RESUMO
Allosteric modulators of the metabotropic group II receptors, mGluR2 and mGluR3, have been widely explored due to their ability to modulate cognitive and neurological functions in mood disorders, although none have been approved yet. In our search for new and selective mGluR2 negative allosteric modulators (NAMs), series of 6,7-dihydropyrazolo[1,5-a]pyrazin-4(5H)-one derivatives were identified from our published series of 1,3,5-trisubstituted pyrazoles. SAR evolution of the initial hit resulted in 100-fold improvement in the mGluR2 NAM potency and subsequent selection of compound 11 based on its overall profile, including selectivity and ADMET properties. Further pharmacokinetic-pharmacodynamic (PK-PD) relationship built showed that compound 11 occupied the mGluR2 receptor in a dose-dependent manner. Additionally, the compound revealed in vivo activity in V-maze as a model of cognition from a dose of 0.32 mg/kg. Compound 11 was selected to be evaluated further.
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The imbalance between production and clearance of amyloid ß (Aß) peptides and their resulting accumulation in the brain is an early and crucial step in the pathogenesis of Alzheimer's disease (AD). Therefore, Aß is strongly positioned as a promising and extensively validated therapeutic target for AD. Investigational disease-modifying approaches aiming at reducing cerebral Aß concentrations include prevention of de novo production of Aß through inhibition of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1), and clearance of Aß deposits via passive Aß immunotherapy. We have developed a novel, high affinity antibody against Aß peptides bearing a pyroglutamate residue at amino acid position 3 (3pE), an Aß species abundantly present in plaque deposits in AD brains. Here, we describe the preclinical characterization of this antibody, and demonstrate a significant reduction in amyloid burden in the absence of microhemorrhages in different mouse models with established plaque deposition. Moreover, we combined antibody treatment with chronic BACE1 inhibitor treatment and demonstrate significant clearance of pre-existing amyloid deposits in transgenic mouse brain, without induction of microhemorrhages and other histopathological findings. Together, these data confirm significant potential for the 3pE-specific antibody to be developed as a passive immunotherapy approach that balances efficacy and safety. Moreover, our studies suggest further enhanced treatment efficacy and favorable safety after combination of the 3pE-specific antibody with BACE1 inhibitor treatment.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Peptídeos beta-Amiloides/antagonistas & inibidores , Anticorpos Monoclonais/administração & dosagem , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Imunização Passiva/métodos , Fragmentos de Peptídeos/antagonistas & inibidores , Placa Amiloide/tratamento farmacológico , Secretases da Proteína Precursora do Amiloide/imunologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/imunologia , Peptídeos beta-Amiloides/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Ácido Aspártico Endopeptidases/imunologia , Ácido Aspártico Endopeptidases/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/imunologia , Placa Amiloide/metabolismo , Resultado do TratamentoRESUMO
Neuroimaging endophenotypes in animal models provide an objective and translationally-relevant alternative to cognitive/behavioral traits in human psychopathologies. Metabolic alterations, such as those involved in the glutamate-cycle, have been proposed to play a preponderant role in both depression and schizophrenia. Chronic Mild Unpredictable Stress (CMUS) and sub-chronic administration of NMDA receptor antagonist generate animal models of depression and schizophrenia, respectively. The models are based on etiologically-relevant factors related to the induction and support of these psychopathologies. To test metabolic alterations within the glutamate-cycle and in other major neurochemicals, single-voxel Magnetic Resonance Spectroscopy was recorded within the hippocampus in both rat models and control animals. Surprisingly, altered glutamate-related metabolites were observed in the CMUS model, but not NMDA-based model, as indicated by decreased glutamine and increased GABA levels. However, both models presented elevated total visible choline and inositol levels relative to controls. These results indicate the presence cell membrane metabolic alterations and inflammatory processes shared in both models, comparable to evidence presented in schizophrenia and depression and other comparable animal models. These translationally-relevant biomarkers may thus form the basis for drug-development targets in both psychopathologies.
