do not show alterations in neuropathic and inflammatory pain sensitivity. These findings provide the first genetic in vivo evidence that MAGL is the major regulator of 2-AG levels and signaling and reveal a pivotal role for 2-AG in modulating CB1 receptor sensitization and endocannabinoid tone.
Assuntos
Moduladores de Receptores de Canabinoides/fisiologia , Endocanabinoides , Monoacilglicerol Lipases/metabolismo , Receptor CB1 de Canabinoide/fisiologia , Animais , Ativação Enzimática/genética , Ativação Enzimática/fisiologia , Hidrólise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Monoacilglicerol Lipases/deficiência , Monoacilglicerol Lipases/fisiologia , Medição da Dor/métodosRESUMO
Rimonabant was the first clinically marketed cannabinoid (CB)(1) receptor antagonist developed to treat obesity. Unfortunately, CB(1) receptor antagonism produced adverse psychiatric events in patients. To determine whether this occurs pre-clinically, we investigated the effects of rimonabant in rodent models of mood disorders. Chronic treatment with rimonabant increased immobility time in the rat forced swim test and reduced the consumption of sucrose-sweetened water in an assay postulated to model anhedonia. These responses were similar to the effects elicited by chronic mild stress in these behavioral models, which, taken together, are indicative of a depression-like phenotype. Additionally, chronic treatment with rimonabant produced decreases in frontal cortex serotonin levels, marked reductions in hippocampal cell proliferation, survival, and BDNF levels, and elevations in the concentrations of pro-inflammatory cytokines including interferon gamma and TNF alpha. These preclinical findings mimic clinical reports and implicate possible mechanisms responsible for the unfavorable psychiatric events reported following chronic rimonabant use.
Assuntos
Fenótipo , Piperidinas/efeitos adversos , Pirazóis/efeitos adversos , Receptor CB1 de Canabinoide/antagonistas & inibidores , Estresse Psicológico/induzido quimicamente , Estresse Psicológico/fisiopatologia , Análise de Variância , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Citometria de Fluxo/métodos , Preferências Alimentares/efeitos dos fármacos , Lobo Frontal/efeitos dos fármacos , Lobo Frontal/metabolismo , Resposta de Imobilidade Tônica/efeitos dos fármacos , Masculino , Microdiálise/métodos , Ratos , Ratos Sprague-Dawley , Rimonabanto , Estresse Psicológico/patologia , Sacarose/administração & dosagem , Edulcorantes/administração & dosagem , Natação/psicologiaRESUMO
A cardinal feature of inflammation is heightened pain sensitivity at the site of the inflamed tissue. This results from the local release by immune and injured cells of nociceptor sensitizers, including prostaglandin E(2), bradykinin, and nerve growth factor, that reduce the threshold and increase the excitability of the peripheral terminals of nociceptors so that they now respond to innocuous stimuli: the phenomenon of peripheral sensitization. We show here that the proinflammatory cytokine interleukin-1beta (IL-1beta), in addition to producing inflammation and inducing synthesis of several nociceptor sensitizers, also rapidly and directly activates nociceptors to generate action potentials and induce pain hypersensitivity. IL-1beta acts in a p38 mitogen-activated protein kinase (p38 MAP kinase)-dependent manner, to increase the excitability of nociceptors by relieving resting slow inactivation of tetrodotoxin-resistant voltage-gated sodium channels and also enhances persistent TTX-resistant current near threshold. By acting as an IL-1beta sensor, nociceptors can directly signal the presence of ongoing tissue inflammation.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Interleucina-1beta/farmacologia , Nociceptores/efeitos dos fármacos , Células Receptoras Sensoriais/fisiologia , Potenciais de Ação/fisiologia , Animais , Biofísica , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Gânglios Espinais/citologia , Imidazóis/farmacologia , Proteínas de Filamentos Intermediários/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Fibras Nervosas Amielínicas/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Patch-Clamp/métodos , Periferinas , Estimulação Física/métodos , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia , Fatores de Tempo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Nociceptive pain in response to peripheral noxious stimuli, and inflammatory pain resulting from tissue damage, serve as warnings that normal bodily function cannot resume until the stimulus abates or the tissue repairs. Stimuli cause numerous receptors, ion channels and other cellular machinery to respond, and propagate signals to the central nervous system, where this information is processed and perceived as pain. In healthy individuals, tissue damage results in physiologic--generally reparative--changes that lead to heightened sensory perception and, often, pain. In rheumatic diseases, the joint pain bears much in common with chronic inflammatory pain, but the underlying disease state is typically much more intricate and no reparative function is evident. Addressing the complex pains of rheumatic disease remains an ongoing challenge. Pain signaling pathways involve many molecular components that could potentially be targets for pharmacotherapeutic intervention, but the complexity of this system might also mean that multiple sites must be affected simultaneously to disrupt propagation of pain signals. In addition, to be therapeutically viable, pain drugs must be safe and not alter tactile sensory function, alertness or cognitive function. In this article we review the molecular functions in nociceptive, inflammatory and rheumatic pain pathways, and the therapeutic options they might offer.
Assuntos
Articulações/fisiopatologia , Dor/fisiopatologia , Doenças Reumáticas/fisiopatologia , Humanos , Nociceptores/fisiologia , Dor/etiologia , Doenças Reumáticas/complicações , Reumatologia , Transdução de Sinais/fisiologiaRESUMO
Recent evidence shows that neuroinflammation plays a role in many neurological diseases including mild cognitive impairment (MCI) and Alzheimer's disease (AD), and that free water (FW) modeling from clinically acquired diffusion MRI (DTI-like acquisitions) can be sensitive to this phenomenon. This FW index measures the fraction of the diffusion signal explained by isotropically unconstrained water, as estimated from a bi-tensor model. In this study, we developed a simple but powerful whole-brain FW measure designed for easy translation to clinical settings and potential use as a priori outcome measure in clinical trials. These simple FW measures use a "safe" white matter (WM) mask without gray matter (GM)/CSF partial volume contamination (WM safe) near ventricles and sulci. We investigated if FW inside the WM safe mask, including and excluding areas of white matter damage such as white matter hyperintensities (WMHs) as shown on T2 FLAIR, computed across the whole white matter could be indicative of diagnostic grouping along the AD continuum. After careful quality control, 81 cognitively normal controls (NC), 103 subjects with MCI and 42 with AD were selected from the ADNIGO and ADNI2 databases. We show that MCI and AD have significantly higher FW measures even after removing all partial volume contamination. We also show, for the first time, that when WMHs are removed from the masks, the significant results are maintained, which demonstrates that the FW measures are not just a byproduct of WMHs. Our new and simple FW measures can be used to increase our understanding of the role of inflammation-associated edema in AD and may aid in the differentiation of healthy subjects from MCI and AD patients.
RESUMO
To date, the development of disease-modifying therapies for Alzheimer's disease (AD) has largely focused on the removal of amyloid beta Aß fragments from the CNS. Proteomic profiling of patient fluids may help identify novel therapeutic targets and biomarkers associated with AD pathology. Here, we applied the Olink™ ProSeek immunoassay to measure 270 CSF and plasma proteins across 415 Aß- negative cognitively normal individuals (Aß- CN), 142 Aß-positive CN (Aß+ CN), 50 Aß- mild cognitive impairment (MCI) patients, 75 Aß+ MCI patients, and 161 Aß+ AD patients from the Swedish BioFINDER study. A validation cohort included 59 Aß- CN, 23 Aß- + CN, 44 Aß- MCI and 53 Aß+ MCI. To compare protein concentrations in patients versus controls, we applied multiple linear regressions adjusting for age, gender, medications, smoking and mean subject-level protein concentration, and corrected findings for false discovery rate (FDR, q < 0.05). We identified, and replicated, altered levels of ten CSF proteins in Aß+ individuals, including CHIT1, SMOC2, MMP-10, LDLR, CD200, EIF4EBP1, ALCAM, RGMB, tPA and STAMBP (- 0.14 < d < 1.16; q < 0.05). We also identified and replicated alterations of six plasma proteins in Aß+ individuals OSM, MMP-9, HAGH, CD200, AXIN1, and uPA (- 0.77 < d < 1.28; q < 0.05). Multiple analytes associated with cognitive performance and cortical thickness (q < 0.05). Plasma biomarkers could distinguish AD dementia (AUC = 0.94, 95% CI = 0.87-0.98) and prodromal AD (AUC = 0.78, 95% CI = 0.68-0.87) from CN. These findings reemphasize the contributions of immune markers, phospholipids, angiogenic proteins and other biomarkers downstream of, and potentially orthogonal to, Aß- and tau in AD, and identify candidate biomarkers for earlier detection of neurodegeneration.
Assuntos
Doença de Alzheimer/sangue , Doença de Alzheimer/líquido cefalorraquidiano , Proteômica/métodos , Idoso , Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides/sangue , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Estudos de Coortes , Feminino , Humanos , Imunoensaio/métodos , Masculino , Pessoa de Meia-IdadeRESUMO
Peripheral inflammation induces p38 MAPK activation in the soma of C fiber nociceptors in the dorsal root ganglion (DRG) after 24 hr. Inflammation also increases protein, but not mRNA levels, of the heat-gated ion channel TRPV1 (VR1) in these cells, which is then transported to peripheral but not central C fiber terminals. Inhibiting p38 activation in the DRG reduces the increase in TRPV1 in the DRG and inflamed skin and diminishes inflammation-induced heat hypersensitivity without affecting inflammatory swelling or basal pain sensitivity. p38 activation in the DRG is secondary to peripheral production of NGF during inflammation and is required for NGF-induced increases in TRPV1. The activation of p38 in the DRG following retrograde NGF transport, by increasing TRPV1 levels in nociceptor peripheral terminals in a transcription-independent fashion, contributes to the maintenance of inflammatory heat hypersensitivity.
Assuntos
Hiperalgesia/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fator de Crescimento Neural/metabolismo , Neurônios Aferentes/enzimologia , Receptores de Droga/deficiência , Regulação para Cima/fisiologia , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/enzimologia , Hiperalgesia/fisiopatologia , Imuno-Histoquímica , Inflamação/enzimologia , Inflamação/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Fibras Nervosas Amielínicas/efeitos dos fármacos , Fibras Nervosas Amielínicas/enzimologia , Fator de Crescimento Neural/antagonistas & inibidores , Neuralgia/enzimologia , Neuralgia/fisiopatologia , Neurônios Aferentes/efeitos dos fármacos , Nociceptores/efeitos dos fármacos , Nociceptores/enzimologia , Células do Corno Posterior/enzimologia , Ratos , Ratos Sprague-Dawley , Receptores de Droga/efeitos dos fármacos , Receptores de Droga/genética , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Monoacylglycerol lipase (MAGL) inhibition provides a potential treatment approach to neuroinflammation through modulation of both the endocannabinoid pathway and arachidonoyl signaling in the central nervous system (CNS). Herein we report the discovery of compound 15 (PF-06795071), a potent and selective covalent MAGL inhibitor, featuring a novel trifluoromethyl glycol leaving group that confers significant physicochemical property improvements as compared with earlier inhibitor series with more lipophilic leaving groups. The design strategy focused on identifying an optimized leaving group that delivers MAGL potency, serine hydrolase selectivity, and CNS exposure while simultaneously reducing log D, improving solubility, and minimizing chemical lability. Compound 15 achieves excellent CNS exposure, extended 2-AG elevation effect in vivo, and decreased brain inflammatory markers in response to an inflammatory challenge.