Assuntos
Depressão/metabolismo , Modelos Animais de Doenças , Ácido Glutâmico/metabolismo , Hipocampo/metabolismo , Esquizofrenia/metabolismo , Anedonia , Animais , Colina/metabolismo , Depressão/diagnóstico por imagem , Antagonistas de Aminoácidos Excitatórios/farmacologia , Glutamina/metabolismo , Inositol/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Memantina/farmacologia , Atividade Motora , Ratos , Ratos Wistar , Esquizofrenia/diagnóstico por imagem , Estresse Psicológico/metabolismo , Sacarose , Taurina/metabolismoRESUMO
Coadministration of 2 or more compounds can alter both the pharmacokinetics and pharmacodynamics of individual compounds. While experiments on pharmacodynamic drug-drug interactions are usually performed in an in vitro setting, this experiment focuses on an in vivo setting. The change over time of a safety biomarker is modeled using an indirect response model, in which the virtual pharmacokinetic profile of one compound drives the effect of the other. Several experiments at different dose level combinations were performed sequentially. While a traditional frequentist analysis consists of estimating the model parameters based on all the data simultaneously, in this work, we consider a Bayesian inference framework allowing to incorporate the results from a historical dose-response experiment.
Assuntos
Teorema de Bayes , Modelos Biológicos , Farmacologia , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Quimioterapia Combinada , HumanosRESUMO
The tau spreading hypothesis provides rationale for passive immunization with an anti-tau monoclonal antibody to block seeding by extracellular tau aggregates as a disease-modifying strategy for the treatment of Alzheimer's disease (AD) and potentially other tauopathies. As the biochemical and biophysical properties of the tau species responsible for the spatio-temporal sequences of seeding events are poorly defined, it is not yet clear which epitope is preferred for obtaining optimal therapeutic efficacy. Our internal tau antibody collection has been generated by immunizations with different tau species: aggregated- and non-aggregated tau and human postmortem AD brain-derived tau fibrils. In this communication, we describe and characterize a set of these anti-tau antibodies for their biochemical and biophysical properties, including binding, tissue staining by immunohistochemistry, and epitope. The antibodies bound to different domains of the tau protein and some were demonstrated to be isoform-selective (PT18 and hTau56) or phospho-selective (PT84). Evaluation of the antibodies in cellular- and in vivo seeding assays revealed clear differences in maximal efficacy. Limited proteolysis experiments support the hypothesis that some epitopes are more exposed than others in the tau seeds. Moreover, antibody efficacy seems to depend on the structural properties of fibrils purified from tau Tg mice- and postmortem human AD brain.
Assuntos
Doença de Alzheimer/patologia , Anticorpos Monoclonais/metabolismo , Encéfalo/metabolismo , Proteínas tau/imunologia , Animais , Mapeamento de Epitopos , Feminino , Células HEK293 , Humanos , Imunização Passiva , Masculino , Camundongos , Camundongos Knockout , Mutação/genética , Ressonância de Plasmônio de Superfície , Proteínas tau/deficiência , Proteínas tau/genéticaRESUMO
The guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase II (cGKII) serine/threonine kinase relays signaling through guanylyl cyclase C (GCC) to control intestinal fluid homeostasis. Here, we report the discovery of small-molecule inhibitors of cGKII. These inhibitors were imidazole-aminopyrimidines, which blocked recombinant human cGKII at submicromolar concentrations but exhibited comparatively little activity toward the phylogenetically related protein kinases cGKI and cAMP-dependent protein kinase (PKA). Whereas aminopyrimidyl motifs are common in protein kinase inhibitors, molecular modeling of these imidazole-aminopyrimidines in the ATP-binding pocket of cGKII indicated an unconventional binding mode that directs their amine substituent into a narrow pocket delineated by hydrophobic residues of the hinge and the αC-helix. Crucially, this set of residues included the Leu-530 gatekeeper, which is not conserved in cGKI and PKA. In intestinal organoids, these compounds blocked cGKII-dependent phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). In mouse small intestinal tissue, cGKII inhibition significantly attenuated the anion secretory response provoked by the GCC-activating bacterial heat-stable toxin (STa), a frequent cause of infectious secretory diarrhea. In contrast, both PKA-dependent VASP phosphorylation and intestinal anion secretion were unaffected by treatment with these compounds, whereas experiments with T84 cells indicated that they weakly inhibit the activity of cAMP-hydrolyzing phosphodiesterases. As these protein kinase inhibitors are the first to display selective inhibition of cGKII, they may expedite research on cGMP signaling and may aid future development of therapeutics for managing diarrheal disease and other pathogenic syndromes that involve cGKII.
Assuntos
Proteína Quinase Dependente de GMP Cíclico Tipo II/antagonistas & inibidores , GMP Cíclico/metabolismo , Intestinos/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Sequência de Aminoácidos , Animais , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Cristalografia por Raios X , Humanos , Intestinos/efeitos dos fármacos , Camundongos , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Fosfoproteínas/metabolismo , Conformação Proteica , Homologia de Sequência , Transdução de SinaisRESUMO
Mounting preclinical evidence has implicated the NLRP3 inflammasome in depression-related behaviours elicited by chronic stress or acute lipopolysaccharide (LPS) challenge. However, the relevance of acute LPS as a model of depression has been questioned and behavioural time-courses of its effects can be inconsistent. The aims of this study were (1) to develop a novel protocol for repeated daily LPS administration and (2) to use this model to assess the involvement of NLRP3 inflammasome signalling in sustained inflammation-induced depressive-like behaviour in adult C57BL/6J mice deficient in NLRP3. Acute LPS (0.83mg/kg; i.p.) induced sickness behaviour evident as hypolocomotor activity. However, there was no significant increase in depressive-like behaviour in the forced swim test 24h post-administration. Interestingly, depressive-like behaviours were observed in the female urine sniffing test and in the sucrose preference test at 24h, but not 48h, post-administration of acute LPS. To mimic a period of sustained inflammation, 3-day repeated increasing LPS doses (0.1, 0.42 and 0.83mg/kg; i.p.) was compared to constant LPS doses (0.83mg/kg; i.p.). Sickness behaviour was seen in response to increasing doses, but tolerance developed to repeated constant doses of LPS. Furthermore, 3-day increasing doses of LPS resulted in a significant increase in immobility time in the forced swim test, consistent with depressive-like behaviour. When NLRP3-/- mice received this 3-day increasing dose regimen of LPS, sickness behaviours were attenuated compared to wild-type mice. The behaviour in the forced swim test was not significantly altered in NLRP3-/- mice. We propose that this increasing repeated dosing LPS model of inflammation-induced depressive-like behaviour may better model the sustained inflammation observed in depression and may provide a more translationally relevant paradigm to study the inflammatory mechanisms that contribute to depression.
Assuntos
Transtorno Depressivo/imunologia , Modelos Animais de Doenças , Inflamação , Lipopolissacarídeos , Anedonia/fisiologia , Animais , Transtorno Depressivo/etiologia , Comportamento de Doença/fisiologia , Inflamassomos/metabolismo , Inflamação/complicações , Inflamação/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neuroimunomodulação/fisiologia , Distribuição Aleatória , Comportamento Sexual Animal/fisiologiaRESUMO
The metabotropic glutamate subtypeâ 2 (mGlu2 ) receptor is a presynaptic membrane receptor distributed widely in brain that provides feedback inhibitory control of glutamate release. Inhibition of the mGlu2 receptor function with a negative allosteric modulator (NAM) enhances activity-dependent glutamate release, which may be of therapeutic benefit for the treatment of neurological and psychiatric disorders. An attractive pyrazole hit was identified after a high-throughput screening (HTS) campaign. The evolution of this hit is described by structure-activity relationship (SAR) studies on specific parts of the molecule. From near micromolar potency we could obtain compounds with single-digit nanomolar activity in the mGlu2 NAM GTPγS assay. In addition to SAR on inâ vitro potency, a more detailed overview is given with a specific set of compounds on the excellent agreement between inâ vitro potency, free brain concentration, and exâ vivo mGlu2 receptor occupancy. Finally, to obtain improved drug-like compounds, plans for future research are suggested toward increasing free brain concentration while maintaining high inâ vitro potency.
Assuntos
Pirazóis/farmacologia , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Microssomos Hepáticos/química , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Permeabilidade/efeitos dos fármacos , Pirazóis/química , Ratos , Relação Estrutura-AtividadeRESUMO
Inflammatory processes may cause depression in subsets of vulnerable individuals. Inflammation-associated behavioral changes are commonly modelled in rodents by administration of bacterial lipopolysaccharide (LPS). However, the time frame in which immune activation and depressive-like behavior occur is not very clear. In this study, we showed that systemic administration of LPS robustly increased circulating levels of corticosterone, leptin, pro- and anti-inflammatory cytokines, and chemokines. Serum concentrations of most analytes peaked within the first 6 h after LPS injection and returned to baseline values by 24 h. Chemokine levels, however, remained elevated for up to 96 h. Using an optimized sucrose preference test (SPT) we showed that sickness behavior was present from 2 to 24 h. LPS-induced anhedonia, as measured by decreased sucrose preference, lasted up to 96 h. To mimic the human situation, where depression develops after chronic inflammation, rats were preexposed to repeated LPS administration or subchronic restraint stress and subsequently challenged with LPS. While these procedures did not increase the duration of anhedonia, our results do indicate that inflammation may cause depressive symptoms such as anhedonia. Using our SPT protocol, more elaborate rodent models can be developed to study the mechanisms underlying inflammation-associated depression in humans.
Assuntos
Anedonia/efeitos dos fármacos , Comportamento Animal/efeitos dos fármacos , Citocinas/sangue , Depressão/sangue , Depressão/induzido quimicamente , Lipopolissacarídeos/toxicidade , Animais , Depressão/fisiopatologia , Humanos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Members of the α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) subtype of ionotropic glutamate receptors mediate the majority of fast synaptic transmission within the mammalian brain and spinal cord, representing attractive targets for therapeutic intervention. Here, we describe novel AMPA receptor modulators that require the presence of the accessory protein CACNG8, also known as transmembrane AMPA receptor regulatory protein γ8 (TARP-γ8). Using calcium flux, radioligand binding, and electrophysiological assays of wild-type and mutant forms of TARP-γ8, we demonstrate that these compounds possess a novel mechanism of action consistent with a partial disruption of the interaction between the TARP and the pore-forming subunit of the channel. One of the molecules, 5-[2-chloro-6-(trifluoromethoxy)phenyl]-1,3-dihydrobenzimidazol-2-one (JNJ-55511118), had excellent pharmacokinetic properties and achieved high receptor occupancy following oral administration. This molecule showed strong, dose-dependent inhibition of neurotransmission within the hippocampus, and a strong anticonvulsant effect. At high levels of receptor occupancy in rodent in vivo models, JNJ-55511118 showed a strong reduction in certain bands on electroencephalogram, transient hyperlocomotion, no motor impairment on rotarod, and a mild impairment in learning and memory. JNJ-55511118 is a novel tool for reversible AMPA receptor inhibition, particularly within the hippocampus, with potential therapeutic utility as an anticonvulsant or neuroprotectant. The existence of a molecule with this mechanism of action demonstrates the possibility of pharmacological targeting of accessory proteins, increasing the potential number of druggable targets.
Assuntos
Benzimidazóis/uso terapêutico , Canais de Cálcio/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Receptores de AMPA/efeitos dos fármacos , Animais , Canais de Cálcio/genética , Sinalização do Cálcio/efeitos dos fármacos , Desenho de Fármacos , Eletroencefalografia/efeitos dos fármacos , Células HEK293 , Humanos , Aprendizagem/efeitos dos fármacos , Memória/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Atividade Motora/efeitos dos fármacos , Mutação/genética , Neurônios/efeitos dos fármacos , Equilíbrio Postural/efeitos dos fármacos , Ratos Sprague-Dawley , Receptores de AMPA/genéticaRESUMO
Clinical observations indicate that activation of the TNF-α system may contribute to the development of inflammation-associated depression. Here, we tested the hypothesis that systemic upregulation of TNF-α induces neuroinflammation and behavioral changes relevant to depression. We report that a single intraperitoneal injection of TNF-α in mice increased serum and brain levels of the proinflammatory mediators TNF-α, IL-6, and MCP-1, in a dose- and time-dependent manner, but not IL-1ß. Protein levels of the anti-inflammatory cytokine IL-10 increased in serum but not in the brain. The transient release of immune molecules was followed by glial cell activation as indicated by increased astrocyte activation in bioluminescent Gfap-luc mice and elevated immunoreactivity against the microglial marker Iba1 in the dentate gyrus of TNF-α-challenged mice. Additionally, TNF-α-injected mice were evaluated in a panel of behavioral tests commonly used to study sickness and depressive-like behavior in rodents. Our behavioral data imply that systemic administration of TNF-α induces a strong sickness response characterized by reduced locomotor activity, decreased fluid intake, and body weight loss. Depressive-like behavior could not be separated from sickness at any of the time points studied. Together, these results demonstrate that peripheral TNF-α affects the central nervous system at a neuroimmune and behavioral level.
Assuntos
Comportamento Animal/efeitos dos fármacos , Encéfalo/metabolismo , Encefalite/metabolismo , Fator de Necrose Tumoral alfa/efeitos adversos , Animais , Biomarcadores/metabolismo , Encéfalo/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Quimiocina CCL2/metabolismo , Depressão/metabolismo , Depressão/patologia , Encefalite/induzido quimicamente , Encefalite/patologia , Inflamação/induzido quimicamente , Inflamação/metabolismo , Inflamação/patologia , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
BACKGROUND: Many enterotoxigenic Escherichia coli strains produce the heat-stable toxin, STa, which, by activation of the intestinal receptor-enzyme guanylyl cyclase (GC) C, triggers an acute, watery diarrhea. We set out to identify GCC inhibitors that may be of benefit for the treatment of infectious diarrheal disease. METHODS: Compounds that inhibit STa-induced cyclic guanosine 3',5'-monophosphate (cGMP) production were selected by performing cyclase assays on cells and membranes containing GCC, or the related GCA. The effect of leads on STa/GCC-dependent activation of the cystic fibrosis transmembrane conductance regulator anion channel was investigated in T84 cells, and in porcine and human intestinal tissue. Their effect on STa-provoked fluid transport was assessed in ligated intestinal loops in piglets. RESULTS: Four N-2-(propylamino)-6-phenylpyrimidin-4-one-substituted piperidines were shown to inhibit GCC-mediated cellular cGMP production. The half maximal inhibitory concentrations were ≤ 5 × 10(-7) mol/L, whereas they were >10 times higher for GCA. In T84 monolayers, these leads blocked STa/GCC-dependent, but not forskolin/adenylyl cyclase-dependent, cystic fibrosis transmembrane conductance regulator activity. GCC inhibition reduced STa-provoked anion secretion in pig jejunal tissue, and fluid retention and cGMP levels in STa-exposed loops. These GCC inhibitors blocked STa-provoked anion secretion in rectal biopsy specimens. CONCLUSIONS: We have identified a novel class of GCC inhibitors that may form the basis for development of future therapeutics for (infectious) diarrheal disease.
Assuntos
Toxinas Bacterianas/antagonistas & inibidores , Enterotoxinas/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Proteínas de Escherichia coli/antagonistas & inibidores , Jejuno/efeitos dos fármacos , Piperidinas/farmacologia , Receptores Acoplados a Guanilato Ciclase/antagonistas & inibidores , Receptores de Peptídeos/antagonistas & inibidores , Adenilil Ciclases/metabolismo , Adulto , Animais , Toxinas Bacterianas/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Diarreia , Escherichia coli Enterotoxigênica , Enterotoxinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Células HeLa , Humanos , Jejuno/citologia , Jejuno/metabolismo , Modelos Biológicos , Receptores de Enterotoxina , Receptores Acoplados a Guanilato Ciclase/metabolismo , Receptores de Peptídeos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Suínos , Adulto JovemRESUMO
Neuroinflammation and the accompanying activation of glial cells is an important feature of many neurodegenerative conditions. It is known that factors such as peripheral infections and stress can influence immune processes in the brain. However, the effect of these stressors on astrocyte activation in vivo remains elusive. In this study, transgenic Gfap-luc mice expressing the luciferase gene under the transcriptional control of the glial fibrillary acidic protein promoter were used to quantify the kinetics of in vivo astrocyte activation following immune challenges relevant to clinical inflammation. It was found that astrocytes respond rapidly to peripheral immune activation elicited by either bacterial lipopolysaccharide (LPS) or the viral mimetic polyinosinic:polycytidylic acid (poly(I:C)). By measuring bioluminescence and 18-kDa translocator protein radioligand binding in the same animal it was observed that LPS induces both astrocyte as well as microglial activation at 6 h post-administration. Furthermore, the astrocyte response decreased upon repeated systemic LPS injections, indicating development of tolerance to the LPS challenge. Finally, restraining Gfap-luc mice for 1 h daily on 5 consecutive days did not affect brain bioluminescence, thereby indicating that sub-chronic stress does not influence astrocyte activation under unchallenged conditions. However, stressed animals showed a reduced response to a subsequent systemic LPS injection, suggesting that the immune system is compromised in these animals. Here, we demonstrate that Gfap-luc mice can be used to study astrocyte activation in response to stimuli relevant for clinical inflammation and that this approach may provide a more complete characterization of existing and novel models of neuroinflammation
Assuntos
Astrócitos/fisiologia , Encéfalo/imunologia , Inflamação/fisiopatologia , Neuroimunomodulação/fisiologia , Estresse Psicológico/imunologia , Animais , Modelos Animais de Doenças , Proteína Glial Fibrilar Ácida , Lipopolissacarídeos , Luciferases/genética , Luciferases/metabolismo , Medições Luminescentes , Masculino , Camundongos Transgênicos , Microglia/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Poli I-C , Distribuição Aleatória , Restrição FísicaRESUMO
Neurofibrillary tangles composed of hyperphosphorylated fibrillized tau are found in numerous tauopathies including Alzheimer's disease. Increasing evidence suggests that tau pathology can be transmitted from cell-to-cell; however the mechanisms involved in the initiation of tau fibrillization and spreading of disease linked to progression of tau pathology are poorly understood. We show here that intracerebral injections of preformed synthetic tau fibrils into the hippocampus or frontal cortex of young tau transgenic mice expressing mutant human P301L tau induces tau hyperphosphorylation and aggregation around the site of injection, as well as a time-dependent propagation of tau pathology to interconnected brain areas distant from the injection site. Furthermore, we show that the tau pathology as a consequence of injection of tau preformed fibrils into the hippocampus induces selective loss of CA1 neurons. Together, our data confirm previous studies on the seeded induction and the spreading of tau pathology in a different tau transgenic mouse model and reveals neuronal loss associated with seeded tau pathology in tau transgenic mouse brain. These results further validate the utility of the tau seeding model in studying disease transmission, and provide a more complete in vivo tauopathy model with associated neurodegeneration which can be used to investigate the mechanisms involved in tau aggregation and spreading, as well as aid in the search for disease modifying treatments for Alzheimer's disease and related tauopathies.
Assuntos
Tauopatias , Proteínas tau/administração & dosagem , Proteínas tau/genética , Fatores Etários , Análise de Variância , Animais , Modelos Animais de Doenças , Progressão da Doença , Lateralidade Funcional , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Camundongos , Camundongos Transgênicos , Mutação/genética , Emaranhados Neurofibrilares/metabolismo , Tauopatias/induzido quimicamente , Tauopatias/genética , Tauopatias/patologia , Proteínas tau/químicaRESUMO
Substantial evidence indicates an association between clinical depression and altered immune function. Systemic administration of bacterial lipopolysaccharide (LPS) is commonly used to study inflammation-associated behavioral changes in rodents. In these experiments, we tested the hypothesis that peripheral immune activation leads to neuroinflammation and depressive-like behavior in mice. We report that systemic administration of LPS induced astrocyte activation in transgenic GFAP-luc mice and increased immunoreactivity against the microglial marker ionized calcium-binding adapter molecule 1 in the dentate gyrus of wild-type mice. Furthermore, LPS treatment caused a strong but transient increase in cytokine levels in the serum and brain. In addition to studying LPS-induced neuroinflammation, we tested whether sickness could be separated from depressive-like behavior by evaluating LPS-treated mice in a panel of behavioral paradigms. Our behavioral data indicate that systemic LPS administration caused sickness and mild depressive-like behavior. However, due to the overlapping time course and mild effects on depression-related behavior per se, it was not possible to separate sickness from depressive-like behavior in the present rodent model.
Assuntos
Astrócitos/citologia , Depressão/imunologia , Comportamento de Doença , Inflamação/patologia , Animais , Comportamento Animal , Encéfalo/imunologia , Encéfalo/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Comportamento de Escolha , Citocinas/sangue , Citocinas/metabolismo , Modelos Animais de Doenças , Comportamento Alimentar , Imuno-Histoquímica , Lipopolissacarídeos/química , Luminescência , Masculino , Camundongos , Camundongos Transgênicos , Proteínas dos Microfilamentos/metabolismo , Microglia/metabolismo , Neurônios/metabolismo , Sacarose/químicaRESUMO
Ghrelin is a pleiotropic neuropeptide that has been recently implicated in epilepsy. Animal studies performed to date indicate that ghrelin has anticonvulsant properties; however, its mechanism of anticonvulsant action is unknown. Here we show that the anticonvulsant effects of ghrelin are mediated via the growth hormone secretagogue receptor (GHSR). To our surprise, however, we found that the GHSR knockout mice had a higher seizure threshold than their wild-type littermates when treated with pilocarpine. Using both in vivo and in vitro models, we further discovered that inverse agonism and desensitization/internalization of the GHSR attenuate limbic seizures in rats and epileptiform activity in hippocampal slices. This constitutes a novel mechanism of anticonvulsant action, whereby an endogenous agonist reduces the activity of a constitutively active receptor.
Assuntos
Anticonvulsivantes/uso terapêutico , Grelina/uso terapêutico , Sistema Límbico/efeitos dos fármacos , Receptores de Grelina/metabolismo , Convulsões/tratamento farmacológico , Convulsões/patologia , Análise de Variância , Animais , Anticonvulsivantes/farmacologia , Cálcio/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Relação Dose-Resposta a Droga , Interações Medicamentosas , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Proteínas de Fluorescência Verde/genética , Células HEK293 , Hipocampo/citologia , Humanos , Técnicas In Vitro , Sistema Límbico/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microdiálise , Agonistas Muscarínicos/toxicidade , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Pilocarpina/toxicidade , Piperidinas/uso terapêutico , Pirazóis/uso terapêutico , Ratos , Ratos Wistar , Receptores de Grelina/agonistas , Receptores de Grelina/deficiência , Convulsões/genética , Índice de Gravidade de Doença , Especificidade da Espécie , Transfecção , Ácido gama-Aminobutírico/metabolismoRESUMO
JNJ-26070109 [(R)4-bromo-N-[1-(2,4-difluoro-phenyl)-ethyl]-2-(quinoxaline-5-sulfonylamino)-benzamide] is a representative of a new chemical class of competitive antagonists of cholecystokinin 2 (CCK2) receptors. In this study, the primary in vitro pharmacology of JNJ-26070109 was evaluated along with the pharmacokinetic and pharmacodynamic properties of this compound in rat and canine models of gastric acid secretion. JNJ-26070109 expressed high affinity for human (pK(I) = 8.49 ± 0.13), rat (pK(I) = 7.99 ± 0.08), and dog (pK(I) = 7.70 ± 0.14) CCK2 receptors. The selectivity of JNJ-26070109 at the CCK2 receptor versus the CCK1 receptor was species-dependent, with the greatest degree of selectivity (>1200-fold) measured at the human isoforms of the CCK1 receptor (selectivity at CCK2 versus CCK1 receptors: human, â¼1222-fold; rat, â¼324-fold; dog â¼336-fold). JNJ-26070109 behaved as a surmountable, competitive, antagonist of human CCK2 receptors in a calcium mobilization assay (pK(B) = 8.53 ± 0.05) and in pentagastrin-stimulated gastric acid secretion in the isolated, lumen-perfused, mouse stomach assay (pK(B) = 8.19 ± 0.13). The pharmacokinetic profile of this compound was determined in vivo in rats and dogs. JNJ-26070109 was shown to have high oral bioavailability (%F rat = 73 ± 16; %F dog = 92 ± 12) with half lives of 1.8 ± 0.3 and 1.2 ± 0.1 h in rat and dog, respectively. The pharmacodynamic properties of this compound were investigated using two in vivo models. In conscious rat and dog chronic gastric fistula models of pentagastrin-stimulated acid secretion, JNJ-26070109 had oral EC(50) values of 1.5 and 0.26 µM, respectively. Overall, we have demonstrated that JNJ-26070109 is a high-affinity, selective CCK2 receptor antagonist with good pharmacokinetic properties.
Assuntos
Benzodiazepinonas/administração & dosagem , Benzodiazepinonas/metabolismo , Compostos de Fenilureia/administração & dosagem , Compostos de Fenilureia/metabolismo , Quinoxalinas/administração & dosagem , Receptor de Colecistocinina B/antagonistas & inibidores , Receptor de Colecistocinina B/metabolismo , Sulfonamidas/administração & dosagem , Administração Oral , Animais , Benzodiazepinonas/química , Disponibilidade Biológica , Células CHO , Células CACO-2 , Cricetinae , Cricetulus , Cães , Relação Dose-Resposta a Droga , Feminino , Cobaias , Humanos , Masculino , Camundongos , Compostos de Fenilureia/química , Quinoxalinas/química , Quinoxalinas/metabolismo , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie , Sulfonamidas/química , Sulfonamidas/metabolismoRESUMO
Slow waves are known to originate orally in the stomach and to propagate toward the antrum, but the exact location of the pacemaker and the precise pattern of propagation have not yet been studied. Using assemblies of 240 extracellular electrodes, simultaneous recordings of electrical activity were made on the fundus, corpus, and antrum in open abdominal anesthetized dogs. The signals were analyzed off-line, pathways of slow wave propagation were reconstructed, and slow wave velocities and amplitudes were measured. The gastric pacemaker is located in the upper part of the fundus, along the greater curvature. Extracellularly recorded slow waves in the pacemaker area exhibited large amplitudes (1.8 +/- 1.0 mV) and rapid velocities (1.5 +/- 0.9 cm/s), whereas propagation in the remainder of the fundus and in the corpus was slow (0.5 +/- 0.2 cm/s) with low-amplitude waveforms (0.8 +/- 0.5 mV). In the antrum, slow wave propagation was fast (1.5 +/- 0.6 cm/s) with large amplitude deflections (2.0 +/- 1.3 mV). Two areas were identified where slow waves did not propagate, the first in the oral medial fundus and the second distal in the antrum. Finally, recordings from the entire ventral surface revealed the presence of three to five simultaneously propagating slow waves. High resolution mapping of the origin and propagation of the slow wave in the canine stomach revealed areas of high amplitude and rapid velocity, areas with fractionated low amplitude and low velocity, and areas with no propagation; all these components together constitute the elements of a gastric conduction system.
Assuntos
Relógios Biológicos/fisiologia , Fenômenos Eletrofisiológicos/fisiologia , Motilidade Gastrointestinal/fisiologia , Estômago/fisiologia , Animais , Cães , Eletromiografia , Feminino , Fundo Gástrico/fisiologia , Masculino , Modelos Biológicos , Antro Pilórico/fisiologia , Piloro/fisiologiaRESUMO
BACKGROUND & AIMS: Gastric arrhythmias occur in humans and experimental animals either spontaneously or induced by drugs or diseases. However, there is no information regarding the origin or the propagation patterns of the slow waves that underlie such arrhythmias. METHODS: To elucidate this, simultaneous recordings were made on the antrum and the distal corpus during tachygastrias in open abdominal anesthetized dogs using a 240 extracellular electrode assembly. After the recordings, the signals were analyzed, and the origin and path of slow wave propagations were reconstructed. RESULTS: Several types of arrhythmias could be distinguished, including (1) premature slow waves (25% of the arrhythmias), (2) single aberrant slow waves (4%), (3) bursts (18%), (4) regular tachygastria (11%), and (5) irregular tachygastria (10%). During regular tachygastria, rapid, regular slow waves emerged from the distal antrum or the greater curvature, whereas, during irregular tachygastria, numerous variations occurred in the direction of propagation, conduction blocks, focal activity, and re-entry. In 12 cases, the arrhythmia was initiated in the recorded area. In each case, after a normal propagating slow wave, a local premature slow wave occurred in the antrum. These premature slow waves propagated in various directions, often describing a single or a double loop that re-entered several times, thereby initiating additional slow waves. CONCLUSIONS: Gastric arrhythmias resemble those in the heart and share many common features such as focal origin, re-entry, circular propagation, conduction blocks, and fibrillation-like behavior.
Assuntos
Sistema de Condução Cardíaco/fisiopatologia , Frequência Cardíaca/fisiologia , Gastropatias/complicações , Estômago/fisiopatologia , Taquicardia por Reentrada no Nó Sinoatrial/etiologia , Animais , Modelos Animais de Doenças , Cães , Eletrodiagnóstico/métodos , Feminino , Gastropatias/fisiopatologia , Taquicardia por Reentrada no Nó Sinoatrial/fisiopatologiaRESUMO
BACKGROUND & AIMS: Ghrelin is an orexigenic peptide with gastroprokinetic effects. Mice with streptozotocin (STZ)-induced diabetes exhibit hyperphagia, altered gastric emptying, and increased plasma ghrelin levels. We investigated the causative role of ghrelin herein by comparing changes in ghrelin receptor knockout (growth hormone secretagogue receptor [GHS-R](-/-)) and wild-type (GHS-R(+/+)) mice with STZ-induced diabetes. METHODS: Gastric emptying was measured with the [(13)C]octanoic acid breath test. The messenger RNA (mRNA) expression of neuropeptide Y (NPY), agouti-related peptide (AgRP), and proopiomelanocortin was quantified by real-time reverse-transcription polymerase chain reaction. Neural contractions were elicited by electrical field stimulation in fundic smooth muscle strips. RESULTS: Diabetes increased plasma ghrelin levels to a similar extent in both genotypes. Hyperphagia was more pronounced in GHS-R(+/+) than in GHS-R(-/-) mice between days 12 and 21. Increases in NPY and AgRP mRNA expression were less pronounced in diabetic GHS-R(-/-) than in GHS-R(+/+) mice from day 15 on, whereas decreases in proopiomelanocortin mRNA levels were similar in both genotypes. Gastric emptying was accelerated to a similar extent in both genotypes, starting on day 16. In fundic smooth muscle strips of diabetic GHS-R(+/+) and GHS-R(-/-) mice, neuronal relaxations were reduced, whereas contractions were increased; this increase was related to an increased affinity of muscarinic and tachykinergic receptors. CONCLUSIONS: Diabetic hyperphagia is regulated by central mechanisms in which the ghrelin-signaling pathway affects the expression of NPY and AgRP in the hypothalamus. The acceleration of gastric emptying, which is not affected by ghrelin signaling, is not the cause of diabetic hyperphagia and probably involves local contractility changes in the fundus